Inducing cellular immune responses to her2/neu using peptide and nucleic acid compositions

I. BACKGROUND OF THE INVENTION

[0001] A growing body of evidence suggests that cytotoxic T lymphocytes (CTL) are important in the immune response to tumor cells. CTL recognize peptide epitopes in the context of HLA class I molecules that are expressed on the surface of almost all nucleated cells. Following intracellular processing of endogenously synthesized tumor antigens, antigen-derived peptide epitopes bind to class I HLA molecules in the endoplasmic reticulum, and the resulting complex is then transported to the cell surface. CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms, e.g., activation of lymphokines such as tumor necrosis factor-α (TNF-α) or interferon-γ (IFNγ) which enhance the immune response and facilitate the destruction of the tumor cell.

[0002] Tumor-specific helper T lymphocytes (HTLs) are also known to be important for maintaining effective antitumor immunity. Their role in antitumor immunity has been demonstrated in animal models in which these cells not only serve to provide help for induction of CTL and antibody responses, but also provide effector functions, which are mediated by direct cell contact and also by secretion of lymphokines (e.g., IFNγ and TNF-α).

[0003] A fundamental challenge in the development of an efficacious tumor vaccine is immune suppression or tolerance that can occur. There is therefore a need to establish vaccine embodiments that elicit immune responses of sufficient breadth and vigor to prevent progression and/or clear the tumor.

[0004] The epitope approach employed in the present invention represents a solution to this challenge, in that it allows the incorporation of various antibody, CTL and HTL epitopes, from discrete regions of a target tumor-associated antigen (TAA), and/or regions of other TAAs, in a single vaccine composition. Such a composition can simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population.

[0005] HER2/neu (or erbB-2) is a 185 kD transmembrane protein with tyrosine kinase activity that has a structure similar to the epidermal growth factor receptor (Coussens et al., Science 230:113-119, 1985; Bargmann et al., Nature 319:226-230, 1986; Yamamoto et al., Nature 319:230-234, 1986). Amplification of the Her2/neu gene and/or overexpression of the protein have been reported in many human adenocarcinomas of the breast, ovary, uterus, prostate, stomach, esophagus, pancreas, kidney, and lung (see, e.g., Slamon et al., Science 235:177-182, 1987 and Science 244:707-712, 1989; Borg et al., Cancer Res. 50:4332-4337, 1990; Lukes et al., Cancer 73:2380-2385, 1994; Kuhn et al., J. Urol. 150:1427-1433, 1993; Sadasivan et al., J. Urol. 150:126-131, 1993; Yonemura et al., Cancer Res. 51:1034-1038, 1991; Kameda et al., Cancer Res. 50:8002-8009, 1990; Houldsworth et al., Cancer Res. 50:6417-6422, 1990; Yamanaka et al., Human Path. 24:1127-1134, 1993; Weidner et al., Cancer Res. 50:4504-4509, 1990; Kem et al., Cancer Res. 50:5184-5187, 1990; and Rachwal et al., Br. J. Cancer 72:56-64, 1995). This widespread expression on cancer cells makes HER2/neu an important target for immunotherapy.

[0006] The information provided in this section is intended to disclose the presently understood state of the art as of the filing date of the present application. Information is included in this section which was generated subsequent to the priority date of this application. Accordingly, information in this section is not intended, in any way, to delineate the priority date for the invention.

II. SUMMARY OF THE INVENTION

[0007] This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards TAAs. More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of cancer.

[0008] Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccines, particularly when compared to the use of whole antigens in vaccine compositions. For example, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines. Such immunosuppressive epitopes may, e.g., correspond to immunodominant epitopes in whole antigens, which may be avoided by selecting peptide epitopes from non-dominant regions (see, e.g., Disis et al., J. Immunol. 156:3151-3158, 1996).

[0009] An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes (CTL and HTL), and further, to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.

[0010] Another major benefit of epitope-based immune-stimulating vaccines is their safety. The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated.

[0011] An epitope-based vaccine also provides the ability to direct and focus an immune response to multiple selected antigens from the same pathogen (a “pathogen” may be an infectious agent or a tumor-associated molecule). Thus, patient-by-patient variability in the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from the pathogen in a vaccine composition.

[0012] Furthermore, an epitope-based anti-tumor vaccine also provides the opportunity to combine epitopes derived from multiple tumor-associated molecules. This capability can therefore address the problem of tumor-to tumor variability that arises when developing a broadly targeted anti-tumor vaccine for a given tumor type and can also reduce the likelihood of tumor escape due to antigen loss. For example, a breast cancer tumor in one patient may express a target TAA that differs from a breast cancer tumor in another patient. Epitopes derived from multiple TAAs can be included in a polyepitopic vaccine that will target both breast cancer tumors.

[0013] One of the most formidable obstacles to the development of broadly efficacious epitope-based immunotherapeutics, however, has been the extreme polymorphism of HLA molecules. To date, effective non-genetically biased coverage of a population has been a task of considerable complexity; such coverage has required that epitopes be used that are specific for HLA molecules corresponding to each individual HLA allele. Impractically large numbers of epitopes would therefore have to be used in order to cover ethnically diverse populations. Thus, there has existed a need for peptide epitopes that are bound by multiple HLA antigen molecules 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.

[0014] Furthermore, as described herein in greater detail, a need has existed to modulate peptide binding properties, e.g., so that peptides that are able to bind to multiple HLA molecules do so with an affinity that will stimulate an immune response. Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor to prevent or clear an infection in a diverse segment of the population. Such a response can also target a broad array of epitopes. The technology disclosed herein provides for such favored immune responses.

[0015] In a preferred embodiment, epitopes for inclusion in vaccine compositions of the invention are selected by a process whereby protein sequences of known antigens are evaluated for the presence of motif or supermotif-bearing epitopes. Peptides corresponding to a motif- or supermotif-bearing epitope are then synthesized and tested for the ability to bind to the HLA molecule that recognizes the selected motif. Those peptides that bind at an intermediate or high affinity i.e., an IC50 (or a KD value) of 500 nM or less for HLA class I molecules or an IC50 of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response. Immunogenic peptide epitopes are selected for inclusion in vaccine compositions.

[0016] Supermotif-bearing peptides may additionally be tested for the ability to bind to multiple alleles within the HLA supertype family. Moreover, peptide epitopes may be analogued to modify binding affinity and/or the ability to bind to multiple alleles within an HLA supertype.

[0017] The invention also includes embodiments comprising methods for monitoring or evaluating an immune response to a TAA in a patient having a known HLA-type. Such methods comprise incubating a T lymphocyte sample from the patient with a peptide composition comprising a TAA epitope that has an amino acid sequence described in, for example, Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI which binds the product of at least one HLA allele present in the patient, and detecting for the presence of a T lymphocyte that binds to the peptide. A CTL peptide epitope can, for example, be used as a component of a tetrameric complex for this type of analysis.

[0018] An alternative modality for defining the peptide epitopes in accordance with the invention is to recite the physical properties, such as length; primary structure; or charge, which are correlated with binding to a particular allele-specific HLA molecule or group of allele-specific HLA molecules. A further modality for defining peptide epitopes is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e.g. pocket configuration and charge distribution) and reciting that the peptide epitope fits and binds to the pocket or pockets.

[0019] 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.

III. BRIEF DESCRIPTION OF THE FIGURES

[0020] not applicable

IV. DETAILED DESCRIPTION OF THE INVENTION

[0021] The peptide epitopes and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to a TAA by stimulating the production of CTL or HTL responses. The peptide epitopes, which are derived directly or indirectly from native TAA protein amino acid sequences, are able to bind to HLA molecules and stimulate an immune response to the TAA. The complete sequence of the TAA proteins to be analyzed can be obtained from GenBank. Peptide epitopes and analogs thereof can also be readily determined from sequence information that may subsequently be discovered for heretofore unknown variants of particular TAAs, as will be clear from the disclosure provided below.

[0022] A list of target TAA includes, but is not limited to, the following antigens: MAGE 1, MAGE 2, MAGE 3, MAGE-11, MAGE-A10, BAGE, GAGE, RAGE, MAGE-C1, LAGE-1, CAG-3, DAM, MUC1, MUC2, MUC18, NY-ESO-1, MUM-1, CDK4, BRCA2, NY-LU-1, NY-LU-7, NY-LU-12, CASP8, RAS, KIAA-2-5, SCCs, p53, p73, CEA, Her 2/neu, Melan-A, gp100, tyrosinase, TRP2, gp75/TRP1, kallikrein, PSM, PAP, PSA, PT1-1, B-catenin, PRAME, Telomerase, FAK, cyclin D1 protein, NOEY2, EGF-R, SART-1, CAPB, HPVE7, p15, Folate receptor CDC27, PAGE-1, and PAGE4.

[0023] The peptide epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that analog peptides have been derived and the binding activity for HLA molecules modulated by modifying specific amino acid residues to create peptide analogs exhibiting altered immunogenicity. Further, the present invention provides compositions and combinations of compositions that enable epitope-based vaccines that are capable of interacting with HLA molecules encoded by various genetic alleles to provide broader population coverage than prior vaccines.

IV.A. Definitions

[0024] The invention can be better understood with reference to the following definitions, which are listed alphabetically:

[0025] A “computer” or “computer system” generally includes: a processor; at least one information storage/retrieval apparatus such as, for example, a hard drive, a disk drive or a tape drive; at least one input apparatus such as, for example, a keyboard, a mouse, a touch screen, or a microphone; and display structure. Additionally, the computer may include a communication channel in communication with a network. Such a computer may include more or less than what is listed above.

[0026] A “construct” as used herein generally denotes a composition that does not occur in nature. A construct can be produced by synthetic technologies, e.g., recombinant DNA preparation and expression or chemical synthetic techniques for nucleic or amino acids. A construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form.

[0027] “Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.

[0028] A “cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein which comprises the epitope is used as an antigen.

[0029] A “dominant epitope” is an epitope that induces an immune response upon immunization with a whole native antigen (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.

[0030] 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.

[0031] 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 within the bounds of the invention. In certain embodiments, there is a limitation on the length of a peptide of the invention which is not otherwise a construct as defined herein. An 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 a recited definition of epitope from reading, e.g., on whole natural molecules, the length of any region that has 100% identity with a native peptide sequence is limited. Thus, for a peptide comprising an epitope of the invention and a region with 100% identity with a native peptide sequence (and which is not otherwise a construct), 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 which is not a construct 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 of (50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) down to 5 amino acids.

[0032] Certain peptide or protein sequences longer than 600 amino acids are within the scope of the invention. Such longer sequences 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, or if longer than 600 amino acids, they are a construct. 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 of the invention be less than 600 residues long in any increment down to eight amino acid residues. “Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, Calif., 1994).

[0033] An “HLA supertype or family”, as used herein, describes sets of HLA molecules grouped on the basis of shared peptide-binding specificities. HLA class I 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.

[0034] Throughout this disclosure, results are expressed in terms of “IC50's.” IC50 is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate KD values. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. It should be noted that IC50 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 IC50 of a given ligand.

[0035] Alternatively, binding is expressed relative to a reference peptide. Although as a particular assay becomes more, or less, sensitive, the IC50's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC50 of the reference peptide increases 10-fold, the IC50 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 IC50, relative to the IC50 of a standard peptide.

[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, 19990; 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), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476, 1990; Schumacher et al., Cell 62:563, 1990; Townsend et al., Cell 62:285, 1990; Parker et al., J. Immunol. 149:1896, 1992).

[0037] As used herein, “high affinity” with respect to HLA class I molecules is defined as binding with an IC50, or KD value, of 50 nM or less; “intermediate affinity” is binding with an IC50 or KD value of between about 50 and about 500 nM. “High affinity” with respect to binding to HLA class II molecules is defined as binding with an IC50 or KD value of 100 nM or less; “intermediate affinity” is binding with an IC50 or KD value of between about 100 and about 1000 nM.

[0038] The terms “identical” or percent “identity,” in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0039] An “immunogenic peptide” or “peptide epitope” is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.

[0040] The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.

[0041] “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.

[0042] “Major Histocompatibility Complex” or “MHC” is a cluster of genes that 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, FUNDAMENTAL IMMUNOLOGY, 3RD ED., Raven Press, New York, 1993.

[0043] The term “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

[0044] A “negative binding residue” or “deleterious residue” is an amino acid which, if present at certain positions (typically not primary anchor positions) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.

[0045] 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 α-amino and carboxyl groups of adjacent amino acids. The preferred CTL-inducing peptides of the invention are 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues. The preferred HTL-inducing oligopeptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.

[0046] “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition.

[0047] A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

[0048] A “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves. In one embodiment, for example, the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9-residue peptide epitope in accordance with the invention. The primary anchor positions for each motif and supermotif are set forth in Table 1. For example, analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to modulate the binding affinity of a peptide comprising a particular motif or supermotif.

[0049] “Promiscuous recognition” is where a distinct peptide is recognized by the same T cell clone in the context of various HLA molecules. Promiscuous recognition or binding is synonymous with cross-reactive binding.

[0050] A “protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression. The immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.

[0051] The term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.

[0052] A “secondary anchor residue” is an amino acid at a position other than a primary anchor position in a peptide which may influence peptide binding. A secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position. The secondary anchor residues are said to occur at “secondary anchor positions.” A secondary anchor residue can be identified as a residue which is present at a higher frequency among high or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate affinity binding. For example, analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif.

[0053] 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 with an isolated peptide, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.

[0054] 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.

[0055] “Synthetic peptide” refers to a peptide that is man-made using such methods as chemical synthesis or recombinant DNA technology.

[0056] As used herein, a “vaccine” is a composition that contains one or more peptides of the invention. There are numerous embodiments of vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more epitopes 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 I-binding peptides of the invention can be admixed with, or linked to, HLA class II-binding peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. Vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.

[0057] 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. When amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal end of the epitope, or the peptide or protein of which it may be a part. 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 formulae, 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. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol. (A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.) In addition to these symbols, “B” in the single letter abbreviations used herein designates α-amino butyric acid.

IV.B. Stimulation of CTL and HTL Responses

[0058] The mechanism by which T cells recognize antigens has been delineated during the past ten years. Based on our understanding of the immune system we have developed efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to a TAA in a broad population. For an understanding of the value and efficacy of the claimed compositions, a brief review of immunology-related technology is provided. The review is intended to disclose the presently understood state of the art as of the filing date of the present application. Information is included in this section which was generated subsequent to the priority date of this application. Accordingly, information in this section is not intended, in any way, to delineate the priority date for the invention.

[0059] 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). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are described herein and are set forth in Tables I, II, and III (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via web at: http://134.2.96.221/scripts.hlaserver.dll/home.htm; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immmunogenetics 1999 November;50(3-4):201-12, Review).

[0060] Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets within the peptide binding cleft of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)

[0061] Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that have the potential of binding particular HLA molecules.

[0062] The present inventors have found that the correlation of binding affinity with immunogenicity, which is disclosed herein, is an important factor to be considered when evaluating candidate peptides. Thus, by a combination of motif searches and HLA-peptide binding assays, candidates for epitope-based vaccines have been identified. 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.

[0063] Various strategies can be utilized to evaluate immunogenicity, including:

[0064] 1) Evaluation of primary T cell cultures from normal individuals (see, eg., 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 using, e.g., a 51Cr-release assay involving peptide sensitized target cells.

[0065] 2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et a!, 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'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 using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

[0066] 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 using assays for T cell activity including 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

[0067] The following describes the peptide epitopes and corresponding nucleic acids of the invention.

IV.C. Binding Affinity of Peptide Epitopes for HLA Molecules

[0068] As indicated herein, 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.

[0069] CTL-inducing peptides of interest for vaccine compositions preferably include those that have an IC50 or binding affinity value for class I HLA molecules of 500 nM or better (i.e., the value is ≦500 nM). HTL-inducing peptides preferably include those that have an IC50 or binding affinity value for class II HLA molecules of 1000 nM or better, (i.e., the value is ≦1,000 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 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.

[0070] As disclosed herein, higher HLA binding affinity is correlated with greater immunogenicity. Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds 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 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. 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 or intermediate affinity binding peptide is used. Thus, in preferred embodiments of the invention, high or intermediate affinity binding epitopes are particularly useful.

[0071] The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens has been determined for the first time in the art by the present inventors. The correlation between binding affinity and immunogenicity was analyzed in two different experimental approaches (see, e.g., Sette, et al., J. Immunol. 153:5586-5592, 1994). In the first approach, the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL from acute hepatitis patients. Pursuant to these approaches, it was determined that an affinity threshold value of approximately 500 nM (preferably 50 nM or less) determines the capacity of a peptide epitope to elicit a CTL response. These data are true for class I binding affinity measurements for naturally processed peptides and for synthesized T cell epitopes. These data also indicate the important role of determinant selection in the shaping of T cell responses (see, e.g., Schaeffer et al., Proc. Natl. Acad Sci. USA 86:4649-4653, 1989).

[0072] An affinity threshold associated with immunogenicity in the context of HLA class II DR molecules has also been delineated (see, e.g., Southwood et al. J. Immunology 160:3363-3373,1998, and co-pending U.S. Ser. No. 09/009,953 filed Jan. 21, 1998). In order to define a biologically significant threshold of DR binding affinity, a database of the binding affinities of 32 DR-restricted epitopes for their restricting element (i.e., the HLA molecule that binds the motif) was compiled. In approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e. binding affinity values of 100 nM or less. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.

[0073] In the case of tumor-associated antigens, many CTL peptide epitopes that have been shown to induce CTL that lyse peptide-pulsed target cells and tumor cell targets endogenously expressing the epitope exhibit binding affinity or IC50 values of 200 nM or less. In a study that evaluated the association of binding affinity and immunogenicity of such TAA epitopes, 100% (10/10) of the high binders, i.e., peptide epitopes binding at an affinity of 50 nM or less, were immunogenic and 80% (8/10) of them elicited CTLs that specifically recognized tumor cells. In the 51 to 200 nM range, very similar figures were obtained. CTL inductions positive for peptide and tumor cells were noted for 86% (6/7) and 71% (5/7) of the peptides, respectively. In the 201-500 nM range, most peptides (4/5 wildtype) were positive for induction of CTL recognizing wildtype peptide, but tumor recognition was not detected.

[0074] The binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.

IV.D. Peptide Epitope Binding Motifs and Supermotifs

[0075] Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues required for allele-specific binding to HLA molecules have been identified. The presence of these residues correlates with binding affinity for HLA molecules. The identification of motifs and/or supermotifs that correlate with high and intermediate affinity binding is an important issue with respect to the identification of immunogenic peptide epitopes for the inclusion in a vaccine. Kast et al. (J. Immunol. 152:3904-3912, 1994) have shown that motif-bearing peptides account for 90% of the epitopes that bind to allele-specific HLA class I molecules. In this study all possible peptides of 9 amino acids in length and overlapping by eight amino acids (240 peptides), which cover the entire sequence of the E6 and E7 proteins of human papillomavirus type 16, were evaluated for binding to five allele-specific HLA molecules that are expressed at high frequency among different ethnic groups. This unbiased set of peptides allowed an evaluation of the predictive value of HLA class I motifs. From the set of 240 peptides, 22 peptides were identified that bound to an allele-specific HLA molecule with high or intermediate affinity. Of these 22 peptides, 20 (i.e. 91%) were motif-bearing. Thus, this study demonstrates the value of motifs for the identification of peptide epitopes for inclusion in a vaccine: application of motif-based identification techniques will identify about 90% of the potential epitopes in a target antigen protein sequence.

[0076] Such peptide epitopes are identified in the Tables described below.

[0077] Peptides of the present invention also comprise epitopes that bind to MHC 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 6th position towards the C-terminus, relative to P1, for binding to various DR molecules.

[0078] 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 (see, e.g., Tables I-III), 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.”

[0079] The peptide motifs and supermotifs described below, and summarized in Tables I-III, provide guidance for the identification and use of peptide epitopes in accordance with the invention.

[0080] Examples of peptide epitopes bearing a respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif below. The Tables include a binding affinity ratio listing for some of the peptide epitopes. The ratio may be converted to IC50 by using the following formula: IC50 of the standard peptide/ratio=IC50 of the test peptide (i.e., the peptide epitope). The IC50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV. The IC50 values of standard peptides used to determine binding affinites for Class II peptides are shown in Table V. The peptides used as standards for the binding assays described herein are examples of standards; alternative standard peptides can also be used when performing binding studies.

[0081] To obtain the peptide epitope sequences listed in each of Tables VII-XX, the amino acid sequence of HER2/neu was evaluated for the presence of the designated supermotif or motif, i.e., the amino acid sequence was searched for the presence of the primary anchor residues as set out in Table I (for Class I motifs) or Table III (for Class II motifs) for each respective motif or supermotif.

[0082] In the Tables, motif- and/or supermotif-bearing epitopes in the HER2/neu sequence are indicated by position number and length of the epitope with reference to the HER2/neu sequence and numbering provided below. The “pos” (position) column designates the amino acid position in the HER2/neu protein sequence that corresponds to the first amino acid residue of the epitope. The “number of amino acids” indicates the number of residues in the epitope sequence and hence the length of the epitope. For example, the first peptide epitope listed in Table VII is a sequence of 8 residues in length starting at position 66. Accordingly, the amino acid sequence of the epitope is PTNASLSF.

[0083] Binding data presented in Tables VII-XX is expressed as a relative binding ratio, supra.

1HER2/neu amino acid sequence1MELAALCRWG LLLALLPPGA ASTQVCTGTD MKLRLPASPE THLDMLRHLY QGCQVVQGNL60ELTYLPTNAS LSFLQDIQEV QGYVLIAHNQ VRQVPLQRLR IVRGTQLFED NYALAVLDNG120DPLNNTTPVT GASPGGLREL QLRSLTEILK GGVLIQRNPQ LCYQDTILWK DIFHKNWQLA180LTLIDTNRSR ACHPCSPMCK GSRCWGESSE DCQSLTRTVC AGGCARCKGP LPTDCCHEQC240AAGCTGPKHS DCLACLHFNH SGICELHCPA LVTYNTDTFE SMPNPEGRYT FGASCVTACP300YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQR CEKCSKPCAR VCYGLGMEHL REVRAVTSAN360IQEFAGCKKI FGSLAFLPES FDGDPASNTA PLQPEQLQVF ETLEEITGYL YISAWPDSLP420DISVFQNLQV IRGRILHNGA YSLTLQGLGI SWLGLRSLRE LGSGLALIHH NTHLCFVHTV480PWDQLFRNPH QALLHTANRP EDECVGEGLA CHQLCARGHC WGPGPTQCVN CSQFLRGQEC540VEECRVLQGL PREYVNARHC LPCHPECQPQ NGSVTCFGPE ADQCVACAHY KDPPFCVARC600PSGVKPDLSY MPIWKFPDEE GACQPCPINC THSCVDLDDK GCPAEQRASP LTSIISAVVG660ILLVVVLGVV FGTLIKRRQQ KIRKYTMRRL LQETELVEPL TPSGANPNQA QMRILKETEL720RKVKVLGSGA FGTVYKGIWI PDGENVKIPV AIKVLRENTS PKANKEILDE AYVMAGVGSP780YVSRLLGICL TSTVQLVTQL MPYGCLLDHV RENRGRLGSQ DLLNWCMQIA KGMSYLEDVR840LVHRDLAARN VLVKSPNHVK ITDFGLARLL DIDETEYHAD GGKVPIKWMA LESILRRRFT900HQSDVWSYGV TVWELMTFGA KPYDGIPARE IPDLLEKGER LPQPPICTID VYMIMVKCWM960IDSECRPRFR ELVSEFSRMA RDPQRFVVIQ NEDLGPASPL DSTFYRSLLE DDDMGDLVDA1020EEYLVPQQGF FCPDPAPGAG GMVHHRHRSS STRSGGGDLT LGLEPSEEEA PRSPLAPSEG1080AGSDVFDGDL GMGAAKGLQS LPTRDPSPLQ RYSEDPTVPL PSETDGYVAP LTCSPQPEYV1140NQPDVRPQPP SPREGPLPAA RPAGATLERP KTLSPGKNGV VKDVFAFGGA VENPEYLTPQ1200GGAAPQPHPP PAFSPAFDNL YYWDQDPPER GAPPSTFKGT PTAENPEYLG LDVPV 1255

[0084] HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:

[0085] The primary anchor residues of the HLA class I peptide epitope supermotifs and motifs delineated below are summarized in Table I. The HLA class I motifs set out in Table I(a) are those most particularly relevant to the invention claimed here. Primary and secondary anchor positions are summarized in Table II. Allele-specific HLA molecules that comprise HLA class I supertype families are listed in Table VI. In some cases, peptide epitopes may be listed in both a motif and a supermotif Table. The relationship of a particular motif and respective supermotif is indicated in the description of the individual motifs.

[0086] IV.D.1. HLA-A1 Supermotif

[0087] The HLA-A1 supermotif is characterized by the presence in peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M) primary anchor residue in position 2, and an aromatic (Y, F, or W) primary anchor residue at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind to the A1 supermotif (i.e., the HLA-A1 supertype) is comprised of at least: A*0101, A*2601, A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M. et al., J. Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994; Kondo, A. et al., Immunogenetics 45:249, 1997). Other allele-specific HLA molecules predicted to be members of the A1 superfamily are shown in Table VI. Peptides binding to each of the individual HLA proteins can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

[0088] Representative peptide epitopes that comprise the A1 supermotif are set forth in Table VII.

[0089] IV.D.2. HLA-A2 Supermotif

[0090] Primary anchor specificities for allele-specific HLA-A2.1 molecules (see, e.g., Falk et al., Nature 351:290-296, 1991; Hunt et al., Science 255:1261-1263, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992; Ruppert et al., Cell 74:929-937, 1993) and cross-reactive binding among HLA-A2 and -A28 molecules have been described. (See, e.g., Fruci et al., Human Immunol. 38:187-192, 1993; Tanigaki et al., Human Immunol. 39:155-162, 1994; Del Guercio et al., J. Immunol. 154:685-693, 1995; Kast et al., J. Immunol. 152:3904-3912, 1994 for reviews of relevant data.) These primary anchor residues define the HLA-A2 supermotif; which presence in peptide ligands corresponds to the ability to bind several different HLA-A2 and -A28 molecules. The HLA-A2 supermotif comprises peptide ligands with L, I, V, M, A, T, or Q as a primary anchor residue at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope.

[0091] The corresponding family of HLA molecules (i.e., the HLA-A2 supertype that binds these peptides) is comprised of at least: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, and A*6901. Other allele-specific HLA molecules predicted to be members of the A2 superfamily are shown in Table VI. As explained in detail below, binding to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

[0092] Representative peptide epitopes that comprise an A2 supermotif are set forth in Table VIII. The motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.

[0093] IV.D.3. HLA-A3 Supermotif

[0094] The HLA-A3 supermotif is characterized by the presence in peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at position 2, and a positively charged residue, R or K, at the C-terminal position of the epitope, e.g., in position 9 of 9-mers (see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996). Exemplary members of the corresponding family of HLA molecules (the HLA-A3 supertype) that bind the A3 supermotif include at least: A*0301, A*1101, A*3101, A*3301, and A*6801. Other allele-specific HLA molecules predicted to be members of the A3 supertype are shown in Table VI. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions of amino acids at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.

[0095] Representative peptide epitopes that comprise the A3 supermotif are set forth in Table IX.

[0096] IV.D.4. HLA-A24 Supermotif

[0097] The HLA-A24 supermotif is characterized by the presence in peptide ligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V, M, or T) residue as a primary anchor in position 2, and Y, F, W, L, I, or M as primary anchor at the C-terminal position of the epitope (see, e.g., Sette and Sidney, Immunogenetics 1999 November; 50(3-4):201-12, Review). The corresponding family of HLA molecules that bind to the A24 supermotif (i.e., the A24 supertype) includes at least: A*2402, A*3001, and A*2301. Other allele-specific HLA molecules predicted to be members of the A24 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

[0098] Representative peptide epitopes that comprise the A24 supermotif are set forth in Table X.

[0099] IV.D.5. HLA-B7 Supermotif

[0100] The HLA-B7 supermotif is characterized by peptides bearing proline in position 2 as a primary anchor, and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminal position of the epitope. The corresponding family of HLA molecules that bind the B7 supermotif (i.e., the HLA-B7 supertype) is comprised of at least twenty six HLA-B proteins comprising at least: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J. Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995; Hill, et al., Nature 360:434, 1992; Rammensee, et al., Immunogenetics 41:178, 1995 for reviews of relevant data). Other allele-specific HLA molecules predicted to be members of the B7 supertype are shown in Table VI. As explained in detail below, peptide binding to each of the individual allele-specific HLA proteins can be modulated by substitutions at the primary and/or secondary anchor positions of the peptide, preferably choosing respective residues specified for the supermotif.

[0101] Representative peptide epitopes that comprise the B7 supermotif are set forth in Table XI.

[0102] IV.D.6. HLA-B27 Supermotif

[0103] The HLA-B27 supermotif is characterized by the presence in peptide ligands of a positively charged (R, H, or K) residue as a primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics 1999 November; 50(3-4):201-12, Review). Exemplary members of the corresponding family of HLA molecules that bind to the B27 supermotif (i.e., the B27 supertype) include at least B*1401, B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301. Other allele-specific HLA molecules predicted to be members of the B27 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

[0104] Representative peptide epitopes that comprise the B27 supermotif are set forth in Table XII.

[0105] IV.D.7. HLA-B44 Supermotif

[0106] The HLA-B44 supermotif is characterized by the presence in peptide ligands of negatively charged (D or E) residues as a primary anchor in position 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney et al., Immunol. Today 17:261, 1996). Exemplary members of the corresponding family of HLA molecules that bind to the B44 supermotif (i.e., the B44 supertype) include at least: B*1801, B*1802, B*3701, B*4001, B*4002, B*4006, B*4402, B*4403, and B*4404. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the supermotif.

[0107] IV.D.8. HLA-B58 Supermotif

[0108] The HLA-B58 supermotif is characterized by the presence in peptide ligands of a small aliphatic residue (A, S, or T) as a primary anchor residue at position 2, and an aromatic or hydrophobic residue (F, W, Y, L, l, V, M, or A) as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics 1999 November; 50(3-4):201-12, Review). Exemplary members of the corresponding family of HLA molecules that bind to the B58 supermotif (i e., the B58 supertype) include at least: B*1516, B*1517, B*5701, B*5702, and B*5801. Other allele-specific HLA molecules predicted to be members of the B58 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

[0109] Representative peptide epitopes that comprise the B58 supermotif are set forth in Table XIII.

