Inducing cellular immune responses to prostate cancer antigens using peptide and nucleic acid compositions

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, as we have described, represents a solution to this challenge, in that it allows the incorporation of various CTL, HTL, and antibody (if desired) epitopes from discrete regions of one or more target tumor-associated antigens (TAAs) in a single vaccine composition. Such a composition may simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population.

[0005] Prostate cancer is the most common malignancy in men. Current therapies, i.e., chemotherapy combined with androgen blockade, antiandrogen withdrawal, and other secondary hormonal therapies, have met with limited success. Thus, there is a need to develop more efficacious therapies. The multiepitopic immunotherapy vaccine compositions of the present invention fulfill this need.

[0006] Antigens that are associated with prostate cancer include, but are not limited to, prostate specific antigen (PSA), prostate specific membrane antigen (PSM), prostatic acid phosphatase (PAP), and human kallikrein2 (hK2 or HuK2). These antigens represent important antigen targets for the polyepitopic vaccine compositions of the invention.

[0007] PSM is also an important candidate for prostate cancer therapy. It is a Type II membrane protein that is expressed at high levels on prostate adenocarcinomas. The levels of expression increase on metastases and in carcinomas that are refractory to hormone therapy. PSM is not generally present on normal tissues, although low levels have been detected in the colonic crypts and in the duodenum, and PSM can be detected in normal male serum and seminal fluid (see, e.g., Silver et al., Clin. Cancer Res. 3:81-85, 1997). CTL responses to PSM have also been documented (see, e.g., Murphy et al., Prostate 29:371-380, 1996; and Salgaller et al., Prostate 35:144-151, 1998).

[0008] PAP is a tissue-specific differentiation antigen that is secreted exclusively by cells in the prostate (see, e.g., Lam et al., Prostate 15:13-21, 1989). It can be detected in serum and levels are increased in patients with prostate carcinoma (see, e.g., Jacobs et al., Curr. Probl. Cancer 15:299-360, 1991). The PAP protein sequence has, at best, a 49% sequence homology with other acid phosphatases with the homologous regions distributed throughout the protein. Accordingly, PAP-specific epitopes can be identified and several different CTL epitopes have been described (see, e.g., Peshwa et al., Prostate 36:129-138, 1998).

[0009] The hK2 protein is functionally a serine protease involved in posttranslational processing of polypeptides. It is expressed by prostate epithelia exclusively, and is found in both benign and malignant prostate cancer tissue. Although it is expressed in 50% of normal prostate cells, the percentage of cells expressing hK2 is increased in adenocarcinomas and prostatic intraepithelial neoplasia (PIN) (see, e.g., Darson et al., Urology 49:857-862, 1997). Based on the preferential expression of this antigen on prostate cancer cells, hK2 is also an important target for immunotherapy.

[0010] Prostate-specific antigen (PSA), also referred to as hK3, is a secreted serine protease and a member of the kallikrein family of proteins. The PSA gene is 80% homologous with the hK2 gene, however, tissue expression of hK2 is regulated independently of PSA (see, e.g., Darson et al., Urology 49:857-862, 1997). Expression of PSA is restricted to prostate epithelial cells, both benign and malignant. The antigen can be detected in the serum of most prostate cancer patients and in seminal plasma. Several T cell epitopes from PSA have been identified and have been found to be immunogenic, and antibody responses have been reported in patients (see, e.g., Correale et al., J. Immunol. 161:3186, 1998; and Alexander et al., Urology 51:150-157, 1998). Thus, based on its prostate-restricted expression and ability to stimulate immune responses, PSA is an attractive target for immunotherapy of prostate cancer.

[0011] 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.

SUMMARY OF THE INVENTION

[0012] 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 identifies epitopes for inclusion in diagnostic and/or pharmaceutical compositions and methods of use of the epitopes for the evaluation of immune responses and for the treatment and/or prevention of cancer.

[0013] 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).

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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, prostate cancer cells in one patient may express target TAAs that differ from the prostate cancer cells in another patient. Epitopes derived from multiple TAAs can be included in a polyepitopic vaccine that will target both prostate cancers.

[0018] 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.

[0019] 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.

[0020] 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 about 500 nM or less for HLA class I molecules or an IC50 of about 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.

[0021] 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 analoged to modify binding affinity and/or the ability to bind to multiple alleles within an HLA supertype.

[0022] 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 comprising a supermotif or motif and 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 may, for example, be used as a component of a tetrameric complex for this type of analysis.

[0023] 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.

[0024] 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.

BRIEF DESCRIPTION OF THE FIGURES

[0025] not applicable

DETAILED DESCRIPTION OF THE INVENTION

[0026] 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.

[0027] A list of target TAAs 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 PAGE-4. Epitopes derived from these antigens may be used in combination with one another to target a specific tumor type, e.g., prostate tumors, or to target multiple types of tumors.

[0028] 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.

[0029] A. Definitions

[0030] The invention can be better understood with reference to the following definitions, which are listed alphabetically:

[0031] 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.

[0032] 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.

[0033] “Cross-reactive binding” indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] “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).

[0040] 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 super family, HLA supertype family, HLA family, and HLA xx-like molecules (where xx denotes a particular HLA type), are synonyms.

[0041] 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.

[0042] 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.

[0043] 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).

[0044] As used herein, “high affinty” 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.

[0045] 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.

[0046] 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 an HLA-restricted cytotoxic or helper T cell response to the antigen from which the immunogenic peptide is derived.

