HK1152076A - Epitope sequences - Google Patents

Description

Epitope sequences

The application is a divisional application of an invention patent application with the application date of 2002, 4/4 and the application number of 02811398.5 and named as an 'epitope sequence'.

Technical Field

The present invention relates generally to peptides, and nucleic acids encoding peptides, which are effective epitopes of target-associated antigens. More specifically, the invention relates to epitopes with high affinity for MHC class I and produced by target-specific proteasomes.

Description of the Related Art

Neoplasia and immune system

A neoplastic disease state commonly referred to as cancer is believed to be caused by a single cell with uncontrolled growth. Uncontrolled growth states are typically caused by a multi-stage process in which a series of cell systems fail, resulting in the development of neoplastic cells. The resulting neoplastic cell rapidly multiplies itself to form one or more tumors, which ultimately can result in the death of the host.

Since progenitors of neoplastic cells share the genetic material of the host, neoplastic cells are mostly not attacked by the host immune system. During immune surveillance, in the process the host immune system monitors and localizes foreign substances, and neoplastic cells appear to the host immune surveillance machinery as "self cells.

Virus and immune system

In contrast to cancer cells, viral infection involves the expression of apparently non-self antigens. As a result, the immune system successfully addresses many viral infections with minimal clinical sequelae. In addition, effective vaccines have been developed for many of those infections that cause severe disease. Various vaccine approaches have been successfully used to combat a variety of diseases. These methods include subunit vaccines, which consist of individual proteins produced by recombinant DNA techniques. Despite these advances, the selection and effective administration of the smallest epitopes for use as viral vaccines remains problematic.

In addition to the difficulties associated with epitope selection, there is a problem with viruses that have evolved to evade the ability of the host immune system. Many viruses, especially those that establish persistent infections, such as members of the herpes and poxvirus families, produce immune modulatory molecules that allow the virus to evade the host immune system. The effect of these immunomodulatory molecules on antigen presentation can be overcome by targeting selected epitopes for administration as immunogenic compositions. To better understand the interaction of neoplastic and virally infected cells with the host immune system, the system components are discussed next below.

The immune system serves to distinguish molecules endogenous to an organism (the "self" molecules) from substances that are foreign or foreign to the organism (the "non-self" molecules). Based on the components that mediate the response, the immune system has two types of adaptive responses to foreign objects: humoral responses and cell-mediated responses. Humoral responses are mediated by antibodies, while cell-mediated responses involve cells classified as lymphocytes. Recent anticancer and antiviral strategies have focused on mobilizing the host immune system as a means of anticancer or antiviral therapy or therapy.

The immune system functions in three phases to protect the host from foreign objects: an identification phase, an activation phase and an effect phase. In the recognition phase, the immune system recognizes and signals the presence of foreign antigens or invaders within the body. The foreign antigen may be, for example, a cell surface marker derived from a neoplastic cell or a viral protein. Once the system detects an invader, antigen-specific cells of the immune system proliferate and differentiate in response to invader-triggered signals. The final phase is the effector phase, in which effector cells of the immune system respond and neutralize the detected invader.

A series of effector cells carry out an immune response to an invader. One type of effector cell, the B cell, produces antibodies that target foreign antigens encountered by the host. In conjunction with the complement system, antibodies direct the destruction of cells or organisms that carry the target antigen. Another class of effector cells are natural killer cells (NK cells), a class of lymphocytes that have the ability to recognize and destroy multiple virally infected cells as well as malignant cell types simultaneously. The method by which NK cells recognize target cells is not well understood.

Another class of effector cells, T cells, have members classified into three subclasses, each of which plays a different role in the immune response. Helper T cells secrete cytokines that stimulate the proliferation of other cells necessary to produce an effective immune response, while suppressor T cells down-regulate the immune response. The third type of T cell, cytotoxic T Cells (CTL), is capable of directly lysing target cells presenting foreign antigens on its surface.

Major histocompatibility complex and T cell targeting

T cells are antigen-specific immune cells that play a role in response to specific antigen signals. B lymphocytes and the antibodies they produce are also antigen-specific entities. However, unlike B lymphocytes, T cells do not respond to antigens in free or soluble form. In order for a T cell to respond to an antigen, the antigen needs to be processed into a peptide, which is then bound to a presentation structure encoded in the Major Histocompatibility Complex (MHC). This requirement is termed "MHC restriction" and is the mechanism by which T cells distinguish "self" from "non-self" cells. If an antigen is not displayed by a recognizable MHC molecule, the T cell will not recognize and act on the antigen signal. Specific T cells that bind peptides that recognize MHC molecules bind to these MHC-peptide complexes and enter the next stage of the immune response.

There are two classes of MHC, MHC class I and MHC class II. T helper cell (CD 4)⁺) Cytolytic T cells, which interact mainly with MHC class II proteins (CD 8)⁺) Mainly interacting with class I MHC proteins. Both classes of MHC proteins are transmembrane proteins, most of which are structured on the outer surface of cells. In addition, two classes of MHC proteins exist in itThey all have a peptide binding cleft (binding cleft) on their exterior. Small fragments of endogenous or exogenous proteins are bound in the cleft and presented in the extracellular environment.

Cells called "professional antigen presenting cells" (pAPCs) display antigens to T cells using MHC proteins, but additionally express various costimulatory molecules, depending on the specific state of differentiation/activation of pAPCs. When specific T cells that recognize MHC protein-binding peptides bind to these MHC-peptide complexes on pAPCs, specific co-stimulatory molecules acting on the T cells direct the differentiation/activation pathway taken by the T cells. That is, the costimulatory molecule affects how T cells will act on antigen signaling when it enters the next stage of the immune response in future encounters.

As discussed above, most neoplastic cells are ignored by the immune system. A great deal of effort is being expended in efforts to control the host immune system to help fight the presence of neoplastic cells in the host. One such area of research involves formulating anti-cancer vaccines.

Anti-cancer vaccine

The immune system of a patient is one of the various weapons available to oncologists in fighting against cancer. Work has been done in various efforts to combat the immune system against cancer or neoplastic disease. Unfortunately, the results to date have been largely disappointing. One area of particular interest relates to the production and use of anti-cancer vaccines.

To produce a vaccine or other immunogenic composition, it is necessary to introduce into a subject an antigen or epitope against which an immune response can be raised. Although neoplastic cells are derived from normal cells and are therefore essentially identical at the genetic level to normal cells, many neoplastic cells are known to present tumor associated antigens (TuAAs). In theory, the subject's immune system can utilize these antigens to recognize these antigens and attack neoplastic cells. However, in practice neoplastic cells often appear to be ignored by the host's immune system.

Many different strategies have been developed in an attempt to produce vaccines with activity against neoplastic cells. These strategies include the use of tumor-associated antigens as immunogens. For example, U.S. patent No.5,993,828 describes a method of generating an immune response against a specific subunit of a Urinary Tumor Associated Antigen (urory Tumor Associated Antigen) by administering to a subject an effective amount of a composition comprising inactivated Tumor cells having a Urinary Tumor Associated Antigen on the cell surface and at least one Tumor Associated Antigen selected from the group consisting of GM-2, GD-2, fetal Antigen and melanoma Associated Antigen. Thus, this patent describes the use of whole killed tumor cells as immunogens in anti-cancer vaccines.

Another strategy for using anti-cancer vaccines involves administering a composition containing the tumor antigen alone. In one approach, the MAGE-A1 antigenic peptide is used as an immunogen (see Chaux, P., et al, "Identification of Five MAGE-A1 Epitopes recognited by Cytolytic TLymphates Obtained by In Vitro Stimulation with a degraded cell transformed with MAGE-A1," J.Immunol., 163 (5): 2928-2936 (1999)). There have been several therapeutic trials using MAGE-a1 peptide for vaccination, although the efficacy of this vaccination therapy is limited. In Vose, J.M., "Tumor antibodies Reccognized by T Lymphocytes," 10^thThe results of some of these assays are discussed in the European Cancer Conference, Day 2, Sept.14, 1999.

In another example of a tumor associated antigen used as a vaccine, Scheinberg et al used 5 injections of a class I related bcr-abl peptide and a helper peptide plus the adjuvant QS-21 to treat 12 Chronic Myelogenous Leukemia (CML) patients who had received Interferon (IFN) or hydroxyurea. Scheinberg, D.A., et al, "BCR-ABL BreakPoint Derived Oncogene Fusion Peptide Vaccines in Patients with Chronic Myelogenogens Leukemia (CML) [ Abstract 1665]，American Society of Clinical Oncology 35^thAnnual Meeting, atlanta (1999). Triggering a proliferative and delayed-type hypersensitivity (DTH) T cell response indicative of T-helper cell activity, but in fresh bloodNo cytolytic killer T cell activity was observed in the samples.

Additional examples of attempts to identify TuAAs for use as vaccines are seen in recent work by Cebon et al and Scheibenbogen et al. Cebon et al use MART-1 administered intradermally_26-35Peptides and IL-12 administered subcutaneously or intravenously in increasing doses immunize patients with metastatic melanoma. Of the first 15 patients, 1 complete remission, 1 partial remission, and 1 mixed response were noted. Immunoassays for T cell production include DTH, which is found in patients with or without IL-12. Positive CTL assays were found in patients with evidence of clinical benefit (clinical benefit), but not in patients without tumor regression. Cebon et al, "Phase I students of Immunization with Melan-A and IL-12in HLA A2+ Positive Patients with Stage III and IV Malignant Melanoma," [ Abstract 1671]，American Society of Clinical Oncology 35^thAnnual Meeting, atlanta 1999).

Scheibenbergen et al immunized 18 patients with 4 HLA class I restricted tyrosinase peptides, 16 of which were immunized with metastatic melanoma and 2 with adjuvant. Scheibenbogen et al, "Vaccination with tyrosine peptides and GM-CSF in Metastatic Melanoma: a Phase II Trial, "[ abstract 1680]，American Society of Clinical Oncology 35^thAnnual Meeting, atlanta (1999). Enhanced CTL activity was observed in 4/15 patients, 2 of whom were immunized with adjuvant and 2 of whom had evidence of tumor regression. Patients with progressive disease (progressive disease) did not show enhanced immunity as in the Cebon et al trial. Despite the various efforts so far spent to produce effective anti-cancer vaccines, such compositions have not been developed.

Antiviral vaccine

Vaccine strategies to protect against viral diseases have met with much success. Perhaps the most significant of these has been the advancement made against the disease smallpox, which has been eradicated. The success of polio vaccines is of similar importance.

Viral vaccines can be divided into three categories: live attenuated virus vaccines, such as vaccinia for smallpox, sabin poliovirus vaccine, and measles mumps and rubella; completely killed or inactivated virus vaccines, such as the solvay poliovirus vaccine, the hepatitis a virus vaccine and the classical influenza virus vaccine; and subunit vaccines, such as hepatitis b. Due to their lack of a complete viral genome, subunit vaccines offer a greater degree of safety than those based on complete viruses.

An example of a successful subunit vaccine is a recombinant hepatitis b vaccine based on viral envelope proteins. Although many researchers are interested in pushing the concept of reduced speaker subunits beyond a single protein to individual epitopes, this effort has not produced much effort. Viral vaccine research has also focused on inducing antibody responses, although cellular responses also occur. However, many subunit preparations are particularly poor at generating CTL responses.

Summary of The Invention

Previous methods for eliciting the display of target cell epitopes by professional antigen presenting cells (pAPCs) have simply relied on making pAPCs express Target Associated Antigens (TAAs), or those epitopes that are believed to have high affinity for MHC class I molecules. However, proteasomal processing of such antigens results in epitopes presented on pAPC that do not correspond to epitopes on the target cell.

Using the knowledge that an effective cellular immune response requires pAPCs to present the same epitope presented by the target cell, the present invention provides epitopes with high affinity for MHC I and corresponding to the processing specificity of housekeeping proteasomes active in surrounding cells. These epitopes thus correspond to those presented on the target cells. The use of such epitopes in vaccines can activate cellular immune responses to recognize properly processed TAAs and can lead to the removal of target cells presenting such epitopes. In some embodiments, the housekeeping epitopes provided herein can be used in conjunction with immune epitopes to generate cellular immune responses that attack target cells both before and after interferon induction. In other embodiments the epitopes are used for diagnosis and monitoring of target-related diseases and for the production of immunological reagents for such purposes.

Embodiments of the invention relate to isolated (epitopes) and antigens or polypeptides comprising the epitopes. Preferred embodiments include epitopes or antigens comprising the sequences disclosed in table 1. Other embodiments may include a cluster comprising epitopes derived from the polypeptides of table 1. In addition, various embodiments include a polypeptide substantially similar to the epitope, polypeptide, antigen or cluster already mentioned. Other preferred embodiments include a polypeptide functionally similar to any of the above. Yet another embodiment relates to a nucleic acid encoding a polypeptide from any one of the epitopes, clusters, antigens and polypeptides mentioned in table 1 and herein. For purposes of the following summary, when referring to an "epitope" or "epitopes," the discussion of other embodiments of the invention refers to all of the foregoing forms of epitopes without limitation.

The epitope may be immunologically active. The polypeptide comprising the epitope can be less than about 30 amino acids in length, more preferably, for example, the polypeptide is 8-10 amino acids in length. Similarity of matter (substential) or function may include, for example, the addition of at least one amino acid, and at least one additional amino acid may be at the N-terminus of the polypeptide. Similarity in substance or function may include substitution of at least one amino acid.

The epitope, cluster or polypeptide comprising it may have affinity for the HLA-a2 molecule. The affinity can be determined by binding assays, epitope recognition restriction assays, predictive algorithms, and the like. The epitope, cluster or polypeptide comprising it may have affinity for HLA-B7, HLA-B51 molecules, and the like.

In a preferred embodiment, the polypeptide may be a housekeeping epitope. The epitope or polypeptide may correspond to an epitope displayed on tumor cells, to an epitope displayed on neovasculature (neovasculature) cells, and the like. The epitope or polypeptide may be an immune epitope. The epitope, cluster and/or polypeptide may be a nucleic acid.

Other embodiments relate to pharmaceutical compositions comprising a plurality of polypeptides comprising an epitope, cluster, or polypeptide comprising the same derived from table 1, and a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, or the like. The adjuvant may be a polynucleotide. The polynucleotide may comprise a dinucleotide, which may be, for example, a CpG. The adjuvant may be encoded by one polynucleotide. The adjuvant may be a cytokine, which may be, for example, GM-CSF.

The pharmaceutical composition may additionally comprise a professional antigen presenting cell (pAPC). pAPC can be, for example, a dendritic cell. The pharmaceutical composition may additionally comprise a second epitope. The second epitope can be a polypeptide, a nucleic acid, a housekeeping epitope, an immunological epitope, or the like.

Still further embodiments relate to pharmaceutical compositions comprising any of the nucleic acids discussed herein, including those encoding polypeptides comprising epitopes or antigens from table 1. Such compositions may include pharmaceutically acceptable adjuvants, carriers, diluents, excipients and the like.

Other embodiments relate to recombinant constructs comprising such nucleic acids as described herein, including those encoding polypeptides comprising epitopes or antigens from table 1. The construct may additionally include plasmids, viral vectors, artificial chromosomes, and the like. The construct may additionally include a sequence encoding at least one feature (feature), e.g., a second epitope, an IRES, an ISS, an NIS, an ubiquitin, etc.

Further embodiments relate to purified antibodies that specifically bind to at least one epitope in table 1. Other embodiments relate to purified antibodies that specifically bind to a peptide-MHC protein complex comprising an epitope disclosed in table 1 or any other suitable epitope. The antibody derived from any of the embodiments may be a monoclonal antibody or a polyclonal antibody.

Still other embodiments relate to multimeric MHC-peptide complexes comprising an epitope, such as, for example, an epitope disclosed in table 1. Likewise, antibodies specific for the complex are contemplated.

Various embodiments relate to isolated T cells expressing a T cell receptor specific for an MHC-peptide complex. The complex may comprise an epitope, such as, for example, the epitopes disclosed in table 1. T cells can be produced by in vitro immunization and can be isolated from immunized animals. Various embodiments relate to T cell cloning, including cloned T cells, such as those discussed above. Various embodiments also relate to polyclonal populations of T cells. The population may include, for example, T cells as described above.

Still further embodiments relate to pharmaceutical compositions comprising T cells such as those described above and pharmaceutically acceptable adjuvants, carriers, diluents, excipients and the like.

Various embodiments of the present invention relate to isolated protein molecules comprising a binding domain of a T cell receptor specific for an MHC-peptide complex. The complex may comprise an epitope disclosed in table 1. The protein may be multivalent. Other embodiments relate to isolated nucleic acids encoding the proteins. Still further embodiments relate to recombinant constructs comprising the nucleic acids.

Other embodiments of the invention relate to host cells expressing recombinant constructs as described herein, including constructs encoding an epitope, cluster or polypeptide comprising the same, e.g., as disclosed in table 1. The host cell may be a dendritic cell, macrophage, tumor cell, tumor-derived cell, bacterium, fungus, protozoan, or the like. Various embodiments also relate to pharmaceutical compositions comprising a host cell, such as those discussed herein, and a pharmaceutically acceptable adjuvant, carrier, diluent, excipient, or the like.

Still other embodiments relate to a vaccine or immunotherapeutic composition comprising at least one component, such as an epitope disclosed in table 1 or otherwise described herein; a cluster comprising the epitope, an antigen or polypeptide comprising the epitope; a composition as described above and herein; a construct as described above and herein, a T cell, or a host cell as described above and herein.

Additional embodiments relate to methods of treating animals. The method may comprise administering to the animal a pharmaceutical composition, such as a vaccine or immunotherapeutic composition, including those as disclosed above and herein. The administration step may include a mode of delivery such as, for example, transdermal, intranodal (intradal), perinodal (perinodal), oral, intravenous, intradermal, intramuscular, intraperitoneal, mucosal, aerosol inhalation, instillation, and the like. The method may additionally comprise an assay step to determine a characteristic representation of the state of a target cell or target cells. The method can include a first assay step and a second assay step, wherein the first assay step precedes the administering step, and wherein the second assay step follows the administering step. The method may further comprise a step of comparing the property determined in the first determining step with the property determined in the second determining step to obtain a result. The result may be, for example, an indication of an immune response, a reduction in the number of target cells, a reduction in the mass or size of a tumor containing the target cells, a reduction in the number and concentration of intracellular parasites infecting the target cells, and the like.

Various embodiments relate to methods of assessing the immunogenicity of a vaccine or immunotherapeutic composition. The method can include administering a vaccine or immunotherapy, such as those described above and elsewhere herein, to the animal and assessing immunogenicity based on a characteristic of the animal. The animal may be an HLA-transgenic animal.

Other embodiments relate to methods of assessing immunogenicity comprising stimulating T cells in vitro with a vaccine or immunotherapeutic composition, such as those described above and elsewhere herein, and assessing immunogenicity based on one characteristic of the T cells. The stimulus may be a primary stimulus.

Still further embodiments relate to methods of performing passive/adoptive immunotherapy. The method may comprise combining T cells or host cells, such as those described above and elsewhere herein, with pharmaceutically acceptable adjuvants, carriers, diluents, excipients, and the like.

Other embodiments relate to methods of determining the frequency of specific T cells, and may include the step of contacting the T cells with an MHC-peptide complex comprising an epitope disclosed in table 1 or a complex comprising a cluster or antigen comprising such an epitope. The contacting step can comprise at least one characteristic, such as, for example, immunization, restimulation (stimulation), detection, counting, and the like. The method may additionally comprise ELISPOT analysis, limiting dilution analysis, flow cytometry, in situ hybridization, polymerase chain reaction, any combination thereof, and the like.

Various embodiments relate to methods of assessing an immune response. The method may comprise the above-described method of determining the frequency of specific T cells carried out before and after the immunization step.

Other embodiments relate to methods of assessing an immune response. The method may comprise determining the frequency, cytokine production or cytolytic activity of T cells before and after stimulation with an MHC-peptide complex comprising an epitope, such as an epitope from table 1, a cluster or polypeptide comprising the epitope.

Additional embodiments relate to methods of diagnosing a disease. The methods comprise contacting a subject tissue with at least one component including, for example, T cells, host cells, antibodies, proteins, including those described above and elsewhere herein; and diagnosing the disease based on a characteristic of the tissue or component. The contacting step can occur, for example, in vivo or in vitro.

Still other embodiments relate to methods of producing vaccines. The methods may comprise contacting at least one component, epitope, composition, construct, T cell, host cell; including any of those described above and elsewhere herein, in combination with pharmaceutically acceptable adjuvants, carriers, diluents, excipients, and the like.

Various embodiments relate to a nucleic acid molecule having recorded thereon SEQ ID NOS: 1-602 in a machine having hardware or software for calculating the physical, biochemical, immunological, molecular genetic characteristics of a molecule comprising said sequence.

Still other embodiments relate to methods of treating animals. The methods may include methods of conjointly treating an animal comprising administering to the animal a vaccine or immunotherapeutic composition, such as described above and elsewhere herein, in combination with at least one treatment modality, including, for example, radiation therapy, chemotherapy, biochemical therapy (biochemotherapy), surgery, and the like.

Further embodiments relate to isolated polypeptides comprising an epitope cluster. In a preferred embodiment, the cluster may be from a target-associated antigen comprising a sequence as disclosed in any one of tables 25-44, wherein the amino acid sequence comprises up to about 80% of the amino acid sequence of the antigen.

Other embodiments relate to a vaccine or immunotherapeutic product comprising an isolated peptide as described above and elsewhere herein. Still other embodiments relate to an isolated polynucleotide encoding a polypeptide as described above and elsewhere herein. Other embodiments relate to vaccines or immunotherapeutic products comprising these polynucleotides. The polynucleotide may be DNA, RNA, etc.

Still other embodiments relate to kits comprising a delivery device and any of the embodiments mentioned above and elsewhere herein. The delivery device may be a catheter, syringe, internal or external pump, reservoir, inhaler, micro-syringe, patch (patch) and any other similar device suitable for any delivery route. As noted above, the kit includes any of the embodiments disclosed herein in addition to the delivery device. For example, without limitation, a kit can include an isolated epitope, polypeptide, cluster, nucleic acid, antigen, pharmaceutical composition comprising any of the foregoing, antibody, T cell receptor, epitope-MHC complex, vaccine, immunotherapeutic agent, and the like. The kit may also include articles such as detailed instructions for use and any other similar articles.

Brief Description of Drawings

FIG. 1 is a sequence alignment of NY-ESO-1 with several similar protein sequences.

Figure 2 illustrates a plasmid vaccine backbone for delivery of nucleic acid encoded epitopes.

FIGS. 3A and 3B are diagrams showing tyrosinase_207-215And tyrosinase_208-216FACS curves of HLA-A2 binding assay results.

Fig. 3C shows cytolytic activity of human CTL against tyrosinase epitopes induced by in vitro immunization.

FIG. 4 is the cleavage of SSX-2 by proteasome_31-68A mass spectrum is generated at the moment when the fragment T is 120min.

FIG. 5 shows HLA-A2: SSX-2_41-49Binding curves to control.

FIG. 6 shows a sample derived from SSX-2_41-49CTL against SSX-2 in immunized HLA-A2 transgenic mice_41-49Specific lysis of pulsed targets.

FIGS. 7A, B and C show PSMA at time T ═ 60min_163-192Results of proteasome digested aliquot N-terminal pool sequencing (pool sequencing).

FIG. 8 shows HLA-A2: PSMA_168-177And HLA-a 2: PSMA_288-297Binding curves to control.

FIG. 9 shows PSMA at time T60 min_281-310Results of sequencing of the proteasome digested aliquot of the N-terminal pool.

FIG. 10 shows HLA-A2: PSMA_461-469，HLA-A2：PSMA_460-469And HLA-a 2: PSMA_663-671Binding curves to control.

FIG. 11 shows detection of PSMA_463-471Reactive HLA-A1⁺CD8⁺Results of γ -IFN-based ELISPOT assay of T cells.

FIG. 12 shows the activity of blocking the T cells used in FIG. 10 with anti-HLA-A1 mAb, which shows HLA-A1-restricted recognition.

FIG. 13 shows HLA-A2: PSMA_663-671Binding curves to control.

FIG. 14 shows HLA-A2: PSMA_662-671Binding curves to control.

Figure 15 compares the anti-peptide CTL response after immunization with different doses of DNA by different injection routes (figure shows lysis of EL4 cells without pulse < open circles > and EL4 cells pulsed with gp33 peptide (solid triangles), symbols representing individual mice and showing one of three similar experiments).

FIG. 16 growth of transplanted gp33 expressing tumors in mice immunized by intralymph node injection expressing gp33 epitopes or control, plasmid (mean tumor volume. + -.1 SD for mice immunized with pEFGPL33A DNA (full circles) or control pEGFP-N3DNA (open triangles), numbers in parentheses indicate the number of mice bearing tumors/total number of mice in the group, showing 1 in 2 similar experiments).

FIG. 17 amount of plasmid DNA in injected or drained (draining) lymph nodes detected by real-time PCR at different times within lymph nodes and after intramuscular injection, respectively.

Detailed description of the preferred embodiments

Definition of

Unless otherwise clear from the context in which the terms herein are used, the terms listed below should generally have the indicated meanings for the purposes of this description.

Professional antigen presenting cells (papcs), cells that have a T cell costimulatory molecule and are capable of inducing a T cell response. Fully characterized pAPCs include dendritic cells, B cells and macrophages.

Surrounding cells-cells that are not pAPC.

Housekeeping proteasomes, proteasomes that are usually active in surrounding cells, are usually absent or not strongly active in pAPCs.

Immunoproteasome-the proteasome normally active in pAPCs; immunoproteasome is also active in some of the surrounding cells of infected tissue.

Epitope-a molecule or substance capable of stimulating an immune response. In a preferred embodiment, an epitope according to the present definition includes, but is not necessarily limited to, a polypeptide and a nucleic acid encoding a polypeptide, wherein the polypeptide is capable of stimulating an immune response. In other preferred embodiments, epitopes according to the present definition include, but are not necessarily limited to, peptides present on the cell surface that bind non-covalently to the MHC class I binding cleft so that they can interact with T cell receptors.

MHC epitope-a polypeptide having known or predicted binding affinity to a mammalian class I or class II Major Histocompatibility Complex (MHC) molecule.

Housekeeping epitope-in a preferred embodiment, a housekeeping epitope is defined as a polypeptide fragment, which is an MHC epitope, and which is displayed on cells in which the activity of the housekeeping proteasome is prominent. In another preferred embodiment, the housekeeping epitope is defined as a polypeptide comprising a housekeeping epitope according to the preceding definition, flanked by one to several additional amino acids. In another preferred embodiment, the housekeeping epitope is defined as a nucleic acid encoding a housekeeping epitope according to the preceding definition.

Immune epitope-in a preferred embodiment, an immune epitope is defined as a polypeptide fragment, which is an MHC epitope, and is displayed on cells in which housekeeping proteasome activity is prominent. In another preferred embodiment, an immune epitope is defined as a polypeptide comprising an immune epitope according to the previous definition flanked by one to several additional amino acids. In another preferred embodiment, an immune epitope is defined as a polypeptide comprising an epitope clustering sequence comprising at least two polypeptide sequences having known or predicted affinity for MHC class I. In yet another preferred embodiment, an immune epitope is defined as a nucleic acid encoding an immune epitope according to any one of the above definitions.

Target cells-cells targeted by vaccines and methods of the invention. Examples of target cells according to the present definition include, but are not necessarily limited to: neoplastic cells and cells containing intracellular parasites, such as viruses, bacteria or protozoa.

Target Associated Antigen (TAA) -a protein or polypeptide present in a target cell.

Tumor associated antigen (TuAA) — TAA, wherein the target cell is a neoplastic cell.

HLA epitope-a polypeptide having known or predicted binding affinity to a human HLA class I or class II complex molecule.

