WO2001035810A2

WO2001035810A2 - Cancer immunotherapy and diagnosis using cytochrome p450 1b1

Info

Publication number: WO2001035810A2
Application number: PCT/US2000/031513
Authority: WO
Inventors: Joachim L. Schultze; Robert H. Vonderheide; David Sherr; Lee M. Nadler; Britta Maecker; Michael Von Bergwelt-Baildon
Original assignee: Dana-Farber Cancer Institute, Inc.; Trustees Of Boston University
Priority date: 1999-11-15
Filing date: 2000-11-15
Publication date: 2001-05-25
Also published as: EP1241945A4; EP1241945A2; CA2390882A1; WO2001035810A3

Abstract

The invention provides methods for conducting cancer immunotherapy and diagnosis using cytochrome P450 1B1 and peptide fragments thereof.

Description

CANCER IMMUNOTHERAPY AND DIAGNOSIS USING CYTOCHROME P450 IBl

Background of the Invention This invention relates to the prevention, treatment, and diagnosis of cancer. The paucity of clinically significant anti-tumor immune responses in cancer patients has long suggested that antigen-specific immunotherapy would not play a significant role in cancer treatment. However, pioneering studies in the early 1990s, using tumor-specific cytotoxic T lymphocytes (CTLs) from cancer patients, showed the existence of human tumor associated antigens (TAAs). This led to the suggestion that such antigens could be used to stimulate therapeutic anti- tumor immune responses in patients. Although these studies focused primarily on melanoma, TAAs have also been characterized in several other malignancies (Van Pel et al., Immunological Reviews 145:229-250, 1995; Rosenberg, Immunol. Today 18:175-182, 1997; Van den Eynde et al., Curr. Opin. Immunol. 9:684-693, 1997), raising the hypothesis that most, if not all, tumors express antigens that can be used to induce CTL-mediated tumor destruction. Consequently, clinical efforts are now underway to target TAAs in strategies, such as vaccination and adoptive T cell therapy, to generate effective anti-tumor CTL responses in patients. The demonstration that TAA-specific immune responses can lead to tumor regression has been borne out extensively in animal models (Rosenberg, Immunity 10:281-287, 1999). Although the identification of TAAs using patients' CTLs has revitalized the field of T cell immunotherapy, these methods are slow, very expensive, and labor-intensive. Moreover, the strategy relies on the generation of tumor-specific T cell clones in vitro, suggesting that only a restricted set of TAAs will be identified by this method. With these limitations in mind, Pfreundschuh and colleagues developed an alternative approach, SEREX (serological identification of antigens by recombinant expression cloning), to identify TAAs (Sahin et al., Curr. Opin. Immunol. 9:709-716, 1997). SEREX makes use of patients' antibody responses to tumor-derived genes and this strategy has accelerated the identification of TAAs significantly. Although several T cell-defined TAAs, such as the MAGE genes, have also been identified by SEREX, there is no information available about CTL epitopes for the vast majority of genes in the SEREX database, and, of course, such epitopes are required to activate a CTL response.

Although there is no doubt that the identification of numerous TAAs by CTL-based approaches or SEREX reflects the existence of an anti-tumor immune response, it remains to be determined if these antigens play a role as tumor regression antigens (Sarma et al., J. Exp. Med. 189:811-820, 1999). Indeed, most T cell epitopes in TAAs identified by patient CTLs have been demonstrated to be of low MHC binding affinity and/or low MHC/peptide complex stability. This quality distinguishes TAA-derived peptides from viral peptides that are almost exclusively of high binding affinity and high MHC/peptide complex stability (Feltkamp et al., Mol., Immunol. 31:1391-1401, 1994; Sette et al., J. Immunol. 153:5586-5592, 1994). Clinical vaccination trials have circumvented this obstacle by utilizing altered peptides with higher MHC binding affinity and higher MHC/peptide complex stability (Rosenberg et al., Nat. Med. 4:321-327, 1998). The low binding affinity of TAA-derived peptides is likely to be one of the reasons why natural CTL responses against such peptides are not successful for tumor eradication. This is in agreement with the finding that large numbers of TAA-specific CTLs co-exist with metastatic tumors in melanoma patients (Romero et al., J. Exp. Med. 188:1641-1650, 1998). A recent study has even demonstrated that despite expansion, such CTLs were hyporesponsive, showing reduced cytotoxic and cytokine responses (Lee et al., Nat. Med. 5:677-685, 1999). In addition, most TAAs described thus far are expressed in only one or a few tumor types, and not all patients with a given tumor type express the associated TAA. As a result, progress in the field of cancer immunotherapy has been relatively slow, because it has not been possible to develop widely useful TAA-specific immunotherapeutic strategies. Not only has it been necessary to tailor such therapies to individual types of malignancies, in some cases (such as the immunoglobulin idiotypic antigen in B cell malignancies), it has been necessary to tailor these therapies to individual patients.

Summary of the Invention The invention provides a method of treating a patient that has or is at risk of having a cell that expresses cytochrome P450 IBl (CYP1B1). This method involves administering to the patient a cytotoxic T lymphocyte (CTL)(autologous or allogeneic) that leads to death of (from here on said as kill) the cell in a CYP IBl -specific, major histocompatibility complex-restricted fashion. The CTL can be generated, for example, by activation with an antigen presenting cell that has been pulsed with CYP IBl, or a peptide of CYP IBl that binds to a major histocompatibility complex molecule. The invention also includes a second method of treating a patient that has or is at risk of having a cell that expresses CYP IBl. This method involves administering to the patient an antigen presenting cell (APC) that activates in the patient a cytotoxic T lymphocyte that kills the cell in a CYP IBl -specific, major histocompatibility complex-restricted fashion. The APC can be pulsed with CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule.

Another method included in the invention is a third method of treating a patient that has or is at risk of having a cell that expresses CYPIBI. This method involves administering to the patient CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule, which is processed by an antigen presenting cell in the patient, which, in turn, activates a cytotoxic T lymphocyte in the patient to induce cell death of the cell that expresses CYPIBI in a CYP IB 1- specific, major histocompatibility complex-restricted fashion. The CYPIBI polypeptide or peptide of CYPIBI used in this method can be administered to the patient in association with an adjuvant. The invention also includes a fourth method of treating a patient that has or is at risk of having a cell that expresses CYPIBI. This method involves administering to the patient a nucleic acid molecule encoding CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule. The nucleic acid molecule is expressed in the patient so that it can be processed by an antigen presenting cell in the patient, which activates a cytotoxic T lymphocyte in the patient to induce cell death of the cell that expresses CYPIBI, in a CYP IBl -specific, major histocompatibility complex-restricted fashion. The nucleic acid molecule encoding CYPIBI or a peptide of CYPIBI can be present in an expression vector.

Each of the methods described above can also include treatment based around a second (or more) tumor associated antigen, e.g., telomerase (hTERT, PCT/US99/25438), or a peptide thereof that binds to MHC (e.g., the 1540 peptide). In any of the methods described above, the patient can have a tumor containing cells that express CYPIBI. APCs used in these methods can be, for example, a dendritic cell or a CD40-activated B cell. The peptide of CYPIBI in these methods can bind to a class I or a class II major histocompatibility complex (MHC) molecule. In the case of a class I MHC molecule, the molecule can be, for example, an HLA-A2 molecule, and the peptide of CYPIB 1 can include the amino acid sequence of CYP239 (SEQ ID NO:l; SLNDVMPWL), CYP246 (SEQ ID ΝO:2; WLQYFPNPI), CYP190 (SEQ ID NO:3; FLDPRPLTV), or CYP528 (SEQ ID NO:4; LLDSAVQNL). Examples of other CYPIBI sequences that can be used in these methods are set forth in the Sequence Appendix and in Tables 3- 10.

The invention also includes a method of assessing the level of immunity of a patient to CYPIBI or a peptide of CYPIBI that binds to a major histocompatibility complex molecule. In this method, the level of cytotoxic T lymphocytes specific for CYPIBI or a peptide of CYPIBI is measured in a sample from a patient. The sample can be obtained from the patient before, during, or after a cancer treatment is administered to the patient. A sample can also be obtained, for example, before and after treatment.

The invention also includes CYPIBI peptides that bind to major histocompatibility complex molecules, for example, a peptide that consists essentially of the amino acid sequence set forth in SEQ ID NO: 1 (CYP239), SEQ

ID NO:2 (CYP246), SEQ ID NO:3 (CYP190), or SEQ ID NO:4 (CYP528). Also included in the invention is an ex vivo generated cytotoxic T lymphocyte that specifically kills a cell expressing CYPIBI in a specific, major histocompatibility complex-restricted fashion, and an ex vivo generated antigen presenting cell (e.g., a dendritic cell or a CD40-activated B cell) that presents a peptide of CYPIBI in the context of a major histocompatibility complex molecule.

As is understood in the art, a "polypeptide" is a chain of amino acids linked to one another by peptide bonds. A "protein" can be made up of one or more polypeptides, while a "peptide" is generally understood to be (or include) a fragment of a polypeptide, and to consist of a chain of peptide bond-linked amino acids that is shorter in length than a full length polypeptide from which it may be derived.

A "tumor associated antigen," such as CYPIBI, is an immunogenic molecule, such as a protein, that is, generally, expressed at a higher level in tumor cells than in non-tumor cells, in which, preferably, it may not be expressed at all, or only at low levels. A tumor associated antigen, or TAA, is said to be

"universal" if it is expressed in tumors of different origins.

A "cytochrome P450 IBl polypeptide," or a "CYPIBI polypeptide" is a full length, non- fragmented polypeptide of CYPIBI, while a "cytochrome P450

IBl peptide," or a "CYPIBI peptide," is (or includes) a fragment of such a

CYPIBI polypeptide. CYPIBI peptides can be of any length, up to just under the full length of a CYPIBI polypeptide. However, preferably, for use in the invention, CYPIBI peptides are of a relatively short length, such as, for example, eight, nine, ten, eleven, or twelve amino acids. Also, a CYPIBI peptide may include sequences that are not present in a corresponding CYPIBI polypeptide, provided that the CYPIBI peptide also includes a stretch of at least, for example, eight, nine, ten, eleven, or twelve consecutive amino acids that have a sequence that is identical to a sequence of eight, nine, ten, eleven, or twelve consecutive amino acids in a CYPIBI polypeptide. Peptides including amino acid substitutions can also be considered as

CYPIBI peptides. For example, a CYPIBI peptide can include a region of at least nine amino acids, of which any six or more are identical to the amino acids within a nine amino acid stretch in CYPIBI. Preferably, at least seven, more preferably, at least eight, and, most preferably, all nine of the amino acids in a CYPIBI peptide nine amino acid region are identical to a nine amino acid region in the CYPIBI.

A CYPIBI polypeptide corresponding to CYPIBI includes 533 amino acids that are substantially identical (see below) to the amino acid sequence of CYPIBI (Sutter et al, J. Biol. Chem. 269:13092-13099, 1994; Tang et al, J. Biol. Chem. 271:28324-28330, 1996; Genbank Accession No. U56438), or such a polypeptide can include the amino acid sequence of CYPIBI, as well as additional sequences.

As is discussed further below, it is preferable that CYPIBI polypeptides of the invention include regions that bind to major histocompatibility complex (MHC) antigens. Preferred examples of CYPIBI peptides that are included in the invention are CYP239 (SEQ ID NO:l), CYP246 (SEQ ID NO:2), CYP190 (SEQ ID NO:3), and CYP528 (SEQ ID NO:4). Additional CYPIBI peptides are listed in the Sequence Appendix, as well in Tables 3-10, and still more CYPIBI peptides can be identified using methods described below (also see PCT/US99/25438).

A CYPIBI peptide or polypeptide can be fused to amino acid sequences that do not naturally occur in CYPIBI. Moreover, a CYPIBI peptide or polypeptide can be attached to the surface of a cell or to a molecule or a macromolecule (e.g., a histocompatibility antigen), or a CYPIBI peptide or polypeptide can be conjugated to immunogens or adjuvants that are known to those of skill in this art, for example, keyhole limpet hemocyanin (KLH), for the purpose of eliciting a CYP IBl -specific immune response. As is noted above, preferred examples of CYPIBI peptides are CYP239 (SEQ ID NO:l), CYP246 (SEQ ID NO:2), CYP190 (SEQ ID NO:3), and CYP528 (SEQ ID NO:4).

By "CYPIBI nucleic acid molecule" is meant a DNA or RNA (e.g., mRNA) molecule that encodes a CYPIBI polypeptide or CYPIBI peptide, as are defined above.

By "CYP IBl -expressing tumor cell" is meant a tumor cell that expresses CYPIBI. A CYP IBl -expressing tumor cell can express a level of CYPIBI that is equal to, or, preferably, greater than the level of CYPIBI expressed by the normal cell type from which the CYP IBl -expressing tumor cell has originated, or other non-tumor cells. Preferably, the tumor cell expresses at least 10% more CYPIBI, more preferably, at least 25% more, still more preferably at least 50% more, and most preferably at least 150% more CYPIBI than the normal cell type from which the CYP IBl -expressing tumor cell has originated, or another non- tumor cell. CYPIBI expression levels in a CYP IB 1- expressing tumor cell can be increased by, for example, increased transcription of the CYPIBI gene, increased CYPIBI mRNA stability or translation, increased CYPIBI polypeptide stability, or increased CYPIBI enzymatic activity. Increasing such CYPIBI expression levels may be useful in the invention to increase the likelihood that a tumor cell will be recognized as a target of the immunotherapeutic methods described herein (see below).

By "histocompatibility antigen" is meant a molecule, such as a major histocompatibility complex (MHC) class I, MHC class II, or minor histocompatibility antigen, that mediates interactions of cells of the immune system with each other and with other cell types. Examples of histocompatibility antigens include MHC class I antigens, such as HLA-A (e.g., Al, A2, A3, Al l, A24, A31, A33, and A38), HLA-B, and HLA-C, MHC class II antigens, such as HLA-DR, HLA-DQ, HLA-DX, HLA-DO, HLA-DZ, and HLA-DP, and minor histocompatibility antigens, such as HA-1. By "generating CTLs" is meant an in vivo, in vitro, or ex vivo process by which CTLs (e.g., CYP IBl -specific CTLs) are activated (e.g., stimulated to grow and divide) and/or selected.

A peptide of CYPIBI is said to "specifically bind" to an MHC antigen if the peptide adheres to a histocompatibility antigen under physiological conditions. For example, such binding can be similar to that of a peptide antigen that is naturally processed and presented in the context of MHC in an antigen presenting cell.

A cytotoxic T lymphocyte (CTL) or antibody is said to "specifically recognize" a CYPIBI polypeptide or a CYPIBI peptide if it binds to the polypeptide or peptide, but does not substantially bind to other, unrelated polypeptides or peptides.

A CTL is said to "specifically kill" a cell if it specifically recognizes and lyses a cell that expresses an antigen (e.g., CYPIBI) to which it has been activated, but does not substantially recognize or lyse cells not expressing the antigen. In the case of CYPIBI, such a CTL is designated as a "CYP IB 1- specific CTL" herein.

By "CYP IBl -specific antibody" is meant an antibody that can specifically recognize and bind to a CYPIBI peptide or polypeptide, and that does not substantially recognize and bind to other, unrelated molecules.

A CYPIBI polypeptide is "presented" if a peptide of CYPIBI is displayed on the extracellular surface of a cell (e.g., an antigen presenting cell), such that it can result in the in vivo, ex vivo, or in vitro generation of CYP IBl - specific CTLs or the lysis of a tumor cell by a CYP IBl -specific CTL. Preferably, the displayed CYPIBI peptide is bound to a histocompatibility antigen.

By "physiological conditions" is meant the in vivo environment in which CYP IBl -specific CTLs are generated (activated and/or selected) and perform their biological functions (e.g., recognition of a CYPIBI peptide and MHC-restricted lysis of CYP IBl -expressing tumor cells), or an in vitro or ex vivo environment that allows CYPIBI -specific CTLs to be generated and to perform their biological functions. By "CYPIBI vaccination" is meant administration of an immunogenic preparation including one or more CYPIBI peptides, CYPIBI polypeptides, CYPIBI nucleic acid molecules, fragments of any of these molecules, CYP IB 1- presenting cells (e.g. , dendritic cells or CD40-activated B cells), or mixtures thereof. Naccination is performed on a subject who has a tumor, has a history of having a tumor or tumors, is likely to develop a tumor, or any healthy individual to prevent tumors, or on a subject in which CYP IBl -specific immune cells (such as CTLs) are to be generated for transfer into a patient. Such vaccination stimulates a CYPIB 1 -specific immune response within the subject. In subjects having tumors, the vaccination can result in partial or complete inhibition of tumor growth, or partial or complete tumor regression, provided that the patient's tumor expresses CYPIBI. In addition, vaccination can provide prophylaxis against the development of new CYPIBI -expressing tumors.

A "vaccine," as used herein, is an immunogenic composition that can be administered in the vaccination method described above. Thus, a vaccine includes, for example, one or more CYPIBI peptides, CYPIBI polypeptides, CYPIBI nucleic acid molecules, fragments of any of these molecules, CYP IB 1- presenting cells (e.g., dendritic cells or CD40-activated B cells), or mixtures thereof. Optionally, a vaccine composition can also include an adjuvant, which is a molecule that stimulates an immune response to a co-administered vaccine antigen. Examples of adjuvants that can be used in the invention are provided below. A vaccine composition can also include other tumor associated antigens (e.g., hTERT) or peptides thereof (PCT/US99/25438).

By "immune cell" is meant any cell that plays a role in cell-mediated or humoral immunity, including CTLs and antigen-presenting cells, e.g., B cells, T helper cells, and dendritic cells.

By "sample" is meant a tumor or tissue biopsy, a lymph node biopsy, bone marrow, cells, blood, serum, urine, stool, sputum, saliva, or other specimen obtained from a patient. A sample can be analyzed to determine the level of CYP IBl -specific CTLs, the level of CYP IBl -specific antibodies, or the level of any other immune response indicator (e.g., a cytokine) in the patient from whom it was taken by methods that are known in the art. For example, ELISA can be used to measure levels of CYPIBI -specific antibodies, and ELISPOT can be used to measure cytokine levels. Also, Cr⁵¹ release (T cell cytotoxicity) assays and assays that test the binding of CTLs to tetrameric CYPIBI peptide/MHC complexes, as described herein, can be used to measure levels of CYPIB 1- specific CTLs.

By "reference sample" is meant a sample in which the level of CYP IBl -specific CTLs or the level of CYP IBl -specific antibodies have been measured, and to which the level of CYPIBI -specific CTLs or the level of CYP IBl -specific antibodies in a test subject's sample are compared. Reference levels can be higher, lower, or the same as patient sample levels. Comparison of a test sample to a reference sample provides an assessment of the CYPIBI -specific immune response in the test subject. In addition, comparison of a patient's sample levels to reference sample levels can allow a diagnosis of cancer and/or a prognosis of a cancer in a patient having a tumor that includes CYP 1 B 1 - expressing cells.

By "cancer treatment" is meant any therapy (e.g., chemotherapy, radiation therapy, administration of a tumor associated antigen (e.g., CYPIBI)- specific CTLs, administration of an APC presenting a peptide of a TAA (e.g., CYPIBI), or vaccination with a TAA (e.g., CYPIBI), a nucleic acid molecule encoding a TAA (e.g., CYPIBI), or a fragment thereof, to enhance an anti-tumor immune response) administered either alone or in combination with other therapies, that alleviates disease in at least some patients to which the treatment is administered. For example, a cancer treatment can reduce or inhibit tumor growth, or can induce partial or complete tumor regression. Furthermore, a cancer treatment can be prophylactic, in that it inhibits or prevents the development of new tumors in healthy individuals, in patients that are in remission from cancer, have metastatic cancer, or have a high risk of developing cancer. By "inhibiting the development of a tumor" is meant administering a protective therapy (such as CYPIB 1 -specific CTLs, CYPIBI peptide presenting APCs, or a vaccine including, for example, one or more CYPIBI peptides, CYPIBI polypeptides, or CYPIBI nucleic acid molecules, or a combination thereof) to a subject adjudged to have a higher than average risk of developing a tumor. Subjects with a relatively high risk of developing a tumor include those having a family history of cancer, those having one or more genetic mutations that are associated with a high risk for cancer (e.g., a mutation that inactivates a tumor suppressor gene), those having relatively high levels of CYPlBl-specific CTLs or CYP IBl -specific antibodies, those who have cancer or are in remission from cancer, and those who have been exposed to agents known or suspected to cause cancer.

By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to a treated patient, while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to those skilled in the art, and are described, for example, in Remington 's Pharmaceutical Sciences (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA. The term "substantially identical" is used herein to describe a polypeptide or nucleic acid molecule exhibiting at least 50%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences is at least 8 amino acids, preferably at least 16 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acid molecules, the length of comparison sequences is at least 24 nucleotides, preferably at least 50 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides. Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). The CYPIBI polypeptides, peptides, and nucleic acid molecules of the invention can be identical or substantially identical to naturally occurring molecules, and thus may or may not include non- wild type sequences. By "substantially pure peptide" or "substantially pure polypeptide" is meant a peptide, polypeptide, or a fragment thereof, which has been separated from the components that naturally accompany it. Typically, the peptide or polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the peptide or polypeptide is a CYPIBI peptide or polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure CYPIBI peptide or polypeptide can be obtained, for example, by extraction from a natural source (e.g., a tumor cell), by expression of a recombinant nucleic acid molecule encoding a CYPIBI peptide or polypeptide, or by chemically synthesizing the peptide or polypeptide. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates is substantially free from its naturally associated components. Accordingly, substantially pure peptides and polypeptides not only include those derived from eukaryotic organisms, but also those synthesized in E. coli or other prokaryotes.

By "substantially pure DNA" or "isolated DNA" is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA is derived, flank the gene. The term thus includes, for example, a recombinant DNA that is incorporated into a vector; an autonomously replicating plasmid or virus; or the genomic DNA of a prokaryote or eukaryote; or DNA that exists as a separate molecule (e.g., a cDNA, or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By "transformation," "transfection," or "transduction" is meant any method for introducing foreign molecules into a cell. Lipofection, DEAE- dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, transduction (e.g., bacteriophage, adenoviral refroviral, or other viral delivery), electroporation, and biolistic fransformation are just a few of the methods known to those skilled in the art that can be used in the invention. By "transformed cell," "transfected cell," or "transduced cell," is meant a cell (or a descendent of a cell) into which a nucleic acid molecule (e.g., a DNA or RNA molecule) encoding a polypeptide of the invention has been introduced by means of recombinant DNA techniques.

By "promoter" is meant a minimal sequence sufficient to direct franscription. Promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal- specific, or inducible by external signals or agents can also be used in the invention; such elements can be located in the 5' or 3' or infron sequence regions of the native gene. By "operably linked" is meant that a gene and one or more regulatory- sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.

By "expression vector" is meant a genetically engineered plasmid or virus, derived from, for example, a bacteriophage, adenovirus, refrovirus, poxvirus, herpesvirus, or artificial chromosome, that is used to transfer a peptide or polypeptide coding sequence (e.g., a CYPIBI peptide coding sequence), operably linked to a promoter, into a host cell, such that the encoded peptide or polypeptide is expressed within the host cell. Other features and advantages of the invention will be apparent from the drawings, following detailed description, and the claims.

Brief Description of the Drawings Fig. 1 is a graph showing the level of peptide binding of MAGE-3,

CYP239, and CYP246 to TAP-deficient T2 cells.

Figs. 2A-2D are graphs showing that CTL derived from healthy donors recognize CYP239 and CYP246 peptides. (A) CTL raised against the CYP239 peptide specifically lyse CYP239 pulsed (■), but not impulsed T2 cells (D), or T2 cells pulsed with an irrelevant peptide (O; F271 from MAGE-3). (B) Similarly, CTL generated against the CYP246 peptide recognize only T2 cells pulsed with CYP246 (■), but not control T2 cells (D, unpulsed; O, pulsed with F271). The diagrams display representative experiments for 11/13 healthy donors positive for CYP239-specific CTL induction and 4/10 healthy donors positive for CYP246- specific CTL induction. (C) CYP239-specific CTL recognize autologous CD40- B cells pulsed with CYP239 peptide (♦), but not unpulsed autologous CD40-B ( ) or allogeneic HLA-A2 mismatched CD40-B unpulsed (O) or pulsed with CYP239 peptide (•). (D) Analogous results were obtained for CYP246-specific CTL using the same target cells unpulsed or pulsed with the CYP246 peptide. Fig. 2E is a series of graphs showing a representative teframer analysis of CYP239- and CYP246-specific CTL after 4 weeks in culture. The A2/TAX teframer served as a negative confrol. Percent teframer⁺ CD8⁺ T cells is shown. Positive teframer staining correlated with specific cytotoxicity in ⁵¹Cr assays.

Fig. 2F is a graph showing the cytotoxicity of expanded CYP239-specific tetramer sorted CTL against T2 cells either unpulsed (D), pulsed with CYP239 (■) or RT-pol476 (O).

Fig. 3 is a graph showing the level of specific lysis of CD40-activated B cells that were titrated with increasing concentrations of peptide before exposure to peptide-specific CTLs. Figs. 4A-4H are graphs showing that CYP239 and CYP246-sρecific CTL are cytotoxic for HLA-A2⁺ melanoma, multiple myeloma and ovarian carcinoma cell lines. Expression of CYPIBI in all tumor cell lines was confirmed by Western blot analysis. HLA-A2⁺ cell lines are shown by solid symbols; HLA-A2^" cell lines by open symbols. Targets for CYP239-specific CTL (upper panel) and CYP246-specific CTL (lower panel) were (A, B) melanoma cell lines K029 (•) and SK-MEL-2 (O), (C, D) multiple myeloma cell lines U266 (•), IM-9 (♦) and HS-Sultan (O), and (E, F) ovarian carcinoma cell lines 36M (■) and SK-OV-3 (D). Normal cells including the HLA-A2⁺ fibroblast cell line GM847 (Δ) and primary monocytes from 3 HLA-A2⁺ (O, O, V) and one HLA-A2^" healthy donors (D) were not lysed by either (G) CYP239-specific or (H) CYP246-specific CTL. Results of one representative experiment are shown. Similar results were obtained for each of 2 to 6 CTL tested per target.

Fig. 5 is a graph showing the specific lysis of tumor cells pulsed with CYP239 by CYP239-specific CTLs.

Figs. 6A-6C are graphs showing lysis of CYPIB 1⁺ HLA-A2⁺ primary lymphoma and acute myeloid leukemia (AML). Two HLA-A2⁺ CYPIB 1⁺ follicular lymphoma samples (■ and •) from lymph node biopsies were lysed by (A) CYP239-specific and (B) CYP246-specific CTL, while no cytotoxicity occurred against an HLA- A2^" CYPIB 1⁺ FL (D). Experiments were performed from two different normal donors with similar results. (C) CYP239-specific CTL lysed primary HLA-A2⁺ (♦), but not HLA-A2^" (O) AML cells.

Figs. 7 A and 7B are each a series of graphs showing the generation of CYP239- and CYP246-sρecific CTL from cancer patients. (A) CYP239-specific CTL from 4 patients (pt 1-4) and CYP246-specific CTL from pt 3 and pt 4 lysed T2 cells pulsed with the immunizing peptide (■), but not unpulsed T2 cells (D) or T2 cells pulsed with the irrelevant F271 peptide from MAGE-3 (O). (B) The same CYP239- or CYP246-specific CTL lysed the CYP1BT HLA-A2⁺ myeloma cell lines IM-9 (♦) and U266 (•) but not the CYPIBI^" HLA-A2^" line HS-Sultan ( ). In two cases, IM-9 cells were not lysed. All experiments shown here were performed twice with similar results. Fig. 8 is a graph showing that the efficacy of a combination of CYPIBI and hTERT-specific CTL in a chromium release assay. CYP239- and 1540- specific CTL were used individually or in combination against a mixture of T2 cells pulsed with either CYP239 or 1540 peptide. Target cells were mixed at a 1 : 1 ratio using a final number of 5000 cells/well. Numbers shown reflect the number of effector cells added to each well.

Fig. 9 is a graph showing the specific lysis of target cells with CTLs specific for heteroclitic peptides CYP239-19 and CYP239-139.

Fig. 10 is a graph showing the stability of HLA-A2/peptide complexes including the indicated peptides, as determined by TAP-deficient T2 cell assays. Fig. 11 is a graph showing the stability of HLA-A2/peptide complexes including CYP 190 and CYP528, as determined by TAP-deficient T2 cell assays.

Figs. 12A-12C are graphs showing that CYP190-specific CTL lyse peptide-pulsed T2 cells (A), HLA-A2⁺ myeloma cell lines, and HLA-A2⁺ primary ALL cells (C).

Figs. 13A and 13B are graphs showing that CYP190-specific CTL can be generated from cancer patients, such as a (A) prostate cancer patient (HLA- A2⁺), and (B) a multiple myeloma patient (HLA-A2⁺).

Fig. 14 is a graph showing the generation and verification of CYPIB 1- specific tetramers including CYP239, CYP246, or a control, Tax 11.

Fig. 15 is a schematic representation of a system to detect CYPIBI T cells by HLA-A2/peptide teframeric complexes.

Fig. 16 is a set of graphs showing the detection of CYP IBl -specific CTL in normal HLA-A2⁺ donors. Fig. 17 is a set of graphs showing the detection of CYP IBl -specific

CTL in HLA-A2⁺ multiple myeloma patients.

Detailed Description We have discovered that cytochrome P450 IBl (CYPIBI) includes peptides that bind to HLA molecules. Antigen presenting cells (APCs) that present such peptides on their surfaces, in complexes with HLA, can activate cytotoxic T lymphocytes (CTLs) to specifically lyse cells expressing CYPIBI, in an MHC-restricted fashion. The invention thus provides methods for immunotherapeutic targeting of CYPIBI -expressing cells, such as cancer cells, and methods of monitoring the efficacy of such therapeutic methods. Based on our observations that CYPIBI is a mediator of dioxin-related effects on tumorigenesis, in combination with searches of public literature databases, such as PubMed, we identified CYPIBI as a potential universal tumor antigen. It is overexpressed in nearly 100% of human tumors (Murray et al, Cancer Res. 57:3026-3031, 1997), whereas the expression in normal tissue is low and limited to steroidogenic and steroid-responsive tissue (Buters et al. , Proc. Natl. Acad. Sci. USA 96:1977-1982, 1999). CYPIBI is a member of the superfamily of monooxygenases responsible for the metabolic activation of environmental carcinogens. Mice lacking CYPIBI have a much lower incidence of lymphoma than wild type mice after challenge with polycyclic aromatic hydrocarbons, further implicating that CYPIBI plays a role in oncogenesis.

T cell mediated anti-tumor immunity

As is noted above, there is considerable evidence that human T cells can specifically lyse tumor cells (Rosenberg, Immunity 10:281-287, 1999). Most attention has been focused on CD8⁺ CTLs as the principle effector cells of antigen-specific anti-tumor immunity. Chief among the recent discoveries that have helped propel clinical efforts has been the characterization of tumor associated antigens (TAAs) (Boon et al., Annual Review of Immunology 12:337-365, 1994). Pioneering studies in the early 1990s demonstrated the existence of human TAAs using patients' CTLs that recognized peptides derived from these antigens (Van Pel et al, Immunological Reviews 145:229-250, 1995; Rosenberg, Immunol. Today 18:175-182, 1997). Although these studies primarily focused on melanoma, TAAs have been subsequently characterized in several other malignancies (Van Pel et al, Immunological Reviews 145:229-250, 1995; Rosenberg, Immunol. Today 18:175-182, 1997; Van den Eynde et al, Curr. Opin. Immunol. 9:684-693, 1997), raising the hypothesis that most, if not all, tumors express antigens that CTL can potentially attack. The demonstration that TAA-specific immune responses can lead to tumor regression has been borne out extensively in animal models (Rosenberg, Immunity 10:281-287, 1999). Although the identification of TAAs using patients' CTLs has revitalized the field of T cell immunotherapy, these methods are slow, very expensive, and labor-intensive. Moreover, the strategy relies on the generation of tumor-specific T cell clones in vitro, suggesting that only a restricted set of TAAs will be identified by this method. With these limitations in mind, Pfreundschuh and colleagues developed an alternative approach, SEREX (serological identification of antigens by recombinant expression cloning), to identify TAAs (Sahin et al, Curr. Opin. Immunol. 9:709-716, 1997). SEREX makes use of patients' antibody responses to tumor-derived genes and this strategy has accelerated the identification of TAAs significantly. Although several T cell-defined TAAs, such as the MAGE genes, have also been identified by SEREX, there is no information available about CTL epitopes for the vast majority of genes in the SEREX database, and, of course, such epitopes are required to activate a CTL response.

Although there is no doubt that the identification of numerous TAAs by CTL-based approaches or SEREX reflects the existence of an anti-tumor immune response, it remains to be determined if these antigens play a role as tumor regression antigens (Sarma et al, J. Exp. Med. 189:811-820, 1999). As is mentioned above, most T cell epitopes in TAAs identified by patient CTLs have been demonstrated to be of low MHC binding affinity and/or low MHC/peptide complex stability. Clinical vaccination trials have circumvented this obstacle by utilizing altered peptides with higher MHC binding affinity and higher MHC/peptide complex stability (Rosenberg et al, Nat. Med. 4:321 -327, 1998). This quality distinguishes TAA-derived peptides from viral peptides that are almost exclusively of high binding affinity and high MHC/peptide complex stability (Feltkamp et al, Mol. Immunol. 31 : 1391-1401, 1994; Sette et al, J. Immunol. 153:5586-5592, 1994). The low binding affinity of TAA-derived peptides is likely to be one of the reasons why natural CTL responses against such peptides are not successful for tumor eradication. This is in agreement with the finding that large numbers of TAA-specific CTLs co-exist with metastatic tumors in melanoma patients (Romero et al, J. Exp. Med. 188: 1641-1650, 1998). A recent study has even demonstrated that despite expansion, such CTLs were hyporesponsive, showing reduced cytotoxic and cytokine responses (Lee et al, Nat. Med. 5:677-685, 1999).

To overcome these limitations of currently known TAAs, we have developed methods to identify more universal TAAs, and, in particular, those containing T cell epitopes with high MHC binding affinity and high MHC/peptide complex stability. Such TAAs and MHC-binding peptides thereof can trigger sufficient CTL responses against a broad range of tumor types. Rather than analyzing tumor-derived T cell clones or tumor-specific antibodies derived from patients, an alternative strategy was used, in which TAA and their CTL epitopes are deduced from genes known to be selectively expressed in tumors. By combining bioinformatics to predict peptides that bind to HLA with high affinity, peptide binding analysis, and a powerful in vitro T cell expansion system, the cytochrome P450 IBl (CYPIBI) was identified (see below). This TAA contains at least two peptide epitopes that (1) bind to HLA-A*0201 with high affinity and high MHC/peptide complex stability, (2) are naturally processed and presented by HLA-A*0201 molecules on the cell surface of a panel of tumor cell lines, (3) elicit peptide-specific HLA-resfricted CTL responses, and (4) are recognized by such CTL on a wide variety of different tumor histologies.

Deducing CTL epitopes in tumor associated antigens (TAAs): Making use of genomics and proteomics for tumor immunology Current developments in genomics and proteomics suggest that numerous TAA candidate genes can be identified. The Human Genome Project (HGP), the Human Cancer Gene Anatomy Project (CGAP), the SEREX database, and other databases, including literature databases such as PubMed, provide an enormous set of data that can be analyzed to identify genes that fulfill the criteria of universal tumor antigens, as are described above. It is clear that entering the post-genomic era, none of the classical approaches to characterize TAA, including T cell cloning and testing of T cell clones against expression libraries (Boon et al, Annual Review of Immunology 12:337-365, 1994), is suitable for the analysis of the ever-growing databases to identify a set of universal tumor antigens.

To overcome the limitations of prior methods in determining CTL epitopes, advances in bioinformatics can be applied. First, database mining and integration can be used to identify of universal tumor antigen candidates, which are genes that are expressed at a much higher level in tumor cells than in normal cells. Then, computational methods are used to predict peptides derived from these proteins for high-affinity binding to MHC molecules. The requirements for peptides to bind to class I HLA molecules and to elicit CTL responses have been studied extensively (Rammensee et al, Annual Review of Immunology 11 :213-244, 1993; Sidney et al, Immunology Today 17:261-266, 1996). The strength of CD8⁺ CTL responses depends upon the binding affinity of the target peptide to MHC, the peptide-MHC complex stability, and the avidity of T cell receptor (TCR) binding for the peptide complex (Sette et al, J. Immunol.