[0110] IV.D.9. HLA-B62 Supermotif

[0111] The HLA-B62 supermotif is characterized by the presence in peptide ligands of the polar aliphatic residue Q or a hydrophobic aliphatic residue (L, V, M, I, or P) as a primary anchor in position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A) as a primary anchor at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics 1999 November; 50(3-4):201-12, Review). Exemplary members of the corresponding family of HLA molecules that bind to the B62 supermotif (i.e., the B62 supertype) include at least: B*1501, B*1502, B*1513, and B5201. Other allele-specific HLA molecules predicted to be members of the B62 supertype are shown in Table VI. Peptide binding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif.

[0112] Representative peptide epitopes that comprise the B62 supermotif are set forth in Table XIV.

[0113] IV.D.10. HLA-A1 Motif

[0114] The HLA-A1 motif is characterized by the presence in peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position of the epitope. An alternative allele-specific A1 motif is characterized by a primary anchor residue at position 3 rather than position 2. This motif is characterized by the presence of D, E, A, or S as a primary anchor residue in position 3, and a Y as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., J. Immunol., 152:620, 1994; Kondo et al., Immunogenetics 45:249, 1997; and Kubo et al., J. Immunol. 152:3913, 1994 for reviews of relevant data). Peptide binding to HLA-A1 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.

[0115] Representative peptide epitopes that comprise either A1 motif are set forth in Table XV. Those epitopes comprising T, S, or M at position 2 and Y at the C-terminal position are also included in the listing of HLA-A1 supermotif-bearing peptide epitopes listed in Table VII, as these residues are a subset of the A1 supermotif primary anchors.

[0116] IV.D.11. HLA-A*0201 Motif

[0117] An HLA-A2*0201 motif was determined to be characterized by the presence in peptide ligands of L or M as a primary anchor residue in position 2, and L or V as a primary anchor residue at the C-terminal position of a 9-residue peptide (see, e.g., Falk et al., Nature 351:290-296, 1991) and was further found to comprise an I at position 2 and I or A at the C-terminal position of a nine amino acid peptide (see, e.g., Hunt et al., Science 255:1261-1263, Mar. 6, 1992; Parker et al., J. Immunol 149:3580-3587, 1992). The A*0201 allele-specific motif has also been defined by the present inventors to additionally comprise V, A, T, or Q as a primary anchor residue at position 2, and M or T as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kast et al., J. Immunol. 152:3904-3912, 1994). Thus, the HLA-A*0201 motif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope. The preferred and tolerated residues that characterize the primary anchor positions of the HLA-A*0201 motif are identical to the residues describing the A2 supermotif. (For reviews of relevant data, see, e.g., del Guercio et al., J. Immunol. 154:685-693, 1995; Ruppert et al., Cell 74:929-937, 1993; Sidney et al., Immunol. Today 17:261-266, 1996; Sette and Sidney, Curr. Opin. in Immunol. 10:478482, 1998). Secondary anchor residues that characterize the A*0201 motif have additionally been defined (see, e.g., Ruppert et al., Cell 74:929-937, 1993). These are shown in Table II. Peptide binding to HLA-A*0201 molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.

[0118] Representative peptide epitopes that comprise an A*0201 motif are set forth in Table VIII. The A*0201 motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.

[0119] IV.D.12. HLA-A3 Motif

[0120] The HLA-A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2, and the presence of K, sY, R, H, F, or A as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al., Proc. Natl. Acad. Sci USA 90:1508, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.

[0121] Representative peptide epitopes that comprise the A3 motif are set forth in Table XVI. Those peptide epitopes that also comprise the A3 supermotif are also listed in Table IX. The A3 supermotif primary anchor residues comprise a subset of the A3- and A11-allele specific motif primary anchor residues.

[0122] IV.D.13. HLA-A11 Motif

[0123] The HLA-A 11 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2, and K, R, Y, or H as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Zhang et al., Proc. Natl. Acad Sci USA 90:2217-2221, 1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-A11 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.

[0124] Representative peptide epitopes that comprise the A11 motif are set forth in Table XVII; peptide epitopes comprising the A3 allele-specific motif are also present in this Table because of the extensive overlap between the A3 and A11 motif primary anchor specificities. Further, those peptide epitopes that comprise the A3 supermotif are also listed in Table IX.

[0125] IV.D.14. HLA-A24 Motif

[0126] The HLA-A24 motif is characterized by the presence in peptide ligands of Y, F, W, or M as a primary anchor residue in position 2, and F, L, I, or W as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Kondo et al., J. Immunol. 155:43074312, 1995; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide binding to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions; preferably choosing respective residues specified for the motif.

[0127] Representative peptide epitopes that comprise the A24 motif are set forth in Table XVIII. These epitopes are also listed in Table X, which sets forth HLA-A24-supermotif-bearing peptide epitopes, as the primary anchor residues characterizing the A24 allele-specific motif comprise a subset of the A24 supermotif primary anchor residues.

[0128] Motifs Indicative of Class II HTL Inducing Peptide Epitopes

[0129] The primary and secondary anchor residues of the HLA class II peptide epitope supermotifs and motifs delineated below are summarized in Table III.

[0130] IV.D.15. HLA DR-1-4-7 Supermotif

[0131] 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). These are set forth in Table III. 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.

[0132] Potential epitope 9-mer core regions comprising the DR-1-4-7 supermotif, wherein position 1 of the supermotif is at position 1 of the nine-residue core, are set forth in Table XIX. Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise a nine residue core, are also shown, along with cross-reactive binding data for the exemplary 15-residue peptides.

[0133] IV.D.16. HLA DR3 Motifs

[0134] 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.

[0135] 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.

[0136] Potential peptide epitope 9-mer core regions corresponding to a nine residue sequence comprising the DR3a submotif (wherein position 1 of the motif is at position 1 of the nine residue core) are set forth in Table XXa. Respective exemplary peptide epitopes of 15 amino acid residues in length, each of which comprise the nine residue core, are also shown in Table XXa along with binding data for the exemplary peptides.

[0137] Potential peptide epitope 9-mer core regions comprising the DR3b submotif and respective exemplary 15-mer peptides comprising the DR3 submotif-b epitope are set forth in Table XXb along with binding data for the exemplary peptides.

[0138] Each of the HLA class I or class II peptide epitopes set out in the Tables herein are deemed singly to be an inventive aspect of this application. Further, it is also an inventive aspect of this application that each peptide epitope may be used in combination with any other peptide epitope.

IV.E. Enhancing Population Coverage of the Vaccine

[0139] Vaccines that have broad population coverage are preferred because they are more commercially viable and generally applicable to the most people. Broad population coverage can be obtained using the peptides of the invention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bind to HLA alleles which, when considered in total, are present in most of the population. Table XXI lists the overall frequencies of the HLA class I supertypes in various ethnicities (Table XXIa) and the combined population coverage achieved by the A2-, A3-, and B7-supertypes (Table XXIb). The A2-, A3-, and B7 supertypes are each present on the average of over 40% in each of these five major ethnic groups. Coverage in excess of 80% is achieved with a combination of these supermotifs. These results suggest that effective and non-ethnically biased population coverage is achieved upon use of a limited number of cross-reactive peptides. Although the population coverage reached with these three main peptide specificities is high, coverage can be expanded to reach 95% population coverage and above, and more easily achieve truly multispecific responses upon use of additional supermotif or allele-specific motif bearing peptides.

[0140] The B44-, A1-, and A24-supertypes are each present, on average, in a range from 25% to 40% in these major ethnic populations (Table XXIa). While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency >25% in at least one major ethnic group (Table XXIa). Table XXIb summarizes the estimated prevalence of combinations of HLA supertypes that have been identified in five major ethnic groups. The incremental coverage obtained by the inclusion of A1,-A24-, and B44-supertypes to the A2, A3, and B7 coverage and coverage obtained with all of the supertypes described herein, is shown.

[0141] The data presented herein, together with the previous definition of the A2-, A3-, and B7-supertypes, indicates that all antigens, with the possible exception of A29, B8, and B46, can be classified into a total of nine HLA supertypes. By including epitopes from the six most frequent supertypes, an average population coverage of 99% is obtained for five major ethnic groups.

IV.F. Immune Response-Stimulating Peptide Analogs

[0142] In general, CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few “immunodominant” determinants (Zinkernagel, et al., Adv. Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991). It has been recognized that immunodominance (Benacerraf, et al., Science 175:273-279, 1972) could be explained by either the ability of a given epitope to selectively bind a particular HLA protein (determinant selection theory) (Vitiello, et al., J. Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977), or to be selectively recognized by the existing TCR (T cell receptor) specificities (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OF SELF/NONSELF DISCRIMINATION, John Wiley & Sons, New York, pp. 270-310, 1982). It has been demonstrated that additional factors, mostly linked to processing events, can also play a key role in dictating, beyond strict immunogenicity, which of the many potential determinants will be presented as immunodominant (Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993).

[0143] Because tissue specific and developmental TAAs are expressed on normal tissue at least at some point in time or location within the body, it may be expected that T cells to them, particularly dominant epitopes, are eliminated during immunological surveillance and that tolerance is induced. However, CTL responses to tumor epitopes in both normal donors and cancer patient has been detected, which may indicate that tolerance is incomplete (see, e.g., Kawashima et al., Hum. Immunol. 59:1, 1998; Tsang, J. Natl. Cancer Inst. 87:82-90, 1995; Rongcun et al., J. Immunol. 163:1037, 1999). Thus, immune tolerance does not completely eliminate or inactivate CTL precursors capable of recognizing high affinity HLA class I binding peptides.

[0144] An additional strategy to overcome tolerance is to use analog peptides. Without intending to be bound by theory, it is believed that because T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow existing T cells to be recruited, which will then lead to a therapeutic or prophylactic response. However, the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide, for example to prepare analog peptides which elicit a more vigorous response.

[0145] Although peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification (i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed. More specifically, peptides which exhibit the broadest cross-reactivity patterns, can be produced in accordance with the teachings herein. The present concepts related to analog generation are set forth in greater detail in co-pending U.S. Ser. No. 09/226,775 filed Jan. 6, 1999.

[0146] In brief, the strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA molecules. The motifs or supermotifs are defined by having primary anchors, and in many cases secondary anchors. 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. Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively.

[0147] 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 (Tables II and III). 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). 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.

[0148] To ensure that an analog peptide, when used as a vaccine, actually elicits a CTL response to the native epitope in vivo (or, in the case of class II epitopes, elicits helper T cells that cross-react with the wild type peptides), the analog peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated. It will be desirable to use as antigen presenting cells, cells that have been either infected, or transfected with the appropriate genes, or, in the case of class II epitopes only, cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells.

[0149] Another embodiment of the invention is to create analogs of weak binding peptides, to thereby ensure adequate numbers of cross-reactive cellular binders. Class I binding peptides exhibiting binding affinities of 500-5000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be “fixed” by substituting preferred anchor residues in accordance with the respective supertype. The analog peptides can then be tested for crossbinding activity.

[0150] 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. For example, a cysteine can be substituted out in favor of α-amino butyric acid (“B” in the single letter abbreviations for peptide sequences listed herein). 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 cysteine 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 & Sons, England, 1999).

[0151] Representative analog peptides are set forth in Tables XXII-XXVI. The Table indicates the length and sequence of the analog peptide as well as the motif or supermotif, if appropriate. The “source” column indicates the residues substituted at the indicated position numbers for the respective analog.

IV.G. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif- or Motif-Bearing Peptides

[0152] In order to identify supermotif- or motif-bearing epitopes in a target antigen, a native protein sequence, e.g., a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation, is screened using a means for computing, such as an intellectual calculation or a computer, to determine the presence of a supermotif or motif within the sequence. The information obtained from the analysis of native peptide can be used directly to evaluate the status of the native peptide or may be utilized subsequently to generate the peptide epitope.

[0153] Computer programs that allow the rapid screening of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present invention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified amino acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof; analogs can be simultaneously determined as well. Generally, the identified sequences will be from a pathogenic organism or a tumor-associated peptide. For example, the target TAA molecules include, without limitation, CEA, MAGE, p53 and HER2/neu.

[0154] It is important that the selection criteria utilized for prediction of peptide binding are as accurate as possible, to correlate most efficiently with actual binding. Prediction of peptides that bind, for example, to HLA-A*0201, on the basis of the presence of the appropriate primary anchors, is positive at about a 30% rate (see, e.g., Ruppert, J. et al. Cell 74:929, 1993). However, by extensively analyzing peptide-HLA binding data disclosed herein, data in related patent applications, and data in the art, the present inventors have developed a number of allele-specific polynomial algorithms that dramatically increase the predictive value over identification on the basis of the presence of primary anchor residues alone. These algorithms take into account not only the presence or absence of primary anchors, but also consider the positive or deleterious presence of secondary anchor residues (to account for the impact of different amino acids at different positions). The algorithms are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA interactions can be approximated as a linear polynomial function of the type:

ΔG=a1i×a2i×a3i . . . ×ani

[0155] where ad is a coefficient that represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. An important assumption of this method is that the effects at each position are essentially independent of each other. This assumption is justified by studies that demonstrated that peptides are bound to HLA molecules and recognized by T cells in essentially an extended conformation. Derivation of specific algorithm coefficients has been described, for example, in Gulukota, K. et al., J. Mol. Biol. 267:1258, 1997.

[0156] Additional methods to identify preferred peptide sequences, which also make use of specific motifs, include the use of neural networks and molecular modeling programs (see, e.g. Milik et al., Nature Biotechnology 16:753, 1998; Altuvia et al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244, 1995; Buus, S. Curr. Opin. Immunol. 11:209-213, 1999; Brusic, V. et al., Bioinformatics 14:121-130, 1998; Parker et al., J. Immunol. 152:163, 1993; Meister et al., Vaccine 13:581, 1995; Hammer et al., J. Exp. Med. 180:2353, 1994; Sturniolo et al., Nature Biotechnol. 17:555 1999).

[0157] For example, it has been shown that in sets of A*0201 motif-bearing peptides containing at least one preferred secondary anchor residue while avoiding the presence of any deleterious secondary anchor residues, 69% of the peptides will bind A*0201 with an IC50 less than 500 nM (Ruppert, J. et al. Cell 74:929, 1993). These algorithms are also flexible in that cut-off scores may be adjusted to select sets of peptides with greater or lower predicted binding properties, as desired.

[0158] In utilizing computer screening to identify peptide epitopes, a protein sequence or translated sequence may be analyzed using software developed to search for motifs, for example the “FINDPATTERNS” program (Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown, San Diego, Calif.) to identify potential peptide sequences containing appropriate HLA binding motifs. The identified peptides can be scored using customized polynomial algorithms to predict their capacity to bind specific HLA class I or class II alleles. As appreciated by one of ordinary skill in the art, a large array of computer programming software and hardware options are available in the relevant art which can be employed to implement the motifs of the invention in order to evaluate (e.g., without limitation, to identify epitopes, identify epitope concentration per peptide length, or to generate analogs) known or unknown peptide sequences.

[0159] In accordance with the procedures described above, HER2/neu peptide epitopes and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules have been identified (Tables VII-XX; Table XXII-XXXI).

IV.H. Preparation of Peptide Epitopes

[0160] Peptides in accordance with the invention can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or from natural sources such as native tumors or pathogenic organisms. Peptide epitopes may be synthesized individually or as polyepitopic peptides. Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles.

[0161] 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, subject to the condition that modifications do not destroy the biological activity of the peptides as described herein.

[0162] When possible, it may be desirable to optimize HLA class I binding epitopes of the invention, such as can be used in a polyepitopic construct, to a length of about 8 to about 13 amino acid residues, often 8 to 11, preferably 9 to 10. HLA class II binding peptide epitopes of the invention may be optimized to a length of about 6 to about 30 amino acids in length, preferably to between about 13 and about 20 residues. Preferably, the peptide epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules, however, the identification and preparation of peptides that comprise epitopes of the invention can also be carried out using the techniques described herein.

[0163] In alternative embodiments, epitopes of the invention can be linked as a polyepitopic peptide, or as a minigene that encodes a polyepitopic peptide.

[0164] In another embodiment, it is preferred to identify native peptide regions that contain a high concentration of class I and/or class II epitopes. Such a sequence is generally selected on the basis that it contains the greatest number of epitopes per amino acid length. It is to be appreciated that epitopes can be present in a nested or overlapping manner, e.g. a 10 amino acid long peptide could contain two 9 amino acid long epitopes and one 10 amino acid long epitope; upon intracellular processing, each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. This larger, preferably multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source.

[0165] The peptides of the invention can be prepared in a wide variety of ways. For the preferred 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 & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984). Further, individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.

[0166] Alternatively, recombinant DNA technology can 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. (1989). Thus, recombinant polypeptides which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.

[0167] The nucleotide coding sequence for peptide epitopes of the preferred lengths contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Peptide analogs can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence; exemplary nucleic acid substitutions are those that encode an amino acid defined by the motifs/supermotifs herein. 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, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences.

IV.I. Assays to Detect T-Cell Responses

[0168] Once HLA binding peptides are identified, they can be tested for the ability to elicit a T-cell response. The preparation and evaluation of motif-bearing peptides are described in PCT publications WO 94/20127 and WO 94/03205. Briefly, peptides comprising epitopes from a particular antigen are synthesized and tested for their ability to bind to the appropriate HLA proteins. These assays may involve evaluating the binding of a peptide of the invention to purified HLA class I molecules in relation to the binding of a radioiodinated reference peptide. Alternatively, cells expressing empty class I molecules (i.e. lacking peptide therein) may be evaluated for peptide binding by immunofluorescent staining and flow microfluorimetry. Other assays that may be used to evaluate peptide binding include peptide-dependent class I assembly assays and/or the inhibition of CTL recognition by peptide competition. Those peptides that bind to the class I molecule, typically with an affinity of 500 nM or less, are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with selected target cells associated with a disease. Corresponding assays are used for evaluation of HLA class II binding peptides. HLA class II motif-bearing peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses.

[0169] Conventional assays utilized to detect T cell responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays. For example, antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells. Alternatively, mutant non-human mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene, may be used to test for the capacity of the peptide to induce in vitro primary CTL responses.

[0170] Peripheral blood mononuclear cells (PBMCs) may be used as the responder cell source of CTL precursors. The appropriate antigen-presenting cells are incubated with peptide, after which the peptide-loaded antigen-presenting cells are then incubated with the responder cell population under optimized culture conditions. Positive CTL activation can be determined by assaying the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the antigen from which the peptide sequence was derived.

[0171] More recently, a method has been devised which allows direct quantification of antigen-specific T cells by staining with Fluorescein-labelled HLA tetrameric complexes (Altman; J. D. et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996). Other relatively recent technical developments include staining for intracellular lymphokines, and interferon-y release assays or ELISPOT assays. Tetramer staining, intracellular lymphokine staining and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity 8:177, 1998).

[0172] HTL activation may also be assessed using such techniques known to those in the art such as T cell proliferation and secretion of lymphokines, e.g. IL-2 (see, e.g. Alexander et al., Immunity 1:751-761, 1994).

[0173] Alternatively, immunization of HLA transgenic mice can be used to determine immunogenicity of peptide epitopes. Several transgenic mouse models including mice with human A2.1, A11 (which can additionally be used to analyze HLA-A3 epitopes), and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed. Additional transgenic mouse models with other HLA alleles may be generated as necessary. Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resulting T cells tested for their capacity to recognize peptide-pulsed target cells and target cells transfected with appropriate genes. CTL responses may be analyzed using cytotoxicity assays described above. Similarly, HTL responses may be analyzed using such assays as T cell proliferation or secretion of lymphokines.

IV.J. Use of Peptide Epitopes as Diagnostic Agents and for Evaluating Immune Responses

[0174] In one embodiment of the invention, HLA class I and class II binding peptides as described herein are used as reagents to evaluate an immune response. The immune response to be evaluated is induced by using as an immunogen any agent that may result in the production of antigen-specific CTLs or HTLs that recognize and bind to the peptide epitope(s) to be employed as the reagent The peptide reagent need not be used as the immunogen. Assay systems that are used for such an analysis include relatively recent technical developments such as tetramers, staining for intracellular lymphokines and interferon release assays, or ELISPOT assays.

[0175] For example, peptides of the invention are used in tetramer staining assays to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a tumor cell antigen or an 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 is generated as follows: A peptide that binds to an HLA molecule is refolded in the presence of the corresponding HLA heavy chain and β2-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 can then be identified, for example, by flow cytometry. Such an analysis may be used for diagnostic or prognostic purposes. Cells identified by the procedure can also be used for therapeutic purposes.

[0176] Peptides of the invention are also used as reagents to evaluate immune recall responses (see, e.g., Bertoni et al., J. Clin. Invest. 100:503-513, 1997 and Penna et al., J. Exp. Med. 174:1565-1570, 1991). For example, patient PBMC samples from individuals with cancer are analyzed for the presence of antigen-specific CTLs or HTLs using specific peptides. A blood sample containing mononuclear cells can be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the invention. After an appropriate cultivation period, the expanded cell population can be analyzed, for example, for CTL or for HTL activity.

[0177] The peptides are also used as reagents to evaluate the efficacy of a vaccine. PBMCs obtained from a patient vaccinated with an immunogen are analyzed using, for example, either of the methods described above. The patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present in that patient are selected for the analysis. The immunogenicity of the vaccine is indicated by the presence of epitope-specific CTLs and/or HTLs in the PBMC sample.

[0178] The peptides of the invention are also used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose or monitor cancer. Such antibodies include those that recognize a peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.

IV.K. Vaccine Compositions

[0179] Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more peptides as described herein are further embodiments of the invention. Once appropriately immunogenic epitopes have been defined, they can be sorted and 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. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. 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. H. 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. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. 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; Robinson, 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.

[0180] Vaccines 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).

[0181] For therapeutic or prophylactic immunization purposes, the peptides of the invention can also be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host bearing a tumor, 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.

[0182] Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. 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 can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.

[0183] 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's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS).

[0184] 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 CTLs and/or HTLs 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.

[0185] In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses to the target antigen of interest. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross-binding HLA class II epitope such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described, for example, in U.S. Pat. No. 5,736,142).

[0186] A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo.

[0187] Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

[0188] Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or 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 CTL or HTL responses to a particular tumor-associated antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells, such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

[0189] The vaccine compositions of the invention can also be used in combination with other treatments used for cancer, including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

[0190] Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. Examples of epitopes that can be utilized in a vaccine to treat or prevent cancer are provided in Tables XXII-XXVII and XI. It is preferred that each of the following principles are balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition can be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

[0191] 1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one TAA. For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs as described, e.g., in Example 15.

[0192] 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, or for Class II an IC50 of 1000 nM or less.

[0193] 3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

[0194] 4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope. When selecting epitopes for infectious disease-related antigens it is preferable to select either native or analoged epitopes.

[0195] 5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise both HLA class I and HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

[0196] 6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

[0197] IV.K.1. Minigene Vaccines

[0198] A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

[0199] The use of multi-epitope minigenes is described below and in, e.g., co-pending application U.S. Ser. No. 09/311,784; Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing HER2/neu epitopes derived from multiple regions of HER2/neu, a universal helper T epitope, e.g., PADRE™ (or multiple HTL epitopes from HER2/neu), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes, in addition to HER2/neu epitopes, that are derived from other TAAs.

[0200] The immunogenicity of a multi-epitopic minigene can be tested in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.

[0201] For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

[0202] The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.

[0203] Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: 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, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0204] 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 and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

[0205] 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.

[0206] In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.

[0207] In some embodiments, a bi-cistronic expression vector which allows production of both 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., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

[0208] Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, 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 QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

[0209] 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-buffered saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (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.

[0210] Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

[0211] In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (IP) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.

[0212] Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

[0213] Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.

[0214] IV.K2. Combinations of CTL Peptides with Helper Peptides

[0215] Vaccine compositions comprising the peptides of the present invention, or analogs thereof, which have immunostimulatory activity may be modified to provide desired attributes, such as improved serum half-life, or to enhance immunogenicity.

[0216] For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. The use of T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in the co-pending applications U.S. Ser. No. 08/820,360, U.S. Ser. No. 08/197,484, and U.S. Ser. No. 08/464,234.

[0217] Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope 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 and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

[0218] In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in the majority of the population. This can be accomplished by selecting amino acid sequences that bind to many, most, or all of the HLA class II molecules. These are known as “loosely HLA-restricted” or “promiscuous” T helper sequences. Examples of peptides that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS), and Streptococcus 18 kD protein at positions 116 (GAVDSILGGVATYGAA). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

[0219] Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-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 (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed to most preferrably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa, 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. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

[0220] HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

[0221] IV.K.3. Combinations of CTL Peptides with T Cell Priming Agents

[0222] In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes cytotoxic T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo against viral antigens. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine 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 administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. A preferred immunogenic composition comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

[0223] As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

[0224] CTL and/or HTL peptides can also be modified by the addition of amino acids 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, particularly class I peptides. However, it is to be noted that modification at the carboxyl terminus of a CTL epitope may, in some cases, alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH2 acylation, e.g., by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

[0225] IV.K.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

[0226] 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's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. 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 complexed with HLA molecules on their surfaces.

[0227] The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL response to one or more antigens of interest, e.g., CEA, p53, Her2/neu, MAGE, prostate cancer-associated antigens and the like. Optionally, a helper T cell peptide such as a PADRE™ family molecule, can be included to facilitate the CTL response.

[0228] IV.L. Administration of Vaccines for Therapeutic or Prophylactic Purposes

[0229] The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are typically used therapeutically to treat cancer. Vaccine compositions containing the peptides of the invention are typically administered to a cancer patient who has a malignancy associated with expression of one or more tumor-associated antigens. Alternatively, vaccine compositions can be administered to an individual susceptible to, or otherwise at risk for developing a particular type of cancer, e.g., breast cancer.

[0230] In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective CTL and/or HTL response to the tumor antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, 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.

[0231] As noted above, peptides comprising CTL and/or HTL epitopes of the invention induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide. The manner in which the peptide is contacted with the CTL or HTL is not critical to the invention. For instance, the peptide can be contacted with the CTL or 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 peptides, viral vectors encoding the peptide(s), liposomes and the like, can be used, as described herein.

[0232] When the peptide is contacted in vitro, the vaccinating agent can comprise a population of cells, e.g., peptide-pulsed dendritic cells, or TAA-specific CTLs, which have been induced by pulsing antigen-presenting cells in vitro with the peptide. Such a cell population is subsequently administered to a patient in a therapeutically effective dose.

[0233] For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already diagnosed with cancer. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.

[0234] For therapeutic use, administration should generally begin at the first diagnosis of cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs) delivered to the patient may vary according to the stage of the disease. For example, a vaccine comprising TAA-specific CTLs may be more efficacious in killing tumor cells in patients with advanced disease than alternative embodiments.

[0235] The vaccine compositions of the invention may also be used therapeutically in combination with treatments such as surgery. An example is a situation in which a patient has undergone surgery to remove a primary tumor and the vaccine is then used to slow or prevent recurrence and/or metastasis.

[0236] Where susceptible individuals, e.g., individuals who may be diagnosed as being genetically pre-disposed to developing a particular type of tumor, for example breast cancer, are identified prior to diagnosis of cancer, the composition can be targeted to them, thus minimizing the need for administration to a larger population.

[0237] The dosage for an initial immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood.

[0238] Administration should continue until at least clinical symptoms or laboratory tests indicate that the tumor has been eliminated or that the tumor cell burden has been substantially reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

[0239] In certain embodiments, peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

[0240] The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration. Preferably, the pharmaceutical compositions are administered parentally, 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, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0241] The concentration of 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.

[0242] 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 (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).

[0243] 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 to target selectively to infected cells, as well as to increase the half-life of the peptide composition. Liposomes include emulsions, foams, miceiles, 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 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 peptide compositions. Liposomes for use in accordance with 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), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0244] For targeting cells of the immune system, 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.

[0245] 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%.

[0246] 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.

IV.M. HLA Expression: Implications for T Cell-Based Immunotherapy

[0247] Disease Progression in Cancer and Infectious Disease

[0248] It is well recognized that a dynamic interaction between exists between host and disease, both in the cancer and infectious disease settings. In the infectious disease setting, it is well established that pathogens evolve during disease. The strains that predominate early in HIV infection are different from the ones that are associated with AIDS and later disease stages (NS versus S strains). It has long been hypothesized that pathogen forms that are effective in establishing infection may differ from the ones most effective in terms of replication and chronicity.

[0249] Similarly, it is widely recognized that the pathological process by which an individual succumbs to a neoplastic disease is complex. During the course of disease, many changes occur in cancer cells. The tumor accumulates alterations which are in part related to dysfunctional regulation of growth and differentiation, but also related to maximizing its growth potential, escape from drug treatment and/or the body's immunosurveillance. Neoplastic disease results in the accumulation of several different biochemical alterations of cancer cells, as a function of disease progression. It also results in significant levels of intra- and inter-cancer heterogeneity, particularly in the late, metastatic stage.

[0250] Familiar examples of cellular alterations affecting treatment outcomes include the outgrowth of radiation or chemotherapy resistant tumors during the course of therapy. These examples parallel the emergence of drug resistant viral strains as a result of aggressive chemotherapy, e.g., of chronic HBV and HIV infection, and the current resurgence of drug resistant organisms that cause Tuberculosis and Malaria. It appears that significant heterogeneity of responses is also associated with other approaches to cancer therapy, including anti-angiogenesis drugs, passive antibody immunotherapy, and active T cell-based immunotherapy. Thus, in view of such phenomena, epitopes from multiple disease-related antigens can be used in vaccines and therapeutics thereby counteracting the ability of diseased cells to mutate and escape treatment.

[0251] The Interplay between Disease and the Immune System

[0252] One of the main factors contributing to the dynamic interplay between host and disease is the immune response mounted against the pathogen, infected cell, or malignant cell. In many conditions such immune responses control the disease. Several animal model systems and prospective studies of natural infection in humans suggest that immune responses against a pathogen can control the pathogen, prevent progression to severe disease and/or eliminate the pathogen. A common theme is the requirement for a multispecific T cell response, and that narrowly focused responses appear to be less effective. These observations guide skilled artisan as to embodiments of methods and compositions of the present invention that provide for a broad immune response.

[0253] In the cancer setting there are several findings that indicate that immune responses can impact neoplastic growth:

[0254] First, the demonstration in many different animal models, that anti-tumor T cells, restricted by MHC class I, can prevent or treat tumors.

[0255] Second, encouraging results have come from immunotherapy trials.

[0256] Third, observations made in the course of natural disease correlated the type and composition of T cell infiltrate within tumors with positive clinical outcomes (Coulie P G, et al. Antitumor immunity at work in a melanoma patient In Advances in Cancer Research, 213-242, 1999).