Antibody-polyclonal or monoclonal native immunoglobulin (Ig), or any molecule consisting wholly or partially of an Ig-binding domain, whether biochemically derived or obtained by using recombinant DNA. Examples include, inter alia, F (ab), single chain Fv and Ig variable region-phage coat protein fusions.

Encoding-an open-ended term such that a nucleic acid encoding a particular amino acid sequence may consist of codons specifying that (poly) peptide, but may also contain additional sequences that are translatable or that are used to control transcription, translation or replication, or that facilitate manipulation of the nucleic acid construct of some hosts.

Material similarity-this term is used to refer to sequences that differ from a reference sequence in an indirect manner, as determined by examination of the sequences. Nucleic acid sequences encoding the same amino acid sequence are substantially similar despite differences in degenerate positions or modest differences in length or composition of any non-coding region. Except that amino acid sequences which differ by conservative substitutions or small length changes are substantially similar. In addition, the amino acid sequences comprising housekeeping epitopes that differ in the number of flanking residues at the N-terminus, or immunological epitopes and epitope clusters that differ in the number of flanking residues at either terminus, are substantially similar. Nucleic acids that encode substantially similar amino acid sequences are themselves also substantially similar.

Functional similarity-the term is used to refer to sequences that differ from a reference sequence in an indirect manner, as determined by assaying for a biological or biochemical property, although the sequences may not be substantially similar. For example, two nucleic acids can be used as hybridization probes for the same sequence but encoding different amino acid sequences. Even if they differ by non-conservative amino acid substitutions (and therefore do not meet the definition of material similarity), the two peptides that induce a cross-reactive CTL response are functionally similar. Paired antibodies or TCRs recognizing the same epitope may be functionally similar to each other, although there are any structural differences. In testing for functional similarity in immunogenicity, individuals are typically immunized with an "altered" antigen and tested for the ability of the priming response (Ab, CTL, cytokine production, etc.) to recognize the target antigen. Thus, two sequences can be designed that differ in some way while retaining the same function. Such designed sequence variants are in embodiments of the invention.

Table 1a. SEQ ID nos. including the epitopes in examples 1-7, 13

Table 1b. SEQ ID nos. including the epitopes in examples 14 and 15

SEQIDNO	Identity	Sequence of
			70	GP100 protein	Registration number: p40967
71	MAGE-1 protein	Registration number: p43355
			72	MAGE-2 protein	Registration number: p43356
73	MAGE-3 protein	Registration number: p43357
			74	NY-ESO-1 protein	Registration number: p78358
75	LAGE-1a proteins	Registration number: CAA11116
			76	LAGE-1b proteins	Registration number: CAA11117
77	PRAME proteins	Registration number: NP 006106
			78	PSA protein	Registration number: p07288

79	PSCA protein	Registration number: o43653
			80	GP100cds	Registration number: u20093
81	MAGE-1cds	Registration number: m77481
			82	MAGE-2cds	Registration number: l18920
83	MAGE-3cds	Registration number: u03735
			84	NY-ESO-1cDNA	Registration number: u87459
85	PRAME cDNA	Registration number: NM _006115
			86	PSAcDNA	Registration number: NM _001648
87	PSCA cDNA	Registration number: AF043498
			88	GP100630-638	LPHSSSHWL
89	GP100629-638	QLPHSSSHWL

[0106]

90	GP100614-622	LIYRRRLMK
			91	GP100613-622	SLIYRRRLMK
92	GP100615-622	IYRRRLMK
			93	GP100630-638	LPHSSSHWL
94	GP100629-638	QLPHSSSHWL
			95	MAGE-195-102	ESLFRAVI
96	MAGE-193-102	ILESLFRAVI
			97	MAGE-193-101	ILESLFRAV
98	MAGE-192-101	CILESLFRAV
			99	MAGE-192-100	CILESLFRA
100	MAGE-1263-271	EFLWGPRAL
			101	MAGE-1264-271	FLWGPRAL
102	MAGE-1264-273	FLWGPRALAE
			103	MAGE-1265-274	LWGPRALAET
104	MAGE-1268-276	PRALAETSY
			105	MAGE-1267-276	GPRALAETSY
106	MAGE-1269-277	RALAETSYV
			107	MAGE-1271-279	LAETSYVKV
108	MAGE-1270-279	ALAETSYVKV
			109	MAGE-1272-280	AETSYVKVL
110	MAGE-1271-280	LAETSYVKVL
			111	MAGE-1274-282	TSYVKVLEY
112	MAGE-1273-282	ETSYVKVLEY
			113	MAGE-1278-286	KVLEYVIKV
114	MAGE-1168-177	SYVLVTCLGL
			115	MAGE-1169-177	YVLVTCLGL
116	MAGE-1170-177	VLVTCLGL
			117	MAGE-1240-248	TQDLVQEKY
118	MAGE-1239-248	LTQDLVQEKY
			119	MAGE-1232-240	YGEPRKLLT
120	MAGE-1243-251	LVQEKYLEY
			121	MAGE-1242-251	DLVQEKYLEY
122	MAGE-1230-238	SAYGEPRKL
			123	MAGE-1278-286	KVLEYVIKV
124	MAGE-1277-286	VKVLEYVIKV

125	MAGE-1276-284	YVKVLEYVI
			126	MAGE-1274-282	TSYVKVLEY
127	MAGE-1273-282	ETSYVKVLEY
			128	MAGE-1283-291	VIKVSARVR
129	MAGE-1282-291	YVIKVSARVR
			130	MAGE-2115-122	ELVHFLLL
131	MAGE-2113-122	MVELVHFLLL

[0107]

132	MAGE-2109-116	ISRKMVEL
			133	MAGE-2108-116	AISRKMVEL
134	MAGE-2107-116	AAISRKMVEL
			135	MAGE-2112-120	KMVELVHFL
136	MAGE-2109-117	ISRKMVELV
			137	MAGE-2108-117	AISRKMVELV
138	MAGE-2116-124	LVHFLLLKY
			139	MAGE-2115-124	ELVHFLLLKY
140	MAGE-2111-119	RKMVELVHF
			141	MAGE-2158-166	LQLVFGIEV
142	MAGE-2157-166	YLQLVFGIEV
			143	MAGE-2159-167	QLVFGIEVV
144	MAGE-2158-167	LQLVFGIEVV
			145	MAGE-2164-172	IEVVEVVPI
146	MAGE-2163-172	GIEVVEVVPI
			147	MAGE-2162-170	FGIEVVEVV
148	MAGE-2154-162	ASEYLQLVF
			149	MAGE-2153-162	KASEYLQLVF
150	MAGE-2218-225	EEKIWEEL
			151	MAGE-2216-225	APEEKIWEEL
152	MAGE-2216-223	APEEKIWE
			153	MAGE-2220-228	KIWEELSML
154	MAGE-2219-228	EKIWEELSML
			155	MAGE-2271-278	FLWGPRAL
156	MAGE-2271-279	FLWGPRALI
			157	MAGE-2278-286	LIETSYVKV
158	MAGE-2277-286	ALIETSYVKV
			159	MAGE-2276-284	RALIETSYV
160	MAGE-2279-287	IETSYVKVL
			161	MAGE-2278-287	LIETSYVKVL
162	MAGE-3271-278	FLWGPRAL
			163	MAGE-3270-278	EFLWGPRAL
164	MAGE-3271-279	FLWGPRALV
			165	MAGE-3276-284	RALVETSYV
166	MAGE-3272-280	LWGPRALVE
			167	MAGE-3271-280	FLWGPRALVE
168	MAGE-3272-281	LWGPRALVET
			169	NY-ESO-182-90	GPESRLLEF
170	NY-ESO-183-91	PESRLLEFY

171	NY-ESO-182-91	GPESRLLEFY
			172	NY-ESO-184-92	ESRLLEFYL
173	NY-ESO-186-94	RLLEFYLAM

[0108]

174	NY-ESO-188-96	LEFYLAMPF
			175	NY-ESO-187-96	LLEFYLAMPF
176	NY-ESO-193-102	AMPFATPMEA
			177	NY-ESO-194-102	MPFATPMEA
178	NY-ESO-1115-123	PLPVPGVLL
			179	NY-ESO-1114-123	PPLPVPGVLL
180	NY-ESO-1116-123	LPVPGVLL
			181	NY-ESO-1103-112	ELARRSLAQD
182	NY-ESO-1118-126	VPGVLLKEF
			183	NY-ESO-1117-126	PVPGVLLKEF
184	NY-ESO-1116-123	LPVPGVLL
			185	NY-ESO-1127-135	TVSGNILTI
186	NY-ESO-1126-135	FTVSGNILTI
			187	NY-ESO-1120-128	GVLLKEFTV
188	NY-ESO-1121-130	VLLKEFTVSG
			189	NY-ESO-1122-130	LLKEFTVSG
190	NY-ESO-1118-126	VPGVLLKEF
			191	NY-ESO-1117-126	PVPGVLLKEF
192	NY-ESO-1139-147	AADHRQLQL
			193	NY-ESO-1148-156	SISSCLQQL
194	NY-ESO-1147-156	LSISSCLQQL
			195	NY-ESO-1138-147	TAADHRQLQL
196	NY-ESO-1161-169	WITQCFLPV
			197	NY-ESO-1157-165	SLLMWITQC
198	NY-ESO-1150-158	SSCLQQLSL
			199	NY-ESO-1154-162	QQLSLLMWI
200	NY-ESO-1151-159	SCLQQLSLL
			201	NY-ESO-1150-159	SSCLQQLSLL
202	NY-ESO-1163-171	TQCFLPVFL
			203	NY-ESO-1162-171	ITQCFLPVFL
204	PRAME 219-227	PMQDIKMIL
			205	PRAME 218-227	MPMQDIKMIL
206	PRAME 428-436	QHLIGLSNL
			207	PRAME 427-436	LQHLIGLSNL
208	PRAME 429-436	HLIGLSNL
			209	PRAME 431-439	IGLSNLTHV
210	PRAME 430-439	LIGLSNLTHV
			211	PSA 53-61	VLVHPQWVL
212	PSA 52-61	GVLVHPQWVL
			213	PSA 52-60	GVLVHPQWV
214	PSA 59-67	WVLTAAHCI
			215	PSA 54-63	LVHPQWVLTA

[0109]

216	PSA 53-62	VLVHPQWVLT
			217	PSA 54-62	LVHPQWVLT
218	PSA 66-73	CIRNKSVI
			219	PSA 65-73	HCIRNKSVI
220	PSA56-64	HPQWVLTAA
			221	PSA 63-72	AAHCIRNKSV
222	PSCA 116-123	LLWGPGQL
			223	PSCA 115-123	LLLWGPGQL
224	PSCA 114-123	GLLLWGPGQL
			225	PSCA 99-107	ALQPAAAIL
226	PSCA 98-107	HALQPAAAIL
			227	Tyr128-137	APEKDKFFAY
228	Tyr129-137	PEKDKFFAY
			229	Tyr 130-138	EKDKFFAYL
230	Tyr 131-138	KDKFFAYL
			231	Tyr205-213	PAFLPWHRL
232	Tyr 204-213	APAFLPWHRL
			233	Tyr214-223	FLLRWEQEIQ
234	TyR212-220	RLFLLRWEQ
			235	Tyr191-200	GSEIWRDIDF
236	Tyr192-200	SEIWRDIDF
			237	Tyr 473-481	RIWSWLLGA
238	Tyr 476-484	SWLLGAAMV
			239	Tyr 477-486	WLLGAAMVGA
240	Tyr478-486	LLGAAMVGA
			241	PSMA 4-12	LLHETDSAV
242	PSMA 13-21	ATARRPRWL
			243	PSMA53-61	TPKHNMKAF
244	PSMA 64-73	ELKAENIKKF
			245	PSMA69-77	NIKKFLH1NF
246	PSMA 68-77	ENIKKFLH1NF
			247	PSMA 220-228	AGAKGVILY
248	PSMA468-477	PLMYSLVHNL
			249	PSMA 469-477	LMYSLVHNL
250	PSMA 463-471	RVDCTPLMY
			251	PSMA 465-473	DCTPLMYSL
252	PSMA 507-515	SGMPRISKL
			253	PSMA 506-515	FSGMPRISKL
254	NY-ESO-1136-163R	LTAADHRQLQLSISSCLQQLSLLMWIT
			255	NY-ESO-1150-177	SSCLQQLSLLMWITQCFLPVFLAQPPSG

[0110] ¹This H is reported as Y in the SWISSPROT database.

²Depending on the database, the amino acid at position 274 may be Pro or Leu. The specific assay presented here uses Pro.

Table 1c. SEQ ID nos. including the epitope in example 14

Any of SEQ ID nos.1, 8, 9, 11-23, 26-29, 32-44, 47-54, 56-63, 66-68, 88-253, and 256-588 may be used as an epitope in any of the various embodiments of the invention. As described in various embodiments of the invention, SEQ ID nos.10, 30, 31, 45, 46, 55, 64, 65, 69, 254, and 255 can be used as sequences containing epitopes or clusters of epitopes.

All accession numbers used here and throughout may be accessed through the NCBI database, for example through the Entrez search and retrieval system on the world wide web.

Note that the following discussion sets forth an understanding of the inventors' operation of the present invention. However, the discussion is not intended to limit the patent to any particular theory of operation not set forth in the claims below.

In the development of epitope vaccines, others have generated a list of predicted epitopes based on MHC binding motifs. Such peptides may be immunogenic, but may not correspond to any naturally occurring antigenic fragment. Thus, the whole antigen will not elicit a similar response or sensitize the target cells to cytolysis by CTLs. This list therefore does not distinguish between those sequences that can be used as vaccines and those that cannot. Efforts to determine which of these predicted epitopes are actually naturally occurring have often relied on screening for their reactivity with Tumor Infiltrating Lymphocytes (TILs). However, although tumors (and chronically infected cells) will typically present housekeeping epitopes, TIL strongly favors the recognition of immune epitopes. Thus, unless the epitope is produced by both housekeeping and immunoproteasome, the target cell will not normally be recognized by CTLs induced with TIL-identified epitopes. In contrast, the epitopes of the present invention are produced by the action of specific proteasomes, suggesting that they can be produced naturally and given their appropriate utility. The significance of the differences between housekeeping and immune epitopes for vaccine design is more fully elucidated in PCT publication WO 01/82963a 2.

The epitopes of the invention include or encode polypeptide fragments of TAAs, which are precursors or products of proteasomal cleavage by housekeeping or immunoproteasome, and contain or include sequences having known or predicted affinity for at least one MHC I allele. In some embodiments, the epitope comprises or encodes a polypeptide of about 6 to 25 amino acids in length, preferably about 7 to 20 amino acids in length, more preferably about 8 to 15 amino acids in length, and still more preferably about 9 or 10 amino acids in length. However, it will be appreciated that the polypeptides may be larger as long as the N-terminal trim can generate MHC epitopes or they do not contain sequences that cause the polypeptide to be directed away from or destroyed by the proteasome. For immune epitopes, larger peptides can be processed in pAPC for immunoproteasome if they do not contain such sequences. Housekeeping epitopes may also be embedded in longer sequences if the sequence is adapted to promote release of the C-terminus of the epitope by immunoproteasome action. The above discussion has assumed that processing of longer epitopes is performed by the action of the pAPC immunoproteasome. However, processing can also be accomplished by designing some other mechanism, such as the release of MHC epitopes by the action of proteases, which provides exogenous protease activity and adapted sequences. These epitope sequences can be subjected to computer analysis to calculate physical, biochemical, immunological or molecular genetic properties, such as mass, isoelectric point, predicted electrophoretic mobility, predicted binding to other MHC molecules, melting temperature of nucleic acid probes, reverse translation, similarity or homology to other sequences, etc.

In the construction of a polypeptide encoding the present inventionIn the polynucleotide of the epitope, the gene sequence of the relevant TAA may be used, or the polynucleotide may be assembled from any corresponding codons. For an epitope of 10 amino acids, this may constitute about 10⁶And (ii) different sequences, depending on the particular amino acid composition. Although large, this is a distinct and easily defined set, which represents > 10¹⁸This length may be a small percentage of the possible polynucleotides, and thus in some embodiments, equivalents to the specific sequences disclosed herein include such divergent and readily definable variations on the listed sequences. In selecting a particular one of these sequences for use in a vaccine, conditions such as codon usage, self-complementarity, restriction sites, chemical stability, etc., may be utilized as will be apparent to those skilled in the art.

The present invention is intended to produce peptide epitopes. In particular, these epitopes are derived from the TAA sequence and have a known or predicted affinity for at least one MHC I allele. Such epitopes are typically identical to those produced on target cells or pAPCs.

Compositions comprising active epitopes

Embodiments of the invention provide polypeptide compositions, including vaccines, therapeutics, diagnostics, pharmacology and pharmaceutical compositions. Various compositions include newly identified epitopes of TAAs as well as variants of these epitopes. Other embodiments of the invention provide polynucleotides encoding epitopes of the polypeptides of the invention. The invention further provides vectors for expressing polypeptide epitopes suitable for purification. In addition, the present invention provides a vector for expressing a polypeptide epitope in APC for use as an anti-tumor vaccine. Any epitope or antigen derived from table 1, or nucleic acid encoding it, may be used. Other embodiments relate to methods of making and using the various compositions.

The overall structure of MHC class I-binding epitopes can be described in Madden, d.r.annu.rev.immunol.13: 587 and 622, 1995. Many of the binding events can arise from the backbone linkage between conserved residues in MHC molecules and the N-and C-termini of peptidesAnd (4) contacting. Additional backbone contacts were generated but were different in the MHC alleles. Sequence specificity is conferred by the side chain of the so-called anchor residue in contact with a pocket that in turn varies in the MHC allele. Anchor residues can be divided into primary (primary) and secondary (secondary). The primary anchor positions show a strongly preferred set of relatively well-defined amino acid residues. Minor sites show weaker and/or less strictly defined preferred residues, which can often be better described in terms of less preferred than more preferred. In addition, residues in some minor anchor sites are not always located in contact with pockets on MHC molecules at all. Thus, there is one subtype of peptide that binds to a particular MHC molecule and presents a side-pocket contact at the site in question, and another subtype that exhibits binding to the same MHC molecule independent of the conformation the peptide presents in the MHC molecule peptide-binding groove. The C-terminal residue (P; ω) is preferably the primary anchor residue. The second position (P2) is also an anchor residue for many of the better studied HLA molecules (e.g. a2, a68, B27, B7, B35 and B53). However, central anchor residues have also been observed, including P3 and P5 in HLA-B8, and H-2D, respectively, in the murine MHC molecule^bAnd H-2K^bP5 and P (ω) -3 in (1). Because more stable binding will generally improve immunogenicity, regardless of their position, it is preferred that the anchor residues be conserved or optimized in designing variants.

Since the anchor residue is usually located near the end of the epitope, the peptide bends upward out of the peptide-binding groove, which allows some variation in length. Epitopes of 8-11 amino acids have been found for HLA-A68, and up to 13 amino acids for HLA A2. In addition to the length change between anchor positions, single residue truncation and extension and at the N-and C-termini, respectively, have been reported. Among the non-anchor residues, some protrude outside the groove, do not come into contact with MHC molecules but can be used to contact the TCR, most often P1, P4 and P (ω) -1 for HLA-a2. Other non-anchor residues may become inserted between the upper edge of the peptide binding groove and the TCR, contacting both. The precise location of these side chain residues, and thus their effect on binding, MHC fine conformation and ultimately immunogenicity, is highly sequence dependent. For highly immunogenic epitopes, it must not only promote sufficient TCR binding that is stable for activation to occur, but the TCR must also have a sufficiently high off-rate so that multiple TCR molecules can interact sequentially with the same peptide-MHC complex (Kalergis, A.M. et al, Nature Immunol.2: 229-234, 2001). Thus, without additional information about the ternary complex, both conservative and non-conservative substitutions at these positions are worth considering when designing variants.

Polypeptide epitope variants can be generated, for example, using any of the techniques and guidelines for conservative and non-conservative mutations. Variants may be derived from substitution, deletion or insertion of one or more amino acids compared to the native sequence. An amino acid substitution can be the result of substituting one amino acid with another having similar structural and/or chemical properties, such as substituting threonine with serine. Such substitutions are referred to as conservative amino acid substitutions, and all suitable conservative amino acid substitutions are considered an embodiment of the invention. Insertions or deletions can optionally be of about 1 to 4, preferably 1 to 2, amino acids. It is generally preferred to maintain an "anchor site" for the peptide, which is responsible for binding to the MHC molecule in question. Indeed, in many cases the immunogenicity of peptides can be improved by replacing more preferred residues at the anchor site (Franco, et al, Nature Immunology, 1 (2): 145-150, 2000). The immunogenicity of peptides can often also be improved by substituting small amino acids found at non-anchoring sites with larger volumes of amino acids, while maintaining sufficient cross-reactivity with the original epitope to constitute an effective vaccine. The allowable variation can be determined by conventional insertion, deletion or substitution of amino acids in the sequence and examination of the resulting variants for the activity exhibited by the polypeptide epitope. Since polypeptide epitopes are often 9 amino acids, it is preferred to replace the shortest active epitope, for example an epitope of 9 amino acids.

Variants can also be generated by adding any sequence to the N-terminus of the polypeptide epitope variant. Such an N-terminal increase may be from 1 amino acid up to at least 25 amino acids. Since peptide epitopes are often trimmed by the active N-terminal exopeptidase in pAPC, it must be understood that variations in the added sequence may have no effect on epitope activity. In a preferred embodiment, the amino acid residue between the last upstream proteasome cleavage site and the N-terminus of the MHC epitope does not include a proline residue. Serwold, t, et al, Nature immunol.2: 644-651, 2001. Thus, effective epitopes can be generated from larger precursors than the preferred 9-mer class I motif.

Generally, peptides are effective in the sense that they correspond to the epitope actually displayed by MHC I on the surface of the target cell or pACP. Individual peptides may have different affinities for different MHC molecules, binding well to some, and not binding at all (table 2). MHC alleles have traditionally been classified according to serological reactivity, which does not reflect the structure of peptide-binding grooves that may differ in the same type of allele. Similarly, cross type (across type) may share binding properties; the class group based on the shared binding property has been called supertype. There are many MHC I alleles in the human population; epitopes specific for certain alleles can be selected based on the genotype of the patient.

TABLE 2

Tyrosinase enzyme _207-216 (SEQ ID NO.1) predictive binding to various MHC classes

MHC class I	Dissociation half-life (min)
		A1	0.05
A*0201	1311.
		A*0205	50.4
A3	2.7
		A1101 (part A3 super type)	0.012
A24	6.0
		B7	4.0
B8	8.0
		B14 (part B27 super type)	60.0
B*2702	0.9
		B*2705	30.0

B3501 (part B7 super type)	2.0
		B*4403	0.1
B5101 (part B7 super type)	26.0
		B*5102	55.0
B*5801	0.20
		B60	0.40
B62	2.0

HLA peptide binding prediction (web hypertext transfer protocol "access bimas. dcrt. nih. gov/molbio/HLA _ bin").

In further embodiments of the invention, the epitope, either as a peptide or encoding polynucleotide, may be administered as a pharmaceutical composition, e.g., a vaccine or immunogenic composition, alone or in combination with various adjuvants, carriers or excipients. It should be noted that although the term vaccine may be used throughout the discussion herein, this concept may be administered or used with any other pharmaceutical composition including those mentioned herein. Particularly advantageous adjuvants include various cytokines and oligonucleotides comprising immunostimulatory sequences (as set forth in more detail in the co-pending applications referenced herein). Alternatively, the polynucleotide encoding the epitope may be contained in a virus (e.g., vaccinia or adenovirus) or in a microbial host cell (e.g., Salmonella (Salmonella) or Listeria monocytogenes), which is then used as a vector for the polynucleotide (Dietrrich, G. et al nat. Biotech.16: 181-185, 1998). Alternatively, pAPC can be transformed ex vivo to express the epitope, or pulsed with a peptide epitope to administer itself as a vaccine. To improve the efficiency of these methods, viral or bacterial vectors can be used to carry the encoded epitope or complexed with a ligand for the receptor found on pAPC. Similarly, the peptide epitope can be complexed or conjugated to a pAPC ligand. A vaccine may comprise more than one single epitope.

A particularly advantageous strategy for incorporating EPITOPEs and/or clusters of EPITOPEs into a vaccine or pharmaceutical composition is disclosed in U.S. patent application No. 09/560,465 filed on year 2000, month 4, 28, entitled "EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS". EPITOPE clustering for use with the present invention is disclosed in U.S. patent application No.09/561,571 entitled "EPITOPE CLUSTERS" filed at 28/4 in 2000.

Preferred embodiments of the invention are directed to vaccines and methods that allow the pAPC or pAPCs population to present housekeeping epitopes corresponding to the epitopes displayed on a particular target cell. For example, any epitope or antigen from table 1 can be used. In one embodiment, the housekeeping epitope is a TuAA epitope processed by the housekeeping proteasome of a particular tumor type. In another embodiment, the housekeeping epitope is a virus-associated epitope processed by the housekeeping proteasome of a cell infected with the virus. This promotes a specific T cell response to the target cells. Because they display housekeeping or immune epitopes, simultaneous expression of multiple epitopes corresponding to different induction states (pre-or post-challenge) of pAPCs can elicit CTL responses effective against target cells.

This embodiment can optimize the cytotoxic T cell response to target cells by containing housekeeping and immune epitopes presented on pAPC. Through dual epitope expression, pAPCs can continue to maintain CTL responses to immune-type epitopes when tumor cells are switched from housekeeping proteasomes to immunoproteasomes under induction by IFNs, which can be produced, for example, by tumor infiltrating CTLs.

In a preferred embodiment, the patient is immunized with a vaccine comprising a housekeeping epitope. Many preferred TAAs are specifically associated with a target cell, particularly in the case of infected cells. In another embodiment, many preferred TAAs are the result of deregulated (deregulated) gene expression in transformed cells, but are also found in testis, ovarian tissue and fetuses. In another embodiment, effective TAAs are expressed at higher levels in target cells than in other cells. In other embodiments, TAAs are not differentially expressed in target cells compared to other cells, but are also effective because they are associated with one particular function of the cell and distinguish the target cell from most other surrounding cells; in these embodiments, it was also shown that healthy cells of TAAs may be attacked in parallel by the induced T cell response, but this collateral damage is considered to be far more preferable than what the target cells cause.

The vaccine comprises a housekeeping epitope in an effective concentration that results in a population of pAPCs or pAPCs displaying the housekeeping epitope. Advantageously, the vaccine may comprise a plurality of housekeeping epitopes or one or more housekeeping epitopes optionally in combination with one or more immunological epitopes. The vaccine formulation comprises peptides and/or nucleic acids at a concentration sufficient to cause epitope presentation by pAPCs. The formulation preferably contains the epitope at a total concentration of about 1. mu.g to 1mg per 100. mu.l of vaccine formulation. The present invention may employ conventional dosages and dosages suitable for peptide vaccines and/or nucleic acid vaccines, the method of administration being well understood in the art. In one embodiment, a single dose for an adult may conveniently be from about 1 μ l to about 5000 μ l of the composition, administered one or more times, for example 2, 3, 4 or more doses spaced 1 week, 2 weeks, one month or more apart. Refer to the intra-nodal method patent insulin pump (insulin pump) delivers 1 μ l per hour (lowest frequency).

The compositions and methods of the invention disclosed herein are also intended to incorporate adjuvants into the formulations to enhance the performance of the vaccines. Specifically, adjuvants are designed to be added to the formulation to enhance epitope delivery or uptake of pAPCs. Adjuvants contemplated by the present invention are known to those skilled in the art and include, for example, GMCSF, GCSF, IL-2, IL-12, BCG, tetanus toxin, osteopontin, and ETA-1.

