Abstract:
The present invention provides HLA-DR (MHC class II) binding peptides derived from the ovarian/breast cancer associated antigens, Human Epidermal Growth Factor Receptor 2 (HER-2/neu), Carcinoembryonic Antigen (CEA), Insulin Growth Factor Binding Protein 2 (IGFBP-2), and Cyclin D1. The immunogenic peptides can be used in cancer vaccines.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims benefit of priority from U.S. Provisional Application Ser. No. 60/984,646, filed on Nov. 1, 2007. 
     
    
     BACKGROUND OF THE INVENTION 
     Field of Invention 
       [0002]    The present invention relates to compositions and methods for preventing, treating or diagnosing a number of pathological states such as cancers. In particular, it provides novel peptides capable of binding selected major histocompatibility complex (MHC) molecules and induce an immune response. 
         [0003]    MHC molecules are classified as either Class I or Class II molecules. Class II MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T lymphocytes, B lymphocytes, dendritic cells, macrophages, etc. Class II MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide that is displayed. Complexes between a particular disease-associated antigenic peptide and class II HLA molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of specific CTL and antibody immune responses. 
         [0004]    A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al.,  Cell  47:1071, 1986; Babbitt, B. P. et al.,  Nature  317:359, 1985; Townsend, A. and Bodmer, H.,  Annu. Rev. Immunol.  7:601, 1989; Germain, R. N.,  Annu. Rev. Immunol.  11:403, 1993). 
         [0005]    Peptides of the present invention comprise epitopes that bind to HLA class II DR molecules. A greater degree of heterogeneity in both size and binding frame position of the motif, relative to the N- and C-termini of the peptide, exists for class II peptide ligands. This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends. Crystallographic analysis of HLA class II DRB*0101-peptide complexes showed that the major energy of binding is contributed by peptide residues complexed with complementary pockets on the DRB*0101 molecules. An important anchor residue engages the deepest hydrophobic pocket (see, e.g., Madden, D. R.  Ann. Rev. Immunol.  13:587, 1995) and is referred to as position 1 (P1). P1 may represent the N-terminal residue of a class II binding peptide epitope, but more typically is flanked towards the N-terminus by one or more residues. Other studies have also pointed to an important role for the peptide residue in the sixth position towards the C-terminus, relative to P1, for binding to various DR molecules. 
         [0006]    In the past few years evidence has accumulated to demonstrate that a large fraction of HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets. Thus, peptides of the present invention are identified by any one of several HLA-specific amino acid motifs, or if the presence of the motif corresponds to the ability to bind several allele-specific HLA molecules, a supermotif. The HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA “supertype.” 
         [0007]    Because human population groups, including racial and ethnic groups, have distinct patterns of distribution of HLA alleles it will be of value to identify motifs that describe peptides capable of binding more than one HLA allele, so as to achieve sufficient coverage of all population groups. The present invention addresses these and other needs. 
         [0008]    T lymphocytes recognize an antigen in the form of a peptide fragment bound to the MHC class I or class II molecule rather than the intact foreign antigen itself. Antigens presented by MHC class II molecules are usually soluble antigens that enter the antigen presenting cell via phagocytosis, pinocytosis, or receptor-mediated endocytosis. Once in the cell, the antigen is partially degraded by acid-dependent proteases in endosomes. The resulting fragments or peptide associate with the MHC class II molecule after the release of the CLIP fragment to form a stable complex that is then transported to the surface for potential recognition by specific HTLs. See Blum, et al., Crit. Rev. Immunol., 17: 411-17 (1997); Arndt, et al., Immunol. Res., 16: 261-72 (1997). 
         [0009]    Peptides that bind a particular MHC allele frequently will fit within a motif and have amino acid residues with particular biochemical properties at specific positions within the peptide. Such residues are usually dictated by the biochemical properties of the MHC allele. Peptide sequence motifs have been utilized to screen peptides capable of binding MHC molecules (Sette, et al.,  Proc. Natl. Acad. Sci. USA  86:3296 (1989)), and it has previously been reported that class I binding motifs identified potential immunogenic peptides in animal models (De Bruijn, et al.,  Eur. J. Immunol.  21: 2963-70 (1991); Pamer, et al.,  Nature  353: 852-955 (1991)). Also, binding of a particular peptide to a MHC molecule has been correlated with immunogenicity of that peptide (Schaeffer, et al.,  Proc. Natl. Acad. Sci. USA  86:4649 (1989)). 
         [0010]    Accordingly, while some MHC binding peptides have been identified, there is a need in the art to identify novel MHC binding peptides from tumor associated antigens that can be utilized to generate an immune response in vaccines against these targets. Further, there is a need in the art to identify peptides capable of binding a wide array of different types of MHC molecules such they are immunogenic in a large fraction a human outbred population. 
         [0011]    One of the most formidable obstacles to the development of broadly efficacious peptide-based immunotherapeutics has been the extreme polymorphism of HLA molecules. Effective coverage of a population without bias would thus be a task of considerable complexity if epitopes were used specific for HLA molecules corresponding to each individual allele because a huge number of them would have to be used in order to cover an ethnically diverse population. There exists, therefore, a need to develop peptide epitopes that are bound by multiple HLA antigen molecules at high affinity for use in epitope-based vaccines. The greater the number of HLA antigen molecules bound, the greater the breadth of population coverage by the vaccine. Analog peptides may be engineered based on the information disclosed herein and thereby used to achieve such an enhancement in breadth of population coverage. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1  shows the identification of preexistent immunity to promiscuous HER-2/neu HLA-DR epitopes. Each panel shows a scatter gram of the mean numbers of T cells specific for one of the identified HER-2/neu (see header of each panel) peptides in both healthy volunteer donors and patients. Each panel represents a unique peptide and each data point is derived from one individual. The grey box (i.e. cutoff) delineates the region that constitutes the mean and two standard deviations calculated from the normal healthy individuals. The percentages represent the fraction of patients that had mean T cell values above the cutoff. The peptides that are circled are those in which a higher fraction of the patients responded compared to the normal healthy controls. These peptides are considered to be the best vaccine candidates. However, it is clear that the rigidness of this approach could potentially result in many false negatives. For example, while p885 was shown to be recognized by 25% of patients, p886, a nearly identical peptide was found to be recognized by only 4%. However, if one looks at the graph for p886, there is a healthy individual that showed a robust response. The exclusion of that data point would have resulted in a patient response rate of 15%, which would have been consistent with the p885 response. Despite this, we identified 5 candidates to move forward. The fidelity of this approach is evident from a prior study which has already shown that a HER-2/neu peptide, p884-899, which encompasses the binding motif of p885, is an HLA-DR4 epitope. 
           [0013]      FIG. 2  shows the identification of preexistent immunity to promiscuous CEA HLA-DR epitopes.  FIG. 2  is identical to  FIG. 1  with the only exception that CEA is the antigen. Seven candidate peptides were identified. 
           [0014]      FIG. 3  shows the identification of preexistent immunity to promiscuous IGFBP2 HLA-DR epitopes.  FIG. 3  is identical to  FIG. 1  with the only exception that IGFBP2 is the antigen. Four candidate peptides were identified. Note that only 10 peptides were assessed as explained in the text above. 
           [0015]      FIG. 4  shows the identification of preexistent immunity to promiscuous IGFBP2 HLA-DR epitopes.  FIG. 4  is identical to  FIG. 1  with the only exception that Cyclin D1 is the antigen. Using the more liberal statistical method, 7 potential epitopes were identified. 
           [0016]      FIG. 5  shows the that HER-2/neu peptides, p59, p83, p88 and p885 are naturally processed and presented antigens. IFN-γ ELISpot analysis of short term T cell lines generated against HER-2/neu peptides, p53 (Panel A), p83 (Panel B), p88 (Panel C) and p885 (Panel D). The lines were tested for responses against respective culture peptides, an irrelevant 15-mer peptide, a HER-2/neu protein fragment (amino acids 22-122, Panels A-C; amino acids 676-1255, Panel D), or an irrelevant similar weight protein, ovalbumin. Each shows the results from two lines established from two different breast or ovarian cancer patients who had a positive ELlspot response to the peptide. Each bar is the mean (s.e.m.) of three replicates. 
           [0017]      FIG. 6  shows that IGFBP2 peptides p17, p22, p249, and p293 are naturally processed peptides.  FIG. 6  is identical to  FIG. 5  except that the results were obtained using IGFBP-2 derived helper epitopes. 
       
    
    
     BRIEF SUMMARY OF THE INVENTION 
       [0018]    The present invention relates to compositions and methods for preventing, treating or diagnosing a number of pathological states such as viral diseases and cancers. Thus, provided herein are novel peptides capable of binding selected major histocompatibility complex (MHC) molecules and inducing or modulating an immune response. Some of the peptides disclosed are capable of binding human class II MHC (HLA) molecules, including HLA-DR and HLA-DQ alleles. Also provided are compositions that include immunogenic peptides having binding motifs specific for MHC molecules. The peptides and compositions disclosed can be utilized in methods for inducing an immune response, a helper T lymphocyte (HTL) response, or a cytotoxic T lymphocyte (CTL) response when administered to a system. 
         [0019]    Epitopes on a number of immunogenic tumor associated antigens have been identified. The peptides are thus useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications (e.g., tetramer reagents; Beckman Coulter). 
         [0020]    The peptides are also useful as epitope-based vaccines. The epitope-based vaccines preferably have enhanced, typically broadened, population coverage. The HLA-DR supermotif-bearing epitopes comprising the vaccine composition preferably bind to more than one HLA DR supertype molecule with a K D  of less than 1000 nM or 500 nM, and stimulate a HTL response in patients bearing an HLA DR supertype allele to which the peptide binds. 
         [0021]    Motif-bearing peptides may additionally be used as diagnostic, rather than immunogenic, reagents to evaluate an immune response. For example, an HLA-DR supermotif-bearing peptide epitope may be used prognostically to analyze an immune response for the presence of specific HTL populations from patients who possess an HLA DR supertype allele bound by the peptide epitope. 
         [0022]    The binding affinity of a peptide epitope in accordance with the invention for at least one HLA DR supertype molecule is preferably determined. A preferred peptide epitope has a binding affinity of less than 1000 nM, or more preferably less than 500 nM for the at least one HLA DR supertype molecule, and most preferably less than 50 nM. 
         [0023]    Synthesis of a HLA DR supermotif-containing epitope may occur in vitro or in vivo. In a preferred embodiment, the peptide is encoded by a recombinant nucleic acid and expressed in a cell. The nucleic acid may encode one or more peptides, at least one of which is an epitope of the invention. 
         [0024]    A peptide epitope of the invention, in the context of an HLA DR supertype molecule to which it binds, can be contacted, either in vitro or in vivo, with a cytotoxic T lymphocyte and thereby be used to elicit a T cell response in an HLA-diverse population. 
         [0025]    An HTL epitope may be comprised by a single peptide. Further, the HTL epitope may be lipidated, preferably with palmitic acid, and may be linked by a spacer molecule to another HTL epitope or a CTL epitope. The epitope may be expressed by a nucleotide sequence; in a preferred embodiment the nucleotide sequence is comprised in an attenuated viral host. 
         [0026]    As will be apparent from the discussion below, other embodiments of methods and compositions are also within the scope of the invention. Further, novel synthetic peptides produced by any of the methods described herein are also part of the invention. 
         [0027]    The present invention provides peptides and nucleic acids encoding them for use in vaccines and therapeutics. The invention provides methods of inducing a helper T cell response against a preselected antigen in, a patient, the method comprising contacting a helper T cell with an immunogenic peptide of the invention. The peptides of the invention may be derived from a number of tumor associated antigens. The methods of the invention can be carried out in vitro or in vivo. In a preferred embodiment the peptides are contacted with the helper T cell by administering to the patient a nucleic acid molecule comprising a sequence encoding the immunogenic peptide. 
         [0028]    The present invention is directed to methods of modulating the binding of peptide epitopes to HLA class II molecules. The invention includes a method of modifying binding of an original peptide epitope that bears a motif correlated with binding to an HLA molecule, said motif comprising at least one primary anchor position, said at least one primary anchor position having specified therefore primary anchor amino acid residues consisting essentially of two or more residues, said method comprising exchanging the primary anchor residue of the original peptide epitope for another primary anchor residue, with the proviso that the original primary anchor residue is not the same as the exchanged primary anchor residue. A preferred embodiment of the invention includes a method where the original primary anchor residue is a less preferred residue, and the exchanged residue is a more preferred residue. 
         [0029]    One alternative embodiment of the invention includes a method of modifying binding of an original peptide epitope that bears a motif correlated with binding to an HLA molecule, said motif comprising at least one primary anchor position having specified therefore at least one primary anchor residue, and at least one secondary anchor position having specified therefore at least one secondary residue, said method comprising exchanging the secondary anchor residue of the original peptide epitope for another secondary anchor residue, with the proviso that the original secondary anchor residue is different than the exchanged amino acid residue. In some cases the original secondary residue is a deleterious residue and the exchanged residue is a residue other than a deleterious residue and/or the original secondary anchor residue is a less preferred residue and the exchanged residue is a more preferred residue. 
         [0030]    As will be apparent from the discussion below, other methods and embodiments are also contemplated. Further, novel synthetic peptides produced by any of the methods described herein are also part of the invention. 
       DEFINITIONS 
       [0031]    The following definitions are provided to enable one of ordinary skill in the art to understand some of the preferred embodiments of invention disclosed herein. It is understood, however, that these definitions are exemplary only and should not be used to limit the scope of the invention as set forth in the claims. Those of ordinary skill in the art will be able to construct slight modifications to the definitions below and utilize such modified definitions to understand and practice the invention disclosed herein. Such modifications, which would be obvious to one of ordinary skill in the art, as they may be applicable to the claims set forth below, are considered to be within the scope of the present invention. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in patents, published patent applications and other publications and sequences from GenBank and other databases that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference. 
         [0032]    An “HLA supertype or family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities, rather than serologic supertypes based on shared antigenic determinants. HLA class II molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes. The terms “HLA superfamily,” “HLA supertype family,” “HLA family,” and “HLA xx-like molecules” (where xx denotes a particular HLA type), are synonyms. 
         [0033]    As used herein, the term “IC 50 ” refers to the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Depending on the conditions in which the assays are run (i.e., limiting MHC proteins and labeled peptide concentrations), these values may approximate K D  values. It should be noted that IC 50  values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC 50  of a given ligand. 
         [0034]    Alternatively, binding is expressed relative to a reference peptide. As a particular assay becomes more, or less, sensitive, the IC 50 &#39;s of the peptides tested may change somewhat. However, the binding relative to the reference peptide will not change. For example, in an assay run under conditions such that the IC 50  of the reference peptide increases 10-fold, the IC 50  values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC 50 , relative to the IC 50  of a standard peptide. 
         [0035]    As used herein, “high affinity” with respect to peptide binding to HLA class II molecules is defined as binding with an K D  (or IC 50 ) of less than 50 nM. “Intermediate affinity” is binding with a K D  (or IC 50 ) of between about 50 and about 500 nM. As used herein, “high affinity” with respect to binding to HLA class II molecules is defined as binding with an K D  (or IC 50 ) of less than 100 nM. “Intermediate affinity” is binding with a K D  (or IC 50 ) of between about 100 and about 1000 nM. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. 
         [0036]    Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al.,  Nature  339:392 (1989); Christnick et al.,  Nature  352:67 (1991); Busch et al.,  Int. Immunol.  2:443 (1990); Hill et al.,  J Immunol.  147:189 (1991); del Guercio et al.,  J Immunol.  154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al.,  J Immunol.  21:2069 (1991)), immobilized purified MHC (e.g., Hill et al.,  J Immunol.  152, 2890 (1994); Marshall et al.,  J Immunol.  152:4946 (1994)), ELISA systems (e.g., Reay et al.,  EMBO J  11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al.,  J Biol. Chem.  268:15425 (1993)); high flux soluble phase assays (Hammer et al.,  J. Exp. Med.  180:2353 (1994)). 
         [0037]    The term “peptide” is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. In certain embodiments, the oligopeptides of the invention are less than about 50 residues in length and usually consist of between about 6 and about 25 residues, preferably 14 or 15 residues. Further, an oligopeptide of the invention can be such that it does not comprise more than 50 contiguous amino acids of a native antigen. The preferred HTL-inducing peptides of the invention are 30 residues or less in length, sometimes 20 residues or less and usually consist of between about 6 and about 25 residues, preferably 14 or 15 residues. 
         [0038]    “Synthetic peptide” refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology. 
         [0039]    The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formula, each residue is generally represented by standard three letter or single letter designations. The  L -form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the  D -form for those amino acids having  D -forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or G. Symbols for each amino acids are shown below: 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Amino acids with their abbreviations 
               
             
          
           
               
                   
                 Amino acid 
                 Three letter code 
                 Single letter code 
               
               
                   
                   
               
               
                   
                 Alanine 
                 Ala 
                 A 
               
               
                   
                 Arginine 
                 Arg 
                 R 
               
               
                   
                 Asparagine 
                 Asn 
                 N 
               
               
                   
                 Aspartic acid 
                 Asp 
                 D 
               
               
                   
                 Cysteine 
                 Cys 
                 C 
               
               
                   
                 Glutamine 
                 Gln 
                 Q 
               
               
                   
                 Glutamic acid 
                 Glu 
                 E 
               
               
                   
                 Glycine 
                 Gly 
                 G 
               
               
                   
                 Histidine 
                 His 
                 H 
               
               
                   
                 Isoleucine 
                 Ile 
                 I 
               
               
                   
                 Leucine 
                 Leu 
                 L 
               
               
                   
                 Lysine 
                 Lys 
                 K 
               
               
                   
                 Methionine 
                 Met 
                 M 
               
               
                   
                 Phenylalanine 
                 Phe 
                 F 
               
               
                   
                 Proline 
                 Pro 
                 P 
               
               
                   
                 Serine 
                 Ser 
                 S 
               
               
                   
                 Threonine 
                 Thr 
                 T 
               
               
                   
                 Tryptophan 
                 Trp 
                 W 
               
               
                   
                 Tyrosine 
                 Tyr 
                 Y 
               
               
                   
                 Valine 
                 Val 
                 V 
               
               
                   
                   
               
             
          
         
       