[0257] Finally, tumors commonly have the ability to mutate, thereby changing their immunological recognition. For example, the presence of monospecific CTL was also correlated with control of tumor growth, until antigen loss emerged (Riker A, et al., Immune selection after antigen-specific immunotherapy of melanoma Surgery, August: 126(2):112-20, 1999; Marchand M, et al., Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1 Int. J. Cancer 80(2):219-30, Jan. 18, 1999). Similarly, loss of beta 2 microglobulin was detected in 5/13 lines established from melanoma patients after receiving immunotherapy at the NCI (Restifo N P, et al., Loss of functional Beta2 -microglobulin in metastatic melanomas from five patients receiving immunotherapy Journal of the National Cancer Institute, Vol. 88 (2), 100-108, January. 1996). It has long been recognized that HLA class I is frequently altered in various tumor types. This has led to a hypothesis that this phenomenon might reflect immune pressure exerted on the tumor by means of class I restricted CTL. The extent and degree of alteration in HLA class I expression appears to be reflective of past immune pressures, and may also have prognostic value (van Duinen S G, et al., Level of HLA antigens in locoregional metastases and clinical course of the disease in patients with melanoma Cancer Research 48, 1019-1025, February 1988; Möller P, et al., Influence of major histocompatibility complex class I and II antigens on survival in colorectal carcinoma Cancer Research 51, 729-736, January 1991). Taken together, these observations provide a rationale for immunotherapy of cancer and infectious disease, and suggest that effective strategies need to account for the complex series of pathological changes associated with disease.

[0258] The Three Main Types of Alterations in HLA Expression in Tumors and Their Functional Significance

[0259] The level and pattern of expression of HLA class I antigens in tumors has been studied in many different tumor types and alterations have been reported in all types of tumors studied. The molecular mechanisms underlining HLA class I alterations have been demonstrated to be quite heterogeneous. They include alterations in the TAP/processing pathways, mutations of β-microglobulin and specific HLA heavy chains, alterations in the regulatory elements controlling over class I expression and loss of entire chromosome sections. There are several reviews on this topic, see, e.g.,: Garrido F, et al., Natural history of HLA expression during tumour development Immunol Today 14(10):491-499, 1993; Kaklamanis L, et al., Loss of HLA class-I alleles, heavy chains and 62-microglobulin in colorectal cancer Int. J. Cancer, 51(3):379-85, May 28, 1992. There are three main types of HLA Class I alteration (complete loss, allele-specific loss and decreased expression). The functional significance of each alteration is discussed separately:

[0260] Complete Loss of HLA Expression

[0261] Complete loss of HLA expression can result from a variety of different molecular mechanisms, reviewed in (Algarra I, et al., The HLA crossroad in tumor immunology Human Immunology 61, 65-73, 2000; Browning M, et al., Mechanisms of loss of HLA class I expression on colorectal tumor cells Tissue Antigens 47:364-371, 1996; Ferrone S, et al., Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance Immunology Today, 16(10): 487-494, 1995; Garrido F, et al., Natural history of HLA expression during tumour development Immunology Today 14(10):491-499, 1993; Tait, B D, HLA Class I expression on human cancer cells: Implications for effective immunotherapy Hum Immunol. 61, 158-165, 2000). In functional terms, this type of alteration has several important implications.

[0262] While the complete absence of class I expression will eliminate CTL recognition of those tumor cells, the loss of HLA class I will also render the tumor cells extraordinary sensitive to lysis from NK cells (Ohnmacht, Ga., et al., Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma J. Cellular Phys 182:332-338, 2000; Liunggren H G, et al., Host resistance directed selectively against H-2 deficient lymphoma variants: Analysis of the mechanism J. Exp. Med., December 1;162(6):1745-59, 1985; Maio M, et al., Reduction in susceptibility to natural killer cell-mediated lysis of human FO-1 melanoma cells after induction of HLA class I antigen expression by transfection with B2m gene J. Clin. Invest. 88(1):282-9, July 1991; Schrier P I, et al., Relationship between myc oncogene activation and MHC class I expression Adv. Cancer Res., 60:181-246, 1993).

[0263] The complementary interplay between loss of HLA expression and gain in NK sensitivity is exemplified by the classic studies of Coulie and coworkers (Coulie, P G, et al., Antitumor immunity at work in a melanoma patient. In Advances in Cancer Research, 213-242, 1999) which described the evolution of a patient's immune response over the course of several years. Because of increased sensitivity to NK lysis, it is predicted that approaches leading to stimulation of innate immunity in general and NK activity in particular would be of special significance. An example of such approach is the induction of large amounts of dendritic cells (DC) by various hematopoietic growth factors, such as Flt3 ligand or ProGP. The rationale for this approach resides in the well known fact that dendritic cells produce large amounts of IL-12, one of the most potent stimulators for innate immunity and NK activity in particular. Alternatively, IL-12 is administered directly, or as nucleic acids that encode it. In this light, it is interesting to note that Flt3 ligand treatment results in transient tumor regression of a class I negative prostate murine cancer model (Ciavarra R P, et al., Flt3-Ligand induces transient tumor regression in an ectopic treatment model of major histocompatibility complex-negative prostate cancer Cancer Res 60:2081-84, 2000). In this context, specific anti-tumor vaccines in accordance with the invention synergize with these types of hematopoietic growth factors to facilitate both CTL and NK cell responses, thereby appreciably impairing a cell's ability to mutate and thereby escape efficacious treatment. Thus, an embodiment of the present invention comprises a composition of the invention together with a method or composition that augments functional activity or numbers of NK cells. Such an embodiment can comprise a protocol that provides a composition of the invention sequentially with an NK-inducing modality, or contemporaneous with an NK-inducing modality.

[0264] Secondly, complete loss of HLA frequently occurs only in a fraction of the tumor cells, while the remainder of tumor cells continue to exhibit normal expression. In functional terms, the tumor would still be subject, in part, to direct attack from a CTL response; the portion of cells lacking HLA subject to an NK response. Even if only a CTL response were used, destruction of the HLA expressing fraction of the tumor has dramatic effects on survival times and quality of life.

[0265] It should also be noted that in the case of heterogeneous HLA expression, both normal HLA-expressing as well as defective cells are predicted to be susceptible to immune destruction based on “bystander effects.” Such effects were demonstrated, e.g., in the studies of Rosendahl and colleagues that investigated in vivo mechanisms of action of antibody targeted superantigens (Rosendahl A, et al., Perforin and IFN-gamma are involved in the antitumor effects of antibody-targeted superantigens J. Immunol. 160(11):5309-13, Jun. 1, 1998). The bystander effect is understood to be mediated by cytokines elicited from, e.g., CTLs acting on an HLA-bearing target cell, whereby the cytokines are in the environment of other diseased cells that are concomitantly killed.

[0266] Allele-Specific Loss

[0267] One of the most common types of alterations in class I molecules is the selective loss of certain alleles in individuals heterozygous for HLA. Allele-specific alterations might reflect the tumor adaptation to immune pressure, exerted by an immunodominant response restricted by a single HLA restriction element. This type of alteration allows the tumor to retain class I expression and thus escape NK cell recognition, yet still be susceptible to a CTL-based vaccine in accordance with the invention which comprises epitopes corresponding to the remaining HLA type. Thus, a practical solution to overcome the potential hurdle of allele-specific loss relies on the induction of multispecific responses. Just as the inclusion of multiple disease-associated antigens in a vaccine of the invention guards against mutations that yield loss of a specific disease antigens, simultaneously targeting multiple HLA specificities and multiple disease-related antigens prevents disease escape by allele-specific losses.

[0268] Decrease in Expression (Allele-Specific or Not)

[0269] The sensitivity of effector CTL has long been demonstrated (Brower, R C, et al., Minimal requirements for peptide mediated activation of CD8+ CTL Mol. Immunol., 31;1285-93, 1994; Chriustnick, E T, et al. Low numbers of MHC class I-peptide complexes required to trigger a T cell response Nature 352:67-70, 1991; Sykulev, Y, et al., Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response Immunity, 4(6):565-71, June 1996). Even a single peptide/MHC complex can result in tumor cells lysis and release of anti-tumor lymphokines. The biological significance of decreased HLA expression and possible tumor escape from immune recognition is not fully known. Nevertheless, it has been demonstrated that CTL recognition of as few as one MHC/peptide complex is sufficient to lead to tumor cell lysis.

[0270] Further, it is commonly observed that expression of HLA can be upregulated by gamma IFN, commonly secreted by effector CTL. Additionally, HLA class I expression can be induced in vivo by both alpha and beta IFN (Halloran, et al. Local T cell responses induce widespread MHC expression. J. Immunol 148:3837, 1992; Pestka, S, et al., Interferons and their actions Annu. Rev. Biochem. 56:727-77, 1987). Conversely, decreased levels of HLA class I expression also render cells more susceptible to NK lysis.

[0271] With regard to gamma IFN, Torres et al (Torres, M J, et al., Loss of an HLA haplotype in pancreas cancer tissue and its corresponding tumor derived cell line. Tissue Antigens 47:372-81, 1996) note that HLA expression is upregulated by gamma IFN in pancreatic cancer, unless a total loss of haplotype has occurred. Similarly, Rees and Mian note that allelic deletion and loss can be restored, at least partially, by cytokines such as IFN-gamma (Rees, R., et al. Selective MHC expression in tumours modulates adaptive and innate antitumour responses Cancer Immunol Immunother 48:374-81, 1999). It has also been noted that IFN-gamma treatment results in upregulation of class I molecules in the majority of the cases studied (Browning M, et al., Mechanisms of loss of HLA class I expression on colorectal tumor cells. Tissue Antigens 47:364-71, 1996). Kaklamakis, et al. also suggested that adjuvant immunotherapy with IFN-gamma may be beneficial in the case of HLA class I negative tumors (Kaklamanis L, Loss of transporter in antigen processing 1 transport protein and major histocompatibility complex class I molecules in metastatic versus primary breast cancer. Cancer Research 55:5191-94, November 1995). It is important to underline that IFN-gamma production is induced and self-amplified by local inflammation/immunization (Halloran, et al. Local T cell responses induce widespread MHC expression J. Immunol 148:3837, 1992), resulting in large increases in MHC expressions even in sites distant from the inflammatory site.

[0272] Finally, studies have demonstrated that decreased HLA expression can render tumor cells more susceptible to NK lysis (Ohnmacht, Ga., et al., Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma J Cellular Phys 182:332-38, 2000; Liunggren H G, et al., Host resistance directed selectively against H-2 deficient lymphoma variants: Analysis of the mechanism J. Exp. Med., 162(6):1745-59, Dec. 1, 1985; Maio M, et al., Reduction in susceptibility to natural killer cell-mediated lysis of human FO-1 melanoma cells after induction of HLA class I antigen expression by transfection with β2m gene J. Clin. Invest. 88(1):282-9, July 1991; Schrier P I, et al., Relationship between myc oncogene activation and MHC class I expression Adv. Cancer Res., 60:181-246, 1993). If decreases in HLA expression benefit a tumor because it facilitates CTL escape, but render the tumor susceptible to NK lysis, then a minimal level of HLA expression that allows for resistance to NK activity would be selected for (Garrido F, et al., Implications for immunosurveillance of altered HLA class I phenotypes in human tumours Immunol Today 18(2):89-96, February 1997). Therefore, a therapeutic compositions or methods in accordance with the invention together with a treatment to upregulate HLA expression and/or treatment with high affinity T-cells renders the tumor sensitive to CTL destruction.

[0273] Frequency of Alterations in HLA Expression

[0274] The frequency of alterations in class I expression is the subject of numerous studies (Algarra I, et al., The HLA crossroad in tumor immunology Human Immunology 61, 65-73, 2000). Rees and Mian estimate allelic loss to occur overall in 3-20% of tumors, and allelic deletion to occur in 15-50% of tumors. It should be noted that each cell carries two separate sets of class I genes, each gene carrying one HLA-A and one HLA-B locus. Thus, fully heterozygous individuals carry two different HLA-A molecules and two different HLA-B molecules. Accordingly, the actual frequency of losses for any specific allele could be as little as one quarter of the overall frequency. They also note that, in general, a gradient of expression exists between normal cells, primary tumors and tumor metastasis. In a study from Natali and coworkers (Natali P G, et al., Selective changes in expression of HLA class I polymorphic determinants in human solid tumors PNAS USA 86:6719-6723, September 1989), solid tumors were investigated for total HLA expression, using W6/32 antibody, and for allele-specific expression of the A2 antigen, as evaluated by use of the BB7.2 antibody. Tumor samples were derived from primary cancers or metastasis, for 13 different tumor types, and scored as negative if less than 20%, reduced if in the 30-80% range, and normal above 80%. All tumors, both primary and metastatic, were HLA positive with W6/32. In terms of A2 expression, a reduction was noted in 16.1% of the cases, and A2 was scored as undetectable in 39.4% of the cases. Garrido and coworkers (Garrido F, et al., Natural history of HLA expression during tumour development Immunol Today 14(10):491-99, 1993) emphasize that HLA changes appear to occur at a particular step in the progression from benign to most aggressive. Jiminez et al (Jiminez P, et al., Microsatellite instability analysis in tumors with different mechanisms for total loss of HLA expression. Cancer Immunol Immunother 48:684-90, 2000) have analyzed 118 different tumors (68 colorectal, 34 laryngeal and 16 melanomas). The frequencies reported for total loss of HLA expression were 11% for colon, 18% for melanoma and 13% for larynx. Thus, HLA class I expression is altered in a significant fraction of the tumor types, possibly as a reflection of immune pressure, or simply a reflection of the accumulation of pathological changes and alterations in diseased cells.

[0275] Immunotherapy in the Context of HLA Loss

[0276] A majority of the tumors express HLA class I, with a general tendency for the more severe alterations to be found in later stage and less differentiated tumors. This pattern is encouraging in the context of immunotherapy, especially considering that: 1) the relatively low sensitivity of immunohistochemical techniques might underestimate HLA expression in tumors; 2) class I expression can be induced in tumor cells as a result of local inflammation and lymphokine release; and, 3) class I negative cells are sensitive to lysis by NK cells.

[0277] Accordingly, various embodiments of the present invention can be selected in view of the fact that there can be a degree of loss of HLA molecules, particularly in the context of neoplastic disease. For example, the treating physician can assay a patient's tumor to ascertain whether HLA is being expressed. If a percentage of tumor cells express no class I HLA, then embodiments of the present invention that comprise methods or compositions that elicit NK cell responses can be employed. As noted herein, such NK-inducing methods or composition can comprise a Flt3 ligand or ProGP which facilitate mobilization of dendritic cells, the rationale being that dendritic cells produce large amounts of IL-12. IL-12 can also be administered directly in either amino acid or nucleic acid form. It should be noted that compositions in accordance with the invention can be administered concurrently with NK cell-inducing compositions, or these compositions can be administered sequentially.

[0278] In the context of allele-specific HLA loss, a tumor retains class I expression and may thus escape NK cell recognition, yet still be susceptible to a CTL-based vaccine in accordance with the invention which comprises epitopes corresponding to the remaining HLA type. The concept here is analogous to embodiments of the invention that include multiple disease antigens to guard against mutations that yield loss of a specific antigen. Thus, one can simultaneously target multiple HLA specificities and epitopes from multiple disease-related antigens to prevent tumor escape by allele-specific loss as well as disease-related antigen loss. In addition, embodiments of the present invention can be combined with alternative therapeutic compositions and methods. Such alternative compositions and methods comprise, without limitation, radiation, cytotoxic pharmaceuticals, and/or compositions/methods that induce humoral antibody responses.

[0279] Moreover, it has been observed that expression of HLA can be upregulated by gamma IFN, which is commonly secreted by effector CTL, and that HLA class I expression can be induced in vivo by both alpha and beta IFN. Thus, embodiments of the invention can also comprise alpha, beta and/or gamma IFN to facilitate upregualtion of HLA.

IV.N. Reprieve Periods from Therapies that Induce Side Effects

[0280] “Scheduled Treatment Interruptions or Drug Holidays”

[0281] Recent evidence has shown that certain patients infected with a pathogen, whom are initially treated with a therapeutic regimen to reduce pathogen load, have been able to maintain decreased pathogen load when removed from the therapeutic regimen, i.e., during a “drug holiday” (Rosenberg, E., et al., Immune control of HIV-1 after early treatment of acute infection Nature 407:523-26, Sept. 28, 2000) As appreciated by those skilled in the art, many therapeutic regimens for both pathogens and cancer have numerous, often severe, side effects. During the drug holiday, the patient's immune system keeps the disease in check. Methods for using compositions of the invention are used in the context of drug holidays for cancer and pathogenic infection.

[0282] For treatment of an infection, where therapies are not particularly immunosuppressive, compositions of the invention are administered concurrently with the standard therapy. During this period, the patient's immune system is directed to induce responses against the epitopes comprised by the present inventive compositions. Upon removal from the treatment having side effects, the patient is primed to respond to the infectious pathogen should the pathogen load begin to increase. Composition of the invention can be provided during the drug holiday as well.

[0283] For patients with cancer, many therapies are immunosuppressive. Thus, upon achievement of a remission or identification that the patient is refractory to standard treatment, then upon removal from the immunosuppressive therapy, a composition in accordance with the invention is administered. Accordingly, as the patient's immune system reconstitutes, precious immune resources are simultaneously directed against the cancer. Composition of the invention can also be administered concurrently with an immunosuppressive regimen if desired.

IV.O. Kits

[0284] The peptide and nucleic acid compositions of this invention can be provided in kit form together with instructions for vaccine administration. Typically the kit would include desired peptide compositions in a container, preferably in unit dosage form and instructions for administration. An alternative kit would include a minigene construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL-12 may also be included in the kit. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.

IV.P. Overview

[0285] Epitopes in accordance with the present invention were successfully used to induce an immune response. Immune responses with these epitopes have been induced by administering the epitopes in various forms. The epitopes have been administered as peptides, as nucleic acids, and as viral vectors comprising nucleic acids that encode the epitope(s) of the invention. Upon administration of peptide-based epitope forms, immune responses have been induced by direct loading of an epitope onto an empty HLA molecule that is expressed on a cell, and via internalization of the epitope and processing via the HLA class I pathway; in either event, the HLA molecule expressing the epitope was then able to interact with and induce a CTL response. Peptides can be delivered directly or using such agents as liposomes. They can additionally be delivered using ballistic delivery, in which the peptides are typically in a crystalline form. When DNA is used to induce an immune response, it is administered either as naked DNA, generally in a dose range of approximately 1-5 mg, or via the ballistic “gene gun” delivery, typically in a dose range of approximately 10-100 μg. The DNA can be delivered in a variety of conformations, e.g., linear, circular etc. Various viral vectors have also successfully been used that comprise nucleic acids which encode epitopes in accordance with the invention.

[0286] Accordingly compositions in accordance with the invention exist in several forms. Embodiments of each of these composition forms in accordance with the invention have been successfully used to induce an immune response.

[0287] One composition in accordance with the invention comprises a plurality of peptides. This plurality or cocktail of peptides is generally admixed with one or more pharmaceutically acceptable excipients. The peptide cocktail can comprise multiple copies of the same peptide or can comprise a mixture of peptides. The peptides can be analogs of naturally occurring epitopes. The peptides can comprise artificial amino acids and/or chemical modifications such as addition of a surface active molecule, e.g., lipidation; acetylation, glycosylation, biotinylation, phosphorylation etc. The peptides can be CTL or HTL epitopes. In a preferred embodiment the peptide cocktail comprises a plurality of different CTL epitopes and at least one HTL epitope. The HTL epitope can be naturally or non-naturally (e.g., PADRE®, Epimmune Inc., San Diego, Calif.). The number of distinct epitopes in an embodiment of the invention is generally a whole unit integer from one through two hundred (e.g., 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200).

[0288] An additional embodiment of a composition in accordance with the invention comprises a polypeptide multi-epitope construct, i.e., a polyepitopic peptide. Polyepitopic peptides in accordance with the invention are prepared by use of technologies well-known in the art. By use of these known technologies, epitopes in accordance with the invention are connected one to another. The polyepitopic peptides can be linear or non-linear, e.g., multivalent. These polyepitopic constructs can comprise artificial amino acids, spacing or spacer amino acids, flanking amino acids, or chemical modifications between adjacent epitope units. The polyepitopic construct can be a heteropolymer or a homopolymer. The polyepitopic constructs generally comprise epitopes in a quantity of any whole unit integer between 2-150 (e.g., 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100). The polyepitopic construct can comprise CTL and/or HTL epitopes. One or more of the epitopes in the construct can be modified, e.g., by addition of a surface active material, e.g. a lipid, or chemically modified, e.g., acetylation, etc. Moreover, bonds in the multiepitopic construct can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.

[0289] Alternatively, a composition in accordance with the invention comprises construct which comprises a series, sequence, stretch, etc., of amino acids that have homology to (i.e., corresponds to or is contiguous with) to a native sequence. This stretch of amino acids comprises at least one subsequence of amino acids that, if cleaved or isolated from the longer series of amino acids, functions as an HLA class I or HLA class II epitope in accordance with the invention. In this embodiment, the peptide sequence is modified, so as to become a construct as defined herein, by use of any number of techniques known or to be provided in the art. The polyepitopic constructs can contain homology to a native sequence in any whole unit integer increment from 70-100%, e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100 percent.

[0290] A further embodiment of a composition in accordance with the invention is an antigen presenting cell that comprises one or more epitopes in accordance with the invention. The antigen presenting cell can be a “professional” antigen presenting cell, such as a dendritic cell. The antigen presenting cell can comprise the epitope of the invention by any means known or to be determined in the art. Such means include pulsing of dendritic cells with one or more individual epitopes or with one or more peptides that comprise multiple epitopes, by nucleic acid administration such as ballistic nucleic acid delivery or by other techniques in the art for administration of nucleic acids, including vector-based, e.g. viral vector, delivery of nucleic acids.

[0291] Further embodiments of compositions in accordance with the invention comprise nucleic acids that encode one or more peptides of the invention, or nucleic acids which encode a polyepitopic peptide in accordance with the invention. As appreciated by one of ordinary skill in the art, various nucleic acids compositions will encode the same peptide due to the redundancy of the genetic code. Each of these nucleic acid compositions falls within the scope of the present invention. This embodiment of the invention comprises DNA or RNA, and in certain embodiments a combination of DNA and RNA. It is to be appreciated that any composition comprising nucleic acids that will encode a peptide in accordance with the invention or any other peptide based composition in accordance with the invention, falls within the scope of this invention.

[0292] It is to be appreciated that peptide-based forms of the invention (as well as the nucleic acids that encode them) can comprise analogs of epitopes of the invention generated using priniciples already known, or to be known, in the art. Principles related to analoging are now known in the art, and are disclosed herein; moreover, analoging principles (heteroclitic analoging) are disclosed in co-pending application serial number U.S. Ser. No. 09/226,775 filed 6 Jan. 1999. Generally the compositions of the invention are isolated or purified.

[0293] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield alternative embodiments in accordance with the invention.

V. EXAMPLES

[0294] The following examples illustrate identification, selection, and use of immunogenic Class I and Class II peptide epitopes for inclusion in vaccine compositions.

Example 1

[0295] HLA Class I and Class II Binding Assays

[0296] The following example of peptide binding to HLA molecules demonstrates quantification of binding affinities of HLA class I and class II peptides. Binding assays can be performed with peptides that are either motif-bearing or not motif-bearing.

[0297] HLA class I and class II binding assays using purified HLA molecules were performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) were incubated with various unlabeled peptide inhibitors and 1-10 nM 125I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes were separated from free peptide by gel filtration and the fraction of peptide bound was determined. Typically, in preliminary experiments, each MHC preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays were performed using these HLA concentrations.

[0298] Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation has proven to be the most accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.

[0299] Binding assays as outlined above can be used to analyze supermotif and/or motif-bearing epitopes as, for example, described in Example 2.

Example 2

[0300] Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes

[0301] Vaccine compositions of the invention may include multiple epitopes that comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

[0302] Computer Searches and Algorthims for Identification of Supermotif and/or Motif-Bearing Epitopes

[0303] The searches performed to identify the motif-bearing peptide sequences in Examples 2 and 5 employed protein sequence data for the tumor-associated antigen HER2/neu.

[0304] Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs were performed as follows. All translated protein sequences were analyzed using a text string search software program, e.g., MotifSearch 1.4 (D. Brown, San Diego) to identify potential peptide sequences containing appropriate HLA binding motifs; alternative programs are readily produced in accordance with information in the art in view of the motif/supermotif disclosure herein. Furthermore, such calculations can be made mentally. Identified A2-, A3-, and DR-supermotif sequences were scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms take into account both extended and refined motifs (that is, to account for the impact of different amino acids at different positions), and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a1i×a2i×a3i . . . ×ani

[0305] where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide. This assumption is justified by studies from our laboratories that demonstrated that peptides are bound to MHC and recognized by T cells in essentially an extended conformation (data omitted herein).

[0306] The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

[0307] Selection of HLA-A2 Supertype Cross-Reactive Peptides

[0308] The complete protein sequence from HER2/neu was scanned, utilizing motif identification software, to identify 8-, 9-, 10-, and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity.

[0309] A total of 623 HLA-A2 supermotif-positive sequences were identified. Of these, 73 scored positive in the A2 algorithm and the peptides corresponding to the sequences were then synthesized. An additional 90 A2 supermotif-bearing nonamers and decamers were also synthesized. These 163 peptides were then tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule). Twenty of the peptides bound A*0201 with IC50 values ≦500 nM.

[0310] The twenty A*0201-binding peptides were subsequently tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). As shown in Table XXII, 9 of the 20 peptides were found to be A2-supertype cross-reactive binders, binding at least three of the five A2-supertype alleles tested.

[0311] Selection of HLA-A3 Supermotif-Bearing Epitopes

[0312] The protein sequences scanned above are also examined for the presence of peptides with the HLA-A3-supermotif primary anchors using methodology similar to that performed to identify HLA-A2 supermotif-bearing epitopes.

[0313] Peptides corresponding to the supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the two most prevalent A3-supertype alleles. The peptides that are found to bind one of the two alleles with binding affinities of ≦500 nM are then tested for binding cross-reactivity to the other common A3-supertype alleles (A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested. Examples of HLA-A3 cross-binding supermotif-bearing peptides identified in accordance with this procedure are provided in Table XXIII.

[0314] Selection of HLA-B7 Supermotif Bearing Epitopes

[0315] The same target antigen protein sequences are also analyzed to identify HLA-B7-supermotif-bearing sequences. The corresponding peptides are then synthesized and tested for binding to HLA-B*0702, the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Those peptides that bind B*0702 with IC50 of ≦500 nM are then tested for binding to other common B7-supertype molecules (B*3501, B*5101, B*5301, and B*5401) to identify those peptides that are capable of binding to three or more of the five B7-supertype alleles tested. Examples of HLA-B7 cross-binding supermotif-bearing peptides identified in accordance with this procedure are provided in Table XXIV.

[0316] Selection of A1 and A24 Motif-Bearing Epitopes

[0317] To further increase population coverage, HLA-A1 and -A24 motif-bearing epitopes can also be incorporated into potential vaccine constructs. An analysis of the protein sequence data from the target antigen utilized above is also performed to identify HLA-A1- and A24-motif-containing conserved sequences. The corresponding peptide sequence are then synthesized and tested for binding to the appropriate allele-specific HLA molecule, HLA-A1 or HLA-24. Peptides are identified that bind to the allele-specific HLA molecules at an IC50 of ≦500 nM. Examples of peptides identified in accordance with this procedure are provided in Tables XXV and XXVI.

Example 3

[0318] Confirmation of Immunogenicity

[0319] The nine cross-reactive candidate CTL A2-supermotif-bearing peptides identified in Example 2 were selected for in vitro immunogenicity testing. Testing was performed using the following methodology:

[0320] Target Cell Lines for Cellular Screening:

[0321] The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, was used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. The colon adenocarcinoma cell lines SW403 and HT-29 were obtained from the American Type Culture Collection (ATCC) (Rockville, Md.). The cell lines that were obtained from ATCC were maintained under the culture conditions recommended by the supplier. All other cell lines were grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. The colon cancer cells were treated with 100U/ml IFNγ (Genzyme) for 48 hours at 37° C. before use as targets in the 51Cr release and in situ IFNγ assays.

[0322] Primary CTL Induction Cultures:

[0323] Generation of Dendritic Cells (DC): PBMCs were thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/strpetomycin). The monocytes were purified by plating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells were removed by gently shaking the plates and aspirating the supernatants. The wells were washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 were then added to each well. DC were used for CTL induction cultures following 7 days of culture.

[0324] Induction of CTL with DC and Peptide: CD8+ T-cells were isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC were processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs were thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106cells/ml. The magnetic beads were washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×106 cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells were washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×106 cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads were washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC were collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×106/ml in the presence of 3 μg/ml β2-microglobulin for 4 hours at 20° C. The DC were then irradiated (4,200 rads), washed 1 time with medium and counted again.

[0325] Setting up induction cultures: 0.25 ml cytokine-generated DC (@1×105 cells/ml) were co-cultured with 0.25 ml of CD8+ T-cells (@2×106 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. rHuman IL10 was added the next day at a final concentration of 10 ng/ml and rhuman IL2 was added 48 hours later at 10 IU/ml.

[0326] Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction the cells were restimulated with peptide-pulsed adherent cells. The PBMCS were thawed and washed twice with RPMI and DNAse. The cells were resuspended at 5×106 cells/ml and irradiated at ˜4200 rads. The PBMCs were plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates were washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 ml RPMI/5%AB per well for 2 hours at 37° C. Peptide solution from each well was aspirated and the wells were washed once with RPMI. Most of the media was aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells were then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later rhuman IL10 was added at a final concentration of 10 ng/ml and rhuman IL2 was added the next day and again 2-3 days later at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later the cultures were assayed for CTL activity in a 51Cr release assay. In some experiments the cultures were assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity was measured in both assays for a side by side comparison.

[0327] Measurement of CTL Lytic Activity by 51Cr Release.

[0328] Seven days after the second restimulation, cytotoxicity was determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets were prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.

[0329] Adherent target cells were removed from culture flasks with trypsin-EDTA. Target cells were labelled with 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labelled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and 100 μl of effectors were plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant were collected from each well and percent lysis was determined according formula: [(cpm of the test sample−cpm of the spontaneous 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100. Maximum and spontaneous release were determined by incubating the labelled targets with 1% Trition X-100 and media alone, respectively. A positive culture was defined as one in which the specific lysis (sample-background) was 10% or higher in the case of individual wells and was 15% or more at the 2 highest E:T ratios when expanded cultures were assayed.