153:5586-5592, 1994; van der Burg et al, J. Immunol. 156:3308-3314, 1996; Savage et al, Immunity 10:485-492, 1999; Gallimore et al, Eur. J. Immunol. 28:3301-3311, 1998). These factors directly influence the efficiency of peptide loading and the number of peptides expressed on the cell surface (Gallimore et al, Eur. J. Immunol. 28:3301-3311, 1998). The vast majority of viral-derived immunodominant peptides are of high binding affinity and/or peptide-HLA complex stability (Feltkamp et al, Mol. Immunol. 31:1391-1401, 1994; Sette et al, J. Immunol. 153:5586-5592, 1994). Since only a very small portion of peptides can bind to MHC molecules, rapid and accurate methods to identify them, such as those used in the present invention, can expedite the search for CTL epitopes by orders of magnitude.

A great deal of effort has been expended on the development of computational methods to identify peptides that bind strongly to various MHC alleles. It began with the work of Rammensee and colleagues, who identified motifs in peptide sequences that serve as signatures of the MHC molecules to which they bind (Rammensee et al, Immunogenetics 41:178-228, 1995). Motif- based methods have recently been applied to the identification of CTL epitopes deduced from proteinase 3 (Molldrem et al, Blood 88:2450-2457, 1996), MAGE-3 (Nijman et al, Eur. J. Immunol. 23:1215-1219, 1993), MUC-1 (Brossart et al, Blood 93:4309-4317, 1999), and telomerase (PCT/US99/25438). Typically, only 20% of peptides that carry the motif bind to the respective MHC molecule. The inclusion of "secondary anchor" positions (Ruppert et al, Cell 74:929-937, 1993), the so-called extended motif, significantly improves the specificity of motif-based methods, but they are available only for HLA-A*0201 (Ruppert et al, Cell 74:929-937, 1993) and HLA-B*3501 (Schόnbach et al, J. Immunol. 154:5951-5958, 1995). Many other statistically-based computational methods have been developed (for reviews, see, e.g., Hammer, Curr. Opin. Immunol. 7:263-269, 1995; Parker et al, Immunol. Res. 14:34-57, 1995), including the polynomial method (Gulukota et al, J. Mol. Biol. 267:1258-1267, 1997), methods based on neural nets (Gulukota et al, J. Mol. Biol. 267:1258-1267, 1997; Brusic et al, Bioinformatics 14:121-130, 1998; Brusic et al, Nucleic Acids Res. 26:368-371, 1998), a method that assigns a score for each amino acid at each position as determined experimentally via single residue substitutions (Hammer et al, J. Exp. Med. 180:2353-2358, 1994), and a method developed by Parker et al. based on a database of the half-lives of bound β2 -microglobulin (β2m) in MHC-peptide complexes (Parker et al, J. Immunol. 152:163-175, 1994). The method developed by Parker et al. assumes that the dissociation of β2m is rate-limited by the dissociation of peptide, so that variation in the microglobulin half-life reflects variation in the peptide half-life. The variation is, in turn, assumed to reflect the variation in the binding affinity of the peptide. A weight matrix is then determined to best reflect the half-lives, assuming that the contribution of one peptide position does not depend on its neighboring positions.

Weng and colleagues have recently developed a new statistical method (implemented as a computer program named LPpep; Weng et al, http://bioinformatics.bu.edu/peptides.hfrnl) to predict strong

HLA-A*0201 -binding peptides. It determines the contributions for each of the 20 amino acids at each of the positions of a peptide using a linear programming algorithm. When tested on a data set of over 1000 peptides having known binding affinities, LPpep has a higher sensitivity (>0.75) and specificity (> 0.9) than four other available methods.

High volume analysis of peptide MHC affinity and MHC/peptide complex stability

The basic principles of peptide binding to MHC molecules have been well established in the field (Rammensee et al, Annual Review of Immunology 11:213-244, 1993; Rothbard et al, Annual Review of Immunology 9:527-565, 1991 ; Engelhard, Annual Review of Immunology 12:181 -207, 1994; Madden, Annual Review of Immunology 13:587-622, 1995; Pamer et al, Annual Review of Immunology 16:323-358, 1998; Rock et al, Annual Review of Immunology 17:739-779, 1999), and numerous assay systems have been developed to analyze the binding of any given peptide to MHC molecules. Binding has been analyzed using intact TAP-deficient cells (Salter et al, EMBO J. 5:943-949, 1986;

Schumacher et al, Cell 62:563-567, 1990) and by in vitro assays utilizing purified HLA molecules (Ruppert et al, Cell 74:929-937, 1993; Schumacher et al, Cell 62:563-567, 1990; Townsend et al, Cell 62:285-295, 1990). While most assay systems have focused on the maximal binding affinity, it has recently been suggested that the dissociation rate of MHC and peptide (also measured as MHC/peptide complex stability) may be a more important determinant for characterizing a peptide as a dominant T cell epitope (van der Burg et al, J. Immunol. 156:3308-3314, 1996; Busch et α/., J. Immunol. 160:4441-4448, 1998; Kammer et al, J. Exp. Med. 190:169-176, 1999).

In vitro analysis of CTL responses

The generation of antigen-specific T cells in vitro is a classical immunological technique. Antigen-specific T cells can be generated relatively easily if the peptides used to make such cells are: (1) immunodominant, (2) of viral or other non-self origin, (3) expressed at a reasonably high copy number on the cell surface (Porgador et al, Immunity 6:715-726, 1997), and (4) of high affinity for, and of low dissociation rate (high MHC/peptide complex stability) from, MHC, and if the T cell pool under study has been exposed to the antigen in vivo prior to ex vivo analysis (recall response). The frequency analysis of peptide-specific T cells by teframer technology (see below) revealed a significantly higher frequency than earlier assays based on in vitro expansion had suggested.

It is therefore apparent that only a fraction of specific CTLs are expanded in classical in vitro systems utilizing unstimulated peripheral blood mononuclear cells (PBMC) as antigen presenting cells (McMichael et al, J. Exp. Med. 187:1367-1371, 1998). To circumvent these pitfalls, in vivo systems utilizing transgenic mice carrying human HLA genes have been introduced (Man et al, International Immunology 7:597-605, 1995; Wentworth et al, International Immunology 8:651-659, 1996; Alexander et al, J. Immunol. 159:4753-4761, 1997). However, these systems are expensive and are not suitable for screening multiple peptide epitopes simultaneously. Making use of new findings in basic immunology, it is possible to optimize further currently available in vitro culture technology. The use of an APC instead of PBMC as stimulators is only one example.

We have developed a system that utilizes dendritic cells (DC) for primary activation and CD40-activated B cells (CD40-B) for re-stimulation, thereby mimicking the physiological sequence of events between T cells and APCs during an ongoing immune response (Schultze et al, J. Exp. Med. 89:1-12, 1999; Schultze et al, J. Clin. Invest. 100:2757-2765, 1997). This system has been successfully used for the identification of T cell epitopes derived from hTERT and the clonal immunoglobulin in B cell malignancies (PCT/US99/25438). From a single blood draw, professional APCs, including DCs and CD40-activated B cells, are generated, and the remaining PBMCs are enriched for CD8⁺ T cells. T cells are primarily stimulated with peptide-pulsed DC, and repeatedly stimulated with peptide-pulsed CD40-B cells. Peptide-specificity and HLA-restriction is analyzed after a total of 2-5 stimulations, depending on the antigen under study. This system is not only very powerful in amplifying rare T cells against TAA-derived peptides, but has several other advantages: (1) it is relatively cheap compared to transgenic mice, (2) a single blood draw is sufficient to generate all cellular components necessary, and (3) the use of professional APCs for restimulation is superior to PBMC. Classically, the function of CTLs in vitro has been defined by cytotoxicity assays using radioactive chromium. Clearly, cytotoxicity analysis is an important component of the characterization of a novel TAA, since tumor cell lysis is the ultimate goal of any TAA-directed immunotherapeutic intervention. However, such assays are not suitable to determine the frequency of peptide-specific CTLs. In addition, the sensitivity of cytotoxicity assays to identify very small numbers of specific CTLs is insufficient. To detect very low numbers of specific CTLs and to determine their frequency, two new technologies, namely the teframer technology (Altman et al, Science 274:94-96, 1996) and cytokine ELISPOT analysis (Herr et al, J. Immunol. Methods 203:141-152, 1997), have been developed and applied to tumor immunology. In particular, teframers have been suggested as a tool to enrich CTL lines for peptide-specific CTL (Dunbar et al, Curr. Biol. 8:413-416, 1998; Yee et al, J. Immunol. 162:2227-2234, 1999; Valmori et al, Cancer Res. 59:2167-2173, 1999). Currently, the tetramer technology is still technically demanding and it is not possible to generate numerous teframers in small quantities to screen peptide-specific CTL responses against a larger set of unknown peptides. For this purpose, cytokine ELISPOT is more suitable (Herr et al, J. Immunol. Methods 203:141-152, 1997).

Experimental Results

The dioxin-inducible cytochrome P450 IBl (CYPIBI)

Using the methods described above (also see PCT/US99/25438), we identified the dioxin-inducible cytochrome P450 IBl (CYPIBI) as a potential

TAA. A list of peptides predicted to bind to all HLA alleles available are listed in the Sequence Appendix. The prediction was carried out using three different algorithms that are freely available on the Internet: http://engpub 1.bu.edu/LPpep-cgi/peptide2.cgi http://www.uni-tuebingen.de/uni/kxi http://bimas.dcrt.nih.gov/molbio/hla_bind/ Analysis of the CYPIBI sequence by two independent prediction algorithms 5 (BIMAS and LPpep, see Experimental Methods, below) revealed two peptides (CYP239 and CYP246) predicted to bind to HLA-A*0201, the most common HLA allele (Table 1). These peptide sequences are unique in the public gene databases and in particular are not found within any other member of the cytochrome P450 family. (Also see below for additional CYPIBI peptides (e.g., l o CYP 190 and CYP528) identified according to the invention.)

Peptide binding of CYPIBI derived peptides

Binding of both peptides to HLA-A2, as well as their complex stability, was determined using a cellular assay employing TAP-deficient T2 cells (Table 1 ;

15 PCT/US99/25438). Both peptides stabilized HLA-A2 molecules on the surface of T2 cells to a similar extent as a positive control peptide (F271) derived from the tumor antigen MAGE-3 (Nijman et al, Eur. J. Immunol. 23:1215-1219,1993), which is known to bind HLA-A2 with high affinity. In particular, T2 cells were incubated with peptide in serum- free medium for up to 18 hours, harvested, 0 washed, and subsequently stained with FITC-labeled anti-HLA-A2 mAb BB7.2 (maximum peptide binding). Increase in fluorescence intensity was determined as a function of peptide binding. For analysis of complex stability, T2 cells were cultured in serum-free media for an additional 2, 4, 6, or 24 hours, and subsequently analyzed for HLA-A2 expression by flow cytometry. As is shown 5 in Fig. 1, the MAGE-3 -derived peptide, which induces CTL responses in the majority of all normal donors, demonstrated high binding affinity and complex stability. Although both CYP IBl -derived peptides bound to HLA-A2, CYP246 showed a significantly lower complex stability than CYP239 and MAGE-3. Moreover, attempts to induce CTL responses were successful in 13/15 donors 0 against CYP239, but only 4/9 donors for CYP246. Our data further show that complex stability might be a more important factor than binding affinity for the likelihood to generate peptide-specific CTL responses.

TABLE 1

Binding of CYPIBI and control peptides to human HLA-A*0201

sequence BIMAS¹ LPpep² binding affinity³

JSL

WLQYFPNPV 1216 6.23 3.4

FLWGPRALV 2655 7.63 3.2

1 Peptide prediction at BIMAS (Bioinformatics & Molecular Analysis Section) scores for predicted binding are calculated as half-life of MHC/peptide complexes (peptides with scores > 500 were chosen as potential candidates)

2 LPpep (peptide prediction at Boston University) scores are predicted as arbitrary ln(IC₅₀) concentrations (peptides with scores < 7 were chosen as potential candidates)

3 Mean fluorescence with peptide / mean fluorescence without peptide - 1 Results representative of 4 experiments.

Peptide-specific killing

To test whether CYP239 and CYP246 reactive T cells are present in the human T cell repertoire, CTL lines were generated ex vivo by repetitive stimulation with peptide pulsed autologous APC. CTL specific for CYP239 were induced from peripheral blood mononuclear cells (PBMC) in 11 of 13 healthy HLA-A2⁺ donors (Fig. 2A). These CTL specifically lysed T2 cells pulsed with CYP239 peptide, while no cytotoxicity occurred against unpulsed T2 cells or T2 cells pulsed with the F271 peptide from MAGE-3. CYP246 specific CTL were generated in 4 of 10 healthy HLA-A*0201⁺ donors (Fig. 2B). HLA-A2 restriction was demonsfrated using autologous and HLA-A2 mismatched CD40-activated B cells (CD40-B) as targets (Figs. 2C and 2D). CYP239-specific CTL lysed autologous CD40-B pulsed with CYP239, but not allogeneic HLA-A2- CD40-B pulsed with CYP239 (Fig. 2C). Similar results were obtained for CYP246- specific CTL (Fig. 2D). Experiments titrating the concentration of peptide onto the CD40-activated B cells before the cytotoxicity assay further support the peptide-specificity of the CTL generated against the CYP239 peptide. Comparing the data with published data in the literature the cell line tested in this experiment 5 is of intermediate avidity. Alternatively, the cell line contains both high and low avidity CTL and the curve represents the sum of the actions of these CTLs (Fig. 3).

For both CYP239 and CYP246 CTL, specificity was further demonsfrated using peptide/MHC teframers (Fig. 2E). Frequency analysis using CYP239 l o teframers demonstrated that 1.4-2.4% of all CD8⁺ T cells recognized the CYP239 peptide, a percentage comparable to previously published data for gplOO specific (Yee et al, J. Immunol. 162:2227-2234, 1999) or proteinase-3 specific CTL lines (Molldrem et al, Cancer Res. 59:2675-268.1,1999). CYP246-specific CTLs were detected with CYP246 teframer, but the frequency of specific CTL was lower

15 (0.47%o). To further confirm peptide-specific cytotoxicity, CYP239 teframer- positive CTL were sorted and expanded using phytohemagglutinin (PHA), IL-7, IL-2, and irradiated allogeneic PBMC. These CTL lysed T2 cells pulsed with CYP239 at extremely low E:T ratios, but not unpulsed T2 cells or T2 cells pulsed with an irrelevant HLA-A2 binding peptide (Fig. 2F). Thus, CYPIBI contains at 0 least two HLA-A*0201 binding peptides, and T cells recognizing these peptides are present in the T cell repertoire of healthy donors.

CYPIBI specific CTL lyse CYPIBI expressing tumors in an HLA-A2 restricted fashion 5 Although peptide-specificity of CTL is demonsfrated by lysis of peptide- pulsed target cells, it is important to show that tumor cells themselves process and present the peptide in the groove of their MHC molecules (Yee et al, J. Immunol. 162:2227-2234, 1999). We approached this question by using a panel of HLA- A2⁺ and HLA-A2- tumor cell lines that all express CYPIBI protein. CYP239- and 0 CYP246-specific CTL from healthy donors were then screened for cytotoxicity (Figs. 4A-4H). CYP239 CTL (Fig. 4A) and CYP246 CTL (Fig. 4B) showed specific lysis of HLA-A2⁺ melanoma cell line K029, but not HLA-A2^" SK-MEL-2 cells. Similarly, the HLA-A2⁺ myeloma cell lines IM-9 and U266 were lysed by CYP239 CTL (Fig. AC) and CYP246 CTL (Fig. 4D), while the HLA-A2^" myeloma HS-Sultan cell line was not killed. Finally, specific cytotoxicity by CYP239 CTL (Fig. 4E) and CYP246 CTL (Fig. 4F) was observed against the HLA-A2⁺ ovarian carcinoma cell line 36M, but not the HLA-A2^" line SK-OV-3. These data show that CYPIBI derived peptides are naturally processed and presented by tumor cell lines of different tissue origin.

Since CYPIBI expression has been reported in fibroblasts (Eltom et al, Carcinogenesis 19:1437-1444, 1998) and monocytes (Baron et al, Biochem. Pharmacol. 56:1105-1110, 1998), we analyzed an HLA-A2⁺ fibroblast cell line (GM847) and primary peripheral blood derived monocytes from four healthy donors as targets for CYPIBI -specific CTL. Western blot analysis showed that of these normal cells express low or absent levels of CYPIBI. As is shown in Figs. 4G and 4H, CYP239 and CYP246-specific CTL failed to lyse these normal targets. In contrast, CD40-activated B cells strongly express CYPIBI protein (detected by Western blot), but these normal cells were not lysed by CYP239- or CYP246-specific CTL (Figs. 2C and 2D), suggesting that there is a differential expression of CYPIB peptides on tumor cells.

Methods to improve killing of tumor cell lines

The experiments described so far suggest that CYP239 peptide is most likely expressed at low levels on tumor cell MHC. Alternatively, tumor cells could be more resistant to CTL-mediated lysis. To address these issues and to determine whether the increase of peptide on the cell surface of tumor cells would lead to increase killing of the tumor cells, tumor cells were pulsed with the specific peptide before they were used in chromium release assays. We could demonstrate that peptide-pulsing of tumor cells significantly increased killing of the target cells, suggesting that the level of naturally expressed CYP239 peptide is low on the tumor cells, however, that these cells can be readily killed once the level of peptide is increased. This also suggests that any methodology to increase the expression of CYPIB 1 -derived peptides on the cell surface will make the tumor cell a susceptible target for CYP IBl -specific CTLs (Fig. 5).

Lysis of primary HLA-A2⁺ follicular lymphoma cells CYP IBl -specific CTLs were then evaluated for cytotoxicity against primary tumor tissue. Because CYPIBI^"'^" mice demonstrate a significantly reduced incidence in carcinogen- induced lymphomas (Buters et al, Proc. Natl. Acad. Sci. U.S.A. 96:1977-1982, 1999), we chose to study human primary follicular lymphoma (FL) as a model tumor target for CYPIBI specific CTL. Tumor cells from two HLA-A2⁺ FL samples and one HLA-A2^" FL sample were found to be CYPIB 1⁺ as assessed by Western blot analysis. Using these target cells, we found that CTL lines generated against CYP239 or CYP246 were cytotoxic for the HLA-A2⁺ FL, while no killing of the HLA-A2^" FL was observed (Figs. 6 A and 6B). We also demonsfrated lysis of HLA-A2⁺ primary acute myeloid leukemia (AML) cells, but not HLA-A2^" primary AML cells by CYP239 CTL (Fig. 6C). These data show that both CYPIB 1 -derived peptides are processed and presented by HLA-A2 on primary tumor cells and that HLA-A2 restricted CYPIBI specific CTL from healthy donors can recognize and kill these target cells.

Generation of CYPIBI -specific CTL from patients with multiple myeloma

Similar to experiments described for healthy donors, we next attempted to generate CYP IBl -specific CTL from peripheral blood of cancer patients. HLA-A2⁺ patients with multiple myeloma (n=3) or follicular lymphoma (n=l) (Table 2) were tested for ex vivo generation of CYP239 (n=4) or CYP246 (n=2) specific CTL. Generation of all cellular components of our ex vivo system (i.e., dendritic cells, CD40-B, and CTL), as well as expansion of CTL to CYP239 and CYP246 were similar to results obtained for healthy donors. Using peptide- pulsed T2 cells as targets, we demonsfrated CYP239-specific CTL in all four patients (Fig. 7 A). Due to lower numbers of PBMC available, CYP246-specific CTL cultures were only initiated in patients 3 and 4. CYP246-specific CTLs were detected in both patients These patient-derived lines showed tumor-specific lysis of HLA-A2⁺ myeloma cell lines U266 and IM-9, but not the HLA-A2^" myeloma cell line HS-Sultan (Fig. 7B) Because autologous tumor cells were not available from these patients, we tested the same FL samples described above as primary 5 tumor targets. CYP239-specιfιc CTL from patient 1 lysed both HLA-A2⁺ FL samples but not the HLA- A2 (18% 0% at an E.T ration of 30.1).

TABLE 2

patient age sex disease stage prior treatment CTL induction

CYP239 CYP246

1 41 f Multiple Myeloma I A none yes ND

2 47 f Multiple Myeloma II A none yes ND High-Dose Dexamethasone,

3 40 m Multiple Myeloma III A discontinued >30d prior to yes yes leukapheresis

Non Hodgkin's Lymphoma

4 29 m III A none yes yes (Follicular Lymphoma)

Patient characteristics, prior treatment, and CTL induction

ND = not determined 0

Combining CYPIB X and hTERT-specific CTL

We next analyzed the combination of CYP IBl -specific CTL with hTERT-specific CTL (Vonderheide et al , Immunity 10:673-679, 1999) To 5 normalize for equal susceptibility to T-cell mediated lysis, we used a mixture (1.1 ratio) of T2 cells pulsed with either CYP239 or 1540 hTERT peptide (Vonderheide et al , Immunity 10:673-679, 1999) as a model for heterogeneous antigen expression Equal ⁵¹Cr labeling of both T2 cell populations was assured Under these conditions, it is expected that either CTL line alone can only lyse a 0 maximum of 50% of the target cell population while the combination if effective has the potential to kill >50% of all cells (Janeway, Immunobiology The Immune System in Health and Disease (Garland Publishing c/o Taylor & Francis, Inc , New York, 1999), p. 297). This is true for specific lysis regardless of the E:T ratio used. As postulated, the combination of CYP239- and 1540-specific CTL was superior to each CTL line alone, achieving specific lysis of >50% (Fig. 8). Similar observations were made in independent experiments using CTL generated from two different donors. We also analyzed the effect of combined CYPIBI and hTERT CTL on the HLA-A2⁺ tumor cell line IM-9, which expresses both antigens. In two experiments, we observed additive lysis of CYP239- and 1540- specific CTL across a range of E:T ratios. These data demonstrate the potential of enhancing antigen-specific T cell immunity by targeting multiple antigens, such as CYPIBI and hTERT.

Use of heteroclitic peptides

To improve immunogenicity of CYPIBI derived peptides, we designed heteroclitic peptides optimized for binding affinity to MHC. We have already shown that CTL generated against the CYP239 wild type peptide can recognize and lyse target cells pulsed with the heteroclitic peptide CYP239-19 equally well, while CTL generated with CYP239 do not recognize CYP239-139. These data show that despite similar binding, the change of a third amino acid does not allow for recognition by CTL specific for CYP239. Most likely, the amino acid change induced a change in the three dimensional structure of the peptide not allowing TCR activation (Fig. 9). To design heteroclitic peptides with higher binding affinity, we used an algorithm available on the Internet

(http://engpubl.bu.edu/LPpep-cgi/peptide3.cgi). Two heteroclitic peptides to the immunogenic peptide CYP239 have been designed as examples to improve binding affinity, complex stability, and, potentially, immunogenicity (Table 3).

TABLE 3 Examples of Heteroclitic Peptides Optimized for Binding to HLA-A*0201 heteroclitic 9mers

Peptide binding of heteroclitic CYP239 peptides

The T2 assay described above was used to determine binding and dissociation rate of heteroclitic peptides engineered for optimal binding to HLA molecules. Two heteroclitic peptides to CYP239 were tested and shown to have higher peptide/MHC-complex stabilities, as is shown in Table 4. While the confrol peptide MAGE-3 and the CYP239 peptide showed no significant binding at 24 hours (0.14 resp. 0.12), both heteroclitic peptides still bound to HLA-A*0201 (0.72 resp. 0.73).

TABLE 4 Examples of heteroclitic peptides optimized for binding to HLA-A*0201

The following Tables 5 and 6 show predicted mutations to improve HLA-A2 binding of CYPIBI 239 and CYPIBI 246.

TABLES Predict Mutations to Improve HLA-A2 Binding CYP239

Under each position, a list of possible amino acid mutations is given, followed by the change in the predicted ln(IC50) produced by the mutation with respect to the original peptide's score

POSITION

1 2 3 4 5 6 7 8 9

K(017) * A(131) A (060)

M (203) C(135) 1(136)

F (060) G (057) V(166)

Y (207) H (078)

L(l 63)

M (200)

F (140)

P(019)

S (055)

W

(138)

Y (257) * indicates best amino acid is already present

Original peptide: SLVDVMPWL, predicted ln(IC50) = 2.88 Top scoring peptide under given constraints: YLYDVMPWV, predicted ln(IC50) = -3.42

TABLE 6 Predict Mutations to Improve HLA-A2 Binding CYP246

POSITION

1 2 3 4 5 6 7 8 9

A (1 53) * A (l 50)

R (1 08) D (0 10)

N (0 33) C (1 54)

C (0 88) G (0 76)

G (1 39) H (0 97)

L (0 92) I (0 16) (1 84) L (1 82)

M (3 70) M (2 19)

F (2 27) F (l 59)

S (1 67) P (0 38)

T (0 94) S (0 74)

W

Y (3 74) (1 57)

V (1 21) Y (2 76)

V (0 19)

* indicates best amino acid is already present

Original peptide: WLQYFPNPV, predicted ln(IC50) = 6.23

Top scoring peptide under given constraints: YLYYFPNPV, predicted ln(IC50) =

-0.27

Identification of additional HLA-A2 binding epitopes from CYPIBI

Binding studies were carried out to characterize additional CYPIB 1- deπved peptides that are predicted to bind to HLA-A2. Table 7, below, shows the sequences of additional peptides that are predicted to bind to HLA-A2. TABLE 7

Predicted binding of epitopes to HLA-A2

Nonamers predicted to bind to HLA-A*0201

Parker LPpep SYFPEITHI position peptide score rank score rank score rank

25 LLLSVLATV 1006 3 3 54 4 32 1

88 RLGSCPIW 29 18 4 61 6 20 31

190 FLDPRPLTV 128 11 6 52 15 26 5

239 SLVDVMPWL 1108 2 2 88 2 24 9

246 WLQYFPNPV 1216 1 6 23 12 21 22

292 MMDAFILSA 21 19 3 31 3 20 29

34₄ LLFTRYPDV 656 4 4 69 7 24 7

377 NLPYVLAFL 270 8 7 1 21 25 6

380 YVLAFLYEA 65 14 1 56 1 20 27

479 QLFLFISIL 283 6 5 66 9 26 4

528 LLDSAVQNL 33 16 4 08 5 26 3

Table 1a

Decamers predicted to bind to HLA-A*0201

Parker LPpep SYFPEITHI position peptide score rank score rank score rank

24 LLLLSVLATV 1006 1 4 55 5 24 1

88 RLGSCPIWL 20 22 3 08 2 26 3

343 LLLFTRYPDV 656 2 5 6 9 343 7

477 KMQLFLFISI 50 13 1 29 1 19 31

479 QLFLFISILA 18 24 3 86 3 15 67

486 ILAHQCDFRA 49 14 3 87 4 18 36

Tatøe 1b

Peptides were pulsed onto TAP-deficient T2 cells, and the maximum binding and the stability over time were assessed by flow cytomefry. As is shown in Fig. 10, CYP 190 and CYP528 show the longest half-life on the cell surface. Additional experiments were carried out to characterize these peptides, in particular, CYP 190. As is shown in Fig. 11, further binding studies using TAP- deficient T2 cells showed that CYP190/A2 complexes can be detected as long as 24 hours after peptide withdrawal. Moreover, as is shown in Figs. 12A-12C, CYP190-specific CTL can be generated from normal HLA-A2⁺ donors, and these CTL can lyse peptide-pulsed T2 cells (Fig. 12 A), HLA-A2⁺ myeloma cell lines (Fig. 12B), and HLA-A2^"1" primary ALL cells (Fig. 12C). In addition, as is shown in Figs. 13A and 13B, CYP190-specific CTL can be generated from HLA-A2⁺ cancer patients (Fig. 13 A, prostate cancer patient, and Fig. 13B, multiple myeloma patient), and show specific lysis.

We also identified HLA- A3 binding epitopes from CYPIBI. Using the BIMAS server, for example, we identified the peptides shown in Table 8, in which the positive confrol is a peptide derived from influenza A.

TABLE 8 Peptides predicted to bind to HLA-A3 (BIMAS server)

rank position sequence score

10mers

1 508 GLTIKPKSFK 90 2 445 FLDKDGLINK 60 3 450 GLINKDLTSR 27

9mers 1 150 SMMRNFFTR 54 2 408 SVLGYHIPK 27 positive control NP265 ILRGSVAHK 90

As is shown in Table 9, these peptides were tested in a binding assay to T2 cells transfected with HLA-A3 (NP265= positive control from influenza A). These studies showed that CYP408, CYP445, and CYP 150, which are not homologous to other cytochrome P450 isoenzymes, repeatedly bound to HLA- A3.

TABLE 9 Binding assay of peptides to T2 cells transfected with HLA- A3

In further studies, we detected CYPIBI reactive T cells in HLA-A2+ normal donors HLA-A2+ cancer patients (Fig. 14). Specific binding of teframers with CYP239 and CYP246 peptides was confirmed on T cell lines generated against the respective peptide. No binding could be detected on T cells generated against an irrelevant peptide. A tetramer containing a peptide from HTLV was used as a negative confrol.

We also devised a system for detecting CYPIBI -specific T cells by HLA-A2/peptide teframeric complexes, as is illustrated in Fig. 15. CD8⁺ T cells from normal HLA-A2⁺ myeloma patients (n=10) were isolated and analyzed with HLA-A2/peptide teframeric complexes directly ex vivo and after a 10 day in vitro restimulation period with peptide, cytokines, and irradiated PBMC. Viral peptides were used as positive (influenza A, EBV) and negative (HTLV Tax) controls. As is shown in Fig. 16, T cells from HLA-A2+ healthy donors (n=8) were stained with CYP239 and CYP246 teframers directly ex vivo and 10 days after in vitro restimulation with CYP239 or CYP246 peptides. The level of detection on day 10 is at 0.05% as determined from background staining of HLA-A2^" donors. No expansion of CYP239-specific T cells was detected in healthy donors on day 10 (mean 0.022%^±0.018%). CYP246-specific T cells were detected in 2 healthy donors with one rising to 0.5% (mean 0.032%^±0.022%). As is shown in Fig. 17, T cells from HLA-A2+ multiple myeloma patients (n=10) were stained with CYP239 and CYP246 teframers directly ex vivo and 10 days after in vitro restimulation with CYP239 or CYP246 peptides. The level of detection on day 10 is at 0.05% as determined from background staining of HLA- A2- donors. 4 patients showed T cells reactive against CYP239 >0.05% on day 10 (mean 0.068%^±0.055%), whereas 5 patients showed reactivity against CYP246 (mean 0.098%^±0.080%).

Table 10 shows the sequence of CYPIBI and the sequences of CYPIBI peptides that were identified by LPEP analysis as having binding affinity for HLA-A2.

TABLE 10 Identify HLA-A2 Binding Peptide Fragments. CYPIBI

Input Sequence MGTSLSPNDPWPLNPLSIQQTTLLLLLSVLATVHVGQRLLRQRRRQLRSAPPGPFAWPLIGNAAA VGQAAHLSFARLARRYGDVFQIRLGSCPIVVLNGERAIHQALVQQGSAFADRPAFASFRVVSGGR SMAFGHYSEHWKVQRRAAHSMMRNFFTRQPRSRQVLEGHVLSEARELVALLVRGSADGAFLDP RPLTVVAVANVMSAVCFGCRYSHDDPEFRELLSHNEEFGRTVGAGSLVDVMPWLQYFPNPVRTV FREFEQLNRNFSNFILDKFLRHCESLRPGAAPRDMMDAFILSAE AAGDSHGGGARLDLENVPA TITDIFGASQDTLSTALQWLLLLFTRYPDVQTRVQAELDQVVGRDRLPCMGDQPNLPYVLAFLYE AMRFSSFVPVTIPHATTANTSVLGYHIPKDTVVFVNQWSVNHDPLKWPNPENFDPARFLD DGLI NK_DLTSRVMIFSVGKRRCIGEELSKMQLFLFISILAHQCDFRANPNEPAKMNFSYGLTIKPKSFKVN VTLRESMELLDSAVQNLQAKETCQ Listed below are 9-resιdue peptides predicted to bind to the HLA-A2 allele with a ln(IC50) < 8 The first entry represents the location in the original sequence of the first amino acid of that peptide Following the location is the peptide, for which the predicted ln(IC50) is given as the third entry

22 TLLLLLSVL 708 23 LLLLLSVLA 771

24 LLLLSVLAT 6 05

25 LLLSVLATV 3 54 55 FAWPLIGNA 5 1 1 88 RLGSCPIVV 4 61 95 VVLNGERAI 6 58

190 FLDPRPLTV 6 52

200 AVANVMSAV 6 14

239 SLVDVMPWL 2 88

246 WLQYFPNPV 6 23 292 MMDAFILSA 3 31

312 GARLDLENV 7 87

314 RLDLENVPA 6 27

322 ATITDIFGA 6 74

334 TLSTALQWL 6 64 344 LLFTRYPDV 4 69

377 NLPYVLAFL 7 10

380 YVLAFLYEA 1 56

381 VLAFLYEAM 6 09 394 FVPVTIPHA 7 03 419 VVFVNQWSV 7 35

479 QLFLFISIL 5 66

487 LAHQCDFRA 7 54

510 TIKPKSFKV 7 60

528 LLDSAVQNL 4 08

Listed below are 10-resιdue peptides predicted to bind to the HLA-A2 allele with a ln( IC50) < 8

4 SLSPNDPWPL 526 20 QTTLLLLLSV 675 21 TTLLLLLSVL 701

22 TLLLLLSVLA 5 18

23 LLLLLSVLAT 7 36

24 LLLLSVLATV 4 55 26 LLSVLATVHV 5 86 88 RLGSCPIVVL 3 08 190 FLDPRPLTVV 7 88 199 VAVANVMSAV 7 87 234 TVGAGSLVDV 7 73 255 RTVFREFEQL 7.72

334 TLSTALQWLL 5.85

336 STALQWLLLL 5.96

343 LLLFTRYPDV 5.60 380 YVLAFLYEAM 5.54

388 AMRFSSFVPV 7.39

418 TVVFVNQWSV 6.72

477 KMQLFLFISI 1.29

479 QLFLFISILA 3.86 486 ILAHQCDFRA 3.87

494 RANPNEPAKM 7.06 502 KMNFSYGLTl 7.12

The following Experimental Methods were used to obtain some of the

Experimental Results set forth above.

Experimental Methods Donor and Patient Samples Peripheral blood from healthy blood donors and cancer patients (Table 2) was obtained by leukapheresis and peripheral blood mononuclear cells (PBMC) were purified by Ficoll-density centrifugation (Schultze et al. , J. Clin. Invest. 100:2757-2765, 1997). Primary NHL and AML samples were obtained from discarded specimens. Leukapheresis products and tumor tissue were obtained following informal consent and approval by our institute's Review Board.

Cell Lines

The melanoma cell line K029 was a kind gift of Dr. G. Dranoff (Dana- Farber Cancer Institute, Boston). The fibroblast cell line GM847 was a kind gift of Dr. W. Hahn (Whitehead Institute of Biomedical Research, Cambridge). The 36M ovarian carcinoma cell line was a kind gift of Dr. S. Cannisfra (Beth Israel Deaconess Hospital, Boston). The TAP-deficient T2 cell line; the multiple myeloma cell lines U266, IM9, and HS-Sultan; the melanoma cell line SK-MEL- 2; and the ovarian carcinoma cell line SK-OV-3 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Peptides

The peptides CYP239 (SLVDVMPWL; SEQ ID NO:l) and CYP246 (WLQYFPNPV; SEQ ID NO:2) from CYPIBI, the 1540 peptide from hTERT (ILAKFLHWL), the RT-pol476 (ILKEPVHGV) peptide from HIV, the HTLV- TAXI 1 (LLFGYPVYV), and the peptide F271 (FLWGPRALV) derived from MAGE-3 were purchased from Sigma Genosys Biotechnologies (The Woodlands, TX).

Peptide Prediction Binding of peptides to HLA molecules can be predicted for the most common HLA alleles by computational methods (Parker et al, J. Immunol. 152:163-75, 1994; Gulukota et al, J. Mol. Biol. 267:1258-67, 1997). To increase specificity of peptide prediction we used two independent algorithms: a matrix algorithm available on the BIMAS (Bioinformatics & Molecular Analysis Section at the NIH) web site (Parker et al, J. Immunol. 152:163-75, 1994) and a linear programming algorithm (LPpep) at Boston University (Z. Weng). BIMAS predicts for the half-life of peptides bound to class I molecules, while LPpep predicts an arbitrary half inhibitory concentration (IC₅₀) in competition with a labeled reference peptide. The output value is listed as ln(IC₅₀).