In some embodiments of the invention, the vaccine may comprise a recombinant organism, such as a virus, bacterium or parasite genetically engineered to express an epitope in a host. For example, listeria monocytogenes, a gram-positive, facultative intracellular bacterium, is an effective vehicle for targeting TuAAs to the immune system. In a preferred embodiment, the vector can be engineered to express a housekeeping epitope to induce a therapeutic response. The normal route of infection for this organism is through the intestine and can be delivered orally. In another embodiment, an adenovirus (Ad) vector encoding a housekeeping epitope against TuAA can be used to induce an antiviral or antitumor response. Dendritic cells derived from bone marrow can be transduced with the viral construct and subsequently injected, or the virus can be delivered directly into the animal by subcutaneous injection to induce an effective T-cell response. Another embodiment uses a recombinant vaccinia virus engineered to encode an amino acid sequence corresponding to a housekeeping epitope against TAA. Vaccinia virus carrying constructs containing appropriate nucleotide substitutions in the form of minigene constructs can direct the expression of housekeeping epitopes, resulting in therapeutic T cell responses against the epitopes.

Immunization with DNA requires APCs to take up the DNA and express the encoded protein or peptide. Discrete class I peptides may be encoded on DNA. By immunization with this construct, APCs can be made to express housekeeping epitopes, which are then displayed on cell surface MHC class I to stimulate an appropriate CTL response. A construct that generally relies on translational termination or non-proteasomal proteases to generate appropriate housekeeping epitope termini has been described in U.S. patent application No.09/561,572 filed on year 2000, month 4, 28, entitled EXPRESSION VECTORS OF Target-As recognized antibodies.

As mentioned, it is desirable to express the housekeeping peptide in a larger protein context. Processing can be detected even when a small amount of amino acids are present beyond the epitope end. Small peptide hormones are usually proteolytically processed from longer translation products, often about 60-120 amino acids in size. This fact has led some to assume that this is the smallest size that can be translated efficiently. In some embodiments, the housekeeping peptide may be embedded in the translation product of at least about 60 amino acids. In other embodiments the housekeeping peptide may be embedded in the translation product of at least about 50, 30 or 15 amino acids.

Due to differential proteasomal processing, the immunoproteasome of pAPC produces different peptides than those produced by housekeeping proteasomes in surrounding somatic cells. Thus, in expressing the housekeeping peptide in the context of a larger protein, it is preferred to express it in the APC in a context different from its full-length native sequence, since, as a housekeeping epitope, it is usually only efficiently processed from the native protein by the housekeeping proteasome, which is not active in the APC. To encode the housekeeping epitope in a DNA sequence encoding a larger protein, it is useful to find flanking regions on either side of the sequence encoding the epitope that allow for appropriate cleavage by the immunoproteasome to release the housekeeping epitope. Altering the flanking amino acid residues at the N-and C-termini of the desired housekeeping epitope can facilitate proper cleavage and production of the housekeeping epitope in the APC. Sequences of the embedded housekeeping epitopes can be newly designed and screened to determine which can be successfully processed by the immunoproteasome to release the housekeeping epitope.

Alternatively, another strategy is very effective for identifying sequences that allow the generation of housekeeping epitopes in APCs. A contiguous amino acid sequence can result from the head-to-tail arrangement of one or more housekeeping epitopes. An animal is immunized with a construct expressing the sequence and the resulting T cell response is evaluated to determine its specificity for one or more epitopes in the array. By definition, these immune responses show housekeeping epitopes that are efficiently processed in pAPC. The necessary flanking regions around the epitope are thus determined. The use of flanking regions of about 4-6 amino acids on either side of the desired peptide may provide the information necessary to facilitate proteasomal processing of housekeeping epitopes by immunoproteasome. Thus, a sequence of about 16-22 amino acids that ensures epitope synchronization can be efficiently inserted or fused to any protein sequence to result in the production of housekeeping epitopes in APCs. In alternative embodiments, the entire head-to-tail arrangement of epitopes, or just the epitopes immediately adjacent to the correctly processed housekeeping epitope, can be similarly transferred from the test construct to the vaccine vector.

In a preferred embodiment, housekeeping epitopes may be embedded within known immune epitopes, or between such segments, to provide a context suitable for processing. The juncture of the housekeeping and immunizing epitopes may generate the necessary context for the immunoproteasome to release the housekeeping epitope, or a larger fragment preferably comprising the correct C-terminus. Screening of the construct to confirm production of the desired epitope is useful. Binding sites for housekeeping epitopes may create sites that can be cleaved by immunoproteasome. Some embodiments of the invention use known epitopes to flank a housekeeping epitope in a test substrate; in other embodiments, the screening described below is used to determine whether the flanking regions are any sequence or a mutant of the native flanking sequence, using knowledge of the preference for proteasome cleavage in designing the substrate.

Although advantageous, cleavage at the N-terminus of the mature epitope is not necessary because there are a variety of N-terminal trimming activities in the cell that are capable of producing the mature epitope N-terminus after proteasome processing. Preferably, the N-terminal extension is less than about 25 amino acids in length, and further preferably the extension contains little or no proline residues. Preferably, in the screening, consideration is given not only to cleavage at the end of the epitope (or at least at its C-terminus), but also to ensuring limited cleavage within the epitope.

The shotgun method can be used to design test substrates and can improve screening efficiency. In one embodiment, multiple epitopes can be combined in sequence, and a single epitope can appear multiple times. The substrate can be screened to determine which epitopes can be produced. In cases where a particular epitope is of interest, one can design a substrate in which it occurs in a variety of different context sequences. When a single epitope present in more than one pre-and post-sequence is released from the substrate, an additional second test substrate in which a particular instance (antigenic entities) of the epitope is removed, disabled or unique can be used to determine which is the sequence that is released and indeed constitutes the sequence that ensures epitope simultaneity.

There are several easily practiced screens. One preferred in vitro screen utilizes a proteasome digestion assay that uses purified immunoproteasome to determine whether the desired housekeeping epitope can be released from a synthetic peptide comprising the sequence in question. The position at which cleavage is obtained can be determined by techniques such as mass spectrometry, HPLC and N-terminal pool sequencing; as described in more detail in U.S. patent application entitled METHOD OF EPITOPE DISCOVERY, EPITOPE SYNCHRONIZATION IN ANTIGENPRESENTING CELLS, two provisional U.S. patent applications entitled EPITOPE SEQUENCES.

Alternatively, in vivo screening such as immunization or target sensitization may be used. For immunization nucleic acid constructs are used which can express the sequence in question. The ability of the harvested CTLs to recognize target cells presenting the housekeeping epitope in question can be examined. Such target cells are extremely easy to obtain by pulsing cells expressing appropriate MHC molecules with synthetic peptides containing mature housekeeping epitopes. Alternatively, cells and antigens known to express housekeeping proteasomes can be used, from which housekeeping epitopes can be derived endogenously or by genetic engineering. For the purpose of using target sensitization as a sieve, CTLs recognizing housekeeping epitopes, or preferably CTL clones, can be used. In this case, it is the target cell expressing the embedded housekeeping epitope (rather than pAPC during immunization) and it must express the immunoproteasome. In general, a target cell can be transformed with an appropriate nucleic acid construct to confer expression of an embedded housekeeping epitope. Using peptide-loaded liposomes or protein transfer agents such as BIOPORTER^TM(Gene Therapy Systems, San Diego, Calif.) Loading a synthetic peptide containing an embedded epitope represents an alternative.

Additional guidance regarding nucleic acid constructs for use AS vaccines according to the present invention is disclosed in U.S. patent application No.09/561,572 entitled "EXPRESSION VECTORS OF TARGET-AS contacted antibodies", filed on 28.4.2000. In addition, EXPRESSION VECTORS useful in accordance with the present invention and METHODS FOR their design are disclosed in U.S. patent application No. 60/336,968 (attorney docket number) CTLIMM.022PR entitled "EXPRESSION VECTORS OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN", filed at 11/7/2001.

A preferred embodiment of the invention comprises a method of administering a vaccine comprising an epitope (or epitopes) to induce a therapeutic immune response. The vaccine is administered to the patient in a manner consistent with standard vaccine delivery protocols known in the art. Methods of administering TAAs epitopes include, but are not limited to, transdermal, intranodal, perinodal, oral, intravenous, intradermal, intramuscular, intraperitoneal and mucosal administration, including delivery by injection, instillation or inhalation. Australian patent No. 739189 issued on 17.1.2002; U.S. patent application No. 09/380,534, filed 9/1/1999, entitled "A METHOD OF INDUCING A CTL RESPONSE"; and its continuation-in-part application, U.S. patent application No. 09/776,232 filed on 2/2001, disclose a particularly useful method of delivering a vaccine to elicit a CTL response.

Reagent recognition epitopes

In another aspect of the invention, proteins having binding specificity for an epitope and/or epitope-MHC molecule complex are contemplated, as well as isolated cells by which they may be expressed. In one set of embodiments, these agents take the form of immunoglobulins: polyclonal sera or monoclonal antibodies (mabs), methods for producing which are well known in the art. The generation of mabs specific for peptide-MHC molecule complexes is known in the art. See, e.g., aharoi et al, Nature 351: 147, 150, 1991; andersen et al proc.natl.acad.sci.usa 93: 1820-1824, 1996; dadaglio et al Immunity 6: 727 738, 1997; duc et al int. Immunol.5: 427 gradient 431, 1993; eastman et al eur.j.immunol.26: 385-393, 1996; engberg et al Immunotechnology 4: 273-278, 1999; porgdor et al Immunity 6: 715-726, 1997; puri et al j.immunol.158: 2471-2476, 1997; and Polakova, k., et al, j.immunol.165342-348, 2000.

In other embodiments, the compositions can be used to induce and generate T-cells specific for any epitope and/or epitope-MHC complex in vivo and in vitro. In a preferred embodiment, the epitope may be any one or more of those listed, for example, in table 1. Thus, embodiments also relate to and include isolated T cells, T cell clones, T cell hybridomas or proteins comprising a T Cell Receptor (TCR) binding domain derived from a cloned gene, and recombinant cells expressing the proteins. The TCR-derived protein may be the extracellular domain of a TCR alone, or fused to a portion of another protein to confer a desired property or function. One example of such a fusion is the attachment of a TCR binding domain to a constant region of an antibody molecule in order to produce a bivalent molecule. The construction and activity of molecules according to this general model has been reported, for example Plaksin, d. et al j.immunol.158: 2218-2227, 1997 and Lebowitz, M.S. et al Cell Immunol.192: 175-184, 1999. More general construction AND USE of such molecules is also discussed IN U.S. patent 5,830,755 entitled CELL RECEPTORS AND THEIR USE IN thermal AND DIAGNOSTIC METHODS.

The production of such T cells can be readily achieved by standard immunization of laboratory animals and reactivity to human target cells can be obtained by immunization with human target cells or by immunization of HLA-transgenic animals with antigens/epitopes. For some therapeutic approaches, T cells derived from the same species are desirable. Although this cell can be generated by, for example, cloning a murine TCR into a human T cell as contemplated above, in vitro immunization of human cells offers a potentially faster selection. Even with donors used for the first time in experiments (naive donors), techniques for in vitro immunization are known in the art, e.g. Stauss et al, proc.natl.acad.sci.usa 89: 7871-7875, 1992; salgaleler et al Cancer Res.55: 4972-4979, 1995; tsai et al, j.immunol.158: 1796-; and Chung et al, J immunoher.22: 279-287, 1999.

Any of these molecules can be coupled to enzymes, radiochemical reagents, fluorescent tags and toxins for use in diagnosis (imaging or other detection), monitoring and treatment of epitope-related pathogenic diseases. Thus, toxin conjugates can be administered to kill tumor cells, radiochemical reagents can facilitate imaging of epitope positive tumors, enzyme conjugates can be used in assays like ELISA to diagnose cancer and confirm epitope expression in biopsy tissues. In another embodiment, such T cells as set forth above may be administered to a patient as an adoptive immunotherapy after expansion is completed by stimulation with epitopes and/or cytokines.

Agents comprising epitopes

Another aspect of the invention provides isolated epitope-MHC complexes. In particularly advantageous embodiments of this aspect of the invention, the complex may be a soluble, multi-subunit protein such as those described in U.S. Pat. No.5,635,363 (tetramer) or U.S. Pat. No.6,015,884 (Ig-dimer). The reagents are useful for detecting and monitoring specific T cell responses, and for purifying such T cells.

Isolated MHC molecules complexed with epitope peptides can also be incorporated into planar lipid bilayers or liposomes. Such compositions can be used to stimulate T cells in vitro or, in the case of liposomes, in vivo. Costimulatory molecules (e.g., B7, CD40, LFA-3) can be incorporated into the same composition, or, particularly for in vitro manipulation, costimulation can be provided by anti-co-receptor antibodies (e.g., anti-CD 28, anti-CD 154, anti-CD 2) or cytokines (e.g., IL-2, IL-12). Such T cell stimulation may constitute vaccination in immunotherapy, driving T cell expansion in vitro for subsequent fusion, or constitute a step in an assay for T cell function.

The epitope, or more directly its complex with an MHC molecule, may be an important component of a functional assay of antigen-specific T cells in the activation or readout step or both. Two broad classes can be defined in the Current art for a wide variety of assays for T cell function (detailed methods can be found in standard immunological references such as Current Protocols in Immunology 1999 John Wiley & Sons inc., n.y), those measuring many cellular responses and those measuring individual cellular responses. While the former conveys an overall measure of the intensity of the response, the latter allows the relative frequency of responding cells to be determined. Examples of assays that measure overall response are cytotoxicity assays, ELISA and proliferation assays that detect cytokine secretion. Assays FOR measuring individual cell (or miniclone derived therefrom) responses include Limiting Dilution Analysis (LDA), ELISPOT, flow cytometry detection OF non-secreted cytokines (in U.S. patent No.5,445,939 entitled "METHOD FOR assay OF THE same monomer empty monomer SYSTEM" and U.S. patent No.5,656,446 entitled "METHOD FOR THE ASSESSMENT OF THE same monomer empty monomer SYSTEM"; and 5,843,689, reagents are sold under the trade name 'FASTIMMENE' by Becton, Dickinson & Company and the relative advantages of these techniques are reviewed in Current Opinion in Immunology, 13: 141-, in particular in situ and single cell PCR techniques, may accomplish additional rearrangements or expression of the detected specific TCR.

These functional assays can be used to assess endogenous levels of immunity, response to immune stimuli (e.g., vaccines) and to monitor immune status throughout disease and treatment periods. Any of these assays presumes a prior immunization step, whether in vivo or in vitro, which depends on the nature of the problem in question, except when measuring endogenous levels of immunity. The immunization can be performed using various embodiments of the invention described above or other forms of immunogens (e.g., pAPC-tumor cell fusions) that can elicit similar immunity. In addition to PCR and tetramer/Ig-dimer type assays capable of detecting expression of the cognate tcr (cognate tcr), these assays generally benefit from one-step in vitro antigen stimulation, which can conveniently use the various embodiments of the invention described above in order to detect specific functional activities (and sometimes a highly cytolytic response directly). Finally, detection of cytolytic activity requires target cells displaying epitopes, which can be generated using various embodiments of the present invention. The particular embodiment chosen for any particular step depends on the issues discussed, ease of use, cost, etc., but the advantages of one embodiment over another for any particular set of circumstances will be apparent to one skilled in the art.

Peptide MHC complexes described in this section have traditionally been understood to be non-covalent binding. However, it is possible and may be beneficial to create a covalent bond, for example by encoding the epitope as a single protein and an MHC heavy chain or epitope, β 2-microglobulin, and MHC heavy chain (Yu, Y.L.Y., et al, J.Immunol.168: 3145-opsin 3149, 2002; Mottez, E., et al, J.exp.Med.181: 493, 1995; Dela Cruz, C.S., et al, int.Immunol.12: 1293, 2000; Mage, M.G., 369, et al, Proc.Natl.Acad.Sci.USA 89: 10658, 1992; Toshitani, K., et al, Proc.Natl.Acad.Sci.USA 93: 236, 1996; Lee, L.L, et al, Eur.J.24: 2633, Chung, Whd.H., J.J.163.J.J.J.USA 93: 236, 1996; 1999, J.J.162, 1999, J.162, 1999, J.J.162, 1999, J.A.162, J.162, J.A.162, 1999, et al, and similar approaches have been described for the vaccine, and for the analogous approach to overcome the difficulties in the A.A.A.A.7, J.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A.A..

Tumor associated antigens

The epitopes of the invention are derived from TuAAs tyrosinase (SEQ ID NO.2), SSX-2, (SEQ ID NO.3), PSMA (prostate-specific membrane antigen) (SEQ ID NO.4), GP100, (SEQ ID NO.70), MAGE-1, (SEQ ID NO.71), MAGE-2, (SEQ ID NO.72), MAGE-3, (SEQ ID NO.73), NY-ESO-1, (SEQ ID NO.74), PRAME, (SEQ ID NO.77), PSA, (SEQ ID NO.78), PSCA, (SEQ ID NO.79), fibronectin ED-B domain (SEQ ID NOS 589 and 590), CEA (carcinoembryonic antigen) (SEQ ID NO.592), Her2/Neu (SEQ ID NO.594), SCP-1(SEQ ID NO.596) and SSX-4(SEQ ID NO. 598). The native coding sequence of these eleven proteins, or any segment within them, can be determined from their cDNA or complete coding (cds) sequences, SEQ ID NOS.5-7, 80-87, 591, 593, 595, 597, and 599, respectively.

Tyrosinase is a melanin biosynthetic enzyme that is considered to be one of the most specific markers of melanocyte differentiation. Tyrosinase is expressed in a few cell types, mainly in melanocytes, and is often found at high levels in melanomas. TYROSINASE is taught FOR use as TuAA in U.S. Pat. No.5,747,271 entitled "METHOD FOR IDENTIFYING INDIVIDUALS SUFFERING FROM A CELLULAR ABNORMALITY SOME OF WHOSE ABNORMAL CELLS PRESENT COMPLEXES OF HLA-A2/TYROSINE DERIVED PEPTIDES, ANDMETHODS FOR TREATING SAID INDIVIDUALS".

GP100, also known as PMe117, is also a melanin biosynthetic protein that is expressed at high levels in melanoma. GP100, a TuAA, is disclosed IN U.S. Pat. No.5,844,075 entitled "MELANOMAANTIGENS AND THEIR USE IN DIAGNOSTIC AND THERAPEUTIC METHODS".

SSX-2, also known as Hom-Mel-40, is a member of the highly conserved cancer-testis antigen family (Gure, A.O. et al int.J.cancer 72: 965-971, 1997). The identification as a TuAA is taught in U.S. Pat. No.6,025,191 entitled "ISOLATED NUCLEIC ACID MOLECULES A MELANOMA SPECIFIC ANTIGEN AND USES THEREOF". Cancer-testis antigens are found in a variety of tumors, but are not normally found in normal adult tissues other than the testis. It has been found that different members of the SSX family are expressed differently in tumor cell lines. Because of the high degree of sequence identity between SSX family members, similar epitopes from more than one family member will be produced and can bind to MHC molecules, some vaccines directed to one member of the family can cross-react and be effective against other members of the family (see example 3 below).

MAGE-1, MAGE-2, and MAGE-3 are members of another cancer-testis antigen family that was originally found in melanoma (MAGE is an abbreviation for melanoma-associated antigen) but is found in many tumors. THE identification of MAGE proteins as TuAAs is taught in U.S. Pat. No.5,342,774 and many later patents entitled NUCLEOTIDE SEQUENCE ENCODING THE TUMOR REJECTION ANTIGEN PRECURSOR, MAGE-1. Currently there are 17 records on (human) MAGE in the SWISS protein database. There is extensive similarity among these proteins and thus in many cases epitopes derived from one can induce cross-reactive responses against other family members. Some of these have not been observed in tumors, most notably MAGE-H1 and MAGE-D1, which are expressed in testis and brain, and bone marrow stromal cells, respectively. The possibility of cross-reactivity on normal tissues is improved by the fact that they are among the least similar to other MAGE proteins.

NY-ESO-1, a cancer-testis antigen found in a variety of tumors, is also known as CTAG-1 (cancer-testis antigen-1) and CAG-3 (cancer antigen-3). NY-ESO-1, a TuAA, is disclosed in U.S. Pat. No.5,804,381 entitled Integrated Nuclear ACID Motor engineering AN agricultural chemical ISOLATED plant, THE same ISOLATED plant ITSELF, AND THE United states plant, to TuAA. Paralogous loci (paralogous loci), LAGE-1a/s (SEQ ID NO.75) and LAGE-1b/L (SEQ ID NO.76) encoding antigens with extensive sequence identity have been disclosed in publicly available human genome clusters (associations) and have been deduced to be produced by alternative splicing (alternative splicing). In addition, CT-2 (or CTAG-2, cancer-testis antigen-2) appears to be an allele, mutant or sequencing difference of LAGE-1 b/L. Due to the extensive sequence identity, many epitopes derived from NY-ESO-1 can also induce immunity against tumors expressing these other antigens. See fig. 1. The protein is virtually identical up to amino acid 70. The longest length of identity between 71-134NY-ESO-1 and LAGE is 6 residues, but potentially cross-reactive sequences are present. From 135-180, the NY-ESO and LAGE-1a/s are identical except for a single residue, but are not related due to alternative splicing of LAGE-1 b/L. CAMEL and LAGE-2 antigens appear to be derived from LAGE-1mRNA, but from variable reading frames, thus generating unrelated protein sequences. More recently, GenBank accession No. AF277315.5, human chromosome X clone RP5-865E18, RP5-1087L19, complete sequence, reported three separate loci in this region, labeled lag 1 (corresponding to CTAG-2 of the genomic cluster), plus lag 2-a and lag 2-B (both corresponding to CTAG-1 of the genomic cluster).

PSMA (PROSTATE specific membrane ANTIGEN), a TuAA described in U.S. Pat. No.5,538,866 entitled "PROSTATE-SPECIFICAMEMBRANE ES ANTIGEN", is expressed by normal PROSTATE epithelial cells and at high levels in PROSTATE cancer. It has also been found to be present in neovasculature of non-prostate tumors. PSMA may thus form the basis of a vaccine directed to both the neovasculature of prostate cancer and other tumors. This latter concept is more fully described in provisional U.S. patent application No. 60/274,063 entitled "ANTI-neovasular vehicles FOR cam" filed on 3/7/2001 and U.S. application No. 10/094,699 entitled "ANTI-neovasular devices FOR cam" filed on 3/7/2002, attorney docket No. ctllimm.015 a. Briefly, as tumors grow, they take up (regrait) an ingrowth of new blood vessels (ingowth). This is thought to be necessary for maintenance of growth, as the non-vascularized tumor centers usually necrose and angiogenesis inhibitors have been reported to cause tumor regression. These new blood vessels, or neovasculature, express antigens not found in the established blood vessels and can therefore be specifically targeted. By inducing CTLs against neovascular antigens, blood vessels can be disrupted, interrupting nutrient flow to (and removing waste products from) the tumor, leading to regression.

Alternative splicing of PSMAmRNA also results in a single event at Met 5,935,818, as described in U.S. Pat. No.3,3926 entitled "ISOLATED NUCLEIC ACID Motor ENCODING ALTERNATIVELY SPLICED State-SPECIFIC polynucleotides ANTIGEN AND USES THEREOF₅₈Contains a significant initial protein, thereby deleting the putative anchoring region of the PSMA membrane. A protein called PSMA-like protein, Genbank accession No. AF261715, is nearly identical to amino acid 309-750 of PSMA and has a different expression profile (expression profile). Thus the most preferred epitopes are those containing the N-terminus located at amino acids 58-308.

PRAME, also known as MAPE, DAGE and OIP4, was originally observed as a melanoma antigen. Later, it has been considered a CT antigen, but unlike many CT antigens (e.g. MAGE, GAGE, and BAGE), it is expressed in acute myeloid leukemia. PRAME is a member of the MAPE family, which is composed primarily of hypothetical proteins with which PRAME shares limited sequence similarity. The use of PRAME as a TuAA is taught in U.S. patent 5,830,753 entitled "ISOLATED NUCLEIC ACID MOLECULES CODING FOR turbine detection anti gene precorresor DAGE AND USES theroflf".

PSA, prostate specific antigen, is a peptidase of the kallikrein family and a differentiation antigen of the prostate. Expression in breast tissue has also been reported. Alternative names include gamma-seminoprotein, kallikrein 3, seminogelase, seminin and the P-30 antigen. PSA has a high degree of sequence identity to the various alternative splice products prostate/gland kallikreins-1 and-2 and kallikreins 4, which are also expressed in prostate and breast tissue. Other kallikreins generally share less sequence identity and have different expression profiles. However, cross-reactivity that may be triggered by any particular epitope, as well as the possibility that the epitope will be released by processing in non-target tissues (most commonly by housekeeping proteasomes) should be considered in designing the vaccine.

PSCA, a prostate stem cell antigen, also known as SCAH-2, is a differentiation antigen that is preferentially expressed in prostate epithelial cells and overexpressed in prostate cancer. Low levels of expression are found in some normal tissues including neuroendocrine cells of the digestive tract and the collecting ducts of the kidney. PSCA is described in us patent 5,856,136 entitled "HUMAN STEM CELL ANTIGENS".

Synaptonemal complex protein 1(SCP-1), also known as HOM-TES-14, is a meiosis-associated protein and is also a cancer-testis antigen (Tureci, O., et al Proc. Natl. Acad. Sci. USA 95: 5211-. As a cancer antigen, its expression is not regulated by the cell cycle and is often found in gliomas, breast, kidney cells and ovarian cancers. It has some similarity to myosin, but has a small enough identity that cross-reactive epitopes are not directly searchable (immediatate promoter).

The ED-B domain of fibronectin is also a potential target. Fibronectin undergoes developmentally regulated alternative splicing, with a single exon used primarily in fetal Cancer tissues encoding the ED-B domain (Matsuura, H.and S.Hakomori Proc.Natl.Acad.Sci.USA 82: 6517-.

The ED-B domain is also expressed in fibronectin of the neovasculature (Kaczmarek, J. et al int.J.cancer 59: 11-16, 1994; Castellani, P. et al int.J.cancer 59: 612. minus 618, 1994; Neri, D. et al Nat.Biotech.15: 1271. minus 1275, 1997; Karelina, T.V. and A.Z.Eisen cancer Detect.Prev.22: 438444, 1998; Tarli, L. et al Blood 94: 192. minus 198; 1999; Castellani, P. et al Acta neurohir. (Wien) 142: 277. minus 282, 2000). As a fetal cancer domain, the ED-B domain is commonly found in fibronectin expressed by neoplastic cells in addition to being expressed by neovasculature. Therefore, CTL-inducing vaccines targeting the ED-B domain may exhibit two mechanisms of action: directly lysing tumor cells and disrupting the blood supply to the tumor by disrupting tumor-associated neovasculature. Because CTL activity decays rapidly after vaccine administration, interference with normal angiogenesis can be minimized. Designing and testing VACCINES targeting NEOVASCULATURE is described in provisional U.S. patent application No. 60/274,063 entitled "ANTI-NEOVASCULATURE VACCINES FOR CANCER" and even U.S. patent application No. 10/094,699 entitled "ANTI-NEOVASCULATURE preportions FOR CANCER," filed on even date (3/7/2002) with the present application, attorney docket No. CTLIMM.015A. A tumor cell line is disclosed in provisional U.S. patent application No. 60/363,131, entitled "HLA-TRANSGENIC MURINE TUMOR CELL LINE," attorney docket number CTLIMM.028PR, filed 3/7/2002.

Carcinoembryonic antigen (CEA) is a typical carcinoembryonic protein that was first described in 1965 (Gold and Freedman, J.exp.Med.121: 439-462, 1965. more complete references can be found in Online medial initiative in Man; record 114890). It has been formally renamed carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM 5). Its expression is strongly associated with adenocarcinoma of the epithelial layer (epithelial lining) in the digestive tract and fetal colon. CEA is a member of the immunoglobulin supergene family and is a defining member of the CEA subfamily (defining member).