[0047] 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.

[0048] “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.

[0049] “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.

[0050] 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, often 8 to 11 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.

[0051] 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.

[0052] A “non-native” sequence or “construct” refers to a sequence that is not found in nature, i.e., is “non-naturally occurring”. Such sequences include, e.g. peptides that are lipidated or otherwise modified, and polyepitopic compositions that contain epitopes that are not contiguous in a native protein sequence.

[0053] 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. CTL-inducing peptides of the invention are often 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues. HTL-inducing oligopeptides are often 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.

[0054] “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition.

[0055] 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.

[0056] 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 I. 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.

[0057] “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.

[0058] 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.

[0059] The term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] “Synthetic peptide” refers to a peptide that is man-made using such methods as chemical synthesis or recombinant DNA technology.

[0064] 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.

[0065] 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.

[0066] B. Stimulation of CTL and HTL Responses

[0067] 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.

[0068] 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.d11/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. Immunogenetics November 1999; 50(3-4):201-12, Review 9).

[0069] 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.)

[0070] 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.

[0071] 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.

[0072] Various strategies can be utilized to evaluate immunogenicity, including:

[0073] 1) Evaluation of primary T cell cultures from normal individuals (see, e.g. Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998); This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine-release or a 51Cr cytotoxicity assay involving peptide sensitized target cells.

[0074] 2) Immunization of HLA transgenic mice (see, e.g. Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); In this method, peptides in incomplete Freund'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.

[0075] 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.

[0076] The following describes the peptide epitopes and corresponding nucleic acids of the invention.

[0077] C. Binding Affinity of Peptide Epitopes for HLA Molecules

[0078] 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.

[0079] 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 farther 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.

[0080] High HLA binding affinity is correlated with greater immunogenicity (see, e.g., Sette, et al., J. Immunol. 153:5586-5592, 1994; Chen et al., J. Immunol. 152:2874-2881, 1994; and Ressing et al., J. Immunol. 154:5934-5943, 1995). 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.

[0081] 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).

[0082] 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.

[0083] 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 a small set 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. With respect to analog peptides, CTL inductions positive for wildtype 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.

[0084] The binding affinity of peptides for HLA molecules can be determined as described in Example 1, below.

[0085] D. Peptide Epitope Binding Motifs and Supermotifs

[0086] 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.

[0087] Such peptide epitopes are identified in the Tables described below.

[0088] Peptides of the present invention may 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.

[0089] 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.”

[0090] 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.

[0091] Examples of supermotif and/or motif-bearing peptide epitopes are shown in Tables VII-XX. To obtain the peptide epitope sequences, protein sequence data for the prostate cancer antigens PAP, PSA, PSM, and hK2, which is designated as kallikrein in Tables VII-XX, were evaluated for the presence of the designated supermotif or motif. The “Position” column indicates the position in the protein sequence that corresponds to the first amino acid residue of the putative epitope. The “number of amino acids” indicates the number of residues in the epitope sequence. The tables also include a binding affinity ratio listing for some of the peptide epitopes for the allele-specific HLA molecule indicated in the column heading. 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 affinities 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.

[0092] To obtain the peptide epitope sequences listed in each of Tables VII-XX, the amino acid sequences of PSA, PSM, PAP, and HuK were 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.

[0093] In the Tables, the motif- and/or supermotif-bearing amino acid sequences are identified by the position number and the length of the epitope with reference to the prostate antigen amino acid sequence and numbering provided below. The “protein” indicates the prostate antigen sequence that includes the epitope. The “pos” (position) column designates the amino acid position in the prostate antigen sequence protein sequence below 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 sequence listed in Table VII is a sequence of 11 residues in length starting at position 122 of PAP. Accordingly, the amino acid sequence of the epitope is ALFPPEGVSIW. Similarly, the first kallikrein sequence in Table VII starts at position 147 and is 11 residues in length. Thus the amino acid sequence is ALGTTCYASGW.

[0094] Binding data presented in Tables VII-XX are expressed as a relative binding ratio, supra in the in columns labeled with the allele-specific HLA molecule.

[0095] PSA (Prostate Specific Antigen)

11VVFLTLSVTW IGAAPLILSR IVGGWECEKH SQPWQVLVAS RGRAVCGGVL VHPQWVLTAA60HCIRNKSVIL LGRHSLFHPE DTGQVFQVSH SFPHPLYDMS LLKNRFLRPG DDSSHDLMLL120RLSEPAELTD AVKVMDLPTQ EPALGTTCYA SGWGSIEPEE FLTPKKLQCV DLHVISNDVC180AQVEPQKVTK FMLCAGRWTG GKSTCSGDSG GPLVCNGVLQ GITSWGSEPC ALPERPSLYT240KVVHYRKWIK DTIVANP 257

[0096] PAP (Prostatic Acid Phosphatase)

21MRAAPLLLAR AASLSLGFLF LLFFWLDRSV LAKELKFVTL VFRHGDRSPI DTFPTDPIKE60SSWPQGFGQL TQLGMEQEYE LGEYIRKRYR KFLNESYKHE QVYTRSTDVD RTLMSAMTNL120AALFPPEGVS IWNPILLWQP IPVHTVPLSE DQLLYLPFRN CPRFQELESE TLKSEEFQKR180LHPYKDFIAT LGKLSGLHGQ DLFGIWSKVY DPLYCESVHN FTLPSWATED TMTKLRELSE240LSLLSLYGIH KQKEKSRLQG GVLVNEILNH MKRATQIPSY KKLIMYSAHD TTVSGLQMAL300DVYNGLLPPY ASCHLTELYF EKGEYFVEMY YRNETQHEPY PLMLPGCSPS CPLERFAELV360GPVIPQDWST ECMTTNSHQG TEDSTD 386