HLA-A *0201 binding assay

TAP-deficient T2 cells were pulsed with 40 μg/ml of peptide and 3 μg/ml of β2 -microglobulin (Sigma, St. Louis, MO) for 18 hours in serum-free IMDM (Life Technologies, Rockville, MD) at 37°C. Cells were washed three times in serum-free IMDM and HLA-A*0201 expression was measured by flow cytometry using FITC-conjugated mAb BB7.2 (ATCC). Increase of HLA-A2 expression on T2 cells reflects stabilization of MHC complexes by the addition of exogenous peptides and was quantified using the fluorescence index (FI = (MFI_peptj_{de pUlsed T2}/ MFI_unpUi_{sed T2}) - 1). Western blot analysis

CYPIBI expression was determined in microsomal cell fractions. Microsomal protein was isolated by differential speed centrifugation. Cells were harvested, washed, and resuspended in hypotonic buffer. After mechanical homogenization high-density particles were pelleted by centrifugation for 20 minutes at 15,000g. The supernatant was collected and centrifuged for 1 hour at 180,000g. The pellet was resuspended in TEDG buffer, and 100 μg of microsomal protein was separated by SDS-PAGE and transferred to nifrocellulose membrane. Western blot for CYPIBI was performed according to the manufacturer's recommendations (Gentest, Woburn, MA). Bands were visualized by enhanced chemiluminescent detection (NEN Life Science Products, Boston, MA).

Generation of CTL CTL were generated as previously described (Vonderheide et al. , Immunity

10:673-679,1999), CD8⁺ T cells (>80% CD8⁺, >95% CD3⁺, <2.0% CD4⁺, and <5% CD56⁺) were isolated from PBMC by negative selection using magnetic beads. B cells were activated via CD40, and DC were prepared from peripheral blood monocytes with IL-4 and GM-CSF (Schultze et al, J. Clin. Invest. 100:2757-2765, 1997). DC were harvested after 7 days, pulsed withpeptide (40 μg/ml) and β2-microglobulin (3 μg/ml) for 2 hr at 37°C, irradiated (33 Gy), and added to autologous CD8⁺ T cells at a T:DC ratio of 20:1 in RPMI media supplemented with 10% human AB serum, 2 mM glutamine, 15 μg/ml gentamicin, 20 mM HEPES, and 15 ng/ml IL-7 (Endogen, Woburn, MA). At day 7 and weekly thereafter, T cell cultures were harvested and restimulated with irradiated (33 Gy), peptide-pulsed (10 μg/ml) autologous CD40-activated B cells. IL-2 (50 U/ml; Chiron Corp, Emeryville, CA) was introduced on day 8 and replenished as needed every 3-4 days. Flow cytometry was performed as described (Schultze et al, J Clin. Invest. 100:2757-2765, 1997). Assessment of cytotoxic effector function and tetramer analysis were performed with CTL cultures always >90% CD37CD8⁺, <5% CD4⁺, and <5% CD56⁺. Cytotoxicity Assay

To assess cytolytic function CTL lines were used after at least four antigenic stimulations in standard ⁵¹Cr release assays as previously described (Vonderheide et al, Immunity 10:673-679, 1999). Percent specific lysis was calculated from cpm of (experimental result - spontaneous release)/(maximum release - spontaneous release) xl00%. Monocytes as targets were isolated from PBMC by RosetteSep^® (Stem Cell Technologies, Vancouver) following the manufacturer's recommendations.

Tetramer analysis

Teframeric A2/ρeptide complexes with CYP239, CYP246, and TAXI 1, an immunogenic peptide derived from HTLV-1, were synthesized essentially as described (Altaian et al, Science 274: 94-96, 1996) and conjugated to ALEXA- 488 (Molecular Probes, Eugene, OR). For staining of CTL lines, cells were incubated with the tetramer and CD8-PE (Beckman Coulter, Fullerton, CA) for 30 minutes at room temperature. Teframers were also used to sort CYP239-specific CTL. Tetramer sorted CTL were expanded by mitogen stimulation as described (Nalmori et al, Cancer Res. 59:2167-2173, 1999).

Use

Use of universal tumor associated antigens in therapeutic methods

As is discussed above, the invention provides methods for preventing or treating conditions associated with excessive cell proliferation and expression of CYPIBI, such as cancer.

Examples of conditions that can be prevented or treated using the methods of the invention, include, for example, all cancers, e.g., melanoma, lymphoma, carcinoma, sarcoma, multiple myeloma, leukemia, lung cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, liver cancer, colon cancer, pancreatic cancer, and brain cancer. Pre-cancerous and non-cancerous conditions characterized by excessive cell proliferation, and expression of a CYPIBI, can be treated using the methods of the invention as well. For example, all carcinomas in situ, e.g., ductal carcinoma in situ, lobular carcinoma in situ, and cervical carcinoma in situ, as well as adenoma and benign polyps can be treated using the methods of the invention. Patients that can be treated using the methods of the invention include those whose conditions are at early, intermediate, or advanced stages of development. Patients can receive treatment according to the invention before, during, or after other types of treatment, such as chemotherapy, radiation, or surgery, or can receive the treatment of the invention in the absence of any other type of treatment. The methods of the invention can also be used as general prophylactic measures; to prevent conditions from arising in patients that are at risk, or have early signs, of developing a condition associated with excessive cellular proliferation, such as cancer; or to prevent recurrence of such a condition. Additional persons that can be treated, in particular, using vaccination methods of the invention (see below), are those who are to donate cells, such as cytotoxic T lymphocytes, for use in the treatment of another (see below).

Central to the prophylactic and therapeutic methods of the invention is the pathway of cell-mediated immunity involving cytotoxic T lymphocytes (CTLs). In this pathway, an antigen is taken up and processed by an antigen presenting cell, so that a peptide of the antigen is presented on the surface of the cell, in the context of MHC. Such antigen presenting cells then activate cytotoxic T lymphocytes, in an MHC-restricted fashion, to proliferate and kill target cells that express the antigen.

The prophylactic and therapeutic methods of the invention intervene in this pathway at different levels. For example, in one of these methods, a CYPIBI antigen is administered to a patient, in whom the antigen is taken up by antigen presenting cells, which in turn activate CTLs. In another of these methods, an antigen presenting cell is contacted with a CYPIBI antigen ex vivo, where it takes up, processes, and presents the antigen, in the context of MHC. Such ex vivo stimulated APCs are then administered to a patient, in whom they specifically activate CTLs. In yet another of these methods, CTLs are activated ex vivo with APCs presenting CYPIBI peptides, and the activated CTLs are then administered to a patient. These methods, each of which includes numerous variations, are described in further detail below. Also, it is noted that all of these methods can be carried out with CYPIBI peptides alone or, preferably, in combination with another (or more) tumor associated antigen polypeptides or peptides (e.g., telomerase).

As is noted above, the prophylactic and therapeutic methods of the invention include one in which CYPIBI, or a fragment thereof that binds to MHC, is administered to a patient, in whom the antigen or fragment is taken up by and processed within an antigen presenting cell, which in turn activates a cytotoxic T cell in the patient. This vaccination method can be carried out using CYPIBI, one or more MHC-binding peptides of CYPIBI, and, in addition to these (or a combination thereof), one or more universal TAAs or one or more MHC-binding peptides of more than one universal TAA, or a combination thereof. Optionally, the antigen can be administered in combination with an adjuvant to enhance the anti-TAA immune response, or the antigen can be packaged into a delivery system (see below).

Any reagent including CYPIBI or a MHC-binding peptide thereof can be used for vaccination. These include, without limitation, full length CYPIBI, MHC-binding fragments of CYPIBI, as well as fusion proteins including CYPIBI and MHC-binding fragments thereof. Peptides or polypeptides including CYPIBI peptides and polypeptides can include 8, 9, 10, 11, 12, or more amino acid stretches having sequence identity with a region of CYPIBI. For example, the peptides can include nine amino acid stretches, in which seven, eight, or all nine of the amino acids in the CYPIBI peptide nine amino acid sequence are identical to a region of nine amino acids in CYPIBI. In addition, a CYPIBI peptide or polypeptide can include up to 533 amino acids that are identical to an amino acid sequence found in CYPIBI, for example, 9-20, 20-40, 40-80, 80-200, or 200-533 amino acids that are identical to an amino acid sequence found in CYPIBI. Polypeptides containing CYPIBI peptides can contain additional amino acid stretches that do not correspond to the amino acid sequence of CYPIBI.

To vaccinate a patient to elicit a CYP IBl -specific immune response in the patient, it is necessary to obtain large amounts of a CYPIBI protein or peptide, and this can be accomplished by numerous standard methods, for example, chemical synthesis (e.g., Fmoc methods (Sigma Genosys); see above) or expression in eukaryotic or prokaryotic cells.

Recombinant CYPIBI peptides can be overexpressed in vivo by introducing coding sequences of the peptides into various types of cells, or in vitro, using cell-free expression systems that are known in the art. The peptide products can then be purified for generating CYP IBl -specific CTLs ex vivo and for vaccine production. Purified CYPIBI peptides are also useful for diagnostic assays that measure the presence of CYP IBl -specific CTLs in a test sample. For example, the presence (or increased levels) of CYP IBl -specific CTLs in a sample from a subject who has received an anti-CYPlBl vaccination, relative to the level of CYP IBl -specific CTLs in a reference sample (such as a pre- vaccination sample from the patient), indicates that the patient has mounted a CYP IB 1- specific immune response.

CYPIBI peptides can be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, IL, or by other methods known to those skilled in the art of peptide synthesis).

A wide variety of expression systems can be used to produce recombinant CYPIBI peptides, polypeptides, fragments, fusion proteins, and amino acid sequence variants. CYPIBI peptides can be produced in prokaryotic hosts (e.g., E. coli) or in eukaryotic hosts (e.g., S. cerevisiae, insect cells, such as Sf9 cells, or mammalian cells, such as COS-1, NIH 3T3, or HeLa cells). These cells are commercially available from, for example, the American Type Culture Collection, Rockville, Maryland (also see, e.g., Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998). The method of fransformation and the choice of expression vehicle (e.g., expression vector) depends on the host system selected. Transformation and transfection methods are described, e.g., by Ausubel et al, supra, and expression vehicles can be chosen from the numerous examples that are known in this field.

First, a nucleic acid molecule encoding a CYPIBI peptide is introduced into a plasmid or other vector, which is then used to transform living cells. Constructs in which a cDNA containing the entire CYPIBI coding sequence, a fragment of the CYPIBI coding sequence, amino acid variations of the CYPIBI coding sequence, or fusion proteins of the aforementioned, inserted in the correct orientation into an expression plasmid, can be used for protein expression. Prokaryotic and eukaryotic expression systems allow various immunogenic domains of CYPIBI peptides or polypeptides to be recovered as fusion proteins, and then used for the generation of CYPIBI -specific CTLs. In some cases, for example, when a CYPIBI peptide is to be expressed directly within a patient's cells, it may be desirable to express the CYPIBI peptide under the confrol of an inducible or tissue-specific promoter.

Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted CYPIBI peptide-encoding nucleic acid molecule in the plasmid-bearing cells. They can also include eukaryotic or prokaryotic "origin of replication" sequences, which allow for their autonomous replication within the host organism, sequences that encode genetic traits that allow vector-containing cells to be selected in the presence of otherwise toxic drugs (such as antibiotics), and sequences that increase the efficiency with which the synthesized mRNA is translated. Stable, long-term vectors can be maintained as freely replicating entities within cells by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Barr Virus genome). Cell lines can also be produced that have the vector integrated into genomic DNA, and, in this manner, the gene product is produced on a continuous basis.

Expression of foreign sequences in bacteria such as Escherichia coli requires insertion of a nucleic acid molecule encoding a polypeptide into a bacterial expression vector. Plasmid vectors in this category contain several elements required for propagation of the plasmid in bacteria and expression of inserted DNA of the plasmid by the plasmid-carrying bacteria. Propagation of only plasmid-bearing bacteria is achieved by introducing into the plasmid selectable marker-encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs (e.g., antibiotics). The plasmid also includes a franscriptional promoter that capable of producing large amounts of mRNA from the cloned gene. Such promoters may or may not be inducible promoters. The plasmid also, preferably, contains a polylinker to simplify insertion of the gene in the correct orientation within the vector. For example, in a simple E. coli expression vector utilizing the lac promoter, the expression vector plasmid contains a fragment of the E. coli chromosome containing the lac promoter and the neighboring lacZ gene. In the presence of the lactose analog IPTG, RNA polymerase normally transcribes the lacZ gene, producing lacZ mRNA, which is translated into the encoded protein, β-galactosidase. The lacZ gene can be cut out of the expression vector with restriction endonucleases and replaced by a CYPIBI peptide gene sequence, or a fragment, fusion, or mutant thereof. When the resulting plasmid is transfected into E. coli, addition of IPTG and subsequent franscription from the lac promoter produces mRNA encoding the CYPIBI polypeptide of interest, which is then translated into a polypeptide. Once the appropriate expression vector containing a CYPIBI gene is constructed, it is introduced into an appropriate host cell by fransformation, transfection, or transduction techniques that are known in the art, including calcium chloride transformation, calcium phosphate transfection, DΕAΕ-dexfran transfection, electroporation, microinjection, protoplast fusion, and liposome- mediated transfection. The host cells that are fransformed with the vectors of this invention can include (but are not limited to) E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression), human, mouse, or other animal cells. Mammalian cells can also be used to express CYPIBI peptides using a vaccinia virus expression system, as is described by Ausubel et al, supra. In vitro expression of CYPIBI peptides, proteins, fusions, polypeptide fragments, or mutated versions thereof encoded by cloned DNA is also possible using the T7 late promoter expression system. Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages such as T3, T5, and SP6 can also be used for in vitro production of proteins from cloned DNA. E. coli can also be used for expression using an M13 phage such as mGPI-2. Furthermore, vectors that contain phage lambda regulatory sequences, or vectors that direct the expression of fusion proteins, for example, a maltose- binding protein fusion protein or a glutathione-S-fransferase fusion protein, also can be used for expression in E. coli.

Eukaryotic expression systems permit appropriate post-translational modifications to expressed proteins. Transient transfection of a eukaryotic expression plasmid allows the transient production of CYPIBI peptides by a transfected host cell. CYPIBI peptides can also be produced by a stably- transfected mammalian cell line. A number of vectors suitable for stable transfection of mammalian cells are available to the public (e.g., see Pouwels et al, Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987), as are methods for constructing such cell lines (see, e.g., Ausubel et al, supra). In one example, cDNA encoding a CYPIBI peptide, protein, fragment, mutant, or fusion protein is cloned into an expression vector that includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, integration of the CYPIBI peptide-encoding gene into the host cell chromosome is selected by inclusion of 0.01-300 μM methotrexate in the cell culture medium (as is described by Ausubel et al, supra). This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described by Ausubel et al, supra. These methods generally involve extended culture in medium containing gradually increasing levels of methotrexate. The most commonly used DHFR-containing expression vectors are pCVSEII-DHFR and pAdD26SV(A) (described by Ausubel et al, supra). The host cells described above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR- cells, ATCC Accession No. CRL 9096) are among those most preferred for DHFR selection of a stably-fransfected cell line or DHFR-mediated gene amplification. Other drug markers can be analogously used. Expression of proteins, such as those containing CYPIBI peptides, in eukaryotic cells allows the production of large amounts of normal or mutant proteins for isolation and purification, and the use of cells expressing a CYPIBI peptide-containing protein provides a functional assay system for antibodies generated against a CYPIBI peptide of interest. Another preferred eukaryotic expression system is the baculovirus system using, for example, the vector pBacPAK9, which is available from Clontech (Palo Alto, CA). If desired, this system can be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985). Once a recombinant CYPIBI protein is expressed, it can be isolated from the expressing cells by cell lysis followed by protein purification techniques, such as affinity chromatography. In this example, an anti-CYPlBl peptide antibody, which can be produced by methods that are well-known in the art, can be attached to a column and used to isolate recombinant CYPIBI peptide-containing proteins. Lysis and fractionation of CYPIBI pep tide-harboring cells prior to affinity chromatography can be performed by standard methods (see, e.g., Ausubel et al, supra). Once isolated, the recombinant protein can, if desired, be purified further, e.g., by high performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory Techniques in Biochemistry and Molecular Biology, Work and Burdon, Eds., Elsevier, 1980).

Preferably, CYPIBI or a MHC-binding peptide thereof is administered to a patient in association with an adjuvant. For example, a chemical antigen (e.g., Freund's incomplete adjuvant; cytoxan; an aluminum compound, such as aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate; liposomes; ISCOMS; microspheres; protein chochleates; vesicles consisting of nonionic surfactants; cationic amphiphilic dispersions in water; oil/water emulsions; muramidyldipeptide (MDP) and its derivatives such as glucosyl muramidyldipeptide (GMDP), threonyl-MDP, murametide and murapalmitin; and QuilA and its subfractions; as well as various other compounds such as monophosphoryl-lipid A (MPLA); gamma-inulin; calcifriol; and loxoribine) can be used.

A biological response modifier, which is a soluble mediator that affects induction of an immune response, can also be used as an adjuvant. For example, cytokines (e.g., IL-2 and GM-CSF), chemokines, co-stimulatory molecules (e.g., B7, ICAM, class I monoclonal antibodies, stem cell factor, and stimulated T cells) can be used. Also, bacterial products, such as toxins or, preferably, subunits or fragments thereof that have reduced (if any) toxicity, but maintained adjuvant activity.

Additional types of adjuvant molecules that can be used in the invention include, for example, biological modifiers of the death response (e.g., apoptosis sensitizers) and compounds or treatment that increases the susceptibility of the target cell to treatment, such as radiation and chemotherapy. Also, increasing expression of CYPIBI in the cell can increase susceptibility of the cell to treatment according to the invention.

Finally, as is described above, cellular adjuvants can be used in the immunization methods of the invention. For example, a CYPIBI peptide can be administered to a patient on the surface of an antigen presenting cell, in the context of MHC. In additional to professional antigen presenting cells, e.g., dendritic cells, CD40-activated B cells, irradiated tumor cells (e.g., in association with GM-CSF), alternative antigen presenting cells, synthetic antigen presenting cells (e.g., lipid mycels and artificial APC-like scaffolds), and fusions of any of the above-listed cells can be used.

As an alternative to vaccination with a CYPIBI protein or peptide, vaccination with a nucleic acid molecule that encodes such a protein or peptide can be used for vaccination. Such nucleic acid molecules can be administered as "naked" DNA molecules, present in a plasmid or viral vector, or packaged into a liposome or cell, such as eukaryotic cell, prior to administration. The nucleic acid molecules can be administered to a patient in vivo, or can be used to treat a cell ex vivo (e.g., an antigen presenting cell, such as a dendritic cell or a CD40-activated B cell), which is then administered to the patient. Alternatively, RNA, e.g., mRNA, can be used in these methods (see, e.g., Boczkowski et al, J. Exp. Med. 184:465-472, 1996; J. Exp. Med. 186:1177-1182, 1997).

For in vivo expression, a gene that encodes a polypeptide that includes CYPIBI or an MHC-binding peptide thereof must be delivered to cells in a form that can be taken up by the cells, in which a sufficient level of protein is expressed to induce an effective immune response. Refroviral, adenoviral, lentiviral, poxviral, and other viral vectors are suited as nucleic acid expression vectors for in vivo delivery, because they show efficient infection and/or integration and expression; see, e.g., Cayouette et al, Hum. Gene Therapy, 8:423-430, 1997; Kido et al, Curr. Eye Res. 15:833-844, 1996; Bloomer et al, J. Virol. 71:6641- 6649, 1997; Naldini et al, Science 272:263-267, 1996; Miyoshi et al, Proc. Nat. Acad. Sci., U.S.A., 94:10319-1032, 1997; Vaccines: New Approaches to

Immunological Problems, R.W. Ellis (Ed.), Butterworth-Heinemann, Boston. For example, any DNA fragment that encodes a polypeptide that contains a CYPIBI peptide can be cloned into a refroviral vector and transcribed via its endogenous promoter, via an exogenous promoter, via a promoter specific for the target cell type of interest, or, in the case of refroviral vectors, via the refroviral long terminal repeat. Other viral vectors that can be used include adenovirus, adeno- associated virus, poxviruses, such as vaccinia virus or bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus.

Gene transfer in vivo can also be achieved by non-viral means. For example, a plasmid vector that encodes a polypeptide that contains a CYPIBI peptide can be injected directly into skeletal muscle or cardiac muscle by previously described methods (e.g., Wolff et al, Science, 247:1465-1468, 1990). Expression vectors injected into skeletal muscle in situ are taken up into muscle cell nuclei and used as templates for expression of their encoded proteins. CYPIBI peptide-encoding genes that are engineered to contain a signal peptide are secreted from CYPIBI peptide-expressing muscle cells, after which they induce an immune response. Gene fransfer into cells within the tissues of a living animal also can be achieved by lipofection (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al, Neurosci. Lett. 117:259, 1990; Brigham et al, Am. J. Med. Sci. 298:278, 1989; Staubinger et al, Meth. Enz. 101:512, 1983), or asialoorosomucoid-polylysine conjugation (Wu et al, J. Biol. Chem. 263: 14621, 1988; Wu et al, J. Biol. Chem. 264: 16985, 1989), and analogous methods.

Refroviral vectors, adenoviral vectors, adenovirus-associated viral vectors, or other viral vectors also can be used to deliver genes encoding CYPIBI peptides or polypeptides to cells ex vivo. Numerous vectors useful for this purpose are generally known (see, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al, BioTechniques 6:608-614, 1988; Tolstoshev et al, Curr. Opin. Biotech. 1 :55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al, Nucl. Acid Res. and Mol. Biol. 36:311-322, 1987; Anderson, Science 226: 401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al, Biotech. 7:980-990, 1989; Le Gal La Salle et al, Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Refroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl. J. Med 323:370, 1990; Anderson et al, U.S. Patent No. 5,399,346). Gene fransfer into cells ex vivo can also be achieved by delivery of nonviral vectors, such as expression plasmids, using methods such as calcium phosphate or DEAE dextran transfection, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell.

Cells that are to be transduced or transfected ex vivo can be obtained from a patient (e.g., peripheral blood cells, such as B cells or dendritic cells, bone marrow stem cells, or cells from a tumor biopsy) prior to transfection, and re- introduced after transfection. However, the cells also can be derived from a source other than the patient undergoing gene transfer.

In the constructs described above, CYPIBI peptide expression can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in skeletal muscle cells can be used to direct CYPIBI peptide expression for vaccination in situ. The enhancers used can include, without limitation, those that are characterized as tissue- or cell- specific in their expression.

Conventional pharmaceutical practice can be employed to provide suitable formulations or compositions to administer CYPIBI peptide or nucleic acid vaccinations for treatment of, or prophylaxis against, cancer. CYPIBI peptides, CYPIBI polypeptides, and CYPIBI nucleic acid molecules can be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Administration can begin before a patient is symptomatic. Any appropriate route of administration can be employed, for example, administration can be parenteral, intravenous, infra-arterial, subcutaneous, intramuscular, infracranial, infraorbital, ophthalmic, intravenfricular, infracapsular, infraspinal, intracisternal, infraperitoneal, infranasal, aerosol, by suppositories, or oral administration.

Therapeutic formulations can be in the form of liquid solutions or suspensions; for oral administration, formulations can be in the form of tablets or capsules; and for infranasal formulations, in the form of powders, nasal drops, or aerosols. An adjuvant, e.g., as listed above, can be included with the formulation. Methods well known in the art for making formulations are found, for example, in Remington 's Pharmaceutical Sciences, (18^th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, PA. Formulations for parenteral administration can, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers can be used to confrol the release of the compounds. Other potentially useful parenteral delivery systems for CYPIBI peptides, polypeptides, and CYPIBI nucleic acid molecules include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation can contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or can be oily solutions for administration in the form of nasal drops, or as a gel.

As is mentioned above, in addition to the vaccination methods described above, which result in the activation of antigen-specific, MHC-resfricted CTLs in vivo, such cells (i.e., antigen-specific, MHC-resfricted CTLs) can be generated in vitro, and then administered to patients. Any cell that expresses an endogenous or exogenously-infroduced major histocompatibility antigen-encoding gene can be used to present a CYPIBI peptide to generate CYPIBI- specific CTLs in vitro. In one variation of this approach, a peptide-presenting cell expresses an endogenously or exogenously-infroduced CYPIBI polypeptide-encoding gene. Expression of endogenous CYPIBI in antigen-presenting cells can be stimulated as described in Schultze et al, supra, by cytokines, such as IL-2, or by other molecules that are known to those of skill in this art to stimulate CYPIBI expression.

In another variation, the antigen presenting cells are pulsed with CYPIBI or MHC-binding peptide thereof, and the pulsed cells are then used to generate CTLs for administration to a patient. Preferably, the CTLs used in these methods are obtained from the patient to whom they are to ultimately be administered (i.e., the cells are autologous). Alternatively, donor cells (i.e., allogeneic cells) can be used in this method.

Finally, methods in which any of the above-described immunotherapeutic approaches are combined are included in the invention. For example, a patient may be treated with an ex vivo, CYP IBl -activated CTL and/or an ex vivo, CYP 1 B 1 -pulsed APC (e.g. , a DC or a CD40-activated B cell), and this freatment can be carried out before, during, or after a vaccination approach (see above). In addition to combining the approaches, each approach (or a combination thereof) can employ multiple peptides of CYPIBI, peptides of other TAAs, or a combination thereof. Measurement of CYP IBl -specific CTL levels in patients, CTL donors, and CYP IBl -specific CTL preparations generated ex vivo

Patients who have one or more tumors containing CYP IBl -expressing tumor cells and patients who are at risk for developing such tumors can be vaccinated with compositions containing one or more CYPIBI peptides, CYPIBI polypeptides, CYPIBI nucleic acid molecules, cells presenting a CYPIBI peptide, or mixtures thereof (other TAA (e.g., hTERT) polypeptides, peptides, nucleic acid molecules, or APCs can also be included). Subjects to be used as donors of CYP IBl -specific CTLs for fransfer into patients can be similarly vaccinated. Levels of CYPlBl-specific CTLs that result from CYPIBI -specific vaccination of patients or other subjects, or ex vivo generation of CYPIBI specific CTLs, can be monitored using well-known methods. An increase in the level of CYP IBl -specific CTLs in a test sample from a vaccinated subject or a CTL culture stimulated with CYPIBI ex vivo, relative to a reference sample (e.g., a pre-vaccination or pre-stimulation sample), indicates that a CYPIBI -specific CTL response has been stimulated in a vaccinated subject or CYP IBl -stimulated CTL culture. Preferably the increase is by at least 50%, more preferably, at least 100%, still more preferably, at least 200%, and most preferably, at least 400%. In addition, the efficacy of non-antigen-specific immunotherapies (e.g., administration of IL-2 or interferon) against tumors containing CYP IB 1- expressing cells can be monitored using similar approaches.

Levels of CYP IBl -specific CTLs can also be assessed in naive subjects who have not received CYPIBI vaccinations or other freatment for the purpose of generating CYP IBl -specific CTLs. Since some types of tumors (e.g., malignant melanoma, renal cell carcinoma, and non-Hodgkin's lymphoma) themselves elicit immune responses in their hosts, an increase in the level of CYP IBl -specific CTLs cells in a patient sample, compared to the level in a reference sample from a normal subject who does not have a tumor, or in a reference sample that was previously obtained from the patient, can indicate the development of a tumor in a patient not known to have a tumor or an increase in tumor burden (e.g., increased tumor size, or the development or increase in metastatic tumors) in a patient known to have a tumor.

One approach by which the level of CYP IBl -specific CTLs can be measured is using standard cytotoxicity assays, such as the Cr⁵¹ release assay (Schultze et al, J. Clin. Invest. 100:2757, 1997), which is described above. Another approach for measuring the level of CYPIB 1 -specific CTLs involves measuring the binding of peptide-specific CTLs to a teframeric peptide/MHC complex in vitro, as is described by Altman et al. (Science 274:94-96, 1996). Briefly, a fusion protein containing an HLA heavy chain molecule, such as HLA-A*0201, plus a peptide that is a subsfrate for biotinylation at the C-terminus of the HLA polypeptide, is produced. The fusion protein is folded in vitro in the presence 2-microglobulin and a CYPIBI peptide ligand. The purified MHC/CYPIBI peptide complexes are then biotinylated at the C-terminus of the HLA heavy chain, and teframers are produced by mixing the biotinylated MHC/CYPIBI peptide complexes with phycoerythrin-labeled deglycosylated avidin at a molar ratio of 4: 1. Samples that contain CTLs (such as blood samples or ex vivo cultures) are mixed with the CYPIBI peptide/MHC teframeric complexes and the relative amount of CYP IBl -specific CTLs that bind to the CYPIBI peptide/MHC teframeric complexes can be measured for each sample by flow cytometry, using methods described by Altman et al, supra, and by other methods known to those of skill in this art. Another method that can be used is ELISPOT (Herr et al, J. Immunol. Methods 203: 141-152, 1997).

Other Embodiments All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and can be applied to the essential features hereinbefore set forth, and follows in the scope of the appended claims.

What is claimed is:

APPENDIX A

Search Report

Hl-A-A*0201 Nonamers HLA-A*0201 Decamers HLA-A*0201 Octamers HLA-A*0202 Nonamers HLA-A*0202 Decamers

HLA-A*0203 Nonamers

HLA-A*0203 Decamers

Hl-A-A*0203 Octamers HLA-A1 Nonamers HLA-A1 Decamers HLA-A26 Nonamers HLA-A26 Decamers

HLA-B*0702 Nonamers

Hl-A-B*0702 Decamers

HLA-B*1510 Nonamers

HLA-B*2705 Nonamers HLA-B8 Octamers HLA-B8 Nonamers

HLA-A*0201 Nonamers HLA-A1 Nonamers HLA-B'0702 Nonamers HLA-B*2705 Nonamers 123456789 Pos 123456789 Pos 123456789 Pos 123456789 LLLSVLATV 372 MGDQPNLPY 192 DPRPLTVVA 232 GRTVGAGSL TLLLLLSVL 241 VDV PWLQY 51 PPGPFAWPL 37 QRLLRQRRR LLDSAVQNL 404 TANTSVLGY 414 IPKDTVVFV 443 ARFLDKDGL QLFLFISIL 190 FLDPRPLTV 375 QPNLPYVLA 258 FREFEQLNR FLDPRPLTV 185 SADGAFLDP 288 APRDMMDAF 145 RRAAHSMMR NLPYVLAFL 174 ARELVALLV 399 IPHATTANT 79 RRYGDVFQI LLFTRYPDV 171 LSEARELVA 250 FPNPVRTVF 347 TRYPDVQTR TLSTALQWL 165 VLEGHVLSE 194 RPLTVVAVA 467 KRRCIGEEL SLVDVMPWL 7 PNDPWPLNP 512 KPKSFKVNV 265 NRNFSNFIL PLIGNAAAV 445 FLDKDGLIN 50 APPGPFAWP 116 DRPAFASFR LLLLSVLAT 341 WLLLLFTRY 320 VPATITDIF 514 KSFKVNVTL TTLLLLLSV 522 LRESMELLD 117 RPAFASFRV 469 RCIGEELSK VLSEARELV 336 STALQWLLL 89 LGSCPIVVL 182 VRGSADGAF SIQQTTLLL 218 DPEFRELLS 57 WPLIGNAAA 129 GRSMAFGHY TLRESMELL 206 SAVCFGCRY 474 ELSKMQLFL 47 LRSAPPGPF TIKPKSFKV 129 GRS AFGHY 395 VPVTIPHAT 40 LRQRRRQLR LT VAVANV 439 NFDPARFLD 183 RGSADGAFL 175 RELVALLVR RLARRYGDV 378 LPYVLAFLY 309 HGGGARLDL 144 QRRAAHSMM LLRQRRRQL 324 ITDIFGASQ 14 NPLSIQQTT 479 Q L F L F I S I L SMELLDSAV 258 FREFEQLNR 438 ENFDPARFL 472 GEELSKMQL TALQWLLLL 216 HDDPEFREL 402 ATTANTSVL 383 AFLYEAMRF WLQYFPNPV 73 SFARLARRY 173 EARELVALL 295 AFILSAEKK VGAGSLVDV 499 EPAKMNFSY 64 AAVGQAAHL 289 PRDMMDAFI SEARELVAL 292 MMDAFILSA 48 RSAPPGPFA 163 RQVLEGHVL LLLLLSVLA 137 YSEHWKVQR 514 KSFKVNVTL 43 RRRQLRSAP CIGEELSKM 81 YGDVFQIRL 467 KRRCIGEEL 509 LTIKPKSFK YVLAFLYEA 349 YPDVQTRVQ 370 PCMGDQPNL 370 PCMGDQPNL DLENVPATI 497 PNEPAKMNF 307 DSHGGGARL 307 DSHGGGARL MMDAFILSA 428 NHDPLKWPN 172 SEARELVAL 294 DAFILSAEK EARELVALL 415 PKDTVVFVN 159 QPRSRQVLE 213 RYSHDDPEF RLGSCPIVV 359 ELDQVVGRD 99 GERAIHQAL 188 GAFLDPRPL AAVGQAAHL 228 NEEFGRTVG 69 AAHLSFARL 153 RNFFTRQPR VTLRESMEL 20 QTTLLLLLS 8 NDPWPLNPL 87 IRLGSCPIV STALQWLLL 530 DSAVQNLQA 6 SPNDPWPLN 72 LSFARLARR GARLDLENV 447 DKDGLINKD 501 AKMNFSYGL 44 RRQLRSAPP YFPNPVRTV 316 DLENVPATI 499 EPAKMNFSY 36 GQRLLRQRR AVANVMSAV 215 SHDDPEFRE 496 NPNEPAKMN 22 TLLLLLSVL VLEGHVLSE 33 VHVGQRLLR 424 QWSVNHDPL 520 VTLRESMEL LGSCPIVVL 528 LLDSAVQNL 349 YPDVQTRVQ 494 RANPNEPAK PLSIQQTTL 525 SMELLDSAV 346 FTRYPDVQT 481 FLFISILAH FLFISILAH 505 FSYGLTIKP 336 STALQWLLL 468 RRCIGEELS ELSKMQLFL 456 LTSRVMIFS 331 SQDTLSTAL 461 MIFSVGKRR VVFVNQWSV 393 SFVPVTIPH 327 IFGASQDTL 460 VMIFSVGKR IPKDTVVFV 376 PNLPYVLAF 284 RPGAAPRDM 459 RVMIFSVGK VLAFLYEAM 333 DTLSTALQW 216 HDDPEFREL 451 LINKDLTSR GDQPNLPYV 331 SQDTLSTAL 189 AFLDPRPLT 446 LDKDGLINK RVQAELDQV 314 RLDLENVPA 163 RQVLEGHVL 341 WLLLLFTRY ALQWLLLLF 305 AGDSHGGGA 158 RQPRSRQVL 340 QWLLLLFTR SLRPGAAPR 299 SAEKKAAGD 115 ADRPAFASF 337 TALQWLLLL HVLSEAREL 240 LVDVMPWLQ 53 GPFAWPLIG 313 ARLDLENVP VVLNGERAI 114 FADRPAFAS 47 LRSAPPGPF 283 LRPGAAPRD QIRLGSCPI 21 TTLLLLLSV 39 LLRQRRRQL 260 EFEQLNRNF AAHLSFARL 476 SKMQLFLFI 31 ATVHVGQRL 247 LQYFPNPVR LINKDLTSR 471 IGEELSKMQ 18 IQQTTLLLL 161 RSRQVLEGH GLINKDLTS 453 NKDLTSRVM 17 SIQQTTLLL 158 RQPRSRQVL YHIPKDTVV 397 VTIPHATTA 15 PLSIQQTTL 124 RVVSGGRSM VLGYHIPKD 385 LYEAMRFSS 11 WPLNPLSIQ 89 LGSCPIVVL ATTA.NTSVL 365 GRDRLPCMG 528 LLDSAVQNL 69 AAHLSFARL LLLLFTRYP 357 QAELDQVVG 521 TLRESMELL 64 AAVGQAAHL ATITDIFGA 279 HCESLRPGA 448 KDGLINKDL 16 LSIQQTTLL ILDKFLRHC 272 ILDKFLRHC 443 ARFLDKDGL 508 GLTIKPKSF LDPRPLTVV 260 EFEQLNRNF 436 NPENFDPAR 504 NFSYGLTIK IRLGSCPIV 227 HNEEFGRTV 430 DPLKWPNPE 497 PNEPAKMNF FAWPLIGNA 221 FRELLSHNE 390 RFSSFVPVT 474 ELSKMQLFL IQQTTLLLL 91 SCPIVVLNG 377 NLPYVLAFL 448 KDGLINKDL KSFKVNVTL 90 GSCPIVVLN 337 TALQWLLLL 438 ENFDPARFL AKMNFSYGL 490 QCDFRANPN 334 TLSTALQWL 408 SVLGYHIPK VMIFSVGKR 472 GEELSKMQL 290 RDMMDAFIL 389 MRFSSFVPV TSRVMIFSV 440 FDPARFLDK 229 EEFGRTVGA 382 LAFLYEAMR QAELDQVV 436 NPENFDPAR 218 DPEFRELLS 376 PNLPYVLAF NVPATITDI 426 SVNHDPLKW 148 AHSMMRNFF 361 DQVVGRDRL LDLENVPAT 353 QTRVQAELD 19 QQTTLLLLL 358 AELDQVVGR RLDLENVPA 338 ALQWLLLLF 16 LSIQQTTLL 352 VQTRVQAEL ILSAEKKAA 289 PRDMMDAFI 9 DPWPLNPLS 327 IFGASQDTL FILSAEKKA 271 FILDKFLRH 493 FRANPNEPA 306 GDSHGGGAR RTVGAGSLV 233 RTVGAGSLV 476 SKMQLFLFI 290 RDMMDAFIL GAFLDPRPL