[0330] In situ Measurement of Human γIFN Production as an Indicator of Peptide-Specific and Endogenous Recognition

[0331] Immulon 2 plates were coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates were washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for 2 hours, after which the CTLs (100 μl/well) and targets (100 μl/well) were added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, were used at a concentration of 1×106cells/ml. The plates were incubated for 48 hours at 37° C. with 5% CO2.

[0332] Recombinant human IFNγ was added to the standard wells starting at 400 pg or 1200 pg/100 μl/well and the plate incubated for 2 hours at 37° C. The plates were washed and 100 μl of biotinylated mouse anti-human IFNγ monoclonal antibody (4 μg/ml in PBS/3%FCS/0.05% Tween 20) were added and incubated for 2 hours at room temperature. After washing again, 100 μl HRP-streptavidin were added and the plates incubated for 1 hour at room temperature. The plates were then washed 6× with wash buffer, 100 μl/well developing solution (TMB 1:1) were added, and the plates allowed to develop for 5-15 minutes. The reaction was stopped with 50 μl/well 1M H3PO4 and read at OD450. A culture was considered positive if it measured at least 50 pg of IFNγ/well above background and was twice the background level of expression.

[0333] CTL Expansion. Those cultures that demonstrated specific lytic activity against peptide-pulsed targets and/or tumor targets were expanded over a two week period with anti-CD3. Briefly, 5×104 CD8+ cells were added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. rHuman IL2 was added 24 hours later at a final concentration of 200 IU/ml and every 3 days thereafter with fresh media at 50 IU/ml. The cells were split if the cell concentration exceeded 1×106/ml and the cultures were assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFNγ assay using the same targets as before the expansion.

[0334] Immunogenicity of A2 Supermotif-Bearing Peptides

[0335] A2-supermotif cross-reactive binding peptides were tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide was considered to be an epitope if it induced peptide-specific CTLs in at least 2 donors (unless otherwise noted) and if those CTLs also recognized the endogenously expressed peptide. Peptides that were able to induce a peptide-specific CTL response in at least 2 normal donors are shown in Table XXVII. Further analysis demonstrated those that also recognized target cells pulsed with the wild-type peptide and tumor targets that endogenously express HER2/neu (Table XXVII). An additional wild-type peptide, Her2/neu.5 was selected for evaluation based on its A2.1 binding affinity and, although it binds to only 2 HLA-A2 supertype molecules, it was capable of generating a strong CTL response that was both peptide- and tumor-specific.

[0336] Immunogenicity was additionally confirmed using PBMCs isolated from cancer patients. Briefly, PBMCs were isolated from two patients with ovarian cancer, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen. These data indicated that Her2/neu.435 was recognized in 2 donors as well as Her2/neu.369, Her2/neu.952, and Her2/neu.48. Her2/neu.689 is also an epitope, but not a supertype binder. Of the other peptides tested, Her2/neu.665 and Her2/neu.773 were recognized by CTLs from only one of the two patients and CTLs to Her2/neu.153 and Her2/neu.789 recognized peptide-pulsed targets only.

[0337] Evaluation of A*03/A11 Immunogenicity

[0338] HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides. Using this procedure, peptides that induce an immune response are identified. Examples of such peptides are shown in Table XXIII.

[0339] Evaluation of Immunogenicity of Motif/Supermotif-Bearing Peptides.

[0340] Analogous methodology, as appreciated by one of ordinary skill in the art, is employed to determine immunogenicity of peptides bearing HLA class I motifs and/or supermotifs set out herein. Using such a prodcedure peptides that induce an immune response are identified.

Example 4

[0341] Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

[0342] HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analogued, or “fixed” to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analog peptides that exhibit modulated binding affinity are set forth in this example and provided in Tables XXII through XXVII.

[0343] Analoguing at Primary Anchor Residues

[0344] Peptide engineering strategies were implemented to further increase the cross-reactivity of the epitopes identified above. On the basis of the data disclosed, e.g., in related and co-pending U.S. Ser. No. 09/226,775, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.

[0345] Peptides that exhibit at least weak A*0201 binding (IC50 of 5000 nM or less), and carrying suboptimal anchor residues at either position 2, the C-terminal position, or both, can be fixed by introducing canonical substitutions (L at position 2 and V at the C-terminus). Those analogued peptides that show at least a three-fold increase in A*0201 binding and bind with an IC50 of 500 nM, or less were then tested for A2 cross-reactive binding along with their wild-type (WT) counterparts. Analogued peptides that bind at least three of the five A2 supertype alleles were then selected for cellular screening analysis (Table XXVII).

[0346] Additionally, the selection of analogs for cellular screening analysis was further restricted by the capacity of the WT parent peptide to bind at least weakly, i.e., bind at an IC50 of 500 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analogued peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the WT epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).

[0347] In the cellular screening of these peptide analogs, it is important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, tumor targets that endogenously express the epitope.

[0348] Of the 20 peptides identified in Example 2 that bound to HLA-A*0201 at a high affinity, 15 carried suboptimal primary anchor residues and met the criterion for analoguing at primary anchor residues by introducing a canonical substitution. Analogs of six of the A*0201-binding peptides were created and tested for primary binding to HLA-A*0201 and supertype binding (Table XXII). In 4 of 6 cases, binding to HLA-A*0201 was improved at least three-fold. In 4 cases, crossbinding capability was also improved. In one instance, peptide Her2/neu.153 did not show a three-fold increase in binding to HLA-A*0201, but crossbinding was improved.

[0349] Additionally, 22 peptides that weakly bound to HLA-A*0201 that carry suboptimal anchors were also identified and can also be analogued.

[0350] Two analogs of Her2/neu.5, two analogs of Her2/neu.369, one version of Her2/neu.952, and one version of Her2/neu.665 were selected for cellular screening studies. As shown in Table XXVII, both Her2/neu.369L2V9 and V2V9 induced peptide-specific CTLs and those CTLs also recognized the target tumor cells expressing that endogenously express the antigen. Her2neu.5B3V9 and Her2/neu.952L2B7V10 induced peptide-specific CTLs in at least 2 donors, but when the positive cultures were expanded, no wild-type peptide or endogenous recognition was observed.

[0351] The Her2/neu.665L2V9 analog exhibited binding to four of the five A2 supertype alleles tested, whereas the wildtype peptide only binds two of the five alleles. In the cellular screening analysis, a strong peptide-specific CTL response was observed. The positive cultures were expanded and assayed for peptide and endogenous recognition. Peptide-specific CTL activity was maintained in some of the cultures, but no corresponding endogenous recognition was observed.

[0352] Using methodology similar to that used to develop HLA-A2 analogs, analogs of HLA-A3 and HLA-B7 supermotif-bearing epitopes are also generated. For example, peptides binding at least weakly to ⅗ of the A3-supertype molecules can be engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2. The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then tested for A3-supertype cross-reactivity. Examples of HLA-A3 supermotif analog peptides are provided in Table XXIII.

[0353] B7 supermotif-bearing peptides can, for example, be engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position (see, e.g. Sidney et al. (J. Immunol. 157:3480-3490, 1996). Analoged peptides are then tested for cross-reactive binding to B7 supertype alleles. Examples of B7-supermotif-bearing analog peptides are provided in Table XXIV.

[0354] Similarly, HLA-A 1 and HLA-A24 motif-bearing peptides can be engineered at primary anchor residues to improved binding to the allele-specific HLA molecule or to improve cross-reactive binding. Examples of analoged HLA-A1 and HLA-A24 motif-bearing peptides are provided in Tables XXV and XXVI.

[0355] Analoged peptides that exhibit improved binding and/or or cross-reactivity are evaluated for immunogenicity using methodology similar to that described for the analysis of HLA-A2 supermotif-bearing peptides. Using such a procedure, peptides that induce an immune response are identified, e.g. Table XXIII.

[0356] Analoguing at Secondary Anchor Residues

[0357] Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. Examples of such analoged peptides are provided in Tables XXII-XXIV.

[0358] For example, the binding capacity of a B7 supermotif-bearing peptide representing a discreet single amino acid substitution at position 1 can be analyzed. A peptide can, for example, be analogued to substitute L with F at position 1 and subsequently be evaluated for increased binding affinity/and or increased cross-reactivity. This procedure will identify analogued peptides with modulated binding affinity.

[0359] Analoged peptides that exhibit improved binding and/or or cross-reactivity are evaluated for immunogenicity using methodology similar to that described for the analysis of HLA-A2 supermotif-bearing peptides. Using such a procedure, peptides that induce an immune response are identified.

[0360] Other Analoguing Strategies

[0361] Another form of peptide analoguing, unrelated to the anchor positions, involves the substitution of a cysteine with 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. Subtitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

[0362] Analoged peptides that exhibit improved binding and/or or cross-reactivity are evaluated for immunogenicity using methodology similar to that described for the analysis of HLA-A2 supermotif-bearing peptides. Using such a procedure, peptides that induce an immune response are identified.

[0363] This Example therefore demonstrates that by the use of even single amino acid substitutions, the binding affinity and/or cross-reactivity of peptide ligands for HLA supertype molecules is modulated.

Example 5

[0364] Identification of Peptide Epitope Sequences with HLA-DR Binding Motifs

[0365] Peptide epitopes bearing an HLA class II supermotif or motif may also be identified as outlined below using methodology similar to that described in Examples 1-3.

[0366] Selection of HLA-DR-Supermotif-Bearing Epitopes

[0367] To identify HLA class II HTL epitopes, the HER2/neu protein sequence was analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences were selected comprising a DR-supermotif, further comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

[0368] Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele specific selection tables (see, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

[0369] The HER2/neu-derived peptides identified above were tested for their binding capacity for various common HLA-DR molecules. All peptides were initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least 2 of these 3 DR molecules with an IC50 value of 1000 nM or less, were then tested for binding to DR5*0101, DRB1*1501, DRB1*1101, DRB1*0802, and DRB1*1302. Peptides were considered to be cross-reactive DR supertype binders if they bound at an IC50 value of 1000 nM or less to at least 5 of the 8 alleles tested.

[0370] Following the strategy outlined above, 188 DR supermotif-bearing sequences were identified within the HER2/neu protein sequence. Of those, 41 scored positive in 2 of the 3 combined DR 147 algorithms. These peptides were synthesized and tested for binding to HLA-DRB1*0101, DRB1*0401, DRB1*0701. Of the 41 peptides tested, 18 bound at least 2 of the 3 alleles (Table XXVIII).

[0371] These 18 peptides were then tested for binding to secondary DR supertype alleles: DRB5*0101, DRB1*1501, DRB1*1101, DRB1*0802, and DRB1*1302. Nine peptides were identified that bound at least 5 of the 8 alleles tested, of which 8 occurred in distinct, non-overlapping regions (Table XXIX).

[0372] Selection of DR3 Motif Peptides

[0373] Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is an important criterion in the selection of HTL epitopes. However, data generated previously indicated that DR3 only rarely cross-reacts with other DR alleles (Sidney et al., J. Immunol. 149:2634-2640, 1992; Geluk et al., J. Immunol. 152:5742-5748, 1994; Southwood et al., J. Immunol. 160:3363-3373, 1998). This is not entirely surprising in that the DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles. For maximum efficiency in developing vaccine candidates it would be desirable for DR3 motifs to be clustered in proximity with DR supermotif regions. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the distinct binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

[0374] To efficiently identify peptides that bind DR3, the HER2/neu protein sequence was analyzed for conserved sequences carrying one of the two DR3 specific binding motifs (Table III) reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Forty-six motif-positive peptides were identified. The corresponding peptides were then synthesized and tested for the ability to bind DR3 with an affinity of 1000 nM or better, i.e., less than 1000 nM. Seven peptides were found that met this binding criterion (Table XXX), and thereby qualify as HLA class II high affinity binders.

[0375] Additionally, the 7 DR3 binders were tested for binding to the DR supertype alleles (Table XXXI). Four of the seven DR3 binders bound at least 3 other DR alleles, and one peptide, Her2/neu.886, was a cross-reactive supertype binder as well. Conversely, the DR supertype cross-reactive binding peptides were also tested for DR3 binding capacity. The cross-reactive DR supermotif-bearing peptides showed little capacity to bind DR3 molecules (Table XXXI).

[0376] DR3 binding epitopes identified in this manner may then be included in vaccine compositions with DR supermotif-bearing peptide epitopes.

[0377] In summary, 8 DR supertype cross-reactive binding peptides and 7 DR3 binding peptides were identified from the HER2/neu protein sequence, with one peptide shared between the two motifs. Of these, 5 DR supertype and 5 DR3-binding peptides were located in the intracellular domain.

[0378] Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides may be analogued to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue may improve DR 3 binding. Analoged peptides are evaluated for immunogenicity in accordance with the methodology of Example 6.

Example 6

[0379] Immunogenicity of HTL Epitopes

[0380] This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology in Example 5. Immunogenicity of HTL epitopes are evaluated in a manner analogous to the determination of immunogenicity of CTL epitopes by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from cancer patient PBMCs.

Example 7

[0381] Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

[0382] This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

[0383] In order to analyze population coverage, gene frequencies of HLA alleles were determined. Gene frequencies for each HLA allele were calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies were calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].

[0384] Where frequency data was not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies was assumed. To obtain total potential supertype population coverage no linkage disequilibrium was assumed, and only alleles confirmed to belong to each of the supertypes were included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations were made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

[0385] Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups (see Table XXI). Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.

Example 8

[0386] Recognition of Generation of Endogenous Processed Antigens After Priming

[0387] This example determines that CTL induced by native or analogued peptide epitopes identified and selected as described in Examples 1-6 recognize endogenously synthesized, i.e., native antigens, using a transgenic mouse model.

[0388] Effector cells isolated from transgenic mice that are immunized with peptide epitopes (as described, e.g., in Wentworth et al., Mol. Immunol. 32:603, 1995), for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with TAA expression vectors.

[0389] The result will demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that is being evaluated. In addition to HLA-A*0201/Kb transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 9

[0390] Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

[0391] This example illustrates the induction of CTLs and HTLs in transgenic mice by use of a tumor associated antigen CTL/HTL peptide conjugate whereby the vaccine composition comprises peptides to be administered to a cancer patient. The peptide composition can comprise multiple CTL and/or HTL epitopes and further, can comprise epitopes selected from multiple-tumor associated antigens. The epitopes are identified using methodology as described in Examples 1-6 This analysis demonstrates the enhanced immunogenicity that can be achieved by inclusion of one or more HTL epitopes in a vaccine composition. Such a peptide composition can comprise an HTL epitope conjugated to a preferred CTL epitope containing, for example, at least one CTL epitope selected from Tables XXVII and XXIII-XXVI, or other analogs of that epitope. The HTL epitope is, for example, selected from Table XI. The peptides may be lipidated, if desired.

[0392] Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are useful for the assessment of the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, are primed subcutaneously (base of the tail) with 0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

[0393] The target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991).

[0394] In vitro CTL activation: One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.

[0395] Assay for cytotoxic activity: Target cells (1.0 to 1.5×106) are incubated at 37° C. in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a 6 hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a 6 hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×106=18 LU.

[0396] The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation. The magnitude and frequency of the response can also be compared to the the CTL response achieved using the CTL epitopes by themselves. Analyses similar to this may be performed to evaluate the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 10

[0397] Selection of CTL and HTL Epitopes for Inclusion in a Cancer Vaccine.

[0398] This example illustrates the procedure for the selection of peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or may be single and/or polyepitopic peptides.

[0399] The following principles are utilized when selecting an array of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.

[0400] Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For example, a vaccine can include 3-4 epitopes that come from at least one TAA. Epitopes from one TAA can be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs as described, e.g., in Example 15.

[0401] Epitopes are preferably selected that have a binding affinity (IC50) of 500 nM or less, often 200 nM or less, for an HLA class I molecule, or for a class II molecule, 1000 nM or less.

[0402] Sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.

[0403] When selecting epitopes from cancer-related antigens it is often preferred to select analogs because the patient may have developed tolerance to the native epitope.

[0404] When creating a polyepitopic composition, e.g. a minigene, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest, although spacers or other flanking sequences can also be incorporated. The principles employed are often similar as those employed when selecting a peptide comprising nested epitopes. Additionally, however, upon determination of the nucleic acid sequence to be provided as a minigene, the peptide sequence encoded thereby is analyzed to determine whether any “junctional epitopes” have been created. A junctional epitope is a potential HLA binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are generally to be avoided because the recipient may bind to an HLA molecule and generate an immune response to that epitope, which is not present in a native protein sequence.

[0405] CTL epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Tables XXVII and XXIII-XXVI. Examples of HTL epitopes that can be included in vaccine compositions are provided in Table XXXI. A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response that results in tumor cell killing and reduction of tumor size or mass.

Example 11

[0406] Construction of Minigene Multi-Epitope DNA Plasmids

[0407] This example provides general guidance for the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of CTL and/or HTL epitopes or epitope analogs as described herein. Expression plasmids have been constructed and evaluated as described, for example, in co-pending U.S. Ser. No. 09/311,784 filed May 13, 1999.

[0408] A minigene expression plasmid may include multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. Preferred epitopes are identified, for example, in Tables XXVII, XXII-XXVI, and XXI. HLA class I supermotif or motif-bearing peptide epitopes derived from multiple TAAs are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from multiple tumor antigens to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

[0409] This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

[0410] The minigene DNA plasmid contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

[0411] Overlapping oligonucleotides, for example eight oligonucleotides, averaging approximately 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.

[0412] For the first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: Oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product for 25 additional cycles. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 12

[0413] The Plasmid Construct and the Degree to which It Induces Immunogenicity.

[0414] The degree to which the plasmid construct prepared using the methodology outlined in Example 11 is able to induce immunogenicity is evaluated through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analysed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in U.S. Ser. No. 09/311,784 filed May 13, 1999 and Alexander et al., Immunity 1:751-761, 1994.

[0415] Alternatively, plasmid constructs can be evaluated in vitro by testing for epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by infected or transfected target cells, and then determining the concentration of peptide necessary to obtained equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

[0416] To assess the capacity of the pMin minigene construct (e.g. a pMin minigene construct generated as decribed in U.S. Ser. No. 09/311,784) to induce CTLs in vivo, HLA-A11/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

[0417] Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 51Cr release assay. The results indicate the magnitude of the CTL response directed against the A3-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine. It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A3 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A2 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A2 and HLA-B7 motif or supermotif epitopes.

[0418] To assess the capacity of a class II epitope encoding minigene to induce HTLs in vivo, I-Ab restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

[0419] DNA minigenes, constructed as described in Example 11, may also be evaluated as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent may consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).

[0420] For example, the efficacy of the DNA minigene may be evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 μg of the DNA minigene encoding the immunogenic peptides. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an IFN-γ ELISA. It is found that the minigene utilized in a prime-boost mode elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis is also performed using other HLA-A11 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes.

Example 13

[0421] Peptide Composition for Prophylactic Uses

[0422] Vaccine compositions of the present invention are used to prevent cancer in persons who are at risk for developing a tumor. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in Examples 9 and/or 10, which are also selected to target greater than 80% of the population, is administered to an individual at risk for a cancer, e.g., breast cancer. The composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is administered in an aqueous carrier comprised of Freunds Incomplete Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against cancer.

[0423] Alternatively, the polyepitopic peptide composition can be administered as a nucleic acid in accordance with methodologies known in the art and disclosed herein.

Example 14

[0424] Polyepitopic Vaccine Compositions Derived from Native TAA Sequences

[0425] A native TAA polyprotein sequence is screened, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes and is preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct, even overlapping, epitopes is selected and used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 1000, 500, or 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with frame shifted overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

[0426] The vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from TAAs. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.

[0427] The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent analogs) directs the immune response to multiple peptide sequences that are actually present in native TAAs thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions.

[0428] Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.

Example 15

[0429] Polyepitopic Vaccine Compositions Directed to Multiple Tumors

[0430] The HER2/neu peptide epitopes of the present invention are used in conjunction with peptide epitopes from other target tumor antigens to create a vaccine composition that is useful for the treatment of various types of tumors. For example, a set of TAA epitopes can be selected that allows the targeting of most common epithelial tumors (see, e.g., Kawashima et al., Hum. Immunol. 59:1-14, 1998). Such a composition includes epitopes from CEA, HER-2/neu, and MAGE2/3, all of which are expressed to appreciable degrees (20-60%) in frequently found tumors such as lung, breast, and gastrointestinal tumors.

[0431] The composition can be provided as a single polypeptide that incorporates the multiple epitopes from the various TAAs, or can be administered as a composition comprising one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

[0432] Targeting multiple tumor antigens is also important to provide coverage of a large fraction of tumors of any particular type. A single TAA is rarely expressed in the majority of tumors of a given type. For example, approximately 50% of breast tumors express CEA, 20% express MAGE3, and 30% express HER-2/neu. Thus, the use of a single antigen for immunotherapy would offer only limited patient coverage. The combination of the three TAAs, however, would address approximately 70% of breast tumors. A vaccine composition comprising epitopes from multiple tumor antigens also reduces the potential for escape mutants due to loss of expression of an individual tumor antigen.

Example 16

[0433] Use of Peptides to Evaluate an Immune Response

[0434] Peptides of the invention may be used to analyze an immune response for the presence of specific CTL or HTL populations directed to a TAA. Such an analysis may be performed using multimeric complexes as described, e.g., by Ogg et al., Science 279:2103-2106, 1998 and Greten et al., Proc. Natl. Acad. Sci. USA 95:7568-7573, 1998. In the following example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

[0435] In this example, highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, tumor-associated antigen HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization using a TAA peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.

[0436] For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive uninfected donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the TAA epitope, and thus the stage of tumor progression or exposure to a vaccine that elicits a protective or therapeutic response.

Example 17

[0437] Use of Peptide Epitopes to Evaluate Recall Responses

[0438] The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who are in remission, have a tumor, or who have been vaccinated with a TAA vaccine.

[0439] For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any TAA vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

[0440] PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.

[0441] In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 105 irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific 51Cr release, based on comparison with uninfected control subjects as previously described (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).

[0442] Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).

[0443] Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.

[0444] Cytolytic activity is determined in a standard 4 hour, split-well 51Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release−spontaneous release)/maximum release−spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.

[0445] The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to the TAA or TAA vaccine.

[0446] The class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×105 cells/well and are stimulated with 10 μg/ml synthetic peptide, whole antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10U/ml IL-2. Two days later, 1 μCi 3H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.

Example 18

[0447] Induction of Specific CTL Response in Humans

[0448] A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study. Such a trial is designed, for example, as follows:

[0449] A total of about 27 subjects are enrolled and divided into 3 groups:

[0450] Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

[0451] Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;

[0452] Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

[0453] After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage. Additional booster inoculations can be administered on the same schedule.

[0454] The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

[0455] Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

[0456] Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

[0457] The vaccine is found to be both safe and efficacious.

Example 19

[0458] Therapeutic Use in Cancer Patients

[0459] Evaluation of vaccine compositions are performed to validate the efficacy of the CTL-HTL peptide compositions in cancer patients. The main objectives of the trials are to determine an effective dose and regimen for inducing CTLs in cancer patients, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of cancer patients, as manifested by a reduction in tumor cell numbers. Such a study is designed, for example, as follows:

[0460] The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.

[0461] There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65, include both males and females (unless the tumor is sex-specific, e.g., breast or prostate cancer), and represent diverse ethnic backgrounds.

Example 20

[0462] Induction of CTL Responses Using a Prime Boost Protocol

[0463] A prime boost protocol similar in its underlying principle to that used to evaluate the efficacy of a DNA vaccine in transgenic mice, which was described in Example 12, can also be used for the administration of the vaccine to humans. Such a vaccine regimen may include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

[0464] For example, the initial immunization can be performed using an expression vector, such as that constructed in Example 11, in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples will be obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

[0465] Analysis of the results will indicate that a magnitude of response sufficient to achieve protective immunity against cancer is generated.

Example 21

[0466] Administration of Vaccine Compositions Using Dendritic Cells

[0467] Vaccines comprising peptide epitopes of the invention can be administered using antigen-presenting cells, or “professional” APCs such as dendritic cells. In this example, the peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced lymphocytes then destroy or facilitate destruction of the specific target tumor cells that bear the proteins from which the epitopes in the vaccine are derived.

[0468] For example, a cocktail of epitope-bearing peptides is administered ex vivo to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

[0469] As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of dendritic cells reinfused into the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2-50×106 dendritic cells per patient are typically administered, larger number of dendritic cells, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% dendritic cells.

[0470] In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC containing DC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×106 DC, then the patient will be injected with a total of 2.5×108 peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

[0471] Ex Vivo Activation of CTL/HTL Responses

[0472] Alternatively, ex vivo CTL or HTL responses to a particular tumor-associated antigen can be induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.

Example 22

[0473] Alternative Method of Identifying Motif-Bearing Peptides

[0474] Another way of identifying motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing, have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism or transfected with nucleic acids that express the tumor antigen of interest. Thereafter, peptides produced by endogenous antigen processing of peptides produced consequent to infection (or as a result of transfection) will bind to HLA molecules within the cell and be transported and displayed on the cell surface.

[0475] The peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because, as disclosed herein, the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

[0476] Alternatively, cell lines that do not express any endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells may then be used as described, i.e., they may be infected with a pathogenic organism or transfected with nucleic acid encoding an antigen of interest to isolate peptides corresponding to the pathogen or antigen of interest that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

[0477] As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than infection or transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

[0478] The above examples are provided to illustrate the invention but not to limit its scope. For example, the human terminology for the Major Histocompatibility Complex, namely HLA, is used throughout this document. It is to be appreciated that these principles can be extended to other species as well. Thus, other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, and patent application cited herein are hereby incorporated by reference for all purposes.

2TABLE IPOSITIONPOSITIONPOSITIONC Terminus2 (Primary Anchor)3 (Primary Anchor)(Primary Anchor)SUPER-MOTIFSA1T, I, L, V, M, SF, W, YA2L, I, V, M, A, T,I, V, M, A, T, LQA3V, S, M, A, T, L,R, KIA24Y, F, W, I, V, L,F, I, Y, W, L, MM, TB7PV, I, L, F, M, W,Y, AB27R, H, KF, Y, L, W, M, I,V, AB44E, DF, W, L, I, M, V,AB58A, T, SF, W, Y, L, I, V,M, AB62Q, L, I, V, M, PF, W, Y, M, I, V,L, AMOTIFSA1T, S, MYA1D, E, A, SYA2.1L, M, V, Q, I, A,V, L, I, M, A, TTA3L, M, V, I, S, A,K, Y, R, H, F, AT, F, C, G, DA11V, T, M, L, I, S,K, R, Y, HA, G, N, C, D, FA24Y, F, W, MF, L, I, WA*3101M, V, T, A, L, I,R, KSA*3301M, V, A, L, F, I,R, KS, TA*6801A, V, T, M, S, L,R, KIB*0702PL, M, F, W, Y, A,I, VB*3501PL, M, F, W, Y, I,V, AB51PL, I, V, F, W, Y,A, MB*5301PI, M, F, W, Y, A,L, VB*5401PA, T, I, V, L, M,F, W, YBolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

[0479]

3

TABLE II

POSITION

SUPER-

MOTIFS

A1

1° Anchor

{overscore (T,I,L,V,M,S)}

A2

1° Anchor

L,I,V,M,A,

T
,Q

A3
preferred

1° Anchor

Y,F,W,(4/5)

V,S,M,A,T,

L
,I

deleterious
D,E (3/5); P,(5/5)

D,E,(4/5)

A24

1° Anchor

Y,F,W,I,V,

L
,M,T

B7
preferred
F,W,Y (5/5)

1° Anchor

F,W,Y (4/5)

L,I,V,M,(3/5)
P

deleterious
D,E (3/5); P(5/5);

G(4/5); A(3/5);

Q,N,(3/5)

B27

1° Anchor

R,H,K

B44

1° Anchor

E,D

B58

1° Anchor

A,T,S

B62

1° Anchor

Q,L,I,V,M,

P

MOTIFS

A1
preferred
G,F,Y,W,

1° Anchor

D,E,A,
Y,F,W,

9-mer

S,T,M,

deleterious
D,E,

R,H,K,L,I,V
A,

M,P,

A1
preferred
G,R,H,K
A,S,T,C,L,I

1° Anchor

G,S,T,C,

9-mer

V,M,
D,E,A,S

deleterious
A
R,H,K,D,E,

D,E,

P,Y,F,W,

POSITION

C-terminus

SUPER-

MOTIFS

A1

1° Anchor

F,W,Y

A2

1° Anchor

{overscore (L,I,V,M,A,T)}

A3
preferred

Y,F,W,
Y,F,W,(4/5)
P,(4/5)

1° Anchor

(3/5)

R,K

deleterious

A24

1° Anchor

{overscore (F,I,Y,W,L,M)}

B7
preferred

F,W,Y,
1° Anchor

(3/5)
{overscore (V,I,L,F,M,W,Y,A)}

deleterious
D,E,(3/5)
G,(4/5)
Q,N,(4/5)
D,E,(4/5)

B27

1° Anchor

{overscore (F,Y,L,W,M,V,A)}

B44

1° Anchor

{overscore (F,W,Y,L,I,M,V,A)}

B58

1° Anchor

{overscore (F,W,Y,L,I,V,M,A)}

B62

1° Anchor

{overscore (F,W,Y,M,I,V,L,A)}

MOTIFS

A1
preferred

P,
D,E,Q,N,
Y,F,W,

1° Anchor

9-mer

Y

deleterious
G,
A,

A1
preferred

A,S,T,C,
L,I,V,M,
D,E,

1° Anchor

9-mer

Y

deleterious
P,Q,N,
R,H,K,
P,G,
G,P,

POSITION

A1
preferred
Y,F,W,

1° Anchor

D,E,A,Q,N,
A,
Y,F,W,Q,N,

10-mer

S,T,M

deleterious
G,P,

R,H,K,G,L,I
D,E,
R,H,K,

V,M,

A1
preferred
Y,F,W,
S,T,C,L,I,V

1° Anchor

A,
Y,F,W,

10-mer

M,
D,E,A,S

deleterious
R,H,K,
R,H,K,D,E,

P,

P,Y,F,W,

A2.1
preferred
Y,F,W,

1° Anchor

Y,F,W,
S,T,C,
Y,F,W,

9-mer

L,M,I,V,Q,

A
,T

deleterious
D,E,P,

D,E,R,K,H

A2.1
preferred
A,Y,F,W,

1° Anchor

L,V,I,M,
G,

10-mer

L,M,I,V,Q,

A
,T

deleterious
D,E,P,

D,E,
R,K,H,A,
P,

A3
preferred
R,H,K,

1° Anchor

Y,F,W,
P,R,H,K,Y,
A,

L,M,V,I,S,

F,W,

A,T,F,C,G,

D

deleterious
D,E,P,

D,E

A11
preferred
A,

1° Anchor

Y,F,W,
Y,F,W,
A,

V,T,L,M,I,

S,A,G,N,C,

D
,F

deleterious
D,E,P,

A24
preferred
Y,F,W,R,H,K,

1° Anchor

S,T,C

9-mer

Y,F,W,M

deleterious
D,E,G,

D,E,
G,
Q,N,P,

A24
preferred

1° Anchor

P,
Y,F,W,P,

10-mer

Y,F,W,M

deleterious

G,D,E
Q,N
R,H,K

A3101
preferred
R,H,K,

1° Anchor

Y,F,W,
P,

M,V,T,A,L,

I
,S

deleterious
D,E,P,

D,E,

A,D,E,

A3301
preferred

1° Anchor

Y,F,W

M,V,A,L,F,

I
,S,T

deleterious
G,P

D,E

A6801
preferred
Y,F,W,S,T,C,

1° Anchor

Y,F,W,L,I,

A,V,T,M,S,

V,M

L
,I

deleterious
G,P,

D,E,G,

R,H,K,

B0702
preferred
R,H,K,F,W,Y,

1° Anchor

R,H,K,

P

deleterious
D,E,Q,N,P,

D,E,P,
D,E,
D,E,

B3501
preferred
F,W,Y,L,I,V,M,

1° Anchor

F,W,Y,

P

deleterious
A,G,P,

G,

B51
preferred
L,I,V,M,F,W,Y,

1° Anchor

F,W,Y,
S,T,C,
F,W,Y,

P

deleterious
A,G,P,D,E,R,H,K,

DE,

S,T,C,

B5301
preferred
L,I,V,M,F,W,Y,

1° Anchor

F,W,Y,
S,T,C,
F,W,Y,

P

deleterious
A,G,P,Q,N,

B5401
preferred
F,W,Y,

1° Anchor

F,W,Y,L,I,V,

L,I,V,M,

P
M,

deleterious
G,P,Q,N,D,E,

G,D,E,S,T,C,

R,H,K,D,E,

POSITION

C-terminus
C-terminus

A1
preferred

P,A,S,T,C,
G,D,E,
P,

1° Anchor

10-mer

Y

deleterious
Q,N,A
R,H,K,Y,F,
R,H,K,
A

W,

A1
preferred

P,G,
G,
Y,F,W,

1° Anchor

10-mer

Y

deleterious
G,

P,R,H,K,
Q,N,

A2.1
preferred

A,
P
1° Anchor

9-mer

{overscore (V,L,I,M,A,T)}

deleterious
R,K,H
D,E,R,K,H

A2.1
preferred
G,

F,Y,W,L,

1° Anchor

10-mer

V,I,M,

{overscore (V,L,I,M,A,T)}

deleterious

R,K,H,
D,E,R,K,
R,K,H,

H,

A3
preferred
Y,F,W,

P,
1° Anchor

{overscore (K,Y,R,H,F,A)}

deleterious

A11
preferred
Y,F,W,
Y,F,W,
P,

1° Anchor

K,,RY,H

deleterious

A
G

A24
preferred

Y,F,W,
Y,F,W,

1° Anchor

9-mer

F,L,I,W

deleterious
D,E,R,H,K,
G,
A,Q,N,

A24
preferred

P,

1° Anchor

10-mer

F,L,I,W

deleterious
D,E
A
Q,N,
D,E,A,

A3101
preferred
Y,F,W,
Y,F,W,
A,P,

1° Anchor

R,K

deleterious
D,E,
D,E,
D,E,

A3301
preferred

A,Y,F,W

1° Anchor

R,K

deleterious

A6801
preferred

Y,F,W,
P,

1° Anchor

R,K

deleterious

A,

B0702
preferred
R,H,K,
R,H,K,
R,H,K,
P,A,
1° Anchor

{overscore (L,M,F,W,Y,A,)}

I
,V

deleterious
G,D,E,
Q,N,
D,E,

B3501
preferred

F,W,Y,

1° Anchor

{overscore (L,M,F,W,Y,I,)}

V
,A

deleterious
G,

B51
preferred

G,
F,W,Y,

1° Anchor

L,I,V,F,W,

Y
,A,M

deleterious
G,
D,E,Q,N,
G,D,E,

B5301
preferred

L,I,V,M,F,
F,W,Y,

1° Anchor

W,Y,

I,M,F,W,Y,

A
,L,V

deleterious
G,
R,H,K,Q,N,
D,E,

B5401
preferred

A,L,I,V,M,
F,W,Y,A,P,

1° Anchor

A,T,I,V,L,

M
,F,W,Y

deleterious
D,E,
Q,N,D,G,E,
D,E,

Italicized residues indicate less preferred or “tolerated” residues.