HER2/NEU is an epidermal growth factor receptor-associated oncogene (van de Vijver, et al, New Eng.J.Med.319: 1239-1245, 1988) and apparently identical to the c-ERBB2 oncogene (Di Fiore, et al, Science 237: 178-182, 1987). Overexpression of ERBB2 has been implicated in neoplastic transformation of prostate cancer. As HER2, it was amplified and overexpressed in 25-30% of breast cancers in other tumors where expression levels correlated with tumor aggressiveness (Slamon, et al, New Eng.J.Med.344: 783-792, 2001). In Online medial initiative in Man; a more detailed description is available in the record 164870.

Additional disclosure related to embodiments of the present invention is found in U.S. patent application No. 10/005,905 (attorney docket No. ctlimm.021cp1), entitled "epiope syndrome IN ANTIGEN PRESENTING CELLS," filed on 7.11.2001, and its continuation, filed on 7.12.2000, attorney docket No. ctlimm.21cp1c, also entitled "epiope IN ANTIGEN PRESENTING CELLS.

Effective epitopes were identified and tested as described in the following examples. These examples are intended for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Examples

Specific preferred epitope sequences

Example 1

Preparation of epitopes

A. Artificial production of epitopes

Synthesis of a peptide comprising SEQ ID NO: 1, 8, 9, 11-23, 26-29, 32-44, 47-54, 56-63, 66-68, 88-253, or 256-588. After synthesis, peptides are cleaved from their supports with trifluoroacetic acid or hydrogen fluoride, respectively, in the presence of an appropriate protective scavenger. After removal of the acid by evaporation, the peptide was extracted with ether to remove the scavenger, and then the crude precipitated peptide was lyophilized. The purity of the crude peptide was determined by HPLC, sequence analysis, amino acid analysis, equilibrium ion content analysis and other appropriate methods. If the crude peptides are sufficiently pure (greater than or equal to about 90% purity), they can be used as is. If purification is required to meet pharmaceutical specifications, the peptide is purified using one or a combination of the following: then precipitating; reversed phase, ion exchange, size exclusion or hydrophobic interaction chromatography; or a counter-current distribution.

Pharmaceutical preparation

GMP-grade peptides are formulated in parenterally acceptable aqueous, organic or aqueous-organic buffers or solvent systems in which they remain physically and chemically stable and bioavailable. In general, buffers or combinations of buffers and organic solvents are suitable. The pH range is typically 6-9. Organic modifiers or other excipients may be added to help solubilize and stabilize the peptide. These include detergents, lipids, co-solvents, antioxidants, chelating agents and reducing agents. In the case of a lyophilized product, sucrose or mannitol or other lyophilized acids may be added. Peptide solutions are sterilized by membrane filtration in their final container-sealed system and lyophilized for clinical dissolution or storage until use.

B. Construction of expression vectors for use as nucleic acid vaccines

The construction of three types of expression vectors for epitope expression is described below. Specific advantages OF these designs are set forth in U.S. patent application No.09/561,572 entitled "EXPRESSION VECTORS OF ARGET-ASSOCIATED antibodies".

The appropriate E.coli strain is then transfected with the plasmid and plated on selective medium. Several clones were grown in suspension culture and positive clones were identified by restriction enzyme mapping. Positive clones were then cultured, aliquoted into storage vials and stored at-70 ℃.

Plasmid minipreps (QIAprep Spin Mini-prep: Qiagen, Valencia, Calif.) were then performed from samples of these cells and the constructs were confirmed to contain the desired sequence using automated fluorescent dideoxy sequencing analysis.

B.1 construction of pVAX-EP1-IRES-EP2

For review:

the starting plasmid for this construct was pVAX1 purchased from Invitrogen (Carlsbad, CA). Epitopes EP1 and EP2 were synthesized by GIBCO BRL (Rockville, Md.). IRES was cleaved from pIRES from Clontech (Palo Alto, Calif.).

The method comprises the following steps:

1 pIRES was digested with EcoRI and NotI. The digested fragments were separated by agarose gel electrophoresis and the IRES fragments were purified from the cut bands.

2 pVAX1 was digested with EcoRI and NotI and the pVAX1 fragment was gel purified.

3 the purified pVAX1 and the IRES fragment were then ligated together.

4 transformation of competent E.coli strain DH 5. alpha. with ligation mixture.

5 Mini preparations were performed from the 4 colonies generated.

6 restriction enzyme digestion analysis was performed on miniprep DNA. One recombinant colony containing the IRES insert was used for further EP1 and EP2 insertions. This intermediate construct is called pVAX-IRES.

7 oligonucleotides encoding EP1 and EP2 were synthesized.

8 subcloning EP1 between the AfIII and EcoRI sites of pVAX-IRES to construct pVAX-EP 1-IRES;

9 EP2 was subcloned between the SalI and NotI sites of pVAX-EP1-IRES to construct the final construct pVAX-EP1-IRES-EP 2.

10 the sequence of the EP1-IRES-EP2 insert was confirmed by DNA sequencing.

B2. Construction of pVAX-EP1-IRES-EP 2-ISS-NIS

For review:

the starting plasmid for this construct was pVAX-EP1-IRES-EP2 (example 1). The ISS (immunostimulatory sequence) introduced into this construct was AACGTT and the NIS (representative of the nuclear import sequence) used was SV4072bp repeat. ISS-NIS is synthesized by GIBCO BRL. See fig. 2.

The method comprises the following steps:

1 digestion of pVAX-EP1-IRES-EP2 with NruI; the linearized plasmid was gel purified.

2 ISS-NIS oligonucleotides were synthesized.

3 the purified linearized pVAX-EP1-IRES-EP2 and the synthesized ISS-NIS are ligated together.

4 transformation of competent E.coli strain DH 5. alpha. with the ligation product.

5 miniprep was performed from the colonies generated.

6 restriction enzyme digestion for miniprep.

7 sequencing of the plasmid containing the insert

B3. Construction of pVAX-EP2-UB-EP1

For review:

the starting plasmid for this construct was pVAX1 (Invitrogen). EP2 and EP1 were synthesized by gibbcobrl. The 76 amino acid wild-type ubiquitin protein was encoded in the construct from the yeast clone.

The method comprises the following steps:

1 RT-PCR was performed using yeast mRNA. Primers were designed to amplify the entire coding sequence of the yeast ubiquitin.

2 the RT-PCR products were analysed using agarose gel electrophoresis. The gel purified bands with the predicted size.

3 the purified DNA band was subcloned at the EcoRV site to pZERO 1. The resulting clone was named pZERO-UB.

4 several pZERO-UB clones were sequenced prior to further manipulation to confirm the ubiquitin sequence.

5 Synthesis of EP1 and EP 2.

EP2, ubiquitin and EP1 were ligated and the insert was cloned in between the BamHI and EcoRI sites of pVAX1, under the control of the CMV promoter.

7 the sequence of the insert EP2-UB-EP1 was confirmed by DNA sequencing.

Example 2

Identification of effective epitope variants

FLPWHRLFLL (SEQ ID NO.1) of the 10-mer was identified as a valid epitope. Based on this sequence, a number of variants were prepared. Variants that show activity in HLA binding assays (see example 3, part 6) were identified as effective and subsequently bound to vaccines.

HLA-A2 binding of FLPWHRLFLL length variants has been evaluated. Proteasome digestion analysis indicated that the C-terminus of 9-mer FLPWHRLFL (SEQ ID NO.8) was also produced. In addition, 9-mer LPWHRLFLL (SEQ ID NO.9) could also be generated by N-terminal pruning of the 10-mer. Both were predicted to bind to HLA-a0201 molecules, however of these two 9-mer FLPWHRLFL showed more significant binding and was preferred (see figures 3A and B).

In vitro proteasome digestion and N-terminal pool sequencing elucidate tyrosinase_207-216(SEQ ID NO.1) tyrosinase to tyrosinase_207-215(SEQ ID NO.8) was produced more generally, however the latter peptides showed superior immunogenicity which may be of concern in achieving optimal vaccine design. FLPWHRLFL, tyrosinase_207-215(SEQ ID NO.8) for HLA-A2⁺In vitro immunization of blood to produce CTLs (see CTL induction culture below). Peptide-pulsed T2 cells were used as targets in a standard chromium release assay and were found to be inhibited by tyrosinase_207-215(SEQ ID NO.8) induced CTL recognizes tyrosinase equally well_207-216(SEQ ID NO.1) (see FIG. 3C). These CTLs also recognized HLA-A2⁺Tyrosinase + tumor cell lines 624.38 and HTB64, but did not recognize HLA-a2 of 624.28, 624.38^-Derivatives (FIG. 3C)). The relative number of these two epitopes produced in vivo is therefore not a problem in vaccine design.

CTL induction culture

PBMCs from normal donors were purified from buffy coat by centrifugation in Ficoll-Hypaque. All cultures were performed using Autologous Plasma (AP) to avoid exposure to possible heterologous pathogens and recognition of FBS peptides. To facilitate the in vitro generation of peptide-specific CTLs, we used autologous Dendritic Cells (DCs) as APCs. DCs were generated and CTL was induced with DCs and peptides derived from PBMCs as described (Keogh et al, 2001). Briefly, monocyte-enriched cell fractions were cultured with GM-CSF and IL-4 for 5 days and cultured in medium containing 2. mu.g/ml CD40 ligand for an additional 2 days to induce maturation. Co-culture of 2X10 in 2ml RPMI supplemented with 10% AP, 10ng/ml IL-7 and 20IU/ml IL-2 in 24-well plates⁶Enriched CD8⁺And 2x10⁵DC/well of peptide pulse. Cultures were restimulated with self-irradiated peptide-pulsed DC on days 7 and 14.

Sequence variants of FLPWHRLFL were constructed as follows. Consistent with the table of binding coefficients (see table 3) derived from the NIH/BIMAS MHC binding prediction program (see reference in example 3 below), binding can be improved by changing one anchor position to V by changing L at position 9. Although usually to a lesser extent, the coupling may also be changed by a change in the non-anchoring position. Referring generally to table 3, binding can be improved by using residues with a relatively large index. Regardless of their effect on MHC binding, changes in sequence may also alter immunogenicity. Thus binding and/or immunogenicity may be improved as follows:

by replacing P at position 3 with F, L, M, W, or Y; these are bulky residues that can also improve immunogenicity regardless of the effect on binding. Residues with amine and hydroxyl groups, Q and N, S and T, respectively, can also elicit strong cross-reactive responses.

By replacing W at position 4 with D or E to improve binding, this addition of negative charge can also make the epitope more immunogenic, and in some cases reduce cross-reactivity with the native epitope. Alternatively conservative substitutions F or Y may elicit a cross-reactive response.

Binding was improved by replacing H at position 5 with F. H can be considered to be partially charged and thus the loss of charge can hinder cross-reactivity in some cases. Substitution of a sufficiently charged residue R or K at this position can enhance immunogenicity without destroying charge-dependent cross-reactivity.

By replacing R at position 6 with I, L, M, V, F, W, or Y. The same caveats as for position 5 and alternatives apply here.

Binding was improved by replacing L at position 7 with W or F. The reduction of binding affinity at this position V, I, S, T, Q, or N substitution is not generally predicted by this model (NIH algorithm), however, as discussed above may be advantageous.

Y and W, which are Fs, preferably at positions 1 and 8, can elicit significant cross-reactivity. Finally, although substitutions in the direction of bulk are generally biased toward improved immunogenicity, it may be effective to substitute smaller residues such as A, S and C at positions 3-7, in terms of size contrast, rather than being bulky in nature, which is a theory of importance for immunogenicity. The reactivity of thiol groups in C can be introduced as described in Chen, j. -l., et al j.immunol.165: 948 955, 2000, among other features discussed above.

TABLE 3 9-Link coefficient Table for HLA-A0201

The table and other comparable data available to the public are used to design epitope variants and to determine whether a particular variant is physically or functionally similar.

Example 3

Cluster analysis (SSX-2)_31-68)

1. Epitope clustering region prediction:

and (3) a computer algorithm: book "MHC Ligands and Peptide Motifs" SYFPEITHI based on H.G.Rammensee, J.Bachmann and S.Stevanovic (Internet Access http:// syfpeiti.bmi-heidelberg.com/Scripts/MHCServer.dll/EpPredict.htm); and in Parker, k.c., et al, j.immunol.152: 163, 1994 (NIH) (Internet access http:// bimas. dcrt. NIH. gov/molbio/HLA _ bin); was used to analyze the protein sequence of SSX-2 (GI: 10337583). EPITOPE clustering (regions containing higher than average density of peptide fragments with high predicted MHC affinity) was defined as fully described in U.S. patent No.09/561,571, entitled "epitop CLUSTERS," filed on 28.4.2000. Cutoff using epitope density ratio (cutoff) of 2, 5 and 2 clusters were defined using syfpetii and NIH algorithms, respectively, and peptide score cutoff was 16 (syfpetii) and 5 (NIH). Peptide scoring highest using NIH algorithm, SSX-2_41-49With an estimated dissociation half-life > 1000min, no overlap of any other predicted epitope in the NIH assay but indeed with SSX-2_57-65And (4) clustering.

2. Peptide synthesis and characterization:

SSX-2 Synthesis by MPS (Multiple Peptide Systems, San Diego, CA 92121) Using Standard solid phase chemistry_31-68YFSKEEWEKMKASEKIFYVYMKRKYEAMT KLGFKATLP (SEQ ID NO. 10). According to the "proof of analysis" provided, the peptide was 95% pure.

3. And (3) proteasome digestion:

proteasomes were isolated from human erythrocytes using the proteasome isolation protocol described in U.S. patent application No.09/561,074 entitled "METHOD OF epidopediscovery," filed on 28.4.2000. SDS-PAGE, Western blotting, and ELISA were used as quality control assays. The final concentration of proteasome was 4mg/ml, determined by a non-interfering protein assay (Geno Technologies Inc.). Proteasome was stored in 25 μ l aliquots at-70 ℃.

The mixture is subjected to SSX-2_31-68Dissolved in Milli-Q water to prepare 2mM stock solution and store 20. mu.l aliquots at-20 ℃.

1 tube of proteasome (25 μ l) was removed from the-70 ℃ storage and thawed on ice. It was then mixed thoroughly with 12.5 μ L of 2mM peptide by repeated pipetting (the sample was kept on ice). Immediately after mixing, 5 μ L of sample was taken and transferred to a tube containing 1.25 μ L10% TFA (final TFA concentration of 2%); t ═ 0min samples. The proteasome digestion reaction was then initiated and performed in a programmed thermal controller at 37 ℃. Additional 5 μ L samples were taken at 15, 30, 60, 120, 180 and 240min, respectively, and the reaction was terminated by adding the samples to 1.25 μ L10% TFA as before. The samples were kept on ice or frozen until analysis by MALDI-MS. All samples were retained and stored at-20 ℃ for HPLC analysis and N-terminal sequencing. Peptides alone (without proteasome) were used as blanks: mu.L peptide + 4. mu.L Tris buffer (20mM, pH 7.6) + 1.5. mu.L TFA.

MALDI-TOF MS measurement:

for each time point, 0.3. mu.L of matrix solution (10mg/ml of AcCN/H of alpha-cyano-4-hydroxycinnamic acid) was first introduced₂O solution (70: 30)) was applied to the sample slide, and then an equal volume of digested sample was gently mixed with the matrix solution on the slide. Slides were air dried at room temperature for 3-5 minutes before mass spectra were obtained. MS was performed on a Lasermat 2000MALDI-TOF mass spectrometer calibrated with peptide/protein standards. To improve the accuracy of the measurement, the molecular ion weight (MH) of the peptide substrate was determined⁺) Used as an internal calibration standard. Mass spectra of digested samples are shown in fig. 4 at T120 min.

MS data analysis and epitope identification:

to specify the measured Mass peaks, all possible fragments (N-and C-terminal ions, and internal fragments) and their corresponding molecular weights were generated using the computer program MS-Product, a tool derived from UCSF Mass Spectrometry Facility (accessible at http:// promoter. UCSF. edu/ucsfhtml3.4/msprod. htm). Average molecular weights are used due to the sensitivity of the mass spectrometer. As summarized in table 4, the mass peaks observed during digestion were identified.

The C-terminal fragment with a sequence 8-10 amino acids long predicted to bind to HLA by SYFPEITHI or NIH algorithm was selected for further study. The digestion and prediction steps of the method may be effectively carried out in any order. Although the substrate peptides used in the proteasome digestion described herein are specifically designed to include predicted HLA-a2.1 binding sequences, actual or predicted binding of actual digestion products to other MHC molecules can be detected afterwards. The selected results are shown in table 5.

TABLE 4 SSX-2_31-68Mass peak identification

Bold sequences correspond to peptides predicted to bind to MHC.

This peak can also be assigned to peptides 32-50 based on mass alone, however, it is less likely that proteasomes will remove only the N-terminal amino acid. N-terminal sequencing (below) confirmed that the sequence was 31-49.

Based on mass the fragments may also represent 33-68. The following N-terminal sequencing was consistent with determinations of 31-65.

TABLE 5 prediction of HLA binding by proteasome generated fragments

Unpredicted

As shown in Table 5, the addition of the authentic sequence (the authentic sequence) to the N-terminus of the epitope can generate epitopes against the same or different MHC restriction elements. Of particular note is the pairing of (K) RKYEAMTKL (SEQ ID NOS 19 and (20)) with HLA-B14 in which the 10-mer has a longer predicted dissociation half-life than the co-C-terminal 9-mer. Also note the case of 10-mer KYEAMTKLGF (SEQ ID No.21), which can be used as an effective vaccine against several MHC classes by relying on N-terminal pruning to generate epitopes against HLA-B4403 and-B08.

HLA-A0201 binding assay:

candidate epitopes KASEKIFYYV, SSX-2 were determined using variants of the method of Stauss et al (Proc Natl Acad Sci USA 89 (17): 7871-5 (1992)))_41-49(SEQ ID NO.15) binding to HLA-A2.1. Specifically, T2 cells expressing empty or labile MHC molecules on their surface were washed twice with Iscove's Modified Dulbecco's Medium (IMDM) and plated in 96-well flat-bottom plates at 3x10⁵Cells/200 μ l/well were cultured overnight in serum-free AIM-V medium (Life Technologies, inc., Rockville, MD) supplemented with 3 μ g/ml human β 2-microglobulin, and peptides at 800, 400, 200, 100, 50, 25, 12.5 and 6.25 μ g/ml were added. The peptides were mixed with the cells by repeated pipetting (alternatively the peptides could be added to separate wells) before dispensing to the plate, and the plate was gently shaken for 2 minutes. 5% CO at 37 ℃₂The incubator of (1). The following day unbound peptides were removed by washing twice with serum-free RPMI medium and saturating amounts of anti-HLA class I monoclonal antibody, Fluorescein Isothiocyanate (FITC) -conjugated anti-HLAA 2, a28(One Lambda, candida Park, CA) were added. After incubation at 4 ℃ for 30min, cells were washed 3 times with PBS supplemented with 0.5% BSA, 0.05% (w/v) sodium azide, pH 7.4-7.6 (staining buffer). (alternatively W6/32(Sigma) can be used as an anti-HLA class I monoclonal antibody, cells washed with staining buffer, incubated with Fluorescein Isothiocyanate (FITC) -conjugated sheep F (ab') anti-mouse-IgG (Sigma) for 30min at 4 ℃ and washed 3 times as above). Cells were resuspended in 0.5ml of stainIn a buffer. Analysis of surface HLA-a2.1 molecules stabilized by peptide binding was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA). If flow cytometry is not performed immediately, cells can be fixed by adding one-quarter volume of 2% paraformaldehyde and storing at 4 ℃ in the dark.

The results of the experiment are shown in fig. 5. Discovery of SSX-2_41-49(SEQ ID NO.15) binds to HLA-A2.1 to the extent of a known A2.1 conjugate FLPSDYFPSV (HBV) used as a positive control_18-27(ii) a SEQ ID NO: 24) similarly. HLA-B44 binding peptide, AEMGKYSFY (SEQ ID NO: 25) was used as a negative control. The fluorescence obtained from the negative control was similar to the signal obtained when the peptide was not used in the assay. The positive and negative control peptides are selected from Table 18.3.1 of Current Protocols in Immunology p.18.3.2, John Wiley and Sons, New York, 1998.

7. Immunogenicity:

A. in vivo immunization of mice

Anaesthetize and avoid the lateral tail vein at the caudal base, 100 μ l SSX-2 containing 100nmol was used_41-49(SEQ ID NO.15) and 20. mu.g of HTL epitope peptide in PBS emulsified with 50. mu.l of IFA (incomplete Freund's adjuvant) were injected subcutaneously into HHD1 transgenic A x 0201 mice (Pascolo, S., et al, J.exp.Med.185: 2043-.

B. Preparation of stimulatory cells (LPS blasts)

Spleens from 2 naive mice (nasal mice) were used for each group of immunized mice, the naive mice were sacrificed and the carcasses were placed in alcohol. Using a sterile instrument, the epithelial layer of the skin on the left side (lower middle portion) of the mouse was punctured, exposing the peritoneum. The peritoneum was saturated with ethanol and the spleen was removed aseptically. The spleens were placed in culture dishes containing serum-free medium. Splenocytes were isolated by triturating the spleen using a sterile plunger from a 3ml syringe. Dish wells were rinsed and splenocytes collected in serum-free medium in 50ml conical tubes. Cells were centrifuged (12000rpm, 7min) and washed once with RPMI. In RPMI-10% FCS (tires)Bovine serum) to a concentration of 1x10 per ml⁶A cell. Lipopolysaccharide 25g/ml and dextran sulphate 7. mu.g/ml were added. In the presence of 5% CO₂The cells were incubated in T-75 flasks at 37 ℃ for 3 days. Splenocytes were collected in 50ml tubes, centrifuged (pelleted) (12000rpm, 7min), and resuspended at 3X10 in RPMI⁷And/ml. Embryonic cells were pulsed with 50. mu.g/ml priming peptide (priming peptide) at room temperature for 4 hours, treated with mitomycin at 25. mu.g/ml C37 ℃ for 20min, and washed three times with DMEM.

C. In vitro stimulation

Antigen-exposed mice (14 days post immunization) were sacrificed as above to remove spleens 3 days after LPS stimulation of embryonic cells and on the same day as peptide loading. Mix 3x10⁶Spleen cells and 1x10⁶LPS embryonic cells/well in 24-well plates in DMEM medium at 5% CO₂At 37 ℃ in DMEM medium supplemented with 10% FCS, 5X10^-5M beta-mercaptoethanol, 100. mu.g/ml streptomycin and 100IU/ml penicillin. 5% (vol/vol) ConA supernatant was added to the culture on day 3 and on day 7⁵¹Cytolytic activity was determined in a Cr-release assay.

D. Chromium release assay to measure CTL activity

To assess peptide specific lysis, 2 × 10 was used⁶T2 cells were incubated with 100. mu. Ci of sodium chromate plus 50. mu.g/ml peptide for 1 hour at 37 ℃. During the incubation period, they were gently shaken every 15 minutes. After labeling and loading, cells were washed 3 times with 10ml DMEM-10% FCS and each tube was wiped with a fresh Kimwipe after the supernatant was run off. Target cells were resuspended in DMEM-10% FBS at 1X10⁵And/ml. Effector cells were adjusted to 1x10 in DMEM-10% FCS⁷100 μ l series of 3-fold dilutions of effectors were made in U-bottom 96-well plates. Add 100. mu.l of target cells per well. To determine spontaneous and maximal release, 6 additional wells containing 100 μ l of target cells were prepared for each target. Spontaneous release was shown by incubating the target cells with 100 μ l of medium; maximum release was shown by incubating the target cells with 100 μ l 2% SDS. Then the flat plate 600rpm centrifuge for 5min and 5% CO₂And incubated at 37 ℃ for 4 hours under 80% humidity conditions. After incubation, the plates were then centrifuged at 1200rpm for 5 min. The supernatants were harvested and counted in a gamma counter. Specific lysis was determined as follows: % specific release ═ [ (experimental release-spontaneous release)/(maximum release-spontaneous release)]x100。

The results of the chromium release assay demonstrating target cell-specific lysis of peptide pulses are shown in fig. 6.

8. Cross-reactivity with other SSX proteins:

SSX-2_41-49(SEQ ID NO.15) shares high sequence identity with the same region of other SSX proteins. The surrounding regions are also usually already well conserved. Therefore, the housekeeping proteasome can be at V in all 5 sequences₄₉And then cutting. In addition, SSX-2 is predicted_41-49Binds to HLA-a0201 (see table 6). By using SSX-2_41-49The CTLs produced by immunization cross-react with tumor cells expressing other SSX proteins.

TABLE 6 SSX _41-49 -A x 0201 predicted binding

Example 4

Cluster analysis (PSMA)_163-192)

Synthesis of a peptide Using Standard solid phase F-moc chemistry on a 433A ABI peptide synthesizer, AFSPQGMPEGDLVYVNYARTEDFFKLERDM, PSMA_163-192(SEQ ID NO.30) containing a 1epitope cluster, PSMA, derived from prostate specific membrane antigen_168-190(SEQ ID NO. 31). After side chain deprotection and cleavage from the resin, the following conditions were applied on a reverse phase preparative HPLC C4 column: a linear AB gradient (5% B/min) at a flow rate of 4ml/min, where eluent A was 0.1% TFA waterSolution and eluent B was 0.1% TFA in acetonitrile, running peptide first dissolved in formic acid and then diluted in 30% acetic acid. The fractions containing the desired peptide at time 16.642min according to mass spectrometry were pooled and lyophilized. The peptide was then subjected to proteasomal digestion and mass spectrometry essentially as described above. The significant peaks derived from mass spectra are summarized in table 7.

TABLE 7 PMSA _163-192 Mass peak identification

Bold sequences correspond to peptides predicted to bind to MHC, see table 8.

N-terminal pool sequencing (pool sequence) analysis

One aliquot of proteasome digested for one hour (see example 3, part 3 above) was subjected to N-terminal amino acid sequence analysis by an ABI 473A protein sequencer (Applied Biosystems, Foster City, Calif.). Determination of cleavage sites and efficiency is based on consideration of the sequencing cycle (sequence cycle), the repetitive yield of the protein sequencer and the relative yield of unique amino acids in the analyzed sequence. I.e., if the only (in the sequence analyzed) residue X is present only in the nth cycle, a cleavage site is present N-1 residues before it N-terminal. In addition to helping to resolve any misreadings in the assignment of masses to sequences, these data also provide a more reliable indication of the relative yields of various fragments than mass spectrometry.

For PSMA_163-192(SEQ ID NO.30) the pool sequencing supports sequencing at V₁₇₇Rear single mainCleavage site and several minor cleavage sites, especially in Y₁₇₉The latter one. Review the results presented in fig. 7A-C show the following:

s at cycle 3 indicates the presence of the N-terminus of the substrate.

Q at cycle 5 indicates the presence of the N-terminus of the substrate.

N in cycle 1 is indicated at V₁₇₇And (4) cutting.

N at cycle 3 is indicated at V₁₇₅And (4) cutting. Note fragment 176- & 192 in Table 7.

T at cycle 5 is indicated at V₁₇₇And (4) cutting.

T at cycles 1 to 3 is indicated at R₁₈₁，A₁₈₀And Y₁₇₉Followed by an increasingly common cut.

Only the last of these corresponds to a peak detected by mass spectrometry; 163, 179, and 180, 192, see table 7. The absence of others may indicate that they are on smaller fragments than are detected in mass spectrometry.

K at cycles 4, 8, and 10 indicates at E₁₈₃，Y₁₇₉And V₁₇₇The cutting is carried out after that,

all these correspond to fragments observed by mass spectrometry. See table 7.

A at cycles 1 and 3 indicates the presence of the N-terminus of the substrate and at V, respectively₁₇₇And (4) cutting.

P at cycles 4 and 8 indicates the presence of the N-terminus of the substrate.

G at cycles 6 and 10 indicates the presence of the N-terminus of the substrate.

M at cycle 7 indicates the presence of the N-terminus of the substrate and/or at F₁₈₅And (4) cutting.

M at cycle 15 is indicated at V₁₇₇And (4) cutting.