[0097] PSM (Prostate Specific Membrane Antigen)

31MWNLLHETDS AVATARRPRW LCAGALVLAG GFFLLGFLFG WFIKSSNEAT NTTPKHNMKA60FLDELKAENI KKFLYNFTQI PHIAGTEQNF QLAKQIQSQW KEFGLDSVEL AHYDVLLSYP120NKTHPNYISI INEDGNEIFN TSLFEPPPPG YENVSDTVPP FSAFSPQGMP EGDLVYVNYA180RTEDFFKLER DMKINCSGKI VIAPYGKVFR GNKVKNAQLA GAKGVILYSD PADYFAPGVK240SYPDGWNLPG GGVQRGNILN LNGAGDPLTP GYPANEYAYR RGIAEAVGLP SIPVHPIGYY300DAQKLLEKMG GSAPPDSSWR GSLKVPYNVG PGFTGNFSTQ KVKMHIHSTN EVTRIYNVIG360TLRGAVEPDR YVILGGHRDS WVFGGIDPQS GAAVVHEIVR SFGTLKKEGW RPRRTILFAS420WDAEEFGLLG STEWAEENSR LLQERGVAYT NADSSIEGNY TLRVDCTPLM YSLVHNLTKE480LKSPDEGFEG KSLYESWTKK SPSPEFSGMP RISKLGSGND FEVFFQRLGI ASGRARYTKN540WETNKFSGYP LYHSVYETYE LVEKFYDPMF KYHLTVAQVR GGMVFELANS IVLPFDCRDY600AVVLRKYADK IYSTSMKHPQ EMKTYSVSDD SLFSAVKNFT EIASKFSERL QDFDKSNPIV660LRMMNDQLMF LERAFIDPLG LPDRPFYRHV IYAPSSHNKY AGESFPGIYD ALFDIESKVD720PSKAWGEVKR QIYVAAFTVQ AAAETLSEVA 750

[0098] Kallikrein (Human Kallikrein2, Accession NM005551)

4MNDLVLSIAL SVGCTGAVPL IQSRIVGGWE CEKHSQPWQV AVYSHGWAHC GGVLVHPQWV60LTAAHCLKKN SQVWLGRHNL FEPEDTGQRV PVSHSFPHPL YNMSLLKHQS LRPDEDSSHD120LMLLRLSEPA KITDVVKVLG LPTQEPALGT TCYASGWGSI EPEEFLRPRS LQCVSLHLLS180NDMCAPAYSE KVTEFMLCAG LWTGGKDTCG GDSGGPLVCN GVLQGITSWG PEPCALPEKP240AVYTKVVHYR KWIKDTIAAN P 261

[0099] HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:

[0100] 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.

[0101] D.1. HLA-A1 Supermotif

[0102] 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 super family 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.

[0103] Representative peptide epitopes that comprise an A1 supermotif are set forth on the attached Table VII.

[0104] D.2. HLA-A2 Supermotif

[0105] 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.

[0106] 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 super family 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.

[0107] Representative peptide epitopes that comprise an A2 supermotif are set forth on the attached 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.

[0108] D.3. HLA-A3 Supermotif

[0109] 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.

[0110] Representative peptide epitopes that comprise the A3 supermotif are set forth on the attached Table IX.

[0111] D.4. HLA-A24 Supermotif

[0112] 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 November 1999; 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.

[0113] Representative peptide epitopes that comprise the A24 supermotif are set forth on the attached Table X.

[0114] D.5. HLA-B7 Supermotif

[0115] 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.

[0116] Representative peptide epitopes that comprise the B7 supermotif are set forth on the attached Table XI.

[0117] D.6. HLA-B27 Supermotif

[0118] 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 November 1999; 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.

[0119] Representative peptide epitopes that comprise the B27 supermotif are set forth on the attached Table XII.

[0120] D.7. HLA-B44 Supermotif

[0121] 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.

[0122] D.8. HLA-B58 Supermotif

[0123] 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, I, V, M, or A) as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Sidney and Sette, Immunogenetics November 1999; 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.

[0124] Representative peptide epitopes that comprise the B27 supermotif are set forth on the attached Table XII.

[0125] D.9. HLA-B62 Supermotif

[0126] 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 November 1999; 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.

[0127] Representative peptide epitopes that comprise the B62 supermotif are set forth on the attached Table XIV.

[0128] D.10. HLA-A1 Motif

[0129] 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.

[0130] Representative peptide epitopes that comprise either A1 motif are set forth on the attached 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.

[0131] D.11. HLA-A*0201 Motif

[0132] 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. 10:478-482, 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.

[0133] Representative peptide epitopes that comprise an A*0201 motif are set forth on the attached Table VII. 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.

[0134] D.12. HLA-A3 Motif

[0135] 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, Y, 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.

[0136] Representative peptide epitopes that comprise the A3 motif are set forth on the attached Table XVI. Those epitopes that comprise the A3 supermotif are also listed in Table IX, as the A3 supermotif primary anchor residues comprise a subset of the A3- and A11-allele-specific motifs.