The information in Table II is specific for 9-mers unless otherwise specified.

Secondary anchor specificities are designated for each position independently.

[0480]

4

TABLE III

POSITION

MOTIFS

DR4
preferred
F, M, Y, L, I,
M,
T,

I,
V, S, T, C, P, A,
M, H,

M, H

V, W,

L, I, M,

deleterious

W,

R,

W, D, E

DR1
preferred
M, F, L, I, V,

P, A, M, Q,

V, M, A, T, S, P,
M,

A, V, M

W, Y,

L, I, C,

deleterious

C
C, H
F, D
C, W, D

G, D, E,
D

DR7
preferred
M, F, L, I, V,
M,
W,
A,

I, V, M, S, A, C,
M,

I ,V

W, Y,

T, P, L,

deleterious

C,

G,

G, R, D,
N
G

DR Supermotif
M, F, L, I, V,
V, M, S, T, A, C,

W, Y,
P, L, I,

DR3 MOTIFS

motif a
L, I, V, M, F,

preferred
Y,

D

motif b
L, I, V, M, F,

D, N, Q, E,

preferred
A, Y,

S, T

K, R, H

Italicized residues indicate less preferred or “tolerated” residues. Secondary anchor specificities are designated for each position independently.

[0481]

5

TABLE IV

HLA Class I Standard Peptide Binding Affinity.

STANDARD
SEQUENCE
STANDARD BINDING

ALLELE
PEPTIDE
(SEQ ID NO:)
AFFINITY (nM)

A*0101
944.02
YLEPAIAKY
25

A*0201
941.01
FLPSDYFPSV
5.0

A*0202
941.01
FLPSDYFPSV
4.3

A*0203
941.01
FLPSDYFPSV
10

A*0205
941.01
FLPSDYFPSV
4.3

A*0206
941.01
FLPSDYFPSV
3.7

A*0207
941.01
FLPSDYFPSV
23

A*6802
1072.34
YVIKVSARV
8.0

A*0301
941.12
KVFPYALINK
11

A*1101
940.06
AVDLYHFLK
6.0

A*3101
941.12
KVFPYALINK
18

A*3301
1083.02
STLPETYVVRR
29

A*6801
941.12
KVFPYALINIK
8.0

A*2402
979.02
AYIDNYNKF
12

B*0702
1075.23
APRTLVYLL
5.5

B*3501
1021.05
FPFKYAAAF
7.2

B51
1021.05
FPFKYAAAF
5.5

B*5301
1021.05
FPFKYAAAF
9.3

B*5401
1021.05
FPFKYAAAF
10

[0482]

6

TABLE V

HLA Class II Standard Peptide Binding Affinity.

Stan-

Binding

Nomen-
dard
Sequence
Affinity

Allele
clature
Peptide
(SEQ ID NO:)
(nM)

DRB1*0101
DR1
515.01
PKYVKQNTLKLAT
5.0

DRB1*0301
DR3
829.02
YKTIAFDEEARR
300

DRB1*0401
DR4w4
515.01
PKYVKQNTLKLAT
45

DRB1*0404
DR4w14
717.01
YARFQSQTTLKQKT
50

DRB1*0405
DR4w15
717.01
YARFQSQTTLKQKT
38

DRB1*0701
DR7
553.01
QYIKANSKFIGITE
25

DRB1*0802
DR8w2
553.01
QYIKANSKFIGITE
49

DRB1*0803
DR8w3
553.01
QYIKANSKFIGITE
1600

DRB1*0901
DR9
553.01
QYIKANSKFIGITE
75

DRB1*1101
DR5w11
553.01
QYIKANSKFIGITE
20

DRB1*1201
DR5w12
1200.05
EALIHQLKINPYVLS
298

DRB1*1302
DR6w19
650.22
QYIKANAKFIGITE
3.5

DRB1*1501
DR2w2β1
507.02
GRTQDENPVVHFFKNIV
9.1

TPRTPPP

DRB3*0101
DR52a
511
NGQIGNDPNRDIL
470

DRB4*0101
DRw53
717.01
YARFQSQTTLKQKT
58

DRB5*0101
DR2w2β2
553.01
QYIKANSKFIGITE
20

[0483]

7

TABLE VI

HLA-
Allelle-specific HLA-supertype members

supertype
Verifieda
Predictedb

A1
A*0101, A*2501, A*2601,
A*0102, A*2604, A*3601,

A*2602, A*3201
A*4301, A*8001

A2
A*0201, A*0202, A*0203,
A*0208, A*0210, A*0211,

A*0204, A*0205, A*0206,
A*0212, A*0213

A*0207, A*0209, A*0214,

A*6802, A*6901

A3
A*0301, A*1101, A*3101,
A*0302, A*1102, A*2603,

A*3301, A*6801
A*3302, A*3303, A*3401,

A*3402, A*6601, A*6602,

A*7401

A24
A*2301, A*2402, A*3001
A*2403, A*2404, A*3002,

A*3003

B7
B*0702, B*0703, B*0704,
B*1511, B*4201, B*5901

B*0705, B*1508, B*3501,

B*3502, B*3503, B*3503,

B*3504, B*3505, B*3506,

B*3507, B*3508, B*5101,

B*5102, B*5103, B*5104,

B*5105, B*5301, B*5401,

B*5501, B*5502, B*5601,

B*5602, B*6701, B*7801

B27
B*1401, B*1402, B*1509,
B*2701, B*2707, B*2708,

B*2702, B*2703, B*2704,
B*3802, B*3903, B*3904,

B*2705, B*2706, B*3801,
B*3905, B*4801, B*4802,

B*3901 ,B*3902, B*7301
B*1510, B*1518, B*1503

B44
B*1801, B*1802, B*3701,
B*4101, B*4501, B*4701,

B*4402, B*4403, B*4404,
B*4901, B*5001

B*4001, B*4002, B*4006

B58
B*5701, B*5702, B*5801,

B*5802, B*1516, B*1517

B62
B*1501, B*1502, B*1513,
B*1301, B*1302, B*1504,

B*5201
B*1505, B*1506, B*1507,

B*1515, B*1520, B*1521,

B*1512, B*1514, B*1510

a
Verified alleles include alleles whose specificity has been determined by pooi sequencing analysis, peptide binding assays, or by analysis of the sequences of CTL epitopes.

b
Predicted alleles are alleles whose specificity is predicted on the basis of B and F pocket structure to overlap with the supertype specificity.

[0484]

8

TABLE VII

HER2/NEU A01 Supermotif Peptides with Binding Data

No. of

Position
Amino Acids
A*0101

66
8

272
8

732
8

899
8

296
8
0.1000

916
8
−0.0021

1241
8
0.0030

166
8

369
8

76
8

434
8

828
8
−0.0021

945
8

418
8

1023
8

2
8

607
8

402
8
−0.0021

1016
8

101
8

479
8

664
8

724
8

911
8

1024
8

1180
8

603
8

796
8

952
8

357
8

892
8

962
8

997
8

818
8

906
8

165
9

356
9

478
9

910
9

104
9
0.1800

401
9
0.0430

1131
9
0.1300

100
9

373
9

1023
9

444
9

513
9

42
9
9.100

546
9
0.0050

795
9
0.0024

869
9
7.6000

1119
9
0.0017

271
9

663
9

1179
9

295
9
0.0042

978
9

197
9

281
9
0.0028

773
9
0.0400

915
9
0.0011

417
9

608
9

293
9
0.0550

727
9
0.0011

997
9
0.0290

1213
9
0.0430

525
10

406
10

443
10

899
10
2.7000

1239
10
0.0630

467
10

960
10

154
10
0.0300

64
10

270
10

391
10

607
10

662
10

890
10

160
10

473
10

816
10

265
10
0.0015

402
10
1.1000

868
10
1.3000

914
10
0.0082

1130
10
0.0072

355
10

722
10

909
10

904
10

950
10

55
10
0.0180

545
10
0.0015

772
10
1.1000

826
10
0.3000

249
10

372
10

1077
10

280
10
0.1800

334
10
0.0016

601
10
0.0010

1213
10
5.5000

40
11
0.2800

401
11
0.4400

1102
11
0.0160

405
11

98
11

466
11

661
11

442
11

73
11

153
11

725
11

476
11

54
11

793
11

1117
11

281
11

959
11

1013
11
0.0027

854
11

976
11

195
11

1213
11

293
11
0.1900

[0485]

9

TABLE VII

HER2/NEU A01 Supermotif Peptides with Binding Data

No. of

Position
Amino Acids
A*0101

869
9
7.6000

1119
9
0.0017

271
9

663
9

1179
9

295
9
0.0042

978
9

197
9

281
9
0.0028

773
9
0.0400

915
9
0.0011

417
9

608
9

293
9
0.0550

727
9
0.0011

997
9
0.0290

1213
9
0.0430

525
10

406
10

443
10

899
10
2.7000

1239
10
0.0630

467
10

960
10

154
10
0.0300

64
10

270
10

391
10

607
10

662
10

890
10

160
10

473
10

816
10

265
10
0.0015

402
10
1.1000

868
10
1.3000

914
10
0.0082

1130
10
0.0072

355
10

722
10

909
10

904
10

950
10

55
10
0.0180

545
10
0.0015

772
10
1.1000

826
10
0.3000

249
10

372
10

[0486]

10

TABLE VII

HER2/NEU A01 Supermotif Peptides with Binding Data

No. of

Position
Amino Acids
A*0101

1077
10

280
10
0.1800

334
10
0.0016

601
10
0.0010

1213
10
5.5000

40
11
0.2800

401
11
0.4400

1102
11
0.0160

405
11

98
11

466
11

661
11

442
11

73
11

153
11

725
11

476
11

54
11

793
11

1117
11

281
11

959
11

1013
11
0.0027

854
11

976
11

195
11

1213
11

293
11
0.1900

[0487]

11

TABLE VIII

HER2/NEU A02 Supermotif with Binding Data

No. of

Position
Amino Acids
A*0201
A*0202
A*0203
A*0206
A*6802

1094
8

1094
10

4
8

4
9

4
10
0.0010

4
11

1203
10

1159
8

1159
9
0.0001

20
8

20
10

751
9

113
11

5
8

5
9
0.0310

5
10
0.0360
0.0022
0.8600
0.0019
0.0160

5
11

890
11

466
9
0.0210

14
8

14
10
0.0001

270
9
0.0001

705
8

705
10
0.0007

705
11

710
9

710
11

1165
8

1165
9

1190
8

1190
9

115
9
0.0004

355
11

657
8

657
9
0.0007

657
10
0.0002

657
11

587
11

224
8

224
10

338
8

338
10
0.0011

255
9

789
8

789
9
0.0340

789
10

826
8

826
11

623
9

567
8

567
9

212
8

212
10

53
8

53
10

53
11

244
10

244
11

26
8

26
10

630
8

947
8

947
9

947
10

596
9
0.0004

634
11

540
8

540
11

504
11

528
8

295
10
0.0001

871
9

171
9
0.0002

171
10

171
11

76
9
0.0001

76
10
0.0001

76
11

845
8

845
9
0.0002

636
9

1089
10
0.0001

993
8

993
11

933
9
0.0002

821
9
0.0002

821
10

421
8

421
10
0.0003

421
11

1016
9
0.0002

1016
10
0.0002

1013
8

30
8

1224
9

483
10

483
11

165
8

1183
8

1183
9
0.0002

1084
9

1084
11

307
8

307
11

838
9
0.0002

838
10

838
11

904
8

904
9
0.0002

950
11

580
9

1069
8

770
8

766
8

766
9

766
10

147
8

147
9
0.0001

405
8

405
10

2
10
0.0001

2
11

460
8

460
9
0.0004

265
8

265
9

139
8

139
10

139
11

719
8

61
9

61
11

695
11

971
9
0.0001

971
10
0.0001

238
8

395
8

395
9

645
8

645
10

645
11

1123
9

1123
10

717
9

717
10

693
8

693
9

874
11

40
10

401
10

79
8

79
9

352
8

352
10

321
8

364
11

376
11

73
8

534
8

425
11

476
10
0.0001

986
9
0.0002

1093
9
0.0001

1093
11

1202
11

19
9

19
11

621
8

621
11

729
10
0.0001

1080
11

919
8

919
10

704
9
0.0002

704
11

1231
10

131
10
0.0001

1164
9

1164
10
0.0002

1189
9
0.0002

1189
10

439
10
0.0030

262
9

262
10
0.0005

262
11

787
8

787
10
0.0004

787
11

672
11

660
8

660
10
0.0007

660
11

925
10
0.0001

925
11

449
10
0.0003

737
10
0.0002

508
9
0.0120
0.0001
0.0790
0.0001
0.0044

464
9

464
11

865
8

865
11

1062
9

447
9
0.0018

344
10
0.0017

10
11

549
9

136
10
0.0001

136
11

454
8

346
8

346
10

346
11

1091
8

1091
11

832
8

832
10
0.0017

832
11

537
10

537
11

28
8

28
10

1239
11

104
10

104
11

732
9

776
10
0.0001

776
11

603
9

603
11

152
10
0.0036

909
8

909
9

668
8

1179
8

878
9
0.0002

473
8

42
8

349
8

349
9

349
11

48
8

48
9
0.0340

490
8

901
10

901
11

495
11

478
8

858
9
0.0002

858
10

809
9
0.0002

86
9

86
11

829
8

829
11

654
8

654
9
0.0005

654
10
0.0120

654
11

767
8

767
9
0.0210
0.0001
0.0024
0.0012
0.0003

767
11

435
9
0.2100

435
10

435
11

673
10

714
10
0.0001

148
8

661
9
0.0020

661
10
0.0006

954
8

361
10

77
8

77
9

77
10

77
11

989
9

861
9

861
10

406
9

762
10

369
9
0.1500

747
8

747
9
0.0002

681
10
0.0001

681
11

860
8

860
10
0.0020

860
11

32
10

1171
10

1171
11

722
9

724
10

724
11

753
11

883
8

883
9
0.0002

3
9

3
10
0.0022

3
11

846
8

509
8

253
11

465
8

465
10

13
8

13
9

13
11

179
8

1075
11

866
10

114
10
0.0002

85
10
0.0001

183
9

467
8

467
11

674
9

154
8

12
9
0.0008

12
10
0.0006

869
11

1008
10
0.0001

1008
11

934
8

785
9

785
10
0.0490

11
10
0.0054

11
11

822
8

822
9
0.0046

15
9
0.0007

15
11

690
8

690
11

662
8

662
9
0.0001

800
8

800
11

74
11

691
10

691
11

445
9

445
11

547
9

547
11

140
9

140
10

392
8

392
11

96
10

1109
9

1109
10

397
10

397
11

428
8

428
9

1131
10

145
9

145
10

145
11

181
11

443
8

443
11

1197
8

700
11

215
11

651
8

651
9

651
11

790
8

790
9

790
11

62
8

62
10

313
10
0.0002

313
11

1017
8

1017
9
0.0030

696
10

696
11

841
8

841
11

852
8

852
10

852
11

972
8
0.0001

972
9
0.0001

796
11

271
8

663
8

663
11

774
9
0.0014

979
9
0.0001

979
10

979
11

953
8

953
9
0.0051

45
11

827
10

916
11

1042
11

68
10
0.0001

360
11

59
9

59
11

427
9

427
10
0.0001

708
8

708
11

319
10

177
8

177
10

89
8

89
11

388
10

275
8

471
9

471
10

758
10

758
11

125
8

745
8

745
10
0.0001

745
11

850
10
0.0001

1158
8

1158
9

1158
10
0.0001

643
9
0.0001

643
10

1211
10
0.0001

1162
11

269
8

269
10

1035
8

1035
9

927
8

927
9
0.0001

385
8

36
8

36
10
0.0001

36
11

996
8

945
9

945
10

945
11

885
10

885
11

627
9
0.0002

627
11

612
11

1074
8

999
10
0.0001

999
11

316
8

316
9

122
8

122
9

122
11

1156
8

1156
10

1156
11

1119
10
0.0001

1119
11

391
9
0.0002

95
8

95
11

1108
10

1108
11

1130
11

699
8

650
8

650
9
0.0015

650
10
0.0003

159
8

159
9

159
10

1135
11

1205
8

942
8

942
10

1147
11

1026
11

1241
9

1241
11

232
10

232
11

1102
8

66
9

525
11

1116
9

749
11

128
10
0.0001

709
10

828
9
0.0006

178
9

160
8

160
9
0.0001

106
8

106
9
0.4600

106
10
0.0140
0.0065
1.1000
0.0170
0.5400

106
11

484
9
0.0062

484
10
0.0003

484
11

799
8

799
9
0.0230
0.0044
0.0880
0.0052
0.0031

396
8

396
11

141
8

141
9
0.0008

711
8

711
10
0.0001

1027
10

679
8

679
9

24
8

24
10
0.0001

398
9

398
10

429
8

93
9

93
10
0.0001

90
10
0.0001

54
9
0.0001

54
10

190
9
0.0001

647
8

647
9
0.0002

647
11

354
8

434
10
0.0180

434
11

713
8

713
11

100
8

816
8

868
8

784
8

784
10

784
11

689
8

689
9
0.0910

34
8

34
10
0.0001

940
9
0.0002

940
10

98
8

98
10
0.0001

840
8

840
9
0.0001

978
10
0.0020

978
11

678
9

678
10

92
8

92
10

92
11

217
9

340
8

340
11

545
11

358
8

656
8

656
9

656
10
0.0009

656
11

413
10
0.0002

653
9
0.0720
0.0082
0.2900
0.0130
0.2700

653
10
0.0002

653
11

893
8

1007
8

1007
11

418
11

1100
10
0.0059

457
9
0.0002

457
10

457
11

70
8

70
11

144
10
0.0150

144
11

442
9
0.0003

214
8

281
10

819
9

819
11

532
10

305
8

305
9

305
10
0.0001

1235
8

1235
9

1002
8

22
8

22
10

1051
9

1051
10

1051
11

792
9

792
10

423
8

423
9
0.0017

573
9

297
8

297
10

1242
8

1242
10
0.0001

496
10

389
9
0.0002

389
11

948
8

948
9
0.0005

402
9
0.0018

402
11

1166
8

1060
11

182
10

444
10
0.0011

1172
9
0.0002

1172
10
0.0001

312
11

686
9

686
11

526
10

105
9

105
10

105
11

798
9

798
10

23
9

23
11

218
8

1117
8

793
8

793
9

911
10

733
8

750
10

597
8

988
10

430
11

116
8

116
11

725
9

725
10
0.0007

666
8

666
9
0.0005

666
10

84
8

84
11

153
9
0.0290

546
10
0.0009

754
10

851
9
0.0002

851
11

773
10
0.0180

56
8

56
10

80
8

794
8

296
9

296
11

574
8

129
9

797
10

797
11

356
10

910
8

910
11

272
11

658
8

658
9
0.0005

658
10
0.0009

987
8

987
11

1180
11

665
9
0.3500
0.0001
0.0040
0.0086
0.0200

665
10
0.0027

665
11

55
8

55
9

55
11

664
10
0.0032

664
11

739
8

739
10

452
10
0.0001

888
8

411
9
0.0003

835
8

1248
8

64
8

64
11

303
10
0.0002

303
11

1196
8

1196
9
0.0001

409
11

952
9
0.0230
0.0001
0.0160
0.0014
0.0400

952
10
0.0600
0.0004
0.0300
0.0190
0.0011

163
10

50
11

289
8

289
9

289
10

685
10

83
9
0.0005

772
11

554
8

781
8

781
10
0.0004

781
11

[0488]

12

TABLE IX

HER2/NEU A03 Supermotif with Binding Data

No. of

Position
Amino Acids
A*0301
A*1101
A*3101
A*3301
A*6801

241
8

847
8

1159
11

890
8

890
9
0.0013
0.0006

492
8

180
9
0.0004
0.0005

180
11

705
9
0.0004
0.0006

37
11

997
10
0.0003
0.0670
0.1200
0.0140
0.0520

240
9
0.0021
0.0021

220
9
−0.0002
−0.0002

805
10
0.0003
0.0001

195
9
−0.0008
−0.0001

26
9
0.0002
0.0005

630
11

947
11

584
8

596
10
0.0220
0.0042
0.0008
0.0064
0.0093

528
9
0.0015
0.0310
0.5300
0.5800
0.4400

845
10
0.0018
0.0007

1089
8

933
8

821
11

607
9
0.0005
0.0100
0.0002
0.0880
0.0310

962
9
−0.0002
−0.0002

165
11

1144
10
0.0003
0.0001

950
8

147
11

930
8

930
11

914
8

971
8

971
11

280
9
0.0003
−0.0002

207
11

717
8

874
10
0.0003
0.0001

40
8

321
10
0.0002
0.0001

976
10
−0.0002
0.0010

729
8

1038
9
−0.0002
0.0043

1038
11

919
11

704
10
−0.0002
0.0041

1231
8

131
8

1164
8

672
10
0.0150
0.0014

449
8

449
11

737
11

508
10
0.0110
0.0001

1062
11

447
10
0.0037
0.0001

344
8

344
11

549
10
0.0002
0.0003

136
8

346
9
−0.0002
−0.0002

832
9
−0.0002
0.0002

1041
8

727
10
0.0660
0.1300
0.0014
−0.0013
0.0012

327
10
0.0210
0.6100
0.0140
0.0012
0.0100

776
9
0.0010
0.0066

668
9
0.0047
0.0890
0.0019
0.0025
0.0011

668
10
0.0180
0.0330
0.0590
0.0140
0.4300

668
11

878
10
0.0003
0.0008

632
9
−0.0002
0.0007

478
10
0.0035
0.0720
0.9600
0.3300
2.0000

858
11

809
8

673
9
0.3800
0.0097
0.0760
0.0064
0.0001

673
11

714
8

714
9
0.0190
0.0023
0.0009
0.0010
0.0001

714
11

148
10
0.0400
0.0005
0.7300
0.2400
0.0390

167
9
0.2800
0.3100
0.2200
0.0300
0.0046

450
10
0.0410
0.0027
2.6000
0.1300
0.1100

861
8

747
10
0.0009
0.0099

681
8
0.0010
0.0004

681
9
0.7600
0.0018
1.1000
0.0072
0.0002

860
9
0.1700
0.2400
0.1800
0.0012
0.0049

753
10
0.3800
0.2200
0.0068
0.0012
0.0008

846
9
0.0580
0.0285
−0.0005
−0.0012
0.0160

509
9
−0.0002
0.0003

179
10
−0.0002
0.0003

85
8

183
8

674
8

674
10
0.0002
0.0001

674
11

806
9
0.0370
0.0006
0.0360
0.0890
0.0014

806
11

822
10
0.1400
0.1400
0.0100
0.0088
0.0086

1173
10
−0.0002
0.0003

422
11

608
8

181
8

181
10
0.0002
0.0005

841
9
0.0040
0.0014

852
9
0.4800
0.0700
0.0990
0.0370
0.1100

972
10
0.0072
0.0330
0.3700
0.2300
0.2200

774
11

889
8

889
9
0.0034
0.0237
0.0940
0.2200
0.0630

889
10
0.0011
0.0003

960
9
0.0017
0.0006

960
11

833
8

360
9
0.0002
0.0036

360
10
0.0003
0.0056

427
8

758
8

745
9
0.0058
0.0007
0.0015
0.0820
0.1200

850
11

1162
8

1162
10
−0.0002
−0.0002

927
11

996
11

999
8

95
9
0.0002
0.0001

1065
8

1102
10
0.0003
0.0001

749
8

128
11

491
9
0.0046
0.0010

709
8

178
11

160
11

141
10
0.2000
0.0130
0.0270
0.0047
0.0002

711
11

24
9
0.0007
0.0520
0.0002
0.0006
0.0110

24
11

93
8

93
11

90
9
0.0029
0.0005

90
11

190
11

713
9
0.0007
0.0038

713
10
0.0570
0.1100
0.0055
0.0013
0.0002

840
10
0.1800
0.0001
0.9500
0.0021
0.0036

978
8

143
8

217
10
0.0068
0.0130
0.4500
0.0220
0.0250

545
8

358
11

281
8

208
10
−0.0002
0.0020

22
11

423
10
0.0170
0.0750
0.0340
0.0390
0.2500

323
8

323
11

948
10
0.0130
0.1200
0.0018
0.0120
0.0250

166
10
0.0430
3.6000
0.0370
0.0420
0.0400

182
9
0.0004
0.0005

1172
11

218
9
0.0004
0.0230
0.1400
0.0890
0.0970

218
11

479
9
0.0006
0.0072

911
11

597
9
0.0100
−0.0002

666
11

84
9
0.0033
0.0007

754
9
0.4000
0.0130
0.1400
0.1000
0.0001

851
10
0.0820
0.0072
0.0052
0.0032
0.0005

973
9
−0.0002
0.0021

322
9
0.0002
0.0140
0.0011
0.0037
0.1000

129
10
0.0002
0.0005

669
8

669
9
0.1100
0.7200
1.4000
0.3700
2.0000

669
10
0.0030
0.0160
0.0620
0.1500
0.5400

739
9
0.0002
0.0001

452
8

888
9
−0.0002
−0.0002

888
10
0.0085
0.0016

888
11

959
8

959
10
−0.0002
0.0002

835
10
0.0003
0.0001

83
10
0.0043
0.0013

1139
8

[0489]