Cycle 1 may be indicated at D₁₉₁See table 7 for later cuts.

R at cycles 4 and 13 is shown at V₁₇₇And (4) cutting.

R at cycles 2 and 11 is indicated at Y₁₇₉And (4) cutting.

V at cycles 2, 6 and 13 are indicated at V, respectively₁₇₅，M₁₆₉Cleavage after and the presence of the N-terminus of the substrate. Note the segments beginning at 176 and 170 in table 7.

Y at 1, 2 and 14 cycles is shown at V₁₇₅，V₁₇₇Cleavage after and the presence of the N-terminus of the substrate.

L at cycles 11 and 12 are shown at V, respectively₁₇₇The latter cleavage and the presence of the N-terminus of the substrate are the most consistent explanations with the other data.

Comparing the mass spectra results we found that L at cycles 2, 5 and 9 is compared to that at F, respectively₁₈₆，E₁₈₃Or M₁₆₉And Y is₁₇₉The cut was consistent. See table 7.

Epitope identification

The co-C-terminal fragment with a sequence 8-10 amino acids long predicted to bind to HLA by SYFPEITHI or NIH algorithm was selected for further analysis. The digestion and prediction steps of the method may be effectively carried out in any order. Although the substrate peptides used in the proteasome digestion described herein are specifically designed to include predicted HLA-a1 binding sequences, actual or predicted binding of actual digestion products to other MHC molecules can be detected afterwards. The selected results are shown in table 8.

TABLE 8 prediction of HLA binding by proteasome generated fragments

Unpredicted

HLA-a0201 binding assay:

PSMA was used essentially as described in example 3_168-177GMPEGDLVYV, (SEQ ID NO.33) HLA-A0201 binding studies were performed. As shown in fig. 8, the epitope showed significant binding even at lower concentrations than the positive control peptide. The Melan-A peptide, ELAGIGILTV, used as a control in this assay (and throughout the disclosure) is actually a variant of the native sequence (EAAGIGILTV) and shows high affinity in this assay.

Example 5

Cluster analysis (PSMA)_281-310)

Another peptide was chemically synthesized using standard solid phase F-moc on a 433A ABI peptide synthesizer, RGIAEAVGLPSIPVHPIGYYDAQKLLEKMG, PSMA_281-310(SEQ ID NO.45) which contains an epitope derived from prostate specific membrane antigen A1, PSMA_283-307(SEQ ID NO. 46). After side chain deprotection and cleavage from the resin, the following conditions were applied on a reverse phase preparative HPLC C18 column: a linear AB gradient (5% B/min) at a flow rate of 4ml/min with eluent A being 0.1% TFA in water and eluent B being 0.1% TFA in acetonitrile was run with deionized water (ddH)₂O) is used. The fractions containing the desired peptide at time 17.061min according to mass spectrometry were pooled and lyophilized. The peptides were then subjected to proteasomal digestion and mass spectrometry analysis essentially as described above. The significant peaks derived from mass spectra are summarized in table 9.

TABLE 9 PSMA _281-310 Mass peak identification

Bold sequences correspond to peptides predicted to bind to MHC, see table 10.

The peak may also be 296-.

The peak may also be 298-. The combination of HPLC and mass spectrometry showed that at some later time point the peak was a mixture of two species.

The peak may also be 289-298 on the basis of mass only.

The peak may also be 281-.

The peak may also be 297 § 303 based only on mass.

The peak may also be 285-.

The peak may also be 288-.

N-terminal pool sequencing analysis does not support the assignment of any of these alternatives.

N-terminal pool sequencing analysis

One aliquot of proteasome digested for one hour (see example 3, part 3 above) was subjected to N-terminal amino acid sequence analysis by an ABI 473A protein sequencer (Applied Biosystems, Foster City, Calif.). Determination of cleavage sites and efficiency is based on consideration of the sequencing cycle (sequence cycle), the repetitive yield of the protein sequencer and the relative yield of unique amino acids in the analyzed sequence. I.e., if the only (in the sequence analyzed) residue X is present only in the nth cycle, a cleavage site is present N-1 residues before it N-terminal. In addition to helping to resolve any misreadings in the assignment of masses to sequences, these data also provide a more reliable indication of the relative yields of various fragments than mass spectrometry.

For PSMA _281-310 (SEQ ID NO.45) this pool sequencing supports V in other minor cleavage sites₂₈₇And I₂₉₇The latter two main cleavage sites. Overview the results presented in figure 9 show the following:

s in cycles 4 and 11 are shown at V₂₈₇Cleavage after and the presence of the N-terminus of the substrate.

H at cycle 8 is indicated at V₂₈₇And (4) cutting. The lack of a decrease in peak height at positions 9 and 10 relative to the decrease in height present at 10 to 11, rather than a peak representing latency in the sequencing reaction, may suggest that at a₂₈₆And E₂₈₅And then cutting.

D at cycles 2, 4 and 7 are indicated at Y₂₉₉，I₂₉₇And V₂₉₄And (4) cutting.

This last cut was not observed in any of the fragments in table 10 or in the alternative designations noted below.

Q at cycle 6 is shown at I₂₉₇And (4) cutting.

M at cycles 10 and 12 are indicated at Y₂₉₉And I₂₉₇And (4) cutting.

Epitope identification

The co-C-terminal fragment with a sequence 8-10 amino acids long predicted to bind to HLA by SYFPEITHI or NIH algorithm was selected for further analysis. The digestion and prediction steps of the method may be effectively carried out in any order. Although the substrate peptides used in the proteasome digestion described herein are specifically designed to include predicted HLA-a1 binding sequences, actual or predicted binding of actual digestion products to other MHC molecules can be detected afterwards. The selected results are shown in table 10.

Table 10.

Fragments produced by proteasome: PSMA _281-310 Predicting HLA binding

Unpredicted

As shown in Table 10, appending authentic sequences to the N-terminus of an epitope can often produce an epitope that is still effective against the same or different MHC restriction elements, or even better. Note for example the pairing of (G) LPSIPVHPI with HLA-a0201, where 10-mer can be used as an effective vaccine against several MHC types by relying on N-terminal pruning to generate epitopes against HLA-B7, -B5101, and Cw 0401.

HLA-a0201 binding assay:

use of PSMA substantially as described in examples 3 and 4 above_288-297GLPSIPVHPI, (SEQ ID NO.48) HLA-A0201 binding studies were performed. As shown in fig. 8, the epitope showed significant binding even at lower concentrations than the positive control peptide.

Example 6

Cluster analysis (PSMA)_454-481)

Synthesis of another peptide, S, by MPS (> 95% purity)SIEGNYTLRVDCTPLMYSLVHLTKEL，PSMA_454-481(SEQ ID No.55) containing clusters of epitopes derived from prostate specific membrane antigen, which were subjected to proteasomal digestion and mass spectrometry as described above. The significant peaks derived from mass spectra are summarized in table 11.

TABLE 11.PSMA _454-481 Mass peak identification

Bold sequences correspond to peptides predicted to bind to MHC, see table 12.

This peak can equally well be designated as peptide 455-472 based on mass alone, however it is less likely that proteasome will remove only the N-terminal amino acid. If the problem is significant it can be solved by N-terminal sequencing.

This fragment may also represent 455-464 based on mass.

Epitope identification

The co-C-terminal fragment with a sequence 8-10 amino acids long predicted to bind to HLA by SYFPEITHI or NIH algorithm was selected for further analysis. The digestion and prediction steps of the method may be effectively carried out in any order. Although the substrate peptides used in the proteasome digestion described herein are specifically designed to include predicted HLA-a2.1 binding sequences, actual or predicted binding of actual digestion products to other MHC molecules can be detected afterwards. The selected results are shown in table 12.

TABLE 12 prediction of HLA binding by proteasome generated fragments

Unpredicted

As shown in Table 12, appending authentic sequences to the N-terminus of an epitope can often produce an epitope that is still effective against the same or different MHC restriction elements, or even better. Note for example the pairing of (L) RVDCTPLMY (SEQ ID NOS 62 and (63)) with HLA-B2702/5, where the 10-mer has a specific (substential) predicted dissociation half-life and the co-C-terminal 9-mer does not. Note also SIEGNYTLRV (SEQ ID NO 57), a predicted case of HLA-a0201 epitopes, which can be used as effective vaccines against HLA-B5101 by creating epitopes dependent on N-terminal pruning.

HLA-A0201 binding assay

Use of PSMA substantially as described in example 3 above_460-469TLRVDCTPL, (SEQ ID NO.60) HLA-A0201 binding studies were performed. As shown in FIG. 10, the epitope and HLA-A2.1 were found to bind with a known A2.1 binder FLPSDYFPSV (HBV) used as a positive control_18-27(ii) a SEQ ID NO: 24) similar degrees of binding. In addition, PSMA_461-469(SEQ ID NO.59) almost also bound.

ELISPOT assay:PSMA _463-471 ，(SEQ ID NO.62)

the wells of a nitrocellulose-based (nitrocellulose-backed) microtiter plate were coated with capture antibody by incubating overnight at 4 ℃ with 50. mu.l/well of 4. mu.g/ml murine anti-human gamma-IFN monoclonal antibody in coating buffer (35mM sodium bicarbonate, 15mM sodium carbonate, pH 9.5). Unbound antibody was removed by washing 4 times with PBS for 5 min. Unbound sites on the membrane were then blocked by adding 200. mu.l/well of RPMI medium containing 10% serum and incubating for 1 hour at room temperature. Antigen-stimulated CD8⁺T cells, seeded in wells of microtiter plates at 1: 3 serial dilutions, 100. mu.l/well from 2X10⁵Cells/well start. (previous antigenic stimulation was essentially as described by Scheibenbogen, CInt.j.cancer 71: 932-936, 1997). Mixing PSMA_462-471(SEQ ID NO.62) to a final concentration of 10. mu.g/ml, IL-2 to 100U/ml and at 5% CO₂The cells were cultured in water-saturated air at 37 ℃ for 40 hours. After this incubation, wash 6 times with 200 μ l/well of PBS containing 0.05% Tween-20 (PBS-Tween). Detection antibody, biotinylated mouse anti-human gamma-IFN monoclonal antibody at 2g/ml in PBS + 10% fetal calf serum at 50. mu.l/well was added and the plates were incubated for 2 hours at room temperature. Unbound detection antibody was removed by washing 4 times with 200 μ l PBS-tween. Mu.l avidin-coupled horseradish peroxidase (Pharmingen, San Diego, Calif.) was added to each well and incubated for 1 hour at room temperature. Unbound enzyme was removed by washing 6 times with 200 μ l PBS-tween. The substrate was prepared by dissolving 20mg of 3-amino 9-ethylcobarasole tablet in 2.5ml of N, N-dimethylformamide and adding that solution to 47.5ml of 0.05M phosphate-citrate buffer (pH 5.0). Immediately before the substrate will be dispensed at 100. mu.l/well and the plate incubated at room temperature 25. mu.l of 30% H₂O₂Added to the substrate solution. After development (typically 15-30min), the reaction was stopped by washing the plate with water. The plates were air dried and the spots were counted using a stereomicroscope.

FIG. 11 shows detection of PSMA_463-471(SEQ ID NO.62) -reactive HLA-A1⁺CD8⁺T cells, previously described in HLA-A1⁺CD8⁺T cells were generated in culture with autologous dendritic cells plus peptide. No reactivity was detected from cultures without peptide (data not shown). In this case it can be seen that the peptide reactive T cells are at 2.2x10⁴One-half to 6.7x10⁴One-half of the frequency is present in the culture. The ability of the anti-HLA-A1 monoclonal antibody to block the production of gamma-IFN demonstrates that this is indeed an HLA-A1-restricted response; see fig. 12.

Example 7

Cluster analysis (PSMA)_653-687)

Synthesis of another peptide by MPS (> 95% purity), FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFY PSMA_653-687(SEQ ID NO.64) containing a2 epitope cluster, PSMA, derived from prostate specific membrane antigen_660-681(SEQ ID NO 65) and proteasomal digestion and mass spectrometry analysis were performed as described above. The significant peaks derived from mass spectra are summarized in table 13.

TABLE 13 PSMA_653-687Mass peak identification

Bold sequences correspond to peptides predicted to bind to MHC, see table 13.

This peak can equally well be assigned to the peptide starting at 654 based on mass alone, whereas proteasome removal of only the N-terminal amino acid is considered unlikely. If the problem is important, it can be solved by N-terminal sequencing.

These peaks may have been assigned as internal fragments based on mass alone, but are considered unlikely given the overall pattern of digestion.

Epitope identification

The co-C-terminal fragment with a sequence 8-10 amino acids long predicted to bind to HLA by SYFPEITHI or NIH algorithm was selected for further analysis. The digestion and prediction steps of the method may be effectively carried out in any order. Although the substrate peptides used in the proteasome digestion described herein are specifically designed to include predicted HLA-a2.1 binding sequences, actual or predicted binding of actual digestion products to other MHC molecules can be detected afterwards. The selected results are shown in table 12.

TABLE 14 prediction of HLA binding by proteasome generated fragments

Unpredicted

As shown in Table 14, appending authentic sequences to the N-terminus of an epitope can often produce epitopes that are still effective against the same or different MHC restriction elements, or even better. Note for example the pairing of (R) MMNDQLMFL (SEQ ID nos.66 and (67)) with HLA-a02, where the 10-mer retains a significant (substential) predicted binding potential.

HLA-A0201 binding assay

PSMA was used essentially as described in example 3 above_663-671(SEQ ID NO.66) and PSMA_662-671RMMNDQLMFL (SEQ NO.67) was subjected to HLA-A0201 binding studies. As shown in FIGS. 10, 13 and 14, in comparison with the positive control peptide (FLPSDYFPSV (HBV)_18-27) (ii) a SEQ ID NO: 24) this epitope also showed significant binding at low concentrations. Comparison with controls, although not performed in parallel, suggests PSMA_662-671(which is close to MelanA peptide in affinity) has the superior binding activity of these two PSMA peptides.

Example 8

Vaccination with a epitope vaccine.

1. Vaccination with peptide vaccine:

A. intra-nodal delivery

A formulation comprising a peptide in an aqueous buffer containing an antibacterial agent, an antioxidant and an immunomodulatory cytokine is injected into the inguinal lymph node for several consecutive days using a micropump system (MiniMed; Northbridge, CA) developed for insulin delivery. The infusion period was chosen in order to mimic the kinetics of antigen presentation in natural infections.

B. Controlled release of a substance

Peptide formulations are delivered using controllable PLGA microspheres known in the art that alter the pharmacokinetics of the peptide and improve immunogenicity. The preparation can be injected or orally administered.

C. Particle gun delivery

A peptide formulation was prepared in which the peptide was attached to gold particles as known in the art. The particles are delivered in a gene gun, which is accelerated at high speed to penetrate the skin, carrying the particles into the skin tissue containing pAPCs.

D. Aerosol delivery

The peptide formulation is inhaled as an aerosol known in the art for absorption into the appropriate vascular or lymphatic tissue in the lung.

2. Vaccination with nucleic acid vaccines

Nucleic acid vaccines are injected into lymph nodes using a micropump system, such as a MiniMed insulin pump. To mimic the kinetics of antigen presentation in natural infections, a nucleic acid construct formulated in an aqueous buffer containing an antibacterial agent, an antioxidant and an immunomodulatory cytokine is delivered over an infusion period of several days.

Optionally, the nucleic acid construct is delivered using a controlled release substance, such as PLGA microspheres or other biodegradable substances. These substances are injected or orally administered. Nucleic acid vaccines are administered using oral delivery to elicit an immune response by absorption in GALT tissue. Alternatively, the nucleic acid vaccine is delivered using a gene gun, wherein the nucleic acid vaccine is adhered to fine gold particles. The nucleic acid construct may also be inhaled as an aerosol for uptake into appropriate vascular or lymphatic tissue in the lung

Example 9

Determining the efficacy of epitope vaccines

1. Tetramer analysis:

a class I tetramer assay was used to determine T cell frequency in animals before and after administration of housekeeping epitopes. Clonal expansion of T cells in response to the epitope revealed that the epitope was presented by pAPCs to the T cells. Specific T cell frequencies for housekeeping epitopes were measured before and after administration of the epitopes to animals to determine whether the epitopes were presented on pAPCs. An increase in the frequency of epitope-specific T cells following administration indicates that the epitope is presented on pAPC.

2. Proliferation assay:

approximately 24 hours after inoculating the animals with the housekeeping epitope, pAPCs were harvested from PBMCs, splenocytes, or lymph node cells using monoclonal antibodies against the specific label present on the pAPCs immobilized on magnetic beads for affinity purification. pAPCs are enriched from crude blood or spleen cell preparations using this technique. The enriched pAPCs are then used in a proliferation assay for T cell clones that have been generated and are specific for the housekeeping epitope of interest. pAPCs were co-incubated with T cell clones and T cell proliferation activity was monitored by measuring incorporation of T cell radiolabeled thymidine. Proliferation indicates that T cells specific for the housekeeping epitope are being stimulated by the epitope on pAPCs.

3. Determination of chromium release:

human patients or non-human animals genetically engineered to express human MHC class I are immunized with housekeeping epitopes. T cells derived from immunized subjects were used in standard chromium release assays using human tumor targets or targets engineered to express the same MHC class I. The T cell killing objective indicates that stimulation of T cells in the patient will effectively kill tumors expressing a similar TuAA.

Example 10

Induction of CTL responses with naked DNA by intralymph node immunization is effective.

To quantitatively compare CD8 induced by different routes of immunization⁺CTL responses used a plasmid DNA vaccine (pEGFPL33A) containing a full characterizationcharaterized) derived from LCMV-glycoprotein (G) (gp 33; immunodominant CTL epitopes of amino acids 33-41) (Oehen, S., et al., Immunology99, 163-16992000) because this system allows for a broad assessment of the anti-viral CTL response. Groups of 2C 57BL/6 mice were immunized with a titrated dose (200-0.02 μ g) of pEGFPL33A DNA or the control plasmid pEGFP-N3, which was administered i.m. (intramuscularly), i.d. (intradermally), i.spl. (intrasplenic), or i.ln. (intralymph node). Positive control mice received 500pfuLCMV (intravenously). 10 days after immunization, splenocytes were isolated and assayed for gp 33-specific CTL activity after a second restimulation in vitro. As shown in fig. 15, the i.m. or i.d. immunization induced a weak detectable CTL response when high doses of pEFGPL33A DNA (200 μ g) were administered. In contrast, immunization with 2. mu.g pEFGPL33A DNA alone i.spl. and with as little as 0.2. mu.g pEFGPL33A DNA i.ln. elicited effective gp 33-specific CTL responses (FIG. 15; symbols indicate individual mice, and one of three similar experiments is shown). Immunization with control pEGFP-N3DNA did not elicit any detectable gp 33-specific CTL response (data not shown).

Example 11

DNA immunization in lymph nodes elicits anti-tumor immunity

To examine whether the effective CTL response elicited after i.ln. immunization could confer protection against peripheral tumors, groups of 6C 57BL/6 mice were immunized 3 times with 10 μ g of pEFGPL33A DNA or control pEGFP-N3DNA at 6 day intervals. After 5 days of the last immunization, small pieces of solid tumor expressing the gp33 epitope (EL4-33) were transferred s.c. to the two flanks and tumor growth was measured every 3-4 days. Although the EL4-33 tumors grew well in mice that had been repeatedly immunized with the control pEGFP-N3DNA (fig. 16), mice immunized with pEFGPL33A dnai. ln. quickly eradicated the peripheral EL4-33 tumors (fig. 16).

Example 12

Differences in DNA content in lymph nodes reflect differences in CTL responses within lymph nodes and after intramuscular injection.

i.ln or i.m. injection of pEFGPL33A DNA and purification of the DNA at 6, 12, 24,plasmid content of the injected or draining lymph nodes was assessed by real-time PCR after 48 hours and 4 and 30 days. After i.m. injection, the plasmid DNA content of the injected lymph nodes was approximately 3 orders of magnitude greater than that of the draining lymph nodes at 6, 12 and 24 hours. No plasmid DNA was detected in the draining lymph nodes at the subsequent time points (fig. 17). This is consistent with the use of i.m. injection requiring a3 order greater dose to achieve similar levels of CTL activity compared to i.ln. Same i.ln. injection of CD8^-/-Knockout mice, which do not develop a CTL response against this epitope, show that the clearance of DNA from lymph nodes is not due to CD8⁺CTLs kill cells in lymph nodes. This observation also supports the conclusion that i.ln. administration will not provoke immunopathological damage to lymph nodes.

Example 13

DNA plasmid formulation for therapeutic vaccine against melanoma administered to human

SYNCHROTOPE TA2M, a melanoma vaccine encoding HLA-A2-restriction tyrosinase epitope SEQ ID NO.1 and epitope clustering SEQ ID NO.69, was formulated in 1% benzyl alcohol, 1% ethanol, 0.5mM EDTA, citrate-phosphate, pH 7.6. For loading into the MINIMED 407C infusion pump, aliquots of 80, 160 and 320. mu.g DNA/ml were prepared. The catheter of the silouette infusion device was placed in the inguinal lymph node visualized by ultrasound imaging. The pump and infusion set assembly was originally designed for delivery of insulin to diabetic patients, replacing the usual 17mm catheter with a 31mm catheter for this application. The infusion device was left open for 4 days (about 96 hours) at an infusion rate of about 25 μ l/hour, resulting in a total infusion volume of about 2.4 ml. Thus for the 3 concentrations mentioned above, the total dose administered for each infusion was about 200 and 400 μ g, respectively; and may be 800. mu.g. The infused subjects were given a 10 day rest period before beginning a subsequent infusion. Given the sustained retention of plasmid DNA in lymph nodes following administration (as in example 12) and the general response kinetics of CTLs after antigen elimination, this schedule will be sufficient to maintain immune CTL responses.

Example 14

Epitope tag

The methods described above, particularly in examples 3-7, have been applied to additional synthetic peptide substrates, which resulted in the identification of further epitopes as in tables 15-36 below. The substrate used here is designed to identify the products of housekeeping proteasome processing that produce HLA-a0201 binding epitopes, however, as described above, additional MHC-binding reactivity can be predicted. However, many such reactivities are disclosed, and these lists are intended to be exemplary, rather than exhaustive and limiting. As also discussed above, the individual components of the analyte may be used in varying combinations and sequences. Digestion of the NY-ESO-1 substrates 136-163 and 150-177 (SEQ ID NOS.254 and 255, respectively) produced fragments that did not fly well in MALDI-TOF mass spectrometry. However they are well suited for N-terminal peptide pool sequencing, thereby allowing the identification of cleavage sites. Not all substrates necessarily meet the formal definition of epitope clustering cited in example 3. Some clusters are so large, e.g. NY-ESO-1_86-171To make it more convenient to use a substrate that spans only a portion of the cluster. In other cases, the substrate extends beyond the cluster that satisfies the formal definition to include the adjacent predicted epitope. In some cases, the actual binding activity may have determined what substrates, such as the MAGE epitopes reported here, were prepared prior to designing the synthetic substrates, where HLA binding activity was determined to select peptides with predicted affinity.

Example 15

Assessment of epitope Cross-reactivity to non-target tissues

As mentioned above, PSA is a member of the kallikrein family of proteases, which is itself a subtype of the serine protease family. Although members of this family that share the greatest degree of sequence identity with PSA also share similar expression profiles, proteins that may have significantly different expression profiles share a separate sequence of the expression profile. The first step in assessing unwanted cross-reactivity is to identify shared sequences. One way to accomplish this is to BLAST Search for epitope sequences on SWISSPROT or Entrez non-redundant peptide sequence databases using the "Search for short near exact matches" option; hypertext transfer protocol is accessible over the world wide web (http:// www) "ncbi. Thus, a search of SEQ ID NO.214, WVLTAAHCI (human-limited records) for SWISSPROT found four exact matches, including PSA. The other 3 are derived from kallikrein 1 (tissue kallikrein), and elastase 2A and 2B. Although these 9 amino acid segments are identical, the flanking sequences are completely different, particularly on the C-terminal side, suggesting that different processing can occur and thus the same epitope may not be released from these other proteins. (please note that kallikrein nomenclature is confused). Kallikrein 1[ accession number P06870] is thus a protein different from that mentioned above in relation to the PSA paragraph in the section on tumor-associated antigens [ accession number AAD13817 ]).

This possibility can be checked in several ways. Synthetic peptides containing epitope sequences embedded within the context of each of these proteins can be subjected to in vitro proteasome digestion and analysis as described above. Alternatively, to determine whether an epitope is processed and presented, CD8 recognizing the epitope is being utilized⁺Cells expressing these other proteins by natural or recombinant expression can be used as targets in cytotoxicity (or similar) assays of T cells.

Example 16

Epitope clustering

Known and predicted epitopes are often unevenly distributed in the protein antigen sequence. As described above, we have defined epitope clusters that contain sequence segments that are more dense than the average density of (known or predicted) epitopes. The use of epitope clustering is to incorporate their sequences into the substrate peptides used in the proteasome digestion assay described herein. Epitope clustering can also be used as a vaccine component. A more thorough discussion of EPITOPE clustering definition and use is found in U.S. patent application No.09/561,571, entitled EPITOPE CLUSTERS.

The following tables (37-60) show the epitope densities of the 9-mer epitope and overlapping epitope regions predicted to be bound by HLA-A2 using SYFPEITHI and the NIH algorithm, as well as the epitope densities of the epitopes throughout the protein, and the ratio of these two densities. (the ratio must exceed 1 to cluster by definition above; requiring a higher value for this ratio reflects a preferred embodiment). Individual 9-mer were identified by scoring permutations and by their first amino position in the complete protein sequence. The numbering is derived from each possible cluster of proteins. The range of amino acid positions within the complete sequence that a cluster contains is expressed as the rank of the individual predicted epitope that makes up it.

Watch 37

BIMAS-NIH/Parker algorithm results for gp100

Watch 38

SYFPEITHI (Rammensee algorithm) result of gp100

Watch 39

Cluster prediction of gp100

Total AAs: 661

Total 9-chain links: 653

SYFPEITHI 16: 1099-Link

NIH 5: 409-chain link

Adjacent but non-overlapping epitopes

Watch 40

BIMAS-NIH/Parker algorithm results for PSMA

Table 41

SYFPEITHI (Rammensee algorithm) result of PSMA

Watch 42

Cluster prediction of Prostate Specific Membrane Antigen (PSMA)

Total AAs: 750

Total 9-chain links: 742

SYFPEITHI 16: 889-Link

NIH 5: 309-chain link

Adjacent but non-overlapping epitopes

Watch 43

BIMAS-NIH/Parker algorithm results for PSA

Watch 44

SYFPEITHI (Rammensee algorithm) results for PSA

TABLE 45

Cluster prediction of Prostate Specific Antigen (PSA)

Total AAs: 261

Total 9-chain links: 253

SYFPEITHI 16: 369-Link

NIH 5: 179-chain Link

These clusters are internal to the less preferred cluster # 4.

Comprises one adjacent but non-overlapping epitope.

TABLE 46

BIMAS-NIH/Parker algorithm result of PSCA

Watch 47

SYFPEITHI (Rammensee algorithm) result of PSCA

Watch 48

Cluster prediction of Prostate Stem Cell Antigen (PSCA)

Total AAs: 123

Total 9-chain links: 115

SYFPEITHI 16：33；

SYFPEITHI 20：13

NIH 5：13

This cluster is the interior of the less preferred cluster # 3.

Epitope prediction and clustering data for each algorithm are presented together in a single table in tables 49-60.

