# Patent

## Title
Expression vectors encoding epitopes of target-associated antigens and methods for their design

## Abstract

The invention disclosed herein is directed to methods of identifying a polypeptide suitable for epitope liberation including, for example, the steps of identifying an epitope of interest; providing a substrate polypeptide sequence including the epitope, wherein the substrate polypeptide permits processing by a proteasome; contacting the substrate polypeptide with a composition including the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope. The invention further relates to vectors including a housekeeping epitope expression cassette. The invention relates to epitope cluster regions and to vectors including epitope cluster regions. The invention also relates to a method of activating a T cell comprising contacting a substrate polypeptide with an APC and contacting the APC with a T cell.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 10/292,413, filed on Nov. 7, 2002, entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS AND METHODS FOR THEIR DESIGN, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/336,968, filed on Nov. 7, 2001, having the same title; both of which are hereby incorporated by reference in their entirety.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention disclosed herein is directed to methods for the design of epitope-encoding vectors, and epitope cluster regions, for use in compositions, including for example, pharmaceutical compositions capable of inducing an immune response in a subject to whom the compositions are administered. The invention is further directed to the vectors themselves. The epitope(s) expressed using such vectors can stimulate a cellular immune response against a target cell displaying the epitope(s).
2. Description of the Related Art
The immune system can be categorized into two discrete effector arms. The first is innate immunity, which involves numerous cellular components and soluble factors that respond to all infectious challenges. The other is the adaptive immune response, which is customized to respond specifically to precise epitopes from infectious agents. The adaptive immune response is further broken down into two effector arms known as the humoral and cellular immune systems. The humoral arm is centered on the production of antibodies by B-lymphocytes while the cellular arm involves the killer cell activity of cytotoxic T Lymphocytes.
Cytotoxic T Lymphocytes (CTL) do not recognize epitopes on the infectious agents themselves. Rather, CTL detect fragments of antigens derived from infectious agents that are displayed on the surface of infected cells. As a result antigens are visible to CTL only after they have been processed by the infected cell and thus displayed on the surface of the cell.
The antigen processing and display system on the surface of cells has been well established. CTL recognize short peptide antigens, which are displayed on the surface in non-covalent association with class I major histocompatibility complex molecules (MHC). These class I peptides are in turn derived from the degradation of cytosolic proteins.
SUMMARY OF THE INVENTION
The invention disclosed herein relates to the identification of epitope cluster regions that are used to generate pharmaceutical compositions capable of inducing an immune response from a subject to whom the compositions have been administered. One embodiment of the disclosed invention relates to an epitope cluster, the cluster being derived from an antigen associated with a target, the cluster including or encoding at least two sequences having a known or predicted affinity for an MHC receptor peptide binding cleft, wherein the cluster is an incomplete fragment of the antigen.
In one aspect of the invention, the target is a neoplastic cell.
In another aspect of the invention, the MHC receptor may be a class I HLA receptor.
In yet another aspect of the invention, the cluster includes or encodes a polypeptide having a length, wherein the length is at least 10 amino acids. Advantageously, the length of the polypeptide may be less than about 75 amino acids.
In still another aspect of the invention, there is provided an antigen having a length, wherein the cluster consists of or encodes a polypeptide having a length, wherein the length of the polypeptide is less than about 80% of the length of the antigen. Preferably, the length of the polypeptide is less than about 50% of the length of the antigen. Most preferably, the length of the polypeptide is less than about 20% of the length of the antigen.
Embodiments of the invention particularly relate to epitope clusters identified in the tumor-associated antigen NY-ESO (SEQ ID NO: 11). One embodiment of the invention relates to an isolated nucleic acid containing a reading frame with a first sequence encoding one or more segments of NY-ESO, wherein the whole antigen is not encoded, wherein each segment contains an epitope cluster, and wherein each cluster contains at least two amino acid sequences with a known or predicted affinity for a same MHC receptor peptide binding cleft. In various aspects of the invention the epitope cluster can be amino acids 79-104, 86-171, 108-140, 108-174, 144-171, and 148-167 of NY-ESO. In other aspects the segments can consist of an epitope cluster; the first sequence can be a fragment of NY-ESO. The fragment can consist of a polypeptide having a length, wherein the length of the polypeptide is less than about 90, 80, 60, 50, 25, or 10% of the length of NY-ESO; and/or the fragment can consist essentially of an amino acid sequence beginning at amino acid 79, 86, 108, 144 and 148 and ending at amino acid 104, 140, 167, 171 and 174 of NY-ESO. Also, the NY-ESO fragment can consist essentially of, for example, amino acids 79-140, 79-167, 79-171, or 79-174; amino acids 86-140, 86-167, 86-171 or 86-174; also, amino acids 86-140, 86-167, 86-171 or 86-174; amino acids 108-167 or 108-171; amino acids 144-167 or 144-174; amino acids 148-171 or 148-174; amino acids 79-174; and/or amino acids 77-180. Embodiments relate to a reading frame operably linked to a promoter. Further embodiments of the invention include a second sequence encoding essentially a housekeeping epitope. In one aspect of this embodiment the first and second sequences constitute a single reading frame. In aspects of the invention the reading frame is operably linked to a promoter. Other embodiments of the invention include the polypeptides encoded by the nucleic acid embodiments of the invention and immunogenic compositions containing the nucleic acids or polypeptides of the invention.
Embodiments of the invention provide expression cassettes, for example, for use in vaccine vectors, which encode one or more embedded housekeeping epitopes, and methods for designing and testing such expression cassettes. Housekeeping epitopes can be liberated from the translation product of such cassettes through proteolytic processing by the immunoproteasome of professional antigen presenting cells (pAPC). In one embodiment of the invention, sequences flanking the housekeeping epitope(s) can be altered to promote cleavage by the immunoproteasome at the desired location(s). Housekeeping epitopes, their uses, and identification are described in U.S. patent application Ser. Nos. 09/560,465 and 09/561,074 entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, and METHOD OF EPITOPE DISCOVERY, respectively; both of which were filed on Apr. 28, 2000, and which are both incorporated herein by reference in their entireties.
Examples of housekeeping epitopes are disclosed in provisional U.S. patent applications entitled EPITOPE SEQUENCES, Nos. 60/282,211, filed on Apr. 6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed Mar. 7, 2002; and 60/409,123, filed on Sep. 5, 2002; and U.S. application Ser. No. 10/117,937, filed on Apr. 4, 2002, which is also entitled EPITOPE SEQUENCES; which are all incorporated herein by reference in their entirety.
In other embodiments of the invention, the housekeeping epitope(s) can be flanked by arbitrary sequences or by sequences incorporating residues known to be favored in immunoproteasome cleavage sites. As used herein the term “arbitrary sequences” refers to sequences chosen without reference to the native sequence context of the epitope, their ability to promote processing or immunological function. In further embodiments of the invention multiple epitopes can be arrayed head-to-tail. These arrays can be made up entirely of housekeeping epitopes. Likewise, the arrays can include alternating housekeeping and immune epitopes. Alternatively, the arrays can include housekeeping epitopes flanked by immune epitopes, whether complete or distally truncated. Further, the arrays can be of any other similar arrangement. There is no restriction on placing a housekeeping epitope at the terminal positions of the array. The vectors can additionally contain authentic protein coding sequences or segments thereof containing epitope clusters as a source of immune epitopes. The term “authentic” refers to natural protein sequences.
Epitope clusters and their uses are described in U.S. patent application Ser. Nos. 09/561,571 entitled EPITOPE CLUSTERS, filed on Apr. 28, 2000; Ser. No. 10/005,905, entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS, filed on Nov. 7, 2001; and Ser. No. 10/026,066, filed on Dec. 7, 2001, also entitled EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS; all of which are incorporated herein by reference in their entirety.
Embodiments of the invention can encompass screening the constructs to determine whether the housekeeping epitope is liberated. In constructs containing multiple housekeeping epitopes, embodiments can include screening to determine which epitopes are liberated. In a preferred embodiment, a vector containing an embedded epitope can be used to immunize HLA transgenic mice and the resultant CTL can be tested for their ability to recognize target cells presenting the mature epitope. In another embodiment, target cells expressing immunoproteasome can be transformed with the vector. The target cell may express immunoproteasome either constitutively, because of treatment with interferon (IFN), or through genetic manipulation, for example. CTL that recognize the mature epitope can be tested for their ability to recognize these target cells. In yet another embodiment, the embedded epitope can be prepared as a synthetic peptide. The synthetic peptide then can be subjected to digestion by an immunoproteasome preparation in vitro and the resultant fragments can be analyzed to determine the sites of cleavage. Such polypeptides, recombinant or synthetic, from which embedded epitopes can be successfully liberated, can also be incorporated into immunogenic compositions.
The invention disclosed herein relates to the identification of a polypeptide suitable for epitope liberation. One embodiment of the invention, relates to a method of identifying a polypeptide suitable for epitope liberation including, for example, the steps of identifying an epitope of interest; providing a substrate polypeptide sequence including the epitope, wherein the substrate polypeptide permits processing by a proteasome; contacting the substrate polypeptide with a composition including the proteasome, under conditions that support processing of the substrate polypeptide by the proteasome; and assaying for liberation of the epitope.
The epitope can be embedded in the substrate polypeptide, and in some aspects the substrate polypeptide can include more than one epitope, for example. Also, the epitope can be a housekeeping epitope.
In one aspect, the substrate polypeptide can be a synthetic peptide. Optionally, the substrate polypeptide can be included in a formulation promoting protein transfer. Alternatively, the substrate polypeptide can be a fusion protein. The fusion protein can further include a protein domain possessing protein transfer activity. Further, the contacting step can include immunization with the substrate polypeptide.
In another aspect, the substrate polypeptide can be encoded by a polynucleotide. The contacting step can include immunization with a vector including the polynucleotide, for example. The immunization can be carried out in an HLA-transgenic mouse or any other suitable animal, for example. Alternatively, the contacting step can include transforming a cell with a vector including the polynucleotide. In some embodiments the transformed cell can be a target cell that is targeted by CTL for purposes of assaying for proper liberation of epitope.
The proteasome processing can take place intracellularly, either in vitro or in vivo. Further, the proteasome processing can take place in a cell-free system.
The assaying step can include a technique selected from the group including, but not limited to, mass spectrometry, N-terminal pool sequencing, HPLC, and the like. Also, the assaying step can include a T cell target recognition assay. The T cell target recognition assay can be selected from the group including, but not limited to, a cytolytic activity assay, a chromium release assay, a cytokine assay, an ELISPOT assay, tetramer analysis, and the like.
In still another aspect, the amino acid sequence of the substrate polypeptide including the epitope can be arbitrary. Also, the substrate polypeptide in which the epitope is embedded can be derived from an authentic sequence of a target-associated antigen. Further, the substrate polypeptide in which the epitope is embedded can be conformed to a preferred immune proteasome cleavage site flanking sequence.
In another aspect, the substrate polypeptide can include an array of additional epitopes. Members of the array can be arranged head-to-tail, for example. The array can include more than one housekeeping epitope. The more than one housekeeping epitope can include copies of the same epitope. The array can include a housekeeping and an immune epitope, or alternating housekeeping and immune epitopes, for example. Also, the array can include a housekeeping epitope positioned between two immune epitopes in an epitope battery. The array can include multiple epitope batteries, so that there are two immune epitopes between each housekeeping epitope in the interior of the array. Optionally, at least one of the epitopes can be truncated distally to its junction with an adjacent epitope. The truncated epitopes can be immune epitopes, for example. The truncated epitopes can have lengths selected from the group including, but not limited to, 9, 8, 7, 6, 5, 4 amino acids, and the like.
In still another aspect, the substrate polypeptide can include an array of epitopes and epitope clusters. Members of the array can be arranged head-to-tail, for example.
In yet another aspect, the proteasome can be an immune proteasome.
Another embodiment of the disclosed invention relates to vectors including a housekeeping epitope expression cassette. The housekeeping epitope(s) can be derived from a target-associated antigen, and the housekeeping epitope can be liberatable, that is capable of liberation, from a translation product of the cassette by immunoproteasome processing.
In one aspect of the invention the expression cassette can encode an array of two or more epitopes or at least one epitope and at least one epitope cluster. The members of the array can be arranged head-to-tail, for example. Also, the members of the array can be arranged head-to-tail separated by spacing sequences, for example. Further, the array can include a plurality of housekeeping epitopes. The plurality of housekeeping epitopes can include more than one copy of the same epitope or single copies of distinct epitopes, for example. The array can include at least one housekeeping epitope and at least one immune epitope. Also, the array can include alternating housekeeping and immune epitopes. Further, the array includes a housekeeping epitope sandwiched between two immune epitopes so that there are two immune epitopes between each housekeeping epitope in the interior of the array. The immune epitopes can be truncated distally to their junction with the adjacent housekeeping epitope.
In another aspect, the expression cassette further encodes an authentic protein sequence, or segment thereof, including at least one immune epitope. Optionally, the segment can include at least one epitope cluster. The housekeeping epitope expression cassette and the authentic sequence including at least one immune epitope can be encoded in a single reading frame or transcribed as a single mRNA species, for example. Also, the housekeeping epitope expression cassette and the authentic sequence including at least one immune epitope may not be transcribed as a single mRNA species.
In yet another aspect, the vector can include a DNA molecule or an RNA molecule. The vector can encode, for example, SEQ ID NO. 4, SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 33, and the like. Also, the vector can include SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 30, SEQ ID NO. 34, and the like. Also, the vector can encode SEQ ID NO. 5 or SEQ ID NO. 18, for example.
In still another aspect, the target-associated antigen can be an antigen derived from or associated with a tumor or an intracellular parasite, and the intracellular parasite can be, for example, a virus, a bacterium, a protozoan, or the like.
Another embodiment of the invention relates to vectors including a housekeeping epitope identified according to any of the methods disclosed herein, claimed or otherwise. For example, embodiments can relate to vector encoding a substrate polypeptide that includes a housekeeping epitope by any of the methods described herein.
In one aspect, the housekeeping epitope can be liberated from the cassette translation product by immune proteasome processing.
Another embodiment of the disclosed invention relates to methods of activating a T cell. The methods can include, for example, the steps of contacting a vector including a housekeeping epitope expression cassette with an APC. The housekeeping epitope can be derived from a target-associated antigen, for example, and the housekeeping epitope can be liberatable from a translation product of the cassette by immunoproteasome processing. The methods can further include contacting the APC with a T cell. The contacting of the vector with the APC can occur in vitro or in vivo.
Another embodiment of the disclosed invention relates to a substrate polypeptide including a housekeeping epitope wherein the housekeeping epitope can be liberated by immunoproteasome processing in a pAPC.
Another embodiment of the disclosed invention relates to a method of activating a T cell comprising contacting a substrate polypeptide including a housekeeping epitope with an APC wherein the housekeeping epitope can be liberated by immunoproteasome processing and contacting the APC with a T cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the sequence of Melan-A (SEQ ID NO: 2), showing clustering of class I HLA epitopes.
FIG. 2 depicts the sequence of SSX-2 (SEQ ID NO: 40), showing clustering of class I HLA epitopes.
FIG. 3 depicts the sequence of NY-ESO (SEQ ID NO: 11), showing clustering of class I HLA epitopes.
FIG. 4. An illustrative drawing depicting pMA2M.
FIG. 5. Assay results showing the % of specific lysis of ELAGIGILTV (SEQ ID NO: 1) pulsed and unpulsed T2 target cells by mock immunized CTL.
FIG. 6. Assay results showing the % of specific lysis of ELAGIGILTV (SEQ ID NO: 1) pulsed and unpulsed T2 target cells by pVXM3 immunized CTL.
FIG. 7. Assay results showing the % of specific lysis of ELAGIGILTV (SEQ ID NO: 1) pulsed and unpulsed T2 target cells by pVXM2 immunized CTL.
FIG. 8. Assay results showing the % of specific lysis of ELAGIGILTV (SEQ ID NO: 1) pulsed and unpulsed T2 target cells by pVXMI immunized CTL.
FIG. 9. Illustrates a sequence of SEQ ID NO. 22 from which the NY-ESO-1157-165 epitope is liberated by immunoproteasomal processing.
FIG. 10. Shows the differential processing by immunoproteasome and housekeeping proteasome of the SLLMWITQC (SEQ ID NO. 12) epitope in its native context where the cleavage following the C is more efficiently produced by housekeeping than immunoproteasome.
FIG. 11. 11A: Shows the results of the human immunoproteasome digest of SEQ ID NO. 31. 11B: Shows the comparative results of mouse versus human immunoproteasome digestion of SEQ ID NO. 31.
FIG. 12. Shows the differing processing of SSX-231-68 (SEQ ID NO: 986) by housekeeping and immunoproteasome.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Definitions
Unless otherwise clear from the context of the use of a term herein, the following listed terms shall generally have the indicated meanings for purposes of this description.
PROFESSIONAL ANTIGEN-PRESENTING CELL (pAPC)—a cell that possesses T cell costimulatory molecules and is able to induce a T cell response. Well characterized pAPCs include dendritic cells, B cells, and macrophages.
PERIPHERAL CELL—a cell that is not a pAPC.
HOUSEKEEPING PROTEASOME—a proteasome normally active in peripheral cells, and generally not present or not strongly active in pAPCs.
IMMUNOPROTEASOME—a proteasome normally active in pAPCs; the immunoproteasome is also active in some peripheral cells in infected tissues or following exposure to interferon.
EPITOPE—a molecule or substance capable of stimulating an immune response. In preferred embodiments, epitopes according to this definition include but are not necessarily limited to a polypeptide and a nucleic acid encoding a polypeptide, wherein the polypeptide is capable of stimulating an immune response. In other preferred embodiments, epitopes according to this definition include but are not necessarily limited to peptides presented on the surface of cells, the peptides being non-covalently bound to the binding cleft of class I MHC, such that they can interact with T cell receptors (TCR). Epitopes presented by class I MHC may be in immature or mature form. “Mature” refers to an MHC epitope in distinction to any precursor (“immature”) that may include or consist essentially of a housekeeping epitope, but also includes other sequences in a primary translation product that are removed by processing, including without limitation, alone or in any combination, proteasomal digestion, N-terminal trimming, or the action of exogenous enzymatic activities. Thus, a mature epitope may be provided embedded in a somewhat longer polypeptide, the immunological potential of which is due, at least in part, to the embedded epitope; or in its ultimate form that can bind in the MHC binding cleft to be recognized by TCR, respectively.
MHC EPITOPE—a polypeptide having a known or predicted binding affinity for a mammalian class I or class II major histocompatibility complex (MHC) molecule.
HOUSEKEEPING EPITOPE—In a preferred embodiment, a housekeeping epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which housekeeping proteasomes are predominantly active. In another preferred embodiment, a housekeeping epitope is defined as a polypeptide containing a housekeeping epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, a housekeeping epitope is defined as a nucleic acid that encodes a housekeeping epitope according to the foregoing definitions. Exemplary housekeeping epitopes are provide in U.S. application Ser. No. 10/117,937, filed on Apr. 4, 2002; and U.S. Provisional Application No. 60/282,211, filed on Apr. 6, 2001; 60/337,017, filed on Nov. 7, 2001; 60/363,210 filed Mar. 7, 2002; and 60/409,123, filed on Sep. 5, 2002; all of which are entitled EPITOPE SEQUENCES, and all of which above were incorporated herein by reference in their entireties.
IMMUNE EPITOPE—In a preferred embodiment, an immune epitope is defined as a polypeptide fragment that is an MHC epitope, and that is displayed on a cell in which immunoproteasomes are predominantly active. In another preferred embodiment, an immune epitope is defined as a polypeptide containing an immune epitope according to the foregoing definition, that is flanked by one to several additional amino acids. In another preferred embodiment, an immune epitope is defined as a polypeptide including an epitope cluster sequence, having at least two polypeptide sequences having a known or predicted affinity for a class I MHC. In yet another preferred embodiment, an immune epitope is defined as a nucleic acid that encodes an immune epitope according to any of the foregoing definitions.
TARGET CELL—a cell to be targeted by the vaccines and methods of the invention. Examples of target cells according to this definition include but are not necessarily limited to: a neoplastic cell and a cell harboring an intracellular parasite, such as, for example, a virus, a bacterium, or a protozoan. Target cells can also include cells that are targeted by CTL as a part of assays to determine or confirm proper epitope liberation and processing by a cell expressing immunoproteasome, to determine T cell specificity or immunogenicity for a desired epitope. Such cells may be transformed to express the substrate or liberation sequence, or the cells can simply be pulsed with peptide/epitope.
TARGET-ASSOCIATED ANTIGEN (TAA)—a protein or polypeptide present in a target cell.
TUMOR-ASSOCIATED ANTIGENS (TuAA)—a TAA, wherein the target cell is a neoplastic cell.
HLA EPITOPE—a polypeptide having a known or predicted binding affinity for a human class I or class II HLA complex molecule.
ANTIBODY—a natural immunoglobulin (Ig), poly- or monoclonal, or any molecule composed in whole or in part of an Ig binding domain, whether derived biochemically or by use of recombinant DNA. Examples include inter alia, F(ab), single chain Fv, and Ig variable region-phage coat protein fusions.
ENCODE—an open-ended term such that a nucleic acid encoding a particular amino acid sequence can consist of codons specifying that (poly)peptide, but can also comprise additional sequences either translatable, or for the control of transcription, translation, or replication, or to facilitate manipulation of some host nucleic acid construct.
SUBSTANTIAL SIMILARITY—this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of the sequence. Nucleic acid sequences encoding the same amino acid sequence are substantially similar despite differences in degenerate positions or modest differences in length or composition of any non-coding regions. Amino acid sequences differing only by conservative substitution or minor length variations are substantially similar. Additionally, amino acid sequences comprising housekeeping epitopes that differ in the number of N-terminal flanking residues, or immune epitopes and epitope clusters that differ in the number of flanking residues at either terminus, are substantially similar. Nucleic acids that encode substantially similar amino acid sequences are themselves also substantially similar.
FUNCTIONAL SIMILARITY—this term is used to refer to sequences that differ from a reference sequence in an inconsequential way as judged by examination of a biological or biochemical property, although the sequences may not be substantially similar. For example, two nucleic acids can be useful as hybridization probes for the same sequence but encode differing amino acid sequences. Two peptides that induce cross-reactive CTL responses are functionally similar even if they differ by non-conservative amino acid substitutions (and thus do not meet the substantial similarity definition). Pairs of antibodies, or TCRs, that recognize the same epitope can be functionally similar to each other despite whatever structural differences exist. In testing for functional similarity of immunogenicity one would generally immunize with the “altered” antigen and test the ability of the elicited response (Ab, CTL, cytokine production, etc.) to recognize the target antigen. Accordingly, two sequences may be designed to differ in certain respects while retaining the same function. Such designed sequence variants are among the embodiments of the present invention.
EXPRESSION CASSETTE—a polynucleotide sequence encoding a polypeptide, operably linked to a promoter and other transcription and translation control elements, including but not limited to enhancers, termination codons, internal ribosome entry sites, and polyadenylation sites. The cassette can also include sequences that facilitate moving it from one host molecule to another.
EMBEDDED EPITOPE—an epitope contained within a longer polypeptide, also can include an epitope in which either the N-terminus or the C-terminus is embedded such that the epitope is not in an interior position.
MATURE EPITOPE—a peptide with no additional sequence beyond that present when the epitope is bound in the MHC peptide-binding cleft.
EPITOPE CLUSTER—a polypeptide, or a nucleic acid sequence encoding it, that is a segment of a native protein sequence comprising two or more known or predicted epitopes with binding affinity for a shared MHC restriction element, wherein the density of epitopes within the cluster is greater than the density of all known or predicted epitopes with binding affinity for the shared MHC restriction element within the complete protein sequence, and as disclosed in U.S. patent application Ser. No. 09/561,571 entitled EPITOPE CLUSTERS.
SUBSTRATE OR LIBERATION SEQUENCE—a designed or engineered sequence comprising or encoding a housekeeping epitope (according to the first of the definitions offered above) embedded in a larger sequence that provides a context allowing the housekeeping epitope to be liberated by immunoproteasomal processing, directly or in combination with N-terminal trimming or other processes.
Epitope Clusters
Embodiments of the invention disclosed herein provide epitope cluster regions (ECRs) for use in vaccines and in vaccine design and epitope discovery. Specifically, embodiments of the invention relate to identifying epitope clusters for use in generating immunologically active compositions directed against target cell populations, and for use in the discovery of discrete housekeeping epitopes and immune epitopes. In many cases, numerous putative class I MHC epitopes may exist in a single target-associated antigen (TAA). Such putative epitopes are often found in clusters (ECRs), MHC epitopes distributed at a relatively high density within certain regions in the amino acid sequence of the parent TAA. Since these ECRs include multiple putative epitopes with potential useful biological activity in inducing an immune response, they represent an excellent material for in vitro or in vivo analysis to identify particularly useful epitopes for vaccine design. And, since the epitope clusters can themselves be processed inside a cell to produce active MHC epitopes, the clusters can be used directly in vaccines, with one or more putative epitopes in the cluster actually being processed into an active MHC epitope.
The use of ECRs in vaccines offers important technological advances in the manufacture of recombinant vaccines, and further offers crucial advantages in safety over existing nucleic acid vaccines that encode whole protein sequences. Recombinant vaccines generally rely on expensive and technically challenging production of whole proteins in microbial fermentors. ECRs offer the option of using chemically synthesized polypeptides, greatly simplifying development and manufacture, and obviating a variety of safety concerns. Similarly, the ability to use nucleic acid sequences encoding ECRs, which are typically relatively short regions of an entire sequence, allows the use of synthetic oligonucleotide chemistry processes in the development and manipulation of nucleic acid based vaccines, rather than the more expensive, time consuming, and potentially difficult molecular biology procedures involved with using whole gene sequences.
Since an ECR is encoded by a nucleic acid sequence that is relatively short compared to that which encodes the whole protein from which the ECR is found, this can greatly improve the safety of nucleic acid vaccines. An important issue in the field of nucleic acid vaccines is the fact that the extent of sequence homology of the vaccine with sequences in the animal to which it is administered determines the probability of integration of the vaccine sequence into the genome of the animal. A fundamental safety concern of nucleic acid vaccines is their potential to integrate into genomic sequences, which can cause deregulation of gene expression and tumor transformation. The Food and Drug Administration has advised that nucleic acid and recombinant vaccines should contain as little sequence homology with human sequences as possible. In the case of vaccines delivering tumor-associated antigens, it is inevitable that the vaccines contain nucleic acid sequences that are homologous to those which encode proteins that are expressed in the tumor cells of patients. It is, however, highly desirable to limit the extent of those sequences to that which is minimally essential to facilitate the expression of epitopes for inducing therapeutic immune responses. The use of ECRs thus offers the dual benefit of providing a minimal region of homology, while incorporating multiple epitopes that have potential therapeutic value.
Note that the following discussion sets forth the inventors' understanding of the operation of the invention. However, it is not intended that this discussion limit the patent to any particular theory of operation not set forth in the claims.
ECRs are Processed into MHC-Binding Epitopes in pAPCs
The immune system constantly surveys the body for the presence of foreign antigens, in part through the activity of pAPCs. The pAPCs endocytose matter found in the extracellular milieu, process that matter from a polypeptide form into shorter oligopeptides of about 3 to 23 amino acids in length, and display some of the resulting peptides to T cells via the MHC complex of the pAPCs. For example, a tumor cell upon lysis releases its cellular contents, including various proteins, into the extracellular milieu. Those released proteins can be endocytosed by pAPCs and processed into discrete peptides that are then displayed on the surface of the pAPCs via the MHC. By this mechanism, it is not the entire target protein that is presented on the surface of the pAPCs, but rather only one or more discrete fragments of that protein that are presented as MHC-binding epitopes. If a presented epitope is recognized by a T cell, that T cell is activated and an immune response results.
Similarly, the scavenger receptors on pAPC can take-up naked nucleic acid sequences or recombinant organisms containing target nucleic acid sequences. Uptake of the nucleic acid sequences into the pAPC subsequently results in the expression of the encoded products. As above, when an ECR can be processed into one or more useful epitopes, these products can be presented as MHC epitopes for recognition by T cells.
MHC-binding epitopes are often distributed unevenly throughout a protein sequence in clusters. Embodiments of the invention are directed to identifying epitope cluster regions (ECRs) in a particular region of a target protein. Candidate ECRs are likely to be natural substrates for various proteolytic enzymes and are likely to be processed into one or more epitopes for MHC display on the surface of an pAPC. In contrast to more traditional vaccines that deliver whole proteins or biological agents, ECRs can be administered as vaccines, resulting in a high probability that at least one epitope will be presented on MHC without requiring the use of a full length sequence.
The Use of ECRs in Identifying Discrete MHC-Binding Epitopes
Identifying putative MHC epitopes for use in vaccines often includes the use of available predictive algorithms that analyze the sequences of proteins or genes to predict binding affinity of peptide fragments for MHC. These algorithms rank putative epitopes according to predicted affinity or other characteristics associated with MHC binding. Exemplary algorithms for this kind of analysis include the Rammensee and NIH (Parker) algorithms. However, identifying epitopes that are naturally present on the surface of cells from among putative epitopes predicted using these algorithms has proven to be a difficult and laborious process. The use of ECRs in an epitope identification process can enormously simplify the task of identifying discrete MHC binding epitopes.
In a preferred embodiment, ECR polypeptides are synthesized on an automated peptide synthesizer and these ECRs are then subjected to in vitro digests using proteolytic enzymes involved in processing proteins for presentation of the epitopes. Mass spectrometry and/or analytical HPLC are then used to identify the digest products and in vitro MHC binding studies are used to assess the ability of these products to actually bind to MHC. Once epitopes contained in ECRs have been shown to bind MHC, they can be incorporated into vaccines or used as diagnostics, either as discrete epitopes or in the context of ECRs.
The use of an ECR (which because of its relatively short sequence can be produced through chemical synthesis) in this preferred embodiment is a significant improvement over what otherwise would require the use of whole protein. This is because whole proteins have to be produced using recombinant expression vector systems and/or complex purification procedures. The simplicity of using chemically synthesized ECRs enables the analysis and identification of large numbers of epitopes, while greatly reducing the time and expense of the process as compared to other currently used methods. The use of a defined ECR also greatly simplifies mass spectrum analysis of the digest, since the products of an ECR digest are a small fraction of the digest products of a whole protein.
In another embodiment, nucleic acid sequences encoding ECRs are used to express the polypeptides in cells or cell lines to assess which epitopes are presented on the surface. A variety of means can be used to detect the epitope on the surface. Preferred embodiments involve the lysis of the cells and affinity purification of the MHC, and subsequent elution and analysis of peptides from the MHC; or elution of epitopes from intact cells; (Falk, K. et al. Nature 351:290, 1991, and U.S. Pat. No. 5,989,565, respectively, both of which references are incorporated herein by reference in their entirety). A sensitive method for analyzing peptides eluted in this way from the MHC employs capillary or nanocapillary HPLC ESI mass spectrometry and on-line sequencing.
Target-Associated Antigens that Contain ECRs
TAAs from which ECRs may be defined include those from TuAAs, including oncofetal, cancer-testis, deregulated genes, fusion genes from errant translocations, differentiation antigens, embryonic antigens, cell cycle proteins, mutated tumor suppressor genes, and overexpressed gene products, including oncogenes. In addition, ECRs may be derived from virus gene products, particularly those associated with viruses that cause chronic diseases or are oncogenic, such as the herpes viruses, human papilloma viruses, human immunodeficiency virus, and human T cell leukemia virus. Also ECRs may be derived from gene products of parasitic organisms, such as Trypanosoma, Leishmania, and other intracellular or parasitic organisms.
Some of these TuAA include α-fetoprotein, carcinoembryonic antigen (CEA), esophageal cancer derived NY-ESO-1, and SSX genes, SCP-1, PRAME, MART-1/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR1 and viral antigens, EBNA1, EBNA2, HPV-E6, -E7; prostate specific antigen (PSA), prostate stem cell antigen (PSCA), MAAT-1, GP-100, TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, p185erbB-2, p185erbB-3, c-met, nm-23H1, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p15, and p16.
Numerous other TAAs are also contemplated for both pathogens and tumors. In terms of TuAAs, a variety of methods are available and well known in the art to identify genes and gene products that are differentially expressed in neoplastic cells as compared to normal cells. Examples of these techniques include differential hybridization, including the use of microarrays; subtractive hybridization cloning; differential display, either at the level of mRNA or protein expression; EST sequencing; and SAGE (sequential analysis of gene expression). These nucleic acid techniques have been reviewed by Carulli, J. P. et al., J. Cellular Biochem Suppl. 30/31:286-296, 1998 (hereby incorporated by reference). Differential display of proteins involves, for example, comparison of two-dimensional poly-acrylamide gel electrophoresis of cell lysates from tumor and normal tissue, location of protein spots unique or overexpressed in the tumor, recovery of the protein from the gel, and identification of the protein using traditional biochemical- or mass spectrometry-based sequencing. An additional technique for identification of TAAs is the Serex technique, discussed in Türeci, Ö., Sahin, U., and Pfreundschuh, M., “Serological analysis of human tumor antigens: molecular definition and implications”, Molecular Medicine Today, 3:342, 1997, and hereby incorporated by reference.
Use of these and other methods provides one of skill in the art the techniques necessary to identify genes and gene products contained within a target cell that may be used as potential candidate proteins for generating the epitopes of the invention disclosed. However, it is not necessary, in practicing the invention, to identify a novel TuAA or TAA. Rather, embodiments of the invention make it possible to identify ECRs from any relevant protein sequence, whether the sequence is already known or is new.
Protein Sequence Analysis to Identify Epitope Clusters
In preferred embodiments of the invention, identification of ECRs involves two main steps: (1) identifying good putative epitopes; and (2) defining the limits of any clusters in which these putative epitopes are located. There are various preferred embodiments of each of these two steps, and a selected embodiment for the first step can be freely combined with a selected embodiment for the second step. The methods and embodiments that are disclosed herein for each of these steps are merely exemplary, and are not intended to limit the scope of the invention in any way. Persons of skill in the art will appreciate the specific tools that can be applied to the analysis of a specific TAA, and such analysis can be conducted in numerous ways in accordance with the invention.
Preferred embodiments for identifying good putative epitopes include the use of any available predictive algorithm that analyzes the sequences of proteins or genes to predict binding affinity of peptide fragments for MHC, or to rank putative epitopes according to predicted affinity or other characteristics associated with MHC binding. As described above, available exemplary algorithms for this kind of analysis include the Rammensee and NIH (Parker) algorithms. Likewise, good putative epitopes can be identified by direct or indirect assays of MHC binding. To choose “good” putative epitopes, it is necessary to set a cutoff point in terms of the score reported by the prediction software or in terms of the assayed binding affinity. In some embodiments, such a cutoff is absolute. For example, the cutoff can be based on the measured or predicted half time of dissociation between an epitope and a selected MHC allele. In such cases, embodiments of the cutoff can be any half time of dissociation longer than, for example, 0.5 minutes; in a preferred embodiment longer than 2.5 minutes; in a more preferred embodiment longer than 5 minutes; and in a highly stringent embodiment can be longer than 10, or 20, or 25 minutes. In these embodiments, the good putative epitopes are those that are predicted or identified to have good MHC binding characteristics, defined as being on the desirable side of the designated cutoff point. Likewise, the cutoff can be based on the measured or predicted binding affinity between an epitope and a selected MHC allele. Additionally, the absolute cutoff can be simply a selected number of putative epitopes.
In other embodiments, the cutoff is relative. For example, a selected percentage of the total number of putative epitopes can be used to establish the cutoff for defining a candidate sequence as a good putative epitope. Again the properties for ranking the epitopes are derived from measured or predicted MHC binding; the property used for such a determination can be any that is relevant to or indicative of binding. In preferred embodiments, identification of good putative epitopes can combine multiple methods of ranking candidate sequences. In such embodiments, the good epitopes are typically those that either represent a consensus of the good epitopes based on different methods and parameters, or that are particularly highly ranked by at least one of the methods.
When several good putative epitopes have been identified, their positions relative to each other can be analyzed to determine the optimal clusters for use in vaccines or in vaccine design. This analysis is based on the density of a selected epitope characteristic within the sequence of the TAA. The regions with the highest density of the characteristic, or with a density above a certain selected cutoff, are designated as ECRs. Various embodiments of the invention employ different characteristics for the density analysis. For example, one preferred characteristic is simply the presence of any good putative epitope (as defined by any appropriate method). In this embodiment, all putative epitopes above the cutoff are treated equally in the density analysis, and the best clusters are those with the highest density of good putative epitopes per amino acid residue. In another embodiment, the preferred characteristic is based on the parameter(s) previously used to score or rank the putative epitopes. In this embodiment, a putative epitope with a score that is twice as high as another putative epitope is doubly weighted in the density analysis, relative to the other putative epitope. Still other embodiments take the score or rank into account, but on a diminished scale, such as, for example, by using the log or the square root of the score to give more weight to some putative epitopes than to others in the density analysis.
Depending on the length of the TAA to be analyzed, the number of possible candidate epitopes, the number of good putative epitopes, the variability of the scoring of the good putative epitopes, and other factors that become evident in any given analysis, the various embodiments of the invention can be used alone or in combination to identify those ECRs that are most useful for a given application. Iterative or parallel analyses employing multiple approaches can be beneficial in many cases. ECRs are tools for increased efficiency of identifying true MHC epitopes, and for efficient “packaging” of MHC epitopes into vaccines. Accordingly, any of the embodiments described herein, or other embodiments that are evident to those of skill in the art based on this disclosure, are useful in enhancing the efficiency of these efforts by using ECRs instead of using complete TAAs in vaccines and vaccine design.
Since many or most TAAs have regions with low density of predicted MHC epitopes, using ECRs provides a valuable methodology that avoids the inefficiencies of including regions of low epitope density in vaccines and in epitope identification protocols. Thus, useful ECRs can also be defined as any portion of a TAA that is not the whole TAA, wherein the portion has a higher density of putative epitopes than the whole TAA, or than any regions of the TAA that have a particularly low density of putative epitopes. In this aspect of the invention, therefore, an ECR can be any fragment of a TAA with elevated epitope density. In some embodiments, an ECR can include a region up to about 80% of the length of the TAA. In a preferred embodiment, an ECR can include a region up to about 50% of the length of the TAA. In a more preferred embodiment, an ECR can include a region up to about 30% of the length of the TAA. And in a most preferred embodiment, an ECR can include a region of between 5 and 15% of the length of the TAA.
In another aspect of the invention, the ECR can be defined in terms of its absolute length. Accordingly, by this definition, the minimal cluster for 9-mer epitopes includes 10 amino acid residues and has two overlapping 9-mers with 8 amino acids in common. In a preferred embodiment, the cluster is between about 15 and 75 amino acids in length. In a more preferred embodiment, the cluster is between about 20 and 60 amino acids in length. In a most preferred embodiment, the cluster is between about 30 and 40 amino acids in length.
In practice, as described above, ECR identification can employ a simple density function such as the number of epitopes divided by the number of amino acids spanned by the those epitopes. It is not necessarily required that the epitopes overlap, but the value for a single epitope is not significant. If only a single value for a percentage cutoff is used and an absolute cutoff in the epitope prediction is not used, it is possible to set a single threshold at this step to define a cluster. However, using both an absolute cutoff and carrying out the first step using different percentage cutoffs, can produce variations in the global density of candidate epitopes. Such variations can require further accounting or manipulation. For example, an overlap of 2 epitopes is more significant if only 3 candidate epitopes were considered, than if 30 candidates were considered for any particular length protein. To take this feature into consideration, the weight given to a particular cluster can further be divided by the fraction of possible peptides actually being considered, in order to increase the significance of the calculation. This scales the result to the average density of predicted epitopes in the parent protein.
Similarly, some embodiments base the scoring of good putative epitopes on the average number of peptides considered per amino acid in the protein. The resulting ratio represents the factor by which the density of predicted epitopes in the putative cluster differs from the average density in the protein. Accordingly, an ECR is defined in one embodiment as any region containing two or more predicted epitopes for which this ratio exceeds 2, that is, any region with twice the average density of epitopes. In other embodiments, the region is defined as an ECR if the ratio exceeds 1.5, 3, 4, or 5, or more.
Considering the average number of peptides per amino acid in a target protein to calculate the presence of an ECR highlights densely populated ECRs without regard to the score/affinity of the individual constituents. This is most appropriate for use of score-based cutoffs. However, an ECR with only a small number of highly ranked candidates can be of more biological significance than a cluster with several densely packed but lower ranking candidates, particularly if only a small percentage of the total number of candidate peptides were designated as good putative epitopes. Thus in some embodiments it is appropriate to take into consideration the scores of the individual peptides. This is most readily accomplished by substituting the sum of the scores of the peptides in the putative cluster for the number of peptides in the putative cluster in the calculation described above.
This sum of scores method is more sensitive to sparsely populated clusters containing high scoring epitopes. Because the wide range of scores (i.e. half times of dissociation) produced by the BIMAS-NIH/Parker algorithm can lead to a single high scoring peptide dwarfing the contribution of other potential epitopes, the log of the score rather than the score itself is preferably used in this procedure.
Various other calculations can be devised under one or another condition. Generally speaking, the epitope density function is constructed so that it is proportional to the number of predicted epitopes, their scores, their ranks, and the like, within the putative cluster, and inversely proportional to the number of amino acids or fraction of protein contained within that putative cluster. Alternatively, the function can be evaluated for a window of a selected number of contiguous amino acids. In either case the function is also evaluated for all predicted epitopes in the whole protein. If the ratio of values for the putative cluster (or window) and the whole protein is greater than, for example, 1.5, 2, 3, 4, 5, or more, an ECR is defined.
Analysis of Target Gene Products For MHC Binding
Once a TAA has been identified, the protein sequence can be used to identify putative epitopes with known or predicted affinity to the MHC peptide binding cleft. Tests of peptide fragments can be conducted in vitro, or using the sequence can be computer analyzed to determine MHC receptor binding of the peptide fragments. In one embodiment of the invention, peptide fragments based on the amino acid sequence of the target protein are analyzed for their predicted ability to bind to the MHC peptide binding cleft. Examples of suitable computer algorithms for this purpose include that found at the world wide web page of Hans-Georg Rammensee, Jutta Bachmann, Niels Emmerich, Stefan Stevanovic: SYFPEITHI: An Internet Database for MHC Ligands and Peptide Motifs (access via hypertext transfer protocol: //134.2.96.221/scripts/hlaserver.dll/EpPredict.htm). Results obtained from this method are discussed in Rammensee, et al., “MHC Ligands and Peptide Motifs,” Landes Bioscience Austin, Tex., 224-227, 1997, which is hereby incorporated by reference in its entirety. Another site of interest is found at hypertext transfer protocol: //bimas.dcrt.nih.gov/molbio/hla_bind, which also contains a suitable algorithm. The methods of this web site are discussed in Parker, et al., “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains,” J. Immunol. 152:163-175, which is hereby incorporated by reference in its entirety.
As an alternative to predictive algorithms, a number of standard in vitro receptor binding affinity assays are available to identify peptides having an affinity for a particular allele of MHC. Accordingly, by the method of this aspect of the invention, the initial population of peptide fragments can be narrowed to include only putative epitopes having an actual or predicted affinity for the selected allele of MHC. Selected common alleles of MHC I, and their approximate frequencies, are reported in the tables below.