     
         [0040]    With regard to a particular amino acid sequence, an “epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Throughout this disclosure epitope and peptide are often used interchangeably. 
         [0041]    It is to be appreciated that protein or peptide molecules that comprise an epitope of the invention as well as additional amino acid(s) are still within the bounds of the invention. In certain embodiments, there is a limitation on the length of a peptide of the invention. The embodiment that is length-limited occurs when the protein/peptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% identity with a native peptide sequence. Thus, for a peptide comprising an epitope of the invention and a region with 100% identity with a native peptide sequence, the region with 100% identity to a native sequence generally has a length of: less than or equal to 600 amino acids, often less than or equal to 500 amino acids, often less than or equal to 400 amino acids, often less than or equal to 250 amino acids, often less than or equal to 100 amino acids; often less than or equal to 85 amino acids, often less than or equal to 75 amino acids, often less than or equal to 65 amino acids, and often less than or equal to 50 amino acids. In certain embodiments, an “epitope” of the invention is comprised by a peptide having a region with less than 51 amino acids that has 100% identity to a native peptide sequence, in any increment down to 5 amino acids. 
         [0042]    Accordingly, peptide or protein sequences longer than 600 amino acids are within the scope of the invention, so long as they do not comprise any contiguous sequence of more than 600 amino acids that have 100% identity with a native peptide sequence. For any peptide that has five contiguous residues or less that correspond to a native sequence, there is no limitation on the maximal length of that peptide in order to fall within the scope of the invention. It is presently preferred that a CTL epitope be less than 600 residues long in any increment down to eight amino acid residues. 
         [0043]    A “dominant epitope” induces an immune response upon immunization with whole native antigens which comprise the epitope. (See, e.g., Sercarz, et al.,  Annu. Rev. Immunol.  11:729-766 (1993)). Such a response is cross-reactive in vitro with an isolated peptide epitope. 
         [0044]    A “cryptic epitope” elicits a response by immunization with isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen. 
         [0045]    A “subdominant epitope” is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization in vivo or in vitro with an isolated epitope, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro. 
         [0046]    A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. 
         [0047]    As used herein, the term “pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition. 
         [0048]    As used herein, the term “protective immune response” or “therapeutic immune response” refers to a HTL and/or a CTL response to a tumor associated antigen, which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response may include an antibody response that has been facilitated by the stimulation of helper T cells. 
         [0049]    In certain embodiments, an “immunogenic peptide” is a peptide which comprises an allele-specific motif such that the peptide will bind an MHC (HLA) molecule and induce a HTL response. Immunogenic peptides of the invention are capable of binding to an appropriate class II MHC molecule (e.g., HLA-DR) and inducing a helper T cell response against the antigen from which the immunogenic peptide is derived. 
         [0050]    An “immunogenic response” includes one that stimulates a HTL and/or CTL response in vitro and/or in vivo as well as modulates an ongoing immune response through directed induction of cell death (or apoptosis) in specific T cell populations. 
         [0051]    Immunogenic peptides of the invention are capable of binding to an appropriate HLA-DR molecule and inducing a helper T-cell response against the antigen from which the immunogenic peptide is derived. The immunogenic peptides of the invention are less than about 50 residues in length, often 30 residues or less in length, or 20 residues or less in length and usually consist of between about 6 and about 25 residues, preferably 14 or 15 residues. 
         [0052]    The term “derived” when used to discuss an epitope is a synonym for “prepared.” A derived epitope can be isolated from a natural source, or it can be synthesized in accordance with standard protocols in the art. Synthetic epitopes can comprise artificial amino acids “amino acid mimetics,” such as D isomers of natural occurring L amino acids or non-natural amino acids such as cyclohexylalanine A derived/prepared epitope can be an analog of a native epitope. 
         [0053]    Immunogenic peptides are conveniently identified using the binding motif algorithms described for the specific HLA subtype (e.g., HLA-DR). The algorithms are mathematical procedures that produce a score which enables the selection of immunogenic peptides. Typically one uses the algorithmic score with a “binding threshold” to enable selection of peptides that have a high probability of binding at a certain affinity and will in turn be immunogenic. The algorithm is based upon either the effects on MHC binding of a particular amino acid at a particular position of a peptide or the effects on binding of a particular substitution in a motif containing peptide. 
         [0054]    The term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic. 
         [0055]    A “conserved residue” is an amino acid which occurs in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. Typically a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide. At least one to three or more, preferably two, conserved residues within a peptide of defined length defines a motif for an immunogenic peptide. These residues are typically in close contact with the peptide binding groove, with their side chains buried in specific pockets of the groove itself. Typically, an immunogenic peptide will comprise up to three conserved residues, more usually two conserved residues. 
         [0056]    The term “motif” refers to the pattern of residues in a peptide of defined length, usually about 6 to about 25 amino acids, which is recognized by a particular MHC allele (one or more HLA molecules). The peptide motifs are typically different for each human MHC allele and differ in the pattern of the highly conserved residues and negative residues. Peptide motifs are often unique for the protein encoded by each human HLA allele, differing in their pattern of the primary and secondary anchor residues. Typically as used herein, a “motif” refers to that pattern of residues which is recognized by an HLA molecule encoded by a particular allele. The binding motif for an allele can be defined with increasing degrees of precision. 
         [0057]    The designation of a residue position in an epitope as the “carboxyl terminus” or the “carboxyl terminal position” refers to the residue position at the end of the epitope which is nearest to the carboxyl terminus of a peptide, which is designated using conventional nomenclature as defined below. The “carboxyl terminal position” of the epitope may or may not actually correspond to the end of the peptide or polypeptide. 
         [0058]    The designation of a residue position in an epitope as “amino terminus” or “amino-terminal position” refers to the residue position at the end of the epitope which is nearest to the amino terminus of a peptide, which is designated using conventional nomenclature as defined below. The “amino terminal position” of the epitope may or may not actually correspond to the end of the peptide or polypeptide. 
         [0059]    A “motif bearing peptide” or “peptide which comprises a motif” refers to a peptide that comprises primary anchors specified for a given motif or supermotif. 
         [0060]    In certain embodiments, a “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Preferably, a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA molecules or antigens. 
         [0061]    Alternatively, the term “supermotif” refers to motifs that, when present in an immunogenic peptide, allow the peptide to bind more than one HLA antigen. The supermotif preferably is recognized with high or intermediate affinity (as defined herein) by at least one HLA allele having a wide distribution in the human population, preferably recognized by at least two alleles, more preferably recognized by at least three alleles, and most preferably recognized by more than three alleles. 
         [0062]    “Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, Stites, et al., I MMUNOLOGY,  8 TH  ED., Lange Publishing, Los Altos, Calif. (1994). 
         [0063]    “Major Histocompatibility Complex” or “MHC” is a cluster of genes which plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC and HLA complexes, see, Paul, F UNDAMENTAL  I MMUNOLOGY,  3 RD  ED., Raven Press, New York, 1993. 
         [0064]    The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Thus, the peptides of this invention do not contain materials normally associated with their in situ environment, e.g., MHC class II molecules on antigen presenting cells. Even where a protein has been isolated to a homogenous or dominant band, there are trace contaminants in the range of 5-10% of native protein which co-purify with the desired protein. Isolated peptides of this invention do not contain such endogenous co-purified protein. 
         [0065]    “Peripheral blood mononuclear cells” (PBMCs) are cells found in from the peripheral blood of a patient. PBMCs comprise, e.g., CTLs and HTLs and antigen presenting cells. These cells can contact an antigen in vivo, or be obtained from a mammalian source and contacted with an antigen in vitro. 
         [0066]    “Cross-reactive binding” indicates that a peptide is bound by more than one  HLA molecule; a synonym is degenerate binding. 
         [0067]    “Promiscuous recognition” is where the same peptide bound by different HLA molecules is recognized by the same T cell clone. It may also refer to the ability of a peptide to be recognized by a single T cell receptor in the context of multiple HLA alleles. 
         [0068]    “Link” or “join” refers to any method known in the art for functionally connecting peptides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding. 
         [0069]    A “non-native” sequence or “construct” refers to a sequence that is not found in nature, i.e., is “non-naturally occurring”. Such sequences include, e.g., peptides that are lipidated or otherwise modified, and polyepitopic compositions that contain epitopes that are not contiguous in a native protein sequence. 
         [0070]    As used herein, a “vaccine” is a composition that contains one or more peptides of the invention, see, e.g., TABLE I. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class II-binding peptides of the invention can be linked to HLA class I-binding peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can comprise peptide pulsed antigen presenting cells, e.g., dendritic cells. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0071]    Certain embodiments of the present invention relate in part to an epitope-based approach for vaccine design. Such an approach is based on the well-established finding that the mechanism for inducing HTL immune response comprises the step of presenting a HTL epitope as a peptide of about 6-25 amino acids bound to an HLA molecule displayed on an antigen-presenting cell. 
         [0072]    Certain embodiments of the present invention relate to peptides comprising allele-specific peptide motifs and supermotifs which bind to HLA class II molecules. 
         [0073]    As noted above, high HLA binding affinity is correlated with higher immunogenicity. Higher immunogenicity can be manifested in several different ways. For instance, a higher binding peptide will be immunogenic more often. Close to 90% of high binding peptides are immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. A higher binding peptide will also lead to a more vigorous response. As a result, less peptide is required to elicit a similar biological effect. Thus, in some embodiments of the invention high binding epitopes are particularly desired. 
         [0074]    It has been noted that a significant number of epitopes derived from known non-viral tumor associated antigens (TAA) bind HLA Class II with intermediate affinity (IC 50  in the 50-500 mM range). It has been found that 8 of 15 known TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 mM range. These data are in contrast with estimates that 90% of known viral antigens that were recognized as peptides bound HLA with IC 50  of 50 mM or less while only approximately 10% bound in the 50-500 mM range (Sette, et al., J. Immunol., 153:5586-5592 (1994)). This phenomenon is probably due in the cancer setting to elimination, or functional inhibition of the CTL recognizing several of the highest binding peptides, presumably because of T cell tolerization events. 
         [0075]    Epitope-bearing peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology, or from natural sources such as whole viruses or tumors. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides are synthetically conjugated to native molecules or particles; the peptides can also be conjugated to non-native molecules or particles. 
         [0076]    The peptides in accordance with the invention can be a variety of lengths, and either in their neutral (uncharged) forms or in forms which are salts. The peptides in accordance with the invention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation; or they contain these modifications. 
         [0077]    Desirably, the epitope-bearing peptide will be as small as possible while still maintaining relevant immunologic activity of the large peptide; of course it is particularly desirable with peptides from pathogenic organisms that the peptide be small in order to avoid pathogenic function. When possible, it may be desirable to optimize epitopes of the invention to a length of about 6 to about 25, preferably 14 to 15 amino acid residues for a class II molecule. Preferably, the peptides are commensurate in size with endogenously processed viral peptides or tumor cell peptides that are bound to HLA class I or class II molecules on the cell surface. Nevertheless, the identification and preparation of peptides of other lengths can be carried out using the techniques described here such as the disclosures of primary anchor positions. It is to be appreciated that peptide epitopes in accordance with the invention can be present in peptides or proteins that are longer than the epitope itself. Moreover, multiepitopic peptides can comprise at least one epitope of the invention along with other epitope(s). 
         [0078]    In particular, the invention provides motifs that are common to peptides bound by more than one HLA allele. By a combination of motif identification and MHC-peptide interaction studies, peptides useful for peptide vaccines have been identified. 
         [0079]    Peptides comprising the epitopes from these antigens are synthesized and then tested for their ability to bind to the appropriate MHC molecules in assays using, for example, immunofluorescent staining and flow microfluorometry, peptide-dependent class II assembly assays. Those peptides that bind to the class II molecule are further evaluated for their ability to serve as targets for HTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo HTL responses that can give rise to HTL populations capable of reacting with tumor cells as potential therapeutic agents. 
         [0080]    The starting point, therefore, for the design of effective vaccines is to ensure that the vaccine will generate a large number of epitopes that can successfully be presented. It may be possible to administer the peptides representing the epitopes per se. Such administration is dependent on the presentation of “empty” HLA molecules displayed on the cells of the subject. In one approach to use of the immunogenic peptides per se, these peptides may be incubated with antigen-presenting cells from the subject to be treated ex vivo and the cells then returned to the subject. 
         [0081]    Alternatively, the peptides can be generated in situ by administering a nucleic acid containing a nucleotide sequence encoding it. Means for providing such nucleic acid molecules are described in WO99/58658, the disclosure of which is incorporated herein by reference. Further, the immunogenic peptides can be administered as portions of a larger peptide molecule and cleaved to release the desired peptide. The larger peptide may contain extraneous amino acids, in general the fewer the better. Thus, peptides which contain such amino acids are typically 50 amino acids or less, more typically 30 amino acids or less, and more typically 20 amino acids or less. The precursor may also be a heteropolymer or homopolymer containing a multiplicity of different or same HTL epitopes. Of course, mixtures of peptides and nucleic acids which generate a variety of immunogenic peptides can also be employed. The design of the peptide vaccines, the nucleic acid molecules, or the hetero- or homo-polymers is dependent on the inclusion of the desired epitope. 
         [0082]    In certain embodiments, it is preferred that peptides include an epitope that binds to an HLA-DR supertype allele. These motifs may be used to define T-cell epitopes from any desired antigen, particularly those associated with human cancers for which the amino acid sequence of the potential antigen targets is known. 
         [0083]    The peptides are thus useful in pharmaceutical compositions for both in vivo and ex vivo therapeutic and diagnostic applications. 
         [0084]    Peptides comprising the supermotif sequences can be identified, as noted above, by screening potential antigenic sources. Useful peptides can also be identified by synthesizing peptides with systematic or random substitution of the variable residues in the supermotif, and testing them according to the assays provided. As demonstrated below, it is useful to refer to the sequences of the target HLA molecule, as well. 
         [0085]    For epitope-based vaccines, the peptides of the present invention preferably comprise a supermotif and/or motif recognized by an HLA class II molecule having a wide distribution in the human population. The large degree of HLA polymorphism is an important factor to be taken into account with the epitope-based approach to vaccine development. To address this factor, epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele-specific HLA molecules. 
         [0086]    HTL-inducing peptides of interest for vaccine compositions preferably include those that have an IC 50  or binding affinity value for class II HLA molecules, 1000 nM or better (i.e., the value is greater than or equal to 1000 nM). For example, peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are generally tested on other members of the supertype family. In preferred embodiments, peptides that exhibit cross-reactive binding are then used in cellular screening analyses or vaccines. 
         [0087]    Definition of motifs that are predictive of binding to specific class II alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs and/or supermotifs. 
         [0088]    The previous definition of motifs specific for different class II alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs. The epitopic sequences are then synthesized. The capacity to bind MHC Class II molecules is measured in a variety of different ways. 
         [0089]    The procedures used to identify peptides of the present invention generally follow the methods disclosed in Falk et al., Nature 351:290 (1991), which is incorporated herein by reference. Briefly, the methods involve large-scale isolation of MHC class II molecules, typically by immunoprecipitation or affinity chromatography, from the appropriate cell or cell line. Examples of other methods for isolation of the desired MHC molecule equally well known to the artisan include ion exchange chromatography, lectin chromatography, size exclusion, high performance ligand chromatography, and a combination of all of the above techniques. 
         [0090]    The peptides bound to the peptide binding groove of the isolated MHC molecules are eluted typically using acid treatment. Peptides can also be dissociated from class II molecules by a variety of standard denaturing means, such as heat, pH, detergents, salts, chaotropic agents, or a combination thereof. 
         [0091]    Peptide fractions are further separated from the MHC molecules by reversed-phase high performance liquid chromatography (HPLC) and sequenced. Peptides can be separated by a variety of other standard means well known to the artisan, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectrofocusing, and the like. 
         [0092]    Sequencing of the isolated peptides can be performed according to standard techniques such as Edman degradation (Hunkapiller, M. W., et al.,  Methods Enzymol.  91, 399 [1983]). Other methods suitable for sequencing include mass spectrometry sequencing of individual peptides as previously described (Hunt, et al.,  Science  225:1261 (1992), which is incorporated herein by reference). Amino acid sequencing of bulk heterogenous peptides (e.g., pooled HPLC fractions) from different class I molecules typically reveals a characteristic sequence motif for each class I allele. 
         [0093]    Next, peptides that test positive in the MHC class II binding assay are assayed for the ability of the peptides to induce specific HTL responses in vitro. For instance, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce HTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba, et al.,  J. Exp. Med.  166:182 (1987); Boog,  Eur. J. Immunol,  18:219 (1988)). 
         [0094]    As disclosed herein, higher HLA binding affinity is correlated with greater immunogenicity. Greater immunogenicity can be manifested in several different ways. Immunogenicity can correspond to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a diverse population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with the principles disclosed herein, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, higher binding affinity peptides lead to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used. Thus, in preferred embodiments of the invention, high affinity binding epitopes are particularly useful. Nevertheless, improvements over the prior art are achieved with intermediate or high binding peptides. 
         [0095]    After determining their binding affinity, additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, antigenicity, and immunogenicity. 
         [0096]    Thus, various strategies can be utilized to evaluate immunogenicity, including: 
         [0097]    1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al.,  Mol. Immunol.  32:603, 1995; Celis, E. et al.,  Proc. Natl. Acad. Sci. USA  91:2105, 1994; Tsai, V. et al.,  J. Immunol.  158:1796, 1997; Kawashima, I. et al.,  Human Immunol.  59:1, 1998); This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected. 
         [0098]    2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al.,  J. Immunol.  26:97, 1996; Wentworth, P. A. et al.,  Int. Immunol.  8:651, 1996; Alexander, J. et al.,  J. Immunol.  159:4753, 1997); In this method, peptides in incomplete Freund&#39;s adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected. 
         [0099]    3) Demonstration of recall T cell responses from patients who have been effectively vaccinated or who have a tumor; (see, e.g., Rehermann, B. et al.,  J. Exp. Med.  181:1047, 1995; Doolan, D. L. et al.,  Immunity  7:97, 1997; Bertoni, R. et al.,  J. Clin. Invest.  100:503, 1997; Threlkeld, S. C. et al.,  J. Immunol.  159:1648, 1997; Diepolder, H. M. et al.,  J. Virol.  71:6011, 1997; Tsang et al.,  J. Natl. Cancer Inst.  87:982-990, 1995; Disis et al.,  J. Immunol.  156:3151-3158, 1996). In applying this strategy, recall responses are detected by culturing PBL from patients with cancer who have generated an immune response “naturally”, or from patients who were vaccinated with tumor antigen vaccines. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected. 
         [0100]    An immunogenic peptide epitope of the invention may be included in a polyepitopic vaccine composition comprising additional peptide epitopes of the same antigen, antigens from the same source, and/or antigens from a different source. Moreover, class II epitopes can be included along with class I epitopes. Peptide epitopes from the same antigen may be adjacent epitopes that are contiguous in sequence or may be obtained from different regions of the protein. 
         [0101]    An epitope present in the peptides of the invention can be cross-reactive or non-cross-reactive in its interactions with MHC alleles and alleles subtypes. Cross-reactive binding of an epitope (or peptide) permits an epitope to be bound by more than one HLA molecule. Such cross-reactivity is also known as degenerate binding. A non-cross-reactive epitope would be restricted to binding a particular MHC allele or allele subtype. 
         [0102]    Motifs Indicative of Class II HTL Inducing Peptide Epitope 
         [0103]    The primary anchor residues of the HLA class II supermotifs and motifs are delineated below. 
       HLA DR-1-4-7 Supermotif 
       [0104]    Motifs have also been identified for peptides that bind to three common HLA class II allele-specific HLA molecules: HLA DRB1*0401, DRB1*0101, and DRB1*0701 (see, e.g., the review by Southwood et al.  J. Immunology  160:3363-3373, 1998). Collectively, the common residues from these motifs delineate the HLA DR-1-4-7 supermotif. Peptides that bind to these DR molecules carry a supermotif characterized by a large aromatic or hydrophobic residue (Y, F, W, L, I, V, or M) as a primary anchor residue in position 1, and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as a primary anchor residue in position 6 of a 9-mer core region. Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified (Southwood et al., supra). Peptide binding to HLA-DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif. 
         [0105]    Two alternative motifs (i.e., submotifs) characterize peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk et al.,  J. Immunol.  152:5742, 1994). In the first motif (submotif DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mer core, and D is present as an anchor at position 4, towards the carboxyl terminus of the epitope. As in other class II motifs, core position 1 may or may not occupy the peptide N-terminal position. 
         [0106]    The alternative DR3 submotif provides for lack of the large, hydrophobic residue at anchor position 1, and/or lack of the negatively charged or amide-like anchor residue at position 4, by the presence of a positive charge at position 6 towards the carboxyl terminus of the epitope. Thus, for the alternative allele-specific DR3 motif (submotif DR3B): L, I, V, M, F, Y, A, or Y is present at anchor position 1; D, N, Q, E, S, or T is present at anchor position 4; and K, R, or H is present at anchor position 6. Peptide binding to HLA-DR3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif. 
         [0107]    As with HLA class I binding peptides, motifs have also been defined for HLA class II-binding peptides. Several studies have identified an important role for an aromatic or hydrophobic residue (I, L, M, V, F, W, or Y) at position 1 of a 9-mer core region, typically nested within a longer peptide sequence, in the binding of peptide ligands to several HLA-class II alleles (Hammer et al.  Cell  74:197, (1993); Sette et al.  J. Immunol.  151:3163-70 (1993); O&#39;Sullivan et al.  J. Immunol.  147:2663 (1991); and Southwood et al.  J. Immunol.  160:3363-73 (1998)). A strong role has also been demonstrated for the residue in position 6 of the 9-mer core, where short and/or hydrophobic residues (S, T, C, A, P, V, I, L, or M) are preferred. This position 1-position 6 motif has been described as a DR-supermotif (Southwood et al.  J. Immunol.  160:3363-3373 (1998)) and has been shown to efficiently identify peptides capable of binding a large set of common HLA-class II alleles. 
         [0108]    Peptides binding to class II molecules may also be analyzed with respect to the identification of secondary preferred or deleterious residues. For example, to derive a more detailed DRB1*0401 motif to define secondary residues influencing peptide binding, we employed a strategy similar to that performed with class I peptides. For each peptide analyzed, nine-residue-long core regions were aligned on the basis of the primary class II positions P1 and P6 anchors. Then, the average binding affinity of a peptide carrying a particular residue was calculated for each position, relative to the remainder of the group. Following this method, values showing average relative binding were compiled. These values also present a map of the positive or negative effect of each of the 20 naturally occurring amino acids in DRB1*0401 binding capacity when occupying a particular position relative to the P1-P6 class II motif positions. 
         [0109]    Variations in average relative binding of greater than or equal to fourfold or less than or equal to 0.25 were arbitrarily considered significant and indicative of secondary effects of a given residue on HLA-peptide interactions. Most secondary effects were associated with P4, P7, and P9. These positions correspond to secondary anchors engaging shallow pockets on the DR molecule. Similar studies defining secondary residues were also performed for DRB1*0101 and DRB1*0701. The definitions of secondary residues of motifs for DR1, DR4, and DR7 are shown in TABLE 139. 
         [0110]    Upon definition of allele-specific secondary effects and secondary anchors, allele-specific algorithms were derived and utilized to identify peptides binding DRB1*0101, DRB1*0401, and DRB*0701. Further experiments, identified a large set of HLA class II molecules, which includes at least the DRB1*0101, DRB1*0401, and DRB*0701, DRB1*1501, DRB1*0901 and DRB1*1302 allelic products recognizing the DR supermotif, and is characterized by largely overlapping peptide binding repertoires. 
         [0111]    The data presented above confirm that several common HLA class II types are characterized by largely overlapping peptide binding repertoires. On this basis, in analogy to the case of HLA class 1 molecules, HLA class II molecules can be grouped in a HLA class II supertype, defined and characterized by similar, or largely overlapping (albeit not identical) peptide binding specificities. 
         [0112]    The peptides present in the invention can be identified by any suitable method. For example, peptides are conveniently identified using the algorithms of the invention described in the co-pending U.S. patent application Ser. No. 09/894,018. These algorithms are mathematical procedures that produce a score which enables the selection of immunogenic peptides. Typically one uses the algorithmic score with a binding threshold to enable selection of peptides that have a high probability of binding at a certain affinity and will in turn be immunogenic. The algorithm are based upon either the effects on MHC binding of a particular amino acid at a particular position of a peptide or the effects on binding MHC of a particular substitution in a motif containing peptide. 
         [0113]    Peptide sequences characterized in molecular binding assays and capture assays have been and can be identified utilizing various technologies. Motif-positive sequences are identified using a customized application created at Epimmune. Sequences are also identified utilizing matrix-based algorithms, and have been used in conjunction with a “power” module that generates a predicted 50% inhibitory concentration (PIC) value. These latter methods are operational on Epimmune&#39;s HTML-based Epitope Information System (EIS) database. All of the described methods are viable options in peptide sequence selection for IC 50  determination using binding assays. 
         [0114]    The capacity to bind MHC molecules is measured in a variety of different ways. One means is a MHC binding assay as described in the related applications, noted above. Other alternatives described in the literature include inhibition of antigen presentation (Sette, et al.,  J. Immunol.  141:3893 (1991), in vitro assembly assays (Townsend, et al., Cell 62:285 (1990), and FACS based assays using mutated cells, such as RMA.S (Melief, et al.,  Eur. J. Immunol.  21:2963 (1991)). 
         [0115]    Capture Assay: Unlike the HPLC-based molecular binding assay, noted above, the high throughput screening (“HTS”) Capture assay does not utilize a size-exclusion silica column for separation of bound from unbound radioactive marker. Instead, wells of an opaque white 96-well Optiplate (Packard) are coated with 3 μg (100 μl @ 30 μg/ml) of HLA-specific antibody (Ab) that “capture” complexes of radiolabeled MHC and unlabeled peptide transferred from the molecular binding assay plate in 100 μl of 0.05% NP40/PBS. After a 3-hour incubation period, the supernatant is decanted and scintillation fluid (Microscint 20) added. Captured complexes are then measured on a microplate scintillation and luminescence counter (TopCount NXT™; Packard). 
         [0116]    Additional assays for determining binding are described in detail, i.e., in PCT publications WO 94/20127 and WO 94/03205. Binding data results are often expressed in terms of IC 50  value. IC 50  is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide occurs. Given the conditions in which the assays are preformed (i.e., limiting MHC proteins and labeled peptide concentrations), these values approximate K D  values. It should be noted that IC 50  values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (i.e., MHC preparation, etc.). For example, excessive concentrations of MHC molecules will increase the apparent measured IC 50  of a given ligand. Alternatively, binding is expressed relative to a reference peptide. Although as a particular assay becomes more, or less, sensitive, the IC 50 &#39;s of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change. For example, in an assay preformed under conditions such that the IC 50  of the reference peptide increases 10-fold, the IC 50  values of the test peptides will also increase approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC 50 , relative to the IC 50  of a standard peptide. 
         [0117]    The peptides of the invention may also comprise isosteres of two or more residues in the MHC-binding peptide. An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola,  Chemistry and Biochemistry of Amino Acids, Peptides and Proteins , Vol. VII (Weinstein ed., 1983). 
         [0118]    Modifications of peptides with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef et al.,  Eur. J. Drug Metab. Pharmacokin.  11:291-302 (1986). Half life of the peptides of the present invention is conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows. Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4° C.) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions. 
         [0119]    Such analogs may also possess improved shelf-life or manufacturing properties. More specifically, non-critical amino acids need not be limited to those naturally occurring in proteins, such as  L -α-amino acids, or their  D -isomers, but may include non-natural amino acids as well, such as amino acids mimetics, e.g.  D - or  L -naphylalanine;  D - or  L -phenylglycine;  D - or  L -2-thieneylalanine;  D - or  L -1, -2,3-, or 4-pyreneylalanine;  D - or  L -3 thieneylalanine;  D - or  L -(2-pyridinyl)-alanine;  D - or  L -(3-pyridinyl)-alanine;  D - or  L -(2-pyrazinyl)-alanine;  D - or  L -(4-isopropyl)-phenylglycine;  D -(trifluoromethyl)-phenylglycine;  D -(trifluoromethyl)-phenylalanine;  D -ρ-fluorophenylalanine;  D - or  L -ρ-biphenylphenylalanine;  D - or  L -ρ-methoxybiphenylphenylalanine;  D - or  L -2-indole(alkyl)alanines; and,  D - or  L -alkylalanines, where the alkyl group can be a substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a normatural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. 