98 NGERAIHQA

441 DPARFLDKD

282 SLRPGAAPR 180 LLVRGSADG 48 RSAPPGPFA 434 WPNPENFDP 277 LRHCESLRP 131 SMAFGHYSE 31 ATVHVGQRL 413 HIPKDTVVF 275 KFLRHCESL 102 AIHQALVQQ 17 SIQQTTLLL 376 PNLPYVLAF 269 SNFILDKFL

96 VLNGERAIH 516 FKVNVTLRE 374 DQPNLPYVL 256 TVFREFEQL 32 TVHVGQRLL 379 PYVLAFLYE 369 LPCMGDQPN 250 FPNPVRTVF 31 ATVHVGQRL 291 DMMDAFILS 361 DQVVGRDRL 221 FRELLSHNE 29 VLATVHVGQ 282 SLRPGAAPR 352 VQTRVQAEL 219 PEFRELLSH 27 LSVLATVHV 266 RNFSNFILD 335 LSTALQWLL 208 VCFGCRYSH 16 LSIQQTTLL 250 FPNPVRTV^C 329 GASQDTLST 202 ANVMSAVCF 8 NDPWPLNPL 234 TVGAGSLVD 314 RLDLENVPA 183 RGSADGAFL 452 INKDLTSRV 160 PRSRQVLEG 297 ILSAEKKAA 174 ARELVALLV 01 HATTANTSV 156 FTRQPRSRQ 275 KFLRHCESL 169 HVLSEAREL 397 VTIPHATTA 101 RAIHQALVQ 269 SNFILDKFL 132 MAFGHYSEH 307 DSHGGGARL 12 PLNPLSIQQ 256 TVFREFEQL 106 ALVQQGSAF 275 KFLRHCESL 503 MNFSYGLTI 252 NPVRTVFRE 81 YGDVFQIRL 256 TVFREFEQL 481 FLFISILAH 244 MPWLQYFPN 80 RYGDVFQIR 199 VAVANVMSA 475 LSKMQLFLF 239 SLVDVMPWL 78 ARRYGDVFQ 179 ALLVRGSAD 408 SVLGYHIPK 235 VGAGSLVDV 31 ATVHVGQRL 79 RRYGDVFQI 403 TTANTSVLG 217 DDPEFRELL 15 PLSIQQTTL 19 QQTTLLLLL 402 ATTANTSVL 191 LDPRPLT V 531 SAVQNLQAK 4 SLSPNDPWP 392 SSFVPVTIP 188 GAFLDPRPL 506 SYGLTIKPK 86 ILAHQCDFR 363 VVGRDRLPC 125 VVSGGRSMA 485 SILAHQCDF 77 KMQLFLFIS 346 FTRYPDVQT 113 AFADRPAFA 470 CIGEELSKM 43 ARFLDKDGL 337 TALQWLLLL 81 YGDVFQIRL 458 SRVMIFSVG 11 GYHIPKDTV 318 ENVPATITD 22 TLLLLLSVL 444 RFLDKDGLI 06 NTSVLGYHI 309 HGGGARLDL 5 LSPNDPWPL 421 FVNQWSVNH 94 FVPVTIPHA 307 DSHGGGARL 520 VTLRESMEL 413 HIPKDTVVF 84 FLYEAMRFS 184 GSADGAFLD 513 PKSFKVNVT 365 GRDRLPCMG 52 VQTRVQAEL 121 ASFRVVSGG 479 QLFLFISIL 354 TRVQAELDQ 43 LLLFTRYPD 115 ADRPAFASF 472 GEELSKMQL 338 ALQWLLLLF 31 SQDTLSTAL 71 HLSFARLAR 454 KDLTSRVMI 335 LSTALQWL 29 GASQDTLST 49 SAPPGPFAW 396 PVTIPHATT 331 SQDTLSTAL 27 IFGASQDTL 24 LLLLSVLAT 391 FSSFVPVTI 271 FILDKFLRH 26 DIFGASQDT 19 QQTTLLLLL 389 MRFSSFVPV 270 NFILDKFLR 71 FILDKFLRH 18 IQQTTLLLL 378 LPYVLAFLY 268 FSNFILDKF 36 GAGSLVDVM 16 LSIQQTTLL 338 ALQWLLLLF 267 NFSNFILDK 32 GRTVGAGSL 520 VTLRESMEL 317 LENVPATIT 236 GAGSLVDVM 27 HNEEFGRTV 514 KSFKVNVTL 305 AGDSHGGGA 223 ELLSHNEEF 16 HDDPEFREL 509 LTIKPKSFK 292 MMDAFILSA 173 EARELVALL 193 PRPLTVVAV 478 MQLFLFISI 265 NRNFSNFIL 138 SEHWKVQRR

177 LVALLVRGS 474 ELSKMQLFL 249 YFPNPVRTV 126 VSGGRSMAF 176 ELVALLVRG 469 RCIGEELSK 233 RTVGAGSLV 123 FRVVSGGRS 135 GHYSEHWKV 463 FSVGKRRCI 232 GRTVGAGSL 115 ADRPAFASF 107 LVQQGSAFA 450 GLINKDLTS 213 RYSHDDPEF 112 SAFADRPAF 512 KPKSFKVNV 417 DTVVFVNQW 202 ANVMSAVCF 99 GERAIHQAL 503 MNFSYGLTI 412 YHIPKDTVV 200 AVANVMSAV 93 PIVVLNGER 502 KMNFSYGLT 406 NTSVLGYHI 193 PRPLTVVAV 75 ARLARRYGD 476 SKMQLFLFI 391 FSSFVPVTI 190 FLDPRPLTV 63 AAAVGQAAH 467 KRRCIGEEL 322 ATITDIFGA 181 LVRGSADGA 33 VHVGQRLLR 454 KDLTSRVMI 308 SHGGGARLD 174 ARELVALLV 32 TVHVGQRLL 389 MRFSSFVPV 268 FSNFILDKF 171 LSEARELVA 528 LLDSAVQNL 387 EAMRFSSFV 205 MSAVCFGCR 169 HVLSEAREL 515 SFKVNVTLR 341 WLLLLFTRY 196 LTVVAVANV 126 VSGGRSMAF 501 AKMNFSYGL 339 LQWLLLLFT 175 RELVALLVR 112 SAFADRPAF 495 ANPNEPAKM

330 ASQDTLSTA 83 DVFQIRLGS 106 ALVQQGSAF 473 EELSKMQLF 309 HGGGARLDL 66 VGQAAHLSF 92 CPIVVLNGE 437 PENFDPARF 269 SNFILDKFL 9 DPWPLNPLS 87 IRLGSCPIV 432 LKWPNPENF

181 LVRGSADGA 4 SLSPNDPWP 79 RRYGDVFQI 425 WSVNHDPLK

178 VALLVRGSA

523 RESMELLDS

77 LARRYGDVF

402 ATTANTSVL 162 SRQVLEGHV 510 TIKPKSFKV 62 NAAAVGQAA 377 NLPYVLAFL 150 SMMRNFFTR 480 LFLFISILA 58 PLIGNAAAV 374 DQPNLPYVL 125 VVSGGRSMA 458 SRVMIFSVG 56 AWPLIGNAA 372 MGDQPNLPY 106 ALVQQGSAF 446 LDKDGLINK 32 TVHVGQRLL 367 DRLPCMGDQ 81 YGDVFQIRL 425 WSVNHDPLK 24 LLLLSVLAT 309 HGGGARLDL 63 AAAVGQAAH 407 TSVLGYHIP 530 DSAVQNLQA 263 QLNRNFSNF 28 SVLATVHVG 354 TRVQAELDQ 503 MNFSYGLTI 239 SLVDVMPWL 26 LLSVLATVH 339 LQWLLLLFT 463 FSVGKRRCI 216 HDDPEFREL 527 ELLDSAVQN 335 LSTALQWLL 412 YHIPKDTVV 212 CRYSHDDPE 517 KVNVTLRES 329 GASQDTLST 411 GYHIPKDTV 197 TVVAVANVM 508 GLTIKPKSF 311 GGARLDLEN 406 NTSVLGYHI 193 PRPLTVVAV 485 SILAHQCDF 281 ESLRPGAAP 387 EAMRFSSFV 172 SEARELVAL 478 MQLFLFISI 277 LRHCESLRP 350 PDVQTRVQA 160 PRSRQVLEG 463 FSVGKRRCI 267 NFSNFILDK 339 LQWLLLLFT 157 TRQPRSRQV 455 DLTSRVMIF 255 RTVFREFEQ 315 LDLENVPAT 147 AAHSMMRNF 45 FLDKDGLIN 219 PEFRELLSH 289 PRDMMDAFI 137 YSEHWKVQR 44 RFLDKDGLI 214 YSHDDPEFR 280 CESLRPGAA 100 ERAIHQALV 13 HIPKDTVVF 161 RSRQVLEGH 264 LNRNFSNFI 51 PPGPFAWPL 91 FSSFVPVTI 126 VSGGRSMAF 253 PVRTVFREF 39 LLRQRRRQL 58 AELDQVVGR 125 VVSGGRS A 248 QYFPNPVRT 35 VGQRLLRQR 35 LSTALQWLL 88 RLGSCPIVV 170 VLSEARELV 26 LLSVLATVH 64 LNRNFSNFI 53 GPFAWPLIG 118 PAFASFRVV 19 QQTTLLLLL 24 LLSHNEEFG 50 APPGPFAWP 100 ERAIHQALV 18 IQQTTLLLL 17 DDPEFRELL 41 RQRRRQLRS 88 RLGSCPIVV 17 SIQQTTLLL 194 RPLTVVAVA 34 HVGQRLLRQ 86 QIRLGSCPI 8 NDPWPLNPL 183 RGSADGAFL 10 PWPLNPLSI 67 GQAAHLSFA 521 TLRESMELL 174 ARELVALLV 3 TSLSPNDPW 66 VGQAAHLSF 499 EPAKMNFSY 166 LEGHVLSEA 2 GTSLSPNDP 61 GNAAAVGQA 493 FRANPNEPA 164 QVLEGHVLS 515 SFKVNVTLR 27 LSVLATVHV 455 DLTSRVMIF 157 TRQPRSRQV 465 VGKRRCIGE 524 ESMELLDSA 454 KDLTSRVMI 124 RVVSGGRSM 434 WPNPENFDP 457 TSRVMIFSV 436 NPENFDPAR 120 FASFRVVSG 416 KDTVVFVNQ 452 INKDLTSRV 393 SFVPVTIPH 118 PAFASFRVV 386 YEAMRFSSF 435 PNPENFDPA 386 YEA RFSSF 05 QALVQQGSA 347 TRYPDVQTR 397 VTIPHATTA 378 LPYVLAFLY

99 GERAIHQAL 330 ASQDTLSTA 386 YEAMRFSSF 336 STALQWLLL 59 LIGNAAAVG 328 FGASQDTLS 383 AFLYEAMRF 284 RPGAAPRDM 46 QLRSAPPGP 286 GAAPRDMMD 373 GDQPNLPYV 259 REFEQLNRN 38 RLLRQRRRQ 253 PVRTVFREF 356 VQAELDQVV 253 PVRTVFREF 5 LSPNDPWPL 239 SLVDVMPWL 355 RVQAELDQV 242 DVMPWLQYF 35 NLQAKETCQ 238 GSLVDVMPW 348 RYPDVQTRV 241 VDVMPWLQY 32 AVQNLQAKE 226 SHNEEFGRT 330 ASQDTLSTA 186 ADGAFLDPR 31 SAVQNLQAK 192 DPRPLTVVA 326 DIFGASQDT 167 EGHVLSEAR 24 ESMELLDSA 189 AFLDPRPLT 322 ATITDIFGA 148 AHSMMRNFF 83 FISILAHQC 170 VLSEARELV 319 NVPATITDI 141 WKVQRRAAH 08 SVLGYHIPK 150 SMMRNFFTR 312 GARLDLENV 128 GGRSMAFGH 04 TANTSVLGY 149 HSMMRNFFT 287 AAPRDMMDA 122 SFRVVSGGR 98 TIPHATTAN 130 RSMAFGHYS 279 HCESLRPGA 96 VLNGERAIH 374 DQPNLPYVL 106 ALVQQGSAF 242 DVMPWLQYF 73 SFARLARRY 370 PCMGDQPNL 80 RYGDVFQIR 199 VAVANVMSA 68 QAAHLSFAR 364 VGRDRLPCM 55 FAWPLIGNA 196 LTVVAVANV 66 VGQAAHLSF 361 DQVVGRDRL 54 PFAWPLIGN 182 VRGSADGAF 42 QRRRQLRSA

359 ELDQVVGRD 6 SPNDPWPLN 149 HSMMRNFFT 30 LATVHVGQR 348 RYPDVQTRV 529 LDSAVQNLQ 147 AAHSMMRNF 522 LRESMELLD 346 FTRYPDVQT 524 ESMELLDSA 140 HWKVQRRAA 518 VNVTLRESM 324 ITDIFGASQ 511 IKPKSFKVN 139 EHWKVQRRA 503 MNFSYGLTI 303 KAAGDSHGG 507 YGLTIKPKS 111 GSAFADRPA 486 ILAHQCDFR 290 RDMMDAFIL 494 RANPNEPAK 107 LVQQGSAFA 478 MQLFLFISI 276 FLRHCESLR 488 AHQCDFRAN 94 IVVLNGERA 475 LSKMQLFLF 263 QLNRNFSNF

484 ISILAHQCD

76 RLARRYGDV

453 NKDLTSRVM 243 VMPWLQYFP 468 RRCIGEELS 70 AHLSFARLA 440 FDPARFLDK 223 ELLSHNEEF 460 VMIFSVGKR 42 QRRRQLRSA 405 ANTSVLGYH 189 AFLDPRPLT 457 TSRVMIFSV 23 LLLLLSVLA 404 TANTSVLGY 163 RQVLEGHVL 432 LKWPNPENF 21 TTLLLLLSV 360 LDQVVGRDR 117 RPAFASFRV 420 VFVNQWSVN 10 PWPLNPLSI 348 RYPDVQTRV 113 AFADRPAFA 409 VLGYHIPKD 525 SMELLDSAV 334 TLSTALQWL 100 ERAIHQALV 373 GDQPNLPYV 510 TIKPKSFKV 320 V P A T I T D I F 71 HLSFARLAR 371 CMGDQPNLP 502 KMNFSYGLT 319 NVPATITDI 67 GQAAHLSFA 367 DRLPCMGDQ 497 PNEPAKMNF 288 APRDMMDAF 62 NAAAVGQAA 358 AELDQVVGR 485 SILAHQCDF 276 FLRHCESLR 61 GNAAAVGQA 321 PATITDIFG 478 MQLFLFISI 251 PNPVRTVFR 57 WPLIGNAAA 298 LSAEKKAAG 473 EELSKMQLF 225 LSHNEEFGR 49 SAPPGPFAW 276 FLRHCESLR 455 DLTSRVMIF 217 DDPEFRELL

12 PLNPLSIQQ 263 QLNRNFSNF 444 RFLDKDGLI 206 SAVCFGCRY 495 ANPNEPAKM 249 YFPNPVRTV 437 PENFDPARF 162 SRQVLEGHV 487 LAHQCDFRA 248 QYFPNPVRT 432 LKWPNPENF 155 FFTRQPRSR 482 L F I S I L A H Q 245 PWLQYFPNP 401 HATTANTSV 152 MRNFFTRQP 472 GEELSKMQL 242 DVMPWLQYF 394 FVPVTIPHA 150 SMMRNFFTR 448 KDGLINKDL 237 AGSLVDVMP 380 YVLAFLYEA 134 FGHYSEHWK 38 ENFDPARFL 231 FGRTVGAGS 372 MGDQPNLPY 109 QQGSAFADR 31 PLKWPNPEN 225 LSHNEEFGR 344 LLFTRYPDV 77 LARRYGDVF 426 SVNHDPLKW 194 RPLTVVAVA 316 DLENVPATI 71 HLSFARLAR 24 QWSVNHDPL 178 VALLVRGSA 281 ESLRPGAAP 5 LSPNDPWPL 399 IPHATTANT 164 QVLEGHVLS 263 QLNRNFSNF 476 SKMQLFLFI 390 RFSSFVPVT 157 TRQPRSRQV 260 EFEQLNRNF 463 FSVGKRRCI 382 LAFLYEAMR 131 SMAFGHYSE 246 WLQYFPNPV 424 QWSVNHDPL 371 CMGDQPNLP 122 SFRVVSGGR 227 HNEEFGRTV 364 VGRDRLPCM 368 RLPCMGDQP 118 PAFASFRVV 226 SHNEEFGRT 254 VRTVFREFE 98 LSAEKKAAG 111 GSAFADRPA 223 ELLSHNEEF 222 RELLSHNEE 287 AAPRDMMDA 97 LNGERAIHQ 178 VALLVRGSA 214 YSHDDPEFR 265 NRNFSNFIL 96 VLNGERAIH 166 LEGHVLSEA 205 MSAVCFGCR 42 DVMPWLQYF 82 GDVFQIRLG 157 TRQPRSRQV 194 RPLTVVAVA 04 VMSAVCFGC 72 LSFARLARR 119 AFASFRVVS 143 VQRRAAHSM 192 DPRPLTVVA 69 AAHLSFARL 95 VVLNGERAI 95 VVLNGERAI 158 RQPRSRQVL 64 AAVGQAAHL 78 ARRYGDVFQ 86 QIRLGSCPI

94 IVVLNGERA 51 PPGPFAWPL 55 FAWPLIGNA 450 GLINKDLTS 51 PPGPFAWPL 38 RLLRQRRRQ 52 PGPFAWPLI 447 DKDGLINKD 48 RSAPPGPFA 32 TVHVGQRLL 25 LLLSVLATV 391 FSSFVPVTI 42 QRRRQLRSA 29 VLATVHVGQ 533 VQNLQAKET 381 VLAFLYEAM

13 LNPLSIQQT 28 SVLATVHVG 508 GLTIKPKSF 316 DLENVPATI 509 LTIKPKSFK 27 LSVLATVHV 487 LAHQCDFRA 301 EKKAAGDSH 505 FSYGLTIKP 5 LSPNDPWPL 480 LFLFISILA 285 PGAAPRDMM 494 RANPNEPAK 533 VQNLQAKET 475 LSKMQLFLF 168 GHVLSEARE 459 RVMIFSVGK 532 AVQNLQAKE 449 DGLINKDLT 135 GHYSEHWKV 447 DKDGLINKD 521 TLRESMELL 419 VVFVNQWSV 101 RAIHQALVQ 417 DTVVFVNQW 500 PAKMNFSYG 410 LGYHIPKDT 45 RQLRSAPPG 396 PVTIPHATT 495 ANPNEPAKM 366 RDRLPCMGD 41 RQRRRQLRS 388 AMRFSSFVP 493 FRANPNEPA 296 FILSAEKKA 10 PWPLNPLSI 347 TRYPDVQTR 486 ILAHQCDFR 285 PGAAPRDMM 505 FSYGLTIKP 340 QWLLLLFTR 464 SVGKRRCIG 268 FSNFILDKF 412 YHIPKDTVV 323 T I T D I F G A S 461 MIFSVGKRR 237 AGSLVDVMP 406 NTSVLGYHI

299 SAEKKAAGD 435 PNPENFDPA 185 SADGAFLDP 314 RLDLENVPA 294 DAFILSAE-K 431 PLKWPNPEN 162 SRQVLEGHV 266 RNFSNFILD 291 DMMDAFILS 423 NQWSVNHDP 156 FTRQPRSRQ 264 LNRNFSNFI 286 GAAPRDMMD 418 TVVFVNQWS 135 GHYSEHWKV 233 RTVGAGSLV 248 QYFPNPVRT 405 ANTSVLGYH 105 QALVQQGSA 154 NFFTRQPRS 229 EEFGRTVGA 389 MRFSSFVPV 98 NGERAIHQA 146 RAAHSMMRN 226 SHNEEFGRT 377 NLPYVLAFL 71 HLSFARLAR 38 RLLRQRRRQ 203 NVMSAVCFG

375 QPNLPYVLA

13 LNPLSIQQT

532 AVQNLQAKE 198 VVAVANVMS 362 QVVGRDRLP 523 RESMELLDS 523 RESMELLDS 197 TVVAVANVM 361 DQVVGRDRL 509 LTIKPKSFK 507 YGLTIKPKS 195 PLTVVAVAN 360 LDQVVGRDR 469 RCIGEELSK 491 CDFRANPNE

185 SADGAFLDP 352 VQTRVQAEL 445 FLDKDGLIN 466 GKRRCIGEE 151 MMRNFFTRQ 323 T I T D I F G A S 433 KWPNPENFD 419 VVFVNQWSV 132 MAFGHYSEH 306 GDSHGGGAR 392 SSFVPVTIP 397 VTIPHATTA 112 SAFADRPAF 296 FILSAEKKA 388 AMRFSSFVP 392 SSFVPVTIP

90 GSCPIVVLN 295 AFILSAEKK 363 VVGRDRLPC 390 RFSSFVPVT 72 LSFARLARR 285 PGAAPRDMM 358 AELDQVVGR 366 RDRLPCMGD 70 AHLSFARLA 257 VFREFEQLN 282 SLRPGAAPR 355 RVQAELDQV 34 HVGQRLLRQ 256 TVFREFEQL 247 LQYFPNPVR 329 GASQDTLST 30 LATVHVGQR 247 LQYFPNPVR 241 VDVMPWLQY 325 TDIFGASQD 20 QTTLLLLLS 217 DDPEFRELL 234 TVGAGSLVD 312 GARLDLENV 518 VNVTLRESM 208 VCFGCRYSH 186 ADGAFLDPR 311 GGARLDLEN 493 FRANPNEPA 207 AVCFGCRYS 165 VLEGHVL.SE 278 RHCESLRPG 461 MIFSVGKRR 204 VMSAVCFGC 160 PRSRQVLEG 248 QYFPNPVRT 446 LDKDGLINK 200 AVANVMSAV 120 FASFRVVSG 245 PWLQYFPNP 441 DPARFLDKD 198 VVAVANVMS 114 FADRPAFAS 238 GSLVDVMPW 421 FVNQWSVNH 197 TVVAVANVM 63 AAAVGQAAH 139 EHWKVQRRA 03 TTANTSVLG 195 PLTVVAVAN 43 RRRQLRSAP 121 ASFRVVSGG 392 SSFVPVTIP 180 LLVRGSADG 41 RQRRRQLRS 117 RPAFASFRV 376 PNLPYVLAF 179 ALLVRGSAD 33 VHVGQRLLR 90 GSCPIVVLN 375 QPNLPYVLA 173 EARELVALL 511 IKPKSFKVN 83 DVFQIRLGS 351 DVQTRVQAE 155 FFTRQPRSR 494 RANPNEPAK 82 GDVFQIRLG 317 LENVPATIT 151 MMRNFFTRQ 488 AHQCDFRAN 55 FAWPLIGNA

304 AAGDSHGGG 140 HWKVQRRAA 459 RVMIFSVGK 53 GPFAWPLIG 259 REFEQLNRN 138 SEHWKVQRR 440 FDPARFLDK 52 PGPFAWPLI 201 VANVMSAVC 124 RVVSGGRSM 431 PLKWPNPEN 48 RSAPPGPFA 143 VQRRAAHSM 119 AFASFRVVS 416 KDTVVFVNQ 21 TTLLLLLSV 121 ASFRVVSGG 112 SAFADRPAF 324 ITDIFGASQ 534 QNLQAKETC 115 ADRPAFASF 109 QQGSAFADR 306 GDSHGGGAR 527 ELLDSAVQN

98 NGERAIHQA 95 VVLNGERAI 303 KAAGDSHGG 526 MELLDSAVQ 93 PIVVLNGER 78 ARRYGDVFQ 267 NFSNFILDK 512 KPKSFKVNV 92 CPIVVLNGE 77 LARRYGDVF 251 PNPVRTVFR 462 IFSVGKRRC 68 QAAHLSFAR 70 AHLSFARLA 231 FGRTVGAGS 416 KDTVVFVNQ 56 AWPLIGNAA 65 AVGQAAHLS 230 EFGRTVGAG 411 GYHIPKDTV 14 NPLSIQQTT 63 AAAVGQAAH 215 SHDDPEFRE 409 VLGYHIPKD 10 PWPLNPLSI 62 NAAAVGQAA 179 ALLVRGSAD 373 GDQPNLPYV 533 VQNLQAKET 57 WPLIGNAAA 176 ELVALLVRG 359 ELDQVVGRD 507 YGLTIKPKS 40 LRQRRRQLR 175 RELVALLVR 326 DIFGASQDT 480 LFLFISILA 25 LLLSVLATV 151 MMRNFFTRQ 322 ATITDIFGA 469 RCIGEELSK 23 LLLLLSVLA 130 RSMAFGHYS 302 KKAAGDSHG 466 GKRRCIGEE 15 PLSIQQTTL 102 AIHQALVQQ 286 GAAPRDM D 395 VPVTIPHAT 531 SAVQNLQAK 101 RAIHQALVQ 255 RTVFREFEQ 313 ARLDLENVP 518 VNVTLRESM 96 VLNGERAIH 229 EEFGRTVGA 311 GGARLDLEN 513 PKSFKVNVT 90 GSCPIVVLN 184 GSADGAFLD

305 AGDSHGGGA 506 SYGLTIKPK 75 ARLARRYGD 176 ELVALLVRG 289 PRDMMDAFI 504 NFSYGLTIK 60 IGNAAAVGQ 164 QVLEGHVLS 268 FSNFILDKF 502 KMNFSYGLT 54 PFAWPLIGN 156 FTRQPRSRQ 238 GSLVDVMPW 496 NPNEPAKMN 26 LLSVLATVH 107 LVQQGSAFA 234 TVGAGSLVD 485 SILAHQCDF 7 PNDPWPLNP 102 AIHQALVQQ 208 VCFGCRYSH 473 EELSKMQLF 4 SLSPNDPWP 88 RLGSCPIVV 207 AVCFGCRYS 470 CIGEELSKM 3 TSLSPNDPW 58 PLIGNAAAV 206 SAVCFGCRY 462 IFSVGKRRC 532 AVQNLQAKE 57 WPLIGNAAA

186 ADGAFLDPR 455 DLTSRVMIF 529 LDSAVQNLQ 34 HVGQRLLRQ 175 RELVALLVR 454 KDLTSRVMI 527 ELLDSAVQN 25 LLLSVLATV 142 KVQRRAAHS 449 DGLINKDLT 516 FKVNVTLRE 24 LLLLSVLAT 138 SEHWKVQRR 444 RFLDKDGLI 505 FSYGLTIKP 23 LLLLLSVLA 119 AFASFRVVS

441 DPARFLDKD

504 NFSYGLTIK

14 NPLSIQQTT 114 FADRPAFAS 427 VNHDPLKWP 495 ANPNEPAKM 13 LNPLSIQQT 103 IHQALVQQG 422 VNQWSVNHD 492 DFRANPNEP 2 GTSLSPNDP 101 RAIHQALVQ 390 RFSSFVPVT 486 ILAHQCDFR 516 FKVNVTLRE 91 SCPIVVLNG 388 AMRFSSFVP 481 FLFISILAH 511 IKPKSFKVN 84 VFQIRLGSC 384 FLYEAMRFS 464 SVGKRRCIG 510 TIKPKSFKV 77 LARRYGDVF 382 LAFLYEAMR 462 IFSVGKRRC 487 LAHQCDFRA 65 AVGQAAHLS 381 VLAFLYEAM 456 LTSRVMIFS 482 L F I S I L A H Q 50 APPGPFAWP 380 YVLAFLYEA 447 DKDGLINKD 480 LFLFISILA 11 WPLNPLSIQ 369 LPCMGDQPN 439 NFDPARFLD 452 INKDLTSRV 530 DSAVQNLQA 368 RLPCMGDQP 428 NHDPLKWPN 430 DPLKWPNPE 513 PKSFKVNVT 356 VQAELDQVV 427 VNHDPLKWP 429 HDPLKWPNP 64 SVGKRRCIG 348 RYPDVQTRV 415 PKDTVVFVN 380 YVLAFLYEA 56 LTSRVMIFS 345 LFTRYPDVQ 408 SVLGYHIPK 368 RLPCMGDQP 27 VNHDPLKWP 343 LLLFTRYPD 404 TANTSVLGY 357 QAELDQVVG 422 VNQWSVNHD 332 QDTLSTALQ 403 TTANTSVLG 350 PDVQTRVQA 10 LGYHIPKDT 325 TDIFGASQD 398 TIPHATTAN 333 DTLSTALQW 393 SFVPVTIPH 313 ARLDLENVP 393 SFVPVTIPH 315 LDLENVPAT 386 YEAMRFSSF 310 GGGARLDLE 379 PYVLAFLYE 308 SHGGGARLD 63 VVGRDRLPC 302 KKAAGDSHG 364 VGRDRLPCM 303 KAAGDSHGG 62 QVVGRDRLP 270 NFILDKFLR 359 ELDQVVGRD 299 SAEKKAAGD 33 DTLSTALQW 269 SNFILDKFL 357 QAELDQVVG 292 MMDAFILSA 08 SHGGGARLD 261 FEQLNRNFS 318 ENVPATITD 274 DKFLRHCES 95 AFILSAEKK 259 REFEQLNRN 313 ARLDLENVP 237 AGSLVDVMP 93 MDAFILSAE 254 VRTVFREFE 311 GGARLDLEN 235 VGAGSLVDV 79 HCESLRPGA 244 MPWLQYFPN 308 SHGGGARLD 234 TVGAGSLVD 78 RHCESLRPG 236 GAGSLVDVM 304 AAGDSHGGG 228 NEEFGRTVG 67 NFSNFILDK 235 VGAGSLVDV 302 KKAAGDSHG 196 LTVVAVANV 40 LVDVMPWLQ 232 GRTVGAGSL 301 EKKAAGDSH 191 LDPRPLTVV 184 GSADGAFLD 223 ELLSHNEEF 300 AEKKAAGDS 189 AFLDPRPLT 171 LSEARELVA 210 FGCRYSHDD 298 LSAEKKAAG 187 DGAFLDPRP 156 FTRQPRSRQ 201 VANVMSAVC 283 LRPGAAPRD 180 LLVRGSADG 147 AAHSMMRNF 199 VAVANVMSA 272 ILDKFLRHC 178 VALLVRGSA 146 RAAHSMMRN 191 LDPRPLTVV 271 FILDKFLRH 165 VLEGHVLSE 111 GSAFADRPA 172 SEARELVAL 236 GAGSLVDVM 125 VVSGGRSMA

83 DVFQIRLGS 166 LEGHVLSEA 228 NEEFGRTVG 118 PAFASFRVV 75 ARLARRYGD 163 RQVLEGHVL 224 LLSHNEEFG 105 QALVQQGSA 60 IGNAAAVGQ 162 SRQVLEGHV 220 EFRELLSHN 94 IVVLNGERA 35 VGQRLLRQR 159 QPRSRQVLE 208 VCFGCRYSH 92 CPIVVLNGE 6 SPNDPWPLN 158 RQPRSRQVL 204 VMSAVCFGC 91 SCPIVVLNG 2 GTSLSPNDP 148 AHSMMRNFF 203 NVMSAVCFG 67 GQAAHLSFA 23 RESMELLDS 139 EHWKVQRRA 198 VVAVANVMS 61 GNAAAVGQA 515 SFKVNVTLR 135 GHYSEHWKV 195 PLTVVAVAN 50 APPGPFAWP 506 SYGLTIKPK 133 AFGHYSEHW 161 RSRQVLEGH 12 PLNPLSIQQ 504 NFSYGLTIK 127 SGGRSMAFG 153 RNFFTRQPR 11 WPLNPLSIQ 75 LSKMQLFLF 123 FRVVSGGRS 145 RRAAHSMMR 1 MGTSLSPND 449 DGLINKDLT 120 FASFRVVSG 144 QRRAAHSMM 529 LDSAVQNLQ 32 LKWPNPENF 108 VQQGSAFAD 141 WKVQRRAAH 525 SMELLDSAV 405 ANTSVLGYH 107 LVQQGSAFA 136 HYSEHWKVQ 524 ESMELLDSA 357 QAELDQVVG 105 QALVQQGSA 133 AFGHYSEHW 517 KVNVTLRES 320 V P A T I T D I F 104 HQALVQQGS 129 GRSMAFGHY 513 PKSFKVNVT 310 GGGARLDLE 100 ERAIHQALV 127 SGGRSMAFG 484 ISILAHQCD 302 KKAAGDSHG 92 CPIVVLNGE 121 ASFRVVSGG 477 KMQLFLFIS 283 LRPGAAPRD 89 LGSCPIVVL 110 QGSAFADRP 471 IGEELSKMQ

280 CESLRPGAA 86 QIRLGSCPI 109 QQGSAFADR 457 TSRVMIFSV 255 RTVFREFEQ 85 FQIRLGSCP 108 VQQGSAFAD 433 KWPNPENFD 222 RELLSHNEE 79 RRYGDVFQI 103 IHQALVQQG 431 PLKWPNPEN 219 PEFRELLSH 75 ARLARRYGD 91 SCPIVVLNG 428 NHDPLKWPN 161 RSRQVLEGH 74 FARLARRYG 83 DVFQIRLGS 417 DTVVFVNQW 149 HSMMRNFFT