TABLE 1







Estimated gene frequencies of HLA-A antigens













CAU
AFR
ASI
LAT
NAT

















Antigen
Gfa
SEb
Gf
SE
Gf
SE
Gf
SE
Gf
SE




















A1
15.1843
0.0489
5.7256
0.0771
4.4818
0.0846
7.4007
0.0978
12.0316
0.2533


A2
28.6535
0.0619
18.8849
0.1317
24.6352
0.1794
28.1198
0.1700
29.3408
0.3585


A3
13.3890
0.0463
8.4406
0.0925
2.6454
0.0655
8.0789
0.1019
11.0293
0.2437


A28
4.4652
0.0280
9.9269
0.0997
1.7657
0.0537
8.9446
0.1067
5.3856
0.1750


A36
0.0221
0.0020
1.8836
0.0448
0.0148
0.0049
0.1584
0.0148
0.1545
0.0303


A23
1.8287
0.0181
10.2086
0.1010
0.3256
0.0231
2.9269
0.0628
1.9903
0.1080


A24
9.3251
0.0395
2.9668
0.0560
22.0391
0.1722
13.2610
0.1271
12.6613
0.2590


A9 unsplit
0.0809
0.0038
0.0367
0.0063
0.0858
0.0119
0.0537
0.0086
0.0356
0.0145


A9 total
11.2347
0.0429
13.2121
0.1128
22.4505
0.1733
16.2416
0.1382
14.6872
0.2756


A25
2.1157
0.0195
0.4329
0.0216
0.0990
0.0128
1.1937
0.0404
1.4520
0.0924


A26
3.8795
0.0262
2.8284
0.0547
4.6628
0.0862
3.2612
0.0662
2.4292
0.1191


A34
0.1508
0.0052
3.5228
0.0610
1.3529
0.0470
0.4928
0.0260
0.3150
0.0432


A43
0.0018
0.0006
0.0334
0.0060
0.0231
0.0062
0.0055
0.0028
0.0059
0.0059


A66
0.0173
0.0018
0.2233
0.0155
0.0478
0.0089
0.0399
0.0074
0.0534
0.0178


A10 unsplit
0.0790
0.0038
0.0939
0.0101
0.1255
0.0144
0.0647
0.0094
0.0298
0.0133


A10 total
6.2441
0.0328
7.1348
0.0850
6.3111
0.0993
5.0578
0.0816
4.2853
0.1565


A29
3.5796
0.0252
3.2071
0.0582
1.1233
0.0429
4.5156
0.0774
3.4345
0.1410


A30
2.5067
0.0212
13.0969
0.1129
2.2025
0.0598
4.4873
0.0772
2.5314
0.1215


A31
2.7386
0.0221
1.6556
0.0420
3.6005
0.0761
4.8328
0.0800
6.0881
0.1855


A32
3.6956
0.0256
1.5384
0.0405
1.0331
0.0411
2.7064
0.0604
2.5521
0.1220


A33
1.2080
0.0148
6.5607
0.0822
9.2701
0.1191
2.6593
0.0599
1.0754
0.0796


A74
0.0277
0.0022
1.9949
0.0461
0.0561
0.0096
0.2027
0.0167
0.1068
0.0252


A19 unsplit
0.0567
0.0032
0.2057
0.0149
0.0990
0.0128
0.1211
0.0129
0.0475
0.0168


A19 total
13.8129
0.0468
28.2593
0.1504
17.3846
0.1555
19.5252
0.1481
15.8358
0.2832


AX
0.8204
0.0297
4.9506
0.0963
2.9916
0.1177
1.6332
0.0878
1.8454
0.1925





aGene frequency.


bStandard error.













TABLE 2







Estimated gene frequencies for HLA-B antigens













CAU
AFR
ASI
LAT
NAT

















Antigen
Gfa
SEb
Gf
SE
Gf
SE
Gf
SE
Gf
SE




















B7
12.1782
0.0445
10.5960
0.1024
4.2691
0.0827
6.4477
0.0918
10.9845 
0.2432


B8
9.4077
0.0397
3.8315
0.0634
1.3322
0.0467
3.8225
0.0715
8.5789
0.2176


B13
2.3061
0.0203
0.8103
0.0295
4.9222
0.0886
1.2699
0.0416
1.7495
0.1013


B14
4.3481
0.0277
3.0331
0.0566
0.5004
0.0287
5.4166
0.0846
2.9823
0.1316


B18
4.7980
0.0290
3.2057
0.0582
1.1246
0.0429
4.2349
0.0752
3.3422
0.1391


B27
4.3831
0.0278
1.2918
0.0372
2.2355
0.0603
2.3724
0.0567
5.1970
0.1721


B35
9.6614
0.0402
8.5172
0.0927
8.1203
0.1122
14.6516
0.1329
10.1198 
0.2345


B37
1.4032
0.0159
0.5916
0.0252
1.2327
0.0449
0.7807
0.0327
0.9755
0.0759


B41
0.9211
0.0129
0.8183
0.0296
0.1303
0.0147
1.2818
0.0418
0.4766
0.0531


B42
0.0608
0.0033
5.6991
0.0768
0.0841
0.0118
0.5866
0.0284
0.2856
0.0411


B46
0.0099
0.0013
0.0151
0.0040
4.9292
0.0886
0.0234
0.0057
0.0238
0.0119


B47
0.2069
0.0061
0.1305
0.0119
0.0956
0.0126
0.1832
0.0159
0.2139
0.0356


B48
0.0865
0.0040
0.1316
0.0119
2.0276
0.0575
1.5915
0.0466
1.0267
0.0778


B53
0.4620
0.0092
10.9529
0.1039
0.4315
0.0266
1.6982
0.0481
1.0804
0.0798


B59
0.0020
0.0006
0.0032
0.0019
0.4277
0.0265
0.0055
0.0028
0c   
—


B67
0.0040
0.0009
0.0086
0.0030
0.2276
0.0194
0.0055
0.0028
0.0059
0.0059


B70
0.3270
0.0077
7.3571
0.0866
0.8901
0.0382
1.9266
0.0512
0.6901
0.0639


B73
0.0108
0.0014
0.0032
0.0019
0.0132
0.0047
0.0261
0.0060
0c   
—


B51
5.4215
0.0307
2.5980
0.0525
7.4751
0.1080
6.8147
0.0943
6.9077
0.1968


B52
0.9658
0.0132
1.3712
0.0383
3.5121
0.0752
2.2447
0.0552
0.6960
0.0641


B5 unsplit
0.1565
0.0053
0.1522
0.0128
0.1288
0.0146
0.1546
0.0146
0.1307
0.0278


B5 total
6.5438
0.0435
4.1214
0.0747
11.1160
0.1504
9.2141
0.1324
7.7344
0.2784


B44
13.4838
0.0465
7.0137
0.0847
5.6807
0.0948
9.9253
0.1121
11.8024 
0.2511


B45
0.5771
0.0102
4.8069
0.0708
0.1816
0.0173
1.8812
0.0506
0.7603
0.0670


B12 unsplit
0.0788
0.0038
0.0280
0.0055
0.0049
0.0029
0.0193
0.0051
0.0654
0.0197


B12 total
14.1440
0.0474
11.8486
0.1072
5.8673
0.0963
11.8258
0.1210
12.6281
0.2584


B62
5.9117
0.0320
1.5267
0.0404
9.2249
0.1190
4.1825
0.0747
6.9421
0.1973


B63
0.4302
0.0088
1.8865
0.0448
0.4438
0.0270
0.8083
0.0333
0.3738
0.0471


B75
0.0104
0.0014
0.0226
0.0049
1.9673
0.0566
0.1101
0.0123
0.0356
0.0145


B76
0.0026
0.0007
0.0065
0.0026
0.0874
0.0120
0.0055
0.0028
0   
—


B77
0.0057
0.0010
0.0119
0.0036
0.0577
0.0098
0.0083
0.0034
0c   
0.0059


B15 unsplit
0.1305
0.0049
0.0691
0.0086
0.4301
0.0266
0.1820
0.0158
0.0059
0.0206


B15 total
6.4910
0.0334
3.5232
0.0608
12.2112
0.1344
5.2967
0.0835
0.0715
0.2035











7.4290



B38
2.4413
0.0209
0.3323
0.0189
3.2818
0.0728
1.9652
0.0517
1.1017
0.0806


B39
1.9614
0.0188
1.2893
0.0371
2.0352
0.0576
6.3040
0.0909
4.5527
0.1615


B16 unsplit
0.0638
0.0034
0.0237
0.0051
0.0644
0.0103
0.1226
0.0130
0.0593
0.0188


B16 total
4.4667
0.0280
1.6453
0.0419
5.3814
0.0921
8.3917
0.1036
5.7137
0.1797


B57
3.5955
0.0252
5.6746
0.0766
2.5782
0.0647
2.1800
0.0544
2.7265
0.1260


B58
0.7152
0.0114
5.9546
0.0784
4.0189
0.0803
1.2481
0.0413
0.9398
0.0745


B17 unsplit
0.2845
0.0072
0.3248
0.0187
0.3751
0.0248
0.1446
0.0141
0.2674
0.0398


B17 total
4.5952
0.0284
11.9540
0.1076
6.9722
0.1041
3.5727
0.0691
3.9338
0.1503


B49
1.6452
0.0172
2.6286
0.0528
0.2440
0.0200
2.3353
0.0562
1.5462
0.0953


B50
1.0580
0.0138
0.8636
0.0304
0.4421
0.0270
1.8883
0.0507
0.7862
0.0681


B21 unsplit
0.0702
0.0036
0.0270
0.0054
0.0132
0.0047
0.0771
0.0103
0.0356
0.0145


B21 total
2.7733
0.0222
3.5192
0.0608
0.6993
0.0339
4.3007
0.0755
2.3680
0.1174


B54
0.0124
0.0015
0.0183
0.0044
2.6873
0.0660
0.0289
0.0063
0.0534
0.0178


B55
1.9046
0.0185
0.4895
0.0229
2.2444
0.0604
0.9515
0.0361
1.4054
0.0909


B56
0.5527
0.0100
0.2686
0.0170
0.8260
0.0368
0.3596
0.0222
0.3387
0.0448


B22 unsplit
0.1682
0.0055
0.0496
0.0073
0.2730
0.0212
0.0372
0.0071
0.1246
0.0272


B22 total
2.0852
0.0217
0.8261
0.0297
6.0307
0.0971
1.3771
0.0433
1.9221
0.1060


B60
5.2222
0.0302
1.5299
0.0404
8.3254
0.1135
2.2538
0.0553
5.7218
0.1801


B61
1.1916
0.0147
0.4709
0.0225
6.2072
0.0989
4.6691
0.0788
2.6023
0.1231


B40 unsplit
0.2696
0.0070
0.0388
0.0065
0.3205
0.0230
0.2473
0.0184
0.2271
0.0367


B40 total
6.6834
0.0338
2.0396
0.0465
14.8531
0.1462
7.1702
0.0963
8.5512
0.2168


BX
1.0922
0.0252
3.5258
0.0802
3.8749
0.0988
2.5266
0.0807
1.9867
0.1634





aGene frequency.


bStandard error.


cThe observed gene count was zero.













TABLE 3







Estimated gene frequencies of HLA-DR antigens













CAU
AFR
ASI
LAT
NAT

















Antigen
Gfa
SEb
Gf
SE
Gf
SE
Gf
SE
Gf
SE




















DR1
10.2279
0.0413
6.8200
0.0832
3.4628
0.0747
7.9859
0.1013
8.2512
0.2139


DR2
15.2408
0.0491
16.2373
0.1222
18.6162
0.1608
11.2389
0.1182
15.3932
0.2818


DR3
10.8708
0.0424
13.3080
0.1124
4.7223
0.0867
7.8998
0.1008
10.2549
0.2361


DR4
16.7589
0.0511
5.7084
0.0765
15.4623
0.1490
20.5373
0.1520
19.8264
0.3123


DR6
14.3937
0.0479
18.6117
0.1291
13.4471
0.1404
17.0265
0.1411
14.8021
0.2772


DR7
13.2807
0.0463
10.1317
0.0997
6.9270
0.1040
10.6726
0.1155
10.4219
0.2378


DR8
2.8820
0.0227
6.2673
0.0800
6.5413
0.1013
9.7731
0.1110
6.0059
0.1844


DR9
1.0616
0.0139
2.9646
0.0559
9.7527
0.1218
1.0712
0.0383
2.8662
0.1291


DR10
1.4790
0.0163
2.0397
0.0465
2.2304
0.0602
1.8044
0.0495
1.0896
0.0801


DR11
9.3180
0.0396
10.6151
0.1018
4.7375
0.0869
7.0411
0.0955
5.3152
0.1740


DR12
1.9070
0.0185
4.1152
0.0655
10.1365
0.1239
1.7244
0.0484
2.0132
0.1086


DR5 unsplit
1.2199
0.0149
2.2957
0.0493
1.4118
0.0480
1.8225
0.0498
1.6769
0.0992


DR5 total
12.4449
0.0045
17.0260
0.1243
16.2858
0.1516
10.5880
0.1148
9.0052
0.2218


DRX
1.3598
0.0342
0.8853
0.0760
2.5521
0.1089
1.4023
0.0930
2.0834
0.2037





aGene frequency.


bStandard error.






It has been observed that predicted epitopes often cluster at one or more particular regions within the amino acid sequence of a TAA. The identification of such ECRs offers a simple and practicable solution to the problem of designing effective vaccines for stimulating cellular immunity. For vaccines in which immune epitopes are desired, an ECR is directly useful as a vaccine. This is because the immune proteasomes of the pAPCs can correctly process the cluster, liberating one or more of the contained MHC-binding peptides, in the same way a cell having immune proteasomes activity processes and presents peptides derived from the complete TAA. The cluster is also a useful a starting material for identification of housekeeping epitopes produced by the housekeeping proteasomes active in peripheral cells.
Identification of housekeeping epitopes using ECRs as a starting material is described in copending U.S. patent application Ser. No. 09/561,074 entitled “METHOD OF EPITOPE DISCOVERY,” filed Apr. 28, 2000, which is incorporated herein by reference in its entirety. Epitope synchronization technology and vaccines for use in connection with this invention are disclosed in copending U.S. patent application Ser. No. 09/560,465 entitled “EPITOPE SYNCHRONIZATION IN ANTIGEN PRESENTING CELLS,” filed Apr. 28, 2000, which is incorporated herein by reference in its entirety. Nucleic acid constructs useful as vaccines in accordance with the present invention are disclosed in copending U.S. patent application Ser. No. 09/561,572 entitled “EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS,” filed Apr. 28, 2000, which is incorporated herein by reference in its entirety.
Vector Design and Vectors
Degradation of cytosolic proteins takes place via the ubiquitin-dependent multi-catalytic multi-subunit protease system known as the proteasome. The proteasome degrades cytosolic proteins generating fragments that can then be translocated from the cytosol into the endoplasmic reticulum (ER) for loading onto class I MHC. Such protein fragments shall be referred to as class I peptides. The peptide loaded MHC are subsequently transported to the cell surface where they can be detected by CTL.
The multi-catalytic activity of the proteasome is the result of its multi-subunit structure. Subunits are expressed from different genes and assembled post-translationally into the proteasome complex. A key feature of the proteasome is its bimodal activity, which enables it to exert its protease, or cleavage function, with two discrete kinds of cleavage patterns. This bimodal action of the proteasome is extremely fundamental to understanding how CTL are targeted to recognize peripheral cells in the body and how this targeting requires synchronization between the immune system and the targeted cells.
The housekeeping proteasome is constitutively active in all peripheral cells and tissues of the body. The first mode of operation for the housekeeping proteasome is to degrade cellular protein, recycling it into amino acids. Proteasome function is therefore a necessary activity for cell life. As a corollary to its housekeeping protease activity, however, class I peptides generated by the housekeeping proteasome are presented on all of the peripheral cells of the body.
The proteasome's second mode of function is highly exclusive and occurs specifically in pAPCs or as a consequence of a cellular response to interferons (IFNs). In its second mode of activity the proteasome incorporates unique subunits, which replace the catalytic subunits of the constitutive housekeeping proteasome. This “modified” proteasome has been called the immunoproteasome, owing to its expression in pAPC and as a consequence of induction by IFN in body cells.
APC define the repertoire of CTL that recirculate through the body and are potentially active as killer cells. CTL are activated by interacting with class I peptide presented on the surface of a pAPC. Activated CTL are induced to proliferate and caused to recirculate through the body in search of diseased cells. This is why the CTL response in the body is defined specifically by the class I peptides produced by the pAPC. It is important to remember that pAPCs express the immunoproteasome, and that as a consequence of the bimodal activity of the proteasome, the cleavage pattern of proteins (and the resultant class I peptides produced) are different from those in peripheral body cells which express housekeeping proteasome. The differential proteasome activity in pAPC and peripheral body cells, therefore, is important to consider during natural infection and with therapeutic CTL vaccination strategies.
All cells of the body are capable of producing IFN in the event that they are infected by a pathogen such as a virus. IFN production in turn results in the expression of the immunoproteasome in the infected cell. Viral antigens are thereby processed by the immunoproteasome of the infected cell and the consequent peptides are displayed with class I MHC on the cell surface. At the same time, pAPC are sequestering virus antigens and are processing class I peptides with their immunoproteasome activity, which is normal for the pAPC cell type. The CTL response in the body is being stimulated specifically by the class I peptides produced by the pAPC. Fortunately, the infected cell is also producing class I peptides from the immunoproteasome, rather than the normal housekeeping proteasome. Thus, virus-related class I peptides are being produced that enable detection by the ensuing CTL response. The CTL immune response is induced by pAPC, which normally produce different class I peptides compared to peripheral body cells, owing to different proteasome activity. Therefore, during infection there is epitope synchronization between the infected cell and the immune system.
This is not the case with tumors and chronic viruses, which block the interferon system. For tumors there is no infection in the tumor cell to induce the immunoproteasome expression, and chronic virus infection either directly or indirectly blocks immunoproteasome expression. In both cases the diseased cell maintains its display of class I peptides derived from housekeeping proteasome activity and avoids effective surveillance by CTL.
In the case of therapeutic vaccination to eradicate tumors or chronic infections, the bimodal function of the proteasome and its differential activity in APC and peripheral cells of the body is significant. Upon vaccination with protein antigen, and before a CTL response can occur, the antigen must be acquired and processed into peptides that are subsequently presented on class I MHC on the pAPC surface. The activated CTL recirculate in search of cells with similar class I peptide on the surface. Cells with this peptide will be subjected to destruction by the cytolytic activity of the CTL. If the targeted diseased cell does not express the immunoproteasome, which is present in the pAPC, then the epitopes are not synchronized and CTL fail to find the desired peptide target on the surface of the diseased cell.
Preferably, therapeutic vaccine design takes into account the class I peptide that is actually present on the target tissue. That is, effective antigens used to stimulate CTL to attack diseased tissue are those that are naturally processed and presented on the surface of the diseased tissue. For tumors and chronic infection this generally means that the CTL epitopes are those that have been processed by the housekeeping proteasome. In order to generate an effective therapeutic vaccine, CTL epitopes are identified based on the knowledge that such epitopes are, in fact, produced by the housekeeping proteasome system. Once identified, these epitopes, embodied as peptides, can be used to successfully immunize or induce therapeutic CTL responses against housekeeping proteasome expressing target cells in the host.
However, in the case of DNA vaccines, there can be an additional consideration. The immunization with DNA requires that APCs take up the DNA and express the encoded proteins or peptides. It is possible to encode a discrete class I peptide on the DNA. By immunizing with this construct, APCs can be caused to express a housekeeping epitope, which is then displayed on class I MHC on the surface of the cell for stimulating an appropriate CTL response. Constructs for generation of proper termini of housekeeping epitopes have been described in U.S. patent application Ser. No. 09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OF TARGET-ASSOCIATED ANTIGENS, filed on Apr. 28, 2000, which is incorporated herein by reference in its entirety.
Embodiments of the invention provide expression cassettes that encode one or more embedded housekeeping epitopes, and methods for designing and testing such expression cassettes. The expression cassettes and constructs can encode epitopes, including housekeeping epitopes, derived from antigens that are associated with targets. Housekeeping epitopes can be liberated from the translation product(s) of the cassettes. For example, in some embodiments of the invention, the housekeeping epitope(s) can be flanked by arbitrary sequences or by sequences incorporating residues known to be favored in immunoproteasome cleavage sites. In further embodiments of the invention multiple epitopes can be arrayed head-to-tail. In some embodiments, these arrays can be made up entirely of housekeeping epitopes. Likewise, the arrays can include alternating housekeeping and immune epitopes. Alternatively, the arrays can include housekeeping epitopes flanked by immune epitopes, whether complete or distally truncated. In some preferred embodiments, each housekeeping epitope can be flanked on either side by an immune epitope, such that an array of such arrangements has two immune epitopes between each housekeeping epitope. Further, the arrays can be of any other similar arrangement. There is no restriction on placing a housekeeping epitope at the terminal positions of the array. The vectors can additionally contain authentic protein coding sequences or segments thereof containing epitope clusters as a source of immune epitopes.
Several disclosures make reference to polyepitopes or string-of-bead arrays. See, for example, WO0119408A1, Mar. 22, 2001; WO9955730A2, Nov. 4, 1999; WO0040261A2, Jul. 13, 2000; WO9603144A1, Feb. 8, 1996; EP1181314A1, Feb. 27, 2002; WO0123577A3, April 5; U.S. Pat. No. 6,074,817, Jun. 13, 2000; U.S. Pat. No. 5,965,381, Oct. 12, 1999; WO9741440A1, Nov. 6, 1997; U.S. Pat. No. 6,130,066, Oct. 10, 2000; U.S. Pat. No. 6,004,777, Dec. 21, 1999; U.S. Pat. No. 5,990,091, Nov. 23, 1999; WO9840501A1, Sep. 17, 1998; WO9840500A1, Sep. 17, 1998; WO0118035A2, Mar. 15, 2001; WO02068654A2, Sep. 6, 2002; WO0189281A2, Nov. 29, 2001; WO0158478A, Aug. 16, 2001; EP1118860A1, Jul. 25, 2001; WO0111040A1, Feb. 15, 2001; WO0073438A1, Dec. 7, 2000; WO0071158A1, Nov. 30, 2000; WO0066727A1, Nov. 9, 2000; WO0052451A1, Sep. 8, 2000; WO0052157A1, Sep. 8, 2000; WO0029008A2, May 25, 2000; WO0006723A1, Feb. 10, 2000; all of which are incorporated by reference in their entirety. Additional disclosures, all of which are hereby incorporated by reference in their entirety, include Palmowski M J, et al—J Immunol 2002; 168(9):4391-8; Fang Z Y, et al—Virology 2001; 291(2):272-84; Firat H, et al—J Gene Med 2002; 4(1):38-45; Smith S G, et al—Clin Cancer Res 2001; 7(12):4253-61; Vonderheide R H, et al—Clin Cancer Res 2001; 7(11):3343-8; Firat H, et al—Eur J Immunol 2001; 31(10):3064-74; Le T T, et al—Vaccine 2001; 19(32):4669-75; Fayolle C, et al—J Virol 2001; 75(16):7330-8; Smith SG—Curr Opin Mol Ther 1999; 1(1):10-5; Firat H, et al—Eur J Immunol 1999; 29(10):3112-21; Mateo L, et al—J Immunol 1999; 163(7):4058-63; Heemskerk M H, et al—Cell Immunol 1999; 195(1):10-7; Woodberry T, et al—J Virol 1999; 73(7):5320-5; Hanke T, et al—Vaccine 1998; 16(4):426-35; Thomson S A, et al—J Immunol 1998; 160(4):1717-23; Toes R E, et al—Proc Natl Acad Sci USA 1997; 94(26):14660-5; Thomson S A, et al—J Immunol 1996; 157(2):822-6; Thomson S A, et al—Proc Natl Acad Sci USA 1995; 92(13):5845-9; Street M D, et al—Immunology 2002; 106(4):526-36; Hirano K, et al—Histochem Cell Biol 2002; 117(1):41-53; Ward S M, et al—Virus Genes 2001; 23(1):97-104; Liu W J, et al—Virology 2000; 273(2):374-82; Gariglio P, et al—Arch Med Res 1998; 29(4):279-84; Suhrbier A—Immunol Cell Biol 1997; 75(4):402-8; Fomsgaard A, et al—Vaccine 1999; 18(7-8):681-91; An L L, et al—J Virol 1997; 71(3):2292-302; Whitton J L, et al—J Virol 1993; 67(1):348-52; Ripalti A, et al—J Clin Microbiol 1994; 32(2):358-63; and Gilbert, S. C., et al., Nat. Biotech. 15:1280-1284, 1997.
One important feature that the disclosures in the preceding paragraph all share is their lack of appreciation for the desirability of regenerating housekeeping epitopes when the construct is expressed in a pAPC. This understanding was not apparent until the present invention. Embodiments of the invention include sequences, that when processed by an immune proteasome, liberate or generate a housekeeping epitope. Embodiments of the invention also can liberate or generate such epitopes in immunogenically effective amounts. Accordingly, while the preceding references contain disclosures relating to polyepitope arrays, none is enabling of the technology necessary to provide or select a polyepitope capable of liberating a housekeeping epitope by action of an immunoproteasome in a pAPC. In contrast, embodiments of the instant invention are based upon a recognition of the desirability of achieving this result. Accordingly, embodiments of the instant invention include any nucleic acid construct that encodes a polypeptide containing at least one housekeeping epitope provided in a context that promotes its generation via immunoproteasomal activity, whether the housekeeping epitope is embedded in a string-of-beads array or some other arrangement. Some embodiments of the invention include uses of one or more of the nucleic acid constructs or their products that are specifically disclosed in any one or more of the above-listed references. Such uses include, for example, screening a polyepitope for proper liberation context of a housekeeping epitope and/or an immune epitope, designing an effective immunogen capable of causing presentation of a housekeeping epitope and/or an immune epitope on a pAPC, immunizing a patient, and the like. Alternative embodiments include use of only a subset of such nucleic acid constructs or a single such construct, while specifically excluding one or more other such constructs, for any of the purposes disclosed herein. Some preferred embodiments employ these and/or other nucleic acid sequences encoding polyepitope arrays alone or in combination. For example, some embodiments exclude use of polyepitope arrays from one or more of the above-mentioned references. Other embodiments may exclude any combination or all of the polyepitope arrays from the above-mentioned references collectively. Some embodiments include viral and/or bacterial vectors encoding polyepitope arrays, while other embodiments specifically exclude such vectors. Such vectors can encode carrier proteins that may have some immunostimulatory effect. Some embodiments include such vectors with such immunostimulatory/immunopotentiating effects, as opposed to immunogenic effects, while in other embodiments such vectors may be included. Further, in some instances viral and bacterial vectors encode the desired epitope as a part of substantially complete proteins which are not associated with the target cell. Such vectors and products are included in some embodiments, while excluded from others. Some embodiments relate to repeated administration of vectors. In some of those embodiments, nonviral and nonbacterial vectors are included. Likewise, some embodiments include arrays that contain extra amino acids between epitopes, for example anywhere from 1-6 amino acids, or more, in some embodiments, while other embodiments specifically exclude such arrays.
Embodiments of the present invention also include methods, uses, therapies, and compositions directed to various types of targets. Such targets can include, for example, neoplastic cells such as those listed below, for example; and cells infected with any virus, bacterium, protozoan, fungus, or other agents, examples of which are listed below, in Tables 4-8, or which are disclosed in any of the references listed above. Alternative embodiments include the use of only a subset of such neoplastic cells and infected cells listed below, in Tables 4-8, or in any of the references disclosed herein, or a single one of the neoplastic cells or infected cells, while specifically excluding one or more other such neoplastic cells or infected cells, for any of the purposes disclosed herein. The following are examples of neoplastic cells that can be targeted: human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, hepatocellular cancer, brain cancer, stomach cancer, liver cancer, and the like. Examples of infectious agents that infect the target cells can include the following: adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus BK, polyomavirus JC, hepatitis C virus, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, human T cell leukemia virus II, Chlamydia, Listeria, Salmonella, Legionella, Brucella, Coxiella, Rickettsia, Mycobacterium, Leishmania, Trypanasoma, Toxoplasma, Plasmodium, and the like. Exemplary infectious agents and neoplastic cells are also included in Tables 4-8 below.
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Cellular Biochem Suppl., 30/31:286-96 (1998); Türeci, Ö., Sahin, U., and Pfreundschuh, M., “Serological analysis of human tumor antigens: molecular definition and implications,” Molecular Medicine Today, 3:342 (1997); Rammensee et al., MHC Ligands and Peptide Motifs, Landes Bioscience Austin, Tex., 224-27, (1997); Parker et al., “Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains,” J. Immunol. 152:163-175 (1994); Kido & Ohshita, Anal. Biochem., 230:41-47 (1995); Yamada et al., J. Biochem. (Tokyo), 95:1155-60 (1984); Kawashima et al., Kidney Int., 54:275-8 (1998); Nakabayshi & Ikezawa, Biochem. Int 16:1119-25 (1988); Kanaseki Ohkuma, J. Biochem. (Tokyo), 110:541-7 (1991); Wattiaux et al., J. Cell Biol., 78:349-68 (1978); Lisman et al., Biochem. J., 178:79-87 (1979); Dean, B., Arch. Biochem. Biophys., 227:154-63 (1983); Overdijk et al., Adv. Exp. Med. Biol., 101:601-10 (1978); Stromhaug et al., Biochem. 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No. 09/561,571, filed Apr. 28, 2000; Simard et al., U.S. patent application Ser. No. 09/561,572, filed Apr. 28, 2000; Miura et al., WO 99/01283, Jan. 14, 1999; Simard et al., U.S. patent application Ser. No. 09/561,074, filed Apr. 28, 2000; Simard et al., U.S. patent application Ser. No. 10/225,568, filed Aug. 20, 2002; Simard et al., U.S. patent application Ser. No. 10/005,905, filed Nov. 7, 2001; Simard et al., U.S. patent application Ser. No. 09/561,074, filed Apr. 28, 2000.
Additional embodiments of the invention include methods, uses, therapies, and compositions relating to a particular antigen, whether the antigen is derived from, for example, a target cell or an infective agent, such as those mentioned above. Some preferred embodiments employ the antigens listed herein, in Tables 4-8, or in the list below, alone, as subsets, or in any combination. For example, some embodiments exclude use of one or more of those antigens. Other embodiments may exclude any combination or all of those antigens. Several examples of such antigens include MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, CEA, RAGE, NY-ESO, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, as well as any of those set forth in the above mentioned references. Other antigens are included in Tables 4-7 below.
Further embodiments include methods, uses, compositions, and therapies relating to epitopes, including, for example those epitopes listed in Tables 4-8. These epitopes can be useful to flank housekeeping epitopes in screening vectors, for example. Some embodiments include one or more epitopes from Tables 4-8, while other embodiments specifically exclude one or more of such epitopes or combinations thereof.