Watch 49

MAGE-1 Cluster prediction (NIH Algorithm)

Total AAs: 309

Total 9-chain links: 301

NIH 5: 199-Link

Watch 50

Clustering prediction of MAGE-1 (SYFPEITHI algorithm)

Total AAs: 309

Total 9-chain links: 301

SYFPEITHI 16: 469-Link

Watch 51

Clustering prediction of MAGE-2 (NIH algorithm)

Total AAs: 314

Total 9-chain links: 308

NIH > ═ 5: 209-chain link

Table 52

Clustering prediction of MAGE-2 (SYFPEITHI algorithm)

Total AAs: 314

Total 9-chain links: 308

SYFPEITHI 16: 529-chain link

Watch 53

Clustering prediction of MAGE-3 (NIH algorithm)

Total AAs: 314

Total 9-chain links: 308

NIH 5: 229-Link

Watch 54

Clustering prediction of MAGE-3 (SYFPEITHI algorithm)

Total AAs: 314

Total 9-chain links: 308

SYFPEITHI 16: 479 chain link

Watch 55

Cluster prediction of PRAME (NIH algorithm)

Total AAs: 509

Total 9-chain links: 501

NIH 5: 409-chain link

Watch 56

Cluster prediction of PRAME (SYFPEITHI Algorithm)

Total AAs: 509

Total 9-chain links: 501

SYFPEITHI 17: 809-chain link

Cluster prediction of PRAME (SYFPEITHI Algorithm)

Total AAs: 509

Total 9-chain links: 501

SYFPEITHI 17: 809-chain link

Watch 57

Clustering prediction of CEA (NIH algorithm)

Total AAs: 702

Total 9-chain links: 694

NIH 5: 309-chain link

Watch 58

Clustering prediction of CEA (SYFPEITHI algorithm)

Total AAs: 702

Total 9-chain links: 694

SYFPEITHI 16: 819-Link

Watch 59

Cluster prediction of SCP-1 (NIH algorithm)

Total AAs: 976

Total 9-chain links: 968

NIH 5: 379-Link

Watch 60

Cluster prediction for SCP-1

Total AAs: 976

Total 9-chain links: 968

Rammensee 16: 1189-Link

Embodiments of the present invention are applicable to and take into account variations in the target antigen sequences provided herein, including those disclosed in various databases accessible via the world wide web. Specifically for the specific sequences disclosed herein, variations in the sequence can be discovered by accessing information about each antigen using the accession numbers provided.

A tyrosinase protein; SEQ ID NO 2

1 MLLAVLYCLL WSFQTSAGHF PRACVSSKNL MEKECCPPWS GDRSPCGQLSGRGSCQNILL

61 SNAPLGPQFP FTGVDDRESW PSVFYNRTCQ CSGNFMGFNC GNCKFGFWGPNCTERRLLVR

121RNIFDLSAPE KDKFFAYLTL AKHTISSDYV IPIGTYGQMK NGSTPMFNDINIYDLFVWMH

181YYVSMDALLG GSEIWRDIDF AHEAPAFLPW HRLFLLRWEQ EIQKLTGDENFTIPYWDWRD

241AEKCDICTDE YMGGQHPTNP NLLSPASFFS SWQIVCSRLE EYNSHQSLCNGTPEGPLRRN

301PGNHDKSRTP RLPSSADVEF CLSLTQYESG SMDKAANFSF RNTLEGFASPLTGIADASQS

361SMHNALHIYM NGTMSQVQGS ANDPIFLLHH AFVDSIFEQW LRRHRPLQEVYPEANAPIGH

421NRESYMVPFI PLYRNGDFFI SSKDLGYDYS YLQDSDPDSF QDYIKSYLEQASRIWSWLLG

481AAMVGAVLTA LLAGLVSLLC RHKRKQLPEE KQPLLMEKED YHSLYQSHLSSX-2 protein; SEQ ID NO 3

1 MNGDDAFARR PTVGAQIPEK IQKAFDDIAK YFSKEEWEKM KASEKIFYVYMKRKYEAMTK

61 LGFKATLPPF MCNKRAEDFQ GNDLDNDPNR GNQVERPQMT FGRLQGISPKIMPKKPAEEG

121 NDSEEVPEAS GPQNDGKELC PPGKPTTSEK IHERSGPKRG EHAWTHRLRERKQLVIYEEI

181 SDPEEDDEPSMA protein; SEQ ID NO 4

1 MWNLLHETDS AVATARRPRW LCAGALVLAG GFFLLGFLFG WFIKSSNEATNITPKHNMKA

61 FLDELKAENI KKFLYNFTQI PHLAGTEQNF QLAKQIQSQW KEFGLDSVELAHYDVLLSYP

121 NKTHPNYISI INEDGNEIFN TSLFEPPPPG YENVSDIVPP FSAFSPQGMPEGDLVYVNYA

181 RTEDFFKLER DMKINCSGKI VIARYGKVFR GNKVKNAQLA GAKGVILYSDPADYFAPGVK

241 SYPDGWNLPG GGVQRGNILN LNGAGDPLTP GYPANEYAYR RGIAEAVGLPSIPVHPIGYY

301 DAQKLLEKMG GSAPPDSSWR GSLKVPYNVG PGFTGNFSTQ KVKMHIHSTNEVTRIYNVIG

361 TLRGAVEPDR YVILGGHRDS WVFGGIDPQS GAAVVHEIVR SFGTLKKEGWRPRRTILFAS

421 WDAEEFGLLG STEWAEENSR LLQERGVAYI NADSSIEGNY TLRVDCTPLMYSLVHNLTKE

481 LKSPDEGFEG KSLYESWTKK SPSPEFSGMP RISKLGSGND FEVFFQRLGIASGRARYTKN

541 WETNKFSGYP LYHSVYETYE LVEKFYDPMF KYHLTVAQVR GGMVFELANSIVLPFDCRDY

601 AVVLRKYADK IYSISMKHPQ EMKTYSVSFD SLFSAVKNFT EIASKFSERLQDFDKSNPIV

661 LRMMNDQLMF LERAFIDPLG LPDRPFYRHV IYAPSSHNKY AGESFPGIYDALFDIESKVD

721 PSKAWGEVKR QIYVAAFTVQ AAAETLSEVA

(R)，mRNA；

Homo sapiens

ACCESSION NM_000372

VERSION NM_000372.1 GI：4507752

SEQ ID NO 2

' MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRSPCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCGNCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQMKNGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSWQIVCSRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESGSMDKAANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFLLHHAFVDSIFEQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDLGYDYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCRHKRKQLPEEKQPLLMEKEDYHSLYQSHL

SEQ ID NO 5

Start 1 atcactgtag tagtagctgg aaagagaaat ctgtgactcc aattagccagttcctgcaga

61 ccttgtgagg actagaggaa gaatgctcct ggctgttttg tactgcctgctgtggagttt

121 ccagacctcc gctggccatt tccctagagc ctgtgtctcc tctaagaacctgatggagaa

181 ggaatgctgt ccaccgtgga gcggggacag gagtccctgt ggccagctttcaggcagagg

241 ttcctgtcag aatatccttc tgtccaatgc accacttggg cctcaatttcccttcacagg

301 ggtggatgac cgggagtcgt ggccttccgt cttttataat aggacctgccagtgctctgg

361 caacttcatg ggattcaact gtggaaactg caagtttggc ttttggggaccaaactgcac

421 agagagacga ctcttggtga gaagaaacat cttcgatttg agtgccccagagaaggacaa

481 attttttgcc tacctcactt tagcaaagca taccatcagc tcagactatgtcatccccat

541 agggacctat ggccaaatga aaaatggatc aacacccatg tttaacgacatcaatattta

601 tgacctcttt gtctggatgc attattatgt gtcaatggat gcactgcttgggggatctga

661 aatctggaga gacattgatt ttgcccatga agcaccagct tttctgccttggcatagact

721 cttcttgttg cggtgggaac aagaaatcca gaagctgaca ggagatgaaaacttcactat

781 tccatattgg gactggcggg atgcagaaaa gtgtgacatt tgcacagatgagtacatggg

841 aggtcagcac cccacaaatc ctaacttact cagcccagca tcattcttctcctcttggca

901 gattgtctgt agccgattgg aggagtacaa cagccatcag tctttatgcaatggaacgcc

961 cgagggacct ttacggcgta atcctggaaa ccatgacaaa tccagaaccccaaggctccc

1021ctcttcagct gatgtagaat tttgcctgag tttgacccaa tatgaatctggttccatgga

1081taaagctgcc aatttcagct ttagaaatac actggaagga tttgctagtccacttactgg

1141gatagcggat gcctctcaaa gcagcatgca caatgccttg cacatctatatgaatggaac

1201aatgtcccag gtacagggat ctgccaacga tcctatcttc cttcttcaccatgcatttgt

1261tgacagtatt tttgagcagt ggctccgaag gcaccgtcct cttcaagaagtttatccaga

1321agccaatgca cccattggac ataaccggga atcctacatg gttccttttataccactgta

1381cagaaatggt gatttcttta tttcatccaa agatctgggc tatgactatagctatctaca

1441agattcagac ccagactctt ttcaagacta cattaagtcc tatttggaacaagcgagtcg

1501gatctggtca tggctccttg gggcggcgat ggtaggggcc gtcctcactgccctgctggc

1561agggcttgtg agcttgctgt gtcgtcacaa gagaaagcag cttcctgaagaaaagcagcc

1621actcctcatg gagaaagagg attaccacag cttgtatcag agccatttataaaaggctta

1681ggcaatagag tagggccaaa aagcctgacc tcactctaac tcaaagtaatgtccaggttc

1741ccagagaata tctgctggta tttttctgta aagaccattt gcaaaattgtaacctaatac

1801aaagtgtagc cttcttccaa ctcaggtaga acacacctgt ctttgtcttgctgttttcac

1861tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatgctatttggta

1921atgaggaact gttatttgta tgtgaattaa agtgctctta tttt

Human synovial sarcoma, X breakpoint 2(SSX2), mRNA.

ACCESSION NM_003147

VERSION NM_003147.1 GI：10337582

SEQ ID NO 3

' MNGDDAFARRPTVGAQIPEKIQKAFDDIAKYFSKEEWEKMKASEKIFYVYMKRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNRGNQVERPQMTFGRLQGISPKIMPKKPAEEGNDSEEVPEASGPQNDGKELCPPGKPTTSEKIHERSGPKRGEHAWTHRLRERKQLVIYEEISDPEEDDE

SEQ ID NO 6

Start 1 ctctctttcg attcttccat actcagagta cgcacggtct gattttctctttggattctt

61 ccaaaatcag agtcagactg ctcccggtgc catgaacgga gacgacgcctttgcaaggag

121 acccacggtt ggtgctcaaa taccagagaa gatccaaaag gccttcgatgatattgccaa

181 atacttctct aaggaagagt gggaaaagat gaaagcctcg gagaaaatcttctatgtgta

241 tatgaagaga aagtatgagg ctatgactaa actaggtttc aaggccaccctcccaccttt

301 catgtgtaat aaacgggccg aagacttcca ggggaatgat ttggataatgaccctaaccg

361 tgggaatcag gttgaacgtc ctcagatgac tttcggcagg ctccagggaatctccccgaa

421 gatcatgccc aagaagccag cagaggaagg aaatgattcg gaggaagtgccagaagcatc

481 tggcccacaa aatgatggga aagagctgtg ccccccggga aaaccaactacctctgagaa

541 gattcacgag agatctggac ccaaaagggg ggaacatgcc tggacccacagactgcgtga

601 gagaaaacag ctggtgattt atgaagagat cagcgaccct gaggaagatgacgagtaact

661 cccctcaggg atacgacaca tgcccatgat gagaagcaga acgtggtgacctttcacgaa

721 catgggcatg gctgcggacc cctcgtcatc aggtgcatag caagtg

Human folate hydrolase (prostate specific membrane antigen) 1(FOLH1), mRNA.

ACCESSION NM_004476

VERSION NM_004476.1 GI：4758397

' MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQ LAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTOKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKT YSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

SEQ ID NO 7

Start 1 ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctctctcgctcgg

61 attggttcag tgcactctag aaacact gctgtggtggaga aactggaccccaggtctgga

121 gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgagagagactttac

181 cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgggtcccgggag

241 gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactcggctgtggcc

301 accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcgggtggcttcttt

361 ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctactaacattact

421 ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacatcaagaagttc

481 ttatataatt ttacacagat accacattta gcaggaacag aacaaaactttcagcttgca

541 aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagctagcacattat

601 gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaataattaatgaa

661 gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccaggatatgaaaat

721 gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgccagagggcgat

781 ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacgggacatgaaa

841 atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcagaggaaataag

901 gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccgaccctgctgac

961 tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctggaggtggtgtc

1021cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacaccaggttaccca

1081gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttccaagtattcct

1141gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatgggtggctcagca

1201ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttggacctggcttt

1261actggaaact tttctacacaa aaagtcaag atgcacatcc actctaccaatgaagtgaca

1321agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacagatatgtcatt

1381ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagagtggagcagct

1441gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtggagacctaga

1501agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttggttctactgag

1561tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatattaatgctgac

1621tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgatgtacagcttg

1681gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaaggcaaatctctt

1741tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcccaggataagc

1801aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaattgcttcaggc

1861agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatccactgtatcac

1921agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtttaaatatcac

1981ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattccatagtgctc

2041ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaaaatctacagt

2101atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttgattcacttttt

2161tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagactccaggacttt

2221gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtttctggaaaga

2281gcatttattg atccattagg gttaccagac aggccttttt ataggcatgtcatctatgct

2341ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatgatgctctgttt

2401gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagagacagatttat

2461gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagcctaagaggat

2521tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaatcgtaatgggt

2581atattgataa attttaaaat tggtatattt gaaataaagt tgaatattatatataaaaaa

2641aaaaaaaaaa aaa

Human melanocyte-specific (pmel 17) gene, exons 2-5, and full cds.

ACCESSION U20093

VERSION U20093.1GI：1142634

SEQ ID NO 70

' MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAPVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV

SEQ ID NO 80

ORIGIN

1 gtgctaaaaa gatgccttct tcatttggct gtgataggtg ctttgtggctgtgggggcta

61 caaaagtacc cagaaaccag gactggcttg gtgtctcaag gcaactcagaaccaaagcct

121 ggaacaggca gctgtatcca gagtggacag aagcccagag acttgactgctggagaggtg

181 gtcaagtgtc cctcaaggtc agtaatgatg ggcctacact gattggtgcaaatgcctcct

241 tctctattgc cttgaacttc cctggaagcc aaaaggtatt gccagatgggcaggttatct

301 gggtcaacaa taccatcatc aatgggagcc aggtgtgggg aggacagccagtgtatcccc

361 aggaaactga cgatgcctgc atcttccctg atggtggacc ttgcccatctggctcttggt

421 ctcagaagag aagctttgtt tatgtctgga agacctgggg tgagggactcccttctcagc

481 ctatcatcca cacttgtgtt tacttctttc tacctgatca cctttcttttggccgcccct

541 tccaccttaa cttctgtgat tttctctaat cttcattttc ctcttagatcttttctcttt

601 cttagcacct agcccccttc aagctctatc ataattcttt ctggcaactcttggcctcaa

661 ttgtagtcct accccatgga atgcctcatt aggacccctt ccctgtccccccatatcaca

721 gccttccaaa caccctcaga agtaatcata cttcctgacc tcccatctccagtgccgttt

781 cgaagcctgt ccctcagtcc cctttgacca gtaatctctt cttccttgcttttcattcca

841 aaaatgcttc aggccaatac tggcaagttc tagggggccc agtgtctgggctgagcattg

901 ggacaggcag ggcaatgctg ggcacacaca ccatggaagt gactgtctaccatcgccggg

961 gatcccggag ctatgtgcct cttgctcatt ccagctcagc cttcaccattactggtaagg

1021gttcaggaag ggcaaggcca gttgtagggc aaagagaagg cagggaggcttggatggact

1081gcaaaggaga aaggtgaaat gctgtgcaaa cttaaagtag aagggccaggaagacctagg

1141cagagaaatg tgaggcttag tgccagtgaa gggccagcca gtcagcttggagttggaggg

1201tgtggctgtg aaaggagaag ctgtggctca ggcctggttc tcaccttttctggctccaat

1261cccagaccag gtgcctttct ccgtgagcgt gtcccagttg cgggccttggatggagggaa

1321caagcacttc ctgagaaatc agcctctgac ctttgccctc cagctccatgaccccagtgg

1381ctatctggct gaagctgacc tctcctacac ctgggacttt ggagacagtagtggaaccct

1441gatctctcgg gcacctgtgg tcactcatac ttacctggag cctggcccagtcactgccca

1501ggtggtcctg caggctgcca ttcctctcac ctcctgtggc tcctccccagttccaggcac

1561cacagatggg cacaggccaa ctgcagaggc ccctaacacc acagctggccaagtgcctac

1621tacagaagtt gtgggtacta cacctggtca ggcgccaact gcagagccctctggaaccac

1681atctgtgcag gtgccaacca ctgaagtcat aagcactgca cctgtgcagatgccaactgc

1741agagagcaca ggtatgacac ctgagaaggt gccagtttca gaggtcatgggtaccacact

1801ggcagagatg tcaactccag aggctacagg tatgacacct gcagaggtatcaattgtggt

1861gctttctgga accacagctg cacaggtaac aactacagag tgggtggagaccacagctag

1921agagctacct atccctgagc ctgaaggtcc agatgccagc tcaatcatgtctacggaaag

1981tattacaggt tccctgggcc ccctgctgga tggtacagcc accttaaggctggtgaagag

2041acaagtcccc ctggattgtg ttctgtatcg atatggttcc ttttccgtcaccctggacat

2101tgtccagggt attgaaagtg ccgagatcct gcaggctgtg ccgtccggtgagggggatgc

2161atttgagctg actgtgtcct gccaaggcgg gctgcccaag gaagcctgcatggagatctc

2221atcgccaggg tgccagcccc ctgcccagcg gctgtgccag cctgtgctacccagcccagc

2281ctgccagctg gttctgcacc agatactgaa gggtggctcg gggacatactgcctcaatgt

2341gtctctggct gataccaaca gcctggcagt ggtcagcacc cagcttatcatgcctggtag

2401gtccttggac agagactaag tgaggaggga agtggataga ggggacagctggcaagcagc

2461agacatgagt gaagcagtgc ctgggattct tctcacaggt caagaagcaggccttgggca

2521ggttccgctg atcgtgggca tcttgctggt gttgatggct gtggtccttgcatctctgat

2581atataggcgc agacttatga agcaagactt ctccgtaccc cagttgccacatagcagcag

2641tcactggctg cgtctacccc gcatcttctg ctcttgtccc attggtgagaatagccccct

2701cctcagtggg cagcaggtct gagtactctc atatgatgct gtgattttcctggagttgac

2761agaaacacct atatttcccc cagtcttccc tgggagacta ctattaactgaaataaa

//

Human kallikrein 3, (prostate specific antigen) (KLK3), mRNA.

ACCESSION NM_001648

VERSION NM_001648.1 GI：4502172

SEQ ID NO 78

' MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP

SEQ ID NO 86

Start 1 agccccaagc ttaccacctg cacccggaga gctgtgtgtc accatgtgggtcccggttgt

61 cttcctcacc ctgtccgtga cgtggattgg tgctgcaccc ctcatcctgtctcggattgt

121 gggaggctgg gagtgcgaga agcattccca accctggcag gtgcttgtggcctctcgtgg

181 cagggcagtc tgcggcggtg ttctggtgca cccccagtgg gtcctcacagctgcccactg

241 catcaggaac aaaagcgtga tcttgctggg tcggcacagc ctgtttcatcctgaagacac

301 aggccaggta tttcaggtca gccacagctt cccacacccg ctctacgatatgagcctcct

361 gaagaatcga ttcctcaggc caggtgatga ctccagccac gacctcatgctgctccgcct

421 gtcagagcct gccgagctca cggatgctgt gaaggtcatg gacctgcccacccaggagcc

481 agcactgggg accacctgct acgcctcagg ctggggcagc attgaaccag

aggagttctt

541 gaccccaaag aaacttcagt gtgtggacct ccatgttatt tccaatgacgtgtgtgcgca

601 agttcaccct cagaaggtga ccaagttcat gctgtgtgct ggacgctggacagggggcaa

661 aagcacctgc tcgggtgatt ctgggggccc acttgtctgt aatggtgtgcttcaaggtat

721 cacgtcatgg ggcagtgaac catgtgccct gcccgaaagg ccttccctgtacaccaaggt

781 ggtgcattac cggaagtgga tcaaggacac catcgtggcc aacccctgagcacccctatc

841 aaccccctat tgtagtaaac ttggaacctt ggaaatgacc aggccaagactcaagcctcc

901 ccagttctac tgacctttgt ccttaggtgt gaggtccagg gttgctaggaaaagaaatca

961 gcagacacag gtgtagacca gagtgtttct taaatggtgt aattttgtcctctctgtgtc

1021ctggggaata ctggccatgc ctggagacat atcactcaat ttctctgaggacacagatag

1081gatggggtgt ctgtgttatt tgtggggtac agagatgaaa gaggggtgggatccacactg

1141agagagtgga gagtgacatg tgctggacac tgtccatgaa gcactgagcagaagctggag

1201gcacaacgca ccagacactc acagcaagga tggagctgaa aacataacccactctgtcct

1261ggaggcactg ggaagcctag agaaggctgt gagccaagga gggagggtcttcctttggca

1321tgggatgggg atgaagtaag gagagggact ggaccccctg gaagctgattcactatgggg

1381ggaggtgtat tgaagtcctc cagacaaccc tcagatttga tgatttcctagtagaactca

1441cagaaataaa gagctgttat actgtg

//

Human autoimmune cancer/testis antigen NY-ESO-1mRNA, full cds

ACCESSION U87459

VERSION U87459.1 GI：1890098

SEQ ID NO 74

' MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR

SEQ ID NO 84

Initiation of

1 atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccggagccatgcagg

61 ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccaggaggccctggca

121ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgccacgggcggca

181gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggcgccccgcggg

241gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggggccagggggc

301cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacacccatggaagcag

361agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgccaggggtgcttc

421tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgctgcagaccacc

481gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttgatgtggatca

541cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagaggcgctaagccc

601agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtcccagcacgagtg

661gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggcttacatgtttgt

721ttctgtagaa aataaaactg agctacgaaa aa

//

LAGE-1a protein (human)

ACCESSION CAA11116

PID g3255959

VERSION CAA11116.1 GI：3255959

SEQ ID NO 75

Initial 1 mqaegrgtgg stgdadgpggg pgipdgggn aggpgagagaegat ggrgprgagaaaarasgprgga

61 prgphggaas aqdgrcpcga rrpdsrllel hitmpfsspm eaelvrrilsrdaaplprpg

121 avlkdftvsg nllfirltaa dhrqlqlsis sclqqlsllm witqcflpvflaqapsgqrr

181

//

LAGE-1b protein (human)

ACCESSION CAA11117

PID g3255960

VERSION CAA11117.1 GI：3255960

SEQ ID NO 76

Initiation of

1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgagaarasgprgga

61 prgphggaas aqdgrcpcga rrpdsrllel hitmpfsspm eaelvrrilsrdaaplprpg

121avlkdftvsg nllfmsvwdq dregagrmrv vgwglgsasp egqkardlrtpkhkvseqrp

181gtpgppppeg aqgdgcrgva fnvmfsaphi

//

Human antigen (MAGE-1) gene, full cds,

ACCESSION M77481

VERSION M77481.1 GI：416114

SEQ ID NO 71

' MSLEQRSLHCKPEEALEAQQEALGLVCVQAATSSSSPLVLGTLEEVPTAGSTDPPQSPQGASAFPTTINFTRQRQPSEGSSSREEEGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHCFPEIFGKASESLQLVFGIDVKEADPTGHSYVLVTCLGLSYDGLLGDNQIMPKTGFLIIVLVMIAMEGGHAPEEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVRFFFPSLREAALREEEEGV

SEQ ID NO 81

Start 1 ggatccaggc cctgccagga aaaatataag ggccctgcgt gagaacagagggggtcatcc

61 actgcatgag agtggggatg tcacagagtc cagcccaccc tcctggtagcactgagaagc

121 cagggctgtg cttgcggtct gcaccctgag ggcccgtgga ttcctcttcctggagctcca

181 ggaaccaggc agtgaggcct tggtctgaga cagtatcctc aggtcacagagcagaggatg

241 cacagggtgt gccagcagtg aatgtttgcc ctgaatgcac accaagggccccacctgcca

301 caggacacat aggactccac agagtctggc ctcacctccc tactgtcagtcctgtagaat

361 cgacctctgc tggccggctg taccctgagt accctctcac ttcctccttcaggttttcag

421 gggacaggcc aacccagagg acaggattcc ctggaggcca cagaggagcaccaaggagaa

481 gatctgtaag taggcctttg ttagagtctc caaggttcag ttctcagctgaggcctctca

541 cacactccct ctctccccag gcctgtgggt cttcattgcc cagctcctgcccacactcct

601 gcctgctgcc ctgacgagag tcatcatgtc tcttgagcag aggagtctgcactgcaagcc

661 tgaggaagcc cttgaggccc aacaagaggc cctgggcctg gtgtgtgtgcaggctgccac

721 ctcctcctcc tctcctctgg tcctgggcac cctggaggag gtgcccactgctgggtcaac

781 agatcctccc cagagtcctc agggagcctc cgcctttccc actaccatcaacttcactcg

841 acagaggcaa cccagtgagg gttccagcag ccgtgaagag gaggggccaagcacctcttg

901 tatcctggag tccttgttcc gagcagtaat cactaagaag gtggctgatttggttggttt

961 tctgctcctc aaatatcgag ccagggagcc agtcacaaag gcagaaatgctggagagtgt

1021catcaaaaat tacaagcact gttttcctga gatcttcggc aaagcctctgagtccttgca

1081gctggtcttt ggcattgacg tgaaggaagc agaccccacc ggccactcctatgtccttgt

1141cacctgccta ggtctctcct atgatggcct gctgggtgat aatcagatcatgcccaagac

1201aggcttcctg ataattgtcc tggtcatgat tgcaatggag ggcggccatgctcctgagga

1261ggaaatctgg gaggagctga gtgtgatgga ggtgtatgat gggagggagcacagtgccta

1321tggggagccc aggaagctgc tcacccaaga tttggtgcag gaaaagtacctggagtaccg

1381gcaggtgccg gacagtgatc ccgcacgcta tgagttcctg tggggtccaagggccctcgc

1441tgaaaccagc tatgtgaaag tccttgagta tgtgatcaag gtcagtgcaagagttcgctt

1501tttcttccca tccctgcgtg aagcagcttt gagagaggag gaagagggagtctgagcatg

1561agttgcagcc aaggccagtg ggagggggac tgggccagtg caccttccagggccgcgtcc

1621agcagcttcc cctgcctcgt gtgacatgag gcccattctt cactctgaagagagcggtca

1681gtgttctcag tagtaggttt ctgttctatt gggtgacttg gagatttatctttgttctct

1741tttggaattg ttcaaatgtt tttttttaag ggatggttga atgaacttcagcatccaagt

1801ttatgaatga cagcagtcac acagttctgt gtatatagtt taagggtaagagtcttgtgt

1861tttattcaga ttgggaaatc cattctattt tgtgaattgg gataataacagcagtggaat

1921aagtacttag aaatgtgaaa aatgagcagt aaaatagatg agataaagaactaaagaaat

1981taagagatag tcaattcttg ccttatacct cagtctattc tgtaaaatttttaaagatat

2041atgcatacct ggatttcctt ggcttctttg agaatgtaag agaaattaaatctgaataaa

2101gaattcttcc tgttcactgg ctcttttctt ctccatgcac tgagcatctgctttttggaa

2161ggccctgggt tagtagtgga gatgctaagg taagccagac tcatacccacccatagggtc

2221gtagagtcta ggagctgcag tcacgtaatc gaggtggcaa gatgtcctctaaagatgtag

2281ggaaaagtga gagaggggtg agggtgtggg gctccgggtg agagtggtggagtgtcaatg

2341ccctgagctg gggcattttg ggctttggga aactgcagtt ccttctgggggagctgattg

2401taatgatctt gggtggatcc

//

Human MAGE-2 Gene exon 14, full cds

ACCESSION L18920

VERSION L18920.1 GI：436180

SEQ ID NO 72

' MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQQTASSSSTLVEVTLGEVPAADSPSPPHSPQGASSFSTTINYTLWRQSDEGSSNQEEEGPRMFPDLESEFQAAISRKMVELVHFLLLKYRAREPVTKAEMLESVLRNCQDFFPVIFSKASEYLQLVFGIEVVEVVPISHLYILVTCLGLSYDGLLGDNQVMPKTGLLIIVLAIIAIEGDCAPEEKIWEELSMLEVFEGREDSVFAHPRKLLMQDLVQENYLEYRQVPGSDPACYEFLWGPRALIETSYVKVLHHTLKIGGEPHISYPPLHERALREGEE