58 PLIGNAAAV 80 RYGDVFQIR

407 TSVLGYHIP

66 JBSTITUTE SHEET (RULE 26) 78 ARRYGDVFQ 56 AWPLIGNAA 74 FARLARRYG 401 HATTANTSV 74 FARLARRYG 52 PGPFAWPLI 65 AVGQAAHLS 399 IPHATTANT 73 SFARLARRY 42 QRRRQLRSA 59 LIGNAAAVG 394 FVPVTIPHA 54 PFAWPLIGN 39 LLRQRRRQL 46 QLRSAPPGP 388 AMRFSSFVP 52 PGPFAWPLI 26 LLSVLATVH 45 RQLRSAPPG 362 QVVGRDRLP 34 QNLQAKETC 22 TLLLLLSVL 44 RRQLRSAPP 356 VQAELDQVV 26 MELLDSAVQ 535 NLQAKETCQ 36 GQRLLRQRR 346 FTRYPDVQT 16 FKVNVTLRE 527 ELLDSAVQN 34 HVGQRLLRQ 344 LLFTRYPDV 89 HQCDFRANP 526 MELLDSAVQ 29 VLATVHVGQ 339 LQWLLLLFT 84 ISILAHQCD 519 NVTLRESME 28 SVLATVHVG 330 ASQDTLSTA 62 IFSVGKRRC 508 GLTIKPKSF 535 NLQAKETCQ 318 ENVPATITD 53 NKDLTSRVM 501 AKMNFSYGL 526 MELLDSAVQ 317 LENVPATIT 78 LPYVLAFLY 491 CDFRANPNE 522 LRESMELLD 310 GGGARLDLE 69 LPCMGDQPN 489 HQCDFRANP 515 SFKVNVTLR 300 AEKKAAGDS 53 QTRVQAELD 483 FISILAHQC 506 SYGLTIKPK 298 LSAEKKAAG 49 YPDVQTRVQ 479 Q L F L F I S I L 498 NEPAKMNFS 296 FILSAEKKA 06 GDSHGGGAR 467 KRRCIGEEL 490 QCDFRANPN 281 ESLRPGAAP 88 APRDMMDAF 448 KDGLINKDL 489 HQCDFRANP 272 ILDKFLRHC 85 PGAAPRDMM 443 ARFLDKDGL 483 FISILAHQC 262 EQLNRNFSN 84 RPGAAPRDM 438 ENFDPARFL 466 GKRRCIGEE 252 NPVRTVFRE 57 VFREFEQLN 433 KWPNPENFD 458 SRVMIFSVG 243 VMPWLQYFP 52 NPVRTVFRE 421 FVNQWSVNH 453 NKDLTSRVM 226 SHNEEFGRT 50 FPNPVRTVF 419 VVFVNQWSV 451 LINKDLTSR 220 EFRELLSHN 47 LQYFPNPVR 414 IPKDTVVFV 450 GLINKDLTS 211 GCRYSHDDP 37 AGSLVDVMP 413 HIPKDTVVF 442 PARFLDKDG 199 VAVANVMSA 31 FGRTVGAGS 411 GYHIPKDTV 426 SVNHDPLKW 192 DPRPLTVVA 20 EFRELLSHN 401 HATTANTSV 421 FVNQWSVNH 181 LVRGSADGA 15 SHDDPEFRE 396 PVTIPHATT 409 VLGYHIPKD 179 ALLVRGSAD 02 ANVMSAVCF 395 VPVTIPHAT 405 ANTSVLGYH 166 LEGHVLSEA 60 PRSRQVLEG 394 FVPVTIPHA 385 LYEAMRFSS 151 M RNFFTRQ 44 QRRAAHSMM 383 AFLYEAMRF 381 VLAFLYEAM 142 KVQRRAAHS 40 HWKVQRRAA 374 DQPNLPYVL 368 RLPCMGDQP 131 SMAFGHYSE 27 SGGRSMAFG 37.0 PCMGDQPNL 362 QVVGRDRLP 130 RSMAFGHYS 26 VSGGRSMAF 366 RDRLPCMGD 354 TRVQAELDQ 111 GSAFADRPA 08 VQQGSAFAD 364 VGRDRLPCM 353 QTRVQAELD 108 VQQGSAFAD

97 LNGERAIHQ 351 DVQTRVQAE 351 DVQTRVQAE 104 HQALVQQGS

85 FQIRLGSCP 350 PDVQTRVQA 347 TRYPDVQTR 103 IHQALVQQG

82 GDVFQIRLG 344 LLFTRYPDV 343 LLLFTRYPD 98 NGERAIHQA

66 VGQAAHLSF 342 LLLLFTRYP 341 WLLLLFTRY 85 FQIRLGSCP

33 VHVGQRLLR 334 TLSTALQW. 333 DTLSTALQW 76 RLARRYGDV

9 DPWPLNPLS 326 DIFGASQDT 332 QDTLSTALQ 70 AHLSFARLA 29 LDSAVQNLQ 320 VPATITDIF 328 FGASQDTLS 65 AVGQAAHLS 19 NVTLRESME 317 LENVPATIT 323 T I T D I F G A S 56 AWPLIGNAA 96 NPNEPAKMN 315 LDLENVPAT 310 GGGARLDLE 28 SVLATVHVG 88 AHQCDFRAN 304 AAGDSHGGG 299 SAEKKAAGD 20 QTTLLLLLS 71 IGEELSKMQ 303 KAAGDSHGG 295 AFILSAEKK 9 DPWPLNPLS 35 PNPENFDPA 301 EKKAAGDSH 293 MDAFILSAE 7 PNDPWPLNP 34 WPNPENFDP 300 AEKKAAGDS 291 DMMDAFILS 3 TSLSPNDPW 33 KWPNPENFD 297 ILSAEKKAA 286 GAAPRDMMD 535 NLQAKETCQ 28 NHDPLKWPN 288 APRDMMDAF 278 RHCESLRPG 533 VQNLQAKET 18 TVVFVNQWS 287 AAPRDMMDA 277 LRHCESLRP 530 DSAVQNLQA 07 TSVLGYHIP 283 LRPGAAPRD 276 FLRHCESLR 502 KMNFSYGLT 85 LYEAMRFSS 273 LDKFLRHCE 266 RNFSNFILD 496 NPNEPAKMN 83 AFLYEAMRF 265 NRNFSNFIL 261 FEQLNRNFS 489 HQCDFRANP 67 DRLPCMGDQ 262 EQLNRNFSN 258 FREFEQLNR 483 FISILAHQC 50 PDVQTRVQA 252 NPVRTVFRE 257 VFREFEQLN 449 DGLINKDLT 32 QDTLSTALQ 246 WLQYFPNPV 254 VRTVFREFE 445 FLDKDGLIN 28 FGASQDTLS 243 VMPWLQYFP 245 PWLQYFPNP 441 DPARFLDKD 25 TDIFGASQD 230 EFGRTVGAG

238 GSLVDVMPW

434 WPNPENFDP 318 ENVPATITD 229 EEFGRTVGA 219 PEFRELLSH 426 SVNHDPLKW 281 ESLRPGAAP 224 LLSHNEEFG 211 GCRYSHDDP 422 VNQWSVNHD 277 LRHCESLRP 203 NVMSAVCFG 207 AVCFGCRYS 420 VFVNQWSVN 253 PVRTVFREF 202 ANVMSAVCF 205 MSAVCFGCR 418 TVVFVNQWS 241 VDVMPWLQY 193 PRPLTVVAV 201 VANVMSAVC 415 PKDTVVFVN 225 LSHNEEFGR 186 ADGAFLDPR 197 TVVAVANVM 414 IPKDTVVFV 214 YSHDDPEFR 183 RGSADGAFL 187 DGAFLDPRP 410 LGYHIPKDT 213 RYSHDDPEF 182 VRGSADGAF 177 LVALLVRGS 400 PHATTANTS 210 FGCRYSHDD 181 LVRGSADGA 167 EGHVLSEAR 396 PVTIPHATT 168 GHVLSEARE 177 LVALLVRGS 164 QVLEGHVLS 384 FLYEAMRFS 152 MRNFFTRQP 176 ELVALLVRG 150 SMMRNFFTR 379 PYVLAFLYE 148 AHSMMRNFF 153 RNFFTRQPR 146 RAAHSMMRN 375 QPNLPY LA 141 WKVQRRAAH 147 AAHSMMRNF 143 VQRRAAHSM 369 LPCMGDQPN 137 YSEHWKVQR 145 RRAAHSMMR 142 KVQRRAAHS 343 LLLFTRYPD 133 AFGHYSEHW 144 QRRAAHSMM 137 YSEHWKVQR 342 LLLLFTRYP 130 RSMAFGHYS 143 VQRRAAHSM 132 MAFGHYSEH 332 QDTLSTALQ 122 SFRVVSGGR 141 WKVQRRAAH 128 GGRSMAFGH 328 FGASQDTLS 104 HQALVQQGS 136 HYSEHWKVQ 124 RVVSGGRSM 324 ITDIFGASQ 53 GPFAWPLIG 134 FGHYSEHWK 122 SFRVVSGGR 297 ILSAEKKAA 47 LRSAPPGPF 132 MAFGHYSEH 116 DRPAFASFR 293 MDAFILSAE 45 RQLRSAPPG 113 AFADRPAFA 72 LSFARLARR 249 YFPNPVRTV 3 TSLSPNDPW 103 IHQALVQQG 68 QAAHLSFAR 215 SHDDPEFRE 522 LRESMELLD 102 AIHQALVQQ 49 SAPPGPFAW 207 AVCFGCRYS 498 NEPAKMNFS 94 IVVLNGERA 38 RLLRQRRRQ 201 VANVMSAVC 492 DFRANPNEP 93 PIVVLNGER 20 QTTLLLLLS 200 AVANVMSAV 491 CDFRANPNE 87 IRLGSCPIV 2 GTSLSPNDP 195 PLTVVAVAN 468 RRCIGEELS 84 VFQIRLGSC 531 SAVQNLQAK 190 FLDPRPLTV 458 SRVMIFSVG 76 RLARRYGDV 518 VNVTLRESM 185 SADGAFLDP 440 FDPARFLDK 61 GNAAAVGQA 517 KVNVTLRES 177 LVALLVRGS 439 NFDPARFLD 59 LIGNAAAVG 507 YGLTIKPKS 159 QPRSRQVLE 436 NPENFDPAR 47 LRSAPPGPF 500 PAKMNFSYG 120 FASFRVVSG 430 DPLKWPNPE 46 QLRSAPPGP 491 CDFRANPNE 119 AFASFRVVS 429 HDPLKWPNP 43 RRRQLRSAP 484 ISILAHQCD 110 QGSAFADRP 423 NQWSVNHDP 36 GQRLLRQRR 482 LFISILAHQ 97 LNGERAIHQ 416 KDTVVFVNQ 35 VGQRLLRQR 477 KMQLFLFIS 60 IGNAAAVGQ 400 PHATTANTS 30 LATVHVGQR 471 IGEELSKMQ 59 LIGNAAAVG 372 MGDQPNLPY 11 WPLNPLSIQ 470 CIGEELSKM 54 PFAWPLIGN 366 RDRLPCMGD 8 NDPWPLNPL 468 RRCIGEELS 49 SAPPGPFAW 365 GRDRLPCMG 461 MIFSVGKRR 27 LSVLATVHV 360 LDQVVGRDR HLA-A1 Decamers 460 VMIFSVGKR 4 SLSPNDPWP 345 LFTRYPDVQ 429 HDPLKWPNP 519 NVTLRESME 274 DKFLRHCES Pos 1234567890 425 WSVNHDPLK 498 NEPAKMNFS

273 LDKFLRHCE 240 LVDVMPWLQY 422 VNQWSVNHD 492 DFRANPNEP 270 NFILDKFLR 403 TTANTSVLGY 420 VFVNQWSVN 490 QCDFRANPN 261 FEQLNRNFS 439 NFDPARFLDK 417 DTVVFVNQW 464 SVGKRRCIG 230 EFGRTVGAG 371 CMGDQPNLPY 400 PHATTANTS 456 LTSRVMIFS 221 FRELLSHNE 205 MSAVCFGCRY 384 FLYEAMRFS 442 PARFLDKDG 205 MSAVCFGCR 72 LSFARLARRY 382 LAFLYEAMR 427 VNHDPLKWP 187 DGAFLDPRP 377 NLPYVLAFLY 371 CMGDQPNLP 423 NQWSVNHDP 182 VRGSADGAF 340 QWLLLLFTRY 367 DRLPCMGDQ 403 TTANTSVLG 159 QPRSRQVLE 174 ARELVALLVR 365 GRDRLPCMG 398 TIPHATTAN

155 FFTRQPRSR 128 GGRSMAFGHY 360 LDQVVGRDR 395 VPVTIPHAT 154 NFFTRQPRS 216 HDDPEFRELL 345 LFTRYPDVQ 387 EAMRFSSFV 145 RRAAHSMMR 190 FLDPRPLTVV 342 LLLLFTRYP 321 PATITDIFG

139 EHWKVQRRA 137 YSEHWKVQRR 321 PATITDIFG 304 AAGDSHGGG 128 GGRSMAFGH 522 LRESMELLDS 294 DAFILSAEK 287 AAPRDMMDA

109 QQGSAFADR 498 NEPAKMNFSY 273 LDKFLRHCE 279 HCESLRPGA 44 RRQLRSAPP 445 FLDKDGLINK 262 EQLNRNFSN 261 FEQLNRNFS 43 RRRQLRSAP 336 STALQWLLLL 259 REFEQLNRN 231 FGRTVGAGS

40 LRQRRRQLR 324 ITDIFGASQD 255 RTVFREFEQ 227 HNEEFGRTV 37 QRLLRQRRR 218 DPEFRELLSH 243 VMPWLQYFP 198 VVAVANVMS 36 GQRLLRQRR 215 SHDDPEFREL 240 LVDVMPWLQ 171 LSEARELVA

511 IKPKSFKVN 90 GSCPIVVLNG 222 RELLSHNEE 136 HYSEHWKVQ 499 EPAKMNFSY 497 PNEPAKMNFS 214 YSHDDPEFR 133 AFGHYSEHW 65 VGKRRCIGE 428 NHDPLKWPNP 212 CRYSHDDPE 127 SGGRSMAFG 25 WSVNHDPLK 331 SQDTLSTALQ 209 CFGCRYSHD 114 FADRPAFAS 20 VFVNQWSVN 233 RTVGAGSLVD 206 SAVCFGCRY 113 AFADRPAFA 15 PKDTVVFVN 171 LSEARELVAL 184 GSADGAFLD 84 VFQIRLGSC 354 TRVQAELDQ 114 FADRPAFASF 180 LLVRGSADG 29 VLATVHVGQ 300 AEKKAAGDS 81 YGDVFQIRLG 168 GHVLSEARE 6 SPNDPWPLN 266 RNFSNFILD 7 PNDPWPLNPL 154 NFFTRQPRS 500 PAKMNFSYG 62 EQLNRNFSN 528 LLDSAVQNLQ 152 MRNFFTRQP 488 AHQCDFRAN 54 VRTVFREFE 525 S MELLDSAVQ 138 SEHWKVQRR 465 VGKRRCIGE 51 PNPVRTVFR 475 LSKMQLFLFI 131 SMAFGHYSE 439 NFDPARFLD 45 PWLQYFPNP 415 PKDTVVFVNQ 123 FRVVSGGRS 435 PNPENFDPA 44 MPWLQYFPN 385 LYEAMRFSSF 104 HQALVQQGS 385 LYEAMRFSS 12 CRYSHDDPE 349 YPDVQTRVQA 97 LNGERAIHQ 371 CMGDQPNLP 136 HYSEHWKVQ 185 SADGAFLDPR 85 FQIRLGSCP 363 VVGRDRLPC 134 FGHYSEHWK 184 GSADGAFLDP 73 SFARLARRY 353 QTRVQAELD 29 GRSMAFGHY 165 VLEGHVLSEA 40 LRQRRRQLR 351 DVQTRVQAE 123 FRVVSGGRS 472 GEELSKMQLF 30 LATVHVGQR 349 YPDVQTRVQ

110 QGSAFADRP 453 NKDLTSRVMI 12 PLNPLSIQQ 345 LFTRYPDVQ 80 RYGDVFQIR 447 DKDGLINKDL 1 MGTSLSPND 323 T I T D I F G A S

41 RQRRRQLRS 425 WSVNHDPLKW 305 AGDSHGGGA

1 MGTSLSPND 359 ELDQVVGRDR HLA-B*0702 Decamers 291 DMMDAFILS 37 PENFDPARF 353 QTRVQAELDQ 280 CESLRPGAA 167 EGHVLSEAR 335 LSTALQWLLL Pos 1234567890 273 LDKFLRHCE 97 PNEPAKMNF 314 RLDLENVPAT 50 APPGPFAWPL 257 VFREFEQLN 79 PYVLAFLYE 305 AGDSHGGGAR 192 DPRPLTVVAV 246 WLQYFPNPV 01 EKKAAGDSH 299 S AEKKAAGDS 288 APRDMMDAFI 244 MPWLQYFPN 18 DPEFRELLS 272 ILDKFLRHCE 369 LPCMGDQPNL 240 LVDVMPWLQ 60 EFEQLNRNF 227 HNEEFGRTVG 14 NPLSIQQTTL 230 EFGRTVGAG 20 QTTLLLLLSV 375 QPNLPYVLAF 218 DPEFRELLS

HLA-A'0201 Decamers 16 LSIQQTTLLL 349 YPDVQTRVQA 210 FGCRYSHDD

490 QCDFRANPNE 284 RPGAAPRDMM 209 CFGCRYSHD s 1234567890 397 VTIPHATTAN 117 RPAFASFRVV 204 VMSAVCFGC

24 LLLLSVLATV 357 QAELDQVVGR 512 KPKSFKVNVT 203 NVMSAVCFG

190 FLDPRPLTVV 316 DLENVPATIT 434 WPNPENFDPA 170 VLSEARELV

88 RLGSCPIVVL 260 EFEQLNRNFS 51 PPGPFAWPLI 149 HSMMRNFFT

17 SIQQTTLLLL 258 FREFEQLNRN 9 DPWPLNPLSI 74 FARLARRYG

527 ELLDSAVQNL 221 FRELLSHNEE 57 WPLIGNAAAV 62 NAAAVGQAA

413 HIPKDTVVFV 471 IGEELSKMQL 436 NPENFDPARF 46 QLRSAPPGP

343 LLLFTRYPDV 436 NPENFDPARF 395 VPVTIPHATT

26 LLSVLATVHV 392 SSFVPVTIPH 252 NPVRTVFREF HLA-B8 Octamers

23 LLLLLSVLAT 372 MGDQPNLPYV 496 NPNEPAKMNF

4 SLSPNDPWPL 365 GRDRLPCMGD 308 SHGGGARLDL Pos 12345678

336 STALQWLLLL 322 ATITDIFGAS 250 FPNPVRTVFR 159 QPRSRQVL

456 LTSRVMIFSV 308 SHGGGARLDL 159 QPRSRQVLEG 521 TLRESMEL

326 DIFGASQDTL 292 MMDAFILSAE 88 RLGSCPIVVL 510 TIKPKSFK

195 PLTVVAVANV

289 PRDMMDAFIL 513 PKSFKVNVTL 218 DPEFRELL 279 HCESLRPGAA 499 EPAKMNFSYG 173 EARELVAL

228 N EEFGRTVGA 473 EELSKMQLFL 515 SFKVNVTL

98 NGERAIHQAL 388 AMRFSSFVPV 414 IPKDTVVF

17 SIQQTTLLLL 330 ASQDTLSTAL 276 FLRHCESL 9 DPWPLNPLSI 194 RPLTVVAVAN 473 EELSKMQL

521 TLRESMELLD 171 LSEARELVAL 475 LSKMQLFL

520 VTLRESMELL 63 AAAVGQAAHL 455 DLTSRV I 402 ATTANTSVLG 15 PLSIQQTTLL 444 RFLDKDGL 378 LPYVLAFLYE 4 SLSPNDPWPL 39 LLRQRRRQ 290 RDMMDAFILS 447 DKDGLINKDL 465 V G K R R C I G 270 NFILDKFLRH 414 IPKDTVVFVN 310 GGGARLDL 265 NRNFSNFILD 378 LPYVLAFLYE 257 VFREFEQL 255 RTVFREFEQL 376 PNLPYVLAFL 170 VLSEAREL

170 VLSEARELVA 336 STALQWLLLL 362 QVVGRDRL 49 SAPPGPFAWP 335 LSTALQWLLL 40 LRQRRRQL

32 TVHVGQRLLR 306 GDSHGGGARL 6 SPNDPWPL 31 ATVHVGQRLL 216 HDDPEFRELL 508 G L T I K P K S

515 SFKVNVTLRE 182 VRGSADGAFL 450 GLINKDLT 510 TIKPKSFKVN 172 SEARELVALL 378 LPYVLAFL 509 LTIKPKSFKV 157 TRQPRSRQVL 375 QPNLPYVL 457 TSRVMIFSVG 68 QAAHLSFARL 353 QTRVQAEL 407 TSVLGYHIPK 18 IQQTTLLLLL 23 LLLLLSVL 362 QVVGRDRLPC 16 LSIQQTTLLL 535 NLQAKETC 346 FTRYPDVQTR 7 PNDPWPLNPL 512 KPKSFKVN 338 ALQWLLLLFT 527 ELLDSAVQNL 498 NEPAKMNF 337 TALQWLLLLF 466 GKRRCIGEEL 464 SVGKRRCI 276 FLRHCESLRP 442 PARFLDKDGL 431 PLKWPNPE 217 DDPEFRELLS 430 DPLKWPNPEN 338 ALQWLLLL 173 EARELVALLV 401 HATTANTSVL 299 SAEKKAAG 70 AHLSFARLAR 399 IPHATTANTS 17 SIQQTTLL 48 RSAPPGPFAW 373 G DQPNLPYVL 9 DPWPLNPL

33 VHVGQRLLRQ 334 TLSTALQWLL 384 FLYEAMRF 23 LLLLLSVLAT 326 DIFGASQDTL 364 VGRDRLPC 19 QQTTLLLLLS 320 VPATITDIFG 271 FILDKF R

18 IQQTTLLLLL 264 LNRNFSNFIL 251 PNPVRTVF

6 SPNDPWPLNP 255 RTVFREFEQL 190 FLDPRPLT 529 LDSAVQNLQA 244 MPWLQYFPNP 179 ALLVRGSA 514 KSFKVNVTLR 231 FGRTVGAGSL 149 HSMMRNFF

502 KMNFSYGLTI 218 DPEFRELLSH 500 PAKMNFSY 479 QLFLFISILA 215 SHDDPEFREL 474 ELSKMQLF 477 KMQLFLFISI 187 DGAFLDPRPL 344 LLFTRYPD 463 FSVGKRRCIG 80 RYGDVFQIRL 337 TALQWLLL 444 RFLDKDGLIN 38 RLLRQRRRQL 282 SLRPGAAP 434 WPNPENFDPA 31 ATVHVGQRLL 113 AFADRPAF

417 DTVVFVNQWS 17 SIQQTTLLLL 76 RLARRYGD

412 YHIPKDTVVF 6 SPNDPWPLNP 41 RQRRRQLR

375 QPNLPYVLAF 529 LDSAVQNLQA 486 I L A H Q C D F 328 FGASQDTLST 471 IGEELSKMQL 479 Q L F L F I S I 307 DSHGGGARLD 441 DPARFLDKDG 445 FLDKDGLI

282 SLRPGAAPRD 437 PENFDPARFL 377 NLPYVLAF

519 NVTLRESMEL 257 VFREFEQLNR 423 NQWSVNHDPL 336 STALQWLL 494 RANPNEPAKM 225 LSHNEEFGRT 413 HIPKDTVVFV 320 VPATITDI

479 QLFLFISILA 196 LTVVAVANVM 412 YHIPKDTVVF 286 GAAPRDMM 478 MQLFLFISIL 189 AFLDPRPLTV 390 RFSSFVPVTI 120 FASFRVVS 445 FLDKDGLINK 164 QVLEGHVLSE 360 LDQVVGRDRL 96 VLNGERAI 418 TVVFVNQWSV 159 QPRSRQVLEG 351 DVQTRVQAEL 78 ARRYGDVF 410 LGYHIPKDTV 156 FTRQPRSRQV 338 ALQWLLLLFT 53 GPFAWPLI

372 MGDQPNLPYV 149 HSMMRNFFTR 314 RLDLENVPAT 429 HDPLKWPN 342 LLLLFTRYPD 100 ERAIHQALVQ 313 ARLDLENVPA 387 EAMRFSSF

329 GASQDTLSTA 96 VLNGERAIHQ 289 PRDMMDAFIL 312 GARLDLEN 308 SHGGGARLDL 82 GDVFQIRLGS 268 FSNFILDKFL 308 SHGGGARL

297 ILSAEKKAAG 65 AVGQAAHLSF 238 GSLVDVMPWL 301 EKKAAGDS

296 FILSAEKKAA 21 TTLLLLLSVL 235 VGAGSLVDVM 298 LSAEKKAA

282 SLRPGAAPRD 4 SLSPNDPWPL 181 LVRGSADGAF 288 APRDMMDA

215 SHDDPEFREL 2 GTSLSPNDPW 173 EARELVALLV 264 LNRNFSNF

164 QVLEGHVLSE 505 FSYGLTIKPK 170 VLSEARELVA 224 LLSHNEEF

156 FTRQPRSRQV 504 NFSYGLTIKP 162 SRQVLEGHVL 217 DDPEFREL

94 IVVLNGERAI 494 RANPNEPAKM 148 AHSMMRNFFT 192 DPRPLTVV

71 HLSFARLARR 480 LFLFISILAH 125 VVSGGRSMAF 164 QVLEGHVL 57 WPLIGNAAAV 473 EELSKMQLFL 98 NGERAIHQAL 140 HWKVQRRA 30 LATVHVGQRL 468 RRCIGEELSK 78 ARRYGDVFQI 138 SEHWKVQR

16 LSIQQTTLLL 464 SVGKRRCIGE 65 AVGQAAHLSF 100 ERAIHQAL 511 IKPKSFKVNV 456 LTSRVMIFSV 53 GPFAWPLIGN 74 FARLARRY 469 RCIGEELSKM 449 DGLINKDLTS 47 LRSAPPGPFA 46 QLRSAPPG 398 TIPHATTANT 406 NTSVLGYHIP 26 LLSVLATVHV 11 WPLNPLSI 386 YEAMRFSSFV 393 SFVPVTIPHA 21 TTLLLLLSVL 463 FSVGKRRC 381 VLAFLYEAMR 391 FSSFVPVTIP 520 VTLRESMELL 452 INKDLTSR

373 GDQPNLPYVL 333 DTLSTALQWL 519 NVTLRESMEL 332 QDTLSTAL 363 VVGRDRLPCM 332 Q DTLSTALQW 511 IKPKSFKVNV 328 FGASQDTL 355 RVQAELDQVV 310 GGGARLDLEN 500 PAKMNFSYGL 270 NFILDKFL 354 TRVQAELDQV 291 DMMDAFILSA 492 DFRANPNEPA 240 LVDVMPWL

330 ASQDTLSTAL 266 RNFSNFILDK 478 MQLFLFISIL 184 GSADGAFL 238 GSLVDVMPWL 214 YSHDDPEFRE 475 LSKMQLFLFI 174 ARELVALL 177 LVALLVRGSA 157 TRQPRSRQVL 474 ELSKMQLFLF 90 GSCPIVVL

173 EARELVALLV 126 VSGGRSMAFG 462 IFSVGKRRCI 86 QIRLGSCP

102 AIHQALVQQG 87 IRLGSCPIVV 453 NKDLTSRVMI 82 GDVFQIRL

96 VLNGERAIHQ 55 FAWPLIGNAA 333 DTLSTALQWL 33 VHVGQRLL

75 ARLARRYGDV 52 PGPFAWPLIG 328 FGASQDTLST 32 TVHVGQRL

55 FAWPLIGNAA 51 PPGPFAWPLI 274 DKFLRHCESL 20 QTTLLLLL

50 APPGPFAWPL 40 LRQRRRQLRS 234 TVGAGSLVDV 18 IQQTTLLL

25 LLLSVLATVH 27 LSVLATVHVG 191 LDPRPLTVVA 529 LDSAVQNL

14 NPLSIQQTTL 5 LSPNDPWPLN 190 FLDPRPLTVV 522 LRESMELL

7 PNDPWPLNPL 455 DLTSRVMIFS 189 AFLDPRPLTV 513 PKSFKVNV

528 LLDSAVQNLQ 408 SVLGYHIPKD 168 GHVLSEAREL 502 KMNFSYGL

521 TLRESMELLD 373 GDQPNLPYVL 147 AAHSMMRNFF 480 LFLFISIL

471 IGEELSKMQL 367 DRLPCMGDQP 139 EHWKVQRRAA 468 RRCIGEEL 466 GKRRCIGEEL 330 ASQDTLSTAL 111 GSAFADRPAF 449 DGLINKDL

409 VLGYHIPKDT 320 VPATITDIFG

110 QGSAFADRPA

446 LDKDGLIN 401 H ATTA TSVL 268 FSNFILDKFL 106 ALVQQGSAFA 442 PARFLDKD 393 SFVPVTIPHA 250 FPNPVRTVFR 99 GERAIHQALV 439 NFDPARFL 380 YVLAFLYEAM 248 QYFPNPVRTV 92 CPIWLNGER 425 WSVNHDPL 369 LPCMGDQPNL 239 SLVDVMPWLQ 87 IRLGSCPIVV 412 YHIPKDTV 347 TRYPDVQTRV 192 DPRPLTVVAV 56 AWPLIGNAAA 403 TTANTSVL 344 LLFTRYPDVQ 172 SEARELVALL 30 LATVHVGQRL 371 CMGDQPNL 306 GDSHGGGARL 150 SMMRNFFTRQ 23 LLLLLSVLAT 335 LSTALQWL 292 MMDAFILSAE 121 ASFRVVSGGR 11 WPLNPLSIQQ 321 PATITDIF 288 APRDMMDAFI 115 ADRPAFASFR 524 ESMELLDSAV 300 AEKKAAGD 286 GAAPRDMMDA 111 GSAFADRPAF 523 RESMELLDSA 291 DMMDAFIL

272 ILDKFLRHCE 108 VQQGSAFADR 502 KMNFSYGLTI 273 LDKFLRHC 255 RTVFREFEQL 12 PLNPLSIQQT 501 AKMNFSYGLT 266 RNFSNFIL 239 SLVDVMPWLQ 531 SAVQNLQAKE 494 RANPNEPAKM 262 EQLNRNFS 235 VGAGSLVDVM 530 DSAVQNLQAK 469 RCIGEELSKM 233 RTVGAGSL 231 FGRTVGAGSL 524 E SMELLDSAV 389 MRFSSFVPVT 229 EEFGRTVG

224 LLSHNEEFGR 506 SYGLTIKPKS 355 RVQAELDQVV 220 EFRELLSH 182 VRGSADGAFL 484 ISILAHQCDF 318 ENVPATITDI 189 AFLDPRPL

168 GHVLSEAREL 432 LKWPNPENFD 304 AAGDSHGGGA 97 LNGERAIH 162 SRQVLEGHVL 388 AMRFSSFVPV 291 DMMDAFILSA 77 LARRYGDV

131 SMAFGHYSEH 376 PNLPYVLAFL 287 AAPRDMMDAF 70 AHLSFARL

112 SAFADRPAFA 347 TRYPDVQTRV 278 RHCESLRPGA 65 AVGQAAHL

99 GERAIHQALV 318 ENVPATITDI 249 YFPNPVRTVF 52 PGPFAWPL

97 LNGERAIHQA 317 LENVPATITD 228 NEEFGRTVGA 19 QQTTLLLL

78 ARRYGDVFQI 298 LSAEKKAAGD 193 PRPLTVVAVA 16 LSIQQTTL

59 LIGNAAAVGQ 286 GAAPRDMMDA 161 RSRQVLEGHV 519 NVTLRESM

58 PLIGNAAAVG 281 ESLRPGAAPR 114 FADRPAFASF 440 FDPARFLD

9 DPWPLNPLSI 269 SNFILDKFLR 112 SAFADRPAFA 392 S S F V P V T I

517 KVNVTLRESM 249 YFPNPVRTVF 86 QIRLGSCPIV 280 CESLRPGA

500 PAKMNFSYGL 242 DVMPWLQYFP 76 RLARRYGDVF 274 DKFLRHCE

485 SILAHQCDFR 238 GSLVDVMPWL 75 ARLARRYGDV 126 VSGGRSMA

403 TTANTSVLGY 235 VGAGSLVDVM 61 GNAAAVGQAA 122 SFRVVSGG

390 RFSSFVPVTI 204 VMSAVCFGC R 60 IGNAAAVGQA 84 VFQIRLGS 360 LDQVVGRDRL 162 SRQVLEGHVL 46 QLRSAPPGPF 44 RRQLRSAP 341 WLLLLFTRYP 161 RSRQVLEGHV 41 RQRRRQLRSA 37 QRLLRQRR 335 LSTALQWLLL 130 RSMAFGHYSE 532 AVQNLQAKET 34 HVGQRLLR 323 TITDIFGASQ 117 RPAFASFRVV 517 KVNVTLRESM 527 ELLDSAVQ 316 DLENVPATIT 80 RYGDVFQIRL 486 ILAHQCDFRA 499 EPAKMNFS

304 AAGDSHGGGA 79 RRYGDVFQIR 477 KMQLFLFISI 496 NPNEPAKM

276 FLRHCESLRP 64 AAVGQAAHLS 456 LTSRVMIFSV 490 QCDFRANP 274 DKFLRHCESL 53 GPFAWPLIGN 454 KDLTSRVMIF 476 SKMQLFLF 264 LNRNFSNFIL 39 LLRQRRRQLR 452 INKDLTSRVM 438 ENFDPARF 216 HDDPEFRELL 11 WPLNPLSIQQ 451 LINKDLTSRV 409 V L G Y H I P K 185 SADGAFLDPR 3 TSLSPNDPWP 448 KDGLINKDLT 386 YEAMRFSS 176 ELVALLVRGS 501 AKMNFSYGLT 443 ARFLDKDGLI 359 ELDQVVGR 157 TRQPRSRQVL 499 EPAKMNFSYG 411 GYHIPKDTVV 351 DVQTRVQA 142 KVQRRAAHSM 487 LAHQCDFRAN 405 ANTSVLGYHI 346 FTRYPDVQ 134 FGHYSEHWKV 474 ELSKMQLFLF 400 PHATTANTSV 269 S N F I L D K F 119 AFASFRVVSG 469 RCIGEELSKM 398 TIPHATTANT 261 FEQLNRNF

101 RAIHQALVQQ 459 RVMIFSVGKR 393 SFVPVTIPHA 255 RTVFREFE 76 RLARRYGDVF 443 ARFLDKDGLI 386 YEAMRFSSFV 254 VRTVFREF 39 LLRQRRRQLR 440 FDPARFLDKD 385 LYEAMRFSSF 239 SLVDVMPW 532 AVQNLQAKET 438 ENFDPARFLD 380 YVLAFLYEAM 223 ELLSHNEE 525 SMELLDSAVQ 384 FLYEAMRFSS 374 DQPNLPYVLA 211 GCRYSHDD 524 ESMELLDSAV 381 VLAFLYEAMR 372 MGDQPNLPYV 209 CFGCRYSH

474 ELSKMQLFLF 370 PCMGDQPNLP 363 VVGRDRLPCM 176 ELVALLVR 459 RVMIFSVGKR 352 VQTRVQAELD 347 TRYPDVQTRV 171 LSEARELV 443 ARFLDKDGLI 334 TLSTALQWLL 345 LFTRYPDVQT 157 TRQPRSRQ 442 PARFLDKDGL 309 HGGGARLDLE 337 TALQWLLLLF 154 NFFTRQPR 423 NQWSVNHDPL 295 AFILSAEKKA 329 GASQDTLSTA 142 KVQRRAAH 421 FVNQWSVNHD 262' EQLNRNFSNF 319 NVPATITDI F 141 WKVQRRAA 411 GYHIPKDTVV 252 NPVRTVFREF 295 AFILSAEKKA 127 SGGRSMAF

400 PHATTANTSV 246 WLQYFPNPVR 286 GAAPRDMMDA 75 ARLARRYG 394 FVPVTIPHAT 244 MPWLQYFPNP 283 LRPGAAPRDM 72 LSFARLAR 337 TALQWLLLLF 232 GRTVGAGSLV 267 NFSNFILDKF 36 GQRLLRQR 318 ENVPATITDI 226 SHNEEFGRTV 262 EQLNRNFSNF 4 SLSPNDPW 13 ARLDLENVPA 188 GAFLDPRPLT 248 QYFPNPVRTV 504 NFSYG TI 196 LTVVAVANVM 169 HVLSEARELV 247 LQYFPNPVRT 481 FLFISILA

188 GAFLDPRPLT 134 FGHYSEHWKV 201 VANVMSAVCF 477 KMQLFLFI

150 SMMRNFFTRQ 122 SFRVVSGGRS 199 VAVANVMSAV 466 GKRRCIGE

117 RPAFASFRVV 89 LGSCPIVVLN 198 VVAVANVMSA 457 T S R V M I F S

85 FQIRLGSCPI 76 RLARRYGDVF 195 PLTVVAVANV 407 T S V L G Y H I 80 RYGDVFQIRL 73 SFARLARRYG 188 GAFLDPRPLT 399 I PH AT TAN 60 IGNAAAVGQA 69 AAHLSFARLA 177 LVALLVRGSA 395 V P V T I P H A 28 SVLATVHVGQ 68 QAAHLSFARL 165 VLEGHVLSEA 381 VLAFLYEA 31 SAVQNLQAKE 58 PLIGNAAAVG 156 FTRQPRSRQV 366 RDRLPCMG 23 RESMELLDSA 29 VLATVHVGQR 146 RAAHSMMRNF 349 YPDVQTRV 13 PKSFKVNVTL 22 TLLLLLSVLA 143 VQRRAAHSMM 342 LLLLFTRY 08 GLTIKPKSFK 15 PLSIQQTTLL 124 RVVSGGRSM A 341 WLLLLFTR 75 LSKMQLFLFI 8 NDPWPLNPLS 94 IVVLNGERAI 339 LQWLLLLF 73 EELSKMQLFL 527 E LLDSAVQNL 69 AAHLSFARLA 317 LENVPATI