TABLE 4







AA
T cell epitope MHC



Virus
Protein
Position
ligand (Antigen)
MHC molecule




















Adenovirus 3
E3 9Kd
30-38
LIVIGILIL
(SEQ. ID NO.:44)
HLA-A*0201


 


Adenovirus 5
EIA
234-243
SGPSNTPPEI
(SEQ. ID NO.:45)
H2-Db


 


Adenovirus 5
E1B
192-200
VNIRNCCYl
(SEQ. ID NO.:46)
H2-Db


 


Adenovirus 5
EIA
234-243
SGPSNIPPEI (T>I)
(SEQ. ID NO.:47)
H2-Db


 


CSFV
NS
2276-2284
ENALLVALF
(SEQ. ID NO.:48)
SLA, haplotype d/d



polyprotein






 


Dengue virus 4
NS3
500-508
TPEGIIPTL
(SEQ. ID NO.:49)
HLA-B*3501


 


EBV
LMP-2
426-434
CLGGLLTMV
(SEQ. ID NO.:50)
HLA-A*0201


 


EBV
EBNA-1
480-484
NIAEGLRAL
(SEQ. ID NO.:51)
HLA-A*0201


 


EBV
EBNA-1
519-527
NLRRGTALA
(SEQ. ID NO.:52)
HLA-A*0201


 


EBV
EBNA-1
525-533
ALAIPQCRL
(SEQ. ID NO.:53)
HLA-A*0201


 


EBV
EBNA-1
575-582
VLKDAIKDL
(SEQ. ID NO.:54)
HLA-A*0201


 


EBV
EBNA-1
562-570
FMVFLQTHI
(SEQ. ID NO.:55)
HLA-A*0201


 


EBV
EBNA-2
15-23
HLIVDTDSL
(SEQ. ID NO.:56)
HLA-A*0201


 


EBV
EBNA-2
22-30
SLGNPSLSV
(SEQ. ID NO.:57)
HLA-A*0201


 


EBV
EBNA-2
126-134
PLASAMRML
(SEQ. ID NO.:58)
HLA-A*0201


 


EBV
EBNA-2
132-140
RMLWMANYl
(SEQ. ID NO.:59)
HLA-A*0201


 


EBV
EBNA-2
133-141
MLWMANYIV
(SEQ. ID NO.:60) 
HLA-A*0201


 


EBV
EBNA-2
151-159
ILPQGPQTA
(SEQ. ID NO.:61)
HLA-A*0201


 


EBV
EBNA-2
171-179
PLRPTAPTI
(SEQ. ID NO.:62)
HLA-A*0201


 


EBV
EBNA-2
205-213
PLPPATLTV
(SEQ. ID NO.:63)
HLA-A*0201


 


EBV
EBNA-2
246-254
RMHLPVLHV
(SEQ. ID NO.:64)
HLA-A*0201


 


EBV
EBNA-2
287-295
PMPLPPSQL
(SEQ. ID NO.:65)
HLA-A*0201


 


EBV
EBNA-2
294-302
QLPPPAAPA
(SEQ. ID NO.:66)
HLA-A*0201


 


EBV
EBNA-2
381-389
SMPELSPVL
(SEQ. ID NO.:67)
HLA-A*0201


 


EBV
EBNA-2
453-461
DLDESWDYI
(SEQ. ID NO.:68)
HLA-A*0201


 


EBV
BZLF1
43-51
PLPCVLWPV
(SEQ. ID NO.:69)
HLA-A*0201


 


EBV
BZLF1
167-175
SLEECDSEL
(SEQ. ID NO.:70)
HLA-A*0201


 


EBV
BZLF1
176-184
EIKRYKNRV
(SEQ. ID NO.:71)
HLA-A*0201


 


EBV
BZLF1
195-203
QLLQHYREV
(SEQ. ID NO.:72)
HLA-A*0201


 


EBV
BZLF1
196-204
LLQHYREVA
(SEQ. ID NO.:73)
HLA-A*0201


 


EBV
BZLFI
217-225
LLKQMCPSL
(SEQ. ID NO.:74)
HLA-A*0201


 


EBV
BZLF1
229-237
SIIPRTPDV
(SEQ. ID NO.:75)
HLA-A*0201


 


EBV
EBNA-6
284-293
LLDFVRFMGV
(SEQ. ID NO.:76)
HLA-A*0201


 


EBV
EBNA-3
464-472
SVRDRLARL
(SEQ. ID NO.:77)
HLA-A*0203


 


EBV
EBNA-4
416-424
IVTDFSVIK
(SEQ. ID NO.:78)
HLA-A*1101


 


EBV
EBNA-4
399-408
AVFDRKSDAK
(SEQ. ID NO.:79)
HLA-A*0201


 


EBV
EBNA-3
246-253
RYSIFFDY
(SEQ. ID NO.:80)
HLA-A24


 


EBV
EBNA-6
881-889
QPRAPIRPI
(SEQ. ID NO.:81)
HLA-B7


 


EBV
EBNA-3
379-387
RPPIFIRRI.
(SEQ. ID NO.:82)
HLA-B7


 


EBV
EBNA-1
426-434
EPDVPPGAI
(SEQ. ID NO.:83)
HLA-B7


 


EBV
EBNA-1
228-236
IPQCRLTPL
(SEQ. ID NO.:84)
HLA-B7


 


EBV
EBNA-1
546-554
GPGPQPGPL
(SEQ. ID NO.:85)
HLA-B7


 


EBV
EBNA-1
550-558
QPGPLRESI
(SEQ. ID NO.:86)
HLA-B7


 


EBV
EBNA-1
72-80
R.PQKRPSCI
(SEQ. ID NO.:87)
HLA-B7


 


EBV
EBNA-2
224-232
PPTPLLTVL
(SEQ. ID NO.:88)
HLA-B7


 


EBV
EBNA-2
241-249
TPSPPRMHL
(SEQ. ID NO.:89)
HLA-B7


 


EBV
EBNA-2
244-252
PPRMHLPVL
(SEQ. ID NO.:90)
HLA-B7


 


EBV
EBNA-2
254-262
VPDQSMIHPL
(SEQ. ID NO.:91)
HLA-B7


 


EBV
EBNA-2
446-454
PPSIDPADL
(SEQ. ID NO.:92)
HLA-B7


 


EBV
BZLFI
44-52
LPCVLWPVL
(SEQ. ID NO.:93)
HLA-B7


 


EBV
BZLF1
222-231
CPSLDVDSII
(SEQ. ID NO.:94)
HLA-B7


 


EBV
BZLFI
234-242
TPDVLHEDL
(SEQ. ID NO.:95)
HLA-B7


 


EBV
EBNA-3
339-347
FLRGRAYGL
(SEQ. ID NO.:96)
HLA-B8


 


EBV
EBNA-3
26-34
QAKWRLQTL
(SEQ. ID NO.:97)
HLA-B8


 


EBV
EBNA-3
325-333
AYPLHEQHG
(SEQ. ID NO.:98)
HLA-B8


 


EBV
EBNA-3
158-166
YlKSFVSDA
(SEQ. ID NO.:99)
HLA-B8


 


EBV
LMP-2
236-244
RRRWRRLTV
(SEQ. ID NO.:100)
HLA-B*2704


 


EBV
EBNA-6
258-266
RRIYDLIEL
(SEQ. ID NO.:101)
HLA-B*2705


 


EBV
EBNA-3
458-466
YPLHEQHGM
(SEQ. ID NO.:102)
HLA-B*3501


 


EBV
EBNA-3
458-466
YPLHEQHGM
(SEQ. ID NO.:103)
HLA-B*3503


 


HCV
NS3
389-397
HSKKKCDEL
(SEQ. ID NO.:104)
HLA-B8


 


HCV
env E
44-51
ASRCWVAM
(SEQ. ID NO.:105)
HLA-B*3501


 


HCV
core
27-35
GQIVGGVYL
(SEQ. ID NO.:106)
HLA-B*40012



protein






 


HCV
NSI
77-85
PPLTDFDQGW
(SEQ. ID NO.:107)
HLA-B*5301


 


HCV
core
18-27
LMGYIPLVGA
(SEQ. ID NO.:108)
H2-Dd



protein






 


HCV
core
16-25
ADLMGYIPLV
(SEQ. ID NO.:109)
H2-Dd



protein






 


HCV
NS5
409-424
MSYSWTGALVTPCAEE
(SEQ. ID NO.:110)
H2-Dd


 


HCV
NS1
205-213
KHPDATYSR
(SEQ. ID NO.:111)
Papa-A06


 


HCV-1
NS3
400-409
KLVALGINAV
(SEQ. ID NO.:112)
HLA-A*0201


 


HCV-1
NS3
440-448
GDFDSVIDC
(SEQ. ID NO.:113)
Patr-B16


 


HCV-1
env E
118-126
GNASRCWVA
(SEQ. ID NO.:114)
Patr-BI6


 


HCV-1
NSI
159-167
TRPPLGNWF
(SEQ. ID NO.:115)
Patr-B13


 


HCV-1
NS3
351-359
VPHPNIEEV
(SEQ. ID NO.:116)
Patr-B13


 


HCV-1
NS3
438-446
YTGDFDSVI
(SEQ. ID NO.:117)
Patr-B01


 


HCV-1
NS4
328-335
SWAIKWEY
(SEQ. ID NO.:118)
Patr-A1 1


 


HCV-1
NSI
205-213
KHPDATYSR
(SEQ. ID NO.:119)
Patr-A04


 


HCV-1
NS3
440-448
GDFDSVIDC
(SEQ. ID NO.:120)
Patr-A04


 


HIV
gp41
583-591
RYLKDQQLL
(SEQ. ID NO.:121)
HLA_A24


 


HIV
gagp24
267-275
IVGLNKLVR
(SEQ. ID NO.:122)
HLA-A*3302


 


HIV
gagp24
262-270
EIYKRWIIL
(SEQ. ID NO.:123)
HLA-B8


 


HIV
gagp24
261-269
GElYKRWIl
(SEQ. ID NO.:124)
HLA-B8


 


HIV
gagp17
 93-101
EIKDTKEAL
(SEQ. ID NO.:125)
HLA-B8


 


HIV
gp41
586-593
YLKDQQLL
(SEQ. ID NO.:126)
HLA-B8


 


HIV
gagp24
267-277
ILGLNKIVRMY
(SEQ. ID NO.:127)
HLA-B*1501


 


HIV
gp41
584-592
ERYLKDQQL
(SEQ. ID NO.:128)
HLA-B14


 


HIV
nef
115-125
YHTQGYFPQWQ
(SEQ. ID NO.:129)
HLA-B17


 


HIV
nef
117-128
TQGYFPQWQNYT
(SEQ. ID NO.:130)
HLA-B17


 


HIV
gp120
314-322
GRAFVTlGK
(SEQ. ID NO.:131)
HLA-B*2705


 


HIV
gagp24
263-271
KRWIILGLN
(SEQ. ID NO.:132)
HLA-B*2702


 


HIV
nef
72-82
QVPLRPMTYK
(SEQ. ID NO.:133)
HLA-B*3501


 


HIV
nef
117-125
TQGYFPQWQ
(SEQ. ID NO.:134)
HLA-B*3701


 


HIV
gagp24
143-151
HQAISPRTI,
(SEQ. ID NO.:135)
HLA-Cw*0301


 


HIV
gagp24
140-151
QMVHQAISPRTL
(SEQ. ID NO.:136)
HLA-Cw*0301


 


HIV
gp120
431-440
MYAPPIGGQI
(SEQ. ID NO.:137)
H2-Kd


 


HIV
gp160
318-327
RGPGRAFVTI
(SEQ. ID NO.:138)
H2-Dd


 


HIV
gp120
17-29
MPGRAFVTI
(SEQ. ID NO.:139)
H2-Ld


 


HIV-1
RT
476-484
ILKEPVHGV
(SEQ. ID NO.:140)
HLA-A*0201


 


HIV-1
nef
190-198
AFHHVAREL
(SEQ. ID NO.:141)
HLA-A*0201


 


HIV-1
gpI60
120-128
KLTPLCVTL
(SEQ. ID NO.:142)
HLA-A*0201


 


HIV-1
gp]60
814-823
SLLNATDIAV
(SEQ. ID NO.:143)
HLA-A*0201


 


HIV-1
RT
179-187
VIYQYMDDL
(SEQ. ID NO.:144)
HLA-A*0201


 


HIV-1
gagp17
77-85
SLYNTVATL
(SEQ. ID NO.:145)
HLA-A*0201


 


HIV-1
gp160
315-329
RGPGRAFVTl
(SEQ. ID NO.:146)
HLA-A*0201


 


HIV-1
gp41
768-778
RLRDLLLIVTR
(SEQ. ID NO.:147)
HLA-A3


 


HIV-1
nef
73-82
QVPLRPMTYK
(SEQ. ID NO.:148)
HLA-A3


 


HIV-1
gp120
36-45
TVYYGVPVWK
(SEQ. ID NO.:149)
HLA-A3


 


HIV-1
gagp17
20-29
RLRPGGKKK
(SEQ. ID NO.:150)
HLA-A3


 


HIV-1
gp120
38-46
VYYGVPVWK
(SEQ. ID NO.:151)
HLA-A3


 


HIV-1
nef
74-82
VPLRPMTYK
(SEQ. ID NO.:152)
HLA-a*1101


 


HIV-1
gagp24
325-333
AIFQSSMTK
(SEQ. ID NO.:153)
HLA-A*1101


 


HIV-1
nef
73-82
QVPLRPMTYK
(SEQ. ID NO.:154)
HLA-A*1101


 


HIV-1
nef
83-94
AAVDLSHFLKEK
(SEQ. ID NO.:155)
HLA-A*1101


 


HIV-1
gagp24
349-359
ACQGVGGPGGHK
(SEQ. ID NO.:156)
HLA-A*1101


 


HIV-1
gagp24
203-212
ETINEEAAEW
(SEQ. ID NO.:157)
HLA-A25


 


HIV-1
nef
128-137
TPGPGVRYPL
(SEQ. ID NO.:158)
HLA-B7


 


HIV-1
gagp17
24-31
GGKKKYKL
(SEQ. ID NO.:159)
HLA-B8


 


HIV-1
gp120
 2-10
RVKEKYQHL
(SEQ. ID NO.:160)
HLA-B8


 


HIV-1
gagp24
298-306
DRFYKTLRA
(SEQ. ID NO.:161)
HLA-B14


 


HIV-1
NEF
132-147
GVRYPLTFGWCYKLVP
(SEQ. ID NO.:162)
HLA-B18


 


HIV-1
gagp24
265-24 
KRWIILGLNK
(SEQ. ID NO.:163)
HLA-B*2705


 


HIV-1
nef
190-198
AFHHVAREL
(SEQ. ID NO.:164)
HLA-B*5201


 


EBV
EBNA-6
335-343
KEHVIQNALF
(SEQ. ID NO.:165)
HLA-B44


 


EBV
EBNA-6
130-139
EENLLDFVRF
(SEQ. ID NO.:166)
HLA-B*4403


 


EBV
EBNA-2
42-51
DTPLIPLTIF
(SEQ. ID NO.:167)
HLA-B51


 


EBV
EBNA-6
213-222
QNGALAINTF
(SEQ. ID NO.:168)
HLA-1362


 


EBV
EBNA-3
603-611
RLRAEAGVK
(SEQ. ID NO.:169)
HLA-A3


 


HBV
sAg
348-357
GLSPTVWLSV
(SEQ. ID NO.:170)
HLA-A*0201


 


HBV
SAg
335-343
WLSLLVPFV
(SEQ. ID NO.:171)
HLA-A*0201


 


HBV
cAg
18-27
FLPSDFFPSV
(SEQ. ID NO.:172)
HLA-A*0201


 


HBV
cAg
18-27
FLPSDFFPSV
(SEQ. ID NO.:173)
HLA-A*0202


 


HBV
cAg
18-27
FLPSDFFPSV
(SEQ. ID NO.:174)
HLA-A*0205


 


HBV
cAg
18-27
FLPSDFFPSV
(SEQ. ID NO.:175)
HLA-A*0206


 


HBV
pol
575-583
FLLSLGIHL
(SEQ. ID NO.:176)
HLA-A*0201


 


HBV
pol
816-824
SLYADSPSV
(SEQ. ID NO.:177)
HLA-A*0201


 


HBV
pol
455-463
GLSRYVARL
(SEQ. ID NO.:178)
HLA-A*0201


 


HBV
env
338-347
LLVPFVQWFV
(SEQ. ID NO.:179)
HLA-A*0201


 


HBV
pol
642-650
ALMPLYACI
(SEQ. ID NO.:180)
HLA-A*0201


 


HBV
env
378-387
LLPIFFCLWV
(SEQ. ID NO.:181)
HLA-A*0201


 


HBV
pol
538-546
YMDDVVLGA
(SEQ. ID NO.:182)
HLA-A*0201


 


HBV
env
250-258
LLLCLIFLL
(SEQ. ID NO.:183)
HLA-A*0201


 


HBV
env
260-269
LLDYQGMLPV
(SEQ. ID NO.:184)
HLA-A*0201


 


HBV
env
370-379
SIVSPFIPLL
(SEQ. ID NO.:185)
HLA-A*0201


 


HBV
env
183-191
FLLTRILTI
(SEQ. ID NO.:186)
HLA-A*0201


 


HBV
cAg
88-96
YVNYNMGLK
(SEQ. ID NO.:187)
HLA-A*1101


 


HBV
cAg
141-151
STLPETTVVRR
(SEQ. ID NO.:188)
HLA-A*3101


 


HBV
cAg
141-151
STLPETTVVRR
(SEQ. ID NO.:189)
HLA-A*6801


 


HBV
cAg
18-27
FLPSDFFPSV
(SEQ. ID NO.:190)
HLA-A*6801


 


HBV
sAg
28-39
IPQSLDSWWTSL
(SEQ. ID NO.:191)
H2-Ld


 


HBV
cAg
 93-100
MGLKFRQL
(SEQ. ID NO.:192)
H2-Kb


 


HBV
preS
141-149
STBXQSGXQ
(SEQ. ID NO.:193)
HLA-A*0201


 


HCMV
gp B
618-628
FIAGNSAYEYV
(SEQ. ID NO.:194)
HLA-A*0201


 


HCMV
E1
978-989
SDEEFAIVAYTL
(SEQ. ID NO.:195)
HLA-B18


 


HCMV
pp65
397-411
DDVWTSGSDSDEELV
(SEQ. ID NO.:196)
HLA-b35


 


HCMV
pp65
123-131
IPSINVKHY
(SEQ. ID NO.:197)
HLA-B*3501


 


HCMV
pp65
495-504
NLVPMVATVO
(SEQ. ID NO.:198)
HLA-A*0201


 


HCMV
pp65
415-429
RKTPRVTOGGAMAGA
(SEQ. ID NO.:199)
HLA-B7


 


HCV
MP
17-25
DLMGYIPLV
(SEQ. ID NO.:200)
HLA-A*0201


 


HCV
MP
63-72
LLALLSCLTV
(SEQ. ID NO.:201)
HLA-A*0201


 


HCV
MP
105-112
ILHTPGCV
(SEQ. ID NO.:202)
HLA-A*0201


 


HCV
env E
66-75
QLRRHIDLLV
(SEQ. ID NO.:203)
HLA-A*0201


 


HCV
env E
88-96
DLCGSVFLV
(SEQ. ID NO.:204)
HLA-A*0201


 


HCV
env E
172-180
SMVGNWAKV
(SEQ. ID NO.:205)
HLA-A*0201


 


HCV
NSI
308-316
HLIIQNIVDV
(SEQ. ID NO.:206)
HLA-A*0201


 


HCV
NSI
340-348
FLLLADARV
(SEQ. ID NO.:207)
HLA-A*0201


 


HCV
NS2
234-246
GLRDLAVAVEPVV
(SEQ. ID NO.:208)
HLA-A*0201


 


HCV
NSI
18-28
SLLAPGAKQNV
(SEQ. ID NO.:209)
HLA-A*0201


 


HCV
NSI
19-28
LLAPGAKQNV
(SEQ. ID NO.:210)
HLA-A*0201


 


HCV
NS4
192-201
LLFNILGGWV
(SEQ. ID NO.:211)
HLA-A*0201


 


HCV
NS3
579-587
YLVAYQATV
(SEQ. ID NO.:212)
HLA-A*0201


 


HCV
core
34-43
YLLPRRGPRL
(SEQ. ID NO.:213)
HLA-A*0201



protein






 


HCV
MP
63-72
LLALLSCLTI
(SEQ. ID NO.:214)
HLA-A*0201


 


HCV
NS4
174-182
SLMAFTAAV
(SEQ. ID NO.:215)
HLA-A*0201


 


HCV
NS3
67-75
CINGVCWTV
(SEQ. ID NO.:216)
HLA-A*0201


 


HCV
NS3
163-171
LLCPAGHAV
(SEQ. ID NO.:217)
HLA-A*0201


 


HCV
NS5
239-247
ILDSFDPLV
(SEQ. ID NO.:218)
HLA-A*0201


 


HCV
NS4A
236-244
IAGYGAGV
(SEQ. ID NO.:219)
HLA-A*0201


 


HCV
NS5
714-722
GLQDCTMLV
(SEQ. ID NO.:220)
HLA-A*0201


 


HCV
NS3
281-290
TGAPVTYSTY
(SEQ. ID NO.:221)
HLA-A*0201


 


HCV
NS4A
149-157
HMWNFISGI
(SEQ. ID NO.:222)
HLA-A*0201


 


HCV
NS5
575-583
RVCEKMALY
(SEQ. ID NO.:223)
HLA-A*0201-A3


 


HCV
NS1
238-246
TINYTIFK
(SEQ. ID NO.:224)
HLA-A*1101


 


HCV
NS2
109-116
YISWCLWW
(SEQ. ID NO.:225)
HLA-A23


 


HCV
core
40-48
GPRLGVRAT
(SEQ. ID NO.:226)
HLA-B7



protein






 


HIV-1
gp120
380-388
SFNCGGEFF
(SEQ. ID NO.:227)
HLA-Cw*0401


 


HIV-1
RT
206-214
TEMEKEGKI
(SEQ. ID NO.:228)
H2-Kk


 


HIV-1
p17
18-26
KIRLRPGGK
(SEQ. ID NO.:229)
HLA-A*031


 


HIV-1
P17
20-29
RLRPGGKKKY
(SEQ. ID NO.:230)
HLA-A*0301


 


HIV-1
RT
325-333
AIFQSSMTK
(SEQ. ID NO.:231)
HLA-A*0301


 


HIV-1
p17
84-92
TLYCVHQRI
(SEQ. ID NO.:232)
HLA-A11


 


HIV-1
RT
508-517
IYQEPFKNLK
(SEQ. ID NO.:233)
HLA-A11


 


HIV-1
p17
28-36
KYKLKHIVW
(SEQ. ID NO.:234)
HLA-A24


 


HIV-1
gp120
53-62
LFCASDAKAY
(SEQ. ID NO.:235)
HLA-A24


 


HIV-1
gagp24
145-155
QAISPRTLNAW
(SEQ. ID NO.:236)
HLA-A25


 


HIV-1
gagp24
167-175
EVIPMFSAL
(SEQ. ID NO.:237)
HLA-A26


 


HIV-1
RT
593-603
ETFYVDGAANR
(SEQ. ID NO.:238)
HLA-A26


 


HIV-1
gp41
775-785
RLRDLLLIVTR
(SEQ. ID NO.:239)
HLA-A31


 


HIV-1
RT
559-568
PIQKETWETW
(SEQ. ID NO.:240)
HLA-A32


 


HIV-1
gp120
419-427
RIKQIINMW
(SEQ. ID NO.:241)
HLA-A32


 


HIV-1
RT
71-79
ITLWQRPLV
(SEQ. ID NO.:242)
HLA-A*6802


 


HIV-1
RT
85-93
DTVLEEMNL
(SEQ. ID NO.:243)
HLA-A*6802


 


HIV-1
RT
71-79
ITLWQRPLV
(SEQ. ID NO.:244)
HLA-A*7401


 


HIV-1
gag p24
148-156
SPRTLNAWV
(SEQ. ID NO.:245)
HLA-B7


 


HIV-1
gagp24
179-187
ATPQDLNTM
(SEQ. ID NO.:246)
HLA-B7


 


HIV-1
gp120
303-312
RPNNNTRKSI
(SEQ. ID NO.:247)
HLA-B7


 


HIV-1
gp41
843-851
IPRRIRQGL
(SEQ. ID NO.:248)
HLA-B7


 


HIV-1
p17
74-82
ELRSLYNTV
(SEQ. ID NO.:249)
HLA-B8


 


HIV-1
nef
13-20
WPTVRERM
(SEQ. ID NO.:250)
HLA-B8


 


HIV-1
nef
90-97
FLKEKGGL
(SEQ. ID NO.:251)
HLA-B8


 


HIV-1
gag p24
183-191
DLNTMLNTV
(SEQ. ID NO.:252)
HLA-B14


 