[0137] D.13. HLA-A11 Motif

[0138] The HLA-A11 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.

[0139] Representative peptide epitopes that comprise the A11 motif are set forth on the attached 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.

[0140] D.14. HLA-A24 Motif

[0141] 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. 155:4307-4312, 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.

[0142] Representative peptide epitopes that comprise the A24 motif are set forth on the attached 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.

[0143] Motifs Indicative of Class II HTL Inducing Peptide Epitopes

[0144] The primary and secondary anchor residues of the HLA class II peptide epitope supermotifs and motifs delineated below are summarized in Table III.

[0145] D.15. HLA DR-1-4-7 Supermotif

[0146] 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, P, 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.

[0147] Representative 9-mer peptide sequences 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. For each sequence, the “protein” column indicates the prostate-associated antigen, i.e., PSA, PSM, PAP, or HuK2 (kallikrein). The “position” column designates the amino acid position in the prostate antigen protein sequence that corresponds to the first amino acid residue of the core sequence. The core sequences are all 9 residues in length. For example, the first PSM sequence listed in Table XIX is a core sequence of nine residues in length that starts at position 611 of the PSM amino acid sequence provided herein. Accordingly, the amino acid sequence of the core sequence is IYSISMKHP. Exemplary epitopes of 15 amino acids in length that comprises the nine residue core include the three residues on either side that flank the nine residue core. For example, the exemplary epitope of 15 amino acids in length that comprises the core epitope at position 611 of PSM is ADKIYSISMKHPQEM.

[0148] HTL epitopes that comprise the core sequences can also be of lengths other than 15 amino acids, supra. For example, epitopes of the invention include sequences that comprise the nine residue core plus the 1, 2, 3 (as in the exemplary 15-mer), 4, or 5 flanking residues immediately adjacent to the nine residue core on each side.

[0149] D.16. HLA-DR3 Motifs

[0150] 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.

[0151] 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.

[0152] Peptide epitope 9-mer core regions corresponding to a nine residue sequence comprising the DR3a or the DR3b submotifs (wherein position 1 of the motif is at position 1 of the nine residue core) are set forth in Table XXa and b. For each sequence, the “protein” column indicates the prostate-associated antigen, i.e., PSA, PSM, PAP, or HuK2 (kallikrein). The “position” column designates the amino acid position in the prostate antigen protein sequence that corresponds to the first amino acid residue of the core sequence. The core sequences are all 9 residues in length. For example, the first sequence listed in Table XXa is a core sequence of nine residues in length that starts at position 124 of the PAP amino acid sequence provided herein. Accordingly, the amino acid sequence of the core sequence is FPPEGVSIW. Exemplary epitopes of 15 amino acids in length that comprises the nine residue core include the three residues on either side that flank the nine residue core. For example, the exemplary epitope of 15 amino acids in length that comprises the core epitope at position 124 of PAP is AALFPPEGVSIWNPI.

[0153] HTL epitopes that comprise the core sequences can also be of lengths other than 15 amino acids, supra. For example, epitopes of the invention include sequences that comprise the nine residue core plus the 1, 2, 3 (as in the exemplary 15-mer), 4, or 5 flanking residues immediately adjacent to the nine residue core on each side.

[0154] Each of the HLA class I or class II peptide epitopes identified as described herein is 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.

[0155] E. Enhancing Population Coverage of the Vaccine

[0156] 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/or 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 shows the overall frequencies of HLA class I supertypes in various ethnicities (Table XXIa) and the combined population coverage achieved by the A2-, A3-, and B7-supertypes (Table XXI). The A2-, A3-, and B7 supertypes are each present on 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.

[0157] 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.

[0158] 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.

[0159] F. Immune Response-Stimulating Peptide Analogs

[0160] In general, CTL and HTL responses to whole antigens 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).

[0161] 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 have 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.

[0162] 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.

[0163] Although peptides with suitable cross-reactivity among all alleles of a super family 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.

[0164] 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.

[0165] 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 super family are inserted.

[0166] 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, cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells.

[0167] 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.

[0168] 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).

[0169] G. Computer Screening of Protein Sequences from Disease-Related Antigens for Supermotif- or Motif-Bearing Peptides

[0170] 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.

[0171] 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. In the present invention, the target TAA molecules include, without limitation, PSA, PSM, PAP, and hK2.

[0172] 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

[0173] where aji 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.

[0174] 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).

[0175] 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

[0176] 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.

[0177] In accordance with the procedures described above, prostate cancer-associated antigen peptide epitopes and analogs thereof that are able to bind HLA supertype groups or allele-specific HLA molecules are identified.

[0178] H. Preparation of Peptide Epitopes

[0179] 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.

[0180] 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.

[0181] 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.

[0182] In alternative embodiments, epitopes of the invention can be linked as a polyepitopic peptide, or as a minigene that encodes a polyepitopic peptide.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] I. Assays to Detect T-Cell Responses

[0188] 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.

[0189] Analogous 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.

[0190] 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.

[0191] 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.

[0192] Additionally, 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-γ 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 at, 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).

[0193] 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).

[0194] 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. The 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 lymphoklnes.

[0195] J. Use of Peptide Epitopes as Diagnostic Agents and for Evaluating Immune Responses

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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, N.Y.; 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.

[0201] K Vaccine Compositions

[0202] 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.

[0203] 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).

[0204] 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.