462 IFSVGKRRCI 518 VNVTLRESME 66 VGQAAHLSFA 314 RLDLENVP 453 NKDLTSRVMI 512 KPKSFKVNVT 55 FAWPLIGNAA 297 I L S A E K K A 447 DKDGLINKDL 507 YGLTIKPKSF 22 TLLLLLSVLA 272 I L D K F L R H 426 SVNHDPLKWP 495 ANPNEPAKMN 20 QTTLLLLLSV 250 FPNPVRTV 405 ANTSVLGYHI 488 AHQCDFRANP 509 LTIKPKSFKV 246 WLQYFPNP 397 VTIPHATTAN 485 SILAHQCDFR 484 ISILAHQCDF 244 MPWLQYFP 389 MRFSSFVPVT 481 FLFISILAHQ 409 VLGYHIPKDT 243 VMPWLQYF 371 CMGDQPNLPY 476 SKMQLFLFIS 396 PVTIPHATTA 231 FGRTVGAG 368 RLPCMGDQPN 467 KRRCIGEELS 394 FVPVTIPHAT 195 PLTVVAVA 357 QAELDQVVGR 460 VMIFSVGKRR 343 LLLFTRYPDV 165 VLEGHVLS 346 FTRYPDVQTR 458 SRVMIFSVGK 316 DLENVPATIT 156 FTRQPRSR 328 FGASQDTLST 448 KDGLINKDLT 315 LDLENVPATI 143 VQRRAAHS 303 KAAGDSHGGG 446 LDKDGLINKD 311 GGARLDLENV 128 GGRSMAFG 268 FSNFILDKFL 426 SVNHDPLKWP 296 FILSAEKKAA 99 GERAIHQA 246 WLQYFPNPVR 424 QWSVNHDPLK 279 HCESLRPGAA 92 CPIVVLNG

245 PWLQYFPNPV

422 VNQWSVNHDP

263 QLNRNFSNFI 87 IRLGSCPI 232 GRTVGAGSLV 421 FVNQWSVNHD 259 REFEQLNRNF 71 HLSFARLA

200 AVANVMSAVC 419 VVFVNQWSVN 245 PWLQYFPNPV 67 GQAAHLSF

187 DGAFLDPRPL 414 IPKDTVVFVN 225 LSHNEEFGRT 29 VLATVHVG

161 RSRQVLEGHV 409 VLGYHIPKDT 222 RELLSHNEEF 26 LLSVLATV

98 NGERAIHQAL 405 ANTSVLGYHI 212 CRYSHDDPEF 24 LLLLSVLA

83 DVFQIRLGSC 389 MRFSSFVPVT 196 LTVVAVANVM 22 TLLLLLSV 69 AAHLSFARLA 386 YEAMRFSSFV 169 HVLSEARELV 531 SAVQNLQA

49 SAPPGPFAWP 380 YVLAFLYEAM 142 KVQRRAAHSM 528 LLDSAVQN 46 QLRSAPPGPF 374 DQPNLPYVLA 116 DRPAFASFRV 509 LTIKPKSF

41 RQRRRQLRSA 364 VGRDRLPCMG 105 QALVQQGSAF 492 DFRANPNE

512 KPKSFKVNVT 361 DQVVGRDRLP 104 HQALVQQGSA 485 SI AHQCD 483 FISILAHQCD 358 AELDQVVGRD 97 LNGERAIHQA 470 CIGEELSK 80 LFLFISILAH 356 VQAELDQVVG 85 FQIRLGSCPI 467 KRRCIGEE 70 CIGEELSKMQ 344^' LLFTRYPDVQ 54 PFAWPLIGNA 456 LTSRVMIF 61 MIFSVGKRRC 342 LLLLFTRYPD 24 LLLLSVLATV 441 DPARFLDK 60 VMIFSVGKRR 327 IFGASQDTLS 12 PLNPLSIQQT 436 NPENFDPA 46 LDKDGLINKD 313 ARLDLENVPA 507 YGLTIKPKSF 434 WPNPENFD 04 TANTSVLGYH 304 AAGDSHGGGA 479 QLFLFISILA 433 KWPNPENF 95 VPVTIPHATT 300 AEKKAAGDSH 472 GEELSKMQLF 430 DPLKWPNP 77 NLPYVLAFLY 294 DAFILSAEKK 431 PLKWPNPENF 388 AMRFSSFV 58 AELDQVVGRD 284 RPGAAPRDMM 418 TVVFVNQWSV 369 LPCMGDQP 39 LQWLLLLFTR 275 KFLRHCESLR 410 LGYHIPKDTV 368 RLPCMGDQ 22 ATITDIFGAS 267 NFSNFILDKF 382 LAFLYEAMRF 343 LLLFTRYP 21 PATITDIFGA 261 FEQLNRNFSN 379 PYVLAFLYEA 334 TLSTALQW 98 LSAEKKAAGD 256 TVFREFEQLN 362 QVVGRDRLPC 316 DLENVPAT 95 AFILSAEKKA 237 AGSLVDVMPW 354 TRVQAELDQV 290 RDMMDAFI 43 VMPWLQYFPN 229 EEFGRTVGAG 325 TDIFGASQDT 289 PRDMMDAF 23 ELLSHNEEFG 208 VCFGCRYSHD 321 PATITDIFGA 284 RPGAAPRD 04 VMSAVCFGCR 207 AVCFGCRYSH 297 ILSAEKKAAG 265 NRNFSNFI

181 LVRGSADGAF 206 SAVCFGCRYS ^•241 VDVMPWLQYF 263 QLNRNFSN

120 FASFRVVSGG 202 ANVMSAVCFG 237 AGSLVDVMPW 253 PVRTVFRE

116 DRPAFASFRV 200 AVANVMSAVC 233 RTVGAGSLVD 252 NPVRTVFR

114 FADRPAFASF 198 VVAVANVMSA 232 GRTVGAGSLV 214 YSHDDPEF

95 VVLNGERAIH 197 TVVAVANVMS 226 SHNEEFGRTV 206 SAVCFGCR

93 PIVVLNGERA 195 PLTVVAVANV 180 LLVRGSADGA 203 NVMSAVCF

66 VGQAAHLSFA 193 PRPLTVVAVA 138 SEHWKVQRRA 201 VANVMSAV

65 AVGQAAHLSF 183 RGSADGAFLD 134 FGHYSEHWKV 194 RPLTVVAV

64 AAVGQAAHLS 182 VRGSADGAFL 123 FRVVSGGRSM 188 GAFLDPRP

62 NAAAVGQAAH

179 ALLVRGSADG 119 AFASFRVVSG 185 SADGAFLD AWPLIGNAAA 178 VALLVRGSAD 115 ADRPAFASFR 183 RGSADGAF TIKPKSFKVN 176 ELVALLVRGS 93 PIVVLNGERA 181 LVRGSADG DLTSRVMIFS 163 RQVLEGHVLS 70 AHLSFARLAR 180 LLVRGSAD YHIPKDTVVF 158 RQPRSRQVLE 13 LNPLSIQQTT 178 VALLVRGS PYVLAFLYEA 155 FFTRQPRSRQ 439 NFDPARFLDK 161 RSRQVLEG YPDVQTRVQA 148 AHSMMRNFFT 402 ATTANTSVLG 151 MMRNFFTR LRPGAAPRDM 147 AAHSMMRNFF 229 EEFGRTVGAG 148 AHSMMRNF RHCESLRPGA 143 VQRRAAHSMM 200 AVANVMSAVC 144 QRRAAHSM LQYFPNPVRT 138 SEHWKVQRRA 186 ADGAFLDPRP 117 RPAFASFR AVCFGCRYSH 131 SMAFGHYSEH 174 ARELVALLVR 116 DRPAFASF VANVMSAVCF 127 SGGRSMAFGH 126 VSGGRSMAFG 115 ADRPAFAS LDPRPLTVVA 125 VVSGGRSMAF 118 PAFASFRVVS 112 SAFADRPA VALLVRGSAD 124 RVVSGGRSMA 100 ERAIHQALVQ 107 LVQQGSAF RELVALLVRG 123 FRVVSGGRSM 89 LGSCPIVVLN 106 ALVQQGSA MMRNFFTRQP 120 FASFRVVSGG 77 LARRYGDVFQ 88 RLGSCPIV VVSGGRSMAF 119 AFASFRVVSG 43 RRRQLRSAPP 80 RYGDVFQI RVVSGGRSMA 118 PAFASFRVVS 521 TLRESMELLD 58 PLIGNAAA HQALVQQGSA 112 SAFADRPAFA 514 KSFKVNVTLR 57 WPLIGNAA SCPIVVLNGE 106 ALVQQGSAFA 488 AHQCDFRANP 51 PPGPFAWP GSCPIVVLNG 102 AIHQALVQQG 457 TSRVMIFSVG 50 APPGPFAW LGSCPIVVLN 99 GERAIHQALV 415 PKDTVVFVNQ 49 SAPPGPFA LARRYGDVFQ 95 VVLNGERAIH 403 TTANTSVLGY 48 RSAPPGPF GNAAAVGQAA 91 SCPIVVLNGE 397 VTIPHATTAN 43 RRRQLRSA PPGPFAWPLI 88 RLGSCPIVVL 391 FSSFVPVTIP 42 QRRRQLRS HVGQRLLRQR 84 VFQIRLGSCP 371 CMGDQPNLPY 38 RLLRQRRR VHVGQRLLRQ 78 ARRYGDVFQI 356 VQAELDQVVG 25 LLLSVLAT LNPLSIQQTT 74 FARLARRYGD 353 QTRVQAELDQ 15 PLSIQQTT LDSAVQNLQA 56 AWPLIGNAAA 327 IFGASQDTLS 14 NPLSIQQT FKVNVTLRES 50 APPGPFAWPL 322 ATITDIFGAS 12 PLNPLSIQ FSYGLTIKPK 38 RLLRQRRRQL 310 GGGARLDLEN 483 FISILAHQ AKMNFSYGLT 35 VGQRLLRQRR 305 AGDSHGGGAR 413 HIPKDTVV LAHQCDFRAN 28 SVLATVHVGQ 303 KAAGDSHGGG 401 HATTANTS SKMQLFLFIS 25 LLLSVLATVH 282 SLRPGAAPRD 382 LAFLYEAM SVGKRRCIGE 533 VQNLQAKETC 280 CESLRPGAAP 357 QAELDQVV KDLTSRVMIF 532 AVQNLQAKET 276 FLRHCESLRP 329 GASQDTLS FDPARFLDKD 517 KVNVTLRESM 272 ILDKFLRHCE 296 FILSAEKK PENFDPARFL 516 FKVNVTLRES 257 VFREFEQLNR 236 GAGSLVDV WPNPENFDPA 513 PKSFKVNVTL 253 PVRTVFREFE 199 VAVANVMS ATTANTSVLG 508 GLTIKPKSFK 213 RYSHDDPEFR 114 FADRPAFA LAFLYEAMRF 493 FRANPNEPAK 204 VMSAVCFGCR 105 QALVQQGS QPNLPYVLAF 492 DFRANPNEPA 202 ANVMSAVCFG 93 PIVVLNGE QVVGRDRLPC 486 ILAHQCDFRA 185 SADGAFLDPR 69 AAHLSFAR ELDQVVGRDR 483 FISILAHQCD 184 GSADGAFLDP 63 AAAVGQAA VQAELDQVVG 470 CIGEELSKMQ 183 RGSADGAFLD 55 FAWPLIGN LFTRYPDVQT

466 GKRRCIGEEL 136 HYSEHWKVQR 30 LATVHVGQ

289 PRDMMDAFIL 465 VGKRRCIGEE 113 AFADRPAFAS 524 ESMELLDS

287 AAPRDMMDAF 462 IFSVGKRRCI 108 VQQGSAFADR 494 RANPNEPA 242 DVMPWLQYFP 454 KDLTSRVMIF 90 GSCPIVVLNG 487 LAHQCDFR 240 LVDVMPWLQY 452 INKDLTSRVM 79 RRYGDVFQIR 461 MIFSVGKR

236 GAGSLVDVMP 450 GLINKDLTSR 64 AAVGQAAHLS 451 LINKDLTS

233 RTVGAGSLVD 433 KWPNPENFDP 62 NAAAVGQAAH 404 TANTSVLG 193 PRPLTVVAVA 431 PLKWPNPENF 58 PLIGNAAAVG 398 TIPHATTA 159 QPRSRQVLEG 430 DPLKWPNPEN 48 RSAPPGPFAW 326 DIFGASQD 147 AAHSMMRNFF 427 VNHDPLKWPN 42 QRRRQLRSAP 323 T I T D I F G A

132 MAFGHYSEHW 420 VFVNQWSVNH 33 VHVGQRLLRQ 304 AAGDSHGG

123 FRV VSGGRSM 416 KDTVVFVNQW 515 SFKVNVTLRE 303 KAAGDSHG

107 LVQ QGSAFAD 413 HIPKDTVVFV 510 TIKPKSFKVN 294 DAFILSAE

105 QAL VQQGSAF 411 GYHIPKDTVV 505 FSYGLTIKPK 287 AAPRDMMD

74 FAR LARRYGD 401 H ATTA TSVL 504 NFSYGLTIKP 226 SHNEEFGR

54 PFA WPLIGNA 400 PHATTANTSV 493 FRANPNEPAK 147 AAHSMMRN

53 GPF AWPLIGN 395 VPVTIPHATT 480 LFLFISILAH 146 RAAHSMMR

47 LRS APPGPFA 394 FVPVTIPHAT 468 RRCIGEELSK 132 MAFGHYSE

27 LSV LATVHVG 387 EAMRFSSFVP 467 KRRCIGEELS 118 PAFASFRV

530 DSA VQNLQAK 383 AFLYEAMRFS 463 FSVGKRRCIG 102 AIHQALVQ

522 LRE SMELLDS 368 RLPCMGDQPN 459 RVMIFSVGKR 101 RAIHQALV

514 KSF KVNVTLR 363 VVGRDRLPCM 444 RFLDKDGLIN 68 QAAHLSFA

465 V G K RRCIGEE 360 LDQVVGRDRL 428 NHDPLKWPNP 64 AAVGQAAH

452 INKDLTSRVM 355 RVQAELDQVV 424 QWSVNHDPLK 62 NAAAVGQA 432 LKWPNPENFD 350 PDVQTRVQAE 408 SVLGYHIPKD 59 LIGNAAAV 431 PLKWPNPENF 348 RYPDVQTRVQ 387 EAMRFSSFVP 525 SMELLDSA

419 VVFVNQWSVN 343 LLLFTRYPDV 383 AFLYEAMRFS 506 SYGLTIKP 414 IPKDTVVFVN 341 WLLLLFTRYP 370 PCMGDQPNLP 471 I GE ELS KM 396 PVTIPHATTA 326 DIFGASQDTL 366 RDRLPCMGDQ 356 VQAELDQV

324 ITDIFGASQD 323 TITDIFGASQ 358 AELDQVVGRD 227 HNEEFGRT

312 GARLDLENVP 312 GARLDLENVP 357 QAELDQVVGR 167 EGHVLSEA

309 HGGGARLDLE 306 GDSHGGGARL 348 RYPDVQTRVQ 162 SRQVLEGH

294 DAFILSAEKK 303 KAAGDSHGGG 346 FTRYPDVQTR 136 HYSEHWKV 277 LRHCESLRPG 297 ILSAEKKAAG 331 SQDTLSTALQ 533 VQNLQAKE 267 NFSNFILDKF 296 FILSAEKKAA 312 GARLDLENVP 484 ISILAHQC 266 RNFSNFILDK 288 APRDMMDAFI 309 HGGGARLDLE 472 GEELSKMQ

250 FPNPVRTVFR 287 AAPRDMMDAF 302 KKAAGDSHGG 462 IFSVGKRR

237 AGSLVDVMPW 285 PGAAPRDMMD 300 AEKKAAGDSH 458 SRVMIFSV

225 LSHNEEFGRT 283 LRPGAAPRDM 292 MMDAFILSAE 435 PNPENFDP

206 SAVCFGCRYS 280 CESLRPGAAP 290 RDMMDAFILS 426 SVNHDPLK

203 NVMSAVCFGC 278 RHCESLRPGA 281 ESLRPGAAPR 419 VVFVNQWS

202 ANVMSAVCFG 271 FILDKFLRHC 270 NFILDKFLRH 408 SVLGYHIP

197 TVVAVANVMS 264 LNRNFSNFIL 266 RNFSNFILDK 394 FVPVTIPH

174 ARELVALLVR 263 QLNRNFSNFI 260 EFEQLNRNFS 393 SFVP TIP

148 AHSMMRNFFT 259 REFEQLNRNF

242 DVMPWLQYFP 331 SQDTLSTA 146 RAAHSMMRNF 254 VRTVFREFEQ 240 LVDVMPWLQY 318 ENVPATIT

138 SEHWKVQRRA 253 PVRTVFREFE 236 GAGSLVDVMP 315 LDLENVPA

113 AFADRPAFAS 251 PNPVRTVFRE 230 EFGRTVGAGS 281 ESLRPGAA

81 YGDVFQIRLG 243 VMPWLQYFPN 227 HNEEFGRTVG 278 RHCESLRP

70 AHLSFARLAR 241 VDVMPWLQYF 220 EFRELLSHNE 260 EFEQLNRN 507 YGLTIKPKSF 236 GAGSLVDVMP 211 GCRYSHDDPE 259 REFEQLNR

506 SYGLTIKPKS 231 FGRTVGAGSL 207 AVCFGCRYSH 238 GSLVDVMP

503 MNFSYGLTIK 230 EFGRTVGAGS 197 TVVAVANVMS 232 GRTVGAGS

493 FRANPNEPAK 224 LLSHNEEFGR 179 ALLVRGSADG 230 EFGRTVGA 82 LFISILAHQC 223 ELLSHNEEFG 176 ELVALLVRGS 221 FRELLSHN 458 SRVMIFSVGK 222 RELLSHNEEF 175 RELVALLVRG 215 SHDDPEFR 48 KDGLINKDLT 210 FGCRYSHDDP 164 QVLEGHVLSE 198 VVAVANVM 16 KDTVVFVNQW 201 VANVMSAVCF 163 RQVLEGHVLS 172 SEARELVA 06 NTSVLGYHIP 199 VAVANVMSAV 158 RQPRSRQVLE 168 GHVLSEAR 99 IPHATTANTS 191 LDPRPLTVVA 151 MMRNFFTRQP 150 SMMRNFFT 74 DQPNLPYVLA 186 ADGAFLDPRP 150 SMMRNFFTRQ 139 EHWKVQRR 53 QTRVQAELDQ 181 LVRGSADGAF 145 RRAAHS MRN 134 FGHYSEHW 25 TDIFGASQDT 180 LLVRGSADGA 144 QRRAAHSMMR 131 SMAFGHYS 17 LENVPATITD 177 LVALLVRGSA 140 HWKVQRRAAH 123 FRVVSGGR 02 KKAAGDSHGG 152 MRNFFTRQPR 133 AFGHYSEHWK 104 HQALVQQG 99 SAEKKAAGDS 144 QRRAAHSMMR 130 RSMAFGHYSE 94 IVVLNGER 93 MDAFILSAEK 140 HWKVQRRAAH 128 GGRSMAFGHY 91 SCPIVVLN

256 TVFREFEQLN 139 EHWKVQRRAA 120 FASFRVVSGG 73 SFARLARR

241 VDVMPWLQYF 135 GHYSEHWKVQ 102 AIHQALVQQG 28 SVLATVHV 229 EEFGRTVGAG 133 AFGHYSEHWK 101 RAIHQALVQQ 2 GTSLSPND

228 NEEFGRTVGA 132 MAFGHYSEHW 82 GDVFQIRLGS 530 DSAVQNLQ 194 RPLTVVAVAN 129 GRSMAFGHYS 74 FARLARRYGD 526 MELLDSAV 184 GSADGAFLDP 113 AFADRPAFAS 71 HLSFARLARR 520 VTLRESME

143 VQRRAAHSMM 105 QALVQQGSAF 67 GQAAHLSFAR 518 VNVTLRES

137 YSEHWKVQRR 93 PIVVLNGERA 59 LIGNAAAVGQ 517 KVNVTLRE

126 VSGGRSMAFG 92 CPIWLNGER 49 SAPPGPFAWP 516 FKVNVTLR

108 VQQGSAFADR 85 FQIRLGSCPI 44 RRQLRSAPPG 511 IKPKSFKV

48 RSAPPGPFAW 75 ARLARRYGDV 39 LLRQRRRQLR 505 FSYGLTIK

32 TVHVGQRLLR 71 HLSFARLARR 32 TVHVGQRLLR 493 FRANPNEP

6 SPNDPWPLNP 66 VGQAAHLSFA 28 SVLATVHVGQ 489 HQCDFRAN

2 GTSLSPNDPW 63 AAAVGQAAHL 2 GTSLSPNDPW 460 V M I F S V G K

533 VQNLQAKETC 61 GNAAAVGQAA 530 DSAVQNLQAK 453 NKDLTSRV 526 MELLDSAVQ N

60 IGNAAAVGQA 526 MELLDSAVQN 448 KDGLINKD 491 CDFRANPNEP 47 LRSAPPGPFA 525 SMELLDSAVQ 443 ARFLDKDG 488 AHQCDFRANP 46 QLRSAPPGPF 522 LRESMELLDS 427 VNHDPLKW 484 ISILAHQCDF 42 QRRRQLRSAP 506 SYGLTIKPKS 424 QWSVNHDP 449 DGLINKDLTS 37 QRLLRQRRRQ 497 PNEPAKMNFS 422 VNQWSVNH 444 RFLDKDGLIN 30 LATVHVGQRL 495 ANPNEPAKMN 421 FVNQWSVN 430 DPLKWPNPEN 26 LLSVLATVHV 490 QCDFRANPNE 420 VFVNQWSV

391 FSSFVPVTIP 24 LLLLSVLATV 487 LAHQCDFRAN 417 DTWFVNQ 378 LPYVLAFLYE 485 SILAHQCDFR 416 KDTWFVN 331 SQDTLSTALQ HLA-A26 Nonamers 483 FISILAHQCD 411 GYHIPKDT

319 NVPATITDIF 464 SVGKRRCIGE 406 NTSVLGYH 310 GGGARLDLEN Pos 123456789 458 SRVMIFSVGK 397 VTIPHATT 284 RPGAAPRDMM 242 DVMPWLQYF 449 DGLINKDLTS 391 FSSFVPVT 279 HCESLRPGAA 455 DLTSRVMIF 445 FLDKDGLINK 383 AFLYEAMR 258 FREFEQLNRN 470 CIGEELSKM 438 ENFDPARFLD 379 PYVLAFLY 257 VFREFEQLNR 223 ELLSHNEEF 435 PNPENFDPAR 373 GDQPNLPY 222 RELLSHNEEF 260 EFEQLNRNF 432 LKWPNPENFD 365 GRDRLPCM

218 DPEFRELLSH 256 TVFREFEQL 425 WSVNHDPLKW 363 VVGRDRLP

212 CRYSHDDPEF 253 PVRTVFREF 420 VFVNQWSVNH 361 DQWGRDR

208 VCFGCRYSHD 528 LLDSAVQNL 416 KDTVVFVNQW 360 LDQWGRD

136 HYSEHWKVQR 341 WLLLLFTRY 406 NTSVLGYHIP 352 VQTRVQAE

130 RSMAFGHYSE 263 QLNRNFSNF 392 SSFVPVTIPH 340 QWLLLLFT

121 ASFRVVSGGR 521 TLRESMELL 381 VLAFLYEAMR 327 IFGASQDT

118 PAFASFRVVS 479 Q L F L F I S I L 368 RLPCMGDQPN 324 I T D I F G A S

115 ADRPAFASFR 474 ELSKMQLFL 364 VGRDRLPCMG 311 GGARLDLE

111 GSAFADRPAF 473 EELSKMQLF 359 ELDQVVGRDR 309 HGGGARLD

110 QGSAFADRPA 413 HIPKDTVVF 350 PDVQTRVQAE 306 GDSHGGGA

92 CPIWLNGER 381 VLAFLYEAM 342 LLLLFTRYPD 295 A F I L S A E K

73 SFARLARRYG 338 ALQWLLLLF 340 QWLLLLFTRY 279 HCESLRPG

67 GQAAHLSFAR 377 NLPYVLAFL 332 QDTLSTALQW 268 FSNFILDK

45 RQLRSAPPGP 351 DVQTRVQAE 324 ITDIFGASQD 258 FREFEQLN

19 QQTTLLLLLS 239 SLVDVMPWL 307 DSHGGGARLD 241 VDVMPWLQ

496 NPNEPAKMNF 173 EARELVALL 301 EKKAAGDSHG 237 AGSLVDVM

492 DFRANPNEPA 106 ALVQQGSAF 298 LSAEKKAAGD 235 VGAGSLVD 468 RRCIGEELSK 22 TLLLLLSVL 293 MDAFILSAEK 222 RELLSHNE

463 FSVGKRRCIG 417 DTVVFVNQW 285 PGAAPRDMMD 216 HDDPEFRE 439 NFDPARFLDK 359 ELDQVVGRD 246 WLQYFPNPVR 210 FGCRYSHD 428 NHDPLKWPNP 334 TLSTALQWL 224 LLSHNEEFGR 208 VCFGCRYS 425 WSVNHDPLKW 326 DIFGASQDT 223 ELLSHNEEFG 205 MSAVCFGC 420 VFVNQWSVNH 220 EFRELLSHN 217 DDPEFRELLS 204 VMSAVCFG 417 DTWFVNQWS 176 ELVALLVRG 208 VCFGCRYSHD 196 LTWAVAN

392 SSFVPVTIPH 520 VTLRESMEL 167 EGHVLSEARE 193 PRPLTVVA 385 LYEAMRFSSF 508 GLTIKPKSF 166 LEGHVLSEAR 182 VRGSADGA 383 AFLYEAMRFS 485 SILAHQCDF 160 PRSRQVLEGH 177 LVALLVRG 367 DRLPCMGDQP 336 STALQWLLL 153 RNFFTRQPRS 175 RELVALLV

320 VPATITDIFG 169 HVLSEAREL 152 MRNFFTRQPR 169 HVLSEARE 305 AGDSHGGGAR 124 RVVSGGRSM 149 HSMMRNFFTR 163 RQVLEGHV

300 AEKKAAGDSH 83 DVFQIRLGS 132 MAFGHYSEHW 155 FFTRQPRS 280 CESLRPGAAP 31 ATVHVGQRL 129 GRSMAFGHYS 135 GHYSEHWK 275 KFLRHCESLR 499 EPAKMNFSY

122 SFRVVSGGRS 129 GRSMAFGH 270 NFILDKFLRH 402 ATTANTSVL 121 ASFRVVSGGR 125 VVSGGRSM

269 SNFILDKFLR 73 SFARLARRY 103 IHQALVQQGS 111 GSAFADRP

261 FEQLNRNFSN 17 SIQQTTLLL 96 VLNGERAIHQ 108 VQQGSAFA

259 REFEQLNRNF 527 ELLDSAVQN 95 VVLNGERAIH 103 IHQALVQQ

221 FRELLSHNEE 383 AFLYEAMRF 73 SFARLARRYG 95 VVLNGERA

214 YSHDDPEFRE 333 DTLSTALQW 52 PGPFAWPLIG 85 FQIRLGSC

186 ADGAFLDPRP 307 DSHGGGARL 45 RQLRSAPPGP 81 YGDVFQIR

166 LEGHVLSEAR 197 TVVAVANVM 40 LRQRRRQLRS 66 VGQAAHLS

154 NFFTRQPRSR 115 ADRPAFASF 36 GQRLLRQRRR 61 GNAAAVGQ

149 HSMMRNFFTR 39 LLRQRRRQL 29 VLATVHVGQR 60 IGNAAAVG

139 EHWKVQRRAA 32 TVHVGQRLL 27 LSVLATVHVG 56 AWPLIGNA 133 AFGHYSEHWK 438 ENFDPARFL 25 LLLSVLATVH 35 VGQRLLRQ 127 SGGRS AFGH 394 FVPVTIPHA 19 QQTTLLLLLS 27 LSVLATVH 103 IHQALVQQGS 386 YEAMRFSSF 534 QNLQAKETCQ 21 TTLLLLLS

79 RRYGDVFQIR 376 PNLPYVLAF 531 SAVQNLQAKE 13 LNPLSIQQ

72 LSFARLARRY 322 ATITDIFGA 528 LLDSAVQNLQ 10 PWPLNPLS

40 LRQRRRQLRS 271 FILDKFLRH 508 GLTIKPKSFK

5 LSPNDPWPLN 230 EFGRTVGAG 503 MNFSYGLTIK HI-A-B8 Nonamers

3 TSLSPNDPWP 217 DDPEFRELL 498 NEPAKMNFSY 34 QNLQAKETCQ 200 AVANVMSAV 489 HQCDFRANPN Pos 123456789 15 SFKVNVTLRE 177 LVALLVRGS 481 FLFISILAHQ 39 LLRQRRRQL 98 NEPAKMNFSY 102 AIHQALVQQ 476 SKMQLFLFIS 173 EARELVALL 89 HQCDFRANPN 15 PLSIQQTTL 470 CIGEELSKMQ 521 TLRESMELL 27 VNHDPLKWPN 481 FLFISILAH 455 DLTSRVMIFS 510 TIKPKSFKV 22 VNQWSVNHDP 475 LSKMQLFLF 450 GLINKDLTSR 508 GLTIKPKSF 87 EAMRFSSFVP 451 LINKDLTSR 440 FDPARFLDKD 455 DLTSRVMIF 65 GRDRLPCMGD 404 TANTSVLGY 433 KWPNPENFDP 159 QPRSRQVLE 64 VGRDRLPCMG 374 DQPNLPYVL 429 HDPLKWPNPE 465 VGKRRCIGE 50 PDVQTRVQAE 323 T I T D I F G A S 427 VNHDPLKWPN 414 IPKDTVVFV 40 QWLLLLFTRY 288 APRDMMDAF 421 FVNQWSVNHD 288 APRDMMDAF 27 IFGASQDTLS 216 HDDPEFREL 419 VVFVNQWSVN 479 Q L F L F I S I L 73 LDKFLRHCES 196 LTVVAVANV 407 TSVLGYHIPK 474 ELSKMQLFL 54 VRTVFREFEQ 147 AAHSMMRNF 384 FLYEAMRFSS 473 EELSKMQLF 52 NPVRTVFREF 34 HVGQRLLRQ 367 DRLPCMGDQP 431 PLKWPNPEN 49 YFPNPVRTVF 21 TTLLLLLSV 365 GRDRLPCMGD 239 SLVDVMPWL 44 MPWLQYFPNP 517 KVNVTLRES 344 LLFTRYPDVQ 99 GERAIHQAL 19 PE FRELLSHN 510 TIKPKSFKV 341 WLLLLFTRYP 528 LLDSAVQNL 17 DDPEFRELLS 482 LFISILAHQ 323 TITDIFGASQ 512 KPKSFKVNV 05 MSAVCFGCRY 447 DKDGLINKD 317 LENVPATITD 467 KRRCIGEEL

163 RQVLEGHVLS 380 YVLAFLYEA 299 SAEKKAAGDS 463 FSVGKRRCI 160 PRSRQVLEGH 361 DQVVGRDRL 277 LRHCESLRPG 450 GLI KDLTS 158 RQPRSRQVLE 355 RVQAELDQV 275 KFLRHCESLR 377 NLPYVLAFL 145 RRAAHSMMRN 344 LLFTRYPDV 265 NRNFSNFILD 86 QIRLGSCPI

141 WKVQRRAAHS 327 IFGASQDTL 251 PNPVRTVFRE 22 TLLLLLSVL

140 HWKVQRRAAH 319 NVPATITDI 243 VMPWLQYFPN 535 NLQAKETCQ 129 GRSMAFGHYS 316 DLENVPATI 209 CFGCRYSHDD 475 LSKMQLFLF

122 SFRVVSGGRS 275 KFLRHCESL 205 MSAVCFGCRY 444 RFLDKDGLI

82 GDVFQIRLGS

272 ILDKFLRHC

203 NVMSAVCFGC 334 TLSTALQWL RRRQLRSAPP 268 FSNFILDKF 178 VALLVRGSAD 299 SAEKKAAGD QRLLRQRRRQ 203 NVMSAVCFG 155 FFTRQPRSRQ 223 ELLSHNEEF WPLNPLSIQQ 172 SEARELVAL 141 WKVQRRAAHS 188 GAFLDPRPL VNVTLRESME 165 VLEGHVLS E 137 YSEHWKVQRR 179 ALLVRGSAD NFSYGLTIKP 129 GRSMAFGHY 135 GHYSEHWKVQ 126 VSGGRSMAF ANPNEPAKMN 69 AAHLSFARL 131 SMAFGHYSEH 77 LARRYGDVF KRRCIGEELS 20 QTTLLLLLS 127 SGGRSMAFGH 51 PPGPFAWPL NPENFDPARF 524 ESMELLDSA 109 QQGSAFADRP 17 SIQQTTLLL PNPENFDPAR 509 LTIKPKSFK 107 LVQQGSAFAD 15 PLSIQQTTL KWPNPENFDP 497 PNEPAKMNF 81 YGDVFQIRLG 364 VGRDRLPCM HDPLKWPNPE 461 MIFSVGKRR 72 LSFARLARRY 271 FILDKFLRH QWSVNHDPLK 456 LTSRVMIFS 37 QRLLRQRRRQ 190 FLDPRPLTV PCMGDQPNLP 409 VLGYHIPKD 35 VGQRLLRQRR 120 FASFRVVSG RYPDVQTRVQ 397 VTIPHATTA 34 HVGQRLLRQR 69 AAHLSFARL DSHGGGARLD 384 FLYEAMRFS 8 NDPWPLNPLS 515 SFKVNVTLR RDMMDAFILS 364 VGRDRLPCM 5 LSPNDPWPLN 500 PAKMNFSYG ESLRPGAAPR 236 GAGSLVDV 3 TSLSPNDPWP 386 YEAMRFSSF PVRTVFREFE 181 LVRGSADGA 344 LLFTRYPDV PNPVRTVFRE 126 VSGGRSMAF HLA-B*1510 Nonamers 337 TALQWLLLL HNEEFGRTVG 125 VVSGGRSMA 282 SLRPGAAPR FGCRYSHDDP 112 SAFADRPAF Pos 123456789 218 DPEFRELLS CFGCRYSHDD 58 PLIGNAAAV 148 AHSMMRNFF 172 SEARELVAL RNFFTRQPRS 28 SVLATVHVG 89 LGSCPIVVL 76 RLARRYGDV GHYSEHWKVQ 25 LLLSVLATV 139 EHWKVQRRA 64 AAVGQAAHL GGRSMAFGHY 24 LLLLSVLAT 438 ENFDPARFL 41 RQRRRQLRS VFQIRLGSCP 532 AVQNLQAKE 412 YHIPKDTVV 448 KDGLINKDL RRQLRSAPPG 492 DFRANPNEP 361 DQVVGRDRL 336 STALQWLLL QRRRQLRSAP 432 LKWPNPENF 216 HDDPEFREL 331 SQDTLSTAL GQRLLRQRRR 421 FVNQWSVNH 32 TVHVGQRLL 320 V PATITDIF VGQRLLRQRR 419 VVFVNQWSV 514 KSFKVNVTL 298 LSAEKKAAG PWPLNPLSIQ 337 TALQWLLLL 308 SHGGGARLD 286 GAAPRDMMD NDPWPLNPLS 324 ITDIFGASQ 307 DSHGGGARL 276 FLRHCESLR EPAKMNFSYG 233 RTVGAGSLV 215 SHDDPEFRE 269 SNFILDKFL PNEPAKMNFS 198 VVAVANVMS 188 GAFLDPRPL 253 PVRTVFREF ENFDPARFLD 164 QVLEGHVLS 172 SEARELVAL 250 FPNPVRTVF PKDTVVFVNQ 156 FTRQPRSRQ 81 YGDVFQIRL 192 DPRPLTVVA DQVVGRDRLP 89 LGSCPIVVL 39 LLRQRRRQL 140 HWKVQRRAA PGAAPRDMMD 76 RLARRYGDV 521 TLRESMELL 138 SEHWKVQRR EQLNRNFSNF 64 AAVGQAAHL 488 AHQCDFRAN 112 SAFADRPAF EGHVLSEARE 19 QQTTLLLLL 474 ELSKMQLFL 106 ALVQQGSAF EFEQLNRNFS 8 NDPWPLNPL 472 GEELSKMQL 520 VTLRESMEL PGPFAWPLIG 519 NVTLRESME 428 NHDPLKWPN 514 KSFKVNVTL EKKAAGDSHG 514 KSFKVNVTL 424 QWSVNHDPL 485 SILAHQCDF