         [0120]    Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in, e.g., a liquid environment. This substitution may occur at any position of the peptide epitope. Analogs of the present invention may include peptides containing substitutions to modify the physical property (e.g., stability or solubility) of the resulting peptide. For example, a cysteine (C) can be substituted out in favor of α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting α-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances (see, e.g., the review by Sette et al., In:  Persistent Viral Infections , Eds. R. Ahmed and I. Chen, John Wiley &amp; Sons, England, 1999). Substitution of cysteine with α-amino butyric acid may occur at any residue of a peptide epitope, i.e. at either anchor or non-anchor positions. 
         [0121]    The binding activity, particularly modification of binding affinity or cross-reactivity among HLA supertype family members, of peptides of the invention can also be altered using analoging, which is described in co-pending U.S. application Ser. No. 09/226,775 filed Jan. 6, 1999. In brief, the analoging strategy utilizes the motifs or supermotifs that correlate with binding to certain HLA molecules. Analog peptides can be created by substituting amino acid residues at primary anchor, secondary anchor, or at primary and secondary anchor positions. Generally, analogs are made for peptides that already bear a motif or supermotif. For a number of the motifs or supermotifs in accordance with the invention, residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind the respective motif or supermotif (see, e.g., Rupert et al.  Cell  74:929, 1993; Sidney, J. et al.,  Hu. Immunol.  45:79, 1996; and Sidney et al.; Sidney, et al.,  J. Immunol.  154:247, 1995). Accordingly, removal of such residues that are detrimental to binding can be performed in accordance with the present invention. For example, in the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of peptides used in the analysis, the incidence of cross-reactivity increased from 22% to 37% (see, e.g., Sidney, J. et al.,  Hu. Immunol.  45:79, 1996). 
         [0122]    Thus, one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small “neutral” residue such as Ala (that may not influence T cell recognition of the peptide). An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, “preferred” residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted. 
         [0123]    In some embodiments, a T helper peptide can used in addition to one of the peptides of the invention. One type of T helper peptide is one that is recognized by T helper cells in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the MHC class II molecules. These are known as “loosely MHC-restricted” T helper sequences. Examples of amino acid sequences that are loosely MHC-restricted include sequences from antigens such as Tetanus toxin at positions 830-843 (QYIKANSKFIGITE (SEQ ID NO:______)),  Plasmodium falciparum  circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS (SEQ ID NO:______)), and  Streptococcus  18 kD protein at positions 1-16 (YGAVDSILGGVATYGAA (SEQ ID NO:______)). 
         [0124]    Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely MHC-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds, called Pan-DR-binding epitopes or PADRE® molecules (Epimmune, San Diego, Calif.), are designed on the basis of their binding activity to most HLA-DR (human MHC class II) molecules (see, e.g., U.S. Ser. No. 08/121,101 (now abandoned) and related U.S. Ser. No. 08/305,871 (now U.S. Pat. No. 5,736,142)). For instance, a pan-DR-binding epitope peptide having the formula: aKXVWANTLKAAa, where X is either cyclohexylalanine, phenylalanine, or tyrosine, and “a” is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. 
         [0125]    Particularly preferred immunogenic peptides and/or T helper conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the HTL peptide may be linked to the T helper peptide without a spacer. 
         [0126]    The immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the HTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated. The T helper peptides used in the invention can be modified in the same manner as HTL peptides. For instance, they may be modified to include D-amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389. 
         [0127]    In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes HTL and CTL. Lipids have been identified as agents capable of priming HTL and CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be injected directly in a micellar form, incorporated into a liposome or emulsified in an adjuvant, e.g., incomplete Freund&#39;s adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid attached to alpha and epsilon amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide. Also in a preferred embodiment a particularly effective immunogen comprises palmitic acid attached to alpha and epsilon amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of a class I restricted peptide having T cell determinants, such as those peptides described herein as well as other peptides which have been identified as having such determinants. 
         [0128]    As another example of lipid priming of HTL and CTL responses,  E. coli  lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P 3 CSS) can be used to prime virus specific HTL CTL when covalently attached to an appropriate peptide. See, Deres et al.,  Nature  342:561-564 (1989), incorporated herein by reference. Peptides of the invention can be coupled to P 3 CSS, for example, and the lipopeptide administered to an individual to specifically prime a HTL response to the target antigen. Further, as the induction of neutralizing antibodies can also be primed with P 3 CSS conjugated to a peptide which displays an appropriate epitope, the two compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection. 
         [0129]    In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide. Modification at the C terminus in some cases may alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH 2  acylation, e.g., by alkanoyl (C 1 -C 20 ) or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule. 
         [0130]    The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young,  Solid Phase Peptide Synthesis,  2d. ed., Pierce Chemical Co. (1984), supra. 
         [0131]    Another aspect of the present invention is directed to vaccines which comprise an immunogenically effective amount of one or more peptides as described herein. Peptides may be introduced into a host using a variety of delivery vehicles known to those of skill in the art including PLG microspheres with entrapped peptides and virus-like particles. Furthermore, epitopes may be introduced as multiple antigen peptides (MAPs) (see e.g., Mora and Tam,  J. Immunol.  161:3616-23 (1998)), or as immunostimulating complexes (ISCOMS) (see e.g., Hu et al.  Clin. Exp. Immunol.  113:235-43 (1998)) as known in the art. 
         [0132]    Vaccines that contain an immunogenically effective amount of one or more peptides as described herein are a further embodiment of the invention. The vaccines of the invention can be used both as a prevantative or therapeutic. Once appropriately immunogenic epitopes have been defined, they can be delivered by various means, herein referred to as “vaccine” compositions. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al.,  J: Clin. Invest.  95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al.,  Molec. Immunol.  28:287-294, 1991: Alonso et al.,  Vaccine  12:299-306, 1994; Jones et al.,  Vaccine  13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al.,  Nature  344:873-875, 1990; Hu et al.,  Clin Exp Immunol.  113:235-24: 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P.,  Proc. Natl. Acaa Sci. U.S.A.  85:5409-5413, 1988; Tam, J. P.,  J Immunol. Methods  196:17-32, 1996), vir delivery vectors (Perkus, M. E. et al., In:  Concepts in vaccine development , Kaufmann H. E., ed., p. 379, 1996; Chakrabarti, S. et al.,  Nature  320:535, 1986; Hu, S. L. et al.,  Nature  320:537, 1986; Kieny, M.-P. et al.,  AIDS Bio/Technology  4:790, 1986; Top, F. et al.,  J Infect. Dis.  124:148, 1971; Chanda, P. K. et al.,  Virology  175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al.,  J Immunol. Methods.  192:2˜1996; Eldridge, J. H. et al.,  Sem. Bematol.  30:16, 1993; Fa10, L. D., Jr. et al.,  Nature Med.  7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A.  Annu. Re Immunol.  4:369, 1986; Gupta, R. K. et al.,  Vaccine  11:293, 1993), liposomes (Reddy, R et al.,  J. Immunol.  148:1585, 1992; Rock, K. L.,  Immunol. Today  17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al.,  Science  259:1745, 1993; Robinsol H. L., Hunt, L. A., and Webster, R. G.,  Vaccine  11:957, 1993; Shiver, J. W. et al., In:  Concepts in vaccine development , Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A.,  Annu. Rev. Immunol.  12:923, 1994 and Eldridge, J. H. et al.,  Sem. Hematol.  30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used. 
         [0133]    Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA encoding one or more of the peptides of the invention can also be administered to a patient. This approach is described, for instance, in Wolff et. al.,  Science  247: 1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687). 
         [0134]    For therapeutic or prophylactic immunization purposes, the peptides of the invention can be expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al.,  Nature  351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors,  Salmonella typhi  vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein. 
         [0135]    Furthermore, vaccines in accordance with the invention can encompass one or more of the peptides of the invention. Accordingly, a peptide can be present in a vaccine individually. Alternatively, the peptide can be individually linked to its own carrier; alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition may be a naturally occurring region of an antigen or may be prepared, e.g., recombinantly or by chemical synthesis. 
         [0136]    Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund&#39;s adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS). 
         [0137]    Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of HTLs and/or CTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later infection, or at least partially resistant to developing an ongoing chronic infection, or derives at least some therapeutic benefit when the antigen was tumor-associated. 
         [0138]    For therapeutic or immunization purposes, the peptides of the invention can also be expressed by vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover, et al.  Nature  351:456-60 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g.,  Salmonella typhi  vectors, retroviral vectors, adenoviral or adeno-associated viral vectors, and the like will be apparent to those skilled in the art from the description herein. 
         [0139]    Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al.,  Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982) (also 1989), which is incorporated herein by reference. Thus, fusion proteins which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope. For example, a coding sequence encoding a peptide of the invention can be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. Expression constructs, i.e., minigenes are described in greater detail in the sections below. Such methodologies are also used to present at least one peptide of the invention along with a substance which is not a peptide of the invention. 
         [0140]    As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, using the phosphotriester method of Matteucci et al.,  J. Am. Chem. Soc.  103:3185 (1981), with modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Of course, yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences. 
         [0141]    The peptides of the present invention and pharmaceutical and vaccine compositions thereof are useful for administration to mammals, particularly humans, to treat and/or prevent cancer. 
         [0142]    In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective HTL response to the tumor antigen and to cure or at least partially arrest symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose” or “unit dose.” Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 μg to about 5000 μg of peptide for a 70 kg patient, followed by boosting dosages of from about 1.0 μg to about 1000 μg of peptide pursuant to a boosting regimen over weeks to months depending upon the patient&#39;s response and condition by measuring specific CTL activity in the patient&#39;s blood. In alternative embodiments, generally for humans the dose range for the initial immunization (that is for therapeutic or prophylactic administration) is from about 1.0 μg to about 20,000 μg of peptide for a 70 kg patient, preferably, 100 μg-, 150 μg-, 200 μg-, 250 μg-, 300 μg-, 400 μg-, or 500 μg-20,000 μg, followed by boosting dosages in the same dose range pursuant to a boosting regimen over weeks to months depending upon the patient&#39;s response and condition by measuring specific HTL activity in the patient&#39;s blood. In embodiments where recombinant nucleic acid administration is used, the administered material is titrated to achieve the appropriate therapeutic response. 
         [0143]    It must be kept in mind that the peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions. 
         [0144]    For therapeutic use, administration should begin at the first sign of tumors or shortly after diagnosis. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. 
         [0145]    Treatment of an affected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developingcancer the compositions are particularly useful in methods for preventing the evolution of cancer. 
         [0146]    The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. 
         [0147]    A pharmaceutical composition of the invention may comprise one or more T cell stimulatory peptides of the invention. For example, a pharmaceutical composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more T cell stimulatory peptides of the invention. Moreover, a pharmaceutical composition of the invention may comprise one or more T cell stimulatory peptides of the invention in combination with one or more other T cell stimulatory peptides. The concentration of each unique T cell stimulatory peptide of the invention in the pharmaceutical formulations can vary widely, e.g., from less than about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, 0.007%, 0.008%, 0.009%, about 0.01%, about 0.02%, about 0.025%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, to about 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. In a preferred embodiment, the concentration of each unique T cell stimulatory peptide of the invention in the pharmaceutical formulations is about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, 0.007%, 0.008%, 0.009%, about 0.01%, about 0.02%, about 0.025%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% by weight. In a more preferred embodiment, the concentration of each unique T cell stimulatory peptide of the invention in the pharmaceutical formulations is about 0.01%, about 0.02%, about 0.025%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1% by weight. 
         [0148]    The concentration of HTL stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. A human unit dose form of the peptide composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans. 
         [0149]    The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al.,  Ann. Rev. Biophys. Bioeng.  9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference. 
         [0150]    For targeting to the immune cells, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated. 
         [0151]    For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%. 
         [0152]    For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery. 
         [0153]    In another aspect the present invention is directed to vaccines which contain as an active ingredient an immunogenically effective amount of an immunogenic peptide as described herein. The peptide(s) may be introduced into a host, including humans, linked to its own carrier or as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of tumor cells. Useful carriers are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant. Adjuvants such as incomplete Freund&#39;s adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of HTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection. 
         [0154]    The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent cancer. Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of cancer to elicit an immune response against the antigen and thus enhance the patient&#39;s own immune response capabilities. Such an amount is defined to be an “immunogenically effective dose.” In this use, the precise amounts again depend on the patient&#39;s state of health and weight, the mode of administration, the nature of the formulation, etc., but generally range from about 1.0 μg to about 5000 μg per 70 kilogram patient, more commonly from about 10 μg to about 500 μg mg per 70 kg of body weight. 
         [0155]    As noted herein, the peptides of the invention induce HTL immune responses when contacted with a HTL specific to an epitope comprised by the peptide. The manner in which the peptide is contacted with the HTL is not critical to the invention. For instance, the peptide can be contacted with the HTL either in vivo or in vitro. If the contacting occurs in vivo, the peptide itself can be administered to the patient or other vehicles, e.g., DNA vectors encoding one or more peptide, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein. 
         [0156]    For therapeutic or immunization purposes, nucleic acids encoding one or more of the peptides of the invention can also be administered to the patient. A number of methods are conveniently used to deliver the nucleic acids to the patient. For instance, the nucleic acid can be delivered directly, as “naked DNA”. This approach is described, for instance, in Wolff et. al.,  Science  247: 1465-68 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. 
         [0157]    The nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372; WO 93/24640; Mannino and Gould-Fogerite (1988)  BioTechniques  6(7): 682-691; Rose U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner et al. (1987)  Proc. Natl. Acad. Sci. USA  84: 7413-14. 
         [0158]    Nucleic acids encoding one or more of the peptides of the invention can also be administered to the patient. This approach is described, for instance, in Wolff, et. al.,  Science,  247:1465-68 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. 
         [0159]    A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding multiple epitopes of the invention. To create a DNA sequence encoding the selected HTL epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes are reverse translated. A human codon usage table is used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences are directly adjoined, creating a continuous polypeptide sequence. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequence that could be reverse translated and included in the minigene sequence include: a leader (signal) sequence, and an endoplasmic reticulum retention signal. In addition, MHC presentation of HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the HTL epitopes. 
         [0160]    The minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the HTL epitope polypeptide, can then cloned into a desired expression vector. 
         [0161]    Standard regulatory sequences well known to those of skill in the art are included in the vector to ensure expression in the target cells. Several vector elements are required: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an  E. coli  origin of replication; and an  E. coli  selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences. 
         [0162]    Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences can also be considered for increasing minigene expression. It has recently been proposed that immunostimulatory sequences (ISSs or CpGs) play a role in the immunogenicity of DNA vaccines. These sequences could be included in the vector, outside the minigene coding sequence, if found to enhance immunogenicity. 
         [0163]    In some embodiments, a bicistronic expression vector, to allow production of the minigene-encoded epitopes and a second protein included to enhance or decrease immunogenicity can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules. Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases. 
         [0164]    Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate  E. coli  strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank. 
         [0165]    Therapeutic quantities of plasmid DNA are produced by fermentation in  E. coli , followed by purification. Aliquots from the working cell bank are used to inoculate fermentation medium (such as Terrific Broth), and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by Quiagen. If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods. 
         [0166]    Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). A variety of methods have been described, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types. 
         [0167]    The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. 
         [0168]    In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. 
         [0169]    An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient&#39;s blood. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes in HLA molecules on their surfaces. 
         [0170]    Dendritic cells can also be transfected, e.g., with a minigene comprising nucleic acid sequences encoding the epitopes in accordance with the invention, in order to elicit immune responses. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. 
         [0171]    Transgenic animals of appropriate haplotypes may additionally provide a useful tool in optimizing the in vivo immunogenicity of minigene DNA. In addition, animals such as monkeys having conserved HLA molecules with cross reactivity to CTL epitopes recognized by human MHC molecules can be used to determine human immunogenicity of CTL epitopes (Bertoni, et al.,  J. Immunol.  161:4447-4455 (1998)). 
         [0172]    Such in vivo studies are required to address the variables crucial for vaccine development, which are not easily evaluated by in vitro assays, such as route of administration, vaccine formulation, tissue biodistribution, and involvement of primary and secondary lymphoid organs. Because of their simplicity and flexibility, HLA transgenic mice represent an attractive alternative, at least for initial vaccine development studies, compared to more cumbersome and expensive studies in higher animal species, such as nonhuman primates. 
         [0173]    Antigenic peptides are used to elicit a HTL response ex vivo, as well. The resulting HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo HTL responses to a particular antigen are induced by incubating in tissue culture the patient&#39;s (HTLp), or genetically compatible, HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days (1-4 weeks)), in which the precursor cells are activated and matured and expanded into effector cells, the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells. In order to optimize the in vitro conditions for the generation of specific helper T cells, the culture of stimulator cells is maintained in an appropriate serum-free medium. 
         [0174]    The peptides may also find use as diagnostic reagents. For example, a peptide of the invention may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, the peptides may also be used to predict which individuals will be at substantial risk for developing chronic infection. 
         [0175]    For example, a peptide of the invention may be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a pathogen or immunogen. The HLA-tetrameric complex is used to directly visualize antigen-specific CTLs (see, e.g., Ogg, et al.  Science  279:2103-2106, 1998; and Altman, et al.  Science  174:94-96, 1996) and determine the frequency of the antigen-specific CTL population in a sample of peripheral blood mononuclear cells. A tetramer reagent using a peptide of the invention may be generated as follows: A peptide that binds to an allele-specific HLA molecule or supertype molecule is refolded in the presence of the corresponding HLA heavy chain and β 2 -microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the heavy chain at a site that was previously engineered into the protein. Tetramer formation is then induced by the addition of streptavidin. By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells. The cells may then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes. In addition, the peptides may also be used to predict which individuals will be at substantial risk for developing chronic infection. 
         [0176]    All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. 
       EXAMPLES 
     Materials and Methods 
       [0177]    The following materials and methods apply generally to all the examples disclosed herein. Specific materials and methods are disclosed in each example, as necessary. 
         [0178]    Reagents: Anti-IFN-γ and biotinylated anti-IFN-γ were obtained from Mabtech (Sweden). Phorbol myristate acetate (PMA), human serum albumin (HSA), polyclonal human IgG, tetanus toxin (TT), and ionomycin were from Sigma (St. Louis, Mo., USA). Goat anti-human horseradish peroxidase (HRP)-conjugated antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Hank&#39;s balanced salts solution (HBSS), RPMI-1640 and phosphate-buffered saline were from Cellgro (Hernden, Va., USA). Ficoll-Paque was from Amersham Biosciences (Uppsala, Sweden). All peptides were synthesized by either the Mayo Clinic Protein Chemistry and Proteomics Core or by Epimmune, Inc. (San Diego, Calif.) and purified to &gt;95% homogeneity by reverse-phase HPLC as previously described (Dzuris J L, Sidney J, Appella E, Chesnut R W, Watkins D I, Sette A. Conserved MHC class I peptide binding motif between humans and rhesus macaques. J Immunol 2000; 164: 283-91). Purity of peptides was determined with reverse-phase HPLC and amino acid analysis, sequencing, and/or mass spectrometry. Lyophilized peptides were resuspended at 20 mg/ml in 100% DMSO and then diluted to required concentrations in PBS. 
         [0179]    Epitope Prediction: The prediction program used, PIC (Predicted IC50), is a modified linear coefficient, or matrix-based method for predicting peptides with HLA-DR binding capacity. PIC is predicated on the assumption that each residue along a peptide molecule can independently contribute to binding affinity (Sette A, et al. Proc Natl Acad Sci USA 1989; 86: 3296-300; Sette A, et al. J Immunol 1989; 142: 35-40). The algorithm yields a predicted IC50 value (designated as PIC) for the corresponding input sequence. Lower PIC values indicate a higher probability of binding to HLA. The program analyzes 15 amino acid long sequences offset by 3 residues encompassing the entire protein. 
         [0180]    Peripheral Blood Mononuclear Cell Preparation (PBMC): PBMC were isolated from blood as described (Disis M L, et al. Clin Cancer Res 1999; 5: 1289-97), and cryopreserved in liquid nitrogen (20×106/ml cells) in freezing media (RPMI with 12.5% HSA, penicillin, streptomycin and 2 mM glutamine) (Disis M L et al. J Immunol Methods 2005.). 
         [0181]    HLA-DR purification. Fifteen distinct HLA-DR molecules were used in quantitative assays to measure the binding of peptides to solubilized HLA-DR molecules. These HLA-DR molecules were chosen to allow balanced population coverage: DRB1*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*0404, DRB1*1101, DRB5*0101, DRB4*0101, DRB3*0101, DRB1*0701, DRB1*0405, DRB1*0802, DRB1*0901, DRB1*1201, and DRB1*1302 (24). MHC molecules utilized were purified from EBV transformed homozygous cell lines or single MHC allele transfected 721.221, C1R, or fibroblast lines. The cell lines were maintained by culture in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U (100 μg/ml) penicillin-streptomycin solution, and 10% heat-inactivated FCS. HLA-DR molecules were purified using antibody-based affinity chromatography from cell lysates prepared in 50 mM Tris-HCL, pH 8.5, containing 1% (v/v) NP-40, 150 mM NaCl, 5 mM EDTA, and 2 mM PMSF. Briefly, columns of inactivated Sepharose CL4B and Protein A Sepharose were used as pre-columns. HLA-DR molecules were captured by passage of lysates over LB3.1 monoclonal antibody (anti-HLA-DRA) columns. Antibody columns were washed with 10 mM Tris-HCL, pH8.0 with 1% (v/v) NP-40, followed by PBS containing 0.4% (w/v) n-octylglucoside. MHC molecules were then eluted with 50 mM diethylamine in 0.15 M NaCl containing 0.4% (w/v) n-octylglucoside, pH 11.5. The pH was reduced to 8.0 and the eluates were concentrated by centrifugation in Centriprep 30 concentrators at 2000 rpm (Amicon, Beverly, Mass.). 
         [0182]    HLA-DR binding assays: Radioligand binding inhibition assays were used to measure the binding of peptides to soluble HLA-DR molecules based on the inhibition of binding of a radiolabeled standard peptide as described previously (Sidney J, Southwood S, Oseroff C, del Guercio M F, Grey H M, Sette A. Measurement of MHC/peptide interactions by gel filtration. Curr Protocols Immunol 1998; 18: 18.3.2-.3.9.). Briefly, 1-10 nM of radiolabeled peptide was co-incubated for 2 days at either room temperature or 37° C. with 1 μM to 1 nM purified HLA-DR molecules in the presence of a cocktail of protease inhibitors. Assays were performed at various pH conditions, ranging from pH 4 to pH 7. The final pH of assay mixtures is adjusted using citrate buffer as described elsewhere (Sidney J, Curr Protocols Immunol 1998). After incubation, the percentage of HLA-DR-bound radioactivity is determined by capturing HLA-DR/peptide complexes on Optiplates (Packard Instruments, Meriden, Conn.) coated with the LB3.1 antibody and determining bound counts per minute using the TopCount microscintillation counter (Packard Instruments). The amount of HLA-DR yielding 10-20% bound radioactivity is used in the inhibition assays in which the concentration of peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Under the conditions used, the measured IC50 values are reasonable approximations of the true Kd values. Competitor peptides are tested in 2-4 complete, independent experiments, at concentrations ranging from 30 μg/mL to 300 pg/mL. As in previous studies, peptides with affinities for specific HLA-DR molecules of 1000 nM or better are defined as binders for the respective antigens. 
         [0183]    Enzyme-linked immunosorbent spot assay. A 10-day ELIspot for detecting low-frequency T cells was used to determine reactivity to the tumor antigen peptides (Table 1) as described (Knutson K L et al. J Clin One 2006; 24: 4254-61). A positive response to a peptide was defined as a frequency that was significantly (p&lt;0.05, two-tailed t test) greater than the mean of control no-antigen wells and detectable (i.e., &gt;1:100,000). PMA/Ionomycin and the CEF pool were used as positive non-tumor related controls as previously described (Knutson, 2006). 
         [0184]    ELISA. ELISAs were done as previously described (Knutson, 2006). Briefly, 96-well plates were coated with 1 μg/ml IGFBP-2 protein, 200 ng/ml tetanus toxin or 1 μg/ml BSA. Human IgG was added at a concentration range of 200 to 0.2 ng/ml to some wells for standard curve generation. After washing and blocking, human sera were added to the plate at a 1:40 dilution in triplicate and plates were incubated for 2 hr at RT. After washing, 100 μL/well of HRP (Santa Cruz Biotechnology) was diluted 1:2000 and incubated for 1 hr at RT. After a final wash, each well was incubated with 100 μL (tetramethylbenzadine) TMB substrate (BD Bioscience). Color development was stopped with diluted HCL and absorbance was read at 450 nm on a plate reader. 
         [0185]    The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results. 
       Example 1 
     Identification of Conserved HLA Class II—Restricted Peptides Derived from Tumor Associated Antigens Using Established Motif Search Algorithms 
       [0186]    To identify epitopes useful for vaccine design, a multidisciplinary approach was used based initially on amino acid motif searching of tumor associated antigen sequences to identify potential HLA Class II motifs (see Table I). This was followed by high throughput synthetic peptide binding assays using purified HLA molecules to determine affinity and breadth of epitope peptide binding. 
         [0187]    Algorithm motif searches: Motif search algorithms were validated for the most common HLA Class II alleles and were focused on the HLA DRB1*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*0404, DRB1*1101, DRB5*0101, DRB4*0101, DRB3*0101, DRB1*0701, DRB1*0405, DRB1*0802, DRB1*0901, DRB1*1201, and DRB1*1302 supertypes in order to attain virtually 100% population coverage. The selected tumor associated antigen sequences were scanned for motif positive amino acid sequences using the motif definitions. 
         [0188]    A total of about 150 Class II-restricted peptide sequences were identified that were specific for various DR supertypes (see Table I). Table I lists for each identified DR antigen peptide, the IC 50  (nM) for each purified HLA. 
       Example 2 
     Identification of HTL Epitopes for Tumor Vaccine Inclusion 
       [0189]    Of the peptides listed in Table I, those peptides that bound to at least 4 different HLA with an IC 50  of less than 1000 nM were identified and are shown in Table II. The peptide sequences of Table II were further evaluated for their binding capacity to purified MHC molecules.  FIGS. 1 through 4  show those peptides of Table II which were immunogenic (shown in a circle). These HTL peptides are candidates for inclusion into a tumor vaccine. 
         [0190]    Using predictive algorithms discussed above, candidate HLA-DR1-binding epitopes were identified. Binding assays targeting 15 different HLA-DR molecules revealed that 10 of the epitopes were indiscriminate, binding (IC 50 &lt;1000 nM) to at least four different HLA-DR variants. An interferon-gamma ELlspot assay was used to assess immunity to the indiscriminate binding peptides in 48 patients with either breast or ovarian cancer and 18 healthy controls. The results showed that elevated T cell immunity in patients was detected to several peptides ( FIGS. 1-4 ). 
         [0191]    Healthy donor and patient samples were obtained. Patients were free from active treatment for at least 30 days when blood (200 ml) was collected. For the T cell studies, the mean (±s.e.m) ages of the healthy donors and patients were 42±11 and 55±2 years, respectively (p&lt;0.0001). Due to sera unavailability, not all of the controls used in the T cell studies were examined for tumor associated antigen antibodies in their sera. However, additional control and patient sera were available for antibody assessment. Additional healthy donor sera was obtained from Bioreclamation (Hicksville, N.Y.). 
         [0192]    Using an ELIspot assay with a limit of detection of approximately 1:100,000 antigen-specific T cells per million PBMC, patient-derived PBMC were screened for reactivity against all of the HLA-DR binding peptides. The immunogenicity data of several peptides is shown in  FIGS. 1-4 . 
       Example 3 
     Design and Development of Multi-Epitope Vaccines 
       [0193]    Peptides that bound to at least 4 different HLA subtypes as described above, and were shown to be immunogenic, as shown in  FIGS. 1-4 , were selected for inclusion in a multi-epitope vaccine. Table III shows the percent of patients that demonstrated positive responses and the binding patterns to specific HLA subtypes of eight vaccine candidates. 
         [0194]    These example and equivalents thereof will become more apparent to those skilled in the art in light of the present disclosure and the accompanying claims. It should be understood, however, that the examples are designed for the purpose of illustration only and not limiting of the scope of the invention in any way. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 HLA-DR binding affinity of Breast/Ovarian-derived peptides 
               