HIV-1
P17
18-27
KIRLRPGGKK
(SEQ. ID NO.:253)
HLA-B27


 


HIV-1
p17
19-27
IRLRPGGKK
(SEQ. ID NO.:254)
HLA-B27


 


HIV-1
gp41
791-799
GRRGWEALKY
(SEQ. ID NO.:255)
HLA-B27


 


HIV-1
nef
73-82
QVPLRPMTYK
(SEQ. ID NO.:256)
HLA-B27


 


HIV-1
GP41
590-597
RYLKDQQL
(SEQ. ID NO.:257)
HLA-B27


 


HIV-1
nef
105-114
RRQDILDLWI
(SEQ. ID NO.:258)
HLA-B*2705


 


HIV-1
nef
134-141
RYPLTFGW
(SEQ. ID NO.:259)
HLA-B*2705


 


HIV-1
p17
36-44
WASRLELERF
(SEQ. ID NO.:260)
HLA-B35


 


HIV-1
GAG P24
262-270
TVLDVGDAY
(SEQ. ID NO.:261)
HLA-B35


 


HIV-1
gp120
42-52
VPVWKEATTTL
(SEQ. ID NO.:262)
HLA-B35


 


HIV-1
P17
36-44
NSSKVSQNY
(SEQ. ID NO.:263)
HLA-B35


 


HIV-1
gag p24
254-262
PPIPVGDIY
(SEQ. ID NO.:264)
HLA-B35


 


HIV-1
RT
342-350
HPDIVIYQY
(SEQ. ID NO.:265)
HLA-B35


 


HIV-1
gp41
611-619
TAVPWNASW
(SEQ. ID NO.:266)
HLA-B35


 


HIV-1
gag
245-253
NPVPVGNlY
(SEQ. ID NO.:267)
HLA-B35


 


HIV-1
nef
120-128
YFPDWQNYT
(SEQ. ID NO.:268)
HLA-B37


 


HIV-1
gag p24
193-201
GHQAAMQML
(SEQ. ID NO.:269)
HLA-B42


 


HIV-1
p17
20-29
RLRPGGKKKY
(SEQ. ID NO.:270)
HLA-B42


 


HIV-1
RT
438-446
YPGIKVRQL
(SEQ. ID NO.:271)
HLA-B42


 


HIV-1
RT
591-600
GAETFYVDGA
(SEQ. ID NO.:272)
HLA-B45


 


HIV-1
gag p24
325-333
NANPDCKTI
(SEQ. ID NO.:273)
HLA-B51


 


HIV-1
gag p24
275-282
RMYSPTSI
(SEQ. ID NO.:274)
HLA-B52


 


HIV-1
gp120
42-51
VPVWKEATTT
(SEQ. ID NO.:275)
HLA-B*5501


 


HIV-1
gag p24
147-155
ISPRTLNAW
(SEQ. ID NO.:276)
HLA-B57


 


HIV-1
gag p24
240-249
TSTLQEQIGW
(SEQ. ID NO.:277)
HLA-B57


 


HIV-1
gag p24
162-172
KAFSPEVIPMF
(SEQ. ID NO.:278)
HLA-B57


 


HIV-1
gagp24
311-319
QASQEVKNW
(SEQ. ID NO.:279)
HLA-B57


 


HIV-1
gagp24
311-319
QASQDVKNW
(SEQ. ID NO.:280)
HLA-B57


 


HIV-1
nef
116-125
HTQGYFPDWQ
(SEQ. ID NO.:281)
HLA-B57


 


HIV-1
nef
120-128
YFPDWQNYT
(SEQ. ID NO.:282)
HLA-B57


 


HIV-1
gag p24
240-249
TSTLQEQIGW
(SEQ. ID NO.:283)
HLA-B58


 


HIV-1
p17
20-29
RLRPGGKKKY
(SEQ. ID NO.:284)
HLA-B62


 


HIV-1
p24
268-277
LGLNKJVRMY
(SEQ. ID NO.:285)
HLA-B62


 


HIV-1
RT
415-426
LVGKLNWASQIY
(SEQ. ID NO.:286)
HLA-B62


 


HIV-1
RT
476-485
ILKEPVHGVY
(SEQ. ID NO.:287)
HLA-B62


 


HIV-1
nef
117-127
TQGYFPDWQNY
(SEQ. ID NO.:288)
HLA-B62


 


HIV-1
nef
84-91
AVDLSHFL
(SEQ. ID NO.:289)
HLA-B62


 


HIV-1
gag p24
168-175
VIPMIFSAL
(SEQ. ID NO.:290)
HLA-Cw*0102


 


HIV-1
gp120
376-384
FNCGGEFFY
(SEQ. ID NO.:291)
HLA-A29


 


HIV-1
gp120
375-383
SFNCGGEFF
(SEQ. ID NO.:292)
HLA-B15


 


HIV-1
nef
136-145
PLTFGWCYKL
(SEQ. ID NO.:293)
HLA-A*0201


 


HIV-1
nef
180-189
VLEWRFDSRL
(SEQ. ID NO.:294)
HLA-A*0201


 


HIV-1
nef
68-77
FPVTPQVPLR
(SEQ. ID NO.:295)
HLA-B7


 


HIV-1
nef
128-137
TPGPGVRYPL
(SEQ. ID NO.:296)
HLA-B7


 


HIV-1
gag p24
308-316
QASQEVKNW
(SEQ. ID NO.:297)
HLA-Cw*0401


 


HIV-1 IIIB
RT
273-282
VPLDEDFRKY
(SEQ. ID NO.:298)
HLA-B35


 


HIV-1 IIIB
RT
25-33
NPDIVIYQY
(SEQ. ID NO.:299)
HLA-B35


 


HIV-1 IIIB
gp41
557-565
RAIEAQAHL
(SEQ. ID NO.:300)
HLA-B51


 


HIV-1 IIIB
RT
231-238
TAFTIPSI
(SEQ. ID NO.:301)
HLA-B51


 


HIV-1 IIIB
p24
215-223
VHPVHAGPIA
(SEQ. ID NO.:302)
HLA-B*5501


 


HIV-1 IIIB
gp120
156-165
NCSFNISTSI
(SEQ. ID NO.:303)
HLA-Cw8


 


HIV-1 IIIB
gp120
241-249
CTNVSTVQC
(SEQ. ID NO.:304)
HLA-Cw8


 


HIV-1 5F2
gp120
312-320
IGPGRAFHT
(SEQ. ID NO.:305)
H2-Dd


 


HIV-1 5F2
pol
25-33
NPDIVIYQY
(SEQ. ID NO.:306)
HLA-B*3501


 


HIV-1 5F2
pol
432-441
EPIVGAETFY
(SEQ. ID NO.:307)
HLA-B*3501


 


HIV-1 5F2
pol
432-440
EPIVGAETF
(SEQ. ID NO.:308)
HLA-B*3501


 


HIV-1 5F2
pol
 6-14
SPAIFQSSM
(SEQ. ID NO.:309)
HLA-B*3501


 


HIV-1 5F2
pol
59-68
VPLDKDFRKY
(SEQ. ID NO.:310)
HLA-B*3501


 


HIV-1 5F2
pol
 6-14
IPLTEEAEL
(SEQ. ID NO.:311)
HLA-B*3501


 


HIV-1 5F2
nef
69-79
RPQVPLRPMTY
(SEQ. ID NO.:312)
HLA-B*3501


 


HIV-1 5F2
nef
66-74
FPVRPQVPL
(SEQ. ID NO.:313)
HLA-B*3501


 


HIV-1 5F2
env
10-18
DPNPQEVVL
(SEQ. ID NO.:314)
HLA-B*3501


 


HIV-1 5F2
env
 7-15
RPIVSTQLL
(SEQ. ID NO.:315)
HLA-B*3501


 


HIV-1 5F2
pol
 6-14
IPLTEEAEL
(SEQ. ID NO.:316)
HLA-B51


 


HIV-1 5F2
env
10-18
DPNPQEVVL
(SEQ. ID NO.:317)
HLA-B51


 


HIV-1 5F2
gagp24
199-207
AMQMLKETI
(SEQ. ID NO.:318)
H2-Kd


 


HIV-2
gagp24
182-190
TPYDrNQML
(SEQ. ID NO.:319)
HLA-B*5301


 


HIV-2
gag
260-269
RRWIQLGLQKV
(SEQ. ID NO.:320)
HLA-B*2703


 


HIV-1 5F2
gp41
593-607
GIWGCSGKLICTTAV
(SEQ. ID NO.:321)
HLA-B17


 


HIV-1 5F2
gp41
753-767
ALIWEDLRSLCLFSY
(SEQ. ID NO.:322)
HLA-B22


 


HPV 6b
E7
21-30
GLHCYEQLV
(SEQ. ID NO.:323)
HLA-A*0201


 


HPV 6b
E7
47-55
PLKQHFQIV
(SEQ. ID NO.:324)
HLA-A*0201


 


HPV11
E7
 4-12
RLVTLKDIV
(SEQ. ID NO.:325)
HLA-A*0201


 


HPV16
E7
86-94
TLGIVCPIC
(SEQ. ID NO.:326)
HLA-A*0201


 


HPV16
E7
85-93
GTLGIVCPI
(SEQ. ID NO.:327)
HLA-A*0201


 


HPV16
E7
12-20
MLDLQPETT
(SEQ. ID NO.:328)
HLA-A*0201


 


HPV16
E7
11-20
YMLDLQPETT
(SEQ. ID NO.:329)
HLA-A*0201


 


HPV16
E6
15-22
RPRKLPQL
(SEQ. ID NO.:330)
HLA-B7


 


HPV16
E6
49-57
RAHYNIVTF
(SEQ. ID NO.:331)
HLA-Db


 


HSV
gp B
498-505
SSIEFARL
(SEQ. ID NO.:332)
H2-Kb


 


HSV-1
gp C
480-488
GIGIGVLAA
(SEQ. ID NO.:333)
HLA-A*0201


 


HSV-1
ICP27
448-456
DYATLGVGV
(SEQ. ID NO.:334)
H2-Kd


 


HSV-1
ICP27
322-332
LYRTFAGNPRA
(SEQ. ID NO.:335)
H2-Kd


 


HSV-1
UL39
822-829
QTFDFGRL
(SEQ. ID NO.:336)
H2-Kb


 


HSV-2
gpC
446-454
GAGIGVAVL
(SEQ. ID NO.:337)
HLA-A*0201


 


HLTV-1
TAX
11-19
LLFGYPVYV
(SEQ. ID NO.:338)
HLA-A*0201


 


Influenza
MP
58-66
GILGFVFTL
(SEQ. ID NO.:339)
HLA-A*0201


 


Influenza
MP
59-68
ILGFVFTLTV
(SEQ. ID NO.:340)
HLA-A*0201


 


Influenza
NP
265-273
ILRGSVAHK
(SEQ. ID NO.:341)
HLA-A3


 


Influenza
NP
91-99
KTGGPIYKR
(SEQ. ID NO.:342)
HLA-A*6801


 


Influenza
NP
380-388
ELRSRYWAI
(SEQ. ID NO.:343)
HLA-B8


 


Influenza
NP
381-388
LRSRYWAI
(SEQ. ID NO.:344)
HLA-B*2702


 


Influenza
NP
339-347
EDLRVLSFI
(SEQ. ID NO.:345)
HLA-B*3701


 


Influenza
NSI
158-166
GEISPLPSL
(SEQ. ID NO.:346)
HLA-B44


 


Influenza
NP
338-346
FEDLRVLSF
(SEQ. ID NO.:347)
HLA-B44


 


Influenza
NSI
158-166
GEISPLPSL
(SEQ. ID NO.:348)
HLA-B*4402


 


Influenza
NP
338-346
FEDLRVLSF
(SEQ. ID NO.:349)
HLA-B*4402


 


Influenza
PBI
591-599
VSDGGPKLY
(SEQ. ID NO.:350)
HLA-A1


 


Influenza A
NP
44-52
CTELKLSDY
(SEQ. ID NO.:351)
HLA-A1


 


Influenza
NSI
122-130
AIMDKNIIL
(SEQ. ID NO.:352)
HLA-A*0201


 


Influenza A
NSI
123-132
IMDKNIILKA
(SEQ. ID NO.:353)
HLA-A*0201


 


Influenza A
NP
383-391
SRYWAIRTR
(SEQ. ID NO.:354)
HLA-B*2705


 


Influenza A
NP
147-155
TYQRTRALV
(SEQ. ID NO.:355)
H2-Kd


 


Influenza A
HA
210-219
TYVSVSTSTL
(SEQ. ID NO.:356)
H2-Kd


 


Influenza A
HA
518-526
IYSTVASSL
(SEQ. ID NO.:357)
H2-Kd


 


Influenza A
HA
259-266
FEANGNLI
(SEQ. ID NO.:358)
H2-Kk


 


Influenza A
HA
10-18
IEGGWTGMl
(SEQ. ID NO.:359)
H2-Kk


 


Influenza A
NP
50-57
SDYEGRLI
(SEQ. ID NO.:360)
H2-Kk


 


Influenza a
NSI
152-160
EEGAIVGEI
(SEQ. ID NO.:361)
H2-Kk


 


Influenza A34
NP
336-374
ASNENMETM
(SEQ. ID NO.:362)
H2Db


 


Influenza A68
NP
366-374
ASNENMDAM
(SEQ. ID NO.:363)
H2Db


 


Influenza B
NP
85-94
KLGEFYNQMM
(SEQ. ID NO.:364)
HLA-A*0201


 


Influenza B
NP
85-94
KAGEFYNQMM
(SEQ. ID NO.:365)
HLA-A*0201


 


Influenza JAP
HA
204-212
LYQNVGTYV
(SEQ. ID NO.:366)
H2Kd


 


Influenza JAP
HA
210-219
TYVSVGTSTL
(SEQ. ID NO.:367)
H2-Kd


 


Influenza JAP
HA
523-531
VYQILATYA
(SEQ. ID NO.:368)
H2-Kd


 


Influenza JAP
HA
529-537
IYATVAGSL
(SEQ. ID NO.:369)
H2-Kd


 


Influenza JAP
HA
210-219
TYVSVGTSTI (L>I)
(SEQ. ID NO.:370)
H2-Kd


 


Influenza JAP
HA
255-262
FESTGNLI
(SEQ. ID NO.:371)
H2-Kk


 


JHMV
cAg
318-326
APTAGAFFF
(SEQ. ID NO.:372)
H2-Ld


 


LCMV
NP
118-126
RPQASGVYM
(SEQ. ID NO.:373)
H2-Ld


 


LCMV
NP
396-404
FQPQNGQFI
(SEQ. ID NO.:374)
H2-Db


 


LCMV
GP
276-286
SGVENPGGYCL
(SEQ. ID NO.:375)
H2-Db


 


LCMV
GP
33-42
KAVYNFATCG
(SEQ. ID NO.:376)
H2-Db


 


MCMV
pp89
168-176
YPHFMPTNL
(SEQ. ID NO.:377)
H2-Ld


 


MHV
spike
510-518
CLSWNGPHL
(SEQ. ID NO.:378)
H2-Db



protein






 


MMTV
env gp 36
474-482
SFAVATTAL
(SEQ. ID NO.:379)
H2-Kd


 


MMTV
gag p27
425-433
SYETFISRL
(SEQ. ID NO.:380)
H2-Kd


 


MMTV
env gp73
544-551
ANYDFICV
(SEQ. ID NO.:381)
H2-Kb


 


MuLV
env p15E
574-581
KSPWFTTL
(SEQ. ID NO.:382)
H2-Kb


 


MuLV
env gp70
189-196
SSWDFITV
(SEQ. ID NO.:383)
H2-Kb


 


MuLV
gag 75K
75-83
CCLCLTVFL
(SEQ. ID NO.:384)
H2-Db


 


MuLV
env gp70
423-431
SPSYVYHQF
(SEQ. ID NO.:385)
H2Ld


 


MV
F protein
437-447
SRRYPDAVYLH
(SEQ. ID NO.:386)
HLA-B*2705


 


Mv
F protein
438-446
RRYPDAVYL
(SEQ. ID NO.:387)
HLA-B*2705


 


Mv
NP
281-289
YPALGLHEF
(SEQ. ID NO.:388)
H2-Ld


 


Mv
HA
343-351
DPVIDRLYL
(SEQ. ID NO.:389)
H2-Ld


 


MV
HA
544-552
SPGRSFSYF
(SEQ. ID NO.:390)
H2-Ld


 


Poliovirus
VP1
111-118
TYKDTVQL
(SEQ. ID NO.:391)
H2-kd


 


Poliovirus
VP1
208-217
FYDGFSKVPL
(SEQ. ID NO.:392)
H2-Kd


 


Pseudorabies
G111
455-463
IAGIGILAI
(SEQ. ID NO.:393)
HLA-A*0201


virus gp







 


Rabiesvirus
NS
197-205
VEAEIAHQI
(SEQ. ID NO.:394)
H2-Kk


 


Rotavirus
VP7
33-40
llYRFLLl
(SEQ. ID NO.:395)
H2-Kb


 


Rotavirus
VP6
376-384
VGPVFPPGM
(SEQ. ID NO.:396)
H2-Kb


 


Rotavirus
VP3
585-593
YSGYIFRDL
(SEQ. ID NO.:397)
H2-Kb


 


RSV
M2
82-90
SYIGSINNI
(SEQ. ID NO.:398)
H2-Kd


 


SIV
gagp11C
179-190
EGCTPYDTNQML
(SEQ. ID NO.:399)
Mamu-A*01


 


SV
NP
324-332
FAPGNYPAL
(SEQ. ID NO.:400)
H2-Db


 


SV
NP
324-332
FAPCTNYPAL
(SEQ. ID NO.:401)
H2-Kb


 


SV40
T
404-411
VVYDFLKC
(SEQ. ID NO.:402)
H2-Kb


 


SV40
T
206-215
SAINNYAQKL
(SEQ. ID NO.:403)
H2-Db


 


SV40
T
223-231
CKGVNKEYL
(SEQ. ID NO.:404)
H2-Db


 


SV40
T
489-497
QGINNLDNL
(SEQ. ID NO.:405)
H2-Db


 


SV40
T
492-500
NNLDNLRDY(L)
(SEQ. ID NO.:406)
H2-Db




(501)





 


SV40
T
560-568
SEFLLEKRI
(SEQ. ID NO.:407)
H2-Kk


 


VSV
NP
52-59
RGYVYQGL
(SEQ. ID NO.:408)
H2-Kb


















TABLE 5





HLA-A1
Position (Antigen)
Source


















T cell epitopes
EADPTGHSY
(SEQ. ID NO.:409)
MAGE-1 161-169


 



VSDGGPNLY
(SEQ. ID NO.:410)
Influenza A PB 1591-599


 



CTELKLSDY
(SEQ. ID NO.:411)
Influenza A NP 44-52


 



EVDPIGHLY
(SEQ. ID NO.:412)
MAGE-3 168-176


 


HLA-A201
MLLSVPLLLG
(SEQ. ID NO.:413)
Calreticulin signal sequence 1-10


 



STBXQSGXQ
(SEQ. ID NO.:414)
HBV PRE-S PROTEIN 141-149


 



YMDGTMSQV
(SEQ. ID NO.:415)
Tyrosinase 369-377


 



ILKEPVHGV
(SEQ. ID NO.:416)
HIV-I RT 476-484


 



LLGFVFTLTV
(SEQ. ID NO.:417)
Influenza MP 59-68


 



LLFGYPVYVV
(SEQ. ID NO.:418)
HTLV-1 tax 11-19


 



GLSPTVWLSV
(SEQ. ID NO.:419)
HBV sAg 348-357


 



WLSLLVPFV
(SEQ. ID NO.:420)
HBV sAg 335-343


 



FLPSDFFPSV
(SEQ. ID NO.:421)
HBV cAg 18-27


 



C L G 0 L L T M V
(SEQ. ID NO.:422)
EBV LMP-2 426-434


 



FLAGNSAYEYV
(SEQ. ID NO.:423)
HCMV gp 618-628B


 



KLGEFYNQMM
(SEQ. ID NO.:424)
Influenza BNP 85-94


 



KLVALGINAV
(SEQ. ID NO.:425)
HCV-1 NS3 400-409


 



DLMGYIPLV
(SEQ. ID NO.:426)
HCV MP 17-25


 



RLVTLKDIV
(SEQ. ID NO.:427)
HPV 11 EZ 4-12


 



MLLAVLYCL
(SEQ. ID NO.:428)
Tyrosinase 1-9


 



AAGIGILTV
(SEQ. ID NO.:429)
Melan A\Mart-127-35


 



YLEPGPVTA
(SEQ. ID NO.:430)
Pmel 17/gp 100 480-488


 



ILDGTATLRL
(SEQ. ID NO.:431)
Pmel 17/gp 100 457-466


 



LLDGTATLRL
(SEQ. ID NO.:432)
Pmel gp1OO 457-466


 



ITDQVPFSV
(SEQ. ID NO.:433)
Pmel gp 100 209-217


 



KTWGQYWQV
(SEQ. ID NO.:434)
Pmel gp 100 154-162


 



TITDQVPFSV
(SEQ. ID NO.:435)
Pmel gp 100 208-217


 



AFHIIVAREL
(SEQ. ID NO.:436)
HIV-I nef 190-198


 



YLNKIQNSL
(SEQ. ID NO.:437)
P. falciparum CSP 334-342


 



MMRKLAILSV
(SEQ. ID NO.:438)
P. falciparum CSP 1-10


 



KAGEFYNQMM
(SEQ. ID NO.:439)
Influenza BNP 85-94


 



NIAEGLRAL
(SEQ. ID NO.:440)
EBNA-1 480-488


 



NLRRGTALA
(SEQ. ID NO.:441)
EBNA-1 519-527


 



ALAIPQCRL
(SEQ. ID NO.:442)
EBNA-1 525-533


 



VLKDAIKDL
(SEQ. ID NO.:443)
EBNA-1 575-582


 



FMVFLQTHI
(SEQ. ID NO.:444)
EBNA-1 562-570


 



HLIVDTDSL
(SEQ. ID NO.:445)
EBNA-2 15-23


 



SLGNPSLSV
(SEQ. ID NO.:446)
EBNA-2 22-30


 



PLASAMRML
(SEQ. ID NO.:447)
EBNA-2 126-134


 



RMLWMANYI
(SEQ. ID NO.:448)
EBNA-2 132-140


 



MLWMANYIV
(SEQ. ID NO.:449)
EBNA-2 133-141


 



ILPQGPQTA
(SEQ. ID NO.:450)
EBNA-2 151-159


 



PLRPTAPTTI
(SEQ. ID NO.:451)
EBNA-2 171-179


 



PLPPATLTV
(SEQ. ID NO.:452)
EBNA-2 205-213


 



R M H L P V L H V
(SEQ. ID NO.:453)
EBNA-2 246-254


 



PMPLPPSQL
(SEQ. ID NO.:454)
EBNA-2 287-295


 



QLPPPAAPA
(SEQ. ID NO.:455)
EBNA-2 294-302


 



SMPELSPVL
(SEQ. ID NO.:456)
EBNA-2 381-389


 



DLDESWDYl
(SEQ. ID NO.:457)
EBNA-2 453-461


 



P L P C V L W P VV
(SEQ. ID NO.:458)
BZLF1 43-51


 



SLEECDSEL
(SEQ. ID NO.:459)
BZLF1 167-175


 



EIKRYKNRV
(SEQ. ID NO.:460)
BZLFI 176-184


 



QLLQFIYREV
(SEQ. ID NO.:461)
BZLF1 195-203


 



LLQHYREVA
(SEQ. ID NO.:462)
BZLFI 196-204


 



LLKQMCPSL
(SEQ. ID NO.:463)
BZLFI 217-225


 



SIIPRTPDV
(SEQ. ID NO.:464)
BZLFI 229-237


 



AIMDKNIIL
(SEQ. ID NO.:465)
Influenza A NS1 122-130


 



IMDKNIILKA
(SEQ. ID NO.:466)
Influenza A NS1 123-132


 



LLALLSCLTV
(SEQ. ID NO.:467)
HCV MP 63-72


 



ILHTPGCV
(SEQ. ID NO.:468)
HCV MP 105-112


 



QLRRHIDLLV
(SEQ. ID NO.:469)
HCV env E 66-75


 



DLCGSVFLV
(SEQ. ID NO.:470)
HCV env E 88-96


 



SMVGNWAKV
(SEQ. ID NO.:471)
HCV env E 172-180


 



HLHQNIVDV
(SEQ. ID NO.:472)
HCV NSI 308-316


 



FLLLADARV
(SEQ. ID NO.:473)
HCV NSI 340-348


 



GLRDLAVAVEPVV
(SEQ. ID NO.:474)
HCV NS2 234-246


 



SLLAPGAKQNV
(SEQ. ID NO.:475)
HCV NS1 18-28


 



LLAPGAKQNV
(SEQ. ID NO.:476)
HCV NS1 19-28


 



FLLSLGIHL
(SEQ. ID NO.:477)
HBV pol 575-583


 



SLYADSPSV
(SEQ. ID NO.:478)
HBV pol 816-824


 



GLSRYVARL
(SEQ. ID NO.:479)
HBV POL 455-463


 



KIFGSLAFL
(SEQ. ID NO.:480)
HER-2 369-377


 



ELVSEFSRM
(SEQ. ID NO.:481)
HER-2 971-979


 



KLTPLCVTL
(SEQ. ID NO.:482)
HIV-I gp 160 120-128


 



SLLNATDIAV
(SEQ. ID NO.:483)
HIV-I GP 160 814-823


 



VLYRYGSFSV
(SEQ. ID NO.:484)
Pmel gp100 476-485


 



YIGEVLVSV
(SEQ. ID NO.:485)
Non-filament forming class I myosin





family (HA-2)**


 



LLFNILGGWV
(SEQ. ID NO.:486)
HCV NS4 192-201


 



LLVPFVQWFW
(SEQ. ID NO.:487)
HBV env 338-347


 



ALMPLYACI
(SEQ. ID NO.:488)
HBV pol 642-650


 



YLVAYQATV
(SEQ. ID NO.:489)
HCV NS3 579-587


 



TLGIVCPIC
(SEQ. ID NO.:490)
HIPV 16 E7 86-94


 



YLLPRRGPRL
(SEQ. ID NO.:491)
HCV core protein 34-43


 



LLPIFFCLWV
(SEQ. ID NO.:492)
HBV env 378-387


 



YMDDVVLGA
(SEQ. ID NO.:493)
HBV Pol 538-546


 



GTLGIVCPI
(SEQ. ID NO.:494)
HPV16 E7 85-93


 



LLALLSCLTI
(SEQ. ID NO.:495)
HCV MP 63-72


 



MLDLQPETT
(SEQ. ID NO.:496)
HPV 16 E7 12-20


 



SLMAFTAAV
(SEQ. ID NO.:497)
HCV NS4 174-182


 



CINGVCWTV
(SEQ. ID NO.:498)
HCV NS3 67-75


 



VMNILLQYVV
(SEQ. ID NO.:499)
Glutarnic acid decarboxylase 114-123


 



ILTVILGVL
(SEQ. ID NO.:500)
Melan A/Mart-32-40


 



FLWGPRALV
(SEQ. ID NO.:501)
MAGE-3 271-279


 



L L C P A G H A V
(SEQ. ID NO.:502)
HCV NS3 163-171


 



ILDSFDPLV
(SEQ. ID NO.:503)
HCV NSS 239-247


 



LLLCLIFLL
(SEQ. ID NO.:504)
HBV env 250-258


 



LIDYQGMLPV
(SEQ. ID NO.:505)
HBV env 260-269


 



SIVSPFIPLL
(SEQ. ID NO.:506)
HBV env 370-379


 



FLLTRILTI
(SEQ. ID NO.:507)
HBV env 183-191


 



HLGNVKYLV
(SEQ. ID NO.:508)
P. faciparum TRAP 3-11


 



GIAGGLALL
(SEQ. ID NO.:509)
P. faciparum TRAP 500-508


 



IILAGYGAGV
(SEQ. ID NO.:510)
HCV NS S4A 236-244


 



GLQDCTMLV
(SEQ. ID NO.:511)
HCV NS5 714-722


 



TGAPVTYSTY
(SEQ. ID NO.:512)
HCV NS3 281-290


 



VIYQYMDDLV
(SEQ. ID NO.:513)
HIV-1RT 179-187


 



VLPDVFIRCV
(SEQ. ID NO.:514)
N-acetylglucosaminyltransferase V Gnt-V





intron


 



VLPDVFIRC
(SEQ. ID NO.:515)
N-acetylglucosaminyltransferase V Gnt-V





intron


 



AVGIGIAVV
(SEQ. ID NO.:516)
Human CD9


 



LVVLGLLAV
(SEQ. ID NO.:517)
Human glutamyltransferase


 



ALGLGLLPV
(SEQ. ID NO.:518)
Human G protein coupled receptor




164-172



 



GIGIGVLAA
(SEQ. ID NO.:519)
HSV-I gp C 480-488


 



GAGIGVAVL
(SEQ. ID NO.:520)
HSV-2 gp C 446-454


 



IAGIGILAI
(SEQ. ID NO.:521)
Pseudorabies gpGIN 455-463


 



LIVIGILIL
(SEQ. ID NO.:522)
Adenovirus 3 E3 9 kD 30-38


 



LAGIGLLIAA
(SEQ. ID NO.:523)
S. Lincolnensis ImrA


 



VDGIGTLTI
(SEQ. ID NO.:524)
Yeast ysa-1 77-85


 



GAGIGVLTA
(SEQ. ID NO.:525)
B. polymyxa, βcndoxylanase 149-157




157



 



AAGIGIIQI
(SEQ. ID NO.:526)
E. coli methionine synthase 590-598


 



QAGIGILLA
(SEQ. ID NO.:527)
E. coli hypothetical protein 4-12


 



KARDPHSGHFV
(SEQ. ID NO.:528)
CDK4w1 22.32


 



KACDPI-ISGIIFV
(SEQ. ID NO.:529)
CDK4-R24C 22-32


 



ACDPFISGHFV
(SEQ. ID NO.:530)
CDK4-R24C 23-32


 



SLYNTVATL
(SEQ. ID NO.:531)
HIV-I gag p 17 77-85


 



ELVSEFSRV
(SEQ. ID NO.:532)
HER-2, m > V substituted 971-979


 



RGPGRAFVTI
(SEQ. ID NO.:533)
HIV-I gp 160 315-329


 



HMWNFISGI
(SEQ. ID NO.:534)
HCV NS4A 149-157


 



NLVPMVATVQ
(SEQ. ID NO.:535)
HCMV pp65 495-504


 



GLHCYEQLV
(SEQ. ID NO.:536)
HPV 6b E7 21-30


 



PLKQHFQIV
(SEQ. ID NO.:537)
HPV 6b E7 47-55


 



LLDFVRFMGV
(SEQ. ID NO.:538)
EBNA-6 284-293


 



AIMEKNIML
(SEQ. ID NO.:539)
Influenza Alaska NS 1 122-130


 



YLKTIQNSL
(SEQ. ID NO.:540)
P. falciparum cp36 CSP


 



YLNKIQNSL
(SEQ. ID NO.:541)
P. falciparurn cp39 CSP


 



YMLDLQPETT
(SEQ. ID NO.:542)
HPV 16 E7 11-20*


 



LLMGTLGIV
(SEQ. ID NO.:543)
HPV16 E7 82-90**


 



TLGIVCPI
(SEQ. ID NO.:544)
HPV 16 E7 86-93


 



TLTSCNTSV
(SEQ. ID NO.:545)
HIV-1 gp120 197-205


 



KLPQLCTEL
(SEQ. ID NO.:546)
HPV 16 E6 18-26


 



TIHDIILEC
(SEQ. ID NO.:547)
HPV16 E6 29-37


 



LGIVCPICS
(SEQ. ID NO.:548)
HPV16 E7 87-95


 



VILGVLLLI
(SEQ. ID NO.:549)
Melan A/Mart-1 35-43


 



ALMDKSLHV
(SEQ. ID NO.:550)
Melan A/Mart-1 56-64


 



GILTVILGV
(SEQ. ID NO.:551)
Melan A/Mart-1 31-39


 


T cell epitopes
MINAYLDKL
(SEQ. ID NO.:552)
P. Falciparum STARP 523-531


 



AAGIGILTV
(SEQ. ID NO.:553)
Melan A/Mart-127-35


 



FLPSDFFPSV
(SEQ. ID NO.:554)
HBV cAg 18-27


 


Motif unknown
SVRDRLARL
(SEQ. ID NO.:555)
EBNA-3 464-472


T cell epitopes





 


T cell epitopes
AAGIGILTV
(SEQ. ID NO.:556)
Melan A/Mart-1 27-35


 



FAYDGKDYI
(SEQ. ID NO.:557)
Human MHC I-ot 140-148


 


T cell epitopes
AAGIGILTV
(SEQ. ID NO.:558)
Melan A/Mart-1 27-35


 



FLPSDFFPSV
(SEQ. ID NO.:559)
HBV cAg 18-27


 


Motif unknown
AAGIGILTV
(SEQ. ID NO.:560)
Meland A/Mart-1 27-35


T cell epitopes





 



FLPSDFFPSV
(SEQ. ID NO.:561)
HBV cAg 18-27


 



AAGIGILTV
(SEQ. ID NO.:562)
Melan A/Mart-1 27-35


 



ALLAVGATK
(SEQ. ID NO.:563)
Pmel17 gp 100 17-25


 


T cell epitopes
R L R D L L L I V T R
(SEQ. ID NO.:564)
HIV-1 gp41 768-778


 



QVPLRPMTYK
(SEQ. ID NO.:565)
HIV-1 nef 73-82


 



TVYYGVPVWK
(SEQ. ID NO.:566)
HIV-1 gp120-36-45


 



RLRPGGKKK
(SEQ. ID NO.:567)
HIV-1 gag p 17 20-29


 



ILRGSVAHK
(SEQ. ID NO.:568)
Influenza NP 265-273


 



RLRAEAGVK
(SEQ. ID NO.:569)
EBNA-3 603-611


 



RLRDLLLIVTR
(SEQ. ID NO.:570)
HIV-1 gp41 770-780


 



VYYGVPVWK
(SEQ. ID NO.:571)
HIV-I GP 120 38-46


 



RVCEKMALY
(SEQ. ID NO.:572)
HCV NS5 575-583


 


Motif unknown
KIFSEVTLK
(SEQ. ID NO.:573)
Unknown; muta melanoma peptide ted (p


T cell epitope


I 83L) 175-183


 



YVNVNMGLK*
(SEQ. ID NO.:574)
HBV cAg 88-96


 


T cell epitopes
IVTDFSVIIK
(SEQ. ID NO.:575)
EBNA-4 416-424


 



ELNEALELK
(SEQ. ID NO.:576)
P53 343-351


 



VPLRPMTYK
(SEQ. ID NO.:577)
HIV-1 NEF 74-82


 



AIFQSSMTK
(SEQ. ID NO.:578)
HIV-I gag p24 325-333


 



QVPLRPMTYK
(SEQ. ID NO.:579)
HIV-1 nef 73-82


 



TINYTIFK HCV
(SEQ. ID NO.:580)
NSI 238-246


 



AAVDLSHFLKEK
(SEQ. ID NO.:581)
HIV-1 nef 83-94


 



ACQ G V G G P G G H K
(SEQ. ID NO.:582)
HIV-1 II 1B p24 349-359


 


HLA-A24
S Y L D S G I H F*
(SEQ. ID NO.:583)
β-catenin, mutated (proto-onocogen)





29-37


 


T cell epitopes
RYLKDQQLL
(SEQ. ID NO.:584)
HIV GP 41 583-591


 



AYGLDFYIL
(SEQ. ID NO.:585)
P15 melanoma Ag 10-18


 



AFLPWHRLFL
(SEQ. ID NO.:586)
Tyrosinase 206-215


 



AFLPWHRLF
(SEQ. ID NO.:587)
Tyrosinase 206-214


 



RYSIFFDY
(SEQ. ID NO.:588)
Ebna-3 246-253


 


T cell epitope
ETINEEAAEW
(SEQ. ID NO.:589)
HIV-1 gag p24 203-212


 


T cell epitopes
STLPETTVVRR
(SEQ. ID NO.:590)
HBV cAg 141-151


 



MSLQRQFLR
(SEQ. ID NO.:591)
ORF 3P-gp75 294-321 (bp)


 



LLPGGRPYR
(SEQ. ID NO.:592)
TRP (tyrosinase rel.) 197-205


 


T cell epitope
IVGLNKIVR
(SEQ. ID NO.:593)
HIV gag p24 267-267-275


 



AAGIGILTV
(SEQ. ID NO.:594)
Melan A/Mart-127 35









Table 6 sets forth additional antigens useful in the invention that are available from the Ludwig Cancer Institute. The Table refers to patents in which the identified antigens can be found and as such are incorporated herein by reference. TRA refers to the tumor-related antigen and the LUD No. refers to the Ludwig Institute number.