[0205] 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.

[0206] 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).

[0207] 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.

[0208] 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 molecule such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described, for example, in U.S. Pat. No. 5,736,142).

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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. 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 may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

[0214] 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.

[0215]

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, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] K.1. Minigene Vaccines

[0221] 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.

[0222] 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 PSA, PSM, PAP, and hK2 epitopes derived from multiple regions of one or more of the prostate cancer-associated antigens, the PADRE™ universal helper T cell epitope (or multiple HTL epitopes from PSA, PSM, PAP, and hK2), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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 immnunogenicity) 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 (e.g., 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] K.2. Combinations of CTL Peptides with Helper Peptides

[0238] Vaccine compositions comprising the peptides of the present invention can be modified to provide desired attributes, such as improved serum half-life, or to enhance immunogenicity.

[0239] 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. Nos. 08/820,360, 08/197,484, and 08/464,234.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] K.3. Combinations of CTL Peptides with T Cell Priming Agents

[0245] 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.

[0246] 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.

[0247] 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.

[0248] K4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

[0249] 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.

[0250] 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., prostate-associated antigens such as PSA, PSM, PAP, kallikrein, and the like. Optionally, a helper T cell peptide such as a PADRE™ family molecule, can be included to facilitate the CTL response.

[0251] L. Administration of Vaccines for Therapeutic or Prophylactic Purposes

[0252] The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are typically used therapeutically to treat cancer, particularly prostate cancer. Vaccine compositions containing the peptides of the invention are typically administered to a prostate cancer patient who has a malignancy associated with expression of one or more prostate-associated antigens. Alternatively, vaccine compositions can be administered to an individual susceptible to, or otherwise at risk for developing prostate cancer.

[0253] 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.

[0254] 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 peptides (or DNA encoding them) can be administered individually or as fusions of one or more peptide sequences. 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.

[0255] 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 or by transfecting antigen-presenting cells with a minigene of the invention. Such a cell population is subsequently administered to a patient in a therapeutically effective dose.

[0256] 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 or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, a vaccine comprising TAA-specific CTLs may be more efficacious in killing tumor cells in patients with advanced disease than alternative embodiments.

[0257] 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.

[0258] Where susceptible individuals, e.g., individuals who may be diagnosed as being genetically pre-disposed to developing a prostate tumor, are identified prior to diagnosis of cancer, the composition can be targeted to them, thus minimizing the need for administration to a larger population.

[0259] The dosage for an initial therapeutic 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 Initial doses followed by boosting doses at established intervals, e.g., from four weeks to six months, may be required, possibly for a prolonged period of time to effectively treat a 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.

[0260] 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.

[0261] 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.

[0262] The vaccine compositions of the invention can also be used as prophylactic agents. For example, the compositions can be administered to individuals at risk of developing prostate cancer. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 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. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine may be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

[0263] 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.

[0264] 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.

[0265] 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).

[0266] 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, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to 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.

[0267] 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.

[0268] 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%.

[0269] 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.

[0270] M. HLA Expression: Implications for T Cell-Based Immunotherapy

[0271] Disease Progression in Cancer and Infectious Disease

[0272] 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.

[0273] 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.

[0274] 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.

[0275] The Interplay between Disease and the Immune System

[0276] 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.

[0277] In the cancer setting there are several findings that indicate that immune responses can impact neoplastic growth:

[0278] First, the demonstration in many different animal models, that anti-tumor T cells, restricted by MHC class I, can prevent or treat tumors.

[0279] Second, encouraging results have come from immunotherapy trials.

[0280] 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).

[0281] 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, Aug: 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, Febuary 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.

[0282] The Three Main Types of Alterations in HLA Expression in Tumors and their Functional Significance

[0283] 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 β2-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 β2-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:

[0284] Complete Loss of HLA Expression

[0285] 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.

[0286] 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, G A, 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., Dec 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 B2 m 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).

[0287] 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.

[0288] 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.

[0289] 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.

[0290] Allele-specific Loss

[0291] 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.

[0292] Decrease in Expression (Allele-specific or not)

[0293] 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.

[0294] 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.

[0295] 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.

[0296] Finally, studies have demonstrated that decreased HLA expression can render tumor cells more susceptible to NK lysis (Ohnmacht, G A, 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 mechanisms 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 β2 m 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.

[0297] Freguency of Alterations in HLA Expression

[0298] 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 Imnmunology 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. Jimmez et al (Jimmez 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.

[0299] Immunotherapy in the Context of HLA Loss

[0300] 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 lympholcine release; and, 3) class I negative cells are sensitive to lysis by NK cells.

[0301] 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.

[0302] 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.

[0303] 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.

[0304] N. Reprieve Periods from Therapies that Induce Side Effects: “Scheduled Treatment Interruptions or Drug Holidays”

[0305] 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, Sep. 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 is keeping the disease in check. Methods for using compositions of the invention are used in the context of drug holidays for cancer and pathogenic infection.

[0306] 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.

[0307] 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.

[0308] O. Kits

[0309] 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.

[0310] P. Overview

[0311] 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 wag 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.

[0312] 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.

[0313] 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 one hundred fifty (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, or, 100).

[0314] 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, eg., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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 principles 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 Jan. 6, 1999. Generally the compositions of the invention are isolated or purified.

[0319] 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.

EXAMPLES

[0320] The following examples illustrate identification, selection, and use of immunogenic Class I and Class II peptide epitopes for inclusion in vaccine compositions.