             
          
           
               
                   
                   
                   
                   
                 Posi- 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB3 
                 DRB4 
                 DRB5 
               
               
                 Peptide 
                 Sequence 
                 Source 
                 Protein 
                 tion 
                 *0101 
                 *0301 
                 *0401 
                 *0404 
                 *0403 
                 *0701 
                 *0802 
                 *0901 
                 *1101 
                 *1301 
                 *1302 
                 *1301 
                 *0101 
                 *0101 
                 *0101 
               
               
                   
               
             
          
           
               
                 9019.0104 
                 RWCIPWQRILLTASL 
                 CEA.10 
                 CEA 
                 10 
                 1467 
                 7860 
                 426 
                 193 
                 221 
                 107 
                 263 
                 4132 
                 191 
                 1558 
                 2326 
                 61 
                 -- 
                 339 
                 4014 
               
               
                   
               
               
                 9019.0105 
                 LLTFWNPPTTAKETI 
                 CEA.24 
                 CEA 
                 24 
                 69 
                 16,313 
                 273 
                 52 
                 258 
                 3.7 
                 174 
                 5779 
                 52 
                 2995 
                 245 
                 46 
                 -- 
                 2171 
                 31 
               
               
                   
               
               
                 9019.0106 
                 TAKLTIESTPFNVAE 
                 CEA.33 
                 CEA 
                 33 
                 72 
                 613 
                 106 
                 41 
                 383 
                 70 
                 1736 
                 4019 
                 5977 
                 2307 
                 35 
                 140 
                 -- 
                 53 
                 3350 
               
               
                   
               
               
                 9019.0107 
                 EVLLLVHNLPQHLFG 
                 CEA.59 
                 CEA 
                 50 
                 27 
                 830 
                 3.4 
                 1.7 
                 30 
                 5.4 
                 59 
                 989 
                 40 
                 54 
                 0.36 
                 4.7 
                 334 
                 50 
                 10888 
               
               
                   
               
               
                 9019.0108 
                 YSWYKGERVDGNRQI 
                 CE3.65 
                 CEA 
                 65 
                 511 
                 -- 
                 34 
                 585 
                 360 
                 866 
                 8432 
                 - 
                 1840 
                 -- 
                 533 
                 306 
                 3902 
                 453 
                 1043 
               
               
                   
               
               
                 9919.0109 
                 NRQIIGYVIGTQQAT 
                 CEA.76 
                 CEA 
                 76 
                 216 
                 -- 
                 108 
                 1.5 
                 129 
                 46 
                 46 
                 345 
                 36 
                 4351 
                 990 
                 2.6 
                 -- 
                 52 
                 2230 
               
               
                   
               
               
                 9019.0110 
                 GYVIGTQQATPGPAY 
                 CEA.81 
                 CEA 
                 81 
                 21 
                   
                 31 
                 34 
                 4210 
                 1705 
                   
                   
                 5877 
                   
                 43 
                 836 
                   
                 63 
                 10,223 
               
               
                   
               
               
                 9019.0111 
                 GPAYSGREIIYPNAS 
                 CEA.92 
                 CEA 
                 92 
                 9435 
                   
                 12,989 
                   
                   
                 5262 
                   
                   
                   
                   
                 154 
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0112 
                 GREIIYPNASLLIQN 
                 CEA.97 
                 CEA 
                 97 
                 62 
                 433 
                 251 
                 88 
                 550 
                 29 
                 1950 
                 1355 
                 2203 
                 212 
                 24 
                 49 
                 4035 
                 43 
                 10,612 
               
               
                   
               
               
                 9019.0113 
                 DTGFYTLHVIKSDLV 
                 CEA.116 
                 CEA 
                 116 
                 64 
                 984 
                 84 
                 260 
                 95 
                 23 
                 90 
                 23 
                 174 
                 14 
                 1972 
                 65 
                 14,943 
                 15 
                 564 
               
               
                   
               
               
                 9019.0114 
                 FYTLHVIKDDLVNEE 
                 CEA.119 
                 CEA 
                 119 
                 101 
                 20 
                 184 
                 169 
                 41 
                 56 
                 514 
                 718 
                 6385 
                 616 
                 1340 
                 14 
                 14,343 
                 22 
                 4501 
               
               
                   
               
               
                 9019.0115 
                 LHVIKSDLVNEEATG 
                 CEA.122 
                 CEA 
                 122 
                 891 
                 46 
                 214 
                 193 
                 37 
                 3393 
                 -- 
                 -- 
                 18,399 
                 -- 
                 394 
                 376 
                 -- 
                 111 
                 -- 
               