TABLE 6






LUD

Date Patent




TRA
No.
Patent No.
Issued
Peptide (Antigen)
HLA




















MAGE-4
5293
5,405,940
11 Apr. 1995
EVDPASNTY
HLA-






(SEQ. ID NO.: 979)
A1


 


MAGE-41
5293
5,405,940
11 Apr. 1995
EVDPTSNTY
HLA-






(SEQ ID NO: 595)
A I


 


MAGE-5
5293
5,405,940
11 Apr. 1995
EADPTSNTY
HLA-






(SEQ ID NO: 596)
A I


 


MAGE-51
5293
5,405,940
11 Apr. 1995
EADPTSNTY
HLA-






(SEQ ID NO: 597)
A I


 


MAGE-6
5294
5,405,940
11 Apr. 1995
EVDPIGHVY
HLA-






(SEQ ID NO: 598)
A1


 



5299.2
5,487,974
30 Jan. 1996
MLLAVLYCLL
HLA-






(SEQ ID NO: 599)
A2


 



5360
5,530,096
25 Jun. 1996
MLLAVLYGL
HLA-






(SEQ ID NO: 600)
B44


 


Tyrosinase
5360.1
5,519,117
21 May 1996
SEIWRDIDFA
HLA-






(SEQ ID NO: 601)
B44


 






SEIWRDIDF







(SEQ ID NO: 602)



 


Tyrosinase
5431
5,774,316
28 Apr. 1998
XEIWRDIDF
HLA-






(SEQ ID NO: 603)
B44


 


MAGE-2
5340
5,554,724
10 Sep. 1996
STLVEVTLGEV
HLA-






(SEQ ID NO: 604)
A2


 






LVEVTLGEV







(SEQ ID NO: 605)



 






VIFSKASEYL







(SEQ ID NO: 606)



 






IIVLAIIAl







(SEQ ID NO: 607)



 






KIWEELSMLEV







(SEQ ID NO: 608)



 






LIETSYVKV







(SEQ ID NO: 609)



 



5327
5,585,461
17 Dec. 1996
FLWGPRALV
HLA-






(SEQ ID NO: 610)
A2


 






TLVEVTLGEV







(SEQ ID NO: 611)



 






ALVETSYVKV







(SEQ ID NO: 612)



 


MAGE-3
5344
5,554,506
10 Sep. 1996
KIWEELSVL
HLA-






(SEQ ID NO: 613)
A2


 


MAGE-3
5393
5,405,940
11 Apr. 1995
EVDPIGHLY
HLA-






(SEQ ID NO: 614)
A1


 


MAGE
5293
5,405,940
11 Apr. 1995
EXDX5Y
HLA-






(SEQ. ID NO.: 615)
A1


 






(but not EADPTGHSY)







(SEQ. ID NO.: 616)



 






E (A/V) D X5 Y







(SEQ. ID NO.: 617)



 






E (A/V) D P X4 Y







(SEQ. ID NO.: 618)



 






E (A/V) D P (I/A/T) X3 Y







(SEQ. ID NO.: 619)



 






E (A/V) D P (I/A/T) (G/S) X2 Y







(SEQ. ID NO.: 620)



 






E (A/V) D P (I/A/T) (G/S) (H/N) X Y







(SEQ. ID NO.: 621)



 






E (A/V) DP (I/A/T) (G/S) (H/N)







(L/T/V) Y (SEQ. ID NO.: 622)



 


MAGE-1
5361
5,558.995
24 Sep. 1996
ELHSAYGEPRKLLTQD
HLA-C






(SEQ ID NO: 623)
Clone







10


 






EHSAYGEPRKLL







(SEQ ID NO: 624)



 






SAYGEPRKL







(SEQ ID NO: 625)



 


MAGE-1
5253.4
TBA
TBA
EADPTGHSY
HLA-






(SEQ ID NO: 626)
A I


 


BAGE
5310.1
TBA
TBA
MAARAVFLALSAQLLQARLMKE
HLA-C






(SEQ ID NO: 627)
Clone







10


 






MAARAVFLALSAQLLQ
HLA-C






(SEQ ID NO: 628)
Clone







10


 






AARAVFLAL
HLA-






(SEQ ID NO: 629)
Clone







10


 


GAGE
5323.2
5,648,226
15 Jul. 1997
YRPRPRRY
HLA-






(SEQ. ID NO.: 630)
CW6






















TABLE 7









T cell epitope
SEQ.





AA
MHC
MHC ligand
ID



Source
Protein
Position
molecules
(Antigen)
NO.:
Ref.







Synthetic
synthetic
synthetic
HLA-A2
ALFAAAAAV
631
Parker, et al., “Scheme


peptides
peptides
peptides



for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIFGGVGGV
632
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLDKGGGV
633
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGFGGV
634
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGAGV
635
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGEGV
636
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGFGV
637
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGL
638
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGV
639
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGVGV
640
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGVGGV
641
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGVGKV
642
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFKGVGGV
643
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLGGGGFGV
644
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLLGGGVGV
645
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLYGGGGGV
646
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GMFGGGGGV
647
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GMFGGVGGV
648
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GQFGGVGGV
649
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GVFGGVGGV
650
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLFGGGGGV
651
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLFGGVGGV
652
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
AILGFVFTL
653
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GAIGFVFTL
654
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GALGFVFTL
655
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GELGFVFTL
656
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIAGFVFTL
657
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIEGFVFTL
658
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILAFVFTL
659
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGAVFTL
660
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGEVFTL
661
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILFGAFTL
662
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFEFTL
663
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFKFTL
664
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVATL
665
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVETL
666
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVFAL
667
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVFEL
668
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVFKL
669
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVFTA
670
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVFTL
671
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVFVL
672
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGFVKTL
673
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILGKVFTL
674
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILKFVFTL
675
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GILPFVFTL
676
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIVGFVFTL
677
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GKLGFVFTL
678
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLLGFVFTL
679
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GQLGFVFTL
680
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KALGFVFTL
681
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KILGFVFTL
682
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KILGKVFTL
683
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
AILLGVFML
684
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
AIYKRWIIL
685
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ALFFFDIDL
686
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ATVELLSEL
687
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
CLFGYPVYV
688
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
FIFPNYTIV
689
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
IISLWDSQL
690
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ILASLFAAV
691
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ILESLFAAV
692
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLGEFFNQM
693
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLGEFYNQM
694
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
LLFGYPVYV
695
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
LLWKGEGAV
696
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
LMFGYPVYV
697
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
LNFGYPVYV
698
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
LQFGYPVYV
699
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
NIVAHTFKV
700
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175





HLA-A2
NLPMVATV
701
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175





HLA-A2
QMLLAIARL
702
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175





HLA-A2
QMWQARLTV
703
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
RLLQTGIHV
704
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
RLVNGSLAL
705
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
SLYNTVATL
706
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
TLNAWVKVV
707
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
WLYRETCNL
708
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
YLFKRMIDL
709
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GAFGGVGGV
710
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GAFGGVGGY
711
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GEFGGVGGV
712
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GGFGGVGGV
713
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIFGGGGGV
714
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIGGFGGGL
715
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIGGGGGGL
716
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLDGGGGGV
717
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLDGKGGGV
718
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLDKKGGGV
719
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGFGF
720
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGFGG
721
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGFGN
722
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGFGS
723
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGI
724
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGM
725
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGT
726
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGY
727
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175





HLA-A2
GLGFGGGGV
728
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLGGFGGGV
729
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLGGGFGGV
730
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLGGGGGFV
731
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLGGGGGGY
732
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLGGGVGGV
733
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLLGGGGGV
734
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLPGGGGGV
735
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GNFGGVGGV
736
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GSFGGVGGV
737
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GTFGGVGGV
738
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
AGNSAYEYV
739
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFPGQFAY
740
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
HILLGVFML
741
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ILESLFRAV
742
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLKKYKLKHI
743
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
MLASTDLKY
744
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
MLERELVRK
745
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLFGFVFTV
746
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ILDKKVEKV
747
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ILKEPVHGV
748
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ALFAAAAAY
749
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIGFGGGGL
750
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GKFGGVGGV
751
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GLFGGGGGK
752
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
EILGFVFTL
753
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GIKGFVFTL
754
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
GQLGFVFTK
755
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
ILGFVFTLT
756
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KILGFVFTK
757
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KKLGFVFTL
758
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
KLFEKVYNY
759
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 





HLA-A2
LRFGYPVYV
760
Parker, et al., “Scheme








for ranking potential








HLA-A2 binding peptides








based on independent








binding of individual








peptide side-chains,” J.








Immunol. 152: 163-175


 


Human
HSP60
140-148
HLA-B27
IRRGVMLAV
761
Rammensee et al. 1997








160


 


Human
HSP60
369-377
HLA-B27
KRIQEIIEQ
762
Rammensee et al. 1997








160


 


Human
HSP60
469-477
HLA-B27
KRTLKIPAM
763
Rammensee et al. 1997








160


 


Yersinia
HSP60
35-43
HLA-B27
GRNVVLDKS
764
Rammensee et al. 1997








160


 


Yersinia
HSP60
117-125
HLA-B27
KRGIDKAVI
765
Rammensee et al. 1997








160


 


Yersinia
HSP60
420-428
HLA-B27
IRAASAITA
766
Rammensee et al. 1997








160


 


Yersinia
HSP 60
284-292
HLA-
RRKAMFEDI
767
169





B*2705





 


P.
LSA-1
1850-1857
HLA-
KPKDELDY
768
170


falciparum


B3501





 


Influenza

379-387
HLA-
LELRSRYWA
769
183


NP


B*4402





 



Tum-P35B
 4-13
HLA-Dd
GPPHSNNFGY
770
230


 


Rotavirus
VP7
33-40

IIYRFLLI
771
262


 



OGDH
104-112
H2-Ld
QLSPYPFDL
772
253



(F108Y)







 



TRP-2
181-188
p287
VYDFFVWL
773
284


 



DEAD box
547-554
p287
SNIFVFAGI
774
283



p 68







 



Vector

p287
SVVEFSSL
775
260



“artefact”







 



Epitope mimic

p287
AHYLFRNL
776
278



of tumor Ag







 





p287
THYLFRNL
777
278


 



Epitope mimic

p287
LIVIYNTL
778
279



of H-3 miHAg”







 





p287
LIYEFNTL
779
279


 





p287
IPYIYNTL
780
279


 





p287
IIYIYHRL
781
279


 





p287
LIYIFNTL
782
279


 



HBV cAg
 93-100
p287
MGLKLFRQL
783
280


 


Human
autoantigen
51-58
p287
IMIKFRNRL
784
281



LA







 


Mouse
UTY protein

H2Db
WMHHNMDLI
785
303


 


Mouse
p53
232-240
H2Db
KYMCNSSCM
786
302


 


MURINE
MDM2
441-449
H2Db
GRPKNGCIV
787
277


 



Epitope mimic

H2Db
AQHPNAELL
788
278



of natural







 



MuLV
75-83
H2Db
CCLCLTVFL
789
301



gag75K







 


P.
CSP
375-383
p290
YENDIEKK
790
315


Falciparum








 


P.
CSP
371-379
p290
DELDYENDI
791
315


Falciparum








 


HIV
−1RT
206-214
p290
TEMEKEGKI
792
316


 


Rabies
NS
197-205
p290
VEAEIAHQI
793
309, 310


 


Influenza A
NS
1152-160
p290
EEGAIVGEI
794
304


 


Murine
SMCY

p291
TENSGKDI
795
317


 



MHC class
 3-11
p293
AMAPRTLLL
796
318



1 leader







 



ND1alpha
 1-12
p293
FFINILTLLVP
797
323


 



ND Beta
 1-12
p293
FFINILTLLVP
798
323


 



ND alpha
 1-17
p293
FFINILTLLVPI
799
324






LIAM




 



ND Beta
 1-17
p293
FFINALTLLVPI
800
324






LIAM




 



COI
 1-6
p293
FINRW
801
325



mitochondrial







 


L.
LemA
 1-6
p293
IGWII
802
326


monocyto-








genes









SW gag
179-190
Mamu-
EGCTPYDINQ
803
334



p11C

A*01
ML




 



MAGE-3

HLA-A2
ALSRKVAEL
804
5,554,506


 





HLA-A2
IMPKAGLLI
805
5,554,506


 





HLA-A2
KIWEELSVL
806
5,554,506


 





HLA-A2
ALVETSYVKV
807
5,554,506


 





HLA-A2
ThrLeuValGluVal
808
5,554,506






ThrLeuGlyGluVal




 





HLA-A2
AlaLeuSerArgLys
809
5,554,506






ValAlaGluLeu




 





HLA-A2
IleMetProLysAla
810
5,554,506






GlyLeuLeuIle




 





HLA-A2
LysIleTrpGluGlu
811
5,554,506






LeuSerValLeu




 





HLA-A2
AlaLeuValGluThr
812
5,554,506






SerTyrValLys








Val




 



peptides

HLA-A2
Lys Gly Ile Leu
813
5,989,565



which bind


Gly Phe Val Phe





to MHCs


Thr Leu Thr Val




 





HLA-A2
Gly Ile Ile Gly
814
5,989,565






Phe Val Phe Thr








Ile




 





HLA-A2
Gly Ile Ile Gly
815
5,989,565






Phe Val Phe Thr








Leu




 





HLA-A2
Gly Ile Leu Gly
816
5,989,565






Phe Val Phe Thr








Leu




 





HLA-A2
Gly Leu Leu Gly
817
5,989,565






Phe Val Phe Thr








Leu




 





HLA-A2
XXTVXXGVX,
818
5,989,565






X=Leu or Ile








(6-37)




 





HLA-A2
Ile Leu Thr Val
819
5,989,565






Ile Leu Gly Val








Leu




 





HLA-A2
Tyr Leu Glu Pro
820
5,989,565






Gly Ala




 





HLA-A2
Gln Val Pro Leu
821
5,989,565






Arg Tyr Lys




 





HLA-A2
Asp Gly Leu Ala
822
5,989,565






Pro Pro Gln His








Leu Ile Arg




 





HLA-A2
Leu Leu Gly Arg
823
5,989,565






Asn Ser Phe Glu








Val




 



Peptides

HLA-C
GluHisSerAlaTyr
824
5,558,995



from

clone 10
GlyGluProArgLys





MAGE-1


LeuLeuThrGlnAsp








Leu




 





HLA-C
GluHisSerAlaTyr
825
5,558,995





clone 10
GlyGluProArgLys








LeuLeu




 





HLA-C
SerAlaTyrGlyGly
826
5,558,995





clone 10
ProArgLysLeu




 



GAGE

HLA-Cw6
TyrArgProArgPro
827
5,648,226






ArgArgTyr




 





HLA-Cw6
ThrTyrArgProArg
828
5,648,226






ProArgArgTyr




 





HLA-Cw6
TyrArgProArgPro
829
5,648,226






ArgArgTyrVal




 





HLA-Cw6
ThrTyrArgProArg
830
5,648,226






ProArgArgTyrVal




 





HLA-Cw6
ArgProArgProArg
831
5,648,226






ArgTyrValGlu




 





HLA-Cw6
MetSerTrpArgGly
832
5,648,226






ArgSerThrTyrArg








ProArgProArgArg




 





HLA-Cw6
ThrTyrArgProArg
833
5,648,226






ProArgArgTyrVal








GluProProGluMet








Ile




 



MAGE

HLA-A1,
Isolated
834
5,405,940





primarily
nonapeptide








having Glu at its








N terminal, Tyr








at its C-terminal,








and Asp at the








third residue from








its N terminal,








with the proviso








that said isolated








nonapeptide is








not Glu Ala Asp








Pro Thr Gly His








Ser Tyr (SEQ ID








NO: 1), and








wherein said








isolated








nonapeptide








binds to a human








leukocyte








antigen molecule








on a cell to form








a complex, said








complex provoking








lysis of said cell








by a cytolytic T








cell specific to








said complex




 





HLA-A1,
GluValValProIle
835
5,405,940





primarily
SerHisLeuTyr




 





HLA-A1,
GluValValArgIle
836
5,405,940





primarily
GlyHisLeuTyr




 





HLA-A1,
GluValAspProIle
837
5,405,940





primarily
GlyHisLeuTyr




 





HLA-A1,
GluValAspProAla
838
5,405,940





primarily
SerAsnThrTyr




 





HLA-A1,
GluValAspProThr
839
5,405,940





primarily
SerAsnThrTyr




 





HLA-A1,
GluAlaAspProThr
840
5,405,940





primarily
SerAsnThrTyr




 





HLA-A1,
GluValAspProIle
841
5,405,940





primarily
GlyHisValTyr




 





HLA-A1,
GAAGTGGTCC
842
5,405,940





primarily
CCATCAGCCA







HLA-A1,
CTTGTAC




 





primarily
GAAGTGGTCC
843
5,405,940





HLA-A1,
GCATCGGCCA







primarily
CTTGTAC




 





HLA-A1,
GAAGTGGAC
844
5,405,940





primarily
CCCATCGGCC







HLA-A1,
ACTTGTAC




 





primarily
GAAGTGGAC
845
5,405,940





HLA-A1,
CCCGCCAGCA







primarily
ACACCTAC




 





HLA-A1,
GAAGTGGAC
846
5,405,940





primarily
CCCACCAGCA







HLA-A1,
ACACCTAC




 





primarily
GAAGCGGAC
847
5,405,940





HLA-A1,
CCCACCAGCA







primarily
ACACCTAC




 





HLA-A1,
GAAGCGGAC
848
5,405,940





primarily
CCCACCAGCA







HLA-A1,
ACACCTAC




 





primarily
GAAGTGGAC
849
5,405,940





HLA-A1,
CCCATCGGCC







primarily
ACGTGTAC




 





HLA-A1,
GluAlaAspProThr
850
5,405,940





primarily
GlyHisSer




 





HLA-A1,
AlaAspProTrpGly
851
5,405,940





primarily
HisSerTyr




 



MAGE

HLA-A2
SerThrLeuValGlu
852
5,554,724



peptides


ValThrLeuGlyGlu








Val




 



MAGE

HLA-A2
LeuValGluValThr
853
5,554,724



peptides


LeuGlyGluVal




 



MAGE

HLA-A2
LysMetValGluLeu
854
5,554,724



peptides


ValHisPheLeu




 



MAGE

HLA-A2
ValIlePheSerLys
855
5,554,724



peptides


AlaSerGluTyrLeu




 



MAGE

HLA-A2
TyrLeuGlnLeuVal
856
5,554,724



peptides


PheGlyIleGluVal




 



MAGE

HLA-A2
GlnLeuValPheGly
857
5,554,724



peptides


IleGluValVal




 



MAGE

HLA-A2
GlnLeuValPheGly
858
5,554,724



peptides


IleGluValValGlu








Val




 



MAGE

HLA-A2
IleIleValLeuAla
859
5,554,724



peptides


IleIleAlaIle




 



MAGE

HLA-A2
LysIleTrpGluGlu
860
5,554,724



peptides


LeuSerMetLeuGlu








Val




 



MAGE

HLA-A2
AlaLeuIleGluThr
861
5,554,724



peptides


SerTyrValLysVal




 



MAGE

HLA-A2
LeuIleGluThrSer
862
5,554,724



peptides


TyrValLysVal




 



MAGE

HLA-A2
GlyLeuGluAlaArg
863
5,554,724



peptides


GlyGluAlaLeuGly








Leu




 



MAGE

HLA-A2
GlyLeuGluAlaArg
864
5,554,724



peptides


GlyGluAlaLeu




 



MAGE

HLA-A2
AlaLeuGlyLeuVal
865
5,554,724



peptides


GlyAlaGlnAla




 



MAGE

HLA-A2
GlyLeuValGlyAla
866
5,554,724



peptides


GlnAlaProAla




 



MAGE

HLA-A2
AspLeuGluSerGlu
867
5,554,724



peptides


PheGlnAlaAla




 



MAGE

HLA-A2
AspLeuGluSerGlu
868
5,554,724



peptides


PheGlnAlaAlaIle




 



MAGE

HLA-A2
AlaIleSerArgLys
869
5,554,724



peptides


MetValGluLeuVal




 



MAGE

HLA-A2
AlaIleSerArgLys
870
5,554,724



peptides


MetValGluLeu




 



MAGE

HLA-A2
LysMetValGluLeu
871
5,554,724



peptides


ValHisPheLeuLeu




 



MAGE

HLA-A2
LysMetValGluLeu
872
5,554,724



peptides


ValHisPheLeuLeu








Leu




 



MAGE

HLA-A2
LeuLeuLeuLysTyr
873
5,554,724



peptides


ArgAlaArgGluPro








Val




 



MAGE

HLA-A2
LeuLeuLysTyrArg
874
5,554,724



peptides


AlaArgGluProVal




 



MAGE

HLA-A2
ValLeuArgAsnCys
875
5,554,724



peptides


GlnAspPhePhePro








Val




 



MAGE

HLA-A2
TyrLeuGlnLeuVal
876
5,554,724



peptides


PheGlyIleGluVal








Val




 



MAGE

HLA-A2
GlyIleGluValVal
877
5,554,724



peptides


GluValValProIle




 



MAGE

HLA-A2
ProIleSerHisLeu
878
5,554,724



peptides


TyrIleLeuVal




 



MAGE

HLA-A2
HisLeuTyrIleLeu
879
5,554,724



peptides


ValThrCysLeu




 



MAGE

HLA-A2
HisLeuTyrIleLeu
880
5,554,724



peptides


ValThrCysLeuGly








Leu




 



MAGE

HLA-A2
TyrIleLeuValThr
881
5,554,724



peptides


CysLeuGlyLeu




 



MAGE

HLA-A2
CysLeuGlyLeuSer
882
5,554,724



peptides


TyrAspGlyLeu




 



MAGE

HLA-A2
CysLeuGlyLeuSer
883
5,554,724



peptides


TyrAspGlyLeu








Leu




 



MAGE

HLA-A2
ValMetProLysThr
884
5,554,724



peptides


GlyLeuLeuIle




 



MAGE

HLA-A2
ValMetProLysThr
885
5,554,724



peptides


GlyLeuLeuIleIle




 



MAGE

HLA-A2
ValMetProLysThr
886
5,554,724



peptides


GlyLeuLeuIleIle








Val




 



MAGE

HLA-A2
GlyLeuLeuIleIle
887
5,554,724



peptides


ValLeuAlaIleIle




 



MAGE

HLA-A2
GlyLeuLeuIleIle
888
5,554,724



peptides


ValLeuAlaIleIle




 



MAGE

HLA-A2
GlyLeuLeuIleIle
889
5,554,724



peptides


ValLeuAlaIleIle








Ala




 



MAGE

HLA-A2
LeuLeuIleIleVal
890
5,554,724



peptides


LeuAlaIleIle




 



MAGE

HLA-A2
LeuLeuIleIleVal
891
5,554,724



peptides


LeuAlaIleIleAla




 



MAGE

HLA-A2
LeuLeullelleVal
892
5,554,724



peptides


LeuAlaIleIleAla








Ile




 



MAGE

HLA-A2
LeuIleIleValLeu
893
5,554,724



peptides


AlaIleIleAla




 



MAGE

HLA-A2
LeuIleIleValLeu
894
5,554,724



peptides


AlaIleIleAlaIle




 



MAGE

HLA-A2
IleIleAlaIleGlu
895
5,554,724



peptides


GlyAspCysAla




 



MAGE

HLA-A2
LysIleTrpGluGlu
896
5,554,724



peptides


LeuSerMetLeu




 



MAGE

HLA-A2
LeuMetGlnAspLeu
897
5,554,724



peptides


ValGlnGluAsnTyr








Leu




 



MAGE

HLA-A2
PheLeuTrpGlyPro
898
5,554,724



peptides


ArgAlaLeuIle




 



MAGE

HLA-A2
LeuIleGluThrSer
899
5,554,724



peptides


TyrValLysVal




 



MAGE

HLA-A2
AlaLeuIleGluThr
900
5,554,724



peptides


SerTyrValLysVal








Leu




 



MAGE

HLA-A2
ThrLeuLysIleGly
901
5,554,724



peptides


GlyGluProHisIle




 



MAGE

HLA-A2
HisIleSerTyrPro
902
5,554,724



peptides


ProLeuHisGluArg








Ala




 



MAGE

HLA-A2
GlnThrAlaSerSer
903
5,554,724



peptides


SerSerThrLeu




 



MAGE

HLA-A2
GlnThrAlaSerSer
904
5,554,724



peptides


SerSerThrLeuVal




 



MAGE

HLA-A2
ValThrLeuGlyGlu
905
5,554,724



peptides


ValProAlaAlaVal




 



MAGE

HLA-A2
ValThrLysAlaGlu
906
5,554,724



peptides


MetLeuGluSerVal




 



MAGE

HLA-A2
ValThrLysAlaGlu
907
5,554,724



peptides


MetLeuGluSerVal








Leu




 



MAGE

HLA-A2
ValThrCysLeuGly
908
5,554,724



peptides


LeuSerTyrAspGly








Leu




 



MAGE

HLA-A2
LysThrGlyLeuLeu
909
5,554,724



peptides


IleIleValLeu




 



MAGE

HLA-A2
LysThrGlyLeuLeu
910
5,554,724



peptides


IleIleValLeuAla




 



MAGE

HLA-A2
LysThrGlyLeuLeu
911
5,554,724



peptides


IleIleValLeuAla








Ile




 



MAGE

HLA-A2
HisThrLeuLysIle
912
5,554,724



peptides


GlyGlyGluProHis








Ile




 



MAGE

HLA-A2
MetLeuAspLeuGln
913
5,554,724



peptides


ProGluThrThr




 



Mage-3

HLA-A2
GlyLeuGluAlaArg
914
5,585,461



peptides


GlyGluAlaLeu




 



Mage-3

HLA-A2
AlaLeuSerArgLys
915
5,585,461



peptides


ValAlaGluLeu




 



Mage-3

HLA-A2
PheLeuTrpGlyPro
916
5,585,461



peptides


ArgAlaLeuVal




 



Mage-3

HLA-A2
ThrLeuValGluVal
917
5,585,461



peptides


ThrLeuGlyGluVal




 



Mage-3

HLA-A2
AlaLeuSerArgLys
918
5,585,461



peptides


ValAlaGluLeuVal




 



Mage-3

HLA-A2
AlaLeuValGluThr
919
5,585,461



peptides


SerTyrValLysVal




 



Tyrosinase

HLA-A2
TyrMetAsnGlyThr
920
5,487,974






MetSerGlnVal




 



Tyrosinase

HLA-A2
MetLeuLeuAlaVal
921
5,487,974






LeuTyrCysLeuLeu




 



Tyrosinase

HLA-A2
MetLeuLeuAlaVal
922
5,530,096






LeuTyrCysLeu




 



Tyrosinase

HLA-A2
LeuLeuAlaValLeu
923
5,530,096






TyrCysLeuLeu




 



Tyrosinase

HLA-A2
SerGluIleTrpArg
924
5,519,117





and HLA-
AspIleAspPheAla







B44
HisGluAla




 



Tyrosinase

HLA-A2
SerGlulleTrpArg
925
5,519,117





and HLA-
AspIleAspPhe







B44





 



Tyrosinase

HLA-A2
GluGluAsnLeuLeu
926
5,519,117





and HLA-
AspPheValArgPhe







B44





 



Melan


EAAGIGILTV
927
Jäger, E. et al.



A/MART-1




Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



Tyrosinase


MLLAVLYCL
928
Jäger, E. et al.








Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



Tyrosinase


YMDGTMSQV
929
Jäger, E. et al.








Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



gp100/Pme117


YLEPGPVTA
930
Jäger, E. et al.








Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



gp100/Pme117


LLDGTATLRL
931
Jäger, E. et al.








Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



Influenza


GILGFVFTL
932
Jäger, E. et al.



matrix




Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



MAGE-1


EADPTGHSY
933
Jäger, E. et al.








Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



MAGE-1

HLA-A1
EADPTGHSY
934



 



BAGE

HLA-C
MAARAVFLAL
935
Jäger, E. et al.






SAQLLQARLM

Granulocyte-macrophage-






KE

colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



BAGE

HLA-C
MAARAVFLAL
936
Jäger, E. et al.






SAQLLQ

Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 



BAGE

HLA-C
AARAVFLAL
937
Jäger, E. et al.








Granulocyte-macrophage-








colony-stimulating








Factor Enhances Immune








Responses To








Melanoma-′associated








Peptides in vivo Int. J








Cancer 67, 54-62 (1996)


 


Influenza
PR8 NP
147-154
Kd
IYQRIRALV
938
Falk et al., Allele-








specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


SELF
P815

Kd
SYFPEITHI
939
Falk et al., Allele-


PEPTIDE





specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Influenza
Jap HA

Kd
IYATVAGSL
940
Falk et al., Allele-



523-549




specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Influenza
Jap HA

Kd
VYQILAIYA
941
Falk et al., Allele-



523-549




specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Influenza
Jap HA

Kd
IYSTVASSL
942
Falk et al., Allele-



523-549




specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Influenza
JAP HA

Kd
LYQNVGTYV
943
Falk et al., Allele-



202-221




specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 



HLA-A24

Kd
RYLENQKRT
944
Falk et al., Allele-








specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 



HLA-Cw3

Kd
RYLKNGKET
945
Falk et al., Allele-








specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 



P815

Kd
KYQAVTTTL
946
Falk et al., Allele-








specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Plasmodium
CSP

Kd
SYIPSAEKI
947
Falk et al., Allele-


berghei





specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Plasmodium
CSP

Kd
SYVPSAFQI
948
Falk et al., Allele-


yoelii





specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Vesicular
NP 52-59

Kb
RGYVYQGL
949
Falk et al., Allele-


stomatitis





specific motifs revealed


virus





by sequencing of self-








by sequencing of self-








peptides eluted from








MHC molecules


 


Ovalbumin


Kb
SIINFEKL
950
Falk et al., Allele-








specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 


Sendai
NP 321-332

Kb
APGNYPAL
951
Falk et al., Allele-


virus





specific motifs revealed








by sequencing of self-








peptides eluted from








MHC molecules


 






VPYGSFKHV
952
Morel et al., Processing








of some antigens by the








standard proteasome








but not by the








immunoproteasome








results in poor








presentation by








dendritic cells,








Immunity, vol. 12: 107-








117, 2000.