13

TABLE X

HER2/NEU A24 Supermotif Peptides with Binding Data

No. of

Position
Amino Acids
A*2401

1216
8
0.0039

1186
11

730
9
0.0002

730
10
0.0010

730
11
0.0008

1212
9
0.0011

1212
10

1212
11

113
11

5
8

5
9

5
11

890
10

466
9

466
11

270
10

705
8

705
10
0.0002

705
11
−0.0003

1165
9

1190
8

115
9

355
10

657
11

414
9
0.0041

440
9
0.1300

440
11
0.0230

771
11

475
8
0.0190

475
11
0.0003

255
9

789
8

826
8

826
10

826
11
−0.0003

244
10

26
8

26
10

630
8

947
8

947
9

540
8

540
11

504
11

528
8

295
9

295
10

342
9
0.0180

162
8
0.0120

162
11
0.0016

863
8
0.0005

863
10
0.0002

171
9

171
11

76
8

76
10

76
11

845
8

1089
10

993
8

933
9

821
9

421
8

421
11

607
8

607
10

1016
8

1016
9

1013
11

165
8

165
9

1084
9

307
11

838
9

904
10

950
10

950
11

363
8
−0.0003

363
9
0.0003

766
9

147
8

147
9

405
8

405
11

2
8

2
10

2
11

460
8

460
9

265
10

914
10

139
10

139
11

719
8

61
11

971
9

1123
9

717
10

693
8

40
10

40
11

401
9

401
10

401
11

79
8

352
10

876
11
−0.0003

1022
9
0.0014

1022
10
0.0120

553
9
0.0061

73
11

899
8

899
10

476
10

476
11

986
9

1004
11

262
10

787
10

672
11

660
8

925
10

925
11

449
10

464
11

865
8

447
9

136
10

454
8

1091
8
−0.0003

1091
11
−0.0003

832
10

28
8

1239
10

1239
11

104
9

104
11

732
8

732
9

776
10

776
11

603
8

603
9

603
11

152
10

909
8

909
10

668
8

1179
9

408
8
0.0044

257
10
0.0002

473
10

42
8

42
9

478
8

478
9

858
9

809
9

370
8
0.0120

172
8
−0.0003

172
10
0.0022

654
8

654
9

654
10

767
8

435
9

435
11

673
10

148
8

661
11

954
8
0.0210

861
9

861
10

406
10

101
8

738
11
0.0027

369
8

369
9

747
9

681
10

681
11

860
10

860
11

722
10

724
8

883
9

887
8
0.0080

887
9
0.0150

684
8
0.0024

107
8
0.0006

107
11
0.0006

485
9
0.0002

485
10
0.0014

467
8

467
10

674
9

154
8

154
10

869
9

1008
10

934
8

822
8

690
11

662
10

800
8
0.0076

915
9
0.0001

1131
9

145
10

145
11

443
8

443
10

443
11

651
11

790
11

62
10

1017
8

696
11

852
10

1024
8

972
8

796
8

796
11

271
9

663
9

663
11

410
10
0.0840

960
10

953
8

953
9

916
8

916
11

360
11

427
9

427
10

388
10

275
8

758
10

758
11

745
8

745
11

824
10
0.0002

945
8

945
9

945
10

945
11

885
10

885
11

627
11

999
10

999
11

1119
9

391
10

1130
10

699
8

197
9
0.0011

1241
8

1241
9

1241
11

1102
8

1102
11

66
8

66
9

525
10

525
11

128
10

922
10
0.0005

780
9
0.1700

780
11
0.0320

828
8

828
9

513
9

160
8

160
9

160
10

106
9

484
10

484
11

799
8

799
9

396
8

396
11

141
8

141
9

795
9

711
10

24
8

24
10

398
9

429
8

93
9

90
10

54
9

54
11

968
9
0.0180

898
9
0.0110

898
11

985
10
0.0002

434
8

434
10

713
8

100
8

100
9

816
8

816
10

868
10

689
8

34
10

940
10

98
10

98
11

978
9
0.0032

340
8

340
11

545
10

8
8
0.0250

8
9
1.3000

1111
10
0.0120

653
9

653
10

653
11

373
9

1007
8

1007
11

418
8

418
11

1100
10

457
9

457
11

70
8

144
11

442
9

442
11

281
9
0.0001

281
11
0.0180

305
9

1002
8

22
10

1051
9

1051
11

792
9

792
10

423
9

451
8
−0.0003

451
11
0.0036

907
9
0.1200

907
10
0.0630

834
8
0.0059

609
8
0.3200

917
10
0.0002

948
8

166
8

402
8

402
9

402
10

402
11

1166
8

444
9

444
10

686
11
−0.0003

1117
11

479
8

793
8

793
9

793
11

911
8

733
8

63
9
0.0380

63
11
8.9000

273
10
0.0074

1085
8

399
8
−0.0003

399
11

424
8
−0.0003

116
8

725
11

666
8

666
9

666
10

153
9

153
11

546
9

851
11

773
9
0.0001

296
8

296
9

129
9

797
10

797
11

356
9

910
9

272
8

272
11

658
10

987
8

1180
8

665
9

665
10

665
11

55
8

55
10

55
11

664
8

664
10

664
11

905
9
0.0800

905
11
0.0920

951
9
0.1600

951
10
0.0220

951
11
1.8000

739
10

452
10

888
8
−0.0003

959
11
0.0011

411
9

64
8

64
10

64
11

303
11

1023
8

1023
9

409
11

952
8
0.0009

952
9

952
10
0.0019

772
10
0.0001

554
8

781
8

781
10

[0490]

14

TABLE XI

HER2/NEU B07 Supermotif Peptides with Binding Data

No. of

Position
Amino Acids
A*2401

1036
8
0.0063

390
8
−0.0006

390
10
0.0001

390
11
0.0011

1129
11
−0.0002

1204
9
0.0056

1204
10
0.0530

1076
10
0.0002

1076
11
0.0006

642
10
0.1500

1032
8
−0.0002

1032
11
−0.0002

626
10
0.0002

315
8
−0.0006

315
10
0.0001

600
9
0.0140

600
11
0.0300

299
10
0.0016

1034
9
0.0001

1034
10
0.0002

384
9
0.0004

121
9
0.0002

982
8
−0.0006

1105
8
−0.0006

698
8
−0.0002

698
9
0.0110

995
10
0.0510

995
11
0.0036

578
8
−0.0006

578
9
0.0001

578
11
−0.0003

522
8
−0.0006

246
8
0.0092

246
9
0.0001

246
11
0.0006

1155
9
0.0900

1155
11
0.0160

524
11
0.0005

564
11
−0.0002

1208
9
0.0093

1208
10
0.0018

926
9
0.0006

926
10
0.0004

740
9
0.0001

740
11
0.0023

931
11
−0.0002

748
8
0.0120

336
8
−0.0006

336
10
0.0370

605
9
0.0720

605
10
0.0001

921
8
0.0150

921
11
0.0430

1157
9
0.0027

1157
11
0.0140

35
9
0.0002

35
11
−0.0002

419
10
0.0003

377
10
0.0001

16
10
0.0002

941
9
0.0280

941
11
0.0032

550
8
0.0012

1120
8
−0.0006

1120
9
0.0002

1120
10
0.0001

231
11
−0.0003

1101
9
0.0460

65
9
0.0260

65
10
0.0190

282
8
−0.0006

282
10
0.0001

706
9
0.0090

706
10
0.0490

801
10
0.0085

284
8
−0.0002

284
10
0.0001

1245
9
0.0001

1245
11
−0.0002

1193
11
−0.0003

488
10
0.0005

158
10
0.0001

158
11
−0.0002

1210
8
−0.0002

1210
11
−0.0002

1227
11
−0.0003

17
9
0.0001

944
8
−0.0006

944
9
0.0001

944
10
0.0004

944
11
0.0064

1209
8
−0.0002

1209
9
0.0002

1149
9
0.0054

1149
11
0.4500

1233
11
−0.0003

393
8
−0.0002

393
11
−0.0002

1136
10
0.0001

1206
8
0.0002

1206
11
0.0003

943
9
0.0001

943
10
0.0001

943
11
0.0020

1148
10
0.0014

568
10
0.0004

499
11
−0.0002

966
8
0.0410

966
11
1.3000

1214
8
−0.0002

1214
9
0.0001

1214
10
0.0001

38
8
0.0014

38
9
0.0005

133
8
0.0550

133
10
0.0580

1174
8
0.0230

1174
11
−0.0002

760
8
0.0580

760
9
0.1200

1073
9
0.0030

998
8
−0.0006

998
11
0.0640

649
9
0.0150

649
10
0.0900

649
11
0.0250

196
10
0.0021

855
10
0.0016

1151
9
0.6400

1151
10
0.4600

779
8
0.0440

779
10
0.1000

701
10
0.0001

1240
9

1240
10
0.0002

127
11
−0.0002

884
8
1.4000

884
11
0.0017

1118
10
0.0001

1118
11
−0.0002

94
8
0.0020

94
9
0.0077

415
8
0.0200

415
10
0.0044

415
11
0.0005

[0491]

15

TABLE XII

HER2/NEU B27 Supermotif Peptides

No. of

Position
Amino Acids

87
10

588
8

598
11

980
10

928
8

867
11

1160
8

339
9

511
11

367
8

367
10

367
11

965
8

544
11

7
9

7
10

808
10

936
11

1229
9

939
8

939
11

173
9

173
11

969
8

969
11

486
8

486
9

1176
10

882
8

882
10

433
9

433
11

815
8

815
9

815
11

469
8

174
8

174
10

174
11

843
10

468
9

675
8

675
11

886
9

886
10

431
11

682
9

682
10

248
9

248
11

368
9

368
10

676
10

266
9

256
8

256
11

436
8

436
10

458
8

458
10

458
11

137
9

99
9

99
10

33
11

455
11

142
8

712
9

687
10

259
8

857
8

857
10

764
9

764
11

813
10

813
11

1207
11

761
8

247
10

551
11

967
10

680
8

680
11

646
9

646
10

984
11

97
11

156
8

1110
11

721
11

683
8

683
9

897
10

688
9

677
9

677
11

896
11

335
9

335
11

189
10

783
8

977
10

1103
10

472
11

41
9

41
10

900
9

1052
8

1052
10

842
11

477
0

477
10

746
10

859
8

859
11

604
8

604
10

604
11

853
9

723
9

353
9

810
8

102
11

839
8

91
9

91
11

169
11

877
10

1005
10

[0492]

16

TABLE XIII

HER2/NEU B58 Supermotif Peptides

No. of

Position
Amino Acids

1094
8

4
8

4
9

4
10

1203
11

1159
9

293
9

293
11

69
9

37
9

37
10

132
9

132
11

997
8

997
9

648
8

648
11

21
11

1165
9

587
9

587
11

224
8

338
8

338
10

334
8

334
10

195
11

1133
8

531
11

244
10

26
8

26
10

630
8

947
8

947
9

947
10

962
8

962
11

417
8

417
9

1001
8

1001
9

165
8

165
9

580
11

770
8

892
8

280
10

207
9

1123
9

717
9

717
10

693
8

874
11

40
10

40
11

401
9

401
10

401
11

364
8

364
11

1213
8

1213
9

1213
10

1213
11

976
11

899
8

899
10

1093
9

621
8

729
10

729
11

1080
11

919
8

704
9

704
11

292
10

131
10

1164
10

1189
8

1189
9

439
10

309
9

1082
9

1082
11

727
8

727
9

372
10

778
8

778
9

778
11

818
8

818
10

28
8

1239
10

1239
11

104
9

104
11

732
8

732
9

878
9

878
11

249
8

249
10

495
11

478
8

478
9

86
9

86
11

829
8

829
11

655
8

655
9

655
10

655
11

412
8

412
11

450
9

861
9

861
10

406
10

762
11

854
8

854
11

1171
10

1171
11

3
9

3
10

3
11

846
8

253
11

374
8

465
10

1075
11

114
10

71
10

1173
8

1173
9

304
10

304
11

422
9

422
10

608
9

1131
9

1131
10

145
9

145
10

145
11

443
8

443
10

443
11

651
8

651
9

651
11

790
8

790
11

62
10

774
8

774
9

889
11

979
8

979
9

979
10

979
11

833
9

833
10

916
8

916
11

68
10

388
10

275
8

471
10

758
10

758
11

1158
10

643
9

1215
8

1215
9

1211
10

1211
11

269
11

1035
8

1035
9

927
8

927
9

385
8

36
8

36
10

36
11

996
9

996
10

1065
11

1077
9

1077
10

1121
8

1121
11

702
11

601
8

601
10

601
11

1150
8

1241
8

1241
9

1241
11

1102
8

1102
11

66
8

66
9

525
10

525
11

902
9

902
11

1099
11

190
9

647
8

647
9

354
8

354
11

1053
9

1053
11

1006
9

456
10

143
11

188
11

656
8

656
9

656
10

656
11

413
10

208
8

1049
11

1050
10

305
9

305
10

1002
8

22
10

1051
9

1051
11

792
9

792
10

297
8

1242
8

1242
10

496
10

389
9

389
11

357
8

652
8

652
10

652
11

759
9

759
10

791
10

791
11

585
11

597
8

782
9

296
8

296
9

129
9

797
10

797
11

356
9

910
9

272
8

272
11

906
8

906
10

906
11

1112
9

441
8

441
10

289
8

[0493]

17

TABLE XIV

HER2/NEU B62 Supermotif Peptides

No. of

Position
Amino Acids

890
10

466
11

270
10

705
8

705
10

1036
8

390
10

390
11

1129
11

1204
10

1076
10

1076
11

355
10

657
8

657
9

657
10

255
9

789
9

826
8

826
10

1032
11

626
10

315
8

600
11

299
10

567
8

567
11

212
8

596
9

295
9

76
8

76
9

76
11

845
9

821
9

421
10

421
11

607
8

607
10

1016
8

1016
10

1013
11

1034
9

1034
10

121
9

982
8

1105
8

582
9

1183
9

1084
9

307
8

904
9

904
10

950
10

950
11

766
8

766
9

147
9

405
8

405
11

2
8

460
9

265
8

265
10

914
10

139
10

971
9

698
9

645
10

645
11

79
8

352
10

73
8

73
11

534
8

425
11

476
11

262
11

787
8

787
11

672
11

660
10

660
11

737
10

865
8

344
10

346
8

346
11

832
8

832
11

995
10

995
11

578
8

522
8

524
11

537
10

603
8

603
9

603
11

909
8

909
10

668
8

1179
9

473
8

473
10

42
9

349
8

48
8

48
9

564
11

1208
10

512
10

901
8

901
10

654
8

654
11

767
8

767
11

673
10

714
10

148
8

661
9

661
10

661
11

954
8

740
9

740
11

361
10

361
11

77
8

77
10

155
9

101
8

369
8

747
8

336
8

605
9

605
10

921
11

722
10

724
8

724
11

85
10

467
10

467
11

674
9

154
10

869
9

1008
11

785
10

822
8

15
11

690
8

662
8

662
9

662
10

800
11

915
9

35
11

16
10

941
9

941
11

1120
8

1120
9

65
9

74
10

74
11

445
8

547
8

547
9

140
9

392
8

392
9

1109
10

397
10

428
8

313
10

1017
9

696
11

841
11

852
8

852
10

1024
8

972
8

796
8

271
9

663
8

663
9

663
11

960
10

953
8

953
9

45
11

282
8

282
10

706
9

801
10

827
9

360
11

427
9

284
8

1245
9

1245
11

158
10

177
8

745
8

745
10

850
10

945
8

945
9

945
10

945
11

885
10

627
9

122
8

1119
9

1119
10

391
9

391
10

95
8

1108
11

1130
10

1130
11

699
8

650
9

650
10

197
9

1210
8

1227
11

17
9

944
8

944
9

944
10

944
11

1209
9

159
9

159
11

569
9

1135
11

1205
9

942
8

942
10

942
11

828
8

513
9

160
8

160
10

106
11

396
11

141
8

795
9

393
8

1136
10

1206
8

943
9

943
10

943
11

568
10

679
9

24
8

398
9

93
9

93
10

54
11

434
8

713
11

100
9

816
10

868
10

784
11

689
9

940
10

98
11

978
9

978
10

978
11

966
8

966
11

678
8

678
10

92
10

92
11

340
8

545
10

545
11

653
9

373
9

1007
8

418
8

70
8

70
11

144
10

442
9

442
11

281
9

281
11

1214
8

1214
9

1214
10

38
8

1174
8

1174
11

760
8

998
8

649
10

649
11

196
10

855
10

779
10

819
9

819
11

532
10

423
8

423
9

948
8

948
9

166
8

402
8

402
10

402
11

444
9

1172
9

1172
10

312
11

1240
9

526
9

105
8

23
9

1117
11

479
8

793
9

793
11

911
8

733
8

725
10

725
11

666
8

666
10

84
8

84
11

153
11

546
9

546
10

851
9

851
11

773
9

773
10

884
11

1118
10

1118
11

[0494]

18

TABLE XV

HER2/NEU B62 Supermotif Peptides

No. of

Position
Amino Acids

94
8

94
9

56
9

794
8

794
10

658
8

658
9

1180
8

665
9

665
11

55
10

664
8

664
10

739
8

739
10

959
11

415
10

415
11

835
8

1248
8

64
10

1023
8

1023
9

952
8

952
9

952
10

163
10

163
11

83
9

772
10

772
11

781
8

HER2/NEU A01 Motif Peptides with Binding Data

No. of

Position
Amino Acids
A*0101

1212
10
0.0010

1212
11
0.0140

293
9
0.0550

293
11
0.1900

997
9
0.0290

826
10
0.3000

600
11

334
10
0.0016

1013
11
0.0027

1105
8

580
11
0.1000

280
10
0.1800

40
11
0.2800

401
9
0.0430

401
11
0.4400

279
11
0.0049

291
11
0.0100

1213
9
0.0430

1213
10
5.5000

899
10
2.7000

292
10
0.0012

1188
9

995
11

727
9
0.0011

1239
10
0.0630

104
9
0.1800

42
9
9.1000

901
8
−0.0021

333
11
−0.0017

403
9
0.0057

726
10
0.0010

869
9
7.6000

915
9
0.0011

1120
8

74
10
0.0015

1131
9
0.1300

1014
10
0.0120

916
8
−0.0021

764
9
0.0017

996
10
0.0150

601
10
0.0010

1241
8
0.0030

1102
11
0.0160

828
8
−0.0021

728
8
−0.0021

281
9
0.0028

1214
8

1214
9

1132
8
−0.0021

1103
10
0.0015

402
8
−0.0021

402
10
1.1000

399
11
0.0045

773
9
0.0400

296
8
0.1000

[0495]

19

TABLE XVI

HER2/NEU A03 Motif Peptides with Binding Data

No. of

Position
Amino Acids
A*0301

241
8

241
9

1094
11

4
11

1203
10

1203
11

847
8

1159
11

586
10

191
10

510
8

510
10

622
11

879
9

581
8

581
9

581
10

581
11

1186
11

1212
10
0.0003

1212
11

1163
9

365
11

242
8

221
8

1039
8

1039
9

1039
10

775
10

5
10

890
8

890
9
0.0013

890
10

466
8

466
11

492
8

14
8

180
9
0.0004

180
11

270
10

705
9
0.0004

293
9
0.0008

293
11

37
11

997
8

997
9
0.0002

997
10
0.0003

648
10

355
10

355
11

240
9
0.0021

240
10

220
9
−0.0002

587
9

235
8

576
11

252
9

805
10
0.0003

826
10
0.0003

334
10
0.0003

195
9
−0.0008

244
11

26
9
0.0002

630
11

947
11

311
8

584
8

596
10
0.0220

634
11

504
9

528
9
0.0015

295
9
0.0002

234
8

234
9

251
8

251
10

211
11

1011
10

638
10

638
11

1012
9

1087
8

1087
9

1087
10

382
9

742
10

880
8

880
11

326
8

326
11

871
8

871
9

171
10

76
8

845
10
0.0018

636
9

1089
8

933
8

821
10

821
11

607
9
0.0005

607
10

1016
8

1013
8

1013
11

30
8

962
8

962
9
−0.0002

417
9

165
9

165
10

165
11

185
9

1183
8

1084
11

838
10

838
11

1144
10
0.0003

950
8

580
9

580
10

580
11

1069
8

543
10

503
8

503
10

210
8

1010
11

501
10

325
9

837
8

837
11

363
9

975
11

1079
8

507
10

507
11

1154
8

1154
10

286
8

766
10

147
11

930
8

930
11

405
10

460
10

460
11

265
10
0.0002

914
8

914
10
0.0002

61
9

695
11

971
8

971
10

971
11

379
8

892
8

892
10

280
9
0.0003

280
10
0.0003

207
11

717
8

874
10
0.0003

40
8

40
9

40
11

401
9
0.0002

401
11

79
9

79
10

352
8

321
10
0.0002

364
8

1031
9

595
11

1086
9

1086
10

1086
11

381
10

1030
10

918
11

291
8

291
11

1187
10

671
8

671
11

577
10

371
11

376
11

73
11

1213
9
0.0002

1213
10
0.0005

976
10
−0.0002

976
11

899
10
0.0003

476
11

1202
11

729
8

1038
8

1038
9
0.0002

1038
10

1038
11

919
10

919
11

704
10
−0.0002

1231
8

292
10
0.0003

131
8

1164
8

1189
8

366
10

366
11

804
8

804
11

641
8

1088
8

1088
9

1015
9

383
8

1029
8

1029
11

1201
8

1188
9
0.0003

881
10

135
9

1040
8

1040
9

262
9

672
10
0.0150

449
8

449
11

737
11

508
9

508
10
0.0110

464
10

1062
9

1062
11

447
10
0.0037

344
8

344
11

10
11

549
9

549
10
0.0002

549
11

1097
8

136
8

346
9
−0.0002

346
10

832
9
−0.0002

1041
8

309
10

727
9
0.0028

727
10
0.0660

462
8

462
9

372
10

572
10

28
10

1239
10
0.0002

104
9
0.0001

104
10

327
10
0.0210

776
9
0.0010

603
8

909
10

668
9
0.0047

668
10
0.0180

668
11

1179
8

1179
9

878
10
0.0003

267
8

1104
8

1104
9

257
11

42
9
0.0370

349
11

632
9
−0.0002

249
9

249
10

260
8

260
11

478
9

478
10
0.0035

858
10

858
11

809
8

263
8

872
8

961
8

961
9

961
10

184
8

184
10

949
9

172
9

767
9

673
9
0.3800

673
11

714
8

714
9
0.0190

714
11

148
10
0.0400

661
11

894
8

167
8

167
9
0.2800

450
10
0.0410

861
8

406
9

101
8

762
10

762
11

333
8

333
11

957
10

170
11

591
8

591
9

1182
9

615
8

640
8

640
9

150
8

1096
9

831
10

228
10

1238
11

369
8

747
10
0.0009

681
8
0.0010

681
9
0.7600

860
8

860
9
0.1700

32
11

854
11

722
9

722
10

724
8

753
10
0.3800

753
11

883
8

846
9
0.0580

509
8

509
9
−0.0002

509
11

253
8

374
8

465
9

13
8

13
9

179
10
−0.0002

6
9

161
10
0.0081

637
8

637
11

768
8

807
8

807
10

870
8

870
9

870
10

43
8

107
9

485
8

485
11

448
9

1061
10

345
10

345
11

994
11

726
10
0.0003

726
11

461
9

461
10

667
10

667
11

85
8

183
8

183
9

183
11

467
10

674
8

674
10
0.0002

674
11

154
10
0.0012

12
9

12
10

806
9
0.0370

806
11

869
9
0.0003

869
10

869
11

11
10

11
11

822
9

822
10
0.1400

662
10

800
10

915
9
0.0002

1173
10
−0.0002

422
11

608
8

608
9

1131
9
0.0001

181
8

181
10
0.0002

181
11

1197
8

700
11

215
11

62
8

696
10

841
8

841
9
0.0040

852
9
0.4800

1024
8

972
9

972
10
0.0072

796
8

271
9

663
9

774
8

774
11

889
8

889
9
0.0034

889
10
0.0011

889
11

979
8

1014
10
0.0002

960
9
0.0017

960
10

960
11

833
8

833
11

916
8

556
9

571
11

1178
8

1178
9

1178
10

360
9
0.0002

360
10
0.0003

59
11

427
8

427
11

471
8

758
8

125
8

745
9
0.0058

850
9

850
11

1158
8

1215
8

1211
11

1162
8

1162
10
−0.0002

269
11

1035
10

1035
11

927
11

996
9

996
10
0.0003

996
11

625
8

194
10

741
11

932
9

606
10

606
11

416
10

1143
11

1037
8

1037
9

1037
10

1037
11

134
10

1175
8

1175
11

945
8

612
11

1074
8

999
8

316
9

122
11

1156
8

1156
10

1119
9
0.0002

1119
11

230
8

391
10

95
9
0.0002

1130
10
0.0002

650
8

1065
8

1077
10

1121
9

702
9

601
10
0.0003

1150
10

1150
11

1234
10

1241
8

232
10

232
11

1102
10
0.0003

1102
11

66
8

525
10

749
8

128
11

491
9
0.0046

709
8

239
10

239
11

583
8

583
9

527
8

527
10

75
9

820
11

1225
8

164
10

164
11

1028
9

1200
9

446
11

548
10

548
11

57
8

81
8

828
8

178
11

160
11

106
8

106
10

484
9

799
11

141
10
0.2000

795
9
0.0110

711
11

213
9

24
9
0.0007

24
11

429
9

93
8

93
11

90
9
0.0029

90
11

54
11

190
11

647
11

354
11

330
10

330
11

844
11

968
9

968
11

898
11

1230
8

1230
9

536
10

432
9

432
10

103
10
0.0003

103
11

434
8

713
9
0.0007

713
10
0.0570

100
9

868
10
0.0017

868
11

34
9

98
11

840
8

840
9

840
10
0.1800

978
8

978
9
0.0001

456
11

143
8

1072
10

217
9

217
10
0.0068

340
10

545
8

545
10
0.0350

358
8

358
11

310
9

633
8

294
8

294
10

250
8

250
9

250
11

380
11

728
8

728
9

703
8

703
11

261
10

463
8

463
11

602
9

893
9

373
9

418
8

457
10

214
8

281
8

281
9
0.0002

281
11

208
10
−0.0002

1235
9

22
11

423
10
0.0170

573
9

323
8

323
11

1132
8

233
9

233
10

29
9

278
11

290
9

1236
8

130
9

245
10

27
8

27
11

407
8

948
10
0.0130

166
8

166
9

166
10
0.0430

402
8

402
10
0.0001

1060
11

182
9
0.0004

182
10

1172
11

357
8

357
9

218
8

218
9
0.0004

218
11

1117
11

479
8

479
9
0.0006

793
11

911
8

911
10

911
11

585
11

597
9
0.0100

219
8

219
10

314
11

25
8

25
10

341
9

341
11

635
10

1085
10

1085
11

399
11

670
8

670
9

424
9

424
11

505
8

308
11

777
8

988
10

430
8

430
11

725
11

666
11

84
9
0.0033

153
11

546
9
0.0012

754
9
0.4000

754
10

851
8

851
10
0.0820

773
9
0.0580

973
8

973
9
−0.0002

296
8

322
9
0.0002

574
8

129
10
0.0002

356
9

356
10

910
9

910
11

272
8

669
8

669
9
0.1100

669
10
0.0030

987
11

1180
8

1180
11

55
10
0.0024

664
8

825
11

1223
8

1223
10

482
9

482
11

739
9
0.0002

452
8

888
9
−0.0002

888
10
0.0085

888
11

959
8

959
10
−0.0002

959
11

803
9

343
9

908
11

835
9

835
10
0.0003

64
10

1196
8

1196
9

1023
8

1023
9

289
10

83
10
0.0043

772
10
0.0100

554
11

1139
8

[0496]

20

TABLE XVII

HER2/NEU A11 Motif Peptides with Binding Data

No. of

Position
Amino Acids
A*1101

241
8

241
9

1094
11

847
8

1159
11

191
10

510
8

510
10

622
11

879
9

581
9

581
10

581
11

1186
11

1212
10
0.0003

1212
11

1163
9

242
8

221
8

1039
8

1039
9

1039
10

775
11

890
8

890
9
0.0006

466
8

492
8

180
9
0.0005

180
11

705
9
0.0006

359
10

359
11

763
10

293
9
0.0074

293
11

37
11

997
9
0.0004

997
10
0.0670

240
9
0.0021

240
10

220
9
−0.0002

252
9

805
10
0.0001

826
10
0.0001

334
10
0.0002

195
9
−0.0001

26
9
0.0005

630
11

947
11

311
8

584
8

596
10
0.0042

504
9

528
9
0.0310

295
9
0.0004

251
10

638
10

1087
10

880
8

326
8

326
11

871
8

76
8

845
10
0.0007

1089
8

933
8

821
11

607
9
0.0100

1016
8

1013
11

962
9
−0.0002

165
10

165
11

185
9

1144
10
0.0001

950
8

580
10

580
11

543
10

503
10

210
8

325
9

837
8

975
11

507
11

1154
8

147
11

930
8

930
11

460
10

460
11

265
10
0.0002

914
8

914
10
0.0002

971
8

971
11

757
9

744
10

892
10

280
9
−0.0002

280
10
0.0003

207
11

717
8

874
10
0.0001

40
8

40
9

40
11

401
9
0.0002

401
11

79
10

321
10
0.0001

595
11

1086
11

291
11

1187
10

671
8

671
11

73
11

258
10

1213
9
0.0002

1213
10
0.0010

976
10
0.0010

899
10
0.0005

729
8

1038
8

1038
9
0.0043

1038
10

1038
11

919
11

704
10
0.0041

1231
8

292
10
0.0001

131
8

1164
8

1189
8

804
8

804
11

1088
9

1015
9

1201
8

1188
9
0.0001

135
9

1040
8

1040
9

672
10
0.0014

449
8

449
11

737
11

508
10
0.0001

464
10

1062
11

447
10
0.0001

344
8

344
11

549
10
0.0003

549
11

1097
8

136
8

346
9
−0.0002

832
9
0.0002

1041
8

309
10

727
9
0.0001

727
10
0.1300

462
8

462
9

1239
10
0.0022

104
9
0.0280

327
10
0.6100

776
9
0.0066

603
8

668
9
0.0890

668
10
0.0330

668
11

878
10
0.0008

267
8

1104
8

1104
9

257
11

42
9
0.0002

88
11

470
9

632
9
0.0007

249
9

260
8

478
10
0.0720

858
11

809
8

961
8

961
10

184
10

949
9

673
9
0.0097

673
11

714
8

714
9
0.0023

714
11

148
10
0.0005

894
8

167
8

167
9
0.3100

450
10
0.0027

861
8

762
11

333
8

333
11

957
10

591
9

640
8

150
8

1096
9

831
10

228
10

1238
11

747
10
0.0099

681
8
0.0004

681
9
0.0018

860
9
0.2400

32
11

753
10
0.2200

846
9
0.0285

509
9
0.0003

509
11

253
8

465
9

179
10
0.0003

161
10
0.0063

637
11

807
8

807
10

870
8

870
9

43
8

485
11

448
9

345
10

726
10
0.0003

726
11

461
9

461
10

667
10

667
11

85
8

183
8

183
11

674
8

674
10
0.0001

674
11

154
10
0.0002

806
9
0.0006

806
11

869
9
0.0001

869
10

822
10
0.1400

800
10

915
9
0.0003

823
9

1173
10
0.0003

422
11

608
8

1131
9
0.0061

181
8

181
10
0.0005

841
9
0.0014

852
9
0.0700

972
10
0.0330

796
8

774
8

774
11

889
8

889
9
0.0237

889
10
0.0003

1014
10
0.0002

960
9
0.0006

960
11

833
8

833
11

916
8

556
9

360
9
0.0036

360
10
0.0056

427
8

427
11

471
8

758
8

745
10
0.0007

850
9

850
11

1215
8

1211
11

1162
8

1162
10
−0.0002

1035
10

1035
11

927
11

996
10
0.0001

996
11

625
8

194
10

932
9

606
10

1143
11

1037
8

1037
9

1037
10

1037
11

134
10

1175
8

945
8

999
8

1119
9
0.0002

230
8

95
9
0.0001

1130
10
0.0002

707
10

1065
8

601
10
0.0003

1241
8

1102
10
0.0001

1102
11

749
8

128
11

491
9
0.0010

709
8

239
10

239
11

583
8

583
9

527
10

75
9

164
11

1200
9

446
11

548
11

57
8

81
8

828
8

178
11

160
11

799
11

141
10
0.0130

795
9
0.0039

711
11

426
9

24
9
0.0520

24
11

429
9

93
8

93
11

90
9
0.0005

90
11

54
11

190
11

330
11

844
11

968
11

898
11

1230
9

536
10

432
10

103
10
0.0015

434
8

713
9
0.0038

713
10
0.1100

868
10
0.0001

868
11

34
9

840
10
0.0001

978
8

487
9

849
10

143
8

217
10
0.0130

340
10

545
8

515
10
0.0050

358
11

310
9

633
8

294
8

294
10

250
8

250
11

728
8

728
9

703
11

463
8

463
11

602
9

893
9

281
8

281
9
0.0003

208
10
0.0020

22
11

423
10
0.0750

323
8

323
11

1132
8

278
11

130
9

27
8

948
10
0.1200

166
9

166
10
3.6000

402
8

402
10
0.0001

182
9
0.0005

1172
11

186
8

218
9
0.0230

218
11

1117
11

479
9
0.0072

793
11

911
11

597
9
−0.0002

219
8

219
10

25
8

25
10

341
9

341
11

399
11

670
8

670
9

424
9

424
11

505
8

308
11

777
8

430
8

725
11

666
11

84
9
0.0007

153
11

546
9
0.0002

754
9
0.0130

851
8

851
10
0.0072

773
9
0.0079

555
10

529
8

973
9
0.0021

296
8

322
9
0.0140

129
10
0.0005

669
8

669
9
0.7200

669
10
0.0160

55
10
0.0110

825
11

1223
8

482
9

730
9
0.0001

452
8

888
9
−0.0002

888
10
0.0016

888
11

959
8

959
10
0.0002

803
9

343
9

835
9

835
10
0.0001

83
10
0.0013

772
10
0.0120

554
11

1139
8

[0497]