               
                   
               
               
                 9012.0115 
                 KSOLVNEEATGQFRV 
                 CE4.129 
                 CEA 
                 126 
                 13,600 
                 2530 
                 236 
                   
                   
                 6338 
                   
                   
                   
                   
                 9963 
                   
                   
                   
                   
               
               
                   
               
               
                 9013.0116 
                 QFRVYPELPKPSISS 
                 CEA.137 
                 CEA 
                 137 
                 1790 
                 1727 
                 2916 
                   
                   
                 7976 
                   
                   
                   
                   
                 786 
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0117 
                 KPSISSNNSKPVEDK 
                 CEA.143 
                 CEA 
                 146 
                 405 
                   
                 919 
                 2111 
                 6959 
                 407 
                   
                   
                 -- 
                   
                 23 
                 97 
                   
                 5977 
                 -- 
               
               
                   
               
               
                 9019.0118 
                 YLWWVNNQSLPVSPR 
                 CEA.176 
                 CEA 
                 176 
                 24 
                 100 
                 832 
                 203 
                 50 
                 17 
                 119 
                 1912 
                 22 
                 1511 
                 22 
                 116 
                 248 
                 555 
                 928 
               
               
                   
               
               
                 9019.0119 
                 SDSVILNVLYGPDAP 
                 CEA.123 
                 CEA 
                 226 
                 111 
                   
                 255 
                 314 
                 1153 
                 5236 
                   
                   
                 9210 
                   
                 83 
                 351 
                   
                 612 
                 -- 
               
               
                   
               
               
                 9019.0120 
                 LNVLYGPDAPTISPL 
                 CEA.231 
                 CEA 
                 231 
                 331 
                   
                 649 
                 1378 
                 -- 
                 410 
                   
                   
                 -- 
                   
                 218 
                 583 
                   
                 10,798 
                 -- 
               
               
                   
               
               
                 9919.0121 
                 APTISPLNTSYRSGE 
                 CEA.239 
                 CEA 
                 239 
                 2431 
                   
                 295 
                 40 
                 5994 
                 10,607 
                   
                   
                 3943 
                   
                 125 
                 1795 
                   
                 1047 
                 51 
               
               
                   
               
               
                 9019.0122 
                 QYSWFVNGTFQQSTQ 
                 CEA.265 
                 CEA 
                 268 
                 5303 
                   
                 21 
                 7830 
                 342 
                 216 
                   
                   
                 230 
                   
                 14,768 
                 -- 
                   
                 11,448 
                 2661 
               
               
                   
               
               
                 9019.0123 
                 QELFIPNITVNNSGS 
                 CEA.292 
                 CEA 
                 282 
                 147 
                 644 
                 25 
                 227 
                 379 
                 1659 
                 293 
                 1096 
                 358 
                 1299 
                 26 
                 740 
                 -- 
                 38130 
                 3869 
               
               
                   
               
               
                 9019.0124 
                 RTTVTTIIVYAEPPK 
                 CEA.310 
                 CEA 
                 310 
                 4115 
                   
                 259 
                 697 
                 32 
                 649 
                   
                   
                 -- 
                   
                 2977 
                 83 
                   
                 6287 
                 14,732 
               
               
                   
               
               
                 9019.0125 
                 TITVYAEPPKPFITS 
                 CEA.315 
                 CEA 
                 315 
                 12,755 
                 539 
                 -- 
                 12,652 
                 5704 
                 8230 
                   
                   
                 -- 
                   
                 2322 
                 62 
                   
                 -- 
                 9255 
               
               
                   
               
               
                 9019.0126 
                 YLWWVNNQSLPVSPR 
                 CEA.374 
                 CEA 
                 354 
                 1123 
                 234 
                 12 
                 248 
                 38 
                 25 
                 243 
                 299 
                 33 
                 1121 
                 1921 
                 227 
                 1083 
                 061 
                 2284 
               
               
                   
               
               
                 9019.0127 
                 SDPVILNVLYGPDDP 
                 CEA.404 
                 CEA 
                 404 
                 384 
                   
                 592 
                 347 
                 3732 
                 2245 
                   
                   
                 5432 
                   
                 161 
                 5999 
                   
                 9262 
                 -- 
               
               
                   
               
               
                 9019.0128 
                 SYTYYRPGVNLSLSC 
                 CEA.423 
                 CEA 
                 423 
                 1.6 
                 4125 
                 6.8 
                 1039 
                 309 
                 5.4 
                 21 
                 1372 
                 776 
                 571 
                 7.2 
                 46 
                 2626 
                 1744 
                 135 
               
               
                   
               
               
                 9015.0129 
                 YSWLIDGNIQQHTQE 
                 CEA.447 
                 CEA 
                 447 
                 1407 
                   
                 95 
                   
                   
                 9427 
                   
                   
                   
                   
                 1312 
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0130 
                 NSGLYTCQANNSASG 
                 CEA.471 
                 CEA 
                 471 
                 49 
                   
                 33 
                 34 
                 96 
                 8139 
                   
                   
                 7338 
                   
                 1924 
                 14,891 
                   
                 150 
                 4717 
               
               
                   
               
               
                 8019.0131 
                 RTTVKTITVSAELPK 
                 CEA.428 
                 CEA 
                 488 
                 89 
                 1267 
                 58 
                 54 
                 11 
                 4.2 
                 3763 
                 367 
                 6938 
                 2755 
                 992 
                 29 
                 -- 
                 1263 
                 1917 
               
               
                   
               
               
                 9919.0132 
                 TITVSAELPKPSISS 
                 CEA.493 
                 CEA 
                 493 
                 223 
                 74 
                 9393 
                 4937 
                 3624 
                 29 
                 -- 
                 6671 
                 -- 
                 -- 
                 1076 
                 1672 
                 -- 
                 10,349 
                 3856 
               
               
                   
               
               
                 9019.0133 
                 KPSISSNNSKPVEDK 
                 CEA.502 
                 CEA 
                 502 
                 146 
                   
                 403 
                 1404 
                 5564 
                 121 
                   
                   
                 -- 
                   
                 4.1 
                 138 
                   
                 -- 
                 -- 
               
               
                   
               
               
                 9019.0134 
                 YLWWVNGQSLPVSPR 
                 CEA.532 
                 CEA 
                 532 
                 1.4 
                   
                 2.5 
                 1356 
                 158 
                 12 
                   
                   
                 3135 
                   
                 314 
                 182 
                   
                 4295 
                 1348 
               
               
                   
               
               
                 9012.0135 
                 VCGIQNSVSANRSDP 
                 CEA.570 
                 CEA 
                 570 
                 44 
                   
                 72 
                 29 
                 1554 
                 1095 
                   
                   
                 -- 
                   
                 921 
                 8979 
                   
                 1051 
                 36 
               
               
                   
               
               
                 9019.0139 
                 QNSVSANRSDPVTLD 
                 CEA.574 
                 CEA 
                 574 
                 240 
                   
                 511 
                 1432 
                 -- 
                 274 
                   
                   
                 -- 
                   
                 2.7 
                 6657 
                   
                 2105 
                 5816 
               
               
                   
               
               
                 9012.0137 
                 SSYLSGANLNLSCHS 
                 CEA.603 
                 CEA 
                 603 
                 40 
                   
                 580 
                 5594 
                 7822 
                 455 
                   
                   
                 -- 
                   
                 37 
                 13,028 
                   
                 42 
                 14,095 
               
               
                   
               
               
                 9019.0138 
                 QYSWRINGIPQQHTQ 
                 CEA.624 
                 CEA 
                 624 
                 472 
                   
                 43 
                 1103 
                 311 
                 11,900 
                   
                   
                 151 
                   
                 70 
                 2074 
                   
                 2286 
                 1121 
               
               
                   
               
               
                 9019.0139 
                 INGIPQQHTQVLFIA 
                 CEA.629 
                 CEA 
                 629 
                 682 
                   
                 181 
                 609 
                 942 
                 31 
                   
                   
                 -- 
                   
                 256 
                 8287 
                   
                 2700 
                 -- 
               
               
                   
               
               
                 9019.0140 
                 NGTYACFVSNLATGR 
                 CEA.650 
                 CEA 
                 650 
                 839 
                 818 
                 11 
                 558 
                 30 
                 29 
                 70 
                 50 
                 2174 
                 2952 
                 30 
                 2355 
                 6963 
                 3247 
                 37 
               
               
                   
               
               
                 9919.0141 
                 YACFVSNLATGRNNS 
                 CEA.653 
                 CEA 
                 653 
                 183 
                 774 
                 225 
                 41 
                 327 
                 531 
                 1774 
                 4520 
                 217 
                 2399 
                 107 
                 1237 
                 -- 
                 1569 
                 17 
               
               
                   
               
               
                 9019.0142 
                 NNSIVKSITVSASGT 
                 CEA.665 
                 CEA 
                 665 
                 34 
                 103 
                 43 
                 1.8 
                 126 
                 34 
                 164 
                 469 
                 265 
                 1328 
                 4.4 
                 274 
                 15,808 
                 26 
                 2280 
               
               
                   
               
               
                 9919.0143 
                 STTVSASGTSPGLSA 
                 CEA.671 
                 CEA 
                 671 
                 2807 
                   
                 75 
                 11 
                 3574 
                 1559 
                   
                   
                 3319 
                   
                 592 
                 3110 
                   
                 174 
                 15,069 
               
               
                   
               
               
                 9019.0144 
                 SPGLSAGATVGIMIG 
                 CEA.680 
                 CEA 
                 680 
                 3507 
                   
                 4191 
                 2009 
                 2389 
                 92 
                   
                   
                 -- 
                   
                 80 
                 3929 
                   
                 2591 
                 -- 
               
               
                   
               
               
                 9019.05 
                 TVGIMIGVLVGVALI 
                 CEA.688 
                 CEA 
                 688 
                 384 
                   
                 -- 
                 -- 
                 -- 
                 4454 
                   
                   
                 -- 
                   
                 5637 
                 7770 
                   
                 12,094 
                 384 
               
               
                   
               
               
                 9019.0009 
                 HQLLCQEVETIRRAY 
                 Cyclin D1.3 
                 Cyclin  
                 3 
                 953 
                 21 
                 746 
                 256 
                 4200 
                 11,553 
                 16,297 
                 5607 
                 3471 
                 5585 
                 349 
                 325 
                 -- 
                 60 
                 2016 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0146 
                 DANLLNDRVLRAMLK 
                 Cyclin D1.19 
                 Cyclin  
                 19 
                 1463 
                   
                 2342 
                   
                   
                 -- 
                   
                   
                   
                   
                 286 
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0147 
                 NDRVLRAMLKAEETC 
                 Cyclin D1.24 
                 Cyclin  
                 24 
                 1171 
                   
                 1963 
                   
                   
                 12,160 
                   
                   
                   
                   
                 319 
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0148 
                 RAMLKAFETCAPSVS 
                 Cyclin D1.29 
                 Cyclin  
                 29 
                 407 
                 -- 
                 360 
                 375 
                 12,517 
                 7793 
                 - 
                 5017 
                 9145 
                 -- 
                 16,129 
                 15,733 
                 -- 
                 313 
                 1341 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0149 
                 FKCVQKEVLPSMRKI 
                 Cyclin D1.45 
                 Cyclin  
                 45 
                 4099 
                   
                 3915 
                   
                   
                 10,059 
                   
                   
                   
                   
                 3587 
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0012 
                 QKEVLPSMRKIVATW 
                 Cyclin D1.49 
                 Cyclin  
                 49 
                 9025 
                 111 
                 12,182 
                 1102 
                 3720 
                 481 
                 1155 
                 1008 
                 50 
                 1121 
                 700 
                 338 
                 -- 
                 108 
                 2019 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0150 
                 LPSMRKIVATWMLEV 
                 Cyclin D1.53 
                 Cyc1in  
                 53 
                 8.5 
                 826 
                 238 
                 28 
                 123 
                 4.6 
                 1482 
                 137 
                 886 
                 145 
                 8.5 
                 7.1 
                 8449 
                 50 
                 47 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0151 
                 MRKIVATWMLEVCEE 
                 Cyclin D1.56 
                 Cyclin  
                 56 
                 764 
                   
                 7953 
                 -- 
                 1982 
                 249 
                   
                   
                 -- 
                   
                 356 
                 87 
                   
                 281 
                 17,021 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0011 
                 MLEVCEEQKGEEEVF 
                 Cyclin D1.64 
                 Cyclin  
                 64 
                   
                 -- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0152 
                 EEFVFPLAMNYLDRF 
                 Cyclin D1.74 
                 Cyclin  
                 74 
                 850 
                   
                 2247 
                 3070 
                 2228 
                 1597 
                   
                   
                 3414 
                   
                 26 
                 77 
                   
                 72 
                 4444 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0153 
                 VFPLAMNYLDRFLSL 
                 Cyclin D1.77 
                 Cyclin  
                 77 
                 146 
                 107 
                 2332 
                 3567 
                 609 
                 3332 
                 259 
                 19,713 
                 27 
                 79 
                 9.4 
                 59 
                 289 
                 2.6 
                 2005 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0007 
                 DRFLSLEPVKKSRLQ 
                 Cyclin D1.86 
                 Cyclin  
                 86 
                 16 
                 290 
                 10 
                 61 
                 159 
                 1057 
                 37 
                 18,378 
                 46 
                 4128 
                 -- 
                 394 
                 -- 
                 359 
                 3.0 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0154 
                 DRFLSLEPVKKSRLQ 
                 Cyc3in D1.86 
                 Cyclin  
                 86 
                 11 
                   
                 63 
                 49 
                 113 
                 1834 
                   
                   
                 51 
                   
                 235 
                 1071 
                   
                 227 
                 2.6 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0155 
                 LEPVKKSRDQLLGAT 
                 Cyclin D1.91 
                 Cyclin  
                 91 
                 102 
                   
                 9112 
                 696 
                 13,609 
                 2152 
                   
                   
                 1248 
                   
                 221 
                 208 
                   
                 102 
                 731 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0156 
                 RDQLLGATCMFVASK 
                 Cyclin D1.95 
                 Cyclin  
                 98 
                 12 
                 -- 
                 91 
                 633 
                 1439 
                 468 
                 3144 
                 -- 
                 831 
                 513 
                 227 
                 277 
                 -- 
                 421 
                 504 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0157 
                 TCMFVASKMKETIPL 
                 Cyclin D1.105 
                 Cyclin  
                 105 
                 202 
                   
                 342 
                 5454 
                 1744 
                 17 
                   
                   
                 61 
                   
                 1031 
                 1443 
                   
                 2423 
                 36 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0158 
                 ASKMKETIPLTAEKL 
                 Cyclin D1.110 
                 Cyclin  
                 110 
                 130 
                   
                 -- 
                 1992 
                 -- 
                 220 
                   
                   
                 321 
                   
                 530 
                 1294 
                   
                 2461 
                 2364 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1622.01 
                 TIPLTAEKLCLYTDN 
                 Cyclin D1.116 
                 Cyclin  
                 116 
                 253 
                 11,708 
                 1230 
                 -- 
                 -- 
                 -- 
                 -- 
                 -- 
                 -- 
                 -- 
                 -- 
                 5360 
                 -- 
                 738 
                 253 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0159 
                 KLCIYTDNSIRPEEL 
                 Cyclin D1.123 
                 Cyclin  
                 123 
                 1915 
                 64 
                 90 
                 241 
                 3472 
                 1105 
                 16,263 
                 15,928 
                 -- 
                 -- 
                 879 
                 25 
                 -- 
                 33 
                 4620 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0009 
                 DNSIRPEELLQMELL 
                 Cyclin D1.129 
                 Cyclin  
                 129 
                 4554 
                 147 
                 4677 
                 2803 
                 16,500 
                 6648 
                   
                   
                 -- 
                   
                 3494 
                 4757 
                   
                 87 
                 18,258 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0160 
                 DNSIRPEELLQMELL 
                 Cyclin D1.129 
                 Cyclin  
                 129 
                 1533 
                   
                 1345 
                   
                   
                 3440 
                   
                   
                   
                   
                 939 
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0161 
                 PEELLQMELLLVNKL 
                 Cyclin D1.134 
                 Cyclin  
                 134 
                 1274 
                   
                 1027 
                 318 
                 603 
                 401 
                   
                   
                 1094 
                   
                 44 
                 1449 
                   
                 11 
                 2802 
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0040 
                 EELLQMELLLVNKLK 
                 Cyclin D1.135 
                 Cyclin  
                 135 
                   
                 3386 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
           
               
                 1622.06 
                 LLQMELLLVNKLKWN 
                 Cyclin D1.137 
                 Cyclin  
                 137 
                 39 
                 -- 
                 3939 
                 6.4 
                 332 
                 2196 
                 761 
                 6097 
                 197 
                 1325 
                 44 
                 29 
                 -- 
                 321 
                 39 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0163 
                 MELLLVNKLKWNLAA 
                 Cyclin D1.140 
                 Cyclin  
                 140 
                 46 
                 9009 
                 177 
                 481 
                 2411 
                 1797 
                 360 
                 3090 
                 41 
                 102 
                 4.9 
                 210 
                 -- 
                 39 
                 19 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0164 
                 VNKLKWNLAAMTPHD 
                 Cyclin D1.145 
                 Cyclin  
                 145 
                 49 
                 1419 
                 06 
                 781 
                 747 
                 382 
                 702 
                 449 
                 1043 
                 210 
                 3.0 
                 359 
                 5897 
                 1039 
                 2519 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0165 
                 KWNLAAMTPHDFIEH 
                 Cyclin D1.149 
                 Cyclin  
                 149 
                 33 
                 6240 
                 3405 
                 653 
                 122 
                 1101 
                 15,625 
                 4876 
                 11,907 
                 194 
                 332 
                 550 
                 -- 
                 5.6 
                 41 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0013 
                 WNLAAMTPHDFIEHF 
                 Cyclin D1.150 
                 Cyclin  
                 150 
                 6425 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0166 
                 PHDFIEHFLSKMPEA 
                 Cyclin D1.157 
                 Cyc1in  
                 157 
                 1246 
                 1299 
                   
                   
                 1160 
                   
                   
                   
                   
                   
                 820 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0167 
                 NKQIIRKHAQTFVAL 
                 Cyclin D1.174 
                 Cyc1in  
                 174 
                 4.7 
                 561 
                 6.5 
                 23 
                 4.4 
                 105 
                 239 
                 34 
                 34 
                 3.9 
                 2.0 
                 3.3 
                 342 
                 8.7 
                 23 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0168 
                 AQTFVALCATDYKFI 
                 Cyclin D1.182 
                 Cyclin  
                 182 
                 8.4 
                 551 
                 26 
                 153 
                 55 
                 15 
                 445 
                 33o 
                 276 
                 132 
                 219 
                 97 
                 -- 
                 46 
                 18 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0169 
                 VKFISNPPSMVAAGS 
                 Cyclin D1.193 
                 Cyclin  
                 193 
                 6.7 
                 4126 
                 12 
                 00 
                 117 
                 304 
                 319 
                 712 
                 88 
                 21 
                 24 
                 84 
                 5290 
                 290 
                 826 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0170 
                 PPSMVAAGSVVAAVQ 
                 Cyclin D1.109 
                 Cyclin  
                 199 
                 6.8 
                 -- 
                 12 
                 26 
                 1718 
                 133 
                 1401 
                 301 
                 466 
                 10,090 
                 55 
                 26 
                 -- 
                 91 
                 957 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0171 
                 VAAVQGLNLRSPNNF 
                 Cyclin D1.209 
                 Cyclin  
                 209 
                 44 
                 18,558 
                 226 
                 532 
                 4248 
                 161 
                 10,850 
                 -- 
                 11,089 
                 2018 
                 171 
                 1205 
                 -- 
                 296 
                 3541 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0172 
                 IEALLESSLRQAQQN 
                 Cyclin D1.251 
                 Cyclin  
                 251 
                 2608 
                   