MOTIFS





Influenza
PR8 NP

Kd
TYQRTRALV
953
5,747,269





restricted








peptide








motif





 


Self
P815

Kd
SYFPEITHI
954
5,747,269


peptide


restricted








peptide








motif





 


Influenza
JAP HA

Kd
IYATVAGSL
955
5,747,269





restricted








peptide








motif





 


Influenza
JAP HA

Kd
VYQILAIYA
956
5,747,269





restricted








peptide








motif





 


Influenza
PR8 HA

Kd
IYSTVASSL
957
5,747,269





restricted








peptide








motif





 


Influenza
JAP HA

Kd
LYQNVGTYV
958
5,747,269





restricted








peptide








motif





 





HLA-A24
RYLENGKETL
959
5,747,269


 





HLA-Cw3
RYLKNGKETL
960
5,747,269


 



P815

HLA-Cw3
KYQAVTTTL
961
5,747,269



tumour








antigen







 


Plasmodium
CSP

HLA-Cw3
SYIPSAEKI
962
5,747,269


berghei








 


Plasmodium
CSP

HLA-Cw3
SYVPSAEQI
963
5,747,269


yoelii








 


Influenza
NP

Db-
ASNENMETM
964
5,747,269





restricted








peptide








motif





 


Adenovirus
E1A

Db-
SGPSNTPPEI
965
5,747,269





restricted








peptide








motif





 


Lymphocytic


Db-
SGVENPGGYC
966
5,747,269


choriomen-


restricted
L




ingitis


peptide








motif





 


Simian
40 T

Db-
SAINNY . . .
967
5,747,269


virus


restricted








peptide








motif





 


HIV
reverse

HLA-
ILKEPVHGV
968
5,747,269



transcriptase

A2.1-








restricted








peptide








motif





 



influenza

HLA-
GILGFVFTL
969
5,747,269



matrix

A2.1-






protein

restricted








peptide








motif





 


Influenza
influenza

HLA-
ILGFVFTLTV
970
5,747,269



matrix

A2.1-






protein

restricted








peptide








motif





 


HIV
Gag protein


FLQSRPEPT
971
5,747,269


 


HIV
Gag protein


AMQMLKE . . .
972
5,747,269


 


HIV
Gag protein


PIAPGQMRE
973
5,747,269


 


HIV
Gag protein


QMKDCTERQ
974
5,747,269


 





HLA-
VYGVIQK
975
5,747,269





A*0205-








restricted








peptide








motif

















TABLE 8








VSV-NP peptide (49-62)



LCMV-NP peptide (118-132)



LCMV glycoprotein peptide. 33-41









Still further embodiments are directed to methods, uses, therapies and compositions related to epitopes with specificity for MHC, including, for example, those listed in Tables 9-13. Other embodiments include one or more of the MHCs listed in Tables 9-13, including combinations of the same, while other embodiments specifically exclude any one or more of the MHCs or combinations thereof. Tables 11-13 include frequencies for the listed HLA antigens.







TABLE 9





Class I MHC Molecules

















Class I



Human



HLA-A1



HLA-A*0101



HLA-A*0201



HLA-A*0202



HLA-A*0203



HLA-A*0204



HLA-A*0205



HLA-A*0206



HLA-A*0207



HLA-A*0209



HLA-A*0214



HLA-A3



HLA-A*0301



HLA-A*1101



HLA-A23



HLA-A24



HLA-A25



HLA-A*2902



HLA-A*3101



HLA-A*3302



HLA-A*6801



HLA-A*6901



HLA-B7



HLA-B*0702



HLA-B*0703



HLA-B*0704



HLA-B*0705



HLA-B8



HLA-B13



HLA-B14



HLA-B*1501 (B62)



HLA-B17



HLA-B18



HLA-B22



HLA-B27



HLA-B*2702



HLA-B*2704



HLA-B*2705



HLA-B*2709



HLA-B35



HLA-B*3501



HLA-B*3502



HLA-B*3701



HLA-B*3801



HLA-B*39011



HLA-B*3902



HLA-B40



HLA-B*40012 (B60)



HLA-B*4006 (B61)



HLA-B44



HLA-B*4402



HLA-B*4403



HLA-B*4501



HLA-B*4601



HLA-B51



HLA-B*5101



HLA-B*5102



HLA-B*5103



HLA-B*5201



HLA-B*5301



HLA-B*5401



HLA-B*5501



HLA-B*5502



HLA-B*5601



HLA-B*5801



HLA-B*6701



HLA-B*7301



HLA-B*7801



HLA-Cw*0102



HLA-Cw*0301



HLA-Cw*0304



HLA-Cw*0401



HLA-Cw*0601



HLA-Cw*0602



HLA-Cw*0702



HLA-Cw8



HLA-Cw*1601 M



HLA-G



Murine



H2-Kd



H2-Dd



H2-Ld



H2-Kb



H2-Db



H2-Kk



H2-Kkml



Qa-1a



Qa-2



H2-M3



Rat



RT1.Aa



RT1.A1



Bovine



Bota-A11



Bota-A20



Chicken



B-F4



B-F12



B-F15



B-F19



Chimpanzee



Patr-A*04



Patr-A*11



Patr-B*01



Patr-B*13



Patr-B*16



Baboon



Papa-A*06



Macaque



Mamu-A*01



Swine



SLA (haplotype d/d)



Virus homolog



hCMV class I homolog UL18
















TABLE 10





Class I MHC Molecules

















Class I



Human



HLA-A1



HLA-A*0101



HLA-A*0201



HLA-A*0202



HLA-A*0204



HLA-A*0205



HLA-A*0206



HLA-A*0207



HLA-A*0214



HLA-A3



HLA-A*1101



HLA-A24



HLA-A*2902



HLA-A*3101



HLA-A*3302



HLA-A*6801



HLA-A*6901



HLA-B7



HLA-B*0702



HLA-B*0703



HLA-B*0704



HLA-B*0705



HLA-B8



HLA-B14



HLA-B*1501 (B62)



HLA-B27



HLA-B*2702



HLA-B*2705



HLA-B35



HLA-B*3501



HLA-B*3502



HLA-B*3701



HLA-B*3801



HLA-B*39011



HLA-B*3902



HLA-B40



HLA-B*40012 (B60)



HLA-B*4006 (B61)



HLA-B44



HLA-B*4402



HLA-B*4403



HLA-B*4601



HLA-B51



HLA-B*5101



HLA-B*5102



HLA-B*5103



HLA-B*5201



HLA-B*5301



HLA-B*5401



HLA-B*5501



HLA-B*5502



HLA-B*5601



HLA-B*5801



HLA-B*6701



HLA-B*7301



HLA-B*7801



HLA-Cw*0102



HLA-Cw*0301



HLA-Cw*0304



HLA-Cw*0401



HLA-Cw*0601



HLA-Cw*0602



HLA-Cw*0702



HLA-G



Murine



H2-Kd



H2-Dd



H2-Ld



H2-Kb



H2-Db



H2-Kk



H2-Kkml



Qa-2



Rat



RT1.Aa



RT1.A1



Bovine



Bota-A11



Bota-A20



Chicken



B-F4



B-F12



B-F15



B-F19



Virus homolog



hCMV class I homolog UL18
















TABLE 11







Estimated gene frequencies of HLA-A antigens













CAU
AFR
ASI
LAT
NAT

















Antigen
Gfa
SEb
Gf
SE
Gf
SE
Gf
SE
Gf
SE




















A1
15.1843
0.0489
5.7256
0.0771
4.4818
0.0846
7.4007
0.0978
12.0316
0.2533


A2
28.6535
0.0619
18.8849
0.1317
24.6352
0.1794
28.1198
0.1700
29.3408
0.3585


A3
13.3890
0.0463
8.4406
0.0925
2.6454
0.0655
8.0789
0.1019
11.0293
0.2437


A28
4.4652
0.0280
9.9269
0.0997
1.7657
0.0537
8.9446
0.1067
5.3856
0.1750


A36
0.0221
0.0020
1.8836
0.0448
0.0148
0.0049
0.1584
0.0148
0.1545
0.0303


A23
1.8287
0.0181
10.2086
0.1010
0.3256
0.0231
2.9269
0.0628
1.9903
0.1080


A24
9.3251
0.0395
2.9668
0.0560
22.0391
0.1722
13.2610
0.1271
12.6613
0.2590


A9 unsplit
0.0809
0.0038
0.0367
0.0063
0.0858
0.0119
0.0537
0.0086
0.0356
0.0145


A9 total
11.2347
0.0429
13.2121
0.1128
22.4505
0.1733
16.2416
0.1382
14.6872
0.2756


A25
2.1157
0.0195
0.4329
0.0216
0.0990
0.0128
1.1937
0.0404
1.4520
0.0924


A26
3.8795
0.0262
2.8284
0.0547
4.6628
0.0862
3.2612
0.0662
2.4292
0.1191


A34
0.1508
0.0052
3.5228
0.0610
1.3529
0.0470
0.4928
0.0260
0.3150
0.0432


A43
0.0018
0.0006
0.0334
0.0060
0.0231
0.0062
0.0055
0.0028
0.0059
0.0059


A66
0.0173
0.0018
0.2233
0.0155
0.0478
0.0089
0.0399
0.0074
0.0534
0.0178


A10 unsplit
0.0790
0.0038
0.0939
0.0101
0.1255
0.0144
0.0647
0.0094
0.0298
0.0133


A10 total
6.2441
0.0328
7.1348
0.0850
6.3111
0.0993
5.0578
0.0816
4.2853
0.1565


A29
3.5796
0.0252
3.2071
0.0582
1.1233
0.0429
4.5156
0.0774
3.4345
0.1410


A30
2.5067
0.0212
13.0969
0.1129
2.2025
0.0598
4.4873
0.0772
2.5314
0.1215


A31
2.7386
0.0221
1.6556
0.0420
3.6005
0.0761
4.8328
0.0800
6.0881
0.1855


A32
3.6956
0.0256
1.5384
0.0405
1.0331
0.0411
2.7064
0.0604
2.5521
0.1220


A33
1.2080
0.0148
6.5607
0.0822
9.2701
0.1191
2.6593
0.0599
1.0754
0.0796


A74
0.0277
0.0022
1.9949
0.0461
0.0561
0.0096
0.2027
0.0167
0.1068
0.0252


A19 unsplit
0.0567
0.0032
0.2057
0.0149
0.0990
0.0128
0.1211
0.0129
0.0475
0.0168


A19 total
13.8129
0.0468
28.2593
0.1504
17.3846
0.1555
19.5252
0.1481
15.8358
0.2832


AX
0.8204
0.0297
4.9506
0.0963
2.9916
0.1177
1.6332
0.0878
1.8454
0.1925





aGene frequency.


bStandard error.













TABLE 12







Estimated gene frequencies for HLA-B antigens













CAU
AFR
ASI
LAT
NAT

















Antigen
Gfa
SEb
Gf
SE
Gf
SE
Gf
SE
Gf
SE




















B7
12.1782
0.0445
10.5960
0.1024
4.2691
0.0827
6.4477
0.0918
10.9845 
0.2432


B8
9.4077
0.0397
3.8315
0.0634
1.3322
0.0467
3.8225
0.0715
8.5789
0.2176


B13
2.3061
0.0203
0.8103
0.0295
4.9222
0.0886
1.2699
0.0416
1.7495
0.1013


B14
4.3481
0.0277
3.0331
0.0566
0.5004
0.0287
5.4166
0.0846
2.9823
0.1316


B18
4.7980
0.0290
3.2057
0.0582
1.1246
0.0429
4.2349
0.0752
3.3422
0.1391


B27
4.3831
0.0278
1.2918
0.0372
2.2355
0.0603
2.3724
0.0567
5.1970
0.1721


B35
9.6614
0.0402
8.5172
0.0927
8.1203
0.1122
14.6516
0.1329
10.1198 
0.2345


B37
1.4032
0.0159
0.5916
0.0252
1.2327
0.0449
0.7807
0.0327
0.9755
0.0759


B41
0.9211
0.0129
0.8183
0.0296
0.1303
0.0147
1.2818
0.0418
0.4766
0.0531


B42
0.0608
0.0033
5.6991
0.0768
0.0841
0.0118
0.5866
0.0284
0.2856
0.0411


B46
0.0099
0.0013
0.0151
0.0040
4.9292
0.0886
0.0234
0.0057
0.0238
0.0119


B47
0.2069
0.0061
0.1305
0.0119
0.0956
0.0126
0.1832
0.0159
0.2139
0.0356


B48
0.0865
0.0040
0.1316
0.0119
2.0276
0.0575
1.5915
0.0466
1.0267
0.0778


B53
0.4620
0.0092
10.9529
0.1039
0.4315
0.0266
1.6982
0.0481
1.0804
0.0798


B59
0.0020
0.0006
0.0032
0.0019
0.4277
0.0265
0.0055
0.0028
0c   
—


B67
0.0040
0.0009
0.0086
0.0030
0.2276
0.0194
0.0055
0.0028
0.0059
0.0059


B70
0.3270
0.0077
7.3571
0.0866
0.8901
0.0382
1.9266
0.0512
0.6901
0.0639


B73
0.0108
0.0014
0.0032
0.0019
0.0132
0.0047
0.0261
0.0060
0c   
—


B51
5.4215
0.0307
2.5980
0.0525
7.4751
0.1080
6.8147
0.0943
6.9077
0.1968


B52
0.9658
0.0132
1.3712
0.0383
3.5121
0.0752
2.2447
0.0552
0.6960
0.0641


B5 unsplit
0.1565
0.0053
0.1522
0.0128
0.1288
0.0146
0.1546
0.0146
0.1307
0.0278


B5 total
6.5438
0.0435
4.1214
0.0747
11.1160
0.1504
9.2141
0.1324
7.7344
0.2784


B44
13.4838
0.0465
7.0137
0.0847
5.6807
0.0948
9.9253
0.1121
11.8024 
0.2511


B45
0.5771
0.0102
4.8069
0.0708
0.1816
0.0173
1.8812
0.0506
0.7603
0.0670


B12 unsplit
0.0788
0.0038
0.0280
0.0055
0.0049
0.0029
0.0193
0.0051
0.0654
0.0197


B12 total
14.1440
0.0474
11.8486
0.1072
5.8673
0.0963
11.8258
0.1210
12.6281
0.2584


B62
5.9117
0.0320
1.5267
0.0404
9.2249
0.1190
4.1825
0.0747
6.9421
0.1973


B63
0.4302
0.0088
1.8865
0.0448
0.4438
0.0270
0.8083
0.0333
0.3738
0.0471


B75
0.0104
0.0014
0.0226
0.0049
1.9673
0.0566
0.1101
0.0123
0.0356
0.0145


B76
0.0026
0.0007
0.0065
0.0026
0.0874
0.0120
0.0055
0.0028
0   
—


B77
0.0057
0.0010
0.0119
0.0036
0.0577
0.0098
0.0083
0.0034
0c   
0.0059


B15 unsplit
0.1305
0.0049
0.0691
0.0086
0.4301
0.0266
0.1820
0.0158
0.0059
0.0206


B15 total
6.4910
0.0334
3.5232
0.0608
12.2112
0.1344
5.2967
0.0835
0.0715
0.2035











7.4290



B38
2.4413
0.0209
0.3323
0.0189
3.2818
0.0728
1.9652
0.0517
1.1017
0.0806


B39
1.9614
0.0188
1.2893
0.0371
2.0352
0.0576
6.3040
0.0909
4.5527
0.1615


B16 unsplit
0.0638
0.0034
0.0237
0.0051
0.0644
0.0103
0.1226
0.0130
0.0593
0.0188


B16 total
4.4667
0.0280
1.6453
0.0419
5.3814
0.0921
8.3917
0.1036
5.7137
0.1797


B57
3.5955
0.0252
5.6746
0.0766
2.5782
0.0647
2.1800
0.0544
2.7265
0.1260


B58
0.7152
0.0114
5.9546
0.0784
4.0189
0.0803
1.2481
0.0413
0.9398
0.0745


B17 unsplit
0.2845
0.0072
0.3248
0.0187
0.3751
0.0248
0.1446
0.0141
0.2674
0.0398


B17 total
4.5952
0.0284
11.9540
0.1076
6.9722
0.1041
3.5727
0.0691
3.9338
0.1503


B49
1.6452
0.0172
2.6286
0.0528
0.2440
0.0200
2.3353
0.0562
1.5462
0.0953


B50
1.0580
0.0138
0.8636
0.0304
0.4421
0.0270
1.8883
0.0507
0.7862
0.0681


B21 unsplit
0.0702
0.0036
0.0270
0.0054
0.0132
0.0047
0.0771
0.0103
0.0356
0.0145


B21 total
2.7733
0.0222
3.5192
0.0608
0.6993
0.0339
4.3007
0.0755
2.3680
0.1174


B54
0.0124
0.0015
0.0183
0.0044
2.6873
0.0660
0.0289
0.0063
0.0534
0.0178


B55
1.9046
0.0185
0.4895
0.0229
2.2444
0.0604
0.9515
0.0361
1.4054
0.0909


B56
0.5527
0.0100
0.2686
0.0170
0.8260
0.0368
0.3596
0.0222
0.3387
0.0448


B22 unsplit
0.1682
0.0055
0.0496
0.0073
0.2730
0.0212
0.0372
0.0071
0.1246
0.0272


B22 total
2.0852
0.0217
0.8261
0.0297
6.0307
0.0971
1.3771
0.0433
1.9221
0.1060


B60
5.2222
0.0302
1.5299
0.0404
8.3254
0.1135
2.2538
0.0553
5.7218
0.1801


B61
1.1916
0.0147
0.4709
0.0225
6.2072
0.0989
4.6691
0.0788
2.6023
0.1231


B40 unsplit
0.2696
0.0070
0.0388
0.0065
0.3205
0.0230
0.2473
0.0184
0.2271
0.0367


B40 total
6.6834
0.0338
2.0396
0.0465
14.8531
0.1462
7.1702
0.0963
8.5512
0.2168


BX
1.0922
0.0252
3.5258
0.0802
3.8749
0.0988
2.5266
0.0807
1.9867
0.1634





aGene frequency.


bStandard error.


cThe observed gene count was zero.













TABLE 13







Estimated gene frequencies of HLA-DR antigens













CAU
AFR
ASI
LAT
NAT

















Antigen
Gfa
SEb
Gf
SE
Gf
SE
Gf
SE
Gf
SE




















DR1
10.2279
0.0413
6.8200
0.0832
3.4628
0.0747
7.9859
0.1013
8.2512
0.2139


DR2
15.2408
0.0491
16.2373
0.1222
18.6162
0.1608
11.2389
0.1182
15.3932
0.2818


DR3
10.8708
0.0424
13.3080
0.1124
4.7223
0.0867
7.8998
0.1008
10.2549
0.2361


DR4
16.7589
0.0511
5.7084
0.0765
15.4623
0.1490
20.5373
0.1520
19.8264
0.3123


DR6
14.3937
0.0479
18.6117
0.1291
13.4471
0.1404
17.0265
0.1411
14.8021
0.2772


DR7
13.2807
0.0463
10.1317
0.0997
6.9270
0.1040
10.6726
0.1155
10.4219
0.2378


DR8
2.8820
0.0227
6.2673
0.0800
6.5413
0.1013
9.7731
0.1110
6.0059
0.1844


DR9
1.0616
0.0139
2.9646
0.0559
9.7527
0.1218
1.0712
0.0383
2.8662
0.1291


DR10
1.4790
0.0163
2.0397
0.0465
2.2304
0.0602
1.8044
0.0495
1.0896
0.0801


DR11
9.3180
0.0396
10.6151
0.1018
4.7375
0.0869
7.0411
0.0955
5.3152
0.1740


DR12
1.9070
0.0185
4.1152
0.0655
10.1365
0.1239
1.7244
0.0484
2.0132
0.1086


DR5 unsplit
1.2199
0.0149
2.2957
0.0493
1.4118
0.0480
1.8225
0.0498
1.6769
0.0992


DR5 total
12.4449
0.0045
17.0260
0.1243
16.2858
0.1516
10.5880
0.1148
9.0052
0.2218


DRX
1.3598
0.0342
0.8853
0.0760
2.5521
0.1089
1.4023
0.0930
2.0834
0.2037





aGene frequency.


bStandard error.






It can be desirable to express housekeeping peptides in the context of a larger protein. Processing can be detected even when a small number of amino acids are present beyond the terminus of an epitope. Small peptide hormones are usually proteolytically processed from longer translation products, often in the size range of approximately 60-120 amino acids. This fact has led some to assume that this is the minimum size that can be efficiently translated. In some embodiments, the housekeeping peptide can be embedded in a translation product of at least about 60 amino acids, in others 70, 80, 90 amino acids, and in still others 100, 110 or 120 amino acids, for example. In other embodiments the housekeeping peptide can be embedded in a translation product of at least about 50, 30, or 15 amino acids.
Due to differential proteasomal processing, the immunoproteasome of the pAPC produces peptides that are different from those produced by the housekeeping proteasome in peripheral body cells. Thus, in expressing a housekeeping peptide in the context of a larger protein, it is preferably expressed in the pAPC in a context other than its full-length native sequence, because, as a housekeeping epitope, it is generally only efficiently processed from the native protein by the housekeeping proteasome, which is not active in the pAPC. In order to encode the housekeeping epitope in a DNA sequence encoding a larger polypeptide, it is useful to find flanking areas on either side of the sequence encoding the epitope that permit appropriate cleavage by the immunoproteasome in order to liberate that housekeeping epitope. Such a sequence promoting appropriate processing is referred to hereinafter as having substrate or liberation sequence function. Altering flanking amino acid residues at the N-terminus and C-terminus of the desired housekeeping epitope can facilitate appropriate cleavage and generation of the housekeeping epitope in the pAPC. Sequences embedding housekeeping epitopes can be designed de novo and screened to determine which can be successfully processed by immunoproteasomes to liberate housekeeping epitopes.
Alternatively, another strategy is very effective for identifying sequences allowing production of housekeeping epitopes in APC. A contiguous sequence of amino acids can be generated from head to tail arrangement of one or more housekeeping epitopes. A construct expressing this sequence is used to immunize an animal, and the resulting T cell response is evaluated to determine its specificity to one or more of the epitopes in the array. These immune responses indicate housekeeping epitopes that are processed in the pAPC effectively. The necessary flanking areas around this epitope are thereby defined. The use of flanking regions of about 4-6 amino acids on either side of the desired peptide can provide the necessary information to facilitate proteasome processing of the housekeeping epitope by the immunoproteasome. Therefore, a substrate or liberation sequence of approximately 16-22 amino acids can be inserted into, or fused to, any protein sequence effectively to result in that housekeeping epitope being produced in an APC. In some embodiments, a broader context of a substrate sequence can also influence processing. In such embodiments, comparisons of a liberaton sequence in a variety of contexts can be useful in further optimizing a particular substrate sequence. In alternate embodiments the whole head-to-tail array of epitopes, or just the epitopes immediately adjacent to the correctly processed housekeeping epitope can be similarly transferred from a test construct to a vaccine vector.
In a preferred embodiment, the housekeeping epitopes can be embedded between known immune epitopes, or segments of such, thereby providing an appropriate context for processing. The abutment of housekeeping and immune epitopes can generate the necessary context to enable the immunoproteasome to liberate the housekeeping epitope, or a larger fragment, preferably including a correct C-terminus. It can be useful to screen constructs to verify that the desired epitope is produced. The abutment of housekeeping epitopes can generate a site cleavable by the immunoproteasome. Some embodiments of the invention employ known epitopes to flank housekeeping epitopes in test substrates; in others, screening as described below is used, whether the flanking regions are arbitrary sequences or mutants of the natural flanking sequence, and whether or not knowledge of proteasomal cleavage preferences are used in designing the substrates.
Cleavage at the mature N-terminus of the epitope, while advantageous, is not required, since a variety of N-terminal trimming activities exist in the cell that can generate the mature N-terminus of the epitope subsequent to proteasomal processing. It is preferred that such N-terminal extension be less than about 25 amino acids in length and it is further preferred that the extension have few or no proline residues. Preferably, in screening, consideration is given not only to cleavage at the ends of the epitope (or at least at its C-terminus), but consideration also can be given to ensure limited cleavage within the epitope.
Shotgun approaches can be used in designing test substrates and can increase the efficiency of screening. In one embodiment multiple epitopes can be assembled one after the other, with individual epitopes possibly appearing more than once. The substrate can be screened to determine which epitopes can be produced. In the case where a particular epitope is of concern, a substrate can be designed in which it appears in multiple different contexts. When a single epitope appearing in more than one context is liberated from the substrate additional secondary test substrates, in which individual instances of the epitope are removed, disabled, or are unique, can be used to determine which are being liberated and truly confer substrate or liberation sequence function.
Several readily practicable screens exist. A preferred in vitro screen utilizes proteasomal digestion analysis, using purified immunoproteasomes, to determine if the desired housekeeping epitope can be liberated from a synthetic peptide embodying the sequence in question. The position of the cleavages obtained can be determined by techniques such as mass spectrometry, HPLC, and N-terminal pool sequencing; as described in greater detail in U.S. patent application Ser. Nos. 09/561,074, 09/560,465 and 10/117,937, and Provisional U.S. Patent Application Nos. 60/282,211, 60/337,017, and 60/363, 210, which were all cited and incorporated by reference above.
Alternatively, in vivo and cell-based screens such as immunization or target sensitization can be employed. For immunization a nucleic acid construct capable of expressing the sequence in question is used. Harvested CTL can be tested for their ability to recognize target cells presenting the housekeeping epitope in question. Such targets cells are most readily obtained by pulsing cells expressing the appropriate MHC molecule with synthetic peptide embodying the mature housekeeping epitope. Alternatively, immunization can be carried out using cells known to express housekeeping proteasome and the antigen from which the housekeeping epitope is derived, either endogenously or through genetic engineering. To use target sensitization as a screen, CTL, or preferably a CTL clone, that recognizes the housekeeping epitope can be used. In this case it is the target cell that expresses the embedded housekeeping epitope (instead of the pAPC during immunization) and it must express immunoproteasome. Generally, the cell or target cell can be transformed with an appropriate nucleic acid construct to confer expression of the embedded housekeeping epitope. Loading with a synthetic peptide embodying the embedded epitope using peptide loaded liposomes, or complexed with cationic lipid protein transfer reagents such as BIOPORTERTM (Gene Therapy Systems, San Diego, Calif.), represents an alternative.
Once sequences with substrate or liberation sequence function are identified they can be encoded in nucleic acid vectors, chemically synthesized, or produced recombinantly. In any of these forms they can be incorporated into immunogenic compositions. Such compositions can be used in vitro in vaccine development or in the generation or expansion of CTL to be used in adoptive immunotherapy. In vivo they can be used to induce, amplify or sustain and active immune response. The uptake of polypeptides for processing and presentation can be greatly enhanced by packaging with cationic lipid, the addition of a tract of cationic amino acids such as poly-L-lysine (Ryser, H. J. et al., J. Cell Physiol. 113:167-178, 1982; Shen, W. C. & Ryser, H. J., Proc. Natl. Aced. Sci. USA 75:1872-1876, 1978), the incorporation into branched structures with importation signals (Sheldon, K. et al., Proc. Natl. Aced. Sci. USA 92:2056-2060, 1995), or mixture with or fusion to polypeptides with protein transfer function including peptide carriers such as pep-1 (Morris, M. C., et al., Nat. Biotech. 19:1173-1176, 2001), the PreS2 translocation motif of hepatitis B virus surface antigen, VP22 of herpes viruses, and HIV-TAT protein (Oess, S. & Hildt, E., Gene Ther. 7:750-758, 2000; Ford, K. G., et al., Gene Ther. 8:1-4, 2001; Hung, C. F. et al., J. Virol. 76:2676-2682, 2002; Oliveira, S. C., et a; Hum. Gene Ther. 12:1353-1359, 2001; Normand, N. et al., J. Biol. Chem. 276:15042-15050, 2001; Schwartz, J. J. & Zhang, S., Curr. Opin. Mol. Ther. 2:162-167, 2000; Elliot G., 7 Hare, P. Cell 88:223-233, 1997), among other methodologies. Particularly for fusion proteins the immunogen can be produced in culture and the purified protein administered or, in the alternative, the nucleic acid vector can be administered so that the immunogen is produced and secreted by cells transformed in vivo. In either scenario the transport function of the fusion protein facilitates uptake by pAPC.
The following examples are intended for illustration purposes only, and should not be construed as limiting the scope of the invention in any way.
EXAMPLES
Example 1
A recombinant DNA plasmid vaccine, pMA2M, which encodes one polypeptide with an HLA A2-specific CTL epitope ELAGIGILTV (SEQ ID NO. 1) from melan-A (26-35A27L), and a portion (amino acids 31-96) of melan-A (SEQ ID NO. 2) including the epitope clusters at amino acids 31-48 and 56-69, was constructed. These clusters were previously disclosed in U.S. patent application Ser. No. 09/561,571 entitled EPITOPE CLUSTERS incorporated by reference above. Flanking the defined melan-A CTL epitope are short amino acid sequences derived from human tyrosinase (SEQ ID NO. 3) to facilitate liberation of the melan-A housekeeping epitope by processing by the immunoproteasome. In addition, these amino acid sequences represent potential CTL epitopes themselves. The cDNA sequence for the polypeptide in the plasmid is under the control of promoter/enhancer sequence from cytomegalovirus (CMVp) (see FIG. 4), which allows efficient transcription of messenger for the polypeptide upon uptake by APCs. The bovine growth hormone polyadenylation signal (BGH polyA) at the 3′ end of the encoding sequence provides a signal for polyadenylation of the messenger to increase its stability as well as for translocation out of nucleus into the cytoplasm for translation. To facilitate plasmid transport into the nucleus after uptake, a nuclear import sequence (NIS) from simian virus 40 (SV40) has been inserted in the plasmid backbone. The plasmid carries two copies of a CpG immunostimulatory motif, one in the NIS sequence and one in the plasmid backbone. Lastly, two prokaryotic genetic elements in the plasmid are responsible for amplification in E. coli, the kanamycin resistance gene (Kan R) and the pMB1 bacterial origin of replication.
Substrate or Liberation Sequence
The amino acid sequence of the encoded polypeptide (94 amino acid residues in length) (SEQ ID NO. 4) containing a 28 amino acid substrate or liberation sequence at its N-terminus (SEQ ID NO. 5) is given below:







MLLAVLYCL-ELAGIGILTV-YMDGTMSQV-GILTVILGVLLLIGCWYCR


 


RRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPV






The first 9 amino acid residues are derived from tyrosinase1-9 (SEQ ID NO. 6), the next ten constitute melan-A (26-35A27L) (SEQ ID NO. 1), and amino acid residues 20 to 29 are derived from tyrosinase369-377 (SEQ ID NO. 7). These two tyrosinase nonamer sequences both represent potential HLA A2-specific CTL epitopes. Amino acid residues 10-19 constitute melan-A (26-35A27L) an analog of an HLA A2-specific CTL epitope from melan-A, EAAGIGILTV (SEQ ID NO. 8), with an elevated potency in inducing CTL responses during in vitro immunization of human PBMC and in vivo immunization in mice. The segment of melan-A constituting the rest of the polypeptide (amino acid residues 30 to 94) contain a number of predicted HLA A2-specific epitopes, including the epitope clusters cited above, and thus can be useful in generating a response to immune epitopes as described at length in the patent applications ‘Epitope Synchronization in Antigen Presenting Cells’ and ‘Epitope Clusters’ cited and incorporated by reference above. This region was also included to overcome any difficulties that can be associated with the expression of shorter sequences. A drawing of pMA2M is shown in FIG. 4.
Plasmid Construction
A pair of long complementary oligonucleotides was synthesized which encoded the first 30 amino acid residues. In addition, upon annealing, these oligonucleotides generated the cohesive ends of Afl II at the 5′ end and that of EcoR I at the 3′ end. The melan A31-96 region was amplified with PCR using oligonucleotides carrying restriction sites for EcoR I at the 5′ end and Not I at the 3′ end. The PCR product was digested with EcoR I and Not I and ligated into the vector backbone, described in Example 1, that had been digested with Afl II and Not I, along with the annealed oligonucleotides encoding the amino terminal region in a three-fragment ligation. The entire coding sequence was verified by DNA sequencing. The sequence of the entire insert, from the Afl II site at the 5′ end to the Not I site at the 3′ end is disclosed as SEQ ID NO. 9. Nucleotides 12-293 encode the polypeptide.
Example 2
Three vectors containing melan-A (26-35A27L) (SEQ ID NO. 1) as an embedded housekeeping epitope were tested for their ability to induce a CTL response to this epitope in HLA-A2 transgenic HHD mice (Pascolo et al. J. Exp. Med. 185:2043-2051, 1997). One of the vectors was pMA2M described above (called pVAXM3 in FIG. 6). In pVAXM2 the same basic group of 3 epitopes was repeated several times with the flanking epitopes truncated by differing degrees in the various repeats of the array. Specifically the cassette consisted of:







(SEQ ID NO. 10)


M-Tyr(5-9)-ELA-Tyr(369-373)-Tyr(4-9)-ELA-Tyr(369-


 


374)-Tyr(3-9)-ELA-Tyr(369-375)-Tyr(2-9)-ELA






    
    
        where ELA represents melan-A (26-35A27L) (SEQ ID NO. 1). This cassette was inserted in the same plasmid backbone as used for pVAXM3. The third, pVAXM1 is identical to pVAXM2 except that the epitope array is followed by an IRES (internal ribosome entry site for encephalomyocarditis virus) linked to a reading frame encoding melan-A 31-70.
    