21

TABLE XVIII

HER2/NEU A24 Motif Peptides with Binding Data

No. of

Position
Amino Acids
A*2401

1216
8
0.0039

730
9
0.0002

730
10
0.0010

730
11
0.0008

1212
9
0.0011

705
10
0.0002

705
11
−0.0003

414
9
0.0041

440
9
0.1300

440
11
0.0230

475
8
0.0190

475
11
0.0003

826
11
−0.0003

342
9
0.0180

162
8
0.0120

162
11
0.0016

863
8
0.0005

863
10
0.0002

363
8
−0.0003

363
9
0.0003

876
11
−0.0003

1022
9
0.0014

1022
10
0.0120

553
9
0.0061

1091
8
−0.0003

1091
11
−0.0003

832
10

408
8
0.0044

257
10
0.0002

370
8
0.0120

172
8
−0.0003

172
10
0.0022

954
8
0.0210

738
11
0.0027

887
8
0.0080

887
9
0.0150

684
8
0.0024

107
8
0.0006

107
11
0.0006

485
9
0.0002

485
10
0.0014

800
8
0.0076

410
10
0.0840

197
9
0.0011

922
10
0.0005

780
9
0.1700

780
11
0.0320

711
10

968
9
0.0180

898
9
0.0110

985
10
0.0002

978
9
0.0032

8
8
0.0250

8
9
1.3000

1111
10
0.0120

281
11
0.0180

451
9
−0.0003

451
11
0.0036

907
9
0.1200

834
8
0.0059

609
8
0.3200

917
10
0.0002

686
11
−0.0003

63
9
0.0380

63
11
8.9000

399
8
−0.0003

424
8
−0.0003

905
9
0.0800

905
11
0.0920

951
9
0.1600

951
11
1.8000

888
8
−0.0003

959
11
0.0011

952
8
0.0009

952
10
0.0019

[0498]

22

TABLE XIX

HER2/NEU DR Super Motif Peptides with Binding Data

Core
Exemplary

Sequence
Sequence
Position
DR1
DR2wB1
DR2w2B2
DR3
DR4w4
DR4w15
DR5w11
DR5w12
DR6w19
DR7
DR8w2
DR9
DRw53
SEQ ID NO.

YNYLSTDVG
ACPYNYLSTDVGSCT
298

3720

VLRENTSPK
AIKVLRENTSPKANK
751

0.0075

3721

LQSLPTHDP
AKGLQSLPTHDPSPL
1095

3722

YDGIPAREI
AKPYDGIPAREIPDL
920

3723

LDIDETEYH
ARLLDIDETEYHADG
867
0.0001
−0.0006
−0.0007
0.3100
−0.0055

−0.0008

−0.0001
−0.0017
−0.0009

3724

VLVKSPNHV
ARNVLVKSPNHVKIT
848

3725

LTSHSAVV
ASPLTSHSAVVGIL
648
0.0890
0.0950
0.0037
0.0010
−0.0025

−0.0005

0.0480
0.0350
−0.0004

3726

LTLOGLGIS
AYSLTLQGLGISWLG
440

3727

MAGVGSPYV
AYVMAGVGSPYVSRL
771

3728

IFGSLAFLP
CKKIFGSLAFLPESF
367
0.2400
0.0070
0.0016
0.0010
−0.0025

−0.0005

0.0034
0.0270
−0.0004

3729

FNHSGICEL
CLHFNHSGICELHCP
255

3730

IAKGMSYLE
CMQIAKGMSYLEDVR
826

3731

LVTYNTDTF
CPALVTYNTDTFESM
268

3732

LTRTVCAGG
CQSLTRTVCAGGCAR
212

3733

LDDKGCPAE
CVDLDDKGCPAEQRA
634

3734

LGMEHLREV
CYGLGMEHLREVRAV
342

0.0083

3735

YVMAGVGSP
DEAYVMAGVGSPYVS
769
0.0500
0.0029
0.0240
0.0010
0.2300

0.0027

0.0020
0.2000
0.0570

3736

VGEGLACHQ
DECVGEGLACHQLCA
502

3737

LGMGAAKGL
DGDLGMGAAKGLQSL
1087

3738

VAPLTCSPQ
DGYVAPLTCSPQPEY
1125

3739

LGLEPSEEE
DLTLGLEPSEEEAPR
1058

−0.0025

3740

LRLPASPET
DMKLRLPASPETHLD
30
0.0010

−0.0025

−0.0013

3741

FCVARCPSG
DPPFCVARCPSGVKP
592

3742

FYRSLLEDD
DSTFYRSLLEDDDMG
1001

3743

LVHRDLAAR
DVRLVHRDLAARNVL
838

3744

MIMVKCWMI
DVYMIMVKCWMIDSE
950
0.0280
0.0047
0.0042
0.0010
0.0570

0.0220

−0.0003
0.1300
0.0450

3745

VLQGLPREY
ECRVLQGLPREYVNA
543

3746

YALAVLDNG
EDNYALAVLDNGDPL
109

3747

LPAARPAGA
EGPLPAARPAGATLE
1154

3748

YTFGASCVT
EGRYTFGASCVTACP
286

3749

YLPTNASLS
ELTYLPTNASLSFLQ
61

3750

LRRRFTHQS
ESILRRRFTHQSDVW
892

3751

LRKVKVLGS
ELELRKVKVLGSGAF
717
0.0160
0.0019
0.0052
0.0045
0.0350

0.0061

0.0014
0.0380
0.0250

3752

LVEPLTPSG
ETELVEPLTPSGAMP
693
0.0060

−0.0025

−0.0013

3753

LVSEFSRMA
FRELVSEFSRMARDP
969

0.0710

3754

IQNEDLGPA
FVVIQNEDLGPASPL
986

−0.0025

3755

LERPKTLSP
GATLERPKTLSPGKN
1164

3756

VVQGNLELT
GCQVVQGNLELTYLP
52

3757

LNNTTPVTG
GDPLNNTTPVTGASP
120

3758

LACHQLCAR
GEGLACHQLCARGHC
506

3759

VKIPVAIKV
GENVKIPVAIKVLRE
743
0.0630
0.0047
0.0034
0.0098
−0.0032

−0.0005

0.0004
0.0310
0.0010

3760

LPQPPICTI
GERLPQPPICTIDVY
938
−0.0005

−0.0032

−0.0011

3761

VPIKWMALE
GGKVPIKWMALESIL
881

3762

LIQRNPQLC
GGVLIQRNPQLCYQD
151

3763

WGPGPTQCV
GHCWGPGPTQCVNCS
518

3764

WLGLRSLRE
GISWLGLRSLRELGS
449

3765

LIHHNTHLC
GLALIHHNTHLCFVH
464

3766

ISWLGLRSL
GLGISWLGLRSLREL
447

3767

LALLPPGAA
GLLLALLPPGAASTQ
10

3768

VFDGDLGMG
GSDVFDGDLGMGAAK
1082

3769

YVSRLLGIC
GSPYVSRLLGICLTS
778

3770

MKLRLPASP
GTDMKLRLPASPETH
28
0.0010

−0.0025

−0.0013

3771

YKGIWIPDG
GTVYKGIWIPDGENV
732

3772

VWELMTFGA
GVTVWELMTFGAKPY
909
1.4000

0.0330

0.0170

3773

YISAWPDSL
GYLYISAWPDSLPDL
408

3774

FVHTVPWDQ
HLCFVHTVPWDQLFR
473

3775

VRQVPLQRL
HNQVRQVPLQRLRIV
88

3776

LAARNVLVK
HRDLAARNVYLVKSPN
843

3777

ICELHCPAL
HSGICELHCPALVTY
260

3778

ITDFGLARL
HVKITDFGLARLLDI
858

3779

LHCPALVTY
ICELHCPALVTYNTD
263

3780

IDVYMIMVK
ICTIDVYMIMVKCWM
946

3781

LRENTSPKA
IKVLRENTSPKANKE
752
−0.0005

−0.0032

−0.0011

3782

MALESILRR
IKWMALESILRRRFT
886
0.9500

0.0400

0.0040

3783

VVVLGVVFG
ILLVVVLGVVFGILI
661

3784

VQGYVLIAH
IQEVQGYVLIAHNQV
77

3785

YTMRRLLQE
IRKYTMRRLLQETEL
682

3786

VYGILLVVV
ISAVVGILLVVVLGV
655

3787

WPDSLPDLS
ISAWPDSLPDLSVFQ
412

3788

LGLRSLREL
ISWLGLRSLRELGSG
450

3789

FGLARLLDI
ITDFGLARLLDIDET
861
0.0048

−0.0032

−0.0011

3790

YLYISAWPD
ITGYLYISAWPDSLP
406

3791

MIDSECRPR
KCWMIDSECRPRFRE
957

3792

FAFGGAVEN
KDVFAFGGAVENPEY
1182

3793

LDEAYVMAG
KEILDEAYVMAGVGS
765
0.0036

0.0073

−0.0011

3794

LPTDCCHEQ
KGPLPTDCCHEQCAA
228

−0.0027

3795

VAIKVLREN
KIPVAIKVLRENTSP
747

3796

LSYMPIWKF
KPDLSYMPIWKFPDE
605
0.0330

−0.0025

0.0029

3797

VLGSGAGFT
KVKVLGSGAFGTVYK
722

3798

IKWMALESI
KVPIKWMALESILRR
883
2.2000
2.7000
2.1000
0.0620
0.0690

0.0073

0.0031
0.0190
0.0079

3799

LCRWGLLLA
LAALCRWGLLLALLP
3

3800

LHFNHSGIC
LACLHFNHSGICELH
253

3801

LPPGAASTQ
LALLPPGAASTQVCT
13

3802

LDNGDPLNN
LAVLDNGDPLNNTTP
114

3803

WGLLLALLP
LCRWGLLLALLPPGA
6
0.0940

−0.0025

0.0021

3804

VFGILIKRR
LGVVFGILIKRRQQK
667

3805

LLPPGAAST
LLALLPPGAASTQVC
12

3806

ICLTSTVQL
LLGICLTSTVQLVTQ
785

3807

WCMQIAKGM
LLNWCMQIAKGMSYL
822
0.8400
0.0057
1.2000
0.0093
0.0011

0.4000

0.0390
0.1200
0.4100

3808

VVLGVVFGI
LLVVVLGVVFGILIK
662
−0.0008

−0.0025

0.0019

3809

LGISWLGLR
LQGLGISWLGLRSLR
445

3810

LPREYVNAR
LQGLPREYVNARHCL
547

−0.0027

3811

YSEDPTVPL
LQRYSEDPTVPLPSE
1109

0.0270

3812

LPTHDPSPL
LQSLPTHDPSPLQRY
1098

3813

IRGRILHNG
LQVIRGRILHNGAYS
428

3814

LGSGLALIH
LRELGSGLALIHHNT
458
0.0310

−0.0025

−0.0013

3815

LQLRSLTEI
LRELQLRSLTEILKG
137

3816

VRAVTSANI
LREVRAVTSANIQEF
350

3817

VRGTQLFED
LRIVRGTQLFEDNYA
99

3818

VKVLGSGAF
LRKVKVLGSGAFGTV
720

3819

LRELGSGLA
LRSLRELGSGLALIH
455

3820

FQNLQVIRG
LSVFQNLQVIRGRIL
422
0.1800
0.0280
0.0740
0.0010
0.0670

0.0100

0.0057
0.2900
0.0330

3821

ILKGGVLIQ
LTEILKGGVLIQRNP
145

3822

IDTNRSRAC
LTLIDTNRSRACHPC
181

3823

HSAVVGIL
LTSHSAVVGILLVV
651
0.1900

−0.0025

0.0049

3824

LPTNASLSF
LTYLPTNASLSFLQD
62
0.4900
0.0100
0.0560
0.0150
0.3300

0.0041

0.0280
0.3200
0.0054

3825

WDQDPPERG
LYYWDQDPPERGAPP
1220

3826

VGSPYVSRL
MAGVGSPYVSRLLGI
774

3827

LREVRAVTS
MEHLREVRAVTSANI
347

3828

0VKCWMIDSE
MIMVKCWMIDSECRP
953

3829

LKETELRKV
MRILKETELRKVKVL
712

3830

LEDVRLVHR
MSYLEDVRLV$$RDLA
833

3831

LGPASPLDS
NEDLGPASPLDSTFY
991

3832

VTCFGPEAD
NGSVTCFGPEADQCV
571

3833

VKDVFAFGG
NGVVKDVFAFGGAVE
1178

3834

LTYLPTNAS
NLELTYLPTNASLSF
59
0.4700
0.0280
0.0090
0.0010
0.3800

0.0050

0.0017
0.0680
0.0220

3835

VIRGRILHN
NLQVIRGRILHNGAY
427

3836

YWDQDPPER
NLYYWDQDPPERGAP
1219

3837

LALTLIDTN
NNQLALTLIDTNRSR
176

3838

LCYQDTILW
NPQLCYQDTILWKDI
158

3839

LCFVHTVPW
NTHLCFVHTVPWDQL
471

3840

INCTHSCVD
PCPINCTHSCVDLDD
625

3841

LPDLSVFQN
PDSLPDLSVFQNLQV
416

3842

LQVFETLEE
PEQLQVFETLEEITG
394

3843

FDGDPASNT
PESFDGDPASNTAPL
378

−0.0027

3844

VNQPDVRPQ
PEYVNQPDVRPQPPS
1137

3845

VARCPSGVK
PECVARCPSGVKPDL
594

3846

LRELQLRSL
PGGLRELQLRSLTEI
134

3847

WMALESILR
PIKWMALESILRRRF
885
0.7900

0.0350

0.0078

3848

VKPDLSYMP
PSGVKPDLSYMPIWK
601

−0.0027

3849

FKGTPTAEN
PSTFKGTPTAENPEY
1234
−0.0005

−0.0032

−0.0011

3850

YLSTDVGSC
PYNYLSTDVGSCTLV
300

3851

ILWKDIFHK
QDTILWKDIFHKNNQ
164

3852

VEECRVLOG
QECVEECRVLQGLPR
538

3853

FCPDPAPGA
QGFFCPDPAPGAGGM
1028
−0.0005

0.0230

−0.0011

3854

LELTYLPTN
QGNLELTYLPTNASL
57

3855

LTLIDTNRS
QLALTLIDTNRSRAC
178

3856

YQDTILWKD
QLCYQDTILWKDIFH
160

3857

VRPQPPSPR
QPDVRPQPPSPREGP
1142
0.0007

−0.0032

−0.0011

3858

ICTIDVYMI
QPPICTIDVYMIMVK
943
0.0670
0.0540
0.0027
0.0976
0.0060

0.0046

0.0013
0.1000
0.0051

3859

FFCPDPAPG
QQGFFCPDPAPGAGG
1027

3860

IRKYTMRRL
QQKIRKYTMRRLLQE
679

3861

VWSYGVTVW
QSDVWSYGVTVWELM
902

3862

LQRLRIVRG
QVPLQRLRIVRGTQL
93

3863

VNARHCLPC
REYVNARHCLPCHPE
552

3864

ILHNGAYSL
RGRILHNGAYSLTLQ
432

3865

LGSQDLLNW
RGRLGSQDLLNWCMQ
814

3866

YQGCQVVQG
RHLYQGCQVVQGNLE
47

3867

FRELVSEFS
RPRFRELVSEFSRMA
966

3868

LQETELVEP
RRLLQETELVEPLTP
688

3869

LEDDMGDL
RSLLEDDDMGDLVDA
1006

0.0080

3870

LLLALLPPG
RWGLLLALLPPGAAS
8
12.0000
0.1300
0.0270
0.0010
0.2800

0.0710

−0.0003
−0.0013
0.1200

3871

FGASCVTAC
RYTFGASCVTACPYN
288

3872

VGILLVVVL
SAVVGILLVVVLGVV
656

3873

WSYGVTVWE
SDVWSYGVTVWELMT
903

3874

LQGLGISWL
SLTLQGLGISWLGLR
442

3875

LLNWCMQIA
SQDLLNWCMQIAKGM
819

3876

LRGQECVEE
SQFLRGQECVEECRV
532

3877

LGICLTSIV
SRLLGICLTSTVQLV
783
0.3500
0.0220
−0.0007
0.0062
0.1200

0.0140

0.3400
0.5600
0.0009

3878

VGSCTLVCP
STDVGSCTLVCPLHN
305

3879

VTVWELMTF
SYGVTVWELMTFGAK
907

3880

LQPEQLQVF
TAPLQPEQLQVFETL
389

0.0023

3881

YVAPLTCSP
TDGYVAPLTCSPQPE
1124
−0.0005

−0.0032

−0.0011

3882

LKGGVLIQR
TEILKGGVLIQRNPQ
146

3883

VEPLTPSGA
TELVEPLTPSGAMPN
694

3884

VYMIMVKCW
TIDVYMIMVKCWMID
948

3885

FEDNYALAV
TQLFEDNYALAVLDN
105
0.0530

−0.0025

0.0160

3886

MPYGCLLDH
TQLMPYGCLLDHVRE
798

3887

VCAGGCARC
TRTVCAGGCARCKGP
216

3888

VTGASPGGL
TTPVTGASPGGLREL
126

3889

LVTQLMPYG
TVQLVTQLMPYGCLL
793
0.2300
0.7500
0.0009
0.0010
0.0460

−0.0005

0.0031
0.0100
0.0069

3890

LHNQEVTAE
VCPLHNQEVTAEDGT
314
−0.0004

−0.0025

−0.0013

3891

LTPSGAMPN
VEPLTPSGAMPNQAQ
697
−0.0008

−0.0025

−0.0011

3892

LLVVVLGVV
VGILLVVVLGVVFGI
659

3893

VPWDQLFRN
VHTVPWDQLFRNPHQ
477

0.0220

3894

VVFGILIKR
VLGVVFGILIKRRQQ
666
0.0700
0.0110
0.0620
0.0021
0.0029

0.4700

0.0150
0.0320
0.6400

3895

VTQLMPYGC
VQLVTQLMPYGCLLD
794

3896

VTSANIQEF
VRAVTSANIQEFAGC
353

3897

VHRDLAARN
VRLVHRDLAARNVLV
839
0.0340
0.0064
0.0033
0.3400
0.0150

0.2700

0.0430
0.0230
0.1000

3898

VPLQRLRIV
VRQVPLQRLRIVRGT
91

3899

LLGICLTST
VSRLLGICLTSTVQL
782

3900

LMPYGCLLD
VTQLMPYGCLLDHVR
797

3901

ILLVVVLGV
VVGILLVVVLGVVFG
658
−0.0004

−0.0025

−0.0013

3902

LMTFGAKPY
VWELMTFGAKPYDGI
912
0.0870
0.0990
0.1000
0.0010
0.0550

0.0054

0.0004
0.0370
0.0089

3903

LLALLPPGA
WGLLLALLPPGAAST
9
5.1000
0.2100
0.0110
0.0013
1.3000

0.2500

−0.0003
−0.0013
0.4500

3904

IPARElPDL
VDGIPARElPDLLEK
923

3905

MVKCWMIDS
YMIMVKCWMIDSECR
952

3906

IAHNQVRQV
YVLIAHNQVRQVPLQ
83

3907

[0499]

23

TABLE XXa

HER2/NEU DR 3a Motif Peptides with Binding Data

Core
Exemplary

Sequence
Sequence
Position
DR1
DR2w2B1
DR2w2B2
DR3
DR4w4
DR4w15
DR5w11
DR5w12
DR6w19
DR7
DR8w2
DR9
DRw53
SEQ ID NO.

VLRENTSPK
AIKVLRENTSPKANK
751

0.0075

3908

LDIDETEYH
ARLLDIDETEYHADG
867
0.0001
−0.0006
−0.0007
0.3100
−0.0055

−0.0008

−0.0001
−0.0017
−0.0009

3909

LGMEHLREV
CYGLGMEHLREVRAV
342

0.0083

3910

LGLEPSEEE
DLTLGLEPSEEEAPR
1058

−0.0025

3911

YYWDQDPPE
DNLYYWDQDPPERGA
1218

−0.0025

3912

LWKDIFHKN
DTILWKDIFHKNNQL
165

−0.0027

3913

YHADGGKVP
ETEYHADGGKVPIKW
874

−0.0027

3914

LVSEFSRMA
FRELVSEFSRMARDP
969

0.0710

3915

MARDPQRFV
FSRMARDPQRFVVIQ
976

0.1600

3916

IQNEDLGPA
FVVIQNEDLGPASPL
986

−0.0025

3917

VDAEEYLVP
GDLVDAEEYLVPQQG
1015

0.0250

3918

LFEDNYALA
GTQLFEDNYALAVLD
104

0.2200

3919

MALESILRR
IKWMALESILRRRFT
886
0.9500

0.0400

0.0040

3920

FPDEEGACQ
IWKFPDEEGACQPCP
613

−0.0027

3921

LPTDCCHEQ
KGPLPTDCCHEQCAA
228

−0.0027

3922

VVKDVFAFG
KNGVVKDVFAFGGAV
1177

−0.0025

3923

LPREYVNAR
LQGLPREYVNARHCL
547

−0.0027

3924

YSEDPTVPL
LQRYSEDPTVPLPSE
1109

0.0270

3925

YNTDTFESM
LVTYNTDTFESMPNP
271

−0.0027

3926

LLQETELVE
MRRLLQETELVEPLT
687

−0.0027

3927

ILDEAYVMA
NKEILDEAYVMAGVG
764

0.0047

3928

VTAEDGTQR
NQEVTAEDGTQRCEK
319

−0.0027

3929

FDGDPASNT
PESFDGDPASNTAPL
378

−0.0027

3930

VKPDLSYMP
PSGVKPDLSYMPIWK
601

−0.0027

3931

FCPDPAPGA
QGFFCPDPAPGAGGM
1028
−0.0005

0.0230

−0.0011

3932

ILKETELRK
QMRILKETELRKVKV
711
0.0419
0.0150
0.5900
0.3200
−0.0055

0.0041

0.0008
0.0130
0.0064

3933

LEDDDMGDL
RSLLEDDDMGDLVDA
1006

0.0080

3934

FDGDLGMGA
SDVFDGDLGMGAAKG
1083

−0.0025

3935

FLPESFDGD
SLAFLPESFDGDPAS
373

−0.0027

3936

FLQDIQEVQ
SLSFLQDIQEVQGYV
70

0.0520

3937

LQPEQLQVF
TAPLQPEQLQVEETL
389

0.0023

3938

LPSETDGYV
TVPLPSETDGYVAPL
1117

−0.0025

3939

VPWDQLFRN
VHTVPWDQLFRNPHQ
477

0.0220

3940

VHRDLAARN
VRLVHRDLAARNVLV
839
0.0340
0.0064
0.0033
0.3400
0.0150

0.2700

0.0430
0.0230
0.1000

3941

FGPEADQCV
VTCFGPEADQCVACA
574

−0.0027

3942

LSTDVGSCT
YNYLSTDVGSCTLVC
301

0.0059

3943

LLEDDDMGD
YRSLLEDDDMGDLVD
1005

0.0630

3944

LIDTNRSRA
ALTLIDTNRSRACHP
180

0.0350

3945

IDSECRPRF
CWMIDSECRPRFREL
958
0.0036
−0.0006
0.0150
0.4500
−0.0055

−0.0008

−0.0001
−0.0014
0.0028

3946

YLEDVRLVH
GMSYLEDVRLVHRDL
832

0.1800

3947

VDLDDKGCP
HSCVDLDDKGCPAEQ
632

−0.0027

3948

IHHNTHLCF
LALIHHNTHLCFVHT
465
0.0140
0.0990
0.0009
0.3100
−0.0055

0.0025

0.7500
0.0200
0.0330

3949

AAPQPHPPP
QGGAAPQPHPPPAFS
1200

−0.0025

3950

ASPETHLDM
RLPASPETHLDMLRH
34

−0.0027

3951

AHNQVRQVP
VLIAHNQVRQVPLQR
84

0.0290

3952

LFRNPHQAL
WDQLFRNPHQALLHT
482
−0.0001
0.0015
−0.0007
0.9000
−0.0055

−0.0008

0.0410
−0.0017
−0.0009

3953

[0500]

24

TABLE XXI

Population coverage with combined HLA Supertypes

PHENOTYPIC FREQUENCY

North

American

HLA-SUPERTYPES
Caucasian
Black
Japanese
Chinese
Hispanic
Average

a. Individual Supertypes

A2
45.8
39.0
42.4
45.9
43.0
43.2

A3
37.5
42.1
45.8
52.7
43.1
44.2

B7
43.2
55.1
57.1
43.0
49.3
49.5

A1
47.1
16.1
21.8
14.7
26.3
25.2

A24
23.9
38.9
58.6
40.1
38.3
40.0

B44
43.0
21.2
42.9
39.1
39.0
37.0

B27
28.4
26.1
13.3
13.9
35.3
23.4

B62
12.6
4.8
36.5
25.4
11.1
18.1

B58
10.0
25.1
1.6
9.0
5.9
10.3

b. Combined Supertypes

A2, A3, B7
84.3
86.8
89.5
89.8
86.8
87.4

A2, A3, B7, A24, B44, A1
99.5
98.1
100.0
99.5
99.4
99.3

A2, A3, B7, A24, B44, A1,
99.9
99.6
100.0
99.8
99.9
99.8

B27, B62, B58

[0501]

25

TABLE XXII

No. A2

A*0201
A*0202
A*0203
A*0206
A*6802
Alleles

Source
AA
Sequence
nM
nM
nM
nM
nM
Crossbound

Crossbinding data of A2 supermotif peptides

Her2/neu.5
9
ALCRWGLLL
100
—
278
—
—
2

Her2/neu.5
10
ALCRWGLLLA
139
1955
12
1947
2500
2

Her2/neu.48
9
HLYQGCQVV
139
307
13
514
1143
3

Her2/neu.106
9
QLFEDNYAL
17
226
11
463
2105
4

Her2/neu.106
10
QLFEDNYALA
357
662
9.1
218
74
4

Her2/neu.144
10
SLTEILKGGV
238
—
22
—
—
2

Her2/neu.153
9
VLIQRNPQL
23
3909
3.3
1057
—
2

Her2/neu.369
9
KIFGSLAFL
36
9.0
19
23
3333
4

Her2/neu.435
9
ILHNGAYSL
75
358
100
569
—
3

Her2/neu.466
9
ALIHHNTHL
278
1265
10
1762
—
2

Her2/neu.508
9
GLACHQLCA
417
—
127
—
9091
2

Her2/neu.653
9
SIISAVVGI
69
524
35
285
148
4

Her2/neu.665
9
VVLGVVFGI
14
—
2500
430
2000
2

Her2/neu.689
9
RLLQETELV
21
—
625
34
—
2

Her2/neu.767
9
ILDEAYVMA
238
—
4167
3083
—
1

Her2/neu.773
10
VMAGVGSPYV
200
391
13
3700
—
3

Her2/neu.789
9
CLTSTVQLV
208
457
6.7
308
8000
4

Her2/neu.799
9
QLMPYGCLL
217
977
114
712
—
2

Her2/neu.952
10
YMIMVKCWMI
20
307
83
116
267
5

Her2/neu.952
9
YMIMVKCWM
217
—
625
2643
1000
1

A2 supermotif analog peptides

Her2/neu.5
9
ALCRWGLLL
100
—
278
—
—
2

Her2/neu.5.T2B3V9
9
ATBRWGLLV
16667
215
323
2177
1739
2

Her2/neu.5V2B3V9
9
AVBRWGLLV
10000
215
141
2177
4706
2

Her2/neu.5.B3
9
ALBRWGLLL
238
1
12
6167
7273
3

Her2/neu.5B3V9
9
ALBRWGLLV
18
33
4.2
285
—
4

Her2/neu.5M2B3V9
9
AMBRWGLLV
36
473
16
726
—
3

Her2/neu.153
9
VLIQRNPQL
23
3909
3.3
1057
—
2

Her2/neu.153V9
9
VLIQRNPQV
55
768
135
385
—
3

Her2/neu.369
9
KIFGSLAFL
36
9.0
19
23
3333
4

Her2/neu.369V2V9
9
KVFGSLAFV
20
19.0
769
15
29
4

Her2/neu.369T2V9
9
KTFGSLAFV
35
13.0
1010
14
17
4

Her2/neu.369L2V9
9
KLFGSLAFV
5.8
7.5
19
17
1270
4

Her2/neu.653
9
SIISAVVGI
69
524
35
285
148
4

Her2/neu.653.L2V9
9
SLISAVVGV
7.1
10
16
20
110
5

Her2/neu.665
9
VVLGVVFGI
14
—
2500
430
2000
2

Her2/neu.665V2V9
9
VVLGVVFGV

Her2/neu.665L2V9
9
VLLGVVFGV
2.4
17
14
6.0
8000
4

Her2/neu.952
10
YMIMVKCWMI
20
307
83
116
267
5

Her2/neu.952L2V10
10
YLIMVKCWMV
13
56
116
18
84
5

Her2/neu.952L2B7V10
10
YLIMVKBWMV
7.2
66
77
11
851
4

— indicates binding affinity = 10,000 nM.