                 18 
                   
                   
                 -- 
                   
                   
                   
                   
                 12,776 
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0014 
                 EALLESSLRQAQQNM 
                 Cyclin D1.252 
                 Cyclin  
                 252 
                 8212 
                 151 
                 605 
                 1019 
                 12,905 
                 -- 
                   
                   
                 756 
                   
                 4755 
                 8176 
                   
                 4028 
                 -- 
                   
               
               
                   
                   
                   
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0024 
                 LAALCRWGLLLALLP 
                 Her/neu.3 
                 Her.neu 
                 3 
                 2044 
                   
                 5394 
                   
                   
                 7995 
                   
                   
                   
                   
                 704 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0025 
                 WGLLLALLPPGAAST 
                 Her/neu.9 
                 Her.neu 
                 9 
                 1687 
                   
                 750 
                 0.93 
                 219 
                 16,366 
                   
                   
                 12 
                   
                 521 
                 21 
                   
                 33 
                 337 
                   
               
               
                   
               
               
                 1622.02 
                 LLALLPPGAASTQVC 
                 Her/neu.12 
                 Her.neu 
                 12 
                 17 
                   
                 5148 
                 118 
                 -- 
                 7183 
                   
                   
                 944 
                   
                 10,697 
                 17,445 
                   
                 19,806 
                 17 
                   
               
               
                   
               
               
                 9019.0027 
                 GTDMKLRLPASPETH 
                 Her/neu.26 
                 Her.neu 
                 28 
                 2117 
                   
                 1693 
                   
                   
                 6014 
                   
                   
                   
                   
                 14,523 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0028 
                 RHLYQGCQVVQGNLE 
                 Her/neu.47 
                 Her.neu 
                 47 
                 1435 
                   
                 422 
                   
                   
                 4940 
                   
                   
                   
                   
                 5043 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0029 
                 GCQVVQGNLELTYLP 
                 Her/neu.52 
                 Her.neu 
                 52 
                 2600 
                   
                 2739 
                   
                   
                 1972 
                   
                   
                   
                   
                 417 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0030 
                 NLELTYLPTNASLSF 
                 Her/neu.59 
                 Her.neu 
                 59 
                 49 
                 7356 
                 6.2 
                 2.7 
                 38 
                 72 
                 94 
                 3055 
                 30 
                 148 
                 105 
                 23 
                 -- 
                 29 
                 189 
                   
               
               
                   
               
               
                 9019.0031 
                 LTYLPTNASLSELQD 
                 Herneu.62 
                 Her.neu 
                 62 
                 9.7 
                 3364 
                 19 
                 26 
                 80 
                 15 
                 426 
                 4081 
                 213 
                 150 
                 47 
                 132 
                 141 
                 1633 
                 173 
                   
               
               
                   
               
               
                 9019.0032 
                 IQEVQGTVLLAHNQV 
                 Her/neu.77 
                 Her.neu 
                 77 
                 57 
                 7763 
                 111 
                 178 
                 102 
                 35 
                 213 
                 302 
                 165 
                 3438 
                 103 
                 75 
                 13,508 
                 546 
                 1361 
                   
               
               
                   
               
               
                 9019.0033 
                 YVLIAHNQVRQVPLQ 
                 Her/neu.83 
                 Her.neu 
                 83 
                 28 
                 454 
                 53 
                 104 
                 1185 
                 62 
                 303 
                 350 
                 200 
                 302 
                 1.9 
                 679 
                 649 
                 124 
                 18 
                   
               
               
                   
               
               
                 9019.0034 
                 HNQVRQVPLQRLRIV 
                 Her/neu.88 
                 Her.neu 
                 88 
                 950 
                 971 
                 840 
                 78 
                 1303 
                 80 
                 09 
                 6941 
                 21 
                 42 
                 270 
                 340 
                 -- 
                 18 
                 173 
                   
               
               
                   
               
               
                 9019.9035 
                 VRQVPLQRIRIVRGT 
                 Her2/neu.91 
                 Her2/neu 
                 91 
                 3065 
                   
                 1798 
                   
                   
                 218 
                   
                   
                   
                   
                 2546 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0001 
                 GTQLFEDNYALAVLD 
                 Her2/neu.104 
                 Her2/neu 
                 104 
                 210 
                 29 
                 640 
                 0923 
                 14,921 
                 129 
                   
                   
                 9195 
                   
                 4.0 
                 450 
                   
                 6955 
                 3744 
                   
               
               
                   
               
               
                 9019.0636 
                 GDPLNNTTPVTGASP 
                 Her2/neu.120 
                 Her2/neu 
                 120 
                 6356 
                   
                 7461 
                   
                   
                 13,172 
                   
                   
                   
                   
                 1030 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0037 
                 TTPVTGASPGGLREL 
                 Her2/neu.126 
                 Her2/neu 
                 126 
                 992 
                   
                 2447 
                 675 
                 13,198 
                 1843 
                   
                   
                 11,554 
                   
                 6055 
                 17,143 
                   
                 2514 
                 3001 
                   
               
               
                   
               
               
                 9019.0030 
                 PGGLRELQLRSLTEI 
                 Her2/neu.134 
                 Her2/neu 
                 134 
                 732 
                   
                 3397 
                 564 
                 110 
                 1541 
                   
                   
                 2207 
                   
                 786 
                 4405 
                   
                 2245 
                 16,273 
                   
               
               
                   
               
               
                 9010.0039 
                 TEILKGGVLIQRNPQ 
                 Her2/neu.146 
                 Her2/neu 
                 146 
                 11 
                   
                 40 
                 32 
                 760 
                 2486 
                   
                   
                 692 
                   
                 343 
                 1183 
                   
                 511 
                 1878 
                   
               
               
                   
               
               
                 9019.0646 
                 GGVLIQRNPQLCYQD 
                 Her2/neu.151 
                 Her2/neu 
                 151 
                 142 
                   
                 196 
                 93 
                 1845 
                 14,279 
                   
                   
                 1720 
                   
                 34 
                 106 
                   
                 351 
                 13,612 
                   
               
               
                   
               
               
                 9019.0041 
                 NPQLCYQDTILWKDI 
                 Her2/neu.158 
                 Her2/neu 
                 158 
                 3653 
                   
                 368 
                   
                   
                 -- 
                   
                   
                   
                   
                 5074 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0042 
                 ICELHCPALVTYNTD 
                 Her2/neu.263 
                 Her2/neu 
                 263 
                 101 
                   
                 754 
                 136 
                 1627 
                 1324 
                   
                   
                 521 
                   
                 3216 
                 748 
                   
                 441 
                 2809 
                   
               
               
                   
               
               
                 9019.0043 
                 STDVGSCTLVCPLHN 
                 Her2/neu.305 
                 Her2/neu 
                 305 
                 2872 
                   
                 -- 
                   
                   
                 2139 
                   
                   
                   
                   
                 6781 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0044 
                 CYGLGMEHLREVRAV 
                 Her2/neu.342 
                 Her2/neu 
                 342 
                 130 
                 265 
                 1027 
                 493 
                 5122 
                 271 
                 -- 
                 -- 
                 3064 
                 18,979 
                 25 
                 690 
                 -- 
                 444 
                 3776 
                   
               
               
                   
               
               
                 9019.0045 
                 MEHLREVRAVTASNI 
                 Her2/neu.347 
                 Her2/neu 
                 347 
                 9.6 
                 2970 
                 533 
                 12 
                 200 
                 -- 
                 95 
                 4345 
                 262 
                 221 
                 23 
                 86 
                 -- 
                 81 
                 216 
                   
               
               
                   
               
               
                 9019.0046 
                 LREVRAVTSANIQEF 
                 Her2/neu.350 
                 Her2/neu 
                 350 
                 17 
                 3913 
                 43 
                 1.2 
                 50 
                 12 
                 456 
                 5110 
                 661 
                 101 
                 1.5 
                 27 
                 -- 
                 163 
                 94 
                   
               
               
                   
               
               
                 9019.0047 
                 CKKIFGSLAFLPESF 
                 Her2/neu.367 
                 Her2/neu 
                 367 
                 119 
                   
                 121 
                 171 
                 310 
                 45 
                   
                   
                 3080 
                   
                 206 
                 635 
                   
                 316 
                 4567 
                   
               
               
                   
               
               
                 9019.0048 
                 PESFDGDPASNTAPL 
                 Her2/neu.378 
                 Her2/neu 
                 378 
                 10,101 
                 989 
                 301 
                 9020 
                 1724 
                 2823 
                   
                   
                 12,591 
                   
                 3905 
                 805 
                   
                 654 
                 -- 
                   
               
               
                   
               
               
                 9019.0049 
                 TAPLQPEQLQVFETL 
                 Her2/neu.389 
                 Her2/neu 
                 389 
                 1112 
                 3674 
                 2633 
                   
                   
                 6994 
                   
                   
                   
                   
                 2322 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0050 
                 ITGYLYISAWPDSLP 
                 Her2/neu.406 
                 Her2/neu 
                 406 
                 96 
                   
                 243 
                 1771 
                 8.5 
                 136 
                   
                   
                 2573 
                   
                 751 
                 152 
                   
                 270 
                 67 
                   
               
               
                   
               
               
                 9019.0051 
                 PDSLPDLSVFQNLQV 
                 Her2/neu.410 
                 Her2/neu 
                 410 
                 -- 
                   
                 -- 
                 -- 
                   
                   
                   
                   
                   
                   
                 -- 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0052 
                 LSVFQNLQVIRGRIL 
                 Her2/neu.422 
                 Her2/neu 
                 422 
                 1.3 
                 345 
                 6.3 
                 33 
                 26 
                 7.1 
                 146 
                 859 
                 9.6 
                 486 
                 80 
                 33 
                 -- 
                 67 
                 17 
                   
               
               
                   
               
               
                 9019.0053 
                 NLQVIRGRILHNGAY 
                 Her2/neu.427 
                 Her2/neu 
                 427 
                 1.9 
                 6979 
                 971 
                 200 
                 3394 
                 12 
                 119 
                 4217 
                 173 
                 47 
                 9.9 
                 3.3 
                 4320 
                 446 
                 28 
                   
               
               
                   
               
               
                 9019.0054 
                 RGRILHNGAYSLTLQ 
                 Her2/neu.432 
                 Her2/neu 
                 432 
                 2.4 
                 710 
                 480 
                 129 
                 2845 
                 5.6 
                 5077 
                 480 
                 773 
                 40 
                 1.3 
                 54 
                 350 
                 562 
                 82 
                   
               
               
                   
               
               
                 9019.0055 
                 SLTLQGLGISWLGLR 
                 Her2/neu.442 
                 Her2/neu 
                 442 
                 93 
                   
                 143 
                 110 
                 409 
                 3096 
                   
                   
                 6307 
                   
                 1597 
                 50 
                   
                 22 
                 -- 
                   
               
               
                   
               
               
                 9019.0056 
                 GLGISWLGLRSLREL 
                 Her2/neu.447 
                 Her2/neu 
                 447 
                 59 
                 122 
                 41 
                 163 
                 3.4 
                 55 
                 1538 
                 1140 
                 2268 
                 436 
                 3030 
                 5.2 
                 -- 
                 154 
                 2048 
                   
               
               
                   
               
               
                 9019.0057 
                 LRSLRELGSGLALIH 
                 Her2/neu.459 
                 Her2/neu 
                 459 
                 7.1 
                 -- 
                 896 
                 14 
                 603 
                 142 
                 1075 
                 594 
                 309 
                 498 
                 16 
                 24 
                 16,142 
                 549 
                 726 
                   
               
               
                   
               
               
                 9019.0058 
                 GLALIHHNTHLCFVH 
                 Her2/neu.464 
                 Her2/neu 
                 464 
                 465 
                   
                 434 
                 171 
                 477 
                 277 
                   
                   
                 2822 
                   
                 18 
                 164 
                   
                 357 
                 6081 
                   
               
               
                   
               
               
                 9019.0059 
                 NTHLCFVHTVPWDQL 
                 Her2/neu.471 
                 Her2/neu 
                 471 
                 416 
                   
                 796 
                 207 
                 116 
                 178 
                   
                   
                 793 
                   
                 175 
                 1431 
                   
                 117 
                 7390 
                   
               
               
                   
               
               
                 9019.0060 
                 DECVGEGLACHQLCA 
                 Her2/neu.502 
                 Her2/neu 
                 502 
                 459 
                   
                 1065 
                 3405 
                 29 37 
                 2007 
                   
                   
                 4976 
                   
                 323 
                 6267 
                   
                 1117 
                 12,277 
                   
               
               
                   
               
               
                 9019.0061 
                 GHCWGPGPTQCVNCS 
                 Her2/neu.518 
                 Her2/neu 
                 518 
                 1913 
                   
                 3740 
                   
                   
                 6742 
                   
                   
                   
                   
                 2912 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0062 
                 SQFKRGQECVEECRV 
                 Her2/neu.532 
                 Her2/neu 
                 532 
                 462 
                   
                 1020 
                 4549 
                 -- 
                 540 
                   
                   
                 16,562 
                   
                 146 
                 285 
                   
                 1523 
                 7043 
                   
               
               
                   
               
               
                 9019.0063 
                 ECRVLQGLPREYVNA 
                 Her2/neu.543 
                 Her2/neu 
                 543 
                 262 
                   
                 706 
                 0351 
                 812 
                 4001 
                   
                   
                 254 
                   
                 548 
                 635 
                   
                 2072 
                 2011 
                   
               
               
                   
               
               
                 9019.0064 
                 LQGLPREYVNARHCL 
                 Her2/neu.547 
                 Her2/neu 
                 547 
                 354 
                 8995 
                 201 
                 1444 
                 603 
                 1903 
                   
                   
                 405 
                   
                 670 
                 423 
                   
                 2551 
                 49 
                   
               
               
                   
               
             
          
           
               
                 9019.0065 
                 PSGVKPDLSYMPIWK 
                 Her2/neu.601 
                 Her2/neu 
                 601 
                 1032 
                 1570 
                 1525 
                   
                   
                 2688 
                   
                   
                   
                   
                   
                   
                   
                 1531 
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0066 
                 ASPLTSIISAVVGIL 
                 Her2/neu.648 
                 Her2/neu 
                 648 
                 15 
                 10,905 
                 20 
                 36 
                 712 
                 24 
                 3089 
                 118 
                 511 
                 1431 
                 14 
                 59 
                 18,926 
                 1500 
                 206 
                   
                   
                   
               
               
                   
               
               
                 9019.0067 
                 ISAVVGILLVVVLGV 
                 Her2/neu.655 
                 Her2/neu 
                 655 
                 5153 
                   
                 420 
                 2996 
                 447 
                 941 
                   
                   
                 5188 
                   
                 443 
                 6005 
                   
                 3109 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0068 
                 ILLVVVLGVVFGILI 
                 Her2/neu.661 
                 Her2/neu 
                 661 
                 -- 
                   
                 635 
                 3532 
                 376 
                 780 
                   
                   
                 2464 
                   
                 478 
                 7117 
                   
                 11,015 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0969 
                 VLGVVFGILIKRRQQ 
                 Her2/neu.666 
                 Her2/neu 
                 666 
                 67 
                 2449 
                 177 
                 335 
                 104 
                 17 
                 35 
                 -- 
                 12 
                 268 
                 17 
                 185 
                 -- 
                 958 
                 38 
                   
                   
                   
               
               
                   
               
               
                 9019.0070 
                 QQKIRKYTMRRLLQE 
                 Her2/neu.679 
                 Her2/neu 
                 679 
                 303 
                   
                 3782 
                 5396 
                 -- 
                 44 
                   
                   
                 40 
                   
                 44 
                 2.0 
                   
                 93 
                 300 
                   
                   
                   
               
               
                   
               
               
                 9019.0071 
                 IRKYTMRRLLQETEL 
                 Her2/neu.682 
                 Her2/neu 
                 682 
                 665 
                   
                 2766 
                 2305 
                 1050 
                 722 
                   
                   
                 46 
                   
                 2513 
                 1.6 
                   
                 303 
                 2207 
                   
                   
                   
               
               
                   
               
               
                 9019.0072 
                 MRILKETELRKVKVL 
                 Her2/neu.712 
                 Her2/neu 
                 712 
                 266 
                   
                 3193 
                 1266 
                 8030 
                 370 
                   
                   
                 2103 
                   
                 1318 
                 329 
                   
                 368 
                 1223 
                   
                   
                   
               
               
                   
               
               
                 9019.0073 
                 ETELRKVKVLGSGAF 
                 Her2/neu.717 
                 Her2/neu 
                 717 
                 214 
                 15,518 
                 246 
                 27 
                 145 
                 101 
                 139 
                 5423 
                 176 
                 2472 
                 980 
                 205 
                 -- 
                 452 
                 1020 
                   
                   
                   
               
               
                   
               
               
                 9019.0074 
                 KVKVLGSGAFGTVYK 
                 Her2/neu.722 
                 Her2/neu 
                 722 
                 64 
                   
                 810 
                 204 
                 10,552 
                 77 
                   
                   
                 1711 
                   
                 1308 
                 244 
                   
                 1380 
                 1369 
                   
                   
                   
               
               
                   
               
               
                 9019.0075 
                 GENVKIPVAIKVLRE 
                 Her2/neu.743 
                 Her2/neu 
                 743 
                 491 
                   
                 1065 
                 488 
                 2093 
                 180 
                   
                   
                 4535 
                   
                 1149 
                 729 
                   
                 34 
                 8353 
                   
                   
                   
               
               
                   
               
               
                 1622.03 
                 KIPVAIKVLRENTSP 
                 Her2/neu.747 
                 Her2/neu 
                 747 
                 1505 
                   
                 3856 
                 125 
                 1178 
                 -- 
                   
                   
                 4108 
                   
                 501 
                 611 
                   
                 7805 
                 1505 
                   
                   
                   
               
               
                   
               
               
                 9019.0077 
                 AYVMAGVGSPYVSRL 
                 Her2/neu.771 
                 Her2/neu 
                 771  
                 92 
                   
                 164 
                 171 
                 596 
                 45 
                   
                   
                 1738 
                   
                 402 
                 24 
                   
                 5060 
                 308 
                   
                   
                   
               
               
                   
               
               
                 9019.0078 
                 MAGVGSPYVSRLLGH 
                 Her2/neu.774 
                 Her2/neu 
                 774 
                 2050 
                   
                 1651 
                   
                   
                 352 
                   
                   
                   
                   
                 1508 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0079 
                 SRLLGICLTSTVQLV 
                 Her2/neu.783 
                 Her2/neu 
                 783 
                 80 
                 2923 
                 85 
                 13 
                 90 
                 9.0 
                 634 
                 137 
                 80 
                 446 
                 4.7 
                 39 
                 3567 
                 481 
                 392 
                   
                   
                   
               
               
                   
               
               
                 9019.0080 
                 TVQLVTQLMPYGCLL 
                 Her2/neu.793 
                 Her2/neu 
                 793 
                 164 
                   
                 215 
                 433 
                 4326 
                 1288 
                   
                   
                 l1,126 
                   
                 3594 
                 94 
                   
                 16 
                 3002 
                   
                   
                   
               
               
                   
               
               
                 9019.0081 
                 RGRLGSQDLLNWCMQ 
                 Her2/neu.214 
                 Her2/neu 
                 214 
                 1039 
                   
                 1412 
                   
                   
                 2029 
                   
                   
                   
                   
                 3367 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0082 
                 LLNWCMQIAKGMSYL 
                 Her2/neu.822 
                 Her2/neu 
                 822 
                 944 
                   
                 558 
                 195 
                 1094 
                 380 
                   
                   
                 159 
                   
                 2082 
                 799 
                   
                 33459 
                 92 
                   
                   
                   
               
               
                   
               
               
                 9019.0083 
                 CMQLAKGMSYLEDVR 
                 Her2/neu.826 
                 Her2/neu 
                 826 
                 959 
                   