SEQ ID NO 82

Start 1 attccttcat caaacagcca ggagtgagga agaggaccct cctgagtgaggactgaggat

61 ccaccctcac cacatagtgg gaccacagaa tccagctcag cccctcttgtcagccctggt

121 acacactggc aatgatctca ccccgagcac acccctcccc ccaatgccacttcgggccga

181 ctcagagtca gagacttggt ctgaggggag cagacacaat cggcagaggatggcggtcca

241 ggctcagtct ggcatccaag tcaggacctt gagggatgac caaaggcccctcccaccccc

301 aactcccccg accccaccag gatctacagc ctcaggatcc ccgtcccaatccctacccct

361 acaccaacac catcttcatg cttaccccca cccccccatc cagatccccatccgggcaga

421 atccggttcc acccttgccg tgaacccagg gaagtcacgg gcccggatgtgacgccactg

481 acttgcacat tggaggtcag aggacagcga gattctcgcc ctgagcaacggcctgacgtc

541 ggcggaggga agcaggcgca ggctccgtga ggaggcaagg taagacgccgagggaggact

601 gaggcgggcc tcaccccaga cagagggccc ccaataatcc agcgctgcctctgctgccgg

661 gcctggacca ccctgcaggg gaagacttct caggctcagt cgccaccacctcaccccgcc

721 accccccgcc gctttaaccg cagggaactc tggcgtaaga gctttgtgtgaccagggcag

781 ggctggttag aagtgctcag ggcccagact cagccaggaa tcaaggtcaggaccccaaga

841 ggggactgag ggcaacccac cccctaccct cactaccaat cccatcccccaacaccaacc

901 ccacccccat ccctcaaaca ccaaccccac ccccaaaccc cattcccatctcctccccca

961 ccaccatcct ggcagaatcc ggctttgccc ctgcaatcaa cccacggaagctccgggaat

1021ggcggccaag cacgcggatc ctgacgttca catgtacggc taagggagggaaggggttgg

1081gtctcgtgag tatggccttt gggatgcaga ggaagggccc aggcctcctggaagacagtg

1141gagtccttag gggacccagc atgccaggac agggggccca ctgtacccctgtctcaaact

1201gagccacctt ttcattcagc cgagggaatc ctagggatgc agacccacttcagcaggggg

1261ttggggccca gcctgcgagg agtcaagggg aggaagaaga gggaggactgaggggacctt

1321ggagtccaga tcagtggcaa ccttgggctg ggggatcctg ggcacagtggccgaatgtgc

1381cccgtgctca ttgcaccttc agggtgacag agagttgagg gctgtggtctgagggctggg

1441acttcaggtc agcagaggga ggaatcccag gatctgccgg acccaaggtgtgcccccttc

1501atgaggactg gggatacccc cggcccagaa agaagggatg ccacagagtctggaagtccc

1561ttgttcttag ctctggggga acctgatcag ggatggccct aagtgacaatctcatttgta

1621ccacaggcag gaggttgggg aaccctcagg gagataaggt gttggtgtaaagaggagctg

1681tctgctcatt tcagggggtt gggggttgag aaagggcagt ccctggcaggagtaaagatg

1741agtaacccac aggaggccat cataacgttc accctagaac caaaggggtcagccctggac

1801aacgcacgtg ggggtaacag gatgtggccc ctcctcactt gtctttccagatctcaggga

1861gttgatgacc ttgttttcag aaggtgactc aggtcaacac aggggccccatctggtcgac

1921agatgcagtg gttctaggat ctgccaagca tccaggtgga gagcctgaggtaggattgag

1981ggtacccctg ggccagaatg cagcaagggg gccccataga aatctgccctgcccctgcgg

2041ttacttcaga gaccctgggc agggctgtca gctgaagtcc ctccattatcctgggatctt

2101tgatgtcagg gaaggggagg ccttggtctg aaggggctgg agtcaggtcagtagagggag

2161ggtctcaggc cctgccagga gtggacgtga ggaccaagcg gactcgtcacccaggacacc

2221tggactccaa tgaatttgga catctctcgt tgtccttcgc gggaggacctggtcacgtat

2281ggccagatgt gggtcccctc atatccttct gtaccatatc agggatgtgagttcttgaca

2341tgagagattc tcaagccagc aaaagggtgg gattaggccc tacaaggagaaaggtgaggg

2401ccctgagtga gcacagaggg gaccctccac ccaagtagag tggggacctcacggagtctg

2461gccaaccctg ctgagacttc tgggaatccg tggctgtgct tgcagtctgcacactgaagg

2521cccgtgcatt cctctcccag gaatcaggag ctccaggaac caggcagtgaggccttggtc

2581tgagtcagtg tcctcaggtc acagagcaga ggggacgcag acagtgccaacactgaaggt

2641ttgcctggaa tgcacaccaa gggccccacc cgcccagaac aaatgggactccagagggcc

2701tggcctcacc ctccctattc tcagtcctgc agcctgagca tgtgctggccggctgtaccc

2761tgaggtgccc tcccacttcc tccttcaggt tctgaggggg acaggctgacaagtaggacc

2821cgaggcactg gaggagcatt gaaggagaag atctgtaagt aagcctttgtcagagcctcc

2881aaggttcagt tcagttctca cctaaggcct cacacacgct ccttctctccccaggcctgt

2941gggtcttcat tgcccagctc ctgcccgcac tcctgcctgc tgccctgaccagagtcatca

3001tgcctcttga gcagaggagt cagcactgca agcctgaaga aggccttgaggcccgaggag

3061aggccctggg cctggtgggt gcgcaggctc ctgctactga ggagcagcagaccgcttctt

3121cctcttctac tctagtggaa gttaccctgg gggaggtgcc tgctgccgactcaccgagtc

3181ctccccacag tcctcaggga gcctccagct tctcgactac catcaactacactctttgga

3241gacaatccga tgagggctcc agcaaccaag aagaggaggg gccaagaatgtttcccgacc

3301tggagtccga gttccaagca gcaatcagta ggaagatggt tgagttggttcattttctgc

3361tcctcaagta tcgagccagg gagccggtca caaaggcaga aatgctggagagtgtcctca

3421gaaattgcca ggacttcttt cccgtgatct tcagcaaagc ctccgagtacttgcagctgg

3481tctttggcat cgaggtggtg gaagtggtcc ccatcagcca cttgtacatccttgtcacct

3541gcctgggcct ctcctacgat ggcctgctgg gcgacaatca ggtcatgcccaagacaggcc

3601tcctgataat cgtcctggcc ataatcgcaa tagagggcga ctgtgcccctgaggagaaaa

3661tctgggagga gctgagtatg ttggaggtgt ttgaggggag ggaggacagtgtcttcgcac

3721atcccaggaa gctgctcatg caagatctgg tgcaggaaaa ctacctggagtaccggcagg

3781tgcccggcag tgatcctgca tgctacgagt tcctgtgggg tccaagggccctcattgaaa

3841ccagctatgt gaaagtcctg caccatacac taaagatcgg tggagaacctcacatttcct

3901acccacccct gcatgaacgg gctttgagag agggagaaga gtgagtctcagcacatgttg

3961cagccagggc cagtgggagg gggtctgggc cagtgcacct tccagggccccatccattag

4021cttccactgc ctcgtgtgat atgaggccca ttcctgcctc tttgaagagagcagtcagca

4081ttcttagcag tgagtttctg ttctgttgga tgactttgag atttatctttctttcctgtt

4141ggaattgttc aaatgttcct tttaacaaat ggttggatga acttcagcatccaagtttat

4201gaatgacagt agtcacacat agtgctgttt atatagttta ggggtaagagtcctgttttt

4261tattcagatt gggaaatcca ttccattttg tgagttgtca cataataacagcagtggaat

4321atgtatttgc ctatattgtg aacgaattag cagtaaaata catgatacaaggaactcaaa

4381agatagttaa ttcttgcctt atacctcagt ctattatgta aaattaaaaatatgtgtatg

4441tttttgcttc tttgagaatg caaaagaaat taaatctgaa taaattcttcctgttcactg

4501gctcatttct ttaccattca ctcagcatct gctctgtgga aggccctggtagtagtggg

//

Human MAGE-3 antigen (MAGE-3) gene, full cds

ACCESSION U03735

VERSION U03735.1 GI：468825

SEQ ID NO 73

(ii)/translation ═ MPLEQRSQHCKPEEGLEARGEALGLVGAQAEQEEQSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEGPSTFPDLESEFQALSRKVAELFLKYRLYLRAPHTHACETEMVGNWQFLYFLVIFSKASSFLQLVFGEVHLYLIGHLYLLGLSATGLYDGLDNQMPKAGLLIGLYLLAAREGDCAPEEKIWEELVFEGFLEXPKKLLTQHFQVQQNYLEPYLEPYLEPHLEPGHVPACYEFLEGFLEGFLEGFLVLVKLVVVKVVHGPPHTHYLESPHTHYLESPHTHEGWVLGEE

SEQ ID NO 83

Start 1 acgcaggcag tgatgtcacc cagaccacac cccttccccc aatgccacttcagggggtac

61 tcagagtcag agacttggtc tgaggggagc agaagcaatc tgcagaggatggcggtccag

121 gctcagccag gcatcaactt caggaccctg agggatgacc gaaggccccgcccacccacc

181 cccaactccc ccgaccccac caggatctac agcctcagga cccccgtcccaatccttacc

241 ccttgcccca tcaccatctt catgcttacc tccaccccca tccgatccccatccaggcag

301 aatccagttc cacccctgcc cggaacccag ggtagtaccg ttgccaggatgtgacgccac

361 tgacttgcgc attggaggtc agaagaccgc gagattctcg ccctgagcaacgagcgacgg

421 cctgacgtcg gcggagggaa gccggcccag gctcggtgag gaggcaaggtaagacgctga

481 gggaggactg aggcgggcct cacctcagac agagggcctc aaataatccagtgctgcctc

541 tgctgccggg cctgggccac cccgcagggg aagacttcca ggctgggtcgccactacctc

601 accccgccga cccccgccgc tttagccacg gggaactctg gggacagagcttaatgtggc

661 cagggcaggg ctggttagaa gaggtcaggg cccacgctgt ggcaggaatcaaggtcagga

721 ccccgagagg gaactgaggg cagcctaacc accaccctca ccaccattcccgtcccccaa

781 cacccaaccc cacccccatc ccccattccc atccccaccc ccacccctatcctggcagaa

841 tccgggcttt gcccctggta tcaagtcacg gaagctccgg gaatggcggccaggcacgtg

901 agtcctgagg ttcacatcta cggctaaggg agggaagggg ttcggtatcgcgagtatggc

961 cgttgggagg cagcgaaagg gcccaggcct cctggaagac agtggagtcctgaggggacc

1021cagcatgcca ggacaggggg cccactgtac ccctgtctca aaccgaggcaccttttcatt

1081cggctacggg aatcctaggg atgcagaccc acttcagcag ggggttggggcccagccctg

1141cgaggagtca tggggaggaa gaagagggag gactgagggg accttggagtccagatcagt

1201ggcaaccttg ggctggggga tgctgggcac agtggccaaa tgtgctctgtgctcattgcg

1261ccttcagggt gaccagagag ttgagggctg tggtctgaag agtgggacttcaggtcagca

1321gagggaggaa tcccaggatc tgcagggccc aaggtgtacc cccaaggggcccctatgtgg

1381tggacagatg cagtggtcct aggatctgcc aagcatccag gtgaagagactgagggagga

1441ttgagggtac ccctgggaca gaatgcggac tgggggcccc ataaaaatctgccctgctcc

1501tgctgttacc tcagagagcc tgggcagggc tgtcagctga ggtccctccattatcctagg

1561atcactgatg tcagggaagg ggaagccttg gtctgagggg gctgcactcagggcagtaga

1621gggaggctct cagaccctac taggagtgga ggtgaggacc aagcagtctcctcacccagg

1681gtacatggac ttcaataaat ttggacatct ctcgttgtcc tttccgggaggacctgggaa

1741tgtatggcca gatgtgggtc ccctcatgtt tttctgtacc atatcaggtatgtgagttct

1801tgacatgaga gattctcagg ccagcagaag ggagggatta ggccctataaggagaaaggt

1861gagggccctg agtgagcaca gaggggatcc tccaccccag tagagtggggacctcacaga

1921gtctggccaa ccctcctgac agttctggga atccgtggct gcgtttgctgtctgcacatt

1981gggggcccgt ggattcctct cccaggaatc aggagctcca ggaacaaggcagtgaggact

2041tggtctgagg cagtgtcctc aggtcacaga gtagaggggg ctcagatagtgccaacggtg

2101aaggtttgcc ttggattcaa accaagggcc ccacctgccc cagaacacatggactccaga

2161gcgcctggcc tcaccctcaa tactttcagt cctgcagcct cagcatgcgctggccggatg

2221taccctgagg tgccctctca cttcctcctt caggttctga ggggacaggctgacctggag

2281gaccagaggc ccccggagga gcactgaagg agaagatctg taagtaagcctttgttagag

2341cctccaaggt tccattcagt actcagctga ggtctctcac atgctccctctctccccagg

2401ccagtgggtc tccattgccc agctcctgcc cacactcccg cctgttgccctgaccagagt

2461catcatgcct cttgagcaga ggagtcagca ctgcaagcct gaagaaggccttgaggcccg

2521aggagaggcc ctgggcctgg tgggtgcgca ggctcctgct actgaggagcaggaggctgc

2581ctcctcctct tctactctag ttgaagtcac cctgggggag gtgcctgctgccgagtcacc

2641agatcctccc cagagtcctc agggagcctc cagcctcccc actaccatgaactaccctct

2701ctggagccaa tcctatgagg actccagcaa ccaagaagag gaggggccaagcaccttccc

2761tgacctggag tccgagttcc aagcagcact cagtaggaag gtggccgagttggttcattt

2821tctgctcctc aagtatcgag ccagggagcc ggtcacaaag gcagaaatgctggggagtgt

2881cgtcggaaat tggcagtatt tctttcctgt gatcttcagc aaagcttccagttccttgca

2941gctggtcttt ggcatcgagc tgatggaagt ggaccccatc ggccacttgtacatctttgc

3001cacctgcctg ggcctctcct acgatggcct gctgggtgac aatcagatcatgcccaaggc

3061aggcctcctg ataatcgtcc tggccataat cgcaagagag ggcgactgtgcccctgagga

3121gaaaatctgg gaggagctga gtgtgttaga ggtgtttgag gggagggaagacagtatctt

3181gggggatccc aagaagctgc tcacccaaca tttcgtgcag gaaaactacctggagtaccg

3241gcaggtcccc ggcagtgatc ctgcatgtta tgaattcctg tggggtccaagggccctcgt

3301tgaaaccagc tatgtgaaag tcctgcacca tatggtaaag atcagtggaggacctcacat

3361ttcctaccca cccctgcatg agtgggtttt gagagagggg gaagagtgagtctgagcacg

3421agttgcagcc agggccagtg ggagggggtc tgggccagtg caccttccggggccgcatcc

3481cttagtttcc actgcctcct gtgacgtgag gcccattctt cactctttgaagcgagcagt

3541cagcattctt agtagtgggt ttctgttctg ttggatgact ttgagattattctttgtttc

3601ctgttggagt tgttcaaatg ttccttttaa cggatggttg aatgagcgtcagcatccagg

3661tttatgaatg acagtagtca cacatagtgc tgtttatata gtttaggagtaagagtcttg

3721ttttttactc aaattgggaa atccattcca ttttgtgaat tgtgacataataatagcagt

3781ggtaaaagta tttgcttaaa attgtgagcg aattagcaat aacatacatgagataactca

3841agaaatcaaa agatagttga ttcttgcctt gtacctcaat ctattctgtaaaattaaaca

3901aatatgcaaa ccaggatttc cttgacttct ttgagaatgc aagcgaaattaaatctgaat

3961aaataattct tcctcttcac tggctcgttt cttttccgtt cactcagcatctgctctgtg

4021ggaggccctg ggttagtagt ggggatgcta aggtaagcca gactcacgcctacccatagg

4081gctgtagagc ctaggacctg cagtcatata attaaggtgg tgagaagtcctgtaagatgt

4141agaggaaatg taagagaggg gtgagggtgt ggcgctccgg gtgagagtagtggagtgtca

4201gtgc

//

Human Prostate Stem Cell Antigen (PSCA) mRNA, full cds

ACCESSION AF043498

VERSION AF043498.1 GI：2909843

SEQ ID NO 79

' MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL

SEQ ID NO 87

Start 1 agggagaggc agtgaccatg aaggctgtgc tgcttgccct gttgatggcaggcttggccc

61 tgcagccagg cactgccctg ctgtgctact cctgcaaagc ccaggtgagcaacgaggact

121 gcctgcaggt ggagaactgc acccagctgg gggagcagtg ctggaccgcgcgcatccgcg

181 cagttggcct cctgaccgtc atcagcaaag gctgcagctt gaactgcgtggatgactcac

241 aggactacta cgtgggcaag aagaacatca cgtgctgtga caccgacttgtgcaacgcca

301 gcggggccca tgccctgcag ccggctgccg ccatccttgc gctgctccctgcactcggcc

361 tgctgctctg gggacccggc cagctatagg ctctgggggg ccccgctgcagcccacactg

421 ggtgtggtgc cccaggcctt tgtgccactc ctcacagaac ctggcccagtgggagcctgt

481 cctggttcct gaggcacatc ctaacgcaag tttgaccatg tatgtttgcaccccttttcc

541 ccnaaccctg accttcccat gggccttttc caggattccn accnggcagatcagttttag

601tganacanat ccgcntgcag atggcccctc caaccntttn tgttgntgtttccatggccc

661agcattttcc acccttaacc ctgtgttcag gcacttnttc ccccaggaagccttccctgc

721ccaccccatt tatgaattga gccaggtttg gtccgtggtg tcccccgcacccagcagggg

781acaggcaatc aggagggccc agtaaaggct gagatgaagt ggactgagtagaactggagg

841acaagagttg acgtgagttc ctgggagttt ccagagatgg ggcctggaggcctggaggaa

901ggggccaggc ctcacatttg tggggntccc gaatggcagc ctgagcacagcgtaggccct

961taataaacac ctgttggata agccaaaaaa

//

Glandular kallikrein 1 precursor (tissue kallikrein) (renal/pancreatic/salivary gland kallikrein)

ACCESSION P06870

PID g125170

VERSION P06870 GI：125170

SEQ ID NO 600

Initiation of

1 mwflvlclal slggtgaapp iqsrivggwe ceqhsqpwqa alyhfstfqcggilvhrqwv

61 ltaahcisdn yqlwlgrhnl fddentaqfv hvsesfphpg fnmsllenhtrqadedyshd

121lmllrltepa dtitdavkvv elptqepevg stclasgwgs iepenfsfpddlqcvdlkil

181pndecekahv qkvtdfmlcv ghleggkdtc vgdsggplmc dgvlqgvtswgyvpcgtpnk

241psvavrvlsy vkwiedtiae ns

//

Elastase 2A precursor

ACCESSION P08217

PID g119255

VERSION P08217 GI：119255

SEQ ID NO 601

Starting with 1 mirtlllsltl vagalscgdp typpyvtrvv ggeearpnsw pwqvsslyssnkgyhtcgg

61 slianswvlt aahcisssrt yrvglgrhnl yvaesgslav svskivvhkdwnsnqiskgn

121 diallklanp vsltdkiqla clppagtilp nnypcyvtgw grlqtngavpdvlqqgrllv

181 vdyatcsssa wwgssvktsm icaggdgvis scngdsggpl ncqasdgrwqvhgivsfgsr

241 lgcnyyhkps vftrvsnyid winsviann

//

Pancreatic elastase IIB (human)

ACCESSION NP_056933

PID g7705648

VERSION NP_056933.1GI：7705648

SEQ ID NO 602

Starting with 1 mirtlllsltl vagalscgvs tyapdmsrml ggeearpnsw pwqvsslssngqwyhtcgg

61 slianswvlt aahcisssri yrvmlgqhnl yvaesgslav svskivvhkdwnsnqvskgn

121diallklanp vsltdkiqla clppagtilp nnypcyvtgw grlqtngalpddlkqgrllv

181vdyatcsssg wwgstvktnm icaggdgvic tcngdsggpl ncqasdgrwevhgigsltsv

241lgcnyyykps iftrvsnynd winsviann

//

Antigens preferentially expressed in PRAME human melanoma

ACCESSION NM_006115

VERSION NM_006115.1 GI：5174640

SEQ ID NO 77

' MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDEALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAWPFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPRRWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAAQPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDELFSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMVQLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIHASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRLDQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLSVLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITDDQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLSNLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELGRPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN

SEQ ID NO 85

Start 1 gcttcagggt acagctcccc cgcagccaga agccgggcct gcagcccctcagcaccgctc

61 cgggacaccc cacccgcttc ccaggcgtga cctgtcaaca gcaacttcgcggtgtggtga

121actctctgag gaaaaaccat tttgattatt actctcagac gtgcgtggcaacaagtgact

181gagacctaga aatccaagcg ttggaggtcc tgaggccagc ctaagtcgcttcaaaatgga

241acgaaggcgt ttgtggggtt ccattcagag ccgatacatc agcatgagtgtgtggacaag

301cccacggaga cttgtggagc tggcagggca gagcctgctg aaggatgaggccctggccat

361tgccgccctg gagttgctgc ccagggagct cttcccgcca ctcttcatggcagcctttga

421 cgggagacac agccagaccc tgaaggcaat ggtgcaggcc tggcccttcacctgcctccc

481 tctgggagtg ctgatgaagg gacaacatct tcacctggag accttcaaagctgtgcttga

541 tggacttgat gtgctccttg cccaggaggt tcgccccagg aggtggaaacttcaagtgct

601 ggatttacgg aagaactctc atcaggactt ctggactgta tggtctggaaacagggccag

661 tctgtactca tttccagagc cagaagcagc tcagcccatg acaaagaagcgaaaagtaga

721 tggtttgagc acagaggcag agcagccctt cattccagta gaggtgctcgtagacctgtt

781 cctcaaggaa ggtgcctgtg atgaattgtt ctcctacctc attgagaaagtgaagcgaaa

841 gaaaaatgta ctacgcctgt gctgtaagaa gctgaagatt tttgcaatgcccatgcagga

901 tatcaagatg atcctgaaaa tggtgcagct ggactctatt gaagatttggaagtgacttg

961 tacctggaag ctacccacct tggcgaaatt ttctccttac ctgggccagatgattaatct

1021gcgtagactc ctcctctccc acatccatgc atcttcctac atttccccggagaaggaaga

1081gcagtatatc gcccagttca cctctcagtt cctcagtctg cagtgcctgcaggctctcta

1141tgtggactct ttatttttcc ttagaggccg cctggatcag ttgctcaggcacgtgatgaa

1201ccccttggaa accctctcaa taactaactg ccggctttcg gaaggggatgtgatgcatct

1261gtcccagagt cccagcgtca gtcagctaag tgtcctgagt ctaagtggggtcatgctgac

1321cgatgtaagt cccgagcccc tccaagctct gctggagaga gcctctgccaccctccagga

1381cctggtcttt gatgagtgtg ggatcacgga tgatcagctc cttgccctcctgccttccct

1441gagccactgc tcccagctta caaccttaag cttctacggg aattccatctccatatctgc

1501cttgcagagt ctcctgcagc acctcatcgg gctgagcaat ctgacccacgtgctgtatcc

1561tgtccccctg gagagttatg aggacatcca tggtaccctc cacctggaga ggcttgccta

1621tctgcatgcc aggctcaggg agttgctgtg tgagttgggg cggcccagcatggtctggct

1681tagtgccaac ccctgtcctc actgtgggga cagaaccttc tatgacccggagcccatcct

1741gtgcccctgt ttcatgccta actagctggg tgcacatatc aaatgcttcattctgcatac

1801ttggacacta aagccaggat gtgcatgcat cttgaagcaa caaagcagccacagtttcag

1861acaaatgttc agtgtgagtg aggaaaacat gttcagtgag gaaaaaacattcagacaaat

1921gttcagtgag gaaaaaaagg ggaagttggg gataggcagatgttgacttgaggagttaat

1981gtgatctttg gggagataca tcttatagag ttagaaatag aatctgaatttctaaaggga

2041gattctggct tgggaagtac atgtaggagt taatccctgt gtagactgttgtaaagaaac

2101tgttgaaaat aaagagaagc aatgtgaagc aaaaaaaaaa aaaaaaaa

ED-B Domain of fibronectin

1 fibronectin Gene ED-B Domain

ACCESSION X07717

VERSION X07717.1 GI：31406

SEQID NO 590

[ SEQ ID NO 591 ] CTFDNLSPGLEYNVSVYTVKDDKESVPISDTIIPEVPQLTDLSFVDITDSSIGLRWTPLNSSTIIGYRITVVAAGEGIPIFEDFVDSSVGYYTVTGLEPGIDYDISVITLINGGESAPTTLTQQTAVPPPTDLRFTNIGPDTMRVTW

Initiation of

1 ctgcactttt gataacctga gtcccggcct ggagtacaat gtcagtgtttacactgtcaa

61 ggatgacaag gaaagtgtcc ctatctctga taccatcatc ccaggtaatagaaaataagc

121tgctatcctg agagtgacat tccaataaga gtggggatta gcatcttaatccccagatgc

181ttaagggtgt caactatatt tgggatttaa ttccgatctc ccagctgcactttccaaaac

241caagaagtca aagcagcgat ttggacaaaa tgcttgctgt taacactgctttactgtctg

301tgcttcactg ggatgctgtg tgttgcagcg agtatgtaat ggagtggcagccatggcttt

361aactctgtat tgtctgctca catggaagta tgactaaaac actgtcacgtgtctgtactc

421agtactgata ggctcaaagt aatatggtaa atgcatccca tcagtacatttctgcccgat

481 tttacaatcc atatcaattt ccaacagctg cctatttcat cttgcagtttcaaatccttc

541 tttttgaaaa ttggatttta aaaaaaagtt aagtaaaagt cacaccttcagggttgttct

601 ttcttgtggc cttgaaagac aacattgcaa aggcctgtcc taaggataggcttgtttgtc

661 cattgggtta taacataatg aaagcattgg acagatcgtg tccccctttggactcttcag

721 tagaatgctt ttactaacgc taattacatg ttttgattat gaatgaacctaaaatagtgg

781 caatggcctt aacctaggcc tgtctttcct cagcctgaat gtgcttttgaatggcacatt

841 tcacaccata cattcataat gcattagcgt tatggccatg atgttgtcatgagttttgta

901 tgggagaaaa aaaatcaatt tatcacccat ttattatttt ttccggttgttcatgcaagc

961 ttattttcta ctaaaacagt tttggaatta ttaaaagcat tgctgatacttacttcagat

1021attatgtcta ggctctaaga atggtttcga catcctaaac agccatatgatttttaggaa

1081tctgaacagt tcaaattgta ccctttaagg atgttttcaa aatgtaaaaaatatatatat

1141atatatatat tccctaaaag aatattcctg tttattcttc tagggaagcaaactgttcat

1201gatgcttagg aagtcttttc agagaattta aaacagattg catattaccatcattgcttt

1261aacattccac caattttact actagtaacc tgatatacac tgctttattttttcctcttt

1321ttttccctct attttccttt tgcctccccc tccctttgct ttgtaactcaatagaggtgc

1381cccaactcac tgacctaagc tttgttgata taaccgattc aagcatcggcctgaggtgga

1441ccccgctaaa ctcttccacc attattgggt accgcatcac agtagttgcggcaggagaag

1501gtatccctat ttttgaagat tttgtggact cctcagtagg atactacacagtcacagggc

1561tggagccggg cattgactat gatatcagcg ttatcactct cattaatggcggcgagagtg

1621cccctactac actgacacaa caaacgggtg aattttgaaa acttctgcgtttgagacata

1681gatggtgttg catgctgcca ccagttactc cggttaaata tggatgtttcatgggggaag

1741tcagcaattg gccaaagatt cagataggtg gaattggggg gataaggaatcaaatgcatc

1801tgctaaactg attggagaaa aacacatgca atatcttcag tacactctcatttaaaccac

1861aagtagatat aaagcctaga gaaatacaga tgtctgctct gttaaatataaaatagcaaa

1921tgttcattca atttgaagac ctagaatttt tcttcttaaa taccaaacacgaataccaaa

1981ttgcgtaagt accaattgat aagaatatat caccaaaatg taccatcatgctcttccttc

2041taccctttga taaactctac catgctcctt ctttgtagct aaaaacccatcaaaatttag

2101ggtagagtgg atgggcattg ttttgaggta ggagaaaagt aaacttgggaccattctagg

2161ttttgttgct gtcactaggt aaagaaacac ctctttaacc acagtctggggacaagcatg

2221caacatttta aaggttctct gctgtgcatg ggaaaagaaa catgctgagaaccaatttgc

2281atgaacatgt tcacttgtaa gtagaattca ctgaatggaa ctgtagctctagatatctca

2341catgggggga agtttaggac cctcttgtct ttttgtctgt gtgcatgtatttctttgtaa

2401agtactgcta tgtttctctt tgctgtgtgg caacttaagc ctcttcggcctgggataaaa

2461taatctgcag tggtattaat aatgtacata aagtcaacat atttgaaagtagattaaaat

2521cttttttaaa tatatcaatg atggcaaaaa ggttaaaggg ggcctaacagtactgtgtgt

2581agtgttttat ttttaacagt agtacactat aacttaaaat agacttagattagactgttt

2641gcatgattat gattctgttt cctttatgca tgaaatattg attttacctttccagctact

2701tcgttagctt taattttaaa atacattaac tgagtcttcc ttcttgttcgaaaccagctg

2761ttcctcctcc cactgacctg cgattcacca acattggtcc agacaccatgcgtgtcacct

2821ggg

//

CEA human carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM5), mRNA.