483 FISILAHQC 402 ATTANTSVL 472 GEELSKMQL

HLA-A*0201 Octamers 459 RVMIFSVGK 374 DQPNLPYVL 438 ENFDPARFL 441 DPARFLDKD 327 IFGASQDTL 429 HDPLKWPNP 12345678 437 PENFDPARF 278 RHCESLRPG 413 HIPKDTVVF SAVQNLQA 408 SVLGYHIPK 239 SLVDVMPWL 352 VQTRVQAEL SMELLDSA 406 NTSVLGYHI 232 GRTVGAGSL 338 ALQWLLLLF RANPNEPA 403 TTANTSVLG 226 SHNEEFGRT 316 DLENVPATI 488 AHQCDFRA 398 TIPHATTAN 173 EARELVALL 312 GARLDLENV 481 F L F I S I L A 390 RFSSFVPVT 169 HVLSEAREL 301 EKKAAGDSH 436 NPENFDPA 372 MGDQPNLPY 168 GHVLSEARE 264 LNRNFSNFI 398 TIPHATTA 370 PCMGDQPNL 158 RQPRSRQVL 263 QLNRNFSNF 395 V P V T I P H A 367 DRLPCMGDQ 103 IHQALVQQG 232 GRTVGAGSL 381 VLAFLYEA 362 QVVGRDRLP 22 TLLLLLSVL 217 DDPEFRELL 376 PNLPYVLA 346 FTRYPDVQT 400 PHATTANTS 216 HDDPEFREL 351 DVQTRVQA 296 FILSAEKKA 370 PCMGDQPNL 169 HVLSEAREL 331 SQDTLSTA 250 FPNPVRTVF 337 TALQWLLLL 115 ADRPAFASF 323 T I T D I F G A 229 EEFGRTVGA 334 TLSTALQWL 89 LGSCPIVVL 315 LDLENVPA 213 RYSHDDPEF 309 HGGGARLDL 74 FARLARRYG 306 GDSHGGGA 209 CFGCRYSHD 217 DDPEFRELL 46 QLRSAPPGP 298 LSAEKKAA 202 ANVMSAVCF 163 RQVLEGHVL 513 PKSFKVNVT 297 ILSAEKKA 182 VRGSADGAF 99 GERAIHQAL 452 INKDLTSRV 293 M D A F I L S A 142 KVQRRAAHS 70 AHLSFARLA 443 ARFLDKDGL 288 APRDMMDA 107 LVQQGSAFA 69 AAHLSFARL 442 PARFLDKDG 281 ESLRPGAA 93 PIWLNGER 51 PPGPFAWPL 424 QWSVNHDPL 280 CESLRPGA 54 PFAWPLIGN 33 VHVGQRLLR 361 DQVVGRDRL 230 EFGRTVGA 18 IQQTTLLLL 31 ATVHVGQRL 327 IFGASQDTL 200 AVANVMSA 16 LSIQQTTLL 18 IQQTTLLLL 309 HGGGARLDL 195 PLTVVAVA 12 PLNPLSIQQ 15 PLSIQQTTL 265 NRNFSNFIL 193 PRPLTVVA 515 SFKVNVTLR 528 LLDSAVQNL 262 EQLNRNFSN 182 VRGSADGA 495 ANPNEPAKM 520 VTLRESMEL 229 EEFGRTVGA 179 ALLVRGSA 472 GEELSKMQL 479 Q L F L F I S I L 163 RQVLEGHVL 172 SEARELVA 464 SVGKRRCIG 467 KRRCIGEEL 81 YGDVFQIRL 167 EGHVLSEA 450 GLINKDLTS 443 ARFLDKDGL 32 TVHVGQRLL 141 WKVQRRAA 443 ARFLDKDGL 413 HIPKDTVVF 18 IQQTTLLLL 140 HWKVQRRA 431 PLKWPNPEN 352 VQTRVQAEL 501 AKMNFSYGL 126 VSGGRSMA 426 SVNHDPLKW 336 STALQWLLL 498 NEPAKMNFS 114 FADRPAFA 418 TVVFVNQWS 331 SQDTLSTAL 464 SVGKRRCIG 112 SAFADRPA 396 PVTIPHATT 260 EFEQLNRNF 446 LDKDGLINK 108 VQQGSAFA 378 LPYVLAFLY 256 TVFREFEQL 412 YHIPKDTVV 106 ALVQQGSA 353 QTRVQAELD 250 FPNPVRTVF 402 ATTANTSVL 99 GERAIHQA 331 SQDTLSTAL 183 RGSADGAFL 374 DQPNLPYVL 95 VVLNGERA 320 VPATITDIF 135 GHYSEHWKV 370 PCMGDQPNL 71 HLSFARLA 294 DAFILSAEK 64 AAVGQAAHL 351 DVQTRVQAE 68 QAAHLSFA 291 DMMDAFILS 17 SIQQTTLLL 335 LSTALQWLL 63 AAAVGQAA 282 SLRPGAAPR 16 LSIQQTTLL 307 DSHGGGARL 62 NAAAVGQA 269 SNFILDKFL 8 NDPWPLNPL 300 AEKKAAGDS 58 PLIGNAAA 255 RTVFREFEQ 5 LSPNDPWPL 290 RDMMDAFIL 57 WPLIGNAA 249 YFPNPVRTV 501 AKMNFSYGL 280 CESLRPGAA 56 AWPLIGNA 241 VDVMPWLQY 497 PNEPAKMNF 275 KFLRHCESL 49 SAPPGPFA 240 LVDVMPWLQ 462 IFSVGKRRC 274 DKFLRHCES 43 RRRQLRSA 206 SAVCFGCRY 453 NKDLTSRVM 273 LDKFLRHCE 24 LLLLSVLA 190 FLDPRPLTV 448 KDGLINKDL 256 TVFREFEQL 158 RQPRSRQVL 432 LKWPNPENF 251 PNPVRTVFR

HLA-A*0202 Nonamers 96 VLNGERAIH 377 NLPYVLAFL 220 EFRELLSHN

95 VVLNGERAI 376 PNLPYVLAF 183 RGSADGAFL s 123456789 94 IVVLNGERA 335 LSTALQWLL 158 RQPRSRQVL 535 NLQAKETCQ 84 VFQIRLGSC 290 RDMMDAFIL 149 HSMMRNFFT 529 LDSAVQNLQ 66 VGQAAHLSF 275 KFLRHCESL 147 AAHSMMRNF 498 NEPAKMNFS 65 AVGQAAHLS 269 SNFILDKFL 97 LNGERAIHQ 492 DFRANPNEP 59 LIGNAAAVG 265 NRNFSNFIL 31 ATVHVGQRL 485 SILAHQCDF 46 QLRSAPPGP 236 GAGSLVDVM 19 QQTTLLLLL 440 FDPARFLDK 29 VLATVHVGQ 19 QQTTLLLLL 16 LSIQQTTLL 402 ATTANTSVL 4 SLSPNDPWP 437 PENFDPARF 8 NDPWPLNPL 399 IPHATTANT 2 GTSLSPNDP 288 APRDMMDAF 5 LSPNDPWPL 385 LYEAMRFSS

518 VNVTLRESM 285 PGAAPRDMM

527 ELLDSAVQN

380 YVLAFLYEA 504 NFSYGLTIK 284 RPGAAPRDM 519 NVTLRESME 355 RVQAELDQV 486 ILAHQCDFR 253 PVRTVFREF 499 EPAKMNFSY 335 LSTALQWLL 445 FLDKDGLIN 213 RYSHDDPEF 496 NPNEPAKMN 327 IFGASQDTL 368 RLPCMGDQP 197 TVVAVANVM 481 FLFISILAH 319 NVPATITDI 363 VVGRDRLPC 147 AAHSMMRNF 466 GKRRCIGEE 310 GGGARLDLE 352 VQTRVQAEL 124 RVVSGGRSM 440 FDPARFLDK 302 KKAAGDSHG 314 RLDLENVPA 112 SAFADRPAF 409 VLGYHIPKD 301 EKKAAGDSH 309 HGGGARLDL 47 LRSAPPGPF 384 FLYEAMRFS 297 ILSAEKKAA 295 AFILSAEKK 508 GLTIKPKSF 359 ELDQVVGRD 292 MMDAFILSA 285 PGAAPRDMM 473 EELSKMQLF 346 FTRYPDVQT 285 PGAAPRDMM 267 NFSNFILDK 386 YEAMRFSSF 314 RLDLENVPA 284 RPGAAPRDM 259 REFEQLNRN 383 AFLYEAMRF 310 GGGARLDLE 234 TVGAGSLVD 234 TVGAGSLVD 381 VLAFLYEAM 297 ILSAEKKAA 204 VMSAVCFGC 207 AVCFGCRYS 364 VGRDRLPCM 260 EFEQLNRNF 199 VAVANVMSA 195 PLTVVAVAN 248 QYFPNPVRT 257 VFREFEQLN 197 TVVAVANVM 192 DPRPLTVVA 242 DVMPWLQYF 255 RTVFREFEQ 186 ADGAFLDPR 188 GAFLDPRPL 223 ELLSHNEEF 195 PLTVVAVAN 183 RGSADGAFL 187 DGAFLDPRP 202 ANVMSAVCF 176 ELVALLVRG 176 ELVALLVRG 179 ALLVRGSAD 126 VSGGRSMAF 170 VLSEARELV 171 LSEARELVA 170 VLSEARELV 115 ADRPAFASF 141 WKVQRRAAH 145 RRAAHSMMR 154 NFFTRQPRS 106 ALVQQGSAF 122 SFRVVSGGR 144 QRRAAHSMM 148 AHSMMRNFF 77 LARRYGDVF 84 VFQIRLGSC 130 RSMAFGHYS 144 QRRAAHSMM 518 VNVTLRESM 44 RRQLRSAPP 118 PAFASFRVV 132 MAFGHYSEH 495 ANPNEPAKM 37 QRLLRQRRR 116 DRPAFASFR 99 GERAIHQAL 470 CIGEELSKM 34 HVGQRLLRQ 112 SAFADRPAF 86 QIRLGSCPI 455 DLTSRVMIF 24 LLLLSVLAT 110 QGSAFADRP 81 YGDVFQIRL 320 VPATITDIF 490 QCDFRANPN 103 IHQALVQQG 77 LARRYGDVF 263 QLNRNFSNF 476 SKMQLFLFI 99 GERAIHQAL 51 PPGPFAWPL 182 VRGSADGAF 457 TSRVMIFSV 75 ARLARRYGD 47 LRSAPPGPF 485 SILAHQCDF 395 VPVTIPHAT 72 LSFARLARR 38 RLLRQRRRQ 475 LSKMQLFLF 391 FSSFVPVTI 67 GQAAHLSFA 23 LLLLLSVLA 414 IPKDTWFV 381 VLAFLYEAM 66 VGQAAHLSF 9 DPWPLNPLS 349 YPDVQTRVQ 376 PNLPYVLAF 62 NAAAVGQAA 535 NLQAKETCQ 338 ALQWLLLLF 375 QPNLPYVLA 61 GNAAAVGQA 530 DSAVQNLQA 268 FSNFILDKF 362 QVVGRDRLP 60 IGNAAAVGQ 501 AKMNFSYGL 249 YFPNPVRTV 211 GCRYSHDDP 53 GPFAWPLIG 467 KRRCIGEE 192 DPRPLTVVA 209 CFGCRYSHD 47 LRSAPPGPF 453 NKDLTSRVM 144 QRRAAHSMM 182 VRGSADGAF 28 SVLATVHVG 439 NFDPARFLD 143 VQRRAAHSM 171 LSEARELVA 389 MRFSSFVPV 119 AFASFRVVS 165 VLEGHVLSE

HLA-A*0202 Decamers 335 LSTALQWLL 90 GSCPIVVLN 157 TRQPRSRQV 297 ILSAEKKAA 66 VGQAAHLSF 154 NFFTRQPRS s 1234567890 276 FLRHCESLR 511 IKPKSFKVN 142 KVQRRAAHS

63 AAAVGQAAHL 274 DKFLRHCES 494 RANPNEPAK 113 AFADRPAFA 303 KAAGDSHGGG 265 NRNFSNFIL 452 INKDLTSRV 95 VVLNGERAI 286 GAAPRDMMDA 257 VFREFEQLN 391 FSSFVPVTI 78 ARRYGDVFQ 146 RAAHSMMRNF 235 VGAGSLVDV 390 RFSSFVPVT 75 ARLARRYGD

68 QAAHLSFARL 232 GRTVGAGSL 373 GDQPNLPYV 72 LSFARLARR

62 NAAAVGQAAH 183 RGSADGAFL 359 ELDQVVGRD 71 HLSFARLAR

200 AVANVMSAVC 180 LLVRGSADG 357 QAELDQVVG 57 WPLIGNAAA 119 AFASFRVVSG 143 VQRRAAHSM 347 TRYPDVQTR 40 LRQRRRQLR 113 AFADRPAFAS 119 AFASFRVVS 315 LDLENVPAT 36 GQRLLRQRR 530 DSAVQNLQAK 113 AFADRPAFA 297 ILSAEKKAA 29 VLATVHVGQ 499 EPAKMNFSYG 88 RLGSCPIVV 272 ILDKFLRHC 23 LLLLLSVLA 493 FRANPNEPAK 71 HLSFARLAR 228 NEEFGRTVG 6 SPNDPWPLN

486 ILAHQCDFRA 26 LLSVLATVH 227 HNEEFGRTV 4 SLSPNDPWP 441 DPARFLDKDG 5 LSPNDPWPL 176 ELVALLVRG 531 SAVQNLQAK 403 TTANTSVLGY 460 VMIFSVGKR 157 TRQPRSRQV 492 DFRANPNEP 400 PHATTANTSV 448 KDGLINKDL 156 FTRQPRSRQ 486 ILAHQCDFR 386 YEAMRFSSFV 444 RFLDKDGLI 140 HWKVQRRAA 454 KDLTSRVMI 381 VLAFLYEAMR 424 QWSVNHDPL 137 YSEHWKVQR 445 FLDKDGLIN 356 VQAELDQVVG 420 VFVNQWSVN 94 IVVLNGERA 436 NPENFDPAR 336 STALQWLLLL 393 SFVPVTIPH 73 SFARLARRY 434 WPNPENFDP 328 FGASQDTLST 358 AELDQVVGR 49 SAPPGPFAW 406 NTSVLGYHI 320 VPATITDIFG 343 LLLFTRYPD 48 RSAPPGPFA 399 IPHATTANT 311 GGARLDLENV 342 LLLLFTRYP 38 RLLRQRRRQ 383 AFLYEAMRF

304 AAGDSHGGGA 318 ENVPATITD 4 SLSPNDPWP 378 LPYVLAFLY 302 KKAAGDSHGG 315 LDLENVPAT 527 ELLDSAVQN 366 RDRLPCMGD 298 LSAEKKAAGD 301 EKKAAGDSH 517 KVNVTLRES 353 QTRVQAELD 293 MDAFILSAEK 290 RDMMDAFIL 513 PKSFKVNVT 349 YPDVQTRVQ 287 AAPRDMMDAF 284 RPGAAPRDM 471 IGEELSKMQ 342 LLLLFTRYP 285 PGAAPRDMMD 281 ESLRPGAAP 463 FSVGKRRCI 341 WLLLLFTRY 235 VGAGSLVDVM 270 NFILDKFLR 411 GYHIPKDTV 319 NVPATITDI 205 MSAVCFGCRY 246 WLQYFPNPV 403 TTANTSVLG 272 ILDKFLRHC

198 VVAVANVMSA 224 LLSHNEEFG 384 FLYEAMRFS 268 FSNFILDKF 187 DGAFLDPRPL 189 AFLDPRPLT 358 AELDQVVGR 252 NPVRTVFRE 184 GSADGAFLDP 167 EGHVLSEAR 356 VQAELDQVV 246 WLQYFPNPV 177 LVALLVRGSA 163 RQVLEGHVL 350 PDVQTRVQA 244 MPWLQYFPN 172 SEARELVALL 155 FFTRQPRSR 318 ENVPATITD 231 FGRTVGAGS 147 AAHSMMRNFF 146 RAAHSMMRN 306 GDSHGGGAR 213 RYSHDDPEF 145 RRAAHSMMRN 139 EHWKVQRRA 298 LSAEKKAAG 202 ANVMSAVCF 131 SMAFGHYSEH 121 ASFRVVSGG 286 GAAPRDMMD 194 RPLTVVAVA 117 RPAFASFRVV 116 DRPAFASFR 283 LRPGAAPRD 156 FTRQPRSRQ 111 GSAFADRPAF 100 ERAIHQALV 247 LQYFPNPVR 143 VQRRAAHSM 104 HQALVQQGSA 72 LSFARLARR 235 VGAGSLVDV 128 GGRSMAFGH 100 ERAIHQALVQ 55 FAWPLIGNA 234 TVGAGSLVD 96 VLNGERAIH 76 RLARRYGDVF 50 APPGPFAWP 229 EEFGRTVGA 93 PIWLNGER 73 SFARLARRYG 13 LNPLSIQQT 198 VVAVANVMS 92 CPIVVLNGE 69 AAHLSFARLA 531 SAVQNLQAK 195 PLTVVAVAN 79 RRYGDVFQI 67 GQAAHLSFAR 511 IKPKSFKVN 193 PRPLTVVAV 66 VGQAAHLSF 64 AAVGQAAHLS 462 IFSVGKRRC 190 FLDPRPLTV 58 PLIGNAAAV

61 GNAAAVGQAA 446 LDKDGLINK 189 AFLDPRPLT 53 GPFAWPLIG 54 PFAWPLIGNA 435 PNPENFDPA 187 DGAFLDPRP 49 SAPPGPFAW 48 RSAPPGPFAW 430 DPLKWPNPE 184 GSADGAFLD 43 RRRQLRSAP 29 VLATVHVGQR 414 IPKDTWFV 177 LVALLVRGS 26 LLSVLATVH 531 SAVQNLQAKE 387 EAMRFSSFV 164 QVLEGHVLS 10 PWPLNPLSI 500 PAKMNFSYGL 373 GDQPNLPYV 146 RAAHSMMRN 9 DPWPLNPLS 494 RANPNEPAKM 347 TRYPDVQTR 136 HYSEHWKVQ 503 MNFSYGLTI 487 LAHQCDFRAN 293 MDAFILSAE 125 VVSGGRSMA 497 PNEPAKMNF 442 PARFLDKDGL 278 RHCESLRPG 120 FASFRVVSG 478 MQLFLFISI 404 TANTSVLGYH 266 RNFSNFILD 118 PAFASFRVV 470 CIGEELSKM 401 HATTANTSVL 262 EQLNRNFSN 111 GSAFADRPA 441 DPARFLDKD 387 EAMRFSSFVP 245 PWLQYFPNP 95 VVLNGERAI 437 PENFDPARF 382 LAFLYEAMRF 226 SHNEEFGRT 87 IRLGSCPIV 432 LKWPNPENF 357 QAELDQVVGR 219 PEFRELLSH 82 GDVFQIRLG 430 DPLKWPNPE 337 TALQWLLLLF ■ 193 PRPLTVVAV 63 AAAVGQAAH 401 HATTANTSV 329 GASQDTLSTA 185 SADGAFLDP 60 IGNAAAVGQ 388 AMRFSSFVP 321 PATITDIFGA 138 SEHWKVQRR 37 QRLLRQRRR 387 EAMRFSSFV 312 GARLDLENVP 133 AFGHYSEHW 36 GQRLLRQRR 369 LPCMGDQPN 299 SAEKKAAGDS 122 SFRVVSGGR 26 LLSVLATVH 368 RLPCMGDQP 294 DAFILSAEKK 118 PAFASFRVV

534 QNLQAKETC

357 QAELDQVVG 236 GAGSLVDVMP 103 IHQALVQQG 531 SAVQNLQAK 343 LLLFTRYPD 206 SAVCFGCRYS 92 CPIVVLNGE 515 SFKVNVTLR 296 FILSAEKKA 201 VANVMSAVCF 90 GSCPIVVLN 512 KPKSFKVNV 289 PRDMMDAFI

199 VAVANVMSAV 79 RRYGDVFQI 510 TIKPKSFKV 284 RPGAAPRDM

188 GAFLDPRPLT 500 PAKMNFSYG 509 LTIKPKSFK 242 DVMPWLQYF

185 SADGAFLDPR 498 NEPAKMNFS 507 YGLTIKPKS 236 GAGSLVDVM

178 VALLVRGSAD 480 LFLFISILA 499 EPAKMNFSY 224 LLSHNEEFG

173 EARELVALLV 478 MQLFLFISI 492 DFRANPNEP 206 SAVCFGCRY

132 MAFGHYSEHW 476 SKMQLFLFI 487 LAHQCDFRA 201 VANVMSAVC

120 FASFRVVSGG 449 DGLINKDLT 486 ILAHQCDFR 199 VAVANVMSA 118 PAFASFRVVS 422 VNQWSVNHD 466 GKRRCIGEE 185 SADGAFLDP

114 FADRPAFASF 416 KDTVVFVNQ 457 TSRVMIFSV 181 LVRGSADGA 112 SAFADRPAFA 392 SSFVPVTIP 454 KDLTSRVMI 180 LLVRGSADG

105 QALVQQGSAF 345 LFTRYPDVQ 439 NFDPARFLD 178 VALLVRGSA

101 RAIHQALVQQ 330 ASQDTLSTA 436 NPENFDPAR 161 RSRQVLEGH

77 LARRYGDVFQ 298 LSAEKKAAG 431 PLKWPNPEN 151 MMRNFFTRQ

74 FARLARRYGD 292 MMDAFILSA 421 FVNQWSVNH 148 AHSMMRNFF 55 FAWPLIGNAA 287 AAPRDMMDA 416 KDTVVFVNQ 144 QRRAAHSMM

49 SAPPGPFAWP 248 QYFPNPVRT 415 PKDTVVFVN 117 RPAFASFRV 30 LATVHVGQRL 218 DPEFRELLS . 397 VTIPHATTA 114 FADRPAFAS

532 AVQNLQAKET 199 VAVANVMSA 394 FVPVTIPHA 88 RLGSCPIVV 501 AKMNFSYGLT 166 LEGHVLSEA 392 SSFVPVTIP 63 AAAVGQAAH 495 ANPNEPAKMN 161 RSRQVLEGH 375 QPNLPYVLA 55 FAWPLIGNA 488 AHQCDFRANP 160 PRSRQVLEG 367 DRLPCMGDQ 52 PGPFAWPLI 443 ARFLDKDGLI 127 SGGRSMAFG 362 QVVGRDRLP 50 APPGPFAWP 405 ANTSVLGYHI 120 FASFRVVSG 360 LDQVVGRDR 47 LRSAPPGPF 402 ATTANTSVLG 109 QQGSAFADR 348 RYPDVQTRV 42 QRRRQLRSA 388 AMRFSSFVPV 91 SCPIVVLNG 346 FTRYPDVQT 38 RLLRQRRRQ 383 AFLYEAMRFS 80 RYGDVFQIR 345 LFTRYPDVQ 30 LATVHVGQR 358 AELDQVVGRD 67 GQAAHLSFA 329 GASQDTLST 25 LLLSVLATV 338 ALQWLLLLFT 61 GNAAAVGQA 323 T I T D I F G A S 14 NPLSIQQTT 330 ASQDTLSTAL 42 QRRRQLRSA 316 DLENVPATI 12 PLNPLSIQQ 322 ATITDIFGAS 35 VGQRLLRQR 313 ARLDLENVP 11 WPLNPLSIQ 313 ARLDLENVPA 513 PKSFKVN T 311 GGARLDLEN 506 SYGLTIKPK 305 AGDSHGGGAR 502 KMNFSYGLT 282 SLRPGAAPR 494 RANPNEPAK 300 AEKKAAGDSH 488 AHQCDFRAN 281 ESLRPGAAP 483 FISILAHQC 295 AFILSAEKKA 469 RCIGEELSK 280 CESLRPGAA 461 MIFSVGKRR 288 APRDMMDAFI 466 GKRRCIGEE 279 HCESLRPGA 404 TANTSVLGY 237 AGSLVDVMPW 458 SRVMIFSVG 271 FILDKFLRH 398 TIPHATTAN 207 AVCFGCRYSH 457 TSRVMIFSV 259 REFEQLNRN 382 LAFLYEAMR 202 ANVMSAVCFG 452 INKDLTSRV 252 NPVRTVFRE 329 GASQDTLST

189 AFLDPRPLTV 440 FDPARFLDK 251 PNPVRTVFR 323 T I T D I F G A S

186 ADGAFLDPRP 427 VNHDPLKWP 238 GSLVDVMPW 294 DAFILSAEK

179 ALLVRGSADG 415 PKDTVVFVN 237 AGSLVDVMP 132 MAFGHYSEH

174 ARELVALLVR 412 YHIPKDTVV 230 EFGRTVGAG 105 QALVQQGSA 148 AHSMMRNFFT 405 ANTSVLGYH 214 YSHDDPEFR 102 AIHQALVQQ

133 AFGHYSEHWK 375 QPNLPYVLA 207 AVCFGCRYS 68 QAAHLSFAR

121 ASFRVVSGGR 356 VQAELDQVV 201 VANVMSAVC 62 NAAAVGQAA

115 ADRPAFASFR 340 QWLLLLFTR 200 AVANVMSAV 533 VQNLQAKET

106 ALVQQGSAFA 339 LQWLLLLFT 194 RPLTVVAVA 525 SMELLDSAV

102 AIHQALVQQG 312 GARLDLENV 191 LDPRPLTVV 524 ESMELLDSA

78 ARRYGDVFQI 286 GAAPRDMMD 175 RELVALLVR 487 LAHQCDFRA

75 ARLARRYGDV 283 LRPGAAPRD 171 LSEARELVA 471 IGEELSKMQ 70 AHLSFARLAR 252 NPVRTVFRE 170 VLSEARELV 451 LINKDLTSR 65 AVGQAAHLSF 244 MPWLQYFPN 167 EGHVLSEAR 392 SSFVPVTIP 56 AWPLIGNAAA 238 GSLVDVMPW 165 VLEGHVLSE 326 DIFGASQDT

50 APPGPFAWPL 208 VCFGCRYSH 160 PRSRQVLEG 321 PATITDIFG 31 ATVHVGQRLL

205 MSAVCFGCR

155 FFTRQPRSR

315 LDLENVPAT 194 RPLTVVAVA 154 NFFTRQPRS 304 AAGDSHGGG

HLA-A^*0203 Nonamers 191 LDPRPLTVV 141 WKVQRRAAH 303 KAAGDSHGG 151 MMRNFFTRQ 138 SEHWKVQRR 287 AAPRDMMDA 123456789 98 NGERAIHQA 123 FRVVSGGRS 230 EFGRTVGAG NAAAVGQAA 82 GDVFQIRLG 117 RPAFASFRV 226 SHNEEFGRT AFADRPAFA 30 LATVHVGQR 113 AFADRPAFA 198 VVAVANVMS DSAVQNLQA 11 WPLNPLSIQ 110 QGSAFADRP 193 PRPLTVVAV FRANPNEPA 523 RESMELLDS 102 AIHQALVQQ 167 EGHVLSEAR AAPRDMMDA 512 KPKSFKVNV 88 RLGSCPIVV 146 RAAHSMMRN GSAFADRPA 506 SYGLTIKPK 78 ARRYGDVFQ 136 HYSEHWKVQ GQAAHLSFA 505 FSYGLTIKP 74 FARLARRYG 118 PAFASFRVV GNAAAVGQA 494 RANPNEPAK 71 HLSFARLAR 101 RAIHQALVQ RSAPPGPFA 491 CDFRANPNE 61 GNAAAVGQA 59 LIGNAAAVG LAHQCDFRA 477 KMQLFLFIS 58 PLIGNAAAV 394 FVPVTIPHA VAVANVMSA 471 IGEELSKMQ 55 FAWPLIGNA 356 VQAELDQVV VALLVRGSA 429 HDPLKWPNP 43 RRRQLRSAP 281 ESLRPGAAP QALVQQGSA 428 NHDPLKWPN 34 HVGQRLLRQ 238 GSLVDVMPW FAWPLIGNA 407 TSVLGYHIP 29 VLATVHVGQ 227 HNEEFGRTV ASQDTLSTA 382 LAFLYEAMR 28 SVLATVHVG 164 QVLEGHVLS ATITDIFGA 379 PYVLAFLYE 9 DPWPLNPLS 162 SRQVLEGHV AGDSHGGGA 325 TDIFGASQD 7 PNDPWPLNP 91 SCPIVVLNG AHLSFARLA 310 GGGARLDLE 6 SPNDPWPLN 90 GSCPIVVLN AWPLIGNAA 303 KAAGDSHGG 2 GTSLSPNDP 28 SVLATVHVG ESMELLDSA 299 SAEKKAAGD 530 DSAVQNLQA 530 DSAVQNLQA LFLFISILA 279 HCESLRPGA 526 MELLDSAVQ 517 KVNVTLRES PNPENFDPA 186 ADGAFLDPR 525 SMELLDSAV 511 IKPKSFKVN VTIPHATTA 175 RELVALLVR 524 ESMELLDSA 493 FRANPNEPA FVPVTIPHA 150 SMMRNFFTR 516 FKVNVTLRE 484 ISILAHQCD YVLAFLYEA 108 VQQGSAFAD 504 NFSYGLTIK 462 IFSVGKRRC QPNLPYVLA 87 IRLGSCPIV 496 NPNEPAKMN 460 VMIFSVGKR PDVQTRVQA 85 FQIRLGSCP 493 FRANPNEPA 458 SRVMIFSVG RLDLENVPA 49 SAPPGPFAW 489 HQCDFRANP 435 PNPENFDPA ILSAEKKAA 48 RSAPPGPFA 484 ISILAHQCD 427 VNHDPLKWP FILSAEKKA 43 RRRQLRSAP 481 FLFISILAH 426 SVNHDPLKW MMDAFILSA 7 PNDPWPLNP 464 SVGKRRCIG 422 VNQWSVNHD CESLRPGAA 1 MGTSLSPND 461 MIFSVGKRR 421 FVNQWSVNH HCESLRPGA 525 SMELLDSAV 459 RVMIFSVGK 419 VVFVNQWSV EEFGRTVGA 503 MNFSYGLTI 458 SRVMIFSVG 417 DTVVFVNQW RPLTVVAVA 496 NPNEPAKMN 456 LTSRVMIFS 416 KDTVVFVNQ DPRPLTVVA 489 HQCDFRANP 450 GLINKDLTS 408 SVLGYHIPK LVRGSADGA 484 ISILAHQCD 449 DGLINKDLT 393 SFVPVTIPH LSEARELVA 463 FSVGKRRCI 447 DKDGLINKD 358 AELDQVVGR LEGHVLSEA 454 KDLTSRVMI 446 LDKDGLINK 318 ENVPATITD HWKVQRRAA 365 GRDRLPCMG 435 PNPENFDPA 311 GGARLDLEN EHWKVQRRA 350 PDVQTRVQA 434 WPNPENFDP 308 SHGGGARLD VVSGGRSMA 348 RYPDVQTRV 430 DPLKWPNPE 306 GDSHGGGAR LVQQGSAFA 328 FGASQDTLS 427 VNHDPLKWP 278 RHCESLRPG NGERAIHQA 311 GGARLDLEN 420 VFVNQWSVN 261 FEQLNRNFS IVVLNGERA 305 AGDSHGGGA 419 VVFVNQWSV 259 REFEQLNRN WPLIGNAAA 304 AAGDSHGGG 417 DTVVFVNQW 254 VRTVFREFE QRRRQLRSA 302 KKAAGDSHG 409 VLGYHIPKD 237 AGSLVDVMP LLLLLSVLA 215 SHDDPEFRE 407 TSVLGYHIP 235 VGAGSLVDV AAAVGQAAH 168 GHVLSEARE 406 NTSVLGYHI 222 RELLSHNEE KAAGDSHGG 157 TRQPRSRQV 404 TANTSVLGY 221 FRELLSHNE GAAPRDMMD 153 RNFFTRQPR 399 IPHATTANT 215 SHDDPEFRE RAAHS MRN 145 RRAAHSMMR 398 TIPHATTAN 196 LTVVAVANV QAAHLSFAR 141 WKVQRRAAH 396 PVTIPHATT 177 LVALLVRGS AVANVMSAV

140 HWKVQRRAA

395 VPVTIPHAT

168 GHVLSEARE 119 AFASFRVVS 104 HQALVQQGS 393 SFVPVTIPH 160 PRSRQVLEG

499 EPAKMNFSY 101 RAIHQALVQ 389 MRFSSFVPV 150 SMMRNFFTR 486 ILAHQCDFR 97 LNGERAIHQ 388 AMRFSSFVP 139 EHWKVQRRA

441 DPARFLDKD 68 QAAHLSFAR 385 LYEAMRFSS 134 FGHYSEHWK

403 TTANTSVLG 63 AAAVGQAAH 380 YVLAFLYEA 131 SMAFGHYSE

400 PHATTANTS 62 NAAAVGQAA 372 MGDQPNLPY 127 SGGRSMAFG

386 YEAMRFSSF 36 GQRLLRQRR 371 CMGDQPNLP 123 FRVVSGGRS

381 VLAFLYEAM 33 VHVGQRLLR 365 GRDRLPCMG 108 VQQGSAFAD

356 VQAELDQVV 533 VQNLQAKET 354 TRVQAELDQ 104 HQALVQQGS

336 STALQWLLL 507 YGLTIKPKS 351 DVQTRVQAE 103 IHQALVQQG

328 FGASQDTLS 487 LAHQCDFRA 342 LLLLFTRYP 100 ERAIHQALV

320 VPATITDIF 465 VGKRRCIGE 341 WLLLLFTRY 94 IVVLNGERA

311 GGARLDLEN 436 NPENFDPAR 330 ASQDTLSTA 82 GDVFQIRLG 304 AAGDSHGGG 434 WPNPENFDP 325 TDIFGASQD 73 SFARLARRY 302 KKAAGDSHG 399 IPHATTANT 324 ITDIFGASQ 61 GNAAAVGQA

298 LSAEKKAAG 371 CMGDQPNLP 317 LENVPATIT 60 IGNAAAVGQ

293 MDAFILSAE 366 RDRLPCMGD 314 RLDLENVPA 56 AWPLIGNAA 285 PGAAPRDMM 349 YPDVQTRVQ 303 KAAGDSHGG 27 LSVLATVHV

235 VGAGSLVDV 332 QDTLSTALQ 302 KKAAGDSHG 13 LNPLSIQQT

205 MSAVCFGCR 329 GASQDTLST 299 SAEKKAAGD 2 GTSLSPNDP 198 VVAVANVMS 313 ARLDLENVP 293 MDAFILSAE 526 MELLDSAVQ

187 DGAFLDPRP 308 SHGGGARLD 277 LRHCESLRP 518 VNVTLRESM

184 GSADGAFLD 300 AEKKAAGDS 267 NFSNFILDK 516 FKVNVTLRE 177 LVALLVRGS 289 PRDMMDAFI 261 FEQLNRNFS 505 FSYGLTIKP

172 SEARELVAL 273 LDKFLRHCE 254 VRTVFREFE 504 NFSYGLTIK 147 AAHSMMRNF 251 PNPVRTVFR 245 PWLQYFPNP 491 CDFRANPNE 145 RRAAHSMMR 243 VMPWLQYFP 240 LVDVMPWLQ 489 HQCDFRANP

131 SMAFGHYSE 237 AGSLVDVMP 224 LLSHNEEFG 488 AHQCDFRAN 117 RPAFASFRV 231 FGRTVGAGS 220 EFRELLSHN 477 KMQLFLFIS 104 HQALVQQGS 227 HNEEFGRTV 208 VCFGCRYSH 459 RVMIFSVGK 100 ERAIHQALV 222 RELLSHNEE 206 SAVCFGCRY 453 NKDLTSRVM