Example 1

[0321] HLA Class I and Class II Binding Assays

[0322] 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.

[0323] 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.

[0324] 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.

[0325] 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

[0326] Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitones

[0327] 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.

[0328] Computer Searches and Algorthims for Identification of Supermotif and/or Motif-bearing Epitopes

[0329] The searches performed to identify the motif-bearing peptide sequences in Examples 2 and 5 employ protein sequence data for prostate cancer-associated antigens.

[0330] 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.

[0331] 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 . . . x ani

[0332] 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 residuej 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).

[0333] 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.

[0334] Selection of HLA-A2 Supertype Cross-Reactive Peptides

[0335] The complete protein sequences of the prostate cancer-associated antigens PAP, PSA, PSM, and hK2 were obtained from GenBank and scanned, utilizing motif identification software, to identify 8-, 9-, 10-, and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity.

[0336] HLA-A2 supermotif-bearing sequences are shown in Table VII. These sequences are then scored using the A2 algorithm and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitio (HLA-A*0201 is considered a prototype A2 supertype molecule).

[0337] Examples of peptides that were identified that bind to HLA-A*0201 with IC50 values ≦500 nM are shown in Tables XXII and XXII. These peptides were then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are deemed A2-supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules. Examples of such peptides are set out in Table XXIII. (Due to the homology described above, a number of CTL and HTL epitopes are represented in both the PSA and hK2 antigens. This is represented in Tables XXIII and XXIV by the headings source and alternate source.)

[0338] Selection of HLA-A3 Supermotif-Bearing Epitopes

[0339] The protein sequences scanned above were 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.

[0340] 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, preferably ≦200 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.

[0341] Selection of HLA-B7 Supermotif Bearing Epitopes

[0342] The same target antigen protein sequences were 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 (ie., the prototype B7 supertype allele). Those peptides that bind B*0702 with IC50 of ≦500 nM, preferably ≦200 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.

[0343] Selection of A1 and A24 Motif-Bearing Epitopes

[0344] To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine constructs. An analysis of the protein sequence data from the target antigens utilized above was performed to identify HLA-A1- and A24-motif-containing sequences. Peptides are then synthesized and tested for binding.

[0345] Peptides that bear other supermotifs and/or motifs can be assessed for binding or cross-reactive binding in an analogous manner.

Example 3

[0346] Confirmation of Immunogenicity

[0347] Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described in Example 2 were selected for in vitro immunogenicity testing. Examples of immunogenic HLA-A2 cross-reactive binding peptides that bind to at least 3/5 HLA-A2 supertype family members at an IC50 of 200 nM or less are shown in Table XXIV. Testing was performed using the following methodology:

[0348] Target Cell Lines for Cellular Screening:

[0349] 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, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to test the ability of peptide-specific CTLs to recognize endogenous antigen.

[0350] Primary CTL Induction Cultures:

[0351] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serun, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are 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 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.

[0352] Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal imnmunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are 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 20×106 cells/ml. The magnetic beads are 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 are 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 are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are 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 are then irradiated (4,200 rads), washed 1 time with medium and counted again.

[0353] Setting up induction cultures: 0.25 ml cytokine-generated DC (@1×105 cells/ml) are 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. Recombinant human IL10 is added the next day at a final concentration of 10 ng/ml and rhuman IL2 is added 48 hours later at 10 IU/mL.

[0354] Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction the cells are restimulated with peptide-pulsed adherent cells. The PBMCS are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×106 cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are 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 is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later rhuman IL10 is added at a final concentration of 10 ng/ml and rhuman IL2 is 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 are assayed for CTL activity in a 51Cr release assay. In some experiments the cultures are 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 is measured in both assays for a side by side comparison.

[0355] Measurement of CTL Lytic Activity by 51Cr Release.

[0356] Seven days after the second restimulation, cytotoxicity is determined in a standard (5hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.

[0357] Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are 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 are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the 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 are determined by incubating the labelled targets with 1% Trition X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample- background) is 10% or higher in the case of individual wells and is 15% or more at the 2 highest E:T ratios when expanded cultures are assayed.

[0358] In situ Measurement of Human γIFN Production as an Indicator of Peptide-Specific and Endogenous Recognition

[0359] Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates are 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) are 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, are used at a concentration of 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO2.

[0360] Recombinant human IFNγ is 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 are washed and 100 μl of biotinylated mouse anti-human IFNγ monoclonal antibody (2 μg/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 μl HRP-streptavidin (1:4000) are added and the plates incubated for 1 hour at room temperature. The plates are then washed 633 with wash buffer, 100 μl/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 μl/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFNγ/well above background and is twice the background level of expression.

[0361] CTL Expansion. Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×104 CD8+ cells are 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 pyrivate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Rhuman IL2 is 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 are split if the cell concentration exceeded 1×106/ml and the cultures are 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.

[0362] Cultures are expanded in the absence of anti-CD3+as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for 2 hours at 37° C. and irradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10%(v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

[0363] Immunogenicity of A2 Supermotif-Bearing Peptides

[0364] 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 is considered to be an epitope if it induces peptide-specific CTLs in at least 2 donors (unless otherwise noted) and preferably, also recognizes the endogenously expressed peptide. Examples of immunogenic peptides are shown in Table XXIV.

[0365] Immunogenicity is additionally confirmed using PBMCs isolated from cancer patients. Briefly, PBMCs are isolated from patients with prostate 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.