ACCESSION NM_004363

VERSION NM_004363.1 GI：11386170

SEQ ID NO 592

' MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNELSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI

SEQ ID NO 593

Initiation of

1 ctcagggcag agggaggaag gacagcagac cagacagtca cagcagccttgacaaaacgt

61 tcctggaact caagctcttc tccacagagg aggacagagc agacagcagagaccatggag

121tctccctcgg cccctcccca cagatggtgc atcccctggc agaggctcctgctcacagcc

181tcacttctaa ccttctggaa cccgcccacc actgccaagc tcactattgaatccacgccg

241ttcaatgtcg cagaggggaa ggaggtgctt ctacttgtcc acaatctgccccagcatctt

301tttggctaca gctggtacaa aggtgaaaga gtggatggca accgtcaaattataggatat

361gtaataggaa ctcaacaagc taccccaggg cccgcataca gtggtcgagagataatatac

421cccaatgcat ccctgctgat ccagaacatc atccagaatg acacaggattctacacccta

481cacgtcataa agtcagatct tgtgaatgaa gaagcaactg gccagttccgggtatacccg

541gagctgccca agccctccat ctccagcaac aactccaaac ccgtggaggacaaggatgct

601gtggccttca cctgtgaacc tgagactcag gacgcaacct acctgtggtgggtaaacaat

661cagagcctcc cggtcagtcc caggctgcag ctgtccaatg gcaacaggaccctcactcta

721ttcaatgtca caagaaatga cacagcaagc tacaaatgtg aaacccagaacccagtgagt

781gccaggcgca gtgattcagt catcctgaat gtcctctatg gcccggatgcccccaccatt

841tcccctctaa acacatctta cagatcaggg gaaaatctga acctctcctgccacgcagcc

901tctaacccac ctgcacagta ctcttggttt gtcaatggga ctttccagcaatccacccaa

961gagctcttta tccccaacat cactgtgaat aatagtggat cctatacgtgccaagcccat

1021aactcagaca ctggcctcaa taggaccaca gtcacgacga tcacagtctatgcagagcca

1081cccaaaccct tcatcaccag caacaactcc aaccccgtgg aggatgaggatgctgtagcc

1141ttaacctgtg aacctgagat tcagaacaca acctacctgt ggtgggtaaataatcagagc

1201ctcccggtca gtcccaggct gcagctgtcc aatgacaaca ggaccctcactctactcagt

1261gtcacaagga atgatgtagg accctatgag tgtggaatcc agaacgaattaagtgttgac

1321cacagcgacc cagtcatcct gaatgtcctc tatggcccag acgaccccaccatttccccc

1381tcatacacct attaccgtcc aggggtgaac ctcagcctct cctgccatgcagcctctaac

1441ccacctgcac agtattcttg gctgattgat gggaacatcc agcaacacacacaagagctc

1501tttatctcca acatcactga gaagaacagc ggactctata cctgccaggccaataactca

1561gccagtggcc acagcaggac tacagtcaag acaatcacag tctctgcggagctgcccaag

1621ccctccatct ccagcaacaa ctccaaaccc gtggaggaca aggatgctgtggccttcacc

1681tgtgaacctg aggctcagaa cacaacctac ctgtggtggg taaatggtcagagcctccca

1741gtcagtccca ggctgcagct gtccaatggc aacaggaccc tcactctattcaatgtcaca

1801agaaatgacg caagagccta tgtatgtgga atccagaact cagtgagtgcaaaccgcagt

1861gacccagtca ccctggatgt cctctatggg ccggacaccc ccatcatttcccccccagac

1921tcgtcttacc tttcgggagc gaacctcaac ctctcctgcc actcggcctctaacccatcc

1981ccgcagtatt cttggcgtat caatgggata ccgcagcaac acacacaagttctctttatc

2041gccaaaatca cgccaaataa taacgggacc tatgcctgtt ttgtctctaacttggctact

2101ggccgcaata attccatagt caagagcatc acagtctctg catctggaacttctcctggt

2161ctctcagctg gggccactgt cggcatcatg attggagtgc tggttggggttgctctgata

2221tagcagccct ggtgtagttt cttcatttca ggaagactga cagttgttttgcttcttcct

2281taaagcattt gcaacagcta cagtctaaaa ttgcttcttt accaaggatatttacagaaa

2341agactctgac cagagatcga gaccatccta gccaacatcg tgaaaccccatctctactaa

2401aaatacaaaa atgagctggg cttggtggcg cgcacctgta gtcccagttactcgggaggc

2461tgaggcagga gaatcgcttg aacccgggag gtggagattg cagtgagcccagatcgcacc

2521actgcactcc agtctggcaa cagagcaaga ctccatctca aaaagaaaagaaaagaagac

2581tctgacctgt actcttgaat acaagtttct gataccactg cactgtctgagaatttccaa

2641aactttaatg aactaactga cagcttcatg aaactgtcca ccaagatcaagcagagaaaa

2701taattaattt catgggacta aatgaactaa tgaggattgc tgattctttaaatgtcttgt

2761ttcccagatt tcaggaaact ttttttcttt taagctatcc actcttacagcaatttgata

2821aaatatactt ttgtgaacaa aaattgagac atttacattt tctccctatgtggtcgctcc

2881agacttggga aactattcat gaatatttat attgtatggt aatatagttattgcacaagt

2941tcaataaaaa tctgctctttgtataacag aaaaa

//

Her2/Neu human tyrosine kinase type receptor (HER2) mRNA, all cds

ACCESSION M11730

VERSION M11730.1 GI：183986

SEQ ID NO 594

' MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKC SKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIVSAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERAKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV

SEQ ID NO 595

Origin chromosome 17q21-q 22.

1 aattctcgag ctcgtcgacc ggtcgacgag ctcgagggtc gacgagctcgagggcgcgcg

61 cccggccccc acccctcgca gcaccccgcg ccccgcgccc tcccagccgggtccagccgg

121 agccatgggg ccggagccgc agtgagcacc atggagctgg cggccttgtgccgctggggg

181 ctcctcctcg ccctcttgcc ccccggagcc gcgagcaccc aagtgtgcaccggcacagac

241 atgaagctgc ggctccctgc cagtcccgag acccacctgg acatgctccgccacctctac

301 cagggctgcc aggtggtgca gggaaacctg gaactcacct acctgcccaccaatgccagc

361 ctgtccttcc tgcaggatat ccaggaggtg cagggctacg tgctcatcgctcacaaccaa

421 gtgaggcagg tcccactgca gaggctgcgg attgtgcgag gcacccagctctttgaggac

481 aactatgccc tggccgtgct agacaatgga gacccgctga acaataccacccctgtcaca

541 ggggcctccc caggaggcct gcgggagctg cagcttcgaa gcctcacagagatcttgaaa

601 ggaggggtct tgatccagcg gaacccccag ctctgctacc aggacacgattttgtggaag

661 gacatcttcc acaagaacaa ccagctggct ctcacactga tagacaccaaccgctctcgg

721 gcctgccacc cctgttctcc gatgtgtaag ggctcccgct gctggggagagagttctgag

781 gattgtcaga gcctgacgcg cactgtctgt gccggtggct gtgcccgctgcaaggggcca

841 ctgcccactg actgctgcca tgagcagtgt gctgccggct gcacgggccccaagcactct

901 gactgcctgg cctgcctcca cttcaaccac agtggcatct gtgagctgcactgcccagcc

961 ctggtcacct acaacacaga cacgtttgag tccatgccca atcccgagggccggtataca

1021ttcggcgcca gctgtgtgac tgcctgtccc tacaactacc tttctacggacgtgggatcc

1081tgcaccctcg tctgccccct gcacaaccaa gaggtgacag cagaggatggaacacagcgg

1141tgtgagaagt gcagcaagcc ctgtgcccga gtgtgctatg gtctgggcatggagcacttg

1201cgagaggtga gggcagttac cagtgccaat atccaggagt ttgctggctgcaagaagatc

1261tttgggagcc tggcatttct gccggagagc tttgatgggg acccagcctccaacactgcc

1321ccgctccagc cagagcagct ccaagtgttt gagactctgg aagagatcacaggttaccta

1381tacatctcag catggccgga cagcctgcct gacctcagcg tcttccagaacctgcaagta

1441atccggggac gaattctgca caatggcgcc tactcgctga ccctgcaagggctgggcatc

1501agctggctgg ggctgcgctc actgagggaa ctgggcagtg gactggccctcatccaccat

1561aacacccacc tctgcttcgt gcacacggtg ccctgggacc agctctttcggaacccgcac

1621caagctctgc tccacactgc caaccggcca gaggacgagt gtgtgggcgagggcctggcc

1681tgccaccagc tgtgcgcccg agggcactgc tggggtccag ggcccacccagtgtgtcaac

1741tgcagccagt tccttcgggg ccaggagtgc gtggaggaat gccgagtactgcaggggctc

1801cccagggagt atgtgaatgc caggcactgt ttgccgtgcc accctgagtgtcagccccag

1861aatggctcag tgacctgttt tggaccggag gctgaccagt gtgtggcctgtgcccactat

1921aaggaccctc ccttctgcgt ggcccgctgc cccagcggtg tgaaacctgacctctcctac

1981atgcccatct ggaagtttcc agatgaggag ggcgcatgcc agccttgccccatcaactgc

2041acccactcct gtgtggacct ggatgacaag ggctgccccg ccgagcagagagccagccct

2101ctgacgtcca tcgtctctgc ggtggttggc attctgctgg tcgtggtcttgggggtggtc

2161tttgggatcc tcatcaagcg acggcagcag aagatccgga agtacacgatgcggagactg

2221ctgcaggaaa cggagctggt ggagccgctg acacctagcg gagcgatgcccaaccaggcg

2281cagatgcgga tcctgaaaga gacggagctg aggaaggtga aggtgcttggatctggcgct

2341tttggcacag tctacaaggg catctggatc cctgatgggg agaatgtgaaaattccagtg

2401gccatcaaag tgttgaggga aaacacatcc cccaaagcca acaaagaaatcttagacgaa

2461gcatacgtga tggctggtgt gggctcccca tatgtctccc gccttctgggcatctgcctg

2521acatccacgg tgcagctggt gacacagctt atgccctatg gctgcctcttagaccatgtc

2581cgggaaaacc gcggacgcct gggctcccag gacctgctga actggtgtatgcagattgcc

2641aaggggatga gctacctgga ggatgtgcgg ctcgtacaca gggacttggccgctcggaac

2701gtgctggtca agagtcccaa ccatgtcaaa attacagact tcgggctggctcggctgctg

2761gacattgacg agacagagta ccatgcagat gggggcaagg tgcccatcaagtggatggcg

2821ctggagtcca ttctccgccg gcggttcacc caccagagtg atgtgtggagttatggtgtg

2881actgtgtggg agctgatgac ttttggggcc aaaccttacg atgggatcccagcccgggag

2941atccctgacc tgctggaaaa gggggagcgg ctgccccagc cccccatctgcaccattgat

3001gtctacatga tcatggtcaa atgttggatg attgactctg aatgtcggccaagattccgg

3061gagttggtgt ctgaattctc ccgcatggcc agggaccccc agcgctttgtggtcatccag

3121aatgaggact tgggcccagc cagtcccttg gacagcacct tctaccgctcactgctggag

3181gacgatgaca tgggggacct ggtggatgct gaggagtatc tggtaccccagcagggcttc

3241ttctgtccag accctgcccc gggcgctggg ggcatggtcc accacaggcaccgcagctca

3301tctaccagga gtggcggtgg ggacctgaca ctagggctgg agccctctgaagaggaggcc

3361cccaggtctc cactggcacc ctccgaaggg gctggctccg atgtatttgatggtgacctg

3421ggaatggggg cagccaaggg gctgcaaagc ctccccacac atgaccccagccctctacag

3481cggtacagtg aggaccccac agtacccctg ccctctgaga ctgatggctacgttgccccc

3541ctgacctgca gcccccagcc tgaatatgtg aaccagccag atgttcggccccagccccct

3601tcgccccgag agggccctct gcctgctgcc cgacctgctg gtgccactctggaaagggcc

3661aagactctct ccccagggaa gaatggggtc gtcaaagacg tttttgcctttgggggtgcc

3721gtggagaacc ccgagtactt gacaccccag ggaggagctg cccctcagccccaccctcct

3781cctgccttca gcccagcctt cgacaacctc tattactggg accaggacccaccagagcgg

3841ggggctccac ccagcacctt caaagggaca cctacggcag agaacccagagtacctgggt

3901ctggacgtgc cagtgtgaac cagaaggcca agtccgcaga agccctgatgtgtcctcagg

3961gagcagggaa ggcctgactt ctgctggcat caagaggtgg gagggccctccgaccacttc

4021caggggaacc tgccatgcca ggaacctgtc ctaaggaacc ttccttcctgcttgagttcc

4081cagatggctg gaaggggtcc agcctcgttg gaagaggaac agcactggggagtctttgtg

4141gattctgagg ccctgcccaa tgagactcta gggtccagtg gatgccacagcccagcttgg

4201ccctttcctt ccagatcctg ggtactgaaa gccttaggga agctggcctgagaggggaag

4261 cggccctaag ggagtgtcta agaacaaaag cgacccattc agagactgtccctgaaacct

4321 agtactgccc cccatgagga aggaacagca atggtgtcag tatccaggctttgtacagag

4381 tgcttttctg tttagttttt actttttttg ttttgttttt ttaaagacga

aataaagacc

4441 caggggagaa tgggtgttgt atggggaggc aagtgtgggg ggtccttctc

cacacccact

4501 ttgtccattt gcaaatatat tttggaaaac

//

Human SCP1 protein mRNA

ACCESSION X9S654

VERSION X95654.1GI：1212982

SEQ ID NO 596

' MEKQKPFALFVPPRSSSSQVSAVKPQTLGGDSTFFKSFNKCTEDDLEFPFAKTNLSKNGENIDSDPALQKVNFLPVLEQVGNSDCHYQEGLKDSDLENSEGLSRVFSKLYKEAEKIKKWKVSTEAELRQKESKLQENRKIIEAQRKAIQELQFGNEKVSLKLEEGIQENKDLIKENNATRHLCNLLKETCARSAEKTKKYEYEREETRQVYMDLNNNIEKMITAHGELRVQAENSRLEMHFKLKEDYEKIQHLEQEYKKEINDKEKQVSLLLIQITEKENKMKDLTFLLEESRDKVNQLEEKTKLQSENLKQSIEKQHHLTKELEDIKVSLQRSVSTQKALEEDLQIATKTICQLTEEKETQMEESNKARAAHSFVVTEFETTVCSLEELLRTEQQRLEKNEDQLKILTMELQKKSSELEEMTKLTNNKEVELEELKKVLGEKETLLYENKQFEKIAEELKGTEQELIGLLQAREKEVHDLEIQLTAITTSEQYYSKEVKDLKTELENEKLKNTELTSHCNKLSLENKELTQETSDMTLELKNQQEDINNNKKQEERMLKQIENLQETETQLRNELEYVREELKQKRDEVKCKLDKSEENCNNLRKQVENKNKYIEELQQENKALKKKGTAESKQLNVYEIKVNKLELELESAKQKFGEITDTYQKEIEDKKISEENLLEEVEKAKVIADEAVKLQKEIDKRCQHKIAEMVALMEKHKHQYDKIIEERDSELGLYKSKEQEQSSLRASLEIELSNLKAELLSVKKQLEIEREEKEKLKREAKENTATLKEKKDKKTQTFLLETPEIYWKLDSKAVPSQTVSRNFTSVDHGISKDKRDYLWTSAKNTLSTPLPKAYTVKTPTKPKLQQRENLNIPIEESKKKRKMAFEFDINSDSSETTDLLSMVSEEETLKTLYRNNNPPASHLCVKTPKKAPSSLTTPGPTLKFGAIRKMREDRWAVIAKMDRKKKLKEAEKLFV

SEQ ID NO 597

Initiation of

1 gccctcatag accgtttgtt gtagttcgcg tgggaacagc aacccacggtttcccgatag

61 ttcttcaaag atatttacaa ccgtaacaga gaaaatggaa aagcaaaagccctttgcatt

121gttcgtacca ccgagatcaa gcagcagtca ggtgtctgcg gtgaaacctcagaccctggg

181aggcgattcc actttcttca agagtttcaa caaatgtact gaagatgatttggagtttcc

241atttgcaaag actaatctct ccaaaaatgg ggaaaacatt gattcagatcctgctttaca

301aaaagttaat ttcttgcccg tgcttgagca ggttggtaat tctgactgtcactatcagga

361aggactaaaa gactctgatt tggagaattc agagggattg agcagagtgttttcaaaact

421gtataaggag gctgaaaaga taaaaaaatg gaaagtaagt acagaagctgaactgagaca

481gaaagaaagt aagttgcaag aaaacagaaa gataattgaa gcacagcgaaaagccattca

541ggaactgcaa tttggaaatg aaaaagtaag tttgaaatta gaagaaggaatacaagaaaa

601taaagattta ataaaagaga ataatgccac aaggcattta tgtaatctactcaaagaaac

661ctgtgctaga tctgcagaaa agacaaagaa atatgaatat gaacgggaagaaaccaggca

721agtttatatg gatctaaata ataacattga gaaaatgata acagctcatggggaacttcg

781tgtgcaagct gagaattcca gactggaaat gcattttaag ttaaaggaagattatgaaaa

841aatccaacac cttgaacaag aatacaagaa ggaaataaat gacaaggaaaagcaggtatc

901actactattg atccaaatca ctgagaaaga aaataaaatg aaagatttaacatttctgct

961 agaggaatcc agagataaag ttaatcaatt agaggaaaag acaaaattacagagtgaaaa

1021cttaaaacaa tcaattgaga aacagcatca tttgactaaa gaactagaagatattaaagt

1081gtcattacaa agaagtgtga gtactcaaaa ggctttagag gaagatttacagatagcaac

1141aaaaacaatt tgtcagctaa ctgaagaaaa agaaactcaa atggaagaatctaataaagc

1201tagagctgct cattcgtttg tggttactga atttgaaact actgtctgcagcttggaaga

1261attattgaga acagaacagc aaagattgga aaaaaatgaa gatcaattgaaaatacttac

1321catggagctt caaaagaaat caagtgagct ggaagagatg actaagcttacaaataacaa

1381agaagtagaa cttgaagaat tgaaaaaagt cttgggagaa aaggaaacacttttatatga

1441aaataaacaa tttgagaaga ttgctgaaga attaaaagga acagaacaagaactaattgg

1501tcttctccaa gccagagaga aagaagtaca tgatttggaa atacagttaactgccattac

1561cacaagtgaa cagtattatt caaaagaggt taaagatcta aaaactgagcttgaaaacga

1621gaagcttaag aatactgaat taacttcaca ctgcaacaag ctttcactagaaaacaaaga

1681gctcacacag gaaacaagtg atatgaccct agaactcaag aatcagcaagaagatattaa

1741taataacaaa aagcaagaag aaaggatgtt gaaacaaata gaaaatcttcaagaaacaga

1801aacccaatta agaaatgaac tagaatatgt gagagaagag ctaaaacagaaaagagatga

1861agttaaatgt aaattggaca agagtgaaga aaattgtaac aatttaaggaaacaagttga

1921aaataaaaac aagtatattg aagaacttca gcaggagaat aaggccttgaaaaaaaaagg

1981 tacagcagaa agcaagcaac tgaatgttta tgagataaag gtcaataaattagagttaga

2041 actagaaagt gccaaacaga aatttggaga aatcacagac acctatcagaaagaaattga

2101 ggacaaaaag atatcagaag aaaatctttt ggaagaggtt gagaaagcaaaagtaatagc

2161 tgatgaagca gtaaaattac agaaagaaat tgataagcga tgtcaacataaaatagctga

2221 aatggtagca cttatggaaa aacataagca ccaatatgat aagatcattgaagaaagaga

2281 ctcagaatta ggactttata agagcaaaga acaagaacag tcatcactgagagcatcttt

2341 ggagattgaa ctatccaatc tcaaagctga acttttgtct gttaagaagcaacttgaaat

2401 agaaagagaa gagaaggaaa aactcaaaag agaggcaaaa gaaaacacagctactcttaa

2461 agaaaaaaaa gacaagaaaa cacaaacatt tttattggaa acacctgaaatttattggaa

2521 attggattct aaagcagttc cttcacaaac tgtatctcga aatttcacatcagttgatca

2581 tggcatatcc aaagataaaa gagactatct gtggacatct gccaaaaatactttatctac

2641 accattgcca aaggcatata cagtgaagac accaacaaaa ccaaaactacagcaaagaga

2701 aaacttgaat atacccattg aagaaagtaa aaaaaagaga aaaatggcctttgaatttga

2761 tattaattca gatagttcag aaactactga tcttttgagc atggtttcagaagaagagac

2821 attgaaaaca ctgtatagga acaataatcc accagcttct catctttgtgtcaaaacacc

2881 aaaaaaggcc ccttcatctc taacaacccc tggacctaca ctgaagtttggagctataag

2941 aaaaatgcgg gaggaccgtt gggctgtaat tgctaaaatg gatagaaaaaaaaaactaaa

3001 agaagctgaa aagttatttg tttaatttca gagaatcagt gtagttaaggagcctaataa

3061 cgtgaaactt atagttaata ttttgttctt atttgccaga gccacattttatctggaagt

3121 tgagacttaa aaaatacttg catgaatgat ttgtgtttct ttatatttttagcctaaatg

3181 ttaactacat attgtctgga aacctgtcat tgtattcaga taattagatgattatatatt

3241 gttgttactt tttcttgtat tcatgaaaac tgtttttact aagttttcaaatttgtaaag

3301 ttagcctttg aatgctagga atgcattatt gagggtcatt ctttattctttactattaaa

3361 atattttgga tgcaaaaaaa aaaaaaaaaa aaa

//

Human synovial sarcoma, X breakpoint 4(SSX4), mRNA.

ACCESSION NM_005636

VERSION NM_005636.1 GI：5032122

SEQ ID NO 598

' MNGDDAFARRPRDDAQISEKLRKAFDDIAKYFSKKEWEKMKSSEKIVYVYMKLNYEVMTKLGFKVTLPPFMRSKRAADFHGNDFGNDRNHRNQVERPQMTFGSLQRIFPKIMPKKPAEEENGLKEVPEASGPQNDGKQLCPPGNPSTLEKINKTSGPKRGKHAWTHRLRERKQLVVYEEISDPEEDDE

SEQ ID NO 599

Initiation of

1 atgaacggag acgacgcctt tgcaaggaga cccagggatg atgctcaaatatcagagaag

61 ttacgaaagg ccttcgatga tattgccaaa tacttctcta agaaagagtgggaaaagatg

121aaatcctcgg agaaaatcgt ctatgtgtat atgaagctaa actatgaggtcatgactaaa

181 ctaggtttca aggtcaccct cccacctttc atgcgtagta aacgggctgcagacttccac

241 gggaatgatt ttggtaacga tcgaaaccac aggaatcagg ttgaacgtcctcagatgact

301 ttcggcagcc tccagagaat cttcccgaag atcatgccca agaagccagcagaggaagaa

361 aatggtttga aggaagtgcc agaggcatct ggcccacaaa atgatgggaaacagctgtgc

421 cccccgggaa atccaagtac cttggagaag attaacaaga catctggacccaaaaggggg

481 aaacatgcct ggacccacag actgcgtgag agaaagcagc tggtggtttatgaagagatc

541 agcgaccctg aggaagatga cgagtaactc ccctcg

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains.

Any element or elements not specifically disclosed herein may be absent to limit or otherwise render the invention illustratively described herein suitable. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof. It will be appreciated that various modifications are possible within the scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims (12)

1. An isolated nucleic acid encoding a polypeptide comprising an epitope cluster from the tumor associated antigen PRAME (SEQ ID NO: 77), said cluster defined as comprising a higher average density of SEQ ID NO: 77, wherein the epitope density ratio of the epitope clusters is greater than 1, wherein the polypeptide is different from the full-length native sequence of PRAME.

2. The isolated nucleic acid of claim 1, wherein the epitope density ratio of the epitope clusters is greater than 1.3.

3. The isolated nucleic acid of claim 1, wherein the epitope density ratio of the epitope clusters is greater than 2.

4. The isolated nucleic acid of claim 1, wherein the epitope cluster is less than 80% of the amino acid sequence of PRAME.

5. The isolated nucleic acid of claim 1, wherein the epitope cluster is selected from the group consisting of: amino acids 18-59, 33-47, 71-81, 78-115, 99-108, 126, 135, 222, 238, 224, 246, 290, 303, 305, 324, 343, 363, 364, 447, 394, 409, 422, 443, 459, 487 of PRAME.

6. The isolated nucleic acid of claim 1, wherein the epitope cluster is less than about 30 amino acids in length.

7. The isolated nucleic acid of claim 1, wherein said epitope cluster is immunologically active.

8. The isolated nucleic acid of claim 1, wherein the epitope cluster comprises a housekeeping epitope.

9. The isolated nucleic acid of claim 1, wherein the epitope cluster comprises an immune epitope.

10. An immunogenic composition comprising the nucleic acid of claim 1.

11. An immunogenic composition comprising the encoded polypeptide of claim 1.

12. An immunogenic composition comprising the encoded epitope cluster of claim 1.