    


Four groups of three HHD A2.1 mice were injected intranodally in surgically exposed inguinal lymph nodes with 25 μl of 1 mg/ml plasmid DNA in PBS on days 0, 3, and 6, each group receiving one of the three vectors or PBS alone. On day 14 the spleens were harvested and restimulated in vitro one time with 3-day LPS blasts pulsed with peptide (melan-A (26-35A27L) (SEQ ID NO. 1)). The in vitro cultures were supplemented with Rat T-Stim (Collaborative Biomedical Products) on the 3rd day and assayed for cytolytic activity on the 7th day using a standard 51Cr-release assay. FIGS. 5 to 8 show % specific lysis obtained using the cells immunized with PBS, pVAXM1, pVAXM2, and pVAXM3, respectively on T2 target cells and T2 target cells pulsed with melan-A (26-35A27L) (ELA) (SEQ ID NO. 1). All three vectors generated strong CTL responses. These data indicated that the plasmids have been taken up by APCs, the encoded polypeptide has been synthesized and proteolytically processed to produce the decamer epitope in question (that is, it had substrate or liberation sequence function), and that the epitope became HLA-A2 bound for presentation. Also, an isolated variant of pVAXM2, that terminates after the 55th amino acid, worked similarly well as the full length version (data not shown). Whether other potential epitopes within the expression cassette can also be produced and be active in inducing CTL responses can be determined by testing for CTL activity against target cells pulsed with corresponding synthetic peptides.
Example 3
An NY-ESO-1 (SE ID NO. 11) Substrate/Liberation Sequence
Six other epitope arrays were tested leading to the identification of a substrate/liberation sequence for the housekeeping epitope NY-ESO-1157-165 (SEQ ID NO. 12). The component epitopes of the arrays were:









SSX-241-49:




KASEKIFYV
(SEQ ID NO. 13)
Array element A


 


NY-ESO-1157-165:




SLLMWITQC
(SEQ ID NO. 12)
Array element B


 


NY-ESO-1163-171:




TQCFLPVFL
(SEQ ID NO. 14)
Array element C


 


PSMA288-297:




GLPSIPVHPI
(SEQ ID NO. 15)
Array element D


 


TYR4-9:




AVLYCL
(SEQ ID NO. 16)
Array element E






The six arrays had the following arrangements of elements after starting with an initiator methionine:









pVAX-PC-A:
B-A-D-D-A-B-A-A
(SEQ ID NO: 980)


 


pVAX-PC-B:
D-A-B-A-A-D-B-A
(SEQ ID NO: 981)


 


pVAX-PC-C:
E-A-D-B-A-B-E-A-A
(SEQ ID NO: 982)


 


pVAX-BC-A:
B-A-C-B-A-A-C-A
(SEQ ID NO: 983)


 


pVAX-BC-B:
C-A-B-C-A-A-B-A
(SEQ ID NO: 984)


 


pVAX-BC-C:
E-A-A-B-C-B-A-A
(SEQ ID NO: 985)






These arrays were inserted into the same vector backbone described in the examples above. The plasmid vectors were used to immunize mice essentially as described in Example 2 and the resulting CTL were tested for their ability to specifically lyse target cells pulsed with the peptide NY-ESO-1157-165, corresponding to element B above. Both pVAX-PC-A and pVAX-BC-A were found to induce specific lytic activity. Comparing the contexts of the epitope (element B) in the various arrays, and particularly between pVAX-PC-A and pVAX-BC-A, between pVAX-PC-A and pVAX-PC-B, and between pVAX-BC-A and pVAX-BC-C, it was concluded that it was the first occurrence of the epitope in pVAX-PC-A and pVAX-BC-A that was being correctly processed and presented. In other words an initiator methionine followed by elements B-A constitute a substrate/liberation sequence for the presentation of element B. On this basis a new expression cassette for use as a vaccine was constructed encoding the following elements:
An initiator methionine,

    
    
        NY-ESO-1157-165 (bold)—a housekeeping epitope,
        SSX241-49 (italic)—providing appropriate context for processing, and
        NY-ESO-177-180—to avoid “short sequence” problems and provide immune epitopes.
    
    


Thus the construct encodes the amino acid sequence:







(SEQ ID NO. 17)


M-SLLMWITQC-KASEKIFYV-RCGARGPESRLLEFYLAMPFATPMEAE


 


LARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSC


 


LQQLSLLMWITQCFLPVFLAQPPSGQ


 


RR






and MSLLMWITQCKASEKIFYV (SEQ ID NO. 18) constitutes the liberation or substrate sequence. A polynucleotide encoding SEQ ID NO. 17 (SEQ ID NO. 19: nucleotides 12-380) was inserted into the same plasmid backbone as used for pMA2M generating the plasmid pN157.

Example 4
A construct similar to pN157 containing the whole epitope array from pVAX-PC-A was also made and designated pBPL. Thus the encoded amino acid sequence in pBPL is:









(SEQ ID NO. 20)



M-SLLMWITQC-KASEKIFYV-GLPSIPVHPI-GLPSIPVHPI-


 



KASEKIFYV-SLLMWITQC-KASEKIFYV-KASEKIFYV-RCGA


 



RGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLK


 



EFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLP


 



VFLAQPPSGQRR.






SEQ ID NO. 21 is the polynucleotide encoding SEQ ID NO. 20 used in pBPL.
A portion of SEQ ID NO. 20, IKASEKIFYVSLLMWITQCKASEKIFYVK (SEQ ID NO. 22) was made as a synthetic peptide and subjected to in vitro proteasomal digestion analysis with human immunoproteasome, utilizing both mass spectrometry and N-terminal pool sequencing. The identification of a cleavage after the C residue indicates that this segment of the construct can function as a substrate or liberation sequence for NY-ESO-1157-165 (SEQ ID NO. 12) epitope (see FIG. 9). FIG. 10 shows the differential processing of the SLLMWITQC epitope (SEQ ID NO. 12) in its native context where the cleavage following the C is more efficiently produced by housekeeping than immunoproteasome. The immunoproteasome also produces a major cleavage internal to the epitope, between the T and the Q when the epitope is in its native context, but not in the context of SEQ ID NO. 22 (compare FIGS. 6 and 7).
Example 5
Screening of further epitope arrays led to the identification of constructs promoting the expression of the epitope SSX-241-49 (SEQ ID NO. 13). In addition to some of the array elements defined in Example 3, the following additional elements were also used:









SSX-457-65:




VMTKLGFKV
(SEQ ID NO. 23)
Array element F.


 


PSMA730-739:




RQIYVAAFTV
(SEQ ID NO. 24)
Array element G.






A construct, denoted CTLA02, encoding an initiator methionine and the array F-A-G-D-C-F-G-A, was found to successfully immunize HLA-A2 transgenic mice to generate a CTL response recognizing the peptide SSX-241-49 (SEQ ID NO. 13).
As described above, it can be desirable to combine a sequence with substrate or liberation sequence function with one that can be processed into immune epitopes. Thus SSX-215-183 (SEQ ID NO. 25) was combined with all or part of the array as follows:









CTLS1:
F-A-G-D-C-F-G-A-SSX-215-183
(SEQ ID NO. 26)


 


CTLS2:
SSX-215-183-F-A-G-D-C-F-G-A
(SEQ ID NO. 27)


 


CTLS3:
F-A-G-D-SSX-215-183
(SEQ ID NO. 28)


 


CTLS4:
SSX-215-183-C-F-G-A.
(SEQ ID NO. 29)






All of the constructs except CTLS3 were able to induce CTL recognizing the peptide SSX-241-49 (SEQ ID NO. 13). CTLS3 was the only one of these four constructs which did not include the second element A from CTLA02 suggesting that it was this second occurrence of the element that provided substrate or liberation sequence function. In CTLS2 and CTLS4 the A element is at the C-terminal end of the array, as in CTLA02. In CTLS1 the A element is immediately followed by the SSX-215-183 segment which begins with an alanine, a residue often found after proteasomal cleavage sites (Toes, R. E. M., et al., J. Exp. Med. 194:1-12, 2001). SEQ ID NO. 30 is the polynucleotide sequence encoding SEQ ID NO. 26 used in CTLS1, also called pCBP.
A portion of CTLS1 (SEQ ID NO. 26), encompassing array elements F-A-SSX-215-23 with the sequence RQIYVAAFTV-KASEKIFYV-AQIPEKIQK (SEQ ID NO. 31), was made as a synthetic peptide and subjected to in vitro proteasomal digestion analysis with human immunoproteasome, utilizing both mass spectrometry and N-terminal pool sequencing. The observation that the C-terminus of the SSX-241-49 epitope (SEQ ID NO. 13) was generated (see FIG. 11) provided further evidence in support of substrate or liberation sequence function. The data in FIG. 12 showed the differential processing of the SSX-241-49 epitope, KASEKIFYV (SEQ ID NO. 13), in its native context, where the cleavage following the V was the predominant cleavage produced by housekeeping proteasome, while the immunoproteasome had several major cleavage sites elsewhere in the sequence. By moving this epitope into the context provided by SEQ ID NO. 31 the desired cleavage became a major one and its relative frequency compared to other immunoproteasome cleavages was increased (compare FIGS. 11 and 12). The data in FIG. 11B also showed the similarity in specificity of mouse and human immunoproteasome lending support to the usefulness of the transgenic mouse model to predict human antigen processing.
Example 6
Screening also revealed substrate or liberation sequence function for a tyrosinase epitope, Tyr207-215 (SEQ ID NO. 32), as part of an array consisting of the sequence [Tyr1-17-Tyr207-215]4, [MLLAVLYCLLWSFQTSA-FLPWHRLFL]4, (SEQ ID NO. 33). The same vector backbone described above was used to express this array. This array differs from those of the other examples in that the Tyr1-17 segment, which was included as a source of immune epitopes, is used as a repeated element of the array. This is in contrast with the pattern shown in the other examples where sequence included as a source of immune epitopes and/or length occurred a single time at the beginning or end of the array, the remainder of which was made up of individual epitopes or shorter sequences.
Plasmid Construction
The polynucleotide encoding SEQ ID NO. 33 was generated by assembly of annealed synthetic oligonucleotides. Four pairs of complementary oligonucleotides were synthesized which span the entire coding sequence with cohesive ends of the restriction sites of Afl II and EcoR I at either terminus. Each complementary pair of oligonucleotides were first annealed, the resultant DNA fragments were ligated stepwise, and the assembled DNA fragment was inserted into the same vector backbone described above pre-digested with Afl II/EcoR I. The construct was called CTLT2/pMEL and SEQ ID NO. 34 is the polynucleotide sequence used to encode SEQ ID NO. 33.
Example 7
Administration of a DNA Plasmid Formulation of a Immunotherapeutic for Melanoma to Humans
An MA2M melanoma vaccine with a sequence as described in Example 1 above, was formulated in 1% Benzyl alcohol, 1% ethyl alcohol, 0.5 mM EDTA, citrate-phosphate, pH 7.6. Aliquots of 200, 400, and 600 μg DNA/ml were prepared for loading into MINIMED 407C infusion pumps. The catheter of a SILHOUETTE infusion set was placed into an inguinal lymph node visualized by ultrasound imaging. The pump and infusion set assembly was originally designed for the delivery of insulin to diabetics. The usual 17 mm catheter was substituted with a 31 mm catheter for this application. The infusion set was kept patent for 4 days (approximately 96 hours) with an infusion rate of about 25 μl/hour resulting in a total infused volume of approximately 2.4 ml. Thus the total administered dose per infusion was approximately 500, and 1000 μg; and can be 1500 μg, respectively, for the three concentrations described above. Following an infusion, subjects were given a 10 day rest period before starting a subsequent infusion. Given the continued residency of plasmid DNA in the lymph node after administration and the usual kinetics of CTL response following disappearance of antigen, this schedule will be sufficient to maintain the immunologic CTL response.
Example 8
SEQ ID NO. 22 is made as a synthetic peptide and packaged with a cationic lipid protein transfer reagent. The composition is infused directly into the inguinal lymph node (see example 7) at a rate of 200 to 600 μg of peptide per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted.
Example 9
A fusion protein is made by adding SEQ ID NO. 34 to the 3′ end of a nucleotide sequence encoding herpes simplex virus 1 VP22 (SEQ ID NO. 42) in an appropriate mammalian expression vector; the vector used above is suitable. The vector is used to transform HEK 293 cells and 48 to 72 hours later the cells are pelleted, lysed and a soluble extract prepared. The fusion protein is purified by affinity chromatography using an anti-VP22 monoclonal antibody. The purified fusion protein is administered intranodally at a rate of 10 to 100 μg per day for seven days, followed by seven days rest. An initial treatment of 3-8 cycles are conducted.
Examples 10-13
The following examples, Examples 10-13, all concern the prediction of 9-mer epitopes presented by HLA-A2.1, although the procedure is equally applicable to any HLA type, or epitope length, for which a predictive algorithm or MHC binding assay is available.
Example 10
Melan-A/MART-1 (SEQ ID NO: 2)
This melanoma tumor-associated antigen (TuAA) is 118 amino acids in length. Of the 110 possible 9-mers, 16 are given a score ≧16 by the SYFPEITHI/Rammensee algorithm. (See Table 14). These represent 14.5% of the possible peptides and an average epitope density on the protein of 0.136 per amino acid. Twelve of these overlap, covering amino acids 22-49 of SEQ ID NO: 2 resulting in an epitope density for the cluster of 0.428, giving a ratio, as described above, of 3.15. Another two predicted epitopes overlap amino acids 56-69 of SEQ ID NO: 2, giving an epitope density for the cluster of 0.143, which is not appreciably different than the average, with a ratio of just 1.05. See FIG. 1.







TABLE 14







SYFPEITHI (Rammensee algorithm) Results for Melan-A/MART-1


(SEQ ID NO: 2)









Rank
Start
Score












1
31
27


2
56
26


3
35
26


4
32
25


5
27
25


6
29
24


7
34
23


8
61
20


9
33
19


10
22
19


11
99
18


12
36
18


13
28
18


14
87
17


15
41
17


16
40
16









Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm leaves only 5. (See Table 15). The average density of epitopes in the protein is now only 0.042 per amino acid. Three overlapping peptides cover amino acids 31-48 of SEQ ID NO: 2 and the other two cover 56-69 of SEQ ID: 2, as before, giving ratios of 3.93 and 3.40, respectively. (See Table 16).







TABLE 15







BIMAS-NIH/Parker algorithm Results for Melan-A/MART-1


(SEQ ID NO: 2)












Rank
Start
Score
Log(Score)















1
40
1289.01
3.11



2
56
1055.104
3.02



3
31
81.385
1.91



4
35
20.753
1.32



5
61
4.968
0.70
















TABLE 16







Predicted Epitope Clusters for Melan-A/MART-1 (SEQ ID NO: 2)










Calculations




(Epitopes/AAs)













Cluster
AA
Peptides
Cluster
Whole protein
Ratio





1
31-48
3, 4, 1
0.17
0.042
3.93


2
56-69
2, 5
0.14
0.042
3.40









Example 11
SSX-2/HOM-MEL-40 (SEQ ID NO: 40)
This melanoma tumor-associated antigen (TuAA) is 188 amino acids in length. Of the 180 possible 9-mers, 11 are given a score ≧16 by the SYFPEITHI/Rammensee algorithm. These represent 6.1% of the possible peptides and an average epitope density on the protein of 0.059 amino acid. Three of these overlap, covering amino acids 99-114 of SEQ ID NO: 40 resulting in an epitope density for the cluster of 0.188, giving a ratio, as described above, of 3.18. There are also overlapping pairs of predicted epitopes at amino acids 16-28, 57-67, and 167-183 of SEQ ID NO: 40, giving ratios of 2.63, 3.11, and 2.01, respectively. There is an additional predicted epitope covering amino acids 5-28. Evaluating the region 5-28 SEQ ID NO: 40 containing three epitopes gives an epitope density of 0.125 and a ratio 2.14.
Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm leaves only 6. The average density of epitopes in the protein is now only 0.032 per amino acid. Only a single pair overlap, at 167-180 of SEQ ID NO: 40, with a ratio of 4.48. However the top ranked peptide is close to another single predicted epitope if that region, amino acids 41-65 of SEQ ID NO: 40, is evaluated the ratio is 2.51, representing a substantial difference from the average. See FIG. 2.







TABLE 17







SYFPEITHI/Rammensee algorithm for SSX-2/HOM-MEL-40


(SEQ ID NO: 40)









Rank
Start
Score












1
103
23


2
167
22


3
41
22


4
16
21


5
99
20


6
59
19


7
20
17


8
5
17


9
175
16


10
106
16


11
57
16
















TABLE 18







Calculations(Epitopes/AAs) (SEQ ID NO: 40)










Calculations




(Epitopes/AAs)













Cluster
AA
Peptides
Cluster
Whole protein
Ratio















1
 5 to 28
8, 4, 7
0.125
0.059
2.14


2
16-28
4, 7
0.15
0.059
2.63


3
57-67
11, 6
0.18
0.059
3.11


4
 99-114
5, 1, 10
0.19
0.059
3.20


5
167-183
2, 9
0.12
0.059
2.01
















TABLE 19







BIMAS-NIH/Parker algorithm (SEQ ID NO: 40)












Rank
Start
Score
Log(Score)















1
41
1017.062
3.01



2
167
21.672
1.34



3
57
20.81
1.32



4
103
10.433
1.02



5
172
10.068
1.00



6
16
6.442
0.81
















TABLE 20







Calculations(Epitopes/AAs) (SEQ ID NO: 40)












Cluster
AA
Peptides
Cluster
Whole protein
Ratio





1
41-65
1, 3
0.08
0.032
2.51


2
167-180
2, 5
0.14
0.032
4.48









Example 12
NY-ESO (SEQ ID NO: 11)
This tumor-associated antigen (TuAA) is 180 amino acids in length. Of the 172 possible 9-mers, 25 are given a score ≧16 by the SYFPEITHI/Rammensee algorithm. Like Melan-A above, these represent 14.5% of the possible peptides and an average epitope density on the protein of 0.136 per amino acid. However the distribution is quite different. Nearly half the protein is empty with just one predicted epitope in the first 78 amino acids. Unlike Melan-A where there was a very tight cluster of highly overlapping peptides, in NY-ESO the overlaps are smaller and extend over most of the rest of the protein. One set of 19 overlapping peptides covers amino acids 108-174 of SEQ ID NO: 11, resulting in a ratio of 2.04. Another 5 predicted epitopes cover 79-104 of SEQ ID NO: 11, for a ratio of just 1.38.
If instead one takes the approach of considering only the top 5% of predicted epitopes, in this case 9 peptides, one can examine whether good clusters are being obscured by peptides predicted to be less likely to bind to MHC. When just these predicted epitopes are considered we see that the region 108-140 of SEQ ID NO: 11 contains 6 overlapping peptides with a ratio of 3.64. There are also 2 nearby peptides in the region 148-167 of SEQ ID NO: 11 with a ratio of 2.00. Thus the large cluster 108-174 of SEQ ID NO: 11 can be broken into two smaller clusters covering much of the same sequence.
Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm brings 14 peptides into consideration. The average density of epitopes in the protein is now 0.078 per amino acid. A single set of 10 overlapping peptides is observed, covering amino acids 144-171 of SEQ ID NO: 11, with a ratio of 4.59. All 14 peptides fall in the region 86-171 of SEQ ID NO: 11 which is still 2.09 times the average density of epitopes in the protein. While such a large cluster is larger than we consider ideal it still offers a significant advantage over working with the whole protein. See FIG. 3.







TABLE 21







SYFPEITHI (Rammensee algorithm) Results for NY-ESO


(SEQ ID NO: 11)









Rank
Start
Score












1
108
25


2
148
24


3
159
21


4
127
21


5
86
21


6
132
20


7
122
20


8
120
20


9
115
20


10
96
20


11
113
19


12
91
19


13
166
18


14
161
18


15
157
18


16
151
18


17
137
18


18
79
18


19
139
17


20
131
17


21
87
17


22
152
16


23
144
16


24
129
16


25
15
16
















TABLE 22







Calculations(Epitopes/AAs) (SEQ ID NO: 11)












Cluster
AA
Peptides
Cluster
Whole protein
Ratio





1
108-140
 1, 9, 8, 7, 4, 6
0.18
0.05
3.64


2
148-167
 2, 3
0.10
0.05
2.00


3
 79-104
 5 12, 10, 18, 21
0.19
0.14
1.38


4
108-174
 1, 11, 9, 8, 7, 4,
0.28
0.14
2.04




 6, 17, 2, 16, 15,







 3, 14, 13, 24,







20, 19, 23, 22
















TABLE 23







BIMAS-NIH/Parker algorithm Results for NY-ESO (SEQ ID NO: 11)












Rank
Start
Score
Log(Score)















1
159
1197.321
3.08



2
86
429.578
2.63



3
120
130.601
2.12



4
161
83.584
1.92



5
155
52.704
1.72



6
154
49.509
1.69



7
157
42.278
1.63



8
108
21.362
1.33



9
132
19.425
1.29



10
145
13.624
1.13



11
163
11.913
1.08



12
144
11.426
1.06



13
148
6.756
0.83



14
152
4.968
0.70
















TABLE 24







Calculations(Epitopes/AAs) (SEQ ID NO: 11)
















Whole



Cluster
AA
Peptides
Cluster
protein
Ratio





1
 86-171
2, 8, 3, 9, 10, 12, 13, 
0.163
0.078
2.09




14, 6, 5, 7, 1, 4, 11





2
144-171
10, 12, 13, 14, 6, 5,
0.36
0.078
4.59




7, 1, 4, 11









Example 13
Tyrosinase (SEQ ID NO: 3)
This melanoma tumor-associated antigen (TuAA) is 529 amino acids in length. Of the 521 possible 9-mers, 52 are given a score ≧16 by the SYFPEITHI/Rammensee algorithm. These represent 10% of the possible peptides and an average epitope density on the protein of 0.098 per amino acid. There are 5 groups of overlapping peptides containing 2 to 13 predicted epitopes each, with ratios ranging from 2.03 to 4.41, respectively. There are an additional 7 groups of overlapping peptides, containing 2 to 4 predicted epitopes each, with ratios ranging from 1.20 to 1.85, respectively. The 17 peptides in the region 444-506 of SEQ ID NO: 3, including the 13 overlapping peptides above, constitutes a cluster with a ratio of 2.20.
Restricting the analysis to the 9-mers predicted to have a half time of dissociation of ≧5 minutes by the BIMAS-NIH/Parker algorithm brings 28 peptides into consideration. The average density of epitopes in the protein under this condition is 0.053 per amino acid. At this density any overlap represents more than twice the average density of epitopes. There are 5 groups of overlapping peptides containing 2 to 7 predicted epitopes each, with ratios ranging from 2.22 to 4.9, respectively. Only three of these clusters are common to the two algorithms. Several, but not all, of these clusters could be enlarged by evaluating a region containing them and nearby predicted epitopes.







TABLE 25







SYFPEITHI/Rammensee algorithm Results


for Tyrosinase (SEQ ID NO: 3)









Rank
Start
Score












1
490
34


2
491
31


3
487
28


4
1
27


5
2
25


6
482
23


7
380
23


8
369
23


9
214
23


10
506
22


11
343
22


12
207
22


13
137
22


14
57
22


15
169
20


16
118
20


17
9
20


18
488
19


19
483
19


20
480
19


21
479
19


22
478
19


23
473
19


24
365
19


25
287
19


26
200
19


27
5
19


28
484
18


29
476
18


30
463
18


31
444
18


32
425
18


33
316
18


34
187
18


35
402
17


36
388
17


37
346
17


38
336
17


39
225
17


40
224
17


41
208
17


42
186
17


43
171
17


44
514
16


45
494
16


46
406
16


47
385
16


48
349
16


49
184
16


50
167
16


51
145
16


52
139
16
















TABLE 26







Calculations(Epitopes/AAs) (SEQ ID NO: 3)
















Whole



Cluster
AA
Peptides
Cluster
protein
Ratio















1
1 to 17
4, 5, 27, 17
0.24
0.098
2.39


2
137-153
13, 52, 51
0.18
0.098
1.80


3
167-179
15, 43, 50
0.23
0.098
2.35


4
184-195
34, 42, 49
0.25
0.098
2.54


5
200-222
26, 41, 9, 12
0.17
0.098
1.77


6
224-233
39, 40
0.20
0.098
2.03


7
336-357
38, 11, 37, 48
0.18
0.098
1.85


8
365-377
24, 8
0.15
0.098
1.57


9
380-396
7, 47, 36
0.18
0.098
1.80


10
402-414
35, 46
0.15
0.098
1.57


11
473-502
29, 28, 23, 22, 21,
0.43
0.098
4.41




20, 6, 19, 3, 18, 1, 2, 45





12
506-522
10, 44
0.12
0.098
1.20



444-522
31, 30, 23, 29, 22, 
0.22
0.098
2.20




21, 20, 6, 19, 28, 3, 18,







1, 2, 45, 10, 44
















TABLE 27







BIMAS-NIH/Parker algorithm Results (SEQ ID NO: 3)












Rank
Start
Score
Log(Score)















1
207
540.469
2.73



2
369
531.455
2.73



3
1
309.05
2.49



4
9
266.374
2.43



5
490
181.794
2.26



6
214
177.566
2.25



7
224
143.451
2.16



8
171
93.656
1.97



9
506
87.586
1.94



10
487
83.527
1.92



11
491
83.527
1.92



12
2
54.474
1.74



13
137
47.991
1.68



14
200
30.777
1.49



15
208
26.248
1.42



16
460
21.919
1.34



17
478
19.425
1.29



18
365
17.14
1.23



19
380
16.228
1.21



20
444
13.218
1.12



21
473
13.04
1.12



22
57
10.868
1.04



23
482
8.252
0.92



24
483
7.309
0.86



25
5
6.993
0.84



26
225
5.858
0.77



27
343
5.195
0.72



28
514
5.179
0.71
















TABLE 28







Calculations(Epitopes/AAs) (SEQ ID NO: 3)
















Whole



Cluster
AA
Peptides
Cluster
protein
Ratio















1
1 to 17
3, 12, 25, 4
0.24
0.053
4.45


2
200-222
14, 1, 15, 6
0.17
0.053
3.29


3
224-233
7, 26
0.20
0.053
3.78


4
365-377
18, 2
0.15
0.053
2.91


5
473-499
21, 17, 23, 24, 10, 5,
0.26
0.053
4.90




11





6
506-522
9, 28
0.12
0.053
2.22


7
365-388
18, 2, 19
0.13
0.053
2.36


8
444-499
20, 16, 21, 17, 23,
0.16
0.053
3.03




24, 10, 5, 11





9
444-522
20, 16, 21, 17, 23, 24,
0.14
0.053
2.63




10, 5, 11, 9, 28





10
200-233
14, 1, 15, 6, 7, 26
0.18
0.053
3.33









All references mentioned herein are hereby incorporated by reference in their entirety. Further, the present invention can utilize various aspects of the following, which are all incorporated by reference in their entirety: U.S. patent application Ser. No. 09/380,534, filed on Sep. 1, 1999, entitled A METHOD OF INDUCING A CTL RESPONSE; Ser. No. 09/776,232, filed on Feb. 2, 2001, entitled METHOD OF INDUCING A CTL RESPONSE; Ser. No. 09/715,835, filed on Nov. 16, 2000, entitled AVOIDANCE OF UNDESIRABLE REPLICATION INTERMEDIATES IN PLASMID PROPAGATION; Ser. No. 09/999,186, filed on Nov. 7, 2001, entitled METHODS OF COMMERCIALIZING AN ANTIGEN; and Provisional U.S. Patent Application No. 60/274,063, filed on Mar. 7, 2001, entitled ANTI-NEOVASCULAR VACCINES FOR CANCER.







TABLE 29





Partial listing of SEQ ID NOS.

