[0366] Evaluation of A*03/A11 Immunogenicity

[0367] 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.

[0368] Evaluation of B7 Immunogenicity

[0369] Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified in Example 2 are evaluated in a manner analogous to the evaluation of A2-and A3-supermotif-bearing peptides.

[0370] Peptides bearing other supermotifs and/or motifs, e.g., HLA-A1, HLA-a24 etc. are also evaluated using similar methodology

Example 4

[0371] Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

[0372] 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 analoged, 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.

[0373] Analoging at Primary Anchor Residues

[0374] Peptide engineering strategies were implemented to further increase the cross-reactivity of the epitopes identified above (see, e.g., Table XXII). 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.

[0375] 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 (typically L at position 2 and V at the C-terminus). Those analoged peptides that show at least a three-fold increase in A*0201 binding and bind with an IC50 of 500 nM, or preferably 200 nM, or less are then tested for A2 cross-reactive binding along with their wild-type (WT) counterparts. Analoged peptides that bind at least three of the five A2 supertype alleles are then selected for cellular screening analysis.

[0376] Additionally, the selection of analogs for cellular screening analysis is further restricted by the capacity of the WT parent peptide to bind at least weakly, i.e., bind at an IC50 of 5000 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. Analoged 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).

[0377] 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.

[0378] Peptides that were analoged at primary anchor residues, generally by adding a preferred residue at a primary anchor position, were synthesized and assessed for enhanced binding to A*0201 and/or enhanced cross-reactive binding. Examples of analoged peptides that exhibit increased binding and/or cross-reactivity are shown in Table XXIII.

[0379] Analogs exhibiting altered binding characteristics are then selected for cellular screening studies. Examples are shown in Table XXIV.

[0380] Using methodology similar to that used to develop HLA-A2 analogs, analogs of HLA-A3 and HLA-B7 supermotif-bearing epitopes are also generated. Analogous strategies can be used for peptides bearing other supermotifs/motifs as well. For example, peptides binding at least weakly to 3/5 of the A3-supertype molecules may 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, often ≦200 nM binding values, are then tested for A3-supertype cross-reactivity. B7 supermotif-bearing peptides may, for example, be engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996) and tested for binding to B7 supertype alleles.

[0381] Analoging at Secondary Anchor Residues

[0382] 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. 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 analoged 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 analoged peptides with modulated binding affinity.

[0383] Engineered analogs with sufficiently improved binding capacity or cross-reactivity are tested for immunogenicity as above.

[0384] Other Analoging Strategies

[0385] Another form of peptide analoging, 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. Substitution 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).

[0386] In conclusion, these data demonstrate that by the use of even single amino acid substitutions, it is possible to increase the binding affinity and/or cross-reactivity of peptide ligands for HLA supertype molecules.

Example 5

[0387] Identification of Peptide Epitope Sequences with HLA-DR Binding Motifs

[0388] 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.

[0389] Selection of HLA-DR-Supermotif-Bearing Epitopes

[0390] To identify HLA class II HTL epitopes, the prostate cancer-associate antigen protein sequences were analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, further comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

[0391] 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.

[0392] The prostate antigen-derived peptides identified above are tested for their binding capacity to various common HLA-DR molecules. All peptides are 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.

[0393] Following the strategy outlined above DR supermotif-bearing sequences were identified within the prostate antigen protein sequence. Generally, these sequences are then scored for the combined DR 1-4-7 algorithms. The positive-scoring peptides are synthesized and tested for binding to HLA-DRB1* 0101, DRB1*0401, DRB1*0701. Those that bind at least 2 of the 3 alleles are then tested for binding to secondary DR supertype alleles: DRB5*0101, DRB1*1501, DRB1*1101, DRB1*0802, and DRB1*1302.

[0394] Selection of DR3 Motif Peptides

[0395] 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.

[0396] To efficiently identify peptides that bind DR3, the PSA, PSM, PAP, and hK2 protein sequences were analyzed for sequences carrying one of the two DR3 specific binding motifs (Table III) reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and tested for the ability to bind DR3 with an affinity of 1000 nM or better, i.e., less than 1000 nM.

[0397] Additionally, the DR3 binders are also tested for binding to the DR supertype alleles. Conversely, the DR supertype cross-reactive binding peptides are also tested for DR3 binding capacity.

[0398] DR3 binding epitopes identified in this manner are then included in vaccine compositions with DR supermotif-bearing peptide epitopes.

[0399] Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged 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 often improves DR 3 binding.

[0400] For example, a number of HLA-DR supermotif and DR-3 motif-bearing prostate antigen-associated sequences have been identified. The number in each category is summarized in Table XXV.

Example 6

[0401] Immunogenicity of HTL Epitopes

[0402] This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology in Example 5.

[0403] 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

[0404] Calculation of Phenotypic Frequencies of HLA-supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

[0405] 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.

[0406] 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].

[0407] 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).

[0408] 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

[0409] Recognition of Generation of Endorenous Processed Antigens after Priming

[0410] This example determines that CTL induced by native or analogued peptide epitopes identified and selected as described in Examples 1-6 recognize endogenously synthesized, ie., native antigens, using a transgenic mouse model.

[0411] 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. prostate tumor cells or cells that are stably transfected with TAA expression vectors.

[0412] 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

[0413] Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

[0414] 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 Table XXIII, or other analogs of that epitope. The peptides may be lipidated, if desired.