76 RLARRYGDV 221 FRELLSHNE 205 MSAVCFGCR 447 DKDGLINKD 73 SFARLARRY 184 GSADGAFLD 199 VAVANVMSA 423 NQWSVNHDP 69 AAHLSFARL 162 SRQVLEGHV 196 LTVVAVANV 420 VFVNQWSVN 64 AAVGQAAHL 159 QPRSRQVLE 179 ALLVRGSAD 418 TVVFVNQWS 54 PFAWPLIGN 137 YSEHWKVQR 159 QPRSRQVLE 415 PKDTVVFVN 29 VLATVHVGQ 136 HYSEHWKVQ 152 MRNFFTRQP 411 GYHIPKDTV 531 SAVQNLQAK 135 GHYSEHWKV 151 MMRNFFTRQ 410 LGYHIPKDT

500 PAKMNFSYG 131 SMAFGHYSE 150 SMMRNFFTR 407 TSVLGYHIP 494 RANPNEPAK 128 GGRSMAFGH 132 MAFGHYSEH 403 TTANTSVLG

442 PARFLDKDG 123 FRVVSGGRS 131 SMAFGHYSE 397 VTIPHATTA

404 TANTSVLGY 117 RPAFASFRV 129 GRSMAFGHY 390 RFSSFVPVT

401 HATTANTSV 114 FADRPAFAS 114 FADRPAFAS 389 MRFSSFVPV

387 EAMRFSSFV 105 QALVQQGSA 108 VQQGSAFAD 380 YVLAFLYEA

382 LAFLYEAMR 60 IGNAAAVGQ 107 LVQQGSAFA 379 PYVLAFLYE

357 QAELDQVVG 57 WPLIGNAAA 101 RAIHQALVQ 373 GDQPNLPYV

337 TALQWLLLL 56 AWPLIGNAA 100 ERAIHQALV 372 MGDQPNLPY

329 GASQDTLST 53 GPFAWPLIG 98 NGERAIHQA 365 GRDRLPCMG

321 PATITDIFG 52 PGPFAWPLI 96 VLNGERAIH 363 VVGRDRLPC

312 GARLDLENV 45 RQLRSAPPG 93 PIWLNGER 360 LDQVVGRDR

299 SAEKKAAGD 27 LSVLATVHV 83 DVFQIRLGS 350 PDVQTRVQA

294 DAFILSAEK 14 NPLSIQQTT 79 RRYGDVFQI 347 TRYPDVQTR

236 GAGSLVDVM 10 PWPLNPLSI 72 LSFARLARR 340 QWLLLLFTR

206 SAVCFGCRY 6 SPNDPWPLN 68 QAAHLSFAR 339 LQWLLLLFT 201 VANVMSAVC 534 QNLQAKETC 67 GQAAHLSFA 332 QDTLSTALQ

188 GAFLDPRPL 526 MELLDSAV 3 62 NAAAVGQAA 328 FGASQDTLS

185 SADGAFLDP 522 LRESMELLD 59 LIGNAAAVG 324 ITDIFGASQ

173 EARELVALL 516 FKVNVTLRE 57 WPLIGNAAA 322 ATITDIFGA

132 MAFGHYSEH

493 FRANPNEPA

54 PFAWPLIGN

317 LENVPATIT 120 FASFRVVSG 490 QCDFRANPN 53 GPFAWPLIG 295 AFILSAEKK 118 PAFASFRVV 442 PARFLDKDG 50 APPGPFAWP 293 MDAFILSAE

114 FADRPAFAS 433 KWPNPENFD 46 QLRSAPPGP 292 MMDAFILSA 112 SAFADRPAF 425 WSVNHDPLK 42 QRRRQLRSA 279 HCESLRPGA

101 RAIHQALVQ 423 NQWSVNHDP 41 RQRRRQLRS 270 NFILDKFLR

77 LARRYGDVF 401 HATTANTSV 35 VGQRLLRQR 267 NFSNFILDK

74 FARLARRYG 400 PHATTANTS 30 LATVHVGQR 258 FREFEQLNR

49 SAPPGPFAW 395 VPVTIPHAT 27 LSVLATVHV 249 YFPNPVRTV

30 LATVHVGQR 391 FSSFVPVTI 24 LLLLSVLAT 248 QYFPNPVRT 532 AVQNLQAKE 388 AMRFSSFVP 23 LLLLLSVLA 247 LQYFPNPVR 501 AKMNFSYGL 385 LYEAMRFSS 12 PLNPLSIQQ 243 VMPWLQYFP 495 ANPNEPAKM 369 LPCMGDQPN 10 PWPLNPLSI 241 VDVMPWLQY 488 AHQCDFRAN 357 QAELDQVVG 3 TSLSPNDPW 240 LVDVMPWLQ 443 ARFLDKDGL 354 TRVQAELDQ 533 VQNLQAKET 210 FGCRYSHDD 405 ANTSVLGYH 306 GDSHGGGAR 529 LDSAVQNLQ 208 VCFGCRYSH 402 ATTANTSVL 280 CESLRPGAA 523 RESMELLDS 205 MSAVCFGCR 388 AMRFSSFVP 277 LRHCESLRP 522 LRESMELLD 204 VMSAVCFGC 383 AFLYEAMRF 258 FREFEQLNR 506 SYGLTIKPK 203 NVMSAVCFG 58 AELDQVVGR 247 LQYFPNPVR 505 FSYGLTIKP 200 AVANVMSAV 338 ALQWLLLLF 225 LSHNEEFG,. 503 MNFSYGLTI 184 GSADGAFLD 313 ARLDLENVP 214 YSHDDPEFR 500 PAKMNFSYG 175 RELVALLVR 300 AEKKAAGDS 212 CRYSHDDPE 498 NEPAKMNFS 174 ARELVALLV 95 AFILSAEKK 211 GCRYSHDDP 491 CDFRANPNE 166 LEGHVLSEA 88 APRDMMDAF 204 VMSAVCFGC 483 FISILAHQC 155 FFTRQPRSR 37 AGSLVDVMP 201 VANVMSAVC 476 SKMQLFLFI 137 YSEHWKVQR 207 AVCFGCRYS 178 VALLVRGSA 469 RCIGEELSK 135 GHYSEHWKV 202 ANVMSAVCF 152 MRNFFTRQP 468 RRCIGEELS 129 GRSMAFGHY 189 AFLDPRPLT 134 FGHYSEHWK 460 VMIFSVGKR 125 VVSGGRSMA 186 ADGAFLDPR 111 GSAFADRPA 451 LINKDLTSR 121 ASFRVVSGG 179 ALLVRGSAD 110 QGSAFADRP 445 FLDKDGLIN 119 AFASFRVVS 174 ARELVALLV 78 ARRYGDVFQ 444 RFLDKDGLI 111 GSAFADRPA 148 AHSMMRNFF 75 ARLARRYGD 441 DPARFLDKD 98 NGERAIHQA 133 AFGHYSEHW 70 AHLSFARLA 433 KWPNPENFD 87 IRLGSCPIV

121 ASFRVVSGG 44 RRQLRSAPP 429 HDPLKWPNP 85 FQIRLGSCP

115 ADRPAFASF 41 RQRRRQLRS 426 SVNHDPLKW 67 GQAAHLSFA 106 ALVQQGSAF 40 LRQRRRQLR 425 WSVNHDPLK 35 VGQRLLRQR

102 AIHQALVQQ 37 QRLLRQRRR 422 VNQWSVNHD 33 VHVGQRLLR

78 ARRYGDVFQ 3 TSLSPNDPW 418 TVVFVNQWS 21 TTLLLLLSV

75 ARLARRYGD 410 LGYHIPKDT 20 QTTLLLLLS 65 AVGQAAHLS HLA-A26 Decamers 408 SVLGYHIPK 3 TSLSPNDPW

50 APPGPFAWP 387 EAMRFSSFV

31 ATVHVGQRL Pos 1234567890 378 LPYVLAFLY 333 DTLSTALQWL 369 LPCMGDQPN

HLA-A^*0203 Decamers 527 E LLDSAVQNL 366 RDRLPCMGD 474 ELSKMQLFLF 363 VVGRDRLPC s 1234567890 326 DIFGASQDTL 353 QTRVQAELD 56 AWPLIGNAAA 403 TTANTSVLGY 344 LLFTRYPDV

296 FILSAEKKAA 351 DVQTRVQAEL 343 LLLFTRYPD

279 HCESLRPGAA 125 VVSGGRSMAF 340 QWLLLLFTR 139 EHWKVQRRAA 83 DVFQIRLGSC 328 FGASQDTLS

61 GNAAAVGQAA 380 YVLAFLYEAM 326 DIFGASQDT

55 FAWPLIGNAA 363 VVGRDRLPCM 322 ATITDIFGA

193 PRPLTVVAVA 336 STALQWLLLL 319 NVPATITDI

112 SAFADRPAFA 255 RTVFREFEQL 312 GARLDLENV

106 ALVQQGSAFA 181 LVRGSADGAF 310 GGGARLDLE

297 ILSAEKKAAG 21 TTLLLLLSVL 304 AAGDSHGGG

280 CESLRPGAAP 17 SIQQTTLLLL 301 EKKAAGDSH 140 HWKVQRRAAH 520 VTLRESMELL 300 AEKKAAGDS 62 NAAAVGQAAH 262 EQLNRNFSNF 294 DAFILSAEK

57 WPLIGNAAAV 240 LVDVMPWLQY 292 MMDAFILSA

529 LDSAVQNLQA 88 RLGSCPIVVL 291 DMMDAFILS 523 RESMELLDSA 65 AVGQAAHLSF 287 AAPRDMMDA 492 DFRANPNEPA 319 NVPATITDIF 274 DKFLRHCES 486 ILAHQCDFRA 267 NFSNFILDKF 273 LDKFLRHCE 479 QLFLFISILA 519 NVTLRESMEL 270 NFILDKFLR 434 WPNPENFDPA 431 PLKWPNPENF 266 RNFSNFILD 396 PVTIPHATTA 517 KVNVTLRESM 262 EQLNRNFSN 393 SFVPVTIPHA 455 DLTSRVMIFS 258 FREFEQLNR 379 PYVLAFLYEA 377 NLPYVLAFLY 257 VFREFEQLN 374 DQPNLPYVLA 176 ELVALLVRGS 255 RTVFREFEQ

349 YPDVQTRVQA 142 KVQRRAAHSM 246 WLQYFPNPV 329 GASQDTLSTA 76 RLARRYGDVF 241 VDVMPWLQY 321 PATITDIFGA 46 QLRSAPPGPF 233 RTVGAGSLV 313 ARLDLENVPA 469 RCIGEELSKM 222 RELLSHNEE

304 AAGDSHGGGA 408 SVLGYHIPKD 221 FRELLSHNE 295 AFILSAEKKA 242 DVMPWLQYFP 218 DPEFRELLS

291 DMMDAFILSA 196 LTVVAVANVM 211 GCRYSHDDP

286 GAAPRDMMDA 31 ATVHVGQRLL 204 VMSAVCFGC

278 RHCESLRPGA 4 SLSPNDPWPL 203 NVMSAVCFG

228 NEEFGRTVGA 510 TIKPKSFKVN 185 SADGAFLDP

198 VVAVANVMSA 447 DKDGLINKDL 181 LVRGSADGA

191 LDPRPLTVVA 417 DTWFVNQWS 180 LLVRGSADG 180 LLVRGSADGA 413 HIPKDTWFV 178 VALLVRGSA 177 LVALLVRGSA 322 ATITDIFGAS 174 ARELVALLV 170 VLSEARELVA 274 DKFLRHCESL 166 LEGHVLSEA 165 VLEGHVLSEA 249 YFPNPVRTVF 162 SRQVLEGHV

138 SEHWKVQRRA 198 VVAVANVMSA 161 RSRQVLEGH 124 RVVSGGRSMA 164 QVLEGHVLSE 153 RNFFTRQPR 110 QGSAFADRPA 114 FADRPAFASF 145 RRAAHSMMR 104 HQALVQQGSA 102 AIHQALVQQG 128 GGRSMAFGH 97 LNGERAIHQA 38 RLLRQRRRQL 127 SGGRSMAFG 93 PIVVLNGERA 481 FLFISILAHQ 122 SFRVVSGGR

69 AAHLSFARLA 421 FVNQWSVNHD 121 ASFRVVSGG 66 VGQAAHLSFA 385 LYEAMRFSSF 105 QALVQQGSA 60 IGNAAAVGQA 359 ELDQVVGRDR 104 HQALVQQGS 54 PFAWPLIGNA 334 TLSTALQWLL 97 LNGERAIHQ 47 LRSAPPGPFA 287 AAPRDMMDAF 92 CPIVVLNGE 41 RQRRRQLRSA 271 FILDKFLRHC 91 SCPIVVLNG 22 TLLLLLSVLA 165 VLEGHVLSEA 86 QIRLGSCPI

530 DSAVQNLQAK 146 RAAHSMMRNF 80 RYGDVFQIR

524 ESMELLDSAV 128 GGRSMAFGHY 76 RLARRYGDV 493 FRANPNEPAK 34 HVGQRLLRQR 75 ARLARRYGD 487 LAHQCDFRAN 15 PLSIQQTTLL 65 AVGQAAHLS 480 LFLFISILAH 12 PLNPLSIQQT 56 AWPLIGNAA

435 PNPENFDPAR 473 EELSKMQLFL 52 PGPFAWPLI

397 VTIPHATTAN 472 GEELSKMQLF 45 RQLRSAPPG

394 FVPVTIPHAT 459 RVMIFSVGKR 40 LRQRRRQLR

380 YVLAFLYEAM 456 LTSRVMIFSV 25 LLLSVLATV

375 QPNLPYVLAF 454 KDLTSRVMIF 21 TTLLLLLSV

350 PDVQTRVQAE 450 GLINKDLTSR 14 NPLSIQQTT

330 ASQDTLSTAL 445 FLDKDGLINK 13 LNPLSIQQT

322 ATITDIFGAS 412 YHIPKDTVVF 11 WPLNPLSIQ

314 RLDLENVPAT 397 VTIPHATTAN 1 MGTSLSPND

305 AGDSHGGGAR 376 PNLPYVLAFL

292 MMDAFILSAE 375 QPNLPYVLAF

287 AAPRDMMDAF 346 FTRYPDVQTR

229 EEFGRTVGAG 340 QWLLLLFTRY

199 VAVANVMSAV 337 TALQWLLLLF 194 RPLTWAVAN 314 RLDLENVPAT 192 DPRPLTWAV 259 REFEQLNRNF 181 LVRGSADGAF 252 NPVRTVFREF

178 VALLVRGSAD 241 VDVMPWLQYF

171 LSEARELVAL 235 VGAGSLVDVM 166 LEGHVLSEAR 234 TVGAGSLVDV 125 VVSGGRSMAF 229 EEFGRTVGAG 113 AFADRPAFAS 223 ELLSHNEEFG 111 GSAFADRPAF 220 EFRELLSHNE 107 LVQQGSAFAD 215 SHDDPEFREL 105 QALVQQGSAF 190 FLDPRPLTVV 98 NGERAIHQAL 187 DGAFLDPRPL 94 IVVLNGERAI 68 QAAHLSFARL 70 AHLSFARLAR 20 Q TTLLLLLSV 67 GQAAHLSFAR 7 PNDPWPLNPL 48 RSAPPGPFAW 509 LTIKPKSFKV 42 QRRRQLRSAP 496 NPNEPAKMNF 23 LLLLLSVLAT 461 MIFSVGKRRC 531 SAVQNLQAKE 426 SVNHDPLKWP 525 SMELLDSAVQ 406 NTSVLGYHIP 494 RANPNEPAKM 393 SFVPVTIPHA 488 AHQCDFRANP 382 LAFLYEAMRF 481 FLFISILAHQ 316 DLENVPATIT 436 NPENFDPARF 230 EFGRTVGAGS 398 TIPHATTANT 195 PLTVVAVANV 395 VPVTIPHATT 192 DPRPLTWAV 381 VLAFLYEAMR 172 SEARELVALL 376 PNLPYVLAFL 171 LSEARELVAL 351 DVQTRVQAEL 119 AFASFRVVSG 331 SQDTLSTALQ 71 HLSFARLARR 323 TITDIFGASQ 29 VLATVHVGQR 315 LDLENVPATI 530 DSAVQNLQAK 306 GDSHGGGARL 499 EPAKMNFSYG 298 LSAEKKAAGD 478 MQLFLFISIL 293 MDAFILSAEK 470 CIGEELSKMQ 288 APRDMMDAFI 451 LINKDLTSRV 281 ESLRPGAAPR 439 NFDPARFLDK

230 EFGRTVGAGS 419 VVFVNQWSVN

200 AVANVMSAVC 398 TIPHATTANT 195 PLTVVAVANV 343 LLLFTRYPDV

182 VRGSADGAFL 324 ITDIFGASQD 179 ALLVRGSADG 323 TITDIFGASQ 172 SEARELVALL 270 NFILDKFLRH 167 EGHVLSEARE 260 EFEQLNRNFS

141 WKVQRRAAHS 256 TVFREFEQLN 126 VSGGRSMAFG 238 GSLVDVMPWL 114 FADRPAFASF 212 CRYSHDDPEF

108 VQQGSAFADR 124 RVVSGGRSMA 99 GERAIHQALV 111 GSAFADRPAF 95 VVLNGERAIH 72 LSFARLARRY 71 HLSFARLARR 58 PLIGNAAAVG 68 QAAHLSFARL 24 LLLLSVLATV

63 AAAVGQAAHL 23 LLLLLSVLAT 58 PLIGNAAAVG 498 NEPAKMNFSY 49 SAPPGPFAWP 494 RANPNEPAKM 43 RRRQLRSAPP 492 DFRANPNEPA 24 LLLLSVLATV 484 ISILAHQCDF 471 IGEELSKMQL

Hl-A-A^*0203 Octamers 464 SVGKRRCIGE 436 NPENFDPARF s 12345678 394 FVPVTIPHAT 31 SAVQNLQA 374 DQPNLPYVLA 25 SMELLDSA 362 QVVGRDRLPC 94 RANPNEPA 338 ALQWLLLLFT 88 AHQCDFRA 318 ENVPATIT I 81 FLFISILA 296 FILSAEKKAA 36 NPENFDPA 291 DMMDAFILSA 98 TIPHATTA 282 SLRPGAAPRD 95 VPVTIPHA 233 RTVGAGSLVD 81 VLAFLYEA 205 MSAVCFGCRY 76 PNLPYVLA 203 NVMSAVCFGC 51 DVQTRVQA 200 AVANVMSAVC 31 SQDTLSTA 197 TVVAVANVMS 23 T I T D I F G A 177 LVALLVRGSA 15 LDLENVPA 156 FTRQPRSRQV 06 GDSHGGGA 107 LVQQGSAFAD 98 LSAEKKAA 105 QALVQQGSAF 297 I L S A E K K A 59 LIGNAAAVGQ 293 MDAFILSA 54 PFAWPLIGNA 288 APRDMMDA 28 SVLATVHVGQ 81 ESLRPGAA 18 IQQTTLLLLL 280 CESLRPGA 532 AVQNLQAKET 230 EFGRTVGA 507 YGLTIKPKSF 200 AVANVMSA 483 FISILAHQCD 195 PLTWAVA 480 LFLFISILAH

193 PRPLTWA 418 TWFVNQWSV 182 VRGSADGA 402 ATTANTSVLG 179 ALLVRGSA 384 FLYEAMRFSS 172 SEARELVA 381 VLAFLYEAMR 167 EGHVLSEA 371 CMGDQPNLPY 141 WKVQRRAA

355 RVQAELDQVV

140 HWKVQRRA 283 LRPGAAPRDM

126 VSGGRSMA 222 RELLSHNEEF

114 FADRPAFA 218 DPEFRELLSH

112 SAFADRPA 216 HDDPEFRELL

108 VQQGSAFA 207 AVCFGCRYSH

106 ALVQQGSA 201 VANVMSAVCF

99 GERAIHQA 169 HVLSEARELV

95 WLNGERA 157 TRQPRSRQVL

71 HLSFARLA 147 AAHSMMRNFF

68 QAAHLSFA 96 VLNGERAIHQ

63 AAAVGQAA 95 VVLNGERAIH

62 NAAAVGQA 94 IVVLNGERAI

58 PLIGNAAA 93 PIVVLNGERA

57 WPLIGNAA 86 QIRLGSCPIV

56 AWPLIGNA 63 AAAVGQAAHL 49 SAPPGPFA 50 APPGPFAWPL 43 RRRQLRSA 16 LSIQQTTLLL 24 LLLLSVLA 528 LLDSAVQNLQ 524 ESMELLDSAV 521 TLRESMELLD 500 PAKMNFSYGL 486 ILAHQCDFRA 485 SILAHQCDFR 482 LFISILAHQC 479 QLFLFISILA 452 INKDLTSRVM 396 PVTIPHATTA 383 AFLYEAMRFS 373 GDQPNLPYVL 369 LPCMGDQPNL 368 RLPCMGDQPN 353 QTRVQAELDQ 344 LLFTRYPDVQ 330 ASQDTLSTAL 308 SHGGGARLDL 306 GDSHGGGARL 297 ILSAEKKAAG 272 ILDKFLRHCE 253 PVRTVFREFE

208 VCFGCRYSHD 182 VRGSADGAFL

170 VLSEARELVA

143 VQRRAAHSMM

106 ALVQQGSAFA 98 NGERAIHQAL 80 RYGDVFQIRL 39 LLRQRRRQLR 32 TVHVGQRLLR 30 LATVHVGQRL 26 LLSVLATVHV 2 GTSLSPNDPW 513 PKSFKVNVTL 508 GLTIKPKSFK 466 GKRRCIGEEL

442 PARFLDKDGL 438 ENFDPARFLD 430 DPLKWPNPEN 423 NQWSVNHDPL 401 HATTANTSVL 367 DRLPCMGDQP 360 LDQVVGRDRL 301 EKKAAGDSHG 295 AFILSAEKKA 294 DAFILSAEKK 289 PRDMMDAFIL 284 RPGAAPRDMM 276 FLRHCESLRP 257 VFREFEQLNR 246 WLQYFPNPVR 239 SLVDVMPWLQ 231 FGRTVGAGSL 224 LLSHNEEFGR 217 DDPEFRELLS 180 LLVRGSADGA 179 ALLVRGSADG 168 GHVLSEAREL

154 NFFTRQPRSR 123 FRVVSGGRSM 113 AFADRPAFAS

25 LLLSVLATVH

22 TLLLLLSVLA

14 NPLSIQQTTL

9 DPWPLNPLSI

515 SFKVNVTLRE

437 PENFDPARFL

420 VFVNQWSVNH 409 VLGYHIPKDT 390 RFSSFVPVTI 389 MRFSSFVPVT 345 LFTRYPDVQT 342 LLLLFTRYPD 341 WLLLLFTRYP 335 LSTALQWLLL 307 DSHGGGARLD

286 GAAPRDMMDA 268 FSNFILDKFL 264 LNRNFSNFIL 263 QLNRNFSNFI 248 QYFPNPVRTV 219 PEFRELLSHN 173 EARELVALLV 167 EGHVLSEARE 162 SRQVLEGHVL

155 FFTRQPRSRQ

139 EHWKVQRRAA 116 DRPAFASFRV 101 RAIHQALVQQ 49 SAPPGPFAWP 514 KSFKVNVTLR 504 NFSYGLTIKP

503 MNFSYGLTIK

446 LDKDGLINKD 444 RFLDKDGLIN 441 DPARFLDKDG 415 PKDTVVFVNQ 372 MGDQPNLPYV 350 PDVQTRVQAE 292 MMDAFILSAE 281 ESLRPGAAPR 275 KFLRHCESLR 266 RNFSNFILDK 189 AFLDPRPLTV 185 SADGAFLDPR 145 RRAAHSMMRN 131 SMAFGHYSEH

126 VSGGRSMAFG 122 SFRVVSGGRS

108 VQQGSAFADR 100 ERAIHQALVQ 97 LNGERAIHQA 90 GSCPIVVLNG 84 VFQIRLGSCP 79 RRYGDVFQIR 73 SFARLARRYG 53 GPFAWPLIGN 33 VHVGQRLLRQ 512 KPKSFKVNVT 505 FSYGLTIKPK 497 PNEPAKMNFS 487 LAHQCDFRAN 465 VGKRRCIGEE 462 IFSVGKRRCI 449 DGLINKDLTS 440 FDPARFLDKD 434 WPNPENFDPA 414 IPKDTVVFVN 387 EAMRFSSFVP 361 DQVVGRDRLP 358 AELDQVVGRD 357 QAELDQVVGR 354 TRVQAELDQV 329 GASQDTLSTA 327 IFGASQDTLS 311 GGARLDLENV 309 HGGGARLDLE 251 PNPVRTVFRE 244 MPWLQYFPNP 209 CFGCRYSHDD

199 VAVANVMSAV 193 PRPLTVVAVA 184 GSADGAFLDP 175 RELVALLVRG 159 QPRSRQVLEG

137 YSEHWKVQRR 133 AFGHYSEHWK

120 FASFRVVSGG 91 SCPIVVLNGE 10 PWPLNPLSIQ 523 RESMELLDSA 522 LRESMELLDS 516 FKVNVTLRES 477 KMQLFLFISI 476 SKMQLFLFIS 475 LSKMQLFLFI 457 TSRVMIFSVG 435 PNPENFDPAR 428 NHDPLKWPNP

416 KDTVVFVNQW 388 AMRFSSFVPV 379 PYVLAFLYEA 321 PATITDIFGA 298 LSAEKKAAGD 258 FREFEQLNRN 243 VMPWLQYFPN

237 AGSLVDVMPW 225 LSHNEEFGRT 160 PRSRQVLEGH 150 SMMRNFFTRQ

132 MAFGHYSEHW 117 RPAFASFRVV 89 LGSCPIVVLN 81 YGDVFQIRLG 78 ARRYGDVFQI 66 VGQAAHLSFA 60 IGNAAAVGQA 41 RQRRRQLRSA 511 IKPKSFKVNV 501 AKMNFSYGLT 404 TANTSVLGYH 391 FSSFVPVTIP 378 LPYVLAFLYE 366 RDRLPCMGDQ

356 VQAELDQVVG 339 LQWLLLLFTR

303 KAAGDSHGGG 290 RDMMDAFILS 278 RHCESLRPGA 277 LRHCESLRPG 265 NRNFSNFILD 204 VMSAVCFGCR

202 ANVMSAVCFG 174 ARELVALLVR 153 RNFFTRQPRS 149 HSMMRNFFTR

136 HYSEHWKVQR 75 ARLARRYGDV 48 RSAPPGPFAW

27 LSVLATVHVG 19 QQTTLLLLLS 531 SAVQNLQAKE 493 FRANPNEPAK 491 CDFRANPNEP 490 QCDFRANPNE 460 VMIFSVGKRR 453 NKDLTSRVMI 365 GRDRLPCMGD 349 YPDVQTRVQA

347 TRYPDVQTRV 328 FGASQDTLST 325 TDIFGASQDT 315 LDLENVPATI

305 AGDSHGGGAR 300 AEKKAAGDSH 293 MDAFILSAEK 285 PGAAPRDMMD 250 FPNPVRTVFR 236 GAGSLVDVMP 188 GAFLDPRPLT

141 WKVQRRAAHS

140 HWKVQRRAAH 121 ASFRVVSGGR 118 PAFASFRVVS 115 ADRPAFASFR 112 SAFADRPAFA 92 CPIWLNGER

62 NAAAVGQAAH

61 GNAAAVGQAA 56 AWPLIGNAAA 55 FAWPLIGNAA 51 PPGPFAWPLI 526 MELLDSAVQN 518 VNVTLRESME 506 SYGLTIKPKS 495 ANPNEPAKMN 488 AHQCDFRANP 463 FSVGKRRCIG 458 SRVMIFSVGK 443 ARFLDKDGLI 432 LKWPNPENFD 405 ANTSVLGYHI 400 PHATTANTSV 392 SSFVPVTIPH 386 YEAMRFSSFV 370 PCMGDQPNLP 364 VGRDRLPCMG

348 RYPDVQTRVQ 331 SQDTLSTALQ 313 ARLDLENVPA

304 AAGDSHGGGA

302 KKAAGDSHGG 288 APRDMMDAFI 280 CESLRPGAAP 279 HCESLRPGAA 273 LDKFLRHCES 269 SNFILDKFLR 247 LQYFPNPVRT 228 NEEFGRTVGA 227 HNEEFGRTVG 226 SHNEEFGRTV 214 YSHDDPEFRE 194 RPLTWAVAN 191 LDPRPLTVVA 186 ADGAFLDPRP 178 VALLVRGSAD 161 RSRQVLEGHV 158 RQPRSRQVLE

144 QRRAAHSMMR 109 QQGSAFADRP

104 HQALVQQGSA 103 IHQALVQQGS 87 IRLGSCPIVV 85 FQIRLGSCPI 70 AHLSFARLAR 67 GQAAHLSFAR

64 AAVGQAAHLS

57 WPLIGNAAAV

45 RQLRSAPPGP

43 RRRQLRSAPP

42 QRRRQLRSAP

35 VGQRLLRQRR

13 LNPLSIQQTT

8 NDPWPLNPLS

6 SPNDPWPLNP

5 LSPNDPWPLN

3 TSLSPNDPWP 34 QNLQAKETCQ 29 LDSAVQNLQA 25 SMELLDSAVQ 89 HQCDFRANPN 68 RRCIGEELSK 33 KWPNPENFDP 29 HDPLKWPNPE 27 VNHDPLKWPN 25 WSVNHDPLKW 24 QWSVNHDPLK 95 VPVTIPHATT 32 QDTLSTALQW 20 VPATITDIFG 10 GGGARLDLEN 99 SAEKKAAGDS 61 FEQLNRNFSN 54 VRTVFREFEQ 45 PWLQYFPNPV 32 GRTVGAGSLV

21 FRELLSHNEE 166 LEGHVLSEAR 163 RQVLEGHVLS 152 MRNFFTRQPR 151 MMRNFFTRQP

138 SEHWKVQRRA

135 GHYSEHWKVQ

134 FGHYSEHWKV

130 RSMAFGHYSE

127 SGGRSMAFGH

99 GERAIHQALV

82 GDVFQIRLGS

77 LARRYGDVFQ

74 FARLARRYGD

52 PGPFAWPLIG

47 LRSAPPGPFA

44 RRQLRSAPPG

40 LRQRRRQLRS

37 QRLLRQRRRQ

36 GQRLLRQRRR

11 WPLNPLSIQQ

1 MGTSLSPNDP

Prediction of HLA binding peptides from cytochrome P450 1 B1 using the algorithm on the BIMAS website

(http://bimas.dcrt.nih.gov/molbio/hla_bind/)

Claims

1. A method of treating a patient that comprises or is at risk of comprising a cell that expresses cytochrome P450 IBl, said method comprising administering to said patient a cytotoxic T lymphocyte that kills said cell in a cytochrome P450 lBl-specific, major histocompatibility complex-restricted fashion.

2. The method of claim 1, wherein said cytotoxic T lymphocyte is autologous to said patient.

3. The method of claim 1, wherein said cytotoxic T lymphocyte is allogeneic to said patient.

4. The method of claim 1, wherein said cytotoxic T lymphocyte is generated by activation with an antigen presenting cell that has been pulsed with cytochrome P450 IBl or a peptide of cytochrome P450 IBl that binds to a major histocompatibility complex molecule.

5. The method of claim 1, further comprising administering to said patient a cytotoxic T lymphocyte that kills a cell in said patient that expresses a second tumor associated antigen.

6. The method of claim 5, wherein said second tumor associated antigen is telomerase.

7. A method of treating a patient that comprises or is at risk of comprising a cell that expresses cytochrome P450 IBl, said method comprising administering to said patient an antigen presenting cell that activates in said patient a cytotoxic T lymphocyte that kills said cell in a cytochrome P450 1B1- specifϊc, major histocompatibility complex-restricted fashion.

8. The method of claim 7, wherein said antigen presenting cell has been pulsed with cytochrome P450 IBl or a peptide of cytochrome P450 IBl that binds to a major histocompatibility complex molecule,

9. The method of claim 7, further comprising administering to said patient an antigen presenting cell that activates in said patient a cytotoxic T lymphocyte that kills a cell in said patient that expresses a second tumor associated antigen.

10. The method of claim 9, wherein said second tumor associated antigen is telomerase.

11. A method of treating a patient that comprises or is at risk of comprising a cell that expresses cytochrome P450 IBl, said method comprising administering to said patient a peptide of cytochrome P450 IBl that binds to a major histocompatibility complex molecule, wherein said peptide of cytochrome P450 IBl is processed by an antigen presenting cell in said patient, which activates a cytotoxic T lymphocyte in said patient to kill said cell that expresses cytochrome P450 IBl in a cytochrome P450 IBl -specific, major histocompatibility complex-restricted fashion.

12. The method of claim 11, wherein said peptide of cytochrome P450 IBl is administered to said patient in association with an adjuvant.

13. The method of claim 11, further comprising administering to said patient a second tumor associated antigen or a peptide thereof that binds to a major histocompatibility complex molecule, wherein said second tumor associated antigen or said peptide thereof is processed by an antigen presenting cell in said patient, which activates a cytotoxic T lymphocyte in said patient to kill cells that express the second tumor associated antigen in a tumor associated antigen-specific, major histocompatibility complex-restricted fashion.

14. The method of claim 13, wherein said second tumor associated antigen is telomerase.

15. A method of treating a patient that comprises or is at risk of comprising a cell that expresses cytochrome P450 IBl, said method comprising administering to said patient a nucleic acid molecule encoding cytochrome P450 IBl or a peptide of cytochrome P450 IBl that binds to a major histocompatibility complex molecule, wherein said nucleic acid molecule is expressed in said patient so that the polypeptide or peptide it encodes can be processed by an antigen presenting cell in said patient, which activates a cytotoxic T lymphocyte in said patient to kill said cell that expresses cytochrome P450 IBl in a cytochrome P450 lBl-specifϊc, major histocompatibility complex-restricted fashion.

16. The method of claim 15, wherein said nucleic acid molecule encoding cytochrome P450 IBl or a peptide of cytochrome P450 IBl is in an expression vector.

17. The method of claim 15, further comprising administering to said patient a nucleic acid molecule encoding a second tumor associated antigen or a peptide thereof that binds to a major histocompatibility complex molecule, wherein said nucleic acid molecule is expressed in said patient so that the polypeptide or peptide that it encodes can be processed by an antigen presenting cell in said patient, which activates a cytotoxic T lymphocyte in said patient to kill cells that express the second tumor associated antigen in a tumor associated antigen-specific, major histocompatibility complex-restricted fashion.

18. The method of claim 17, wherein the second tumor associated antigen is telomerase.

19. The method of claim 1, 7, 11, or 15, wherein said patient comprises a tumor comprising cells that express cytochrome P450 IBl.

20. The method of claim 4 or 7, wherein said antigen presenting cell is a dendritic cell or a CD40-activated B cell.

21. The method of claim 4, 8, 11, or 15, wherein said peptide of cytochrome P450 IBl binds to a class I major histocompatibility complex molecule.

22. The method of claim 21, wherein said class I major histocompatibility complex molecule is an HLA-A2 or an HLA-A3 molecule.

23. The method of claim 22, wherein said peptide of cytochrome P450 IBl comprises the amino acid sequence of CYP239 (SEQ ID NO:l), CYP246 (SEQ ID NO:2), CYP190 (SEQ ID NO:3), or CYP528 (SEQ ID NO:4).

24. A method of assessing the level of immunity of a patient to cytochrome P450 IBl or a peptide of cytochrome P450 IBl that binds to a major histocompatibility complex molecule, said method comprising measuring the level of cytotoxic T lymphocytes specific for cytochrome P450 IBl or said peptide of cytochrome P450 IBl in a sample from said patient.

25. The method of claim 24, wherein said sample is obtained from said patient before or after a cancer treatment is administered to said patient.

26. A cytochrome P450 IBl peptide that binds to a major histocompatibility complex molecule.

27. The peptide of claim 26, consisting essentially of the amino acid sequence of CYP239 (SEQ ID NO:l), CYP246 (SEQ ID NO:2), CYP 190 (SEQ ID NO:3), or CYP528 (SEQ ID NO:4).

28. An ex vivo generated cytotoxic T lymphocyte that specifically kills a cell expressing cytochrome P450 IBl in a specific, major histocompatibility complex-restricted fashion.

29. An e vivo generated antigen presenting cell that presents a peptide of a cytochrome P450 IBl in the context of a major histocompatibility complex molecule.