                 2651 
                 867 
                 1040 
                 116 
                   
                   
                 405 
                   
                 2232 
                 1002 
                   
                 3005 
                 130 
                   
                   
                   
               
               
                   
               
               
                 9019.0002 
                 GMSYLEDVRLVHRDL 
                 Her2/neu.932 
                 Her2/neu 
                 832 
                 123 
                 27 
                 957 
                 1357 
                 4315 
                 5408 
                   
                   
                 244 
                   
                 3789 
                 567 
                   
                 2543 
                 467 
                   
                   
                   
               
               
                   
               
               
                 9019.0084 
                 VRLVHRDLAARNVLV 
                 Her2/neu.839 
                 Her2/neu 
                 839 
                 59 
                 1503 
                 105 
                 245 
                 561 
                 356 
                   
                   
                 16 
                   
                 57 
                 1097 
                   
                 298 
                 2425 
                   
                   
                   
               
               
                   
               
               
                 9019.0085 
                 HRDLAARNVLVKSPN 
                 Her2/neu.843 
                 Her2/neu 
                 843 
                 158 
                   
                 401 
                 765 
                 11,210 
                 1332 
                   
                   
                 1648 
                   
                 116 
                 221 
                   
                 525 
                 302 
                   
                   
                   
               
               
                   
               
               
                 9019.0086 
                 ARNVLVKSPNGVKIT 
                 Her2/neu.848 
                 Her2/neu 
                 848 
                 65 
                 1275 
                 209 
                 197 
                 11,536 
                 118 
                 717 
                 4771 
                 513 
                 628 
                 15 
                 29 
                 16,142 
                 742 
                 4.9 
                   
                   
                   
               
               
                   
               
               
                 9019.0087 
                 PIKWVALESILRRRF 
                 Her2/neu.885 
                 Her2/neu 
                 885 
                 12 
                 30 
                 14 
                 250 
                 161 
                 664 
                 312 
                 3620 
                 133 
                 66 
                 349 
                 3.3 
                 -- 
                 62 
                 3.4 
                   
                   
                   
               
               
                   
               
               
                 9019.0003 
                 IKWMALESILRRRFT 
                 Her2/neu.886 
                 Her2/neu 
                 886 
                 16 
                 10 
                 37 
                 1075 
                 435 
                 1795 
                 515 
                 9282 
                 136 
                 241 
                 1118 
                 11 
                 -- 
                 340 
                 3.3 
                   
                   
                   
               
               
                   
               
               
                 9019.0088 
                 ESILRRRFTHQSDVW 
                 Her2/neu.892 
                 Her2/neu 
                 892 
                 1525 
                   
                 794 
                 7977 
                 2375 
                 9598 
                   
                   
                 1159 
                   
                 16,279 
                 2726 
                   
                 818 
                 5305 
                   
                   
                   
               
               
                   
               
               
                 9019.0089 
                 SDVWSYGVTVWELMT 
                 Her2/neu.903 
                 Her2/neu 
                 903 
                 165 
                   
                 2760 
                 3621 
                 1900 
                 546 
                   
                   
                 2639 
                   
                 467 
                 1879 
                   
                 -- 
                 5423 
                   
                   
                   
               
               
                   
               
               
                 9019.0090 
                 GVTVWELMTFGAKPY 
                 Her2/neu.909 
                 Her2/neu 
                 909 
                 40 
                   
                 377 
                 4.1 
                 2107 
                 558 
                   
                   
                 315 
                   
                 8860 
                 20 
                   
                 1171 
                 34 
                   
                   
                   
               
               
                   
               
               
                 9019.0091 
                 VWELMTFGAKPYDGI 
                 Her2/neu.912 
                 Her2/neu 
                 912 
                 36 
                   
                 676 
                 144 
                 4704 
                 191 
                   
                   
                 1952 
                   
                 7553 
                 245 
                   
                 3565 
                 65 
                   
                   
                   
               
               
                   
               
               
                 9019.0092 
                 AKPYDGIPAREIPDL 
                 Her2/neu.920 
                 Her2/neu 
                 920 
                 136 
                   
                 -- 
                 -- 
                 12,548 
                 41 
                   
                   
                 - 
                   
                 19,575 
                 6957 
                   
                 -- 
                 3044 
                   
                   
                   
               
               
                   
               
               
                 1622.04 
                 ICTIDVYMIMVKCWM 
                 Her2/neu.946 
                 Her2/neu 
                 946 
                 -- 
                   
                 717 
                 -- 
                 955 
                 -- 
                   
                   
                 1480 
                   
                 -- 
                 2652 
                   
                 -- 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0094 
                 DVYMIMVKCWMIDSE 
                 Her2/neu.950 
                 Her2/neu 
                 950 
                 1312 
                   
                 189 
                 7807 
                 274 
                 297 
                   
                   
                 179 
                   
                 395 
                 1303 
                   
                 697 
                 716 
                   
                   
                   
               
               
                   
               
               
                 9019.0095 
                 RPRFRELVSEFSRMA 
                 Her2/neu.966 
                 Her2/neu 
                 966 
                 26 
                 6218 
                 38 
                 62 
                 151 
                 309 
                 376 
                 2321 
                 125 
                 1779 
                 12,182 
                 348 
                 -- 
                 351 
                 26 
                   
                   
                   
               
               
                   
               
               
                 9019.0096 
                 FRELVSEFSRMARDP 
                 Her2/neu.969 
                 Her2/neu 
                 969 
                 20 
                 150 
                 22 
                 51 
                 1497 
                 5055 
                 1714 
                 -- 
                 381 
                 -- 
                 -- 
                 124 
                 4537 
                 123.2 
                 380 
                   
                   
                   
               
               
                   
               
               
                 9019.0004 
                 FSRMARDPQRFVVIQ 
                 Her2/neu.976 
                 Her2/neu 
                 976 
                 29 
                 35 
                 512 
                 2224 
                 555 
                 1422 
                 798 
                 1481 
                 49 
                 6867 
                 240 
                 1408 
                 901 
                 227 
                 45 
                   
                   
                   
               
               
                   
               
               
                 9019.0097 
                 DGDLGMGAAKGLQSL 
                 Her2/neu.1087 
                 Her2/neu 
                 1087 
                 110 
                   
                 254 
                 506 
                 5799 
                 828 
                   
                   
                 2233 
                   
                 3224 
                 1684 
                   
                 896 
                 2141 
                   
                   
                   
               
               
                   
               
               
                 9019.0098 
                 AKGLQSLPTHDPSPL 
                 Her2/neu.1095 
                 Her2/neu 
                 1095 
                 149 
                   
                 194 
                 19 
                 3864 
                 4459 
                   
                   
                 6806 
                   
                 3991 
                 1665 
                   
                 572 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0099 
                 LQRYSEDPTVPLPSE 
                 Her2/neu.1109 
                 Her2/neu 
                 1109 
                 1367 
                 18 
                 25 
                 13,089 
                 226 
                 107 
                   
                   
                 94 
                   
                 280 
                 5310 
                   
                 -- 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0100 
                 PEYVNQPDVRPQPPS 
                 Her2/neu.1137 
                 Her2/neu 
                 1137 
                 6165 
                   
                 150 
                   
                   
                 -- 
                   
                   
                   
                   
                 1714 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0101 
                 EGPLPAARPAGETLE 
                 Her2/neu.1154 
                 Her2/neu 
                 1154 
                 17,288 
                   
                 -- 
                   
                   
                 7463 
                   
                   
                   
                   
                 10,247 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0102 
                 GATLERPKTLSPGKN 
                 Her2/neu.1164 
                 Her2/neu 
                 1164 
                 1812 
                   
                 2792 
                   
                   
                 -- 
                   
                   
                   
                   
                 5332 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9919.0103 
                 KDVFAFGGAVENPEY 
                 Her2/neu.1182 
                 Her2/neu 
                 1182 
                 1505 
                   
                 -- 
                   
                   
                 1577 
                   
                   
                   
                   
                 16,146 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9919.0173 
                 LPRVGCPALPLPPPP 
                 IGFBP2.2 
                 IGFBP2 
                 2 
                 1906 
                   
                 6543 
                   
                   
                 -- 
                   
                   
                   
                   
                 8936 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0174 
                 ALLPLPPPPLLPLLP 
                 IGFBP2.9 
                 IGFBP2 
                 9 
                 174 
                 -- 
                 18 
                 4.2 
                 20 
                 336 
                 1087 
                 -- 
                 41 
                 1337 
                 1524 
                 262 
                 -- 
                 40 
                 9665 
                   
                   
                   
               
               
                   
               
               
                 9019.0175 
                 PPPLLPLLPLLLLLL 
                 IGFBP2.14 
                 IGFBP2 
                 14 
                 15 
                 5816 
                 15 
                 16 
                 65 
                 13 
                 86 
                 307 
                 121 
                 23 
                 1322 
                 5.0 
                 16,239 
                 2.1 
                 121 
                   
                   
                   
               
               
                   
               
               
                 9019.0176 
                 LLPLLPLLLLLLGAS 
                 IGFBP2.17 
                 IGFBP2 
                 17 
                 119 
                 -- 
                 337 
                 35 
                 674 
                 964 
                 213 
                 5893 
                 403 
                 320 
                 2922 
                 182 
                 -- 
                 19 
                 2390 
                   
                   
                   
               
               
                   
               
               
                 9019.0177 
                 PLLLLLLGASGGGGG 
                 IGFBP2.22 
                 IGFBP2 
                 22 
                 20 
                 18,033 
                 339 
                 2144 
                 1422 
                 5882 
                 5696 
                 -- 
                 12,818 
                 18,255 
                 2164 
                 253 
                 -- 
                 316 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0178 
                 AEVLPRCPPCTPERL 
                 IGFBP2.39 
                 IGFBP2 
                 39 
                 1285 
                   
                 1611 
                   
                   
                 15,970 
                   
                   
                   
                   
                 5774 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0179 
                 PERLAACGPPPVAPP 
                 IGFBP2.50 
                 IGFBP2 
                 50 
                 28 
                   
                 5545 
                 163 
                 4353 
                 -- 
                   
                   
                 3541 
                   
                 3618 
                 4054 
                   
                 4455 
                 19,685 
                   
                   
                   
               
               
                   
               
               
                 9019.0180 
                 PPPVAPPAAVAAVAG 
                 IGFBP2.58 
                 IGFBP2 
                 58 
                 178 
                 -- 
                 380 
                 81 
                 -- 
                 2684 
                 -- 
                 317 
                 -- 
                 -- 
                 237 
                 1378 
                 -- 
                 1888 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0181 
                 GARMPGAELVREPGC 
                 IGFBP2.73 
                 IGFBP2 
                 73 
                 164 
                   
                 11,638 
                 13,072 
                 -- 
                 -- 
                   
                   
                 2968 
                   
                 16,279 
                 -- 
                   
                 4697 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0015 
                 CAELVREPGCGCCSV 
                 IGFBP2.78 
                 IGFBP2 
                 78 
                   
                 12,298 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0016 
                 CARLEGEACGVYTPR 
                 IGFBP2.93 
                 IGFBP2 
                 93 
                   
                 -- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0182 
                 ELPLQALVMGEGTCE 
                 IGFBP2.121 
                 IGFBP2 
                 121 
                 1502 
                   
                 6782 
                   
                   
                 15,638 
                   
                   
                   
                   
                 9167 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0017 
                 QALVMGEGTCEKRRD 
                 IGFBP2.125 
                 IGFBP2 
                 125 
                   
                 2240 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0183 
                 SEGGLVENHVDSTMN 
                 IGFBP2.157 
                 IGFBP2 
                 157 
                 1153 
                   
                 1196 
                   
                   
                 4623 
                   
                   
                   
                   
                 2518 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0184 
                 TMNMLGGGGSAGRKP 
                 IGFBP2.169 
                 IGFBP2 
                 169 
                 1021 
                   
                 791 
                   
                   
                 6177 
                   
                   
                   
                   
                 5686 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0185 
                 LKSGMKELAVFREKV 
                 IGFBP2.154 
                 IGFBP2 
                 154 
                 607 
                 -- 
                 881 
                 862 
                 34 
                 -- 
                 260 
                 -- 
                 163 
                 1768 
                 4974 
                 91 
                 -- 
                 417 
                 843 
                   
                   
                   
               
               
                   
               
               
                 9019.0186 
                 KELAVFREKVTEQHR 
                 IGFBP2.189 
                 IGFBP2 
                 189 
                 2045 
                   
                   
                 26 
                   
                   
                 -- 
                   
                   
                   
                   
                 -- 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0018 
                 ELAVFREKVIEQHRQ 
                 IGFBP2.190 
                 IGFBP2 
                 190 
                   
                 8621 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0019 
                 REKVTEQHRQMGKGG 
                 IGFBP2.195 
                 IGFBP2 
                 195 
                   
                 2839 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0187 
                 GKHHLGLEEPKKLRP 
                 IGFBP2.209 
                 IGFBP2 
                 209 
                 238 
                   
                 1654 
                 2756 
                 6494 
                 3697 
                   
                   
                 16,749 
                   
                 3029 
                 3345 
                   
                 1378 
                 6390 
                   
                   
                   
               
               
                   
               
               
                 9019.0020 
                 KHHLGLEEPKKLRPP 
                 IGFBP2.210 
                 IGFBP2 
                 210 
                   
                 4016 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0188 
                 EPKKLRPPPARIPCQ 
                 IGFBP2.217 
                 IGFBP2 
                 217 
                 1258 
                   
                 806 
                   
                   
                 1122 
                   
                   
                   
                   
                 1213 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0189 
                 LDQVLERISTMRLPD 
                 IGFBP2.234 
                 IGFBP2 
                 234 
                 795 
                 452 
                 25 
                 20 
                 18 
                 5.2 
                 1607 
                 52 
                 467 
                 324 
                 1052 
                 84 
                 -- 
                 367 
                 223 
                   
                   
                   
               
               
                   
               
               
                 9019.0021 
                 IMRLPDERGPLEHLV 
                 IGFBP2.243 
                 IGFBP2 
                 243 
                   
                 -- 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0190 
                 ERGPLEHLYSLHIPN 
                 IGFBP2.249 
                 IGFBP2 
                 249 
                 7.7 
                 -- 
                 497 
                 29 
                 110 
                 18 
                 9361 
                 638 
                 1993 
                 50 
                 1149 
                 23 
                 -- 
                 1648 
                 4000 
                   
                   
                   
               
               
                   
               
               
                 9019.0191 
                 GLYNLKQCKMSLNGQ 
                 IGFBP2.268 
                 IGFBP2 
                 269 
                 923 
                 -- 
                 743 
                 2835 
                 5589 
                 1791 
                 204 
                 9434 
                 2600 
                 413 
                 561 
                 223 
                 -- 
                 1613 
                 5609 
                   
                   
                   
               
               
                   
               
               
                 9019.0192 
                 TGKLIQGAPTIRGDP 
                 IGFBP2.293 
                 IGFBP2 
                 293 
                 148 
                 9554 
                 40 
                 27 
                 608 
                 683 
                 5160 
                 700 
                 7989 
                 2691 
                 927 
                 1733 
                 2191 
                 465 
                 36 
                   
                   
                   
               
               
                   
               
               
                 9019.0022 
                 APTIRGDPECHLFYN 
                 IGFBP2.300 
                 IGFBP2 
                 300 
                 4031 
                 31 
                 1643 
                 2908 
                 -- 
                 16,779 
                 -- 
                 -- 
                 -- 
                 16,917 
                 1674 
                 3669 
                 191 
                 2510 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 9019.0023 
                 CHLFYNEQQEARGVH 
                 IGFBP2.309 
                 IGFBP2 
                 309 
                 2490 
                 162 
                 1600 
                 3187 
                 -- 
                 -- 
                 -- 
                 -- 
                 -- 
                 1855 
                 146 
                 -- 
                 18,902 
                 5310 
                 -- 
                   
                   
                   
               
               
                   
               
               
                 -- indicates binding affinity ≧ 20,000nM 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 Breast/Ovarian HLA-DR Supertype Candidates 
               
             
          
           
               
                   
                   
                   
                   
                   
                 IC 50  μM to purified HLA 
               
             
          
           
               
                 Peptide 
                   
                   
                 Pro- 
                 Posi- 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB3 
                 DRB4 
                 DRB5 
                 Total 
               
               
                 No. 
                 Sequence 
                 Source 
                 tein 
                 tion 
                 *0101 
                 *0301 
                 *0401 
                 *0404 
                 *0405 
                 *0701 
                 *0802 
                 *0901 
                 *1101 
                 *1201 
                 *1202 
                 *1501 
                 * 0101 
                 *0101 
                 *0101 
                 XRN 
               
               
                   
               
             
          
           
               
                 9019.0105 
                 LLTFWNPPTTAKLTI 
                 CEA.24 
                 CEA 
                 24 
                 6.9 
                 16,313 
                 273 
                 52 
                 258 
                 3.7 
                 174 
                 5779 
                 52 
                 4995 
                 245 
                 46 
                 -- 
                 2171 
                 13 
                 10 
               
               
                   
               
               
                 9019.0106 
                 TAKLTIESTPENVAE 
                 CEA.33 
                 CEA 
                 33 
                 72 
                 613 
                 106 
                 41 
                 383 
                 70 
                 1736 
                 4019 
                 5997 
                 2907 
                 35 
                 140 
                 -- 
                 53 
                 3550 
                 6 
               
               
                   
               
               
                 9019.0107 
                 EVLLLVHNLPQHLFG 
                 CEA.50 
                 CEA 
                 50 
                 2.7 
                 830 
                 3.4 
                 1.7 
                 30 
                 5.4 
                 59 
                 989 
                 4. 
                 5.4 
                 0.36 
                 4.7 
                 334 
                 50 
                 1088 
                 14 
               
               
                   
               
               
                 9019.0108 
                 YSWYKGERVDGNRQI 
                 CEA.65 
                 CEA 
                 65 
                 511 
                 -- 
                 34 
                 585 
                 360 
                 866 
                 8432 
                 -- 
                 1840 
                 -- 
                 533 
                 306 
                 3002 
                 453 
                 1043 
                 8 
               
               
                   
               
               
                 9019.0109 
                 NRQIIGYVIGTQQAT 
                 CEA.76 
                 CEA 
                 76 
                 216 
                 -- 
                 108 
                 1.5 
                 29 
                 46 
                 46 
                 345 
                 36 
                 4351 
                 990 
                 2.6 
                 -- 
                 5.2 
                 2230 
                 11 
               
               
                   
               
               
                 9019.0112 
                 GREIIYPNASLLIQN 
                 CEA.97 
                 CEA 
                 97 
                 62 
                 433 
                 251 
                 88 
                 550 
                 29 
                 1959 
                 1355 
                 2209 
                 212 
                 24 
                 49 
                 4035 
                 43 
                 10,612 
                 10 
               
               
                   
               
               
                 9019.0113 
                 DTGFYTLHVIKSDLV 
                 CEA.116 
                 CEA 
                 116 
                 64 
                 984 
                 84 
                 260 
                 95 
                 23 
                 90 
                 83 
                 174 
                 174 
                 1072 
                 65 
                 14,943 
                 18 
                 564 
                 13 
               
               
                   
               
               
                 9019.0114 
                 FYTLHVIKSDLVNEE 
                 CEA.119 
                 CEA 
                 119 
                 101 
                 80 
                 184 
                 169 
                 41 
                 56 
                 514 
                 718 
                 6385 
                 616 
                 1340 
                 14 
                 14,343 
                 22 
                 4501 
                 11 
               
               
                   
               
               
                 9019.0118 
                 YLWWVNNQSLPVSPR 
                 CEA.176 
                 CEA 
                 176 
                 2.4 
                 100 
                 832 
                 203 
                 80 
                 17 
                 119 
                 1912 
                 22 
                 1511 
                 22 
                 116 
                 248 
                 555 
                 923 
                 13 
               
               
                   
               
               