[0502]

26

TABLE XXIII

HLA-A3 Supermotif-bearing Peptides

No. of A3

A*
A*
A*
A*
A*
Alleles
CTL

Published
Published

0301
1101
3101
3301
6801
Cross-
Wild-
CTL
CTL
CTL

AA
Sequence
Source
nM
nM
nM
nM
nM
bound
type
Tumor
Wildtype
Tumor

10
QLRSLTEILK
Her2/neu.141
55
462
667
6170
—
2

10
ILKGGVLIQR
Her2/neu.148
275
—
25
121
205
4

10
IVKGGVLIQR
Her2/neu.148.V2
275
7500
72
126
29
4

10
IVKGGVLIQK
Her2/neu.148.V2K10
26
102
450
6591
27
4

10
TILWKDIFHK
Her2/neu.166
256
1.7
487
690
200
4

10
TVLWKDIFHR
Her2/neu.166.V2R10
8462
286
600
76
42
3

9
ILWKDIFHK
Her2/neu.167
39
19
82
967
1739
3

9
IVWKDIFHK
Her2/neu.167.V2
23
40
247
853
178
4

9
IVWKDIFHR
Her2/neu.167.V2R9
143
286
6.0
16
15
5

10
RTVCAGGCAR
Her2/neu.217
1618
462
40
1318
320
3

9
TVCAGGCAR
Her2/neu.218
—
261
129
326
83
4

9
TVBAGGBAR
Her2/neu.218.B3B7
314
111
247
242
8.0
5

9
TVBAGGBAK
Her2/neu.218.B3B7K9
24
29
—
—
7.3
3

10
IVWLGLRSLR
Her2/neu.450.V2
234
1936
11
193
7.3
4

10
IVWLGLRSLK
Her2/neu.450.V2K10
3.9
128
273
2071
12
4

10
ISWLGLRSLR
Her2/neu.450
268
2222
6.9
223
73
4

10
HTVPWDQLFR
Her2/neu.478
3143
83
19
88
4.0
4

10
HVVPWDQLFR
Her2/neu.478.V2
7333
1333
391
193
3.6
3

10
HVVPWDQLFK
Her2/neu.478.V2K10
180
375
—
—
8.9
3

9
CVNCSQFLR
Her2/neu.528
7333
194
34
50
18
4

9
BVNBSQFLR
Her2/neu.528.B1B4
177
80
38
58
10
5

9
BVNBSQFLK
Her2/neu.528.B1B4K9
34
22
60
4265
15
4

10
GVVFGILIKR
Her2/neu.668
611
182
305
2071
19
3

9
VVFGILIKR
Her2/neu.669
100
8.3
13
78
4.0
5
1/3
0/1

10
VVFGILIKRR
Her2/neu.669
3667
375
290
193
15
4

9
VVFGILIKK
Her2/neu.669.K9
22
19
3750
—
35
3

9
KIRKYTMRR
Her2/neu.681
15
—
16
4028
—
2
3/3
2/3
+1)
+

9
VLRENTSPK
Her2/neu.754
28
462
129
290
—
4
4/7
3/6
+1)
+

9
VVRENTSPR
Her2/neu.754.V2R9
200
5455
375
126
178
4

9
LLDHVRENR
Her2/neu.806
297
—
500
326
5714
3

9
LVARNVLVK
Her2/neu.846.V2
42
214
9000
—
205
3

9
LAARNVLVK
Her2/neu.846
190
211
—
—
500
3

9
LVKSPNHVK
Her2/neu.852
23
86
182
784
73
4

+1)

9
LVKSPNHVR
Her2/neu.852.R9
7857
—
198
107
50
3

9
KITDFGLAR
Her2/neu.860
65
25
100
—
1633
3

9
KVTDFGLAR
Her2/neu.860.V2
201
76
106
—
133
4

9
MVLESILRR
Her2/neu.889.V2
216
273
207
153
22
5

9
MVLESILRK
Her2/neu.889.V2K9
61
16
—
2636
381
3

9
MALESILRR
Her2/neu.889
3235
253
192
132
127
4

10
LVSEFSRMAR
Her2/neu.972
1528
182
49
126
36
4

10
LVSEFSRMAK
Her2/neu.972.K10
250
71
2250
5273
62
3

10
AVPLDSTFYR
Her2/neu.997.V2
−110000
88
30000
2636
73
2

10
AVPLDSTFYK
Her2/neU.997.V2K10
550
33
1500
22308
229
2

10
ASPLDSTFYR
Her2/neu.997
—
90
150
2071
154
3

1)
Kawashima et al., Cancer Research 59:431, 1999

— indicates binding affnity > 10,000 nM.

[0503]

27

TABLE XXIV

B7 Supermotif Peptides

No. of B7

Alleles

AA
Sequence
Source
B*0702 nM
B*3501 nM
B*5101 nM
B*5301 nM
B*5401 nM
Crossbound

9
LPTNASLSF
Her2/neu.65
212
114
809
34
—
3

10
LPTNASLSFL
Her2/neu.65
290
—
2500
—
—
1

9
FPTNASLSF
Her2/neu.65.F1
1.1
7.9
85
6.2
556
4

9
FPTNASLSI
Her2/neu.65.F1I9
4.6
300
8.7
14
2.8
5

10
FPTNASLSFI
Her2/neu.65.F1I10
120
424
23
85
53
5

10
FPTNASLSFL
Her2/neu.65.F1
18
200
262
172
119
5

9
FPLNNTTPI
Her2/neu.121.F1I9
1.6
200
13
52
1.6
5

9
FPLNNTTPV
Her2/neu.121.F1
0.20
40
7.1
1069
1.1
4

10
SPGGLRELQL
Her2/neu.133
95
—
—
—
—
1

8
SPGGLREL
Her2/neu.133
100
—
—
—
—
1

10
FPGGLRELQI
Her2/neu.133.F1I10
306
—
1774
310
476
3

10
FPMCKGSRCI
Her2/neu.196.F1
183
—
500
6643
303
3

10
MPNPEGRYTI
Her2/neu.282.I10
34
111
13
9.3
46
5

10
FPNPEGRYTI
Her2/neu.282.F1
324
24
9.3
6.6
3.2
5

10
CPLHNQEVTI
Her2/neu.315.I10
458
—
42
274
556
3

10
KPCARVCYGL
Her2/neu.336
149
—
—
—
—
1

10
FPCARVCYGL
Her2/neu.336.F1
190
450
1000
1691
36
3

8
WPDSLPDI
Her2/neu.415.I8
71
—
5000
—
—
1

10
FPHQALLHTA
Her2/neu.488.F1
393
379
4231
—
1.3
3

10
FPHQALLHTI
Her2/neu.488.F1I10
86
655
19
42
4.0
4

11
CPSGVKPDLSY
Her2/neu.600
183
3600
—
—
—
1

11
CPSGVKPDLSI
Her2/neu.600.I11
290
—
138
344
625
3

9
FPSGVKPDI
Her2/neu.600.F1I9
6.3
9000
204
930
44
3

11
FPSGVKPDLSI
Her2/neu.600.F1I11
196
3273
153
211
208
4

9
KPDLSYMPI
Her2/neu.605
76
—
2500
—
—
1

9
FPDLSYMPI
Her2/neu.605.F1
22
167
10
72
31
5

10
CPAEQRASPL
Her2/neu.642
37
—
—
—
—
1

10
FPAEQRASPI
Her2/neu.642.F1I10
3.1
—
98
4895
56
3

10
FPAEQRASPL
Her2/neu.642.F1
1.4
248
859
8455
286
3

10
SPLTSIISAV
Her2/neu.649
61
—
—
—
667
1

9
SPLTSIISI
Her2/neu.649.I9
86
—
275
3444
217
3

9
FPLTSIISA
Her2/neu.649.F1
290
21
663
3000
1.4
3

9
FPLTSIISI
Her2/neu.649.F1I9
212
118
13
28
1.5
5

10
FPLTSIISAI
Her2/neu.649.F1I10
229
327
26
194
3.3
5

10
FPLTSIISAV
Her2/neu.649.F1
220
300
63
2906
1.2
4

11
FPLTSIISAVI
Her2/neu.649.F1I11
367
96
4.6
66
2.2
5

9
FPLTPSGAI
Her2/neu.698.F1I9
0.90
2057
9.2
1632
1.9
3

9
FPLTPSGAM
Her2/neu.698.F1
2.0
16
71
1525
91
4

10
FPSGAMPNQI
Her2/neu.701.F1
229
6546
131
775
100
3

11
MPNQAQMRILI
Her2/neu.706.I11
344
1800
37
12
303
4

9
FPNQAQMRI
Her2/neu.706.F1
68
108
12
18
4.5
5

10
FPNQAQMRII
Her2/neu.706.F1I10
290
514
76
490
59
4

10
FPNQAQMRIL
Her2/neu.706.F1
81
200
458
443
31
5

8
FPKANKEI
Her2/neu.760.F1
0.16
—
2500
—
3125
1

9
FPKANKEII
Her2/neu.760.F1I9
8.5
—
55
7154
39
3

9
FPKANKEIL
Her2/neu.760.F1
6.7
4500
190
—
77
3

8
FPYVSRLL
Her2/neu.779.F1
61
600
6.9
581
172
3

10
FPYVSRLLGI
Her2/neu.779.F1
112
3600
7.9
358
9.1
4

11
MPYGCLLDHVI
Her2/neu.801.I11
66
4.2
2.4
1.9
7.7
5

8
FPIKWMAI
Her2/neu.884.F1I8
22
1143
122
930
33
3

8
FPIKWMAL
Her2/neu.884.F1
0.60
248
306
547
40
4

8
KPYDGIPA
Her2/neu.921
367
—
—
490
29
3

8
KPYDGIPI
Her2/neu.921.I8
115
—
74
5167
303
3

8
FPYDGIPA
Her2/neu.921.F1
423
206
157
6200
1.0
4

8
FPYDGIPI
Her2/neu.921.F1I8
177
379
10
344
56
5

11
FPYDGIPAREI
Her2/neu.921.F1I11
141
2880
50
465
15
4

9
LPQPPICTI
Her2/neu.941
196
—
4.2
28
19
4

9
FPQPPICTI
Her2/neu.941.F1
20
360
17
11
1.7
5

11
FPRFRELVSEI
Her2/neu.966.F1I11
229
240
167
127
28
5

10
FPASPLDSTF
Her2/neu.995.F1
3.7
10
579
10
200
4

10
FPASPLDSTI
Her2/neu.995.F1I10
20
514
20
30
14
5

11
FPLDSTFYRSI
Her2/neu.998.F1I11
229
2667
46
620
2.4
3

11
FPLDSTFYRSL
Her2/neu.998.F1
42
400
324
1192
2.7
4

9
FPLAPSEGI
Her2/neu.1073.F1I9
100
554
204
239
5.9
4

9
FPTHDPSPI
Her2/neu.1101.F1I9
108
600
56
274
50
4

9
FPTHDPSPL
Her2/neu.1101.F1
10
72
3438
1632
208
3

9
FPSETDGYI
Her2/neu.1120.F1I9
220
655
31
37
156
4

9
FPSETDGYV
Her2/neu.1120.F1
204
809
32
517
26
3

11
PPSPREGPLPI
Her2/neu.1149.I11
22
—
423
85
—
3

11
FPSPREGPLPI
Her2/neu.1149.F1I11
190
—
262
5167
263
3

9
FPREGPLPI
Her2/neu.1151.F1I9
0.30
—
14
2818
4.0
3

10
FPREGPLPAI
Her2/neu.1151.F1I10
20
7200
20
620
3.4
3

9
FPLPAARPA
Her2/neu.1155.F1
34
277
1897
—
1.5
3

9
FPLPAARPI
Her2/neu.1155.F1I9
6.5
600
7.1
282
3.8
4

9
FPAARPAGI
Her2/neu.1157.F1I9
9.3
—
131
4227
48
3

11
FPAARPAGATI
Her2/neu.1157.F1I11
39
4235
13
332
50
4

11
FPAARPAGATL
Her2/neu.1157.F1
3.9
360
239
2906
133
4

8
FPGKNGVI
Her2/neu.1174.F1I8
458
—
177
—
385
3

[0504]

28

TABLE XXV

HLA-A1 Motif-Bearing Peptides

AA
Sequence
Source
A*0101 nM

11
GTDMKLRLPY
Her2/neu.28.Y10
50

11
ETHLDMLRHLY
Her2/neu.40
89

9
HLDMLRHLY
Her2/neu.42
2.7

9
HTDMLRHLY
Her2/neu.42.T2
1.9

9
GTQLFEDNY
Her2/neu.104
139

9
GTDLFEDNY
Her2/neu.104.D3
0.90

10
PTDCCHEQCA
Her2/neu.232
125

10
PTDCCHEQCY
Her2/neu.232.Y10
46

11
PTDCCHEQCAA
Her2/neu.232
58

11
PTDCCHEQCAY
Her2/neu.232.Y11
18

10
ESMPNPEGRY
Her2/neu.280
139

10
ETMPNPEGRY
Her2/neu.280.T2
3.9

9
ASCVTACPY
Her2/neu.293
455

11
ASCVTACPYNY
Her2/neu.293
132

9
ATCVTACPY
Her2/neu.293.T2
49

8
VTACPYNY
Her2/neu.296
250

11
VFETLEEITGY
Her2/neu.399
5556

11
ETLEEITGYLY
Her2/neu.401
57

9
ETDEEITGY
Her2/neu.401.D3
17

10
TLEEITGYLY
Her2/neu.402
23

10
TLDEITGYLY
Her2/neu.402.D3
3.4

11
EADQCVACAHY
Her2/neu.580
250

9
VMDGVGSPY
Her2/neu.773.D3
40

10
CMQIAKGMSY
Her2/neu.826
83

10
CTQIAKGMSY
Her2/neu.826.T2
19

9
LLDIDETEY
Her2/neu.869
3.3

9
LTDIDETEY
Her2/neu.869.T2
5.7

10
FTHQSDVWSY
Her2/neu.899
9.3

10
FTDQSDVWSY
Her2/neu.899.D3
0.60

10
PADPLDSTFY
Her2/neu.996.D3
19

9
ATPLDSTFY
Her2/neu.997.T2
36

10
MTDLVDAEEY
Her2/neu.1014.T2
2.3

9
LTCSPQPEY
Her2/neu.1131
192

9
LTDSPQPEY
Her2/neu.1131.D3
32

10
FSPAFDNLYY
Her2/neu.1213
4.5

10
FTPAFDNLYY
Her2/neu.1213.T2
0.80

9
SPDFDNLYY
Her2/neu 1214.D3
73.50

10
GTPTAENPEY
Her2/neu.1239
397

10
GTDTAENPEY
Her2/neu.1239.D3
26

[0505]

29

TABLE XXVI

HLA-A24 Motif-Bearing Peptides

AA
Sequence
Source
A*2402 nM

8
RWGLLLAL
Her2/neu.8
480

9
RWGLLLALL
Her2/neu.8
9.2

9
RYGLLALF
Her2/neu8.Y2F9
1.3

9
TYLPTNASL
Her2/neu.63
316

11
TYLPTNASLSF
Her2/neu.63
1.3

9
TYLPTNASF
Her2/neu.63.F9
44

9
CYGLGMEHF
Her2/neu.342.F9
164

10
LYISAWPDSL
Her2/neu.410
143

10
LYISAWPDSF
Her2/neu.410.F10
10

9
AYPDSLPDF
Herw/neu414.Y2F9
24

9
AYSLTLQGL
Her2/neu.440
92

9
AYSLTLQGF
Her2/neu.440.F9
52

9
EYVNARHCF
Her2/neu.553.F9
150

8
SYMPIWKF
Her2/neu.609
38

9
PYVSRLLGI
Her2/neu 780
71

11
PYVSRLLGICL
Her2/neu.780
375

9
PYVSRLLGF
Her2/neu.780.F9
9.2

10
GYSYLEDVRF
Her2/neu.832.Y2F10
235.0

9
KYMALESIF
Her2/neu.887.Y2F9
19.0

9
RYTHQSDVF
Her2/neu.898.Y2F9
60.0

9
VWSYGVTVW
Her2/neu.905
150

9
VYSYGVTVF
Her2/neu.905.Y2F9
16

11
VWSYGVTVWEL
Her2/neu.905
130

9
SYGVTVWEL
Her2/neu.907
100

9
SYGVTVWEF
Her2/neu.907.F9
26

9
VYMIMVKCW
Her2/neu.951
75

11
VYMIMVKCWMI
Her2/neu.951
6.7

9
VYMIMVKCF
Her2/neu.951.F9
19

9
RYRELVSEF
Her2/neu.968.Y2
36

9
RYARDPQRF
Her2/neu.978.Y2
120

[0506]

30

TABLE XXVII

HLA-A2 Supermotif-bearing Peptides

A*
A*
A*
No. of A2

A*0201
A*0202
0203
0206
6802
Alleles
CTL
CTL
CTL
CTL

AA
Sequence
Source
nM
nM
nM
nM
nM
Crossbound
Wildtype1
Tumor1
Wildtype2
Tumor2

9
ALCRWGLLL
Her2/neu.5
100
—
278
—
—
2
2/2
2/2

9
ALBRWGLLV
Her2/neu.5.B3V9
18
33
4.2
285
—
4

9
HLYQGCQVV
Her2/neu.48
139
307
13
514
1143
3
1/2
0/2
2/2
1/2

9
VLIQRNPQL
Her2/neu.153
23
3909
3.3
1057
—
2

9
VLIQRNPQV
Her2/neu.153.V9
55
768
135
385
—
3

9
KIFGSLAFL
Her2/neu.369
36
9.0
19
23
3333
4
6/7
4/7

9
KLFGSLAFV
Her2/neu.369.L2V9
5.8
7.5
19
17
1270
4

9
KVFGSLAFV
Her2/neu.369.V2V9
20
19
769
15
29
4

9
KTFGSLAFV
Her2/neu.369.T2V9
35
13
1010
14
17
4

10
RILHNGAYSL
Her2/neu 434
278
1000
6667
1276
—
1

9
ILHNGAYSL
Her2/neu.435
75
358
100
569
—
3
3/3
1/3
2/2
2/2

9
SLISAVVGV
Her2/neu.653.L2V9
7.1
10
16
20
110
5

9
VVLGVVFGI
Her2/neu.665
14
—
2500
430
2000
2

9
VLLGVVFGV
Her2/neu.665.L2V9
2.4
19
14
6.0
8000
4

9
RLLQETELV
Her2/neu.689
21
—
625
34
—
2

10
VMAGVGSPYV
Her2/neu.773
200
391
13
3700
—
3
1/2
0/2
1/2
1/2

9
CLTSTVQLV
Her2/neu.789
208
457
6.7
308
8000
4
1/4
0/4
1/2

10
YMIMVKCWMI
Her2/neu.952
20
307
83
116
267
5
0/1
0/1
2/2
2/2

10
YLIMVKCWMV
Her2/neu.952.L2V10
13
56
116
18
84
5

1
Number of donors yielding a positive response/total tested.

2
Data from ovarian cancer patients.

[0507]

31

TABLE XXVIII

Her2/neu DR supertype primary binding

DR147

DR147

Algo

DR1
DR4w4
DR7
Cross-

Sum
Sequence
Source
nM
nM
nM
binding

2
LCRWGLLLALLPPGA
Her2/neu.6
53
—
—
1

2
RWGLLLALLPPGAAS
Her2/neu.8
0.42
161
—
2

2
WGLLLALLPPGAAST
Her2/neu.9
0.98
35
—
2

2
GTDMKLRLPASPETH
Her2/neu.28
5000
—
—
0

2
DMKLRLPASPETHLD
Her2/neu.30
5000
—
—
0

2
NLELTYLPTNASLSF
Her2/neu.59
11
118
368
3

3
LTYLPTNASLSFLQD
Her2/neu.62
10
136
78
3

2
TQLFEDNYALAVLDN
Her2/neu.105
94
—
1563
1

2
VCPLHNQEVTAEDGT
Her2/neu.314
—
—
—
0

2
CKKIFGSLAFLPESF
Her2/neu.367
21
—
926
2

2
LSVFQNLQVIRGRIL
Her2/neu.422
28
672
86
3

2
LRELGSGLALIHHNT
Her2/neu.458
161
—
—
1

3
KPDLSYMPIWKFPDE
Her2/neu.605
152
—
8621
1

3
ASPLTSIISAVVGIL
Her2/neu.648
56
—
714
2

2
LTSIISAVVGILLVV
Her2/neu.651
26
—
5102
1

3
VVGILLVVVLGVVFG
Her2/neu.658
—
—
—
0

3
LLVVVLGVVFGILIK
Her2/neu.662
>6250
—
—
0

2
VLGVVFGILIKRRQQ
Her2/neu.666
71
—
781
2

2
ETELVEPLTPSGAMP
Her2/neu.693
833
—
—
1

2
VEPLTPSGAMPNQAQ
Her2/neu.697
>6250
—
—
0

2
ETELRKVKVLGSGAF
Her2/neu.717
313
1286
658
2

2
GENVKIPVAIKVLRE
Her2/neu.743
79
—
807
2

2
IKVLRENTSPKANKE
Her2/neu.752
—
—
—
0

3
KEILDEAYVMAGVGS
Her2/neu.765
—
6164
—
0

3
DEAYVMAGVGSPYVS
Her2/neu.769
100
196
125
3

2
SRLLGICLTSTVQLV
Her2/neu.783
14
375
45
3

2
TVQLVTQLMPYGCLL
Her2/neu.793
22
978
2500
2

3
LLNWCMQIAKGMSYL
Her2/neu.822
6.0
—
208
2

2
ITDFGLARLLDIDET
Her2/neu.861
1042
—
—
0

3
KVPIKWMALESILRR
Her2/neu.883
2.3
652
1316
2

3
PIKWMALESILRRRF
Her2/neu.885
6.3
1286
3205
1

2
IKWMALESILRRRFT
Her2/neu.886
5.3
1125
6250
1

2
GVTVWELMTFGAKPY
Her2/neu.909
3.6
1364
1471
1

3
VWELMTFGAKPYDGI
Her2/neu.912
58
818
676
3

2
GERLPQPPICTIDVY
Her2/neu.938
—
—
—
0

2
QPPICTIDVYMIMVK
Her2/neu.943
75
7500
250
2

2
DVYMIMVKCWMIDSE
Her2/neu.950
179
790
192
3

2
QGFFCPDPAPGAGGM
Her2/neu.1028
—
1957
—
0

3
TDGYVAPLTCSPQPE
Her2/neu.1124
—
—
—
0

2
QPDVRPQPPSPREGP
Her2/neu.1142
7143
—
—
0

2
PSTFKGTPTAENPEY
Her2/neu.1234
—
—
—
0

[0508]

32

TABLE XXIX

DR supertype crossbinding

Broad

DR4w4
DR7
DR2w2β1
DR2w2β2
DR6w19
DR5w11
DR8w2
DR147
Binding

Sequence
Source
DR1 nM
nM
nM
nM
nM
nM
nM
nM
Binding
(5/8)

RWGLLLALLPPGAAS
Her2/neu.8
0.40
161
—
70
741
—
282
408
2
6

WGLLLALLPPGAAST
Her2/neu.9
1.0
35
—
43
1818
—
80
109
2
5

NLELTYLPTNASLSF
Her2/neu.59
11
118
368
325
2222
2059
4000
2227
3
4

LTYLPTNASLSFLQD
Her2/neu.62
10
136
78
910
357
125
4878
9074
3
6

CKKIFGSLAFLPESF
Her2/neu.367
21
—
926
1300
—
1029
—
—
2
2

LSVFQNLQVIRGRIL
Her2/neu.422
28
672
86
325
270
614
2000
1485
3
6

ASPLTSIISAVVGIL
Her2/neu.648
56
—
714
96
5405
73
—
—
2
4

VLGVVFGILIKRRQQ
Her2/neu.666
71
—
781
827
323
233
43
77
2
7

ETELRKVKVLGSGAF
Her2/neu.717
313
1286
658
4790
3846
2500
3279
1960
2
2

GENVKIPVAIKVLRE
Her2/neu.743
79
—
807
1936
5882
8750
—
—
2
2

DEAYVMAGVGSPYVS
Her2/neu.769
100
196
125
3138
833
1750
7407
860
3
5

SRLLGICLTSTVQLV
Her2/neu.783
14
375
45
414
—
10
1429
—
3
5

TVQLVTQLMPYGCLL
Her2/neu.793
22
978
2500
12
—
1129
—
7101
2
3

LLNWCMQIAKGMSYL
Her2/neu.822
6.0
—
208
1597
17
90
50
120
2
6

KVPIKWMALESILRR
Her2/neu.883
2.3
652
1316
3.4
9.5
1129
2740
6203
2
4

VWELMTFGAKPYDGI
Her2/neu.912
58
818
676
92
200
8750
3704
5506
3
5

QPPICTIDVYMIMVK
Her2/neu.943
75
7500
250
169
7407
2692
4348
9608
2
3

DVYMIMVKCWMIDSE
Her2/neu.950
179
790
192
1936
4762
—
909
1089
3
4

[0509]

33

TABLE XXX

DR3 binding

Sequence
Source
DR3 nM

RLPASPETHLDMLRH
Her2/neu.34
—

SLSFLQDIQEVQGYV
Her2/neu.70
5769

VLIAHNQVRQVPLQR
Her2/neu.84
—

GTQLFEDNYALAVLD
Her2/neu.104
1364

DTILWKDIFHKNNQL
Her2/neu.165
—

ALTLIDTNRSRACHP
Her2/neu.180
8571

KGPLPTDCCHEQCAA
Her2/neu.228
—

LVTYNTDTFESMPNP
Her2/neu.271
—

YNYLSTDVGSCTLVC
Her2/neu.301
—

NQEVTAEDGTQRCEK
Her2/neu.319
—

CYGLGMEHLREVRAV
Her2/neu.342
—

SLAFLPESFDGDPAS
Her2/neu.373
—

PESFDGDPASNTAPL
Her2/neu.378
—

TAPLQPEQLQVFETL
Her2/neu.389
—

LALIHHNTHLCFVHT
Her2/neu.465
968

VHTVPWDQLFRNPHQ
Her2/neu.477
—

WDQLFRNPHQALLHT
Her2/neu.482
333

LQGLPREYVNARHCL
Her2/neu.547
—

VTCFGPEADQCVACA
Her2/neu.574
—

PSGVKPDLSYMPIWK
Her2/neu.601
—

IWKFPDEEGACQPCP
Her2/neu.613
—

HSCVDLDDKGCPAEQ
Her2/neu.632
—

MRRLLQETELVEPLT
Her2/neu.687
—

QMRILKETELRKVKV
Her2/neu.711
938

AIKVLRENTSPKANK
Her2/neu.751
—

NKEILDEAYVMAGVG
Her2/neu.764
—

GMSYLEDVRLVHRDL
Her2/neu.832
1667

VRLVHRDLAARNVLV
Her2/neu.839
882

ARLLDIDETEYHADG
Her2/neu.867
968

ETEYHADGGKVPIKW
Her2/neu.874
—

IKWMALESILRRRFT
Her2/neu.886
682

CWMIDSECRPRFREL
Her2/neu.958
667

FRELVSEFSRMARDP
Her2/neu.969
4225

FSRMARDPQRFVVIQ
Her2/neu.976
1875

FVVIQNEDLGPASPL
Her2/neu.986
—

YRSLLEDDDMGDLVD
Her2/neu.1005
4762

RSLLEDDDMGDLVDA
Her2/neu.1006
—

GDLVDAEEYLVPQQG
Her2/neu.1015
—

QGFFCPDPAPGAGGM
Her2/neu.1028
—

DLTLGLEPSEEEAPR
Her2/neu.1058
—

SDVFDGDLGMGAAKG
Her2/neu.1083
—

LQRYSEDPTVPLPSE
Her2/neu.1109
—

TVPLPSETDGYVAPL
Her2/neu.1117
—

KNGVVKDVFAFGGAV
Her2/neu.1177
—

QGGAAPQPHPPPAFS
Her2/neu.1200
—

DNLYYWDQDPPERGA
Her2/neu.1218
—

[0510]

34

TABLE XXXI

HLA Class II Supermotif and Motif-Bearing Epitopes

DRB1*
DRB1*
DRB1*
DRB1*
DRB1*
DRB1*
DRB1*

No. of DR

0101
0301
0401
0701
0802
1101
1302
*DRB1
*DRB5
Alleles

Sequence
Source
nM
nM
nM
nM
nM
nM
nM
1501 nM
0101 nM
Crossbound

VLGVVFGILIKRRQQ
Her2/neu.666
71
—
—
781
77
43
233
827
323
7

QMRILKETELRKVKV
Her2/neu.711
119
938
>8182
1923
7656
4878
4375
607
34
3

DEAYVMAGVGSPYVS
Her2/neu.769
100
—
196
125
860
7407
1750
3138
833
5

SRLLGICLTSTVQLV
Her2/neu.783
14
—
375
45
—
1429
10
414
—
5

LLNWCMQIAKGMSYL
Her2/neu.822
6.0
—
—
208
120
50
90
1597
17
6

VRLVHRDLAARNVLV
Her2/neu.839
147
882
3058
1087
490
74
81
1422
6061
4

ARLLDIDETEYHADG
Her2/neu.867
—
968
>8182
—
—
—
—
—
—
0

IKWMALESILRRRFT
Her2/neu.886
17
682
3224
4098
731
370
2500
11
2.5
5

VWELMTFGAKPYDGI
Her2/neu.912
58
—
818
676
5506
3704
8750
92
200
5

CWMIDSECRPRFREL
Her2/neu.958
1389
667
>8182
—
—
—
—
—
1333
0

[0511]

Inducing cellular immune responses to her2/neu using peptide and nucleic acid compositions

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