SEQ ID NO: 1
ELAGIGILTV
MELAN-A 26-35 (A27L)


 


SEQ ID NO: 2
MELAN-A PROTEIN
ACCESSION NUMBER:




NP_005502


 


SEQ ID NO: 3
TYROSINASE PROTEIN
ACCESSION NUMBER:




P14679


 


SEQ ID NO: 4
MLLAVLYCLELAGIGILTVYMDGT
PMA2M EXPRESSION



MSQVGILTVILGVLLLIGCWYCRRR 
PRODUCT



NGYRALMDKSLHVGTQCALTRRCP




QEGFDIARDSKVSLQEKNCEPV



 


SEQ ID NO: 5
MLLAVLYCLELAGIGILTVYMDGT
LIBERATION OR



MSQV
SUBSTRATE SEQUENCE




FOR SEQ ID NO. 1




FROM PMA2M


 


SEQ ID NO: 6
MLLAVLYCL
TYROSINASE 1-9


 


SEQ ID NO: 7
YMDGTMSQV
TYROSINASE 369-377


 


SEQ ID NO: 8
EAAGIGILTV
MELAN-A 26-35


 


SEQ ID NO: 9
CTTAAGCCACCATGTTACTAGCTGTTTTGTACTGCC
PMA2M INSERT



TGGAACTAGCAGGGATCGGCATATTGACAGTGTATA




TGGATGGAACAATGTCCCAGGTAGGAATTCTGACAG




TGATCCTGGGAGTCTTACTGCTCATCGGCTGTTGGT




ATTGTAGAAGACGAAATGGATACAGAGCCTTGATGG




ATAAAAGTCTTCATGTTGGCACTCAATGTGCCTTAA




CAAGAAGATGCCCACAAGAAGGGTTTGATCATCGGG




ACAGCAAAGTGTCTCTTCAAGAGAAAAACTGTGAAC




CTGTGTAGTGAGCGGCCGC



 


SEQ ID NO: 10
MVLYCLELAGIGILTVYMDGIAVL
EPITOPE ARRAY FROM



YCLELAGIGILTVYMDGTMLAVLY
PVAXM2 AND PVAXM1



CLELAGIGILTVYMDGTMSLLAVLY




CLELAGICHLTV



 


SEQ ID NO: 11
NY-ESO-1 PROTEIN
ACCESSION NUMBER:




P78358


 


SEQ ID NO: 12
SLLMWITQC
NY-ESO-1 157-165


 


SEQ ID NO: 13
KASEKIFYV
SSX-2 41-49


 


SEQ ID NO: 14
TQCFLPVFL
NY-ESO-1 163-171


 


SEQ ID NO: 15
GLPSIPVHPI
PSMA 288-297


 


SEQ ID NO: 16
AVLYCL
TYROSINASE 4-9


 


SEQ ID NO: 17
MSLLMWIQCKASEKIFYVRCGAR
PN157 EXPRESSION



GPESRLLEFYLAMPFATPMEAELAR
PRODUCT



RSLAQDAPPLPVPGVLLKEFFVSGN




ILTIRLTAADHRQLQLSISSCLQQLSL




LMWITQCFLPVFLAQPPSGQRR



 


SEQ ID NO: 18
MSLLMWITQCKASEKIFYV
LIBERATION OR




SUBSTRATE SEQUENCE




FOR SEQ ID NO. 12 FROM




PN157


 


SEQ ID NO: 19
CTTAAGCCACCATGTCCCTGTTGATGTGGATCACGC
INSERT FOR PN157



AGTGCAAAGCTTCGGAGAAAATCTTCTACGTACGGT




GCGGTGCCAGGGGGCCGGAGAGCCGCCTGCTTGAGT




TCTACCTCGCCATGCCTTTCGCGACACCCATGGAAG




CAGAGCTGGCCCGCAGGAGCCTGGCCCAGGATGCCC




CACCGCTTCCCGTGCCAGGGGTGCTTCTGAAGGAGT




TCACTGTGTCCGGCAACATACTGACTATCCGACTGA




CTGCTGCAGACCACCGCCAACTGCAGCTCTCCATCA




GCTCCTGTCTCCAGCAGCTTTCCCTGTTGATGTGGA




TCACGCAGTGCTTTCTGCCCGTGTTTTTGGCTCAGC




CTCCCTCAGGGCAGAGGCGCTAGTGAGAATTC



 


SEQ ID NO: 20
MSLLMWITQCKASEKIFYVGLPSIP
PBPL EXPRESSION



VHPIGLPSIPVHPIKASEKIFYVSLLM
PRODUCT



WITQCKASEKIFYVKASEKIFYVRC




GARGPESRLLEFYLAMPFATPMEAE




LARRSLAQDAPPLPVPGVIAKEFTV




SGNILTIRLTAADHRQLQLSISSCLQ




QLSLLMWITQCFLPVFLAQPPSGQR




R



 


SEQ ID NO: 21
ATGTCCCTGTTGATGTGGATCACGCAGTGCAAAGCT
PBPL INSERT CODING



TCGGAGAAAATCTTCTATGTGGGTCTTCCAAGTATT
REGION



CCTGTTCATCCAATTGGTCTTCCAAGTATTCCTGTT




CATCCAATTAAAGCTTCGGAGAAAATCTTCTATGTG




TCCCTGTTGATGTGGATCACGCAGTGCAAAGCTTCG




GAGAAAATCTTCTATGTGAAAGCTTCGGAGAAAATC




TTCTACGTACGGTGCGGTGCCAGGGGGCCGGAGAGC




CGCCTGCTTGAGTTCTACCTCGCCATGCCTTTCGCG




ACACCCATGGAAGCAGAGCTGGCCCGCAGGAGCCTG




GCCCAGGATGCCCCACCGCTTCCCGTGCCAGGGGTG




CTTCTGAAGGAGTTCACTGTGTCCGGCAACATACTG




ACTATCCGACTGACTGCTGCAGACCACCGCCAACTG




CAGCTCTCCATCAGCTCCTGTCTCCAGCAGCTTTCC




CTGTTGATGTGGATCACGCAGTGCTTTCTGCCCGTG




TTTTTGGCTCAGCCTCCCTCAGGGCAGAGGCGCTAG




TGA



 


SEQ ID NO: 22
IKASEKIFYVSLLMWITQCKASEKI
SUBSTRATE IN FIG. 9



FYVK



 


SEQ ID NO: 23
VMTKLGFKV
SSX-457-65


 


SEQ ID NO: 24
RQIYVAAFTV
PSMA730-739


 


SEQ ID NO: 25
AQIPEKIQKAIDDIAKYFSKEENVEK
SSX-215-183



MKASEKIFYVYMKRKYEAMTKLG




FKATLPPFMCNKRAEDFQGNDLDN




DPNRGNQVERPQMTFGRLQGISPKI




MPKKPAEEGNDSEEVPEASGPQND




GKELCPPGKPTTSEKIHERSGPKRG




EHAWTHRLRERKQLVIYEEISDP



 


SEQ ID NO: 26
MVMTKLGFKVKASEKIFYVRQIYV
CTLS1/PCBP EXPRESSION



AAFTVGLPSIPVHPITQCFLPVFLVM
PRODUCT



TKLGFKVRQIYVAAFTVKASEKIFY




VAQIPEKIQKAFDDIAKYFSKEEWE




KMKASEKIFYVYMKRKYEAMTKL




GFKATLPPFMCNKRAEDFQGNDLD




NDPNRGNQVERPQMTFGRLQGISP




KIMPKKPAEEGNDSEEVPEASGPQN




DGKELCPPGKPTISEKIHERSGPKR




GEHAWTHRLRERKQLVIYEEISDP



 


SEQ ID NO: 27
TMAQIPEKIQKAFDDIAKYFSKEEWE
CTLS2 EXPRESSION



KMKASEKIFYVYMKRKYEAMTKL
PRODUCT



GFKATLPPFMCNKRAEDFQGNDLD




NDPNRGNQVERPQMTFGRLQGISP




KIMPKKPAEEGNDSEEVPEASGPQN




DGKELCPPGKPITSEKIHERSGPKR




GEHAWTHRLRERKQLVIYEEISDPV




MTKLGFKVKASEKWYVRQIYVAAF




TVGLPSIPVHPITQCFLPVFLVMTKL




GFKVRQIYVAAFTVKASEKIFYV



 


SEQ ID NO: 28
MVMTKLGFKVKASEKIFYVRQIYV
CTLS3 EXPRESSION



AAFTVGLPSIPVHPIAQIPEKIQKAFD
PRODUCT



DIAKYFSKEEWEKMKASEKIFYVY




MKRKYEAMTKLGFKATLPPFMCN




KRAEDFQGNDLDNDPNRGNQVERP




QMITGRLQGISPKIMPKKPAEEGND




SEEVPEASGPQNDGKELCPPGKPTT




SEKIHERSGPKRGEHAWTHRLRER




KQLVIYEEISDP



 


SEQ ID NO: 29
MAQIPEKIQKAFDDIAKYFSKEEWE
CTLS4 EXPRESSION



KMKASEKIFYVYMKRKYEAMTKL
PRODUCT



GFKATLPPFMCNKRAEDFQGNDLD




NDPNRGNQVERPQMTFGRLQGISP




K1MPKKPAEEGNDSEEVPEASGPQN




DGKELCPPGKPTTSEKIHERSGPKR




GEHAWTHRLRERKQLVIYEEISDPT




QCFLPVFLVMTKLGFKVRQIYVAAF




TVKASEKIFYV



 


SEQ ID NO: 30
ATGGTCATGACTAAACTAGGTTTCAAGGTCAAAGCT
PCBP INSERT CODING



TCGGAGAAAATCTTCTATGTGAGACAGATTTATGTT
REGION



GCAGCCTTCACAGTGGGTCTTCCAAGTATTCCTGTT




CATCCAATTACGCAGTGCTTTCTGCCCGTGTTTTTG




GTCATGACTAAACTAGGTTTCAAGGTCAGACAGAIT




TATGTTGCAGCCTTCACAGTGAAAGCTTCGGAGAAA




ATCTTCTACGTAGCTCAAATACCAGAGAAGATCCAA




AAGGCCTTCGATGATATTGCCAAATACTTCTCTAAG




GAAGAGTGGGAAAAGATGAAAGCCTCGGAGAAAATC




TTCTATGTGTATATGAAGAGAAAGTATGAGGCTATG




ACTAAACTAGGTTTCAAGGCCACCCTCCCACCTTTC




ATGTGTAATAAACGGGCCGAAGACTTCCAGGGGAAT




GATTTGGATAATGACCCTAACCGTGGGAATCAGGTT




GAACGTCCTCAGATGACTTTCGGCAGGCTCCAGGGA




ATCTCCCCGAAGATCATGCCCAAGAAGCCAGCAGAG




GAAGGAAATGATTCGGAGGAAGTGCCAGAAGCATCT




GGCCCACAAAATGATGGGAAAGAGCTGTGCCCCCCG




GGAAAACCAACTACCTCTGAGAAGATTCACGAGAGA




TCTGGACCCAAAAGGGGGGAACATGCCTGGACCCAC




AGACTGCGTGAGAGAAAACAGCTGGTGATTTATGAA




GAGATCAGCGACCCTTAGTGA



 


SEQ ID NO: 31
RQIYVAAFTVKASEKIFYVAQIPEKI
FIG. 11 SUBSTRATE/CTLS1-



QK
2


 


SEQ ID NO: 32
FLPWHRLFL
Tyr207-215


 


SEQ ID NO: 33
MLLAVLYCLIMSFQTSAFLPWHRL
CTLT2/PMEL EXPRESSION



FLMLLAVLYCLLWSFQTSAFLPWH
PRODUCT



RLFLMLLAVLYCLLWSFQTSAFLP




WHRLFLMLLAVLYCLLWSFQTSAF




LPWFIRLFL



 


SEQ ID NO: 34
ATGCTCCTGGCTGTTTTGTACTGCCTGCTGTGGAGT 
CTLT2/PMEL INSERT



TTCCAGACCTCCGCTTTTCTGCCTTGGCATAGACTC
CODING REGION



TTCTTGATGCTCCTGGCTGITTTGTACTGCCTGCTG 




TGGAGTTTCCAGACCTCCGCTTTTCTGCCTTGGCAT




AGACTCTTCTrGATGCTCCTGGCTGTTTTGTACTGC




CTGCTGTGGAGTTTCCAGACCTCCGCTTTTCTGCCT




TGGCATAGACTCTTCTTGATGCTCCTGGCTGTTTTG




TACTGCCTGCTGTGGAGTTTCCAGACCTCCGCTTTT




CTGCCTTGGCATAGACTCTTCTTGTAGTGA



 


SEQ ID NO: 35
MELAN-A CDNA
ACCESSION NUMBER:




NM_005511


 


SEQ ID NO: 36
TYROSINASE CDNA
ACCESSION NUMBER:




NM_000372


 


SEQ ID NO: 37
NY-ESO-1 CDNA
ACCESSION NUMBER:




U87459


 


SEQ ID NO: 38
PSMA PROTEIN
ACCESSION NUMBER:




NP_004467


 


SEQ ID NO: 39
PSMA CDNA
ACCESSION NUMBER:




NM_004476


 


SEQ ID NO: 40
SSX-2 PROTEIN
ACCESSION NUMBER:




NP_003138


 


SEQ ID NO: 41
SSX-2 CDNA
ACCESSION NUMBER:




NM_003147


 


SEQ ID NO: 42
ATGACCTCTCGCCGCTCCGTGAAG
FROM ACCESSION:



TCGGGTCCGCGGGAGGTTCCGCG
NUMBER: DI0879



CGATGAGTACGAGGATCTGTACT
HERPES SIMPLEX VIRUS 1



ACACCCCGTCTTCAGGTATGGCGA
UL49 CODING SEQUENCE



GTCCCGATAGTCCGCCTGACACCT
(V-P22)



CCCGCCGTGGCGCCCTACAGACA




CGCTCGCGCCAGAGGGGCGAGGT




CCGTTTCGTCCAGTACGACGAGTC




GGATTATGCCCTCTACGGGGGCTC




GTCATCCGAAGACGACGAACACC




CGGAGGTCCCCCGGACGCGGCGT




CCCGTTTCCGGGGCGGTTTTGTCC




GGCCCGGGGCCTGCGCGGGCGCC




TCCGCCACCCGCTGGGTCCGGAG




GGGCCGGACGCACACCCACCACC




GCCCCCCGGGCCCCCCGAACCCA




GCGGGTGGCGACTAAGGCCCCQG




CGGCCCCGGCGGCGGAGACCACC




CGCGGCAGGAAATCGGCCCAGCC




AGAATCCGCCGCACTCCCAGACG




CCCCCGCGTCGACGGCGCCAACC




CGATCCAAGACACCCGCGCAGGG




GCTGGCCAGAAAGCTGCACTTTA




GCACCGCCCCCCCAAACCCCGAC




GCGCCATGGACCCCCCGGGTGGC




CGGCTTTAACAAGCGCGTCTTCTG




CGCCGCGGTCGGGCGCCTGGCGG




CCATGCATGCCCGGATGGCGGCG




GTCCAGCTCTGGGACATGTCGCGT




CCGCGCACAGACGAAGACCTCAA




CGAACTCCTTGGCATCACCACCAT




CCGCGTGACGGTCTGCGAGGGCA




AAAACCTGCTTCAGCGCGCCAAC




GAGTTGGTGAATCCAGACGTGGT




GCAGGACGTCGACGCGGCCACGG




CGACTCGAGGGCGTTCTGCGGCG




TCGCGCCCCACCGAGCGACCTCG




AGCCCCAGCCCGCTCCGCTTCTCG




CCCCAGACGGCCCGTCGAG



 


SEQ ID NO: 43
MTSRRSVKSGPREVPRDEYEDLYY
ACCESSION NUMBER:



TPSSGMASPDSPPDTSRRGALFTQT
P10233



RSRQRGEVRFVQYDESDYALYGGS
HERPES SIMPLEX VIRUS 1



SSEDDEHPEVPRTIUOVSGAVLSGP
UL49/VP22 PROTEIN



GPARAPPPFTPAGSGGAGRTPTTAP
SEQUENCE 



RAPRTQRVATKAPAAPAAETTRGR




KSAQPESAALPDAPASTAPTFTRSK




TPAQGLARKLHFSTAPPNPDAPWTP




RVAGFNKRVFCAAVGRLAAMHAR




MAAVQLWDFTMSRPRTDEDLNELL




GITTIRVTVCEGKNLLQRANELVNP




DVVQDVDAATATRGRSAASRFTPT




ERPRAPARSASRPRRPVE









    Melan-A mRNA Sequence
    LOCUS NM—005511 1524 bp mRNA PRI 14 Oct. 2001
    DEFINITION Homo sapiens melan-A (MLANA), mRNA.
    ACCESSION NM—005511
    VERSION NM—005511.1 GI:5031912













/translation = “MPREDAHFIYGYPKKGHGHSYTTAEEAAGIGILTVILGVLLLIGCWYCR
(SEQ ID NO. 2)



 



RRNGYRALMDKSLHVGTQCALTRRCPQEGFDHRDSKVSLQEKNCEPVVPNAPPAYE



 



KLSAEQSPPPYSP”



 









ORIGIN




 










1
agcagacaga ggactctcat taaggaaggt gtcctgtgcc ctgaccctac aagatgccaa
(SEQ ID NO. 35)



 


61
gagaagatgc tcacttcatc tatggttacc ccaagaaggg gcacggccac tcttacacca



 


121
cggctgaaga ggccgctggg atcggcatcc tgacagtgat cctgggagtc ttactgctca



 


181
tcggctgttg gtattgtaga agacgaaatg gatacagagc cttgatggat aaaagtcttc



 


241
atgttggcac tcaatgtgcc ttaacaagaa gatgcccaca agaagggttt gatcatcggg



 


301
acagcaaagt gtctcttcaa gagaaaaact gtgaacctgt ggttcccaat gctccacctg



 


361
cttatgagaa actctctgca gaacagtcac caccacctta ttcaccttaa gagccagcga



 


421
gacacctgag acatgctgaa attatttctc tcacactttt gcttgaattt aatacagaca



 


481
tctaatgttc tcctttggaa tggtgtagga aaaatgcaag ccatctctaa taataagtca



 


541
gtgttaaaat tttagtaggt ccgctagcag tactaatcat gtgaggaaat gatgagaaat



 


601
attaaattgg gaaaactcca tcaataaatg ttgcaatgca tgatactatc tgtgccagag



 


661
gtaatgttag taaatccatg gtgttatttt ctgagagaca gaattcaagt gggtattctg



 


721
gggccatcca atttctcttt acttgaaatt tggctaataa caaactagtc aggttttcga



 


781
accttgaccg acatgaactg tacacagaat tgttccagta ctatggagtg ctcacaaagg



 


841
atacttttac aggttaagac aaagggttga ctggcctatt tatctgatca agaacatgtc



 


901
agcaatgtct ctttgtgctc taaaattcta ttatactaca ataatatatt gtaaagatcc



 


961
tatagctctt tttttttgag atggagtttc gcttttgttg cccaggctgg agtgcaatgg



 


1021
cgcgatcttg gctcaccata acctccgcct cccaggttca agcaattctc ctgccttagc



 


1081
ctcctgagta gctgggatta caggcgtgcg ccactatgcc tgactaattt tgtagtttta



 


1141
gtagagacgg ggtttctcca tgttggtcag gctggtctca aactcctgac ctcaggtgat



 


1201
ctgcccgcct cagcctccca aagtgctgga attacaggcg tgagccacca cgcctggctg



 


1261
gatcctatat cttaggtaag acatataacg cagtctaatt acatttcact tcaaggctca



 


1321
atgctattct aactaatgac aagtattttc tactaaacca gaaattggta gaaggattta



 


1381
aataagtaaa agctactatg tactgcctta gtgctgatgc ctgtgtactg ccttaaatgt



 


1441
acctatggca atttagctct cttgggttcc caaatccctc tcacaagaat gtgcagaaga



 


1501
aatcataaag gatcagagat tctg







Tyrosinase mRNA Sequence

    LOCUS NM—000372 1964 bp mRNA PRI 31 Oct. 2000
    DEFINITION Homo sapiens tyrosinase (oculocutaneous albinism IA) (TYR), mRNA.
    ACCESSION NM—000372
    VERSION NM—000372.1 GI:4507752













/translation = “MLLAVLYCLLWSFQTSAGHFPRACVSSKNLMEKECCPPWSGDRS
(SEQ ID NO. 3)



 



PCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRESWPSVFYNRTCQCSGNFMGFNCG



 



NCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYLTLAKHTISSDYVIPIGTYGQM



 



KNGSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDIDFAHEAPAFLPWHRLFLL



 



RWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPTNPNLLSPASFFSSW



 



QIVCSRLEEYNSHQSLCNGTPEGPLRRNPGNHDKSRTPRLPSSADVEFCLSLTQYESG



 



SMDKAANFSFRNTLEGFASPLTGIADASQSSMHNALHIYMNGTMSQVQGSANDPIFL



 



LHHAFVDSIFEQWLRRHRPLQEVYPEANAPIGHNRESYMVPFIPLYRNGDFFISSKDL



 



GYDYSYLQDSDPDSFQDYIKSYLEQASRIWSWLLGAAMVGAVLTALLAGLVSLLCR



 



HKRKQLP EEKQPLLMEKEDYHSLYQSHL”



 









ORIGIN




 










1
atcactgtag tagtagctgg aaagagaaat ctgtgactcc aattagccag ttcctgcaga
(SEQ ID NO. 36)



 


61
ccttgtgagg actagaggaa gaatgctcct ggctgttttg tactgcctgc tgtggagttt



 


121
ccagacctcc gctggccatt tccctagagc ctgtgtctcc tctaagaacc tgatggagaa



 


181
ggaatgctgt ccaccgtgga gcggggacag gagtccctgt ggccagcttt caggcagagg



 


241
ttcctgtcag aatatccttc tgtccaatgc accacttggg cctcaatttc ccttcacagg



 


301
ggtggatgac cgggagtcgt ggccttccgt cttttataat aggacctgcc agtgctctgg



 


361
caacttcatg ggattcaact gtggaaactg caagtttggc ttttggggac caaactgcac



 


421
agagagacga ctcttggtga gaagaaacat cttcgatttg agtgccccag agaaggacaa



 


481
attttttgcc tacctcactt tagcaaagca taccatcagc tcagactatg tcatccccat



 


541
agggacctat ggccaaatga aaaatggatc aacacccatg tttaacgaca tcaatattta



 


601
tgacctcttt gtctggatgc attattatgt gtcaatggat gcactgcttg ggggatctga



 


661
aatctggaga gacattgatt ttgcccatga agcaccagct tttctgcctt ggcatagact



 


721
cttcttgttg cggtgggaac aagaaatcca gaagctgaca ggagatgaaa acttcactat



 


781
tccatattgg gactggcggg atgcagaaaa gtgtgacatt tgcacagatg agtacatggg



 


841
aggtcagcac cccacaaatc ctaacttact cagcccagca tcattcttct cctcttggca



 


901
gattgtctgt agccgattgg aggagtacaa cagccatcag tctttatgca atggaacgcc



 


961
cgagggacct ttacggcgta atcctggaaa ccatgacaaa tccagaaccc caaggctccc



 


1021
ctcttcagct gatgtagaat tttgcctgag tttgacccaa tatgaatctg gttccatgga



 


1081
taaagctgcc aatttcagct ttagaaatac actggaagga tttgctagtc cacttactgg



 


1141
gatagcggat gcctctcaaa gcagcatgca caatgccttg cacatctata tgaatggaac



 


1201
aatgtcccag gtacagggat ctgccaacga tcctatcttc cttcttcacc atgcatttgt



 


1261
tgacagtatt tttgagcagt ggctccgaag gcaccgtcct cttcaagaag tttatccaga



 


1321
agccaatgca cccattggac ataaccggga atcctacatg gttcctttta taccactgta



 


1381
cagaaatggt gatttcttta tttcatccaa agatctgggc tatgactata gctatctaca



 


1441
agattcagac ccagactctt ttcaagacta cattaagtcc tatttggaac aagcgagtcg



 


1501
gatctggtca tggctccttg gggcggcgat ggtaggggcc gtcctcactg ccctgctggc



 


1561
agggcttgtg agcttgctgt gtcgtcacaa gagaaagcag cttcctgaag aaaagcagcc



 


1621
actcctcatg gagaaagagg attaccacag cttgtatcag agccatttat aaaaggctta



 


1681
ggcaatagag tagggccaaa aagcctgacc tcactctaac tcaaagtaat gtccaggttc



 


1741
ccagagaata tctgctggta tttttctgta aagaccattt gcaaaattgt aacctaatac



 


1801
aaagtgtagc cttcttccaa ctcaggtaga acacacctgt ctttgtcttg ctgttttcac



 


1861
tcagcccttt taacattttc ccctaagccc atatgtctaa ggaaaggatg ctatttggta



 


1921
atgaggaact gttatttgta tgtgaattaa agtgctctta tttt







NY-ESO-1 mRNA Sequence

    LOCUS HSU87459 752 bp mRNA PRI 22 Dec. 1999
    DEFINITION Human autoimmunogenic cancer/testis antigen NY-ESO-1 mRNA, complete cds.
    ACCESSION U87459
    VERSION U87459.1 GI:1890098













/translation = “MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRG
(SEQ ID NO. 11)



 



AGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAE



 



LARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLM



 



WITQCFLPVFLAQPPSGQRR”



 









ORIGIN




 










1
atcctcgtgg gccctgacct tctctctgag agccgggcag aggctccgga gccatgcagg
(SEQ ID NO. 37)



 


61
ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccagga ggccctggca



 


121
ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc acgggcggca



 


181
gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gccccgcggg



 


241
gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gccagggggc



 


301
cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc atggaagcag



 


361
agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca ggggtgcttc



 


421
tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gcagaccacc



 


481
gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg atgtggatca



 


541
cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cgctaagccc



 


601
agcctggcgc cccttcctag gtcatgcctc ctcccctagg gaatggtccc agcacgagtg



 


661
gccagttcat tgtgggggcc tgattgtttg tcgctggagg aggacggctt acatgtttgt



 


721
ttctgtagaa aataaaactg agctacgaaa aa







PSMA cDNA Sequence

    LOCUS NM—004476 2653 bp mRNA PRI 1 Nov. 2000
    DEFINITION Homo sapiens folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1), mRNA.
    ACCESSION NM—004476
    VERSION NM—004476.1 GI:4758397













/translation = “MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNE
(SEQ ID NO. 38)



 



ATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGL



 



DSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSP



 



QGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAG



 



AKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEY



 



AYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFT



 



GNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSG



 



AAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVA



 



YINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEF



 



SGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVE



 



KFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHP



 



QEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFID



 



PLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVA



 



AFTVQAAAETLSEVA”



 









ORIGIN




 










1
ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg
(SEQ ID NO. 39)



 


61
attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga



 


121
gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac



 


181
cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag



 


241
gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc



 


301
accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt



 


361
ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctac taacattact



 


421
ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc



 


481
ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca



 


541
aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat



 


601
gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa



 


661
gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccagg atatgaaaat



 


721
gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat



 


781
ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa



 


841
atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag



 


901
gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac



 


961
tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg aggtggtgtc



 


1021
cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacacc aggttaccca



 


1081
gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct



 


1141
gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca



 


1201
ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt



 


1261
actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca



 


1321
agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag atatgtcatt



 


1381
ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct



 


1441
gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga



 


1501
agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag



 


1561
tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac



 


1621
tcatetatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg



 


1681
gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt



 


1741
tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc



 


1801
aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat tgcttcaggc



 


1861
agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac



 


1921
agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac



 


1981
ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc



 


2041
ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt



 


2101
atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga ttcacttttt



 


2161
tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagact ccaggacttt



 


2221
gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga



 


2281
gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct



 


2341
ccaagcagcc acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt



 


2401
gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag acagatttat



 


2461
gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagc ctaagaggat



 


2521
tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt



 


2581
atattgataa attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa



 


2641
aaaaaaaaaa aaa







    NM 003147 Homo sapiens synovial sarcoma, X breakpoint 2 (SSX2), mRNA
    LOCUS NM—003147 766 bp mRNA PRI 14 Mar. 2001
    DEFINITION Homo sapiens synovial sarcoma, X breakpoint 2 (SSX2), mRNA.
    ACCESSION NM—003147
    VERSION NM—003147.1 GI:10337582













/translation = “MNGDDAFARRPTVGAQIPEKIQKAFDDIAKYFSKEEWEKMKASE
SEQ ID NO. 40



 



KIFYVYMKRKYEAMTKLGFKATLPPFMCNKRAEDFQGNDLDNDPNRGNQVERPQMTFG



 



RLQGISPKIMPKKPAEEGNDSEEVPEASGPQNDGKELCPPGKPTTSEKIHERSGPKRG



 



EHAWTHRLRERKQLVIYEEISDPEEDDE”



 










1
ctctctttcg attcttccat actcagagta cgcacggtct gattttctct ttggattctt
SEQ ID NO 41



 


61
ccaaaatcag agtcagactg ctcccggtgc catgaacgga gacgacgcct ttgcaaggag



 


121
acccacggtt ggtgctcaaa taccagagaa gatccaaaag gccttcgatg atattgccaa



 


181
atacttctct aaggaagagt gggaaaagat gaaagcctcg gagaaaatct tctatgtgta



 


241
tatgaagaga aagtatgagg ctatgactaa actaggtttc aaggccaccc tcccaccttt



 


301
catgtgtaat aaacgggccg aagacttcca ggggaatgat ttggataatg accctaaccg



 


361
tgggaatcag gttgaacgtc ctcagatgac tttcggcagg ctccagggaa tctccccgaa



 


421
gatcatgccc aagaagccag cagaggaagg aaatgattcg gaggaagtgc cagaagcatc



 


481
tggcccacaa aatgatggga aagagctgtg ccccccggga aaaccaacta cctctgagaa



 


541
gattcacgag agatctggac ccaaaagggg ggaacatgcc tggacccaca gactgcgtga



 


601
gagaaaacag ctggtgattt atgaagagat cagcgaccct gaggaagatg acgagtaact



 


661
cccctcaggg atacgacaca tgcccatgat gagaagcaga acgtggtgac ctttcacgaa



 


721
catgggcatg gctgcggacc cctcgtcatc aggtgcatag caagtg

## Claims

1. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of a tumor-associated antigen, wherein the antigen is NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 30% of the length of the antigen, or greater than 55% and less than 60% of the length of the antigen, wherein the reading frame does not encode the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein said first sequence encodes a fragment of NY-ESO, wherein said encoded fragment comprises an amino acid sequence beginning at one of amino acids selected from the group consisting of 77, 79, 86, and 108 of NY-ESO, and ending at one of the amino acids selected from the group consisting of 104, 140, 167, 171, 174, and 180 of NY-ESO.

2. The nucleic acid molecule of claim 1, wherein said reading frame is operably linked to a promoter.

3. An immunogenic composition comprising the nucleic acid molecule of claim 1.

4. The nucleic acid molecule of claim 1, wherein said encoded fragment consists essentially of amino acids 79-174 of NY-ESO.

5. The nucleic acid molecule of claim 4, wherein said first sequence encodes exactly amino acids 77-180 of NY-ESO.

6. The nucleic acid molecule of claim 1, wherein said one or more segments consist of said epitope cluster.

7. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of tumor-associated antigen NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 60% of the length of the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein the first sequence comprises an epitope cluster chosen from the group consisting of amino acids 79-104, 86-171, 108-140, and 108-174 of NY-ESO, and wherein said first sequence encodes a fragment of NY-ESO, and wherein said encoded fragment consists essentially of amino acids 79-174 of NY-ESO.

8. The nucleic acid molecule of claim 7, wherein said first sequence encodes exactly amino acids 77-180 of NY-ESO.

9. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of tumor-associated antigen NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 90% of the length of the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein the first sequence comprises an epitope cluster chosen from the group consisting of amino acids 86-171, 108-140, and 108-174 of NY-ESO.

10. The nucleic acid molecule of claim 9, wherein said first sequence encodes a fragment of NY-ESO.

11. The nucleic acid molecule of claim 10, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 80% of the length of NY-ESO.

12. The nucleic acid molecule of claim 10, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 50% of the length of NY-ESO.

13. The nucleic acid molecule of claim 10, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 25% of the length of NY-ESO.

14. The nucleic acid molecule of claim 10, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 10% of the length of NY-ESO.

15. The nucleic acid molecule of claim 10, wherein said encoded fragment comprises an amino acid sequence beginning at one of amino acids selected from the group consisting of 77, 79, 86, and 108 of NY-ESO, and ending at one of the amino acids selected from the group consisting of 104, 140, 167, 171, 174, and 180 of NY-ESO.

16. The nucleic acid molecule of claim 9, wherein said reading frame is operably linked to a promoter.

17. An immunogenic composition comprising the nucleic acid molecule of claim 16.

18. The nucleic acid molecule of claim 16, wherein said first sequence encodes exactly amino acids 77-180 of NY-ESO.

19. The nucleic acid molecule of claim 9, wherein said one or more segments consist of said epitope cluster.

20. The nucleic acid molecule of claim 9, wherein said wherein said one or more segments of a tumor-associated antigen comprise multiple segments of NY-ESO, each consisting of an epitope cluster.

21. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of tumor-associated antigen NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 60% of the length of the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein the first sequence comprises an epitope cluster chosen from the group consisting of amino acids 79-104, 86-171, 108-140, and 108-174 of NY-ESO, wherein said first sequence further comprises amino acids 148-167 of NY-ESO.

22. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of a tumor-associated antigen, wherein the antigen is NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 30% of the length of the antigen, or greater than 55% and less than 60% of the length of the antigen, wherein the reading frame does not encode the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein at least one cluster is at least 11% of the length of the antigen, wherein said encoded fragment comprises an amino acid sequence beginning at one of amino acids selected from the group consisting of 77, 79, 86, and 108 of NY-ESO, and ending at one of the amino acids selected from the group consisting of 104, 140, 167, 171, 174, and 180 of NY-ESO.

23. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of a tumor-associated antigen, wherein the antigen is NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 30% of the length of the antigen, or greater than 55% and less than 60% of the length of the antigen, wherein the reading frame does not encode the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein at least one cluster is at least 11% of the length of the antigen, wherein said encoded fragment consists essentially of amino acids 79-174 of NY-ESO.

24. The nucleic acid molecule of 23, wherein said first sequence encodes exactly amino acids 77-180 of NY-ESO.

25. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of tumor-associated antigen NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 60% of the length of the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein the first sequence comprises an epitope cluster chosen from the group consisting of amino acids 86-171, 108-140, and 108-174 of NY-ESO.

26. The nucleic acid molecule of 25, wherein said first sequence encodes a fragment of NY-ESO.

27. The nucleic acid molecule of claim 26, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 50% of the length of NY-ESO.

28. The nucleic acid molecule of claim 26, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 25% of the length of NY-ESO.

29. The nucleic acid molecule of claim 26, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 10% of the length of NY-ESO.

30. The nucleic acid molecule of claim 26, wherein said encoded fragment comprises an amino acid sequence beginning at one of amino acids selected from the group consisting of 77, 79, 86, and 108 of NY-ESO, and ending at one of the amino acids selected from the group consisting of 140, 167, 171, 174, and 180 of NY-ESO.

31. The nucleic acid molecule of claim 25, wherein said reading frame is operably linked to a promoter.

32. An immunogenic composition comprising the nucleic acid molecule of claim 31.

33. The nucleic acid molecule of claim 26, wherein said encoded fragment consists essentially of amino acids 79-174 of NY-ESO.

34. The nucleic acid molecule of claim 33, wherein said first sequence encodes exactly amino acids 77-180 of NY-ESO.

35. The nucleic acid molecule of claim 25, wherein said one or more segments consist of said epitope cluster.

36. The nucleic acid molecule of claim 25, wherein said one or more segments of a tumor-associated antigen comprise multiple segments of NY-ESO, each consisting of an epitope cluster.

37. The nucleic acid molecule of 1, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 25% of the length of NY-ESO.

38. The nucleic acid molecule of 1, wherein said encoded fragment consists of a polypeptide having a length, wherein the length of the polypeptide is less than about 10% of the length of NY-ESO.

39. An isolated nucleic acid molecule comprising a reading frame comprising a first sequence, wherein said first sequence encodes one or more segments of tumor-associated antigen NY-ESO (SEQ ID NO: 11), wherein the first sequence comprises less than 60% of the length of the complete NY-ESO antigen, and wherein each segment comprises an epitope cluster, said cluster comprising or encoding at least two amino acid sequences having a known or predicted affinity for a same MHC Class I receptor peptide binding cleft, wherein the first sequence comprises an epitope cluster chosen from the group consisting of amino acids 79-104, 108-140, and 108-174 of NY-ESO, wherein said first sequence further comprises amino acids 144-171 of NY-ESO.