                 9019.0123 
                 QELFIPNITVNNSGS 
                 CEA.282 
                 CEA 
                 282 
                 147 
                 644 
                 25 
                 227 
                 379 
                 1658 
                 293 
                 1086 
                 358 
                 1299 
                 26 
                 740 
                 -- 
                 3880 
                 3869 
                 9 
               
               
                   
               
               
                 9019.0126 
                 YLWWVNNQSLPVSPR 
                 CEA.354 
                 CEA 
                 354 
                 1123 
                 234 
                 12 
                 248 
                 88 
                 28 
                 243 
                 299 
                 33 
                 2121 
                 1921 
                 227 
                 1088 
                 861 
                 2284 
                 10 
               
               
                   
               
               
                 9019.0128 
                 SYTYYRPGVNLSLSC 
                 CEA.423 
                 CEA 
                 423 
                 1.6 
                 4425 
                 68 
                 4036 
                 300 
                 5.4 
                 21 
                 1372 
                 776 
                 371 
                 7.2 
                 46 
                 2626 
                 1784 
                 135 
                 10 
               
               
                   
               
               
                 9019.0131 
                 RTTVKTITVSAELPK 
                 CEA.488 
                 CEA 
                 488 
                 99 
                   
                 58 
                 54 
                 11 
                 4.2 
                   
                   
                 6988.0 
                   
                 962 
                 29 
                   
                 1263 
                 1917 
                 7 
               
               
                   
               
               
                 9019.0140 
                 NGTYACFVSNLATGR 
                 CEA.650 
                 CEA 
                 650 
                 839 
                 818 
                 11 
                 558 
                 30 
                 20 
                 70 
                 377 
                 2174 
                 2052 
                 30 
                 2351 
                 6963 
                 3247 
                 37 
                 10 
               
               
                   
               
               
                 9019.0141 
                 TACFVSNLATGRNNS 
                 CEA.653 
                 CEA 
                 653 
                 183 
                 774 
                 225 
                 41 
                 327 
                 531 
                 1774 
                 4520 
                 217 
                 2399 
                 107 
                 1237 
                 -- 
                 1569 
                 17 
                 9 
               
               
                   
               
               
                 9019.0142 
                 NNSIVKSITVSASGT 
                 CEA.665 
                 CEA 
                 665 
                 34 
                 103 
                 43 
                 1.8 
                 128 
                 34 
                 184 
                 469 
                 209 
                 1328 
                 4.4 
                 274 
                 15,808 
                 26 
                 2280 
                 12 
               
               
                   
               
               
                 9019.0006 
                 HQLLCCEVETIRRAY 
                 Cyclin  
                 Cyclin  
                 3 
                 953 
                 21 
                 746 
                 256 
                 4200 
                 11,553 
                 16,297 
                 5607 
                 3471 
                 5585 
                 349 
                 325 
                 -- 
                 60 
                 2016 
                 7 
               
               
                   
                   
                 D1.3 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0012 
                 QKEVLPSMRKIVATW 
                 Cyclin  
                 Cyclin  
                 49 
                 9825 
                 111 
                 12,182 
                 1102 
                 3720 
                 481 
                 1155 
                 1088 
                 58 
                 1121 
                 700 
                 228 
                 -- 
                 108 
                 2019 
                 6 
               
               
                   
                   
                 D1.49 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0150 
                 LPSMRKIVATWMLEV 
                 Cyclin  
                 Cyclin  
                 53 
                 8.5 
                 826 
                 238 
                 28 
                 123 
                 4.6 
                 1182 
                 137 
                 886 
                 145 
                 8.5 
                 7.1 
                 8449 
                 50 
                 47 
                 13 
               
               
                   
                   
                 D1.53 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0153 
                 VFPLAMNYLDRFLSL 
                 Cyclin  
                 Cyclin  
                 77 
                 146 
                 107 
                 2352 
                 1567 
                 609 
                 3332 
                 259 
                 19,713 
                 27 
                 79 
                 5.4 
                 39 
                 239 
                 2.6 
                 2005 
                 10 
               
               
                   
                   
                 D1.77 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0007 
                 DRFLSLEPVKKSRLQ 
                 Cyclin  
                 Cyclin  
                 86 
                 16 
                 290 
                 18 
                 61 
                 159 
                 1057 
                 37 
                 18,378 
                 46 
                 4128 
                 -- 
                 394 
                 -- 
                 359 
                 3.0 
                 10 
               
               
                   
                   
                 D1.86 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0156 
                 RLQLLGATCMFVASK 
                 Cyclin  
                 Cyclin  
                 98 
                 12 
                 -- 
                 91 
                 633 
                 1439 
                 468 
                 3144 
                 -- 
                 831 
                 513 
                 227 
                 277 
                 -- 
                 421 
                 564 
                 10 
               
               
                   
                   
                 D1.98 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 1622.06 
                 LLQMELLLVNKLSWN 
                 Cyclin  
                 Cyclin  
                 137 
                 39 
                 -- 
                 3539 
                 6.4 
                 332 
                 2196 
                 765 
                 6097 
                 187 
                 1325 
                 44 
                 28 
                 -- 
                 321 
                 39 
                 9 
               
               
                   
                   
                 D1.137 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0163 
                 MELLLVNKLKWNLAA 
                 Cyclin  
                 Cyclin  
                 140 
                 46 
                 9006 
                 177 
                 491 
                 2411 
                 1979 
                 368 
                 3090 
                 48 
                 162 
                 4.9 
                 246 
                 -- 
                 39 
                 19 
                 10 
               
               
                   
                   
                 D1.140 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0164 
                 VNKLKWNLAAMTPHD 
                 Cyclin  
                 Cyclin  
                 145 
                 46 
                 1419 
                 88 
                 781 
                 747 
                 382 
                 702 
                 449 
                 1043 
                 116 
                 3.0 
                 356 
                 5867 
                 1839 
                 2519 
                 10 
               
               
                   
                   
                 D1.145 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0165 
                 KWNLAAMIPHDFIEH 
                 Cyclin  
                 Cyclin  
                 149 
                 33 
                 6249 
                 3405 
                 653 
                 122 
                 1100 
                 18,625 
                 4876 
                 11,907 
                 194 
                 332 
                 356 
                 -- 
                 576 
                 41 
                 8 
               
               
                   
                   
                 D1.149 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0167 
                 NKQIIRKHAQTFVAL 
                 Cyclin  
                 Cyclin  
                 174 
                 4.7 
                 561 
                 65 23 
                 25 
                 4.4 
                 195 
                 236 
                 34 
                 3.9 
                 2.0 
                 3.3 
                 342 
                 8.7 
                 23 
                 15 
                   
               
               
                   
                   
                 D1.174 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0168 
                 AQTFVALCATDVKFI 
                 Cyclin  
                 Cyclin  
                 182 
                 8.4 
                 551 
                 26 
                 133 
                 55 
                 13 
                 445 
                 536 
                 276 
                 132 
                 219 
                 97 
                 -- 
                 46 
                 18 
                 14 
               
               
                   
                   
                 D1.182 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0169 
                 VKFISNPPSMVAAGS 
                 Cyclin  
                 Cyclin  
                 193 
                 6.7 
                 4128 
                 12 
                 18 
                 117 
                 304 
                 319 
                 752 
                 88 
                 21 
                 24 
                 84 
                 5290 
                 290 
                 826 
                 13 
               
               
                   
                   
                 D1.193 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
           
               
                 9019.0170 
                 PPSMVAAGSVVAAVQ 
                 Cyclin  
                 Cyclin  
                 199 
                 6.8 
                 -- 
                 12 
                 26 
                 1718 
                 133 
                 1401 
                 301 
                 466 
                 10,090 
                 55 
                 26 
                 -- 
                 91 
                 957 
                 10 
                   
               
               
                   
                   
                 D1.199 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0171 
                 VAAVQGLNLRSPNNF 
                 Cyclin  
                 Cyclin  
                 209 
                 44 
                 18,558 
                 226 
                 532 
                 4248 
                 161 
                 10,850 
                 -- 
                 11,089 
                 2018 
                 171 
                 1205 
                 -- 
                 296 
                 3541 
                 6 
                   
               
               
                   
                   
                 D1.209 
                 D1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0030 
                 NLELTYLPTNASLSF 
                 Her2/ 
                 Her2/ 
                 59 
                 4.9 
                 7356 
                 6.2 
                 2.7 
                 38 
                 7.2 
                 94 
                 3055 
                 30 
                 141 
                 105 
                 23 
                 -- 
                 29 
                 189 
                 12 
                   
               
               
                   
                   
                 neu.59 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0031 
                 LTYLPTNASLSFLQD 
                 Her2/ 
                 Her2/ 
                 62 
                 9.3 
                 3364 
                 19 
                 16 
                 80 
                 15 
                 426 
                 1081 
                 213 
                 150 
                 47 
                 132 
                 141 
                 1633 
                 173 
                 12 
                   
               
               
                   
                   
                 neu.62 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0032 
                 IQEVQGYVLLAHNQV 
                 Her2/ 
                 Her2/ 
                 77 
                 57 
                 7763 
                 111 
                 178 
                 102 
                 35 
                 213 
                 302 
                 165 
                 3438 
                 103 
                 75 
                 13,508 
                 546 
                 1361 
                 11 
                   
               
               
                   
                   
                 neu.77 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0033 
                 TVLIAHNQVRQVPLQ 
                 Her2/ 
                 Her2/ 
                 83 
                 28 
                 454 
                 53 
                 104 
                 1185 
                 92 
                 300 
                 358 
                 208 
                 302 
                 1.9 
                 679 
                 349 
                 1234 
                 18 
                 14 
                   
               
               
                   
                   
                 neu.83 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0034 
                 HNQVRQVPLQRLRIV 
                 Her2/ 
                 Her2/ 
                 88 
                 950 
                 971 
                 840 
                 78 
                 1303 
                 80 
                 85 
                 6644 
                 21 
                 42 
                 270 
                 340 
                 -- 
                 18 
                 173 
                 12 
                   
               
               
                   
                   
                 neu.88 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0045 
                 MEHLREVRAVTSANI 
                 Her2/ 
                 Her2/ 
                 347 
                 9.6 
                 2970 
                 533 
                 12 
                 200 
                 9.7 
                 95 
                 4345 
                 262 
                 221 
                 23 
                 86 
                 -- 
                 81 
                 216 
                 12 
                   
               
               
                   
                   
                 neu.347 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0046 
                 LREVRAVTSANIQEF 
                 Her2/ 
                 Her2/ 
                 350 
                 17 
                 3913 
                 43 
                 8.2 
                 50 
                 12 
                 456 
                 5187 
                 661 
                 161 
                 1.5 
                 27 
                 -- 
                 163 
                 94 
                 12 
                   
               
               
                   
                   
                 neu.350 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0052 
                 LSVFQNLQVIRGRIL 
                 Her2/ 
                 Her2/ 
                 422 
                 1.3 
                 345 
                 6.3 
                 33 
                 26 
                 7.1 
                 1484 
                 859 
                 9.6 
                 486 
                 80 
                 3.3 
                 -- 
                 67 
                 17 
                 14 
                   
               
               
                   
                   
                 neu.422 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0054 
                 RGRILHNGAYSLTLQ 
                 Her2/ 
                 Her2/ 
                 432 
                 2.4 
                 710 
                 480 
                 129 
                 2845 
                 5.6 
                 5077 
                 430 
                 773 
                 40 
                 1.3 
                 5.4 
                 358 
                 562 
                 82 
                 13 
                   
               
               
                   
                   
                 neu.432 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0057 
                 LRSLRELGSGLALIH 
                 Her2/ 
                 Her2/ 
                 455 
                 7.1 
                 -- 
                 896 
                 14 
                 603 
                 142 
                 1075 
                 594 
                 309 
                 498 
                 16 
                 24 
                 16.142 
                 549 
                 726 
                 12 
                   
               
               
                   
                   
                 neu.455 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0069 
                 VLGVVFGILIKRRQQ 
                 Her2/ 
                 Her2/ 
                 666 
                 67 
                 2449 
                 177 
                 335 
                 101 
                 17 
                 35 
                 -- 
                 12 
                 268 
                 17 
                 185 
                 -- 
                 958 
                 38 
                 12 
                   
               
               
                   
                   
                 neu.666 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0079 
                 SRLLGICLTSTVQLV 
                 Her2/ 
                 Her2/ 
                 783 
                 80 
                 2923 
                 85 
                 13 
                 80 
                 9.0 
                 634 
                 137 
                 80 
                 446 
                 4.7 
                 39 
                 3567 
                 481 
                 392 
                 13 
                   
               
               
                   
                   
                 neu.783 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0087 
                 PIKWMALESHLRRRF 
                 Her2/ 
                 Her2/ 
                 885 
                 12 
                 30 
                 14 
                 250 
                 161 
                 664 
                 312 
                 3620 
                 133 
                 66 
                 349 
                 3.3 
                 -- 
                 62 
                 3.4 
                 13 
                   
               
               
                   
                   
                 neu.885 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0003 
                 IKWMALESILRRRFT 
                 Her2/ 
                 Her2/ 
                 886 
                 16 
                 10 
                 37 
                 1075 
                 435 
                 1795 
                 515 
                 9282 
                 136 
                 241 
                 1118 
                 11 
                 -- 
                 340 
                 3.3 
                 10 
                   
               
               
                   
                   
                 neu.886 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0004 
                 FRSMARDPQRFVVIQ 
                 Her2/ 
                 Her2/ 
                 976 
                 29 
                 35 
                 512 
                 2224 
                 855 
                 1423 
                 798 
                 1481 
                 49 
                 6867 
                 240 
                 1408 
                 901 
                 227 
                 45 
                 10 
                   
               
               
                   
                   
                 neu.976 
                 neu 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0174 
                 ALPLPPPPLLPLLPL 
                 IGFBP 
                 IGFBP2 
                 9 
                 174 
                 -- 
                 18 
                 4.2 
                 20 
                 336 
                 1087 
                 -- 
                 41 
                 1337 
                 1524 
                 262 
                 -- 
                 2.1 
                 9665 
                 8 
                   
               
               
                   
                   
                 2.9 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0175 
                 PPPLLPLLPLLLLLL 
                 IGFBP 
                 IGFBP2 
                 14 
                 15 
                 5816 
                 15 
                 16 
                 65 
                 13 
                 86 
                 307 
                 121 
                 23 
                 1322 
                 5.0 
                 16,239 
                 2.1 
                 121 
                 12 
                   
               
               
                   
                   
                 2.14 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0176 
                 LLPLLPLLLLLLGAS 
                 IGFBP 
                 IGFBP2 
                 17 
                 119 
                 -- 
                 337 
                 35 
                 674 
                 964 
                 213 
                 5893 
                 458 
                 320 
                 2022 
                 182 
                 -- 
                 19 
                 2390 
                 10 
                   
               
               
                   
                   
                 2.17 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0177 
                 PLLLLLLGASGGGGG 
                 IGFBP 
                 IGFBP2 
                 22 
                 20 
                 18,033 
                 339 
                 2144 
                 1422 
                 5882 
                 5696 
                 -- 
                 12,818 
                 18,255 
                 2164 
                 253 
                 -- 
                 316 
                 -- 
                 4 
                   
               
               
                   
                   
                 2.22 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0180 
                 PPPVAPPAAVAAVAG 
                 IGFBP 
                 IGFBP2 
                 58 
                 178 
                 -- 
                 380 
                 81 
                 -- 
                 2684 
                 -- 
                 317 
                 -- 
                 -- 
                 237 
                 1378 
                 -- 
                 1888 
                 -- 
                 5 
                   
               
               
                   
                   
                 2.58 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0185 
                 LKSGMKELAVFREKV 
                 IGFBP 
                 IGFBP2 
                 184 
                 607 
                 -- 
                 881 
                 862 
                 34 
                 -- 
                 260 
                 -- 
                 163 
                 1768 
                 4974 
                 91 
                 -- 
                 417 
                 843 
                 9 
                   
               
               
                   
                   
                 2.184 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0189 
                 LDQVLERISTMRLPD 
                 IGFBP 
                 IGFBP2 
                 234 
                 795 
                 452 
                 25 
                 20 
                 18 
                 5.2 
                 1607 
                 52 
                 467 
                 314 
                 1052 
                 84 
                 -- 
                 367 
                 223 
                 12 
                   
               
               
                   
                   
                 2.234 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0190 
                 ERGPLEHLYSLHIPN 
                 IGFBP 
                 IGFBP2 
                 249 
                 7.7 
                 -- 
                 497 
                 29 
                 110 
                 18 
                 9361 
                 631 
                 1993 
                 50 
                 1149 
                 23 
                 -- 
                 1648 
                 4000 
                 8 
                   
               
               
                   
                   
                 2.249 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0191 
                 GLYNLKQCKMSLNGQ 
                 IGFBP 
                 IGFBP2 
                 268 
                 923 
                 -- 
                 743 
                 2835 
                 5589 
                 1791 
                 204 
                 9434 
                 2600 
                 413 
                 561 
                 223 
                 -- 
                 1613 
                 5609 
                 6 
                   
               
               
                   
                   
                 2.268 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 9019.0192 
                 TGKLIQGAPTIRGDP 
                 IGFBP 
                 IGFBP2 
                 293 
                 148 
                 9554 
                 40 
                 27 
                 608 
                 883 
                 5160 
                 700 
                 7989 
                 2691 
                 927 
                 1733 
                 2191 
                 465 
                 36 
                 9 
                   
               
               
                   
                   
                 2.293 
               
               
                   
               
               
                 -- indicates binding affinity ≧ 20.000nM 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE III 
               
               
                   
               
               
                 Vaccine Candidates 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
               
               
                 EPITOPE 
                 % 
                 *0101 
                 *0301 
                 *0401 
                 *0404 
                 *0405 
                 *0701 
                 *0802 
                 *0901 
               
               
                   
               
               
                 HER2/NEU.59 
                 15 
                 X 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 HER2/NEU.885 
                 25 
                 X 
                 X 
                 X 
                 X 
                 X 
                   
                 X 
               
               
                 CEA.24 
                 17 
                 X 
                   
                 X 
                 X 
                 X 
                 X 
                 X 
               
               
                 CEA.653 
                 25 
                 X 
                   
                 X 
                 X 
                 X 
                   
                   
               
               
                 IGFBP2.17 
                 19 
                 X 
                   
                 X 
                 X 
                   
                   
                 X 
               
               
                 IGFBP2.249 
                 23 
                 X 
                   
                 X 
                 X 
                 X 
                 X 
               
               
                 CYCLIND1.53 
                 13 
                 X 
                   
                 X 
                 X 
                 X 
                 X 
                   
                 X 
               
               
                 CYCLIND1.199 
                 13 
                 X 
                   
                 X 
                 X 
                   
                 X 
                   
                 X 
               
               
                   
               
             
          
           
               
                   
                   
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB1 
                 DRB3 
                 DRB4 
                 DRB5 
               
               
                   
                 EPITOPE 
                 *1101 
                 *1201 
                 *1302 
                 *1501 
                 *0101 
                 *0101 
                 *0101 
               
               
                   
                   
               
               
                   
                 HER2/NEU.59 
                 X 
                 X 
                 X 
                 X 
                   
                 X 
                 X 
               
               
                   
                 HER2/NEU.885 
                 X 
                 X 
                 X 
                 X 
                   
                 X 
                 X 
               
               
                   
                 CEA.24 
                 X 
                   
                 X 
                 X 
                   
                   
                 X 
               
               
                   
                 CEA.653 
                 X 
                   
                 X 
                   
                   
                   
                 X 
               
               
                   
                 IGFBP2.17 
                 X 
                 X 
                   
                 X 
                   
                 X 
               
               
                   
                 IGFBP2.249 
                   
                 X 
                   
                 X 
               
               
                   
                 CYCLIND1.53 
                   
                 X 
                 X 
                 X 
                   
                 X 
                 X 
               
               
                   
                 CYCLIND1.199 
                 X 
                   
                 X 
                 X 
                   
                 X 
               
               
                   
                   
               
               
                   
                 % = Percent of patients with responses