[0415] 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 a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

[0416] 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. 173:107, 1991).

[0417] 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.

[0418] 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.

[0419] 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

[0420] Selection of CTL and HTL Epitopes for Inclusion in a Cancer Vaccine.

[0421] 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 (ie., minigene) that encodes peptide(s), or may be single and/or polyepitopic peptides.

[0422] 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.

[0423] 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 prostate cancer-associated antigen. Epitopes from one prostate cancer-associated antigen 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] 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

[0429] Construction of Minigene Multi-Epitope DNA Plasmids

[0430] 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. Examples of the construction and evaluation of expression plasmids are described, for example, in co-pending U.S. Ser. No. 09/311,784 filed May 13, 1999.

[0431] A minigene expression plasmid may include multiple CTL and HTL peptide epitopes. In this 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. HLA class I supermotif or motif-bearing peptide epitopes derived from multiple prostate cancer-associated antigens are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from multiple prostate cancer-associated antigens to provide broad population coverage, ie. 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.

[0432] 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.

[0433] 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.

[0434] Overlapping oligonucleotides that can, for example, average about 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/Ebmer 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.

[0435] For example, a minigene can be prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, 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. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 12

[0436] The Plasmid Construct and the Degree to which it Induces Immunogenicity.

[0437] The degree to which a plasmid construct, for example a plasmid constructed in accordance with Example 11, is able to induce immunogenicity can be evaluated iib 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).

[0438] Alternatively, immunogenicity can be evaluated through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in co-pending U.S. Ser. No. 09/311,784 filed May 13, 1999 and Alexander et al., Immunity 1:751-761, 1994.

[0439] For example, to assess the capacity of a DNA minigene construct (e.g., a pMin minigene construct generated as described in U.S. Ser. No. 09/311,784) containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/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.

[0440] 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 A2-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-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes.

[0441] To assess the capacity of a class II epitope encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitope that cross react with the appropriate mouse MHC molecule, 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.

[0442] DNA minigene, 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 can consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccine, 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).

[0443] For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. 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.

[0444] It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes.

[0445] The use of prime boost protocols in humans is described in Example 20.

Example 13

[0446] Peptide Composition for Prophylactic Uses

[0447] Vaccine compositions of the present invention are used to prevent cancer in persons who are at high 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 high risk for prostate 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.

[0448] 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

[0449] Polyepitopic Vaccine Compositions Derived from Native TAA Sequences

[0450] A native TAA polyprotein sequence is screened, preferably using computer algorithmns defined for each class I and/or class II supermotif or motif, to identify “relatively shore” 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, ie., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (ie., 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.

[0451] The vaccine composition will preferably include, for example, three CTL epitopes and at least one HTL epitope from multiple prostate cancer-associated antigens. 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.

[0452] 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.

[0453] 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

[0454] Polyepitopic Vaccine Compositions Comprising Epitopes from Multiple Tumor-Associated Antigens

[0455] The prostate cancer-associated antigen peptide epitopes of the present invention are used in combination with each other, or with peptide epitopes from other target tumor-associated antigens to create a vaccine composition that is useful for the treatment of prostate tumors from multiple patients. Furthermore, 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.

[0456] 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.

Example 16

[0457] Use of Peptides to Evaluate an Immune Response

[0458] Peptides of the invention may be used to analyze an immune response for the presence of specific CTL or HTL populations directed to a prostate cancer-associated antigen. 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.

[0459] 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-phycoeryhrin.

[0460] 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

[0461] Use of Peptide Epitopes to Evaluate Recall Responses

[0462] 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 prostate cancer-associated antigen vaccine.

[0463] 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.

[0464] 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 (50 U/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

[0465] 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).

[0466] 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).

[0467] 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.

[0468] 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.

[0469] 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.

[0470] 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 10 U/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-thyridine 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

[0471] Induction of Specific CTL Response in Humans

[0472] 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:

[0473] A total of about 27 male subjects are enrolled and divided into 3 groups:

[0474] Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

[0475] Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;

[0476] Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

[0477] 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.

[0478] 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.

[0479] Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

[0480] 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.

[0481] The vaccine is found to be both safe and efficacious.

Example 19

[0482] Therapeutic Use in Cancer Patients

[0483] 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 prostate 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:

[0484] 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.

[0485] 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 are males, typically above the age of 50, and represent diverse ethnic backgrounds.

Example 20

[0486] Induction of CTL Responses Using a Prime Boost Protocol

[0487] A prime boost protocol similar in its underlying principle to that used to evaluate the efficacy of a DNA vaccine in transgenic mice, such as described in Example 12, can also be used for the administration of the vaccine to humans. Such a vaccine regimen can 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.

[0488] For example, the initial immunization can be performed using an expression vector, such as one constructed in accordance with 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.

[0489] Analysis of the results will indicate that a magnitude of response sufficient to achieve protective immunity against prostate cancer is generated.

Example 21

[0490] Administration of Vaccine Compositions Using Antigen Presenting Cells

[0491] Vaccines comprising peptide epitopes of the invention may be administered using antigen-presenting cells (APCs), or “professional” APCs such as dendritic cells (DC). 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 CTL and HTL then destroy (CTL) or facilitate destruction (HTL) of the specific target tumor cells that bear the proteins from which the epitopes in the vaccine are derived.

[0492] 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/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

[0493] 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.

[0494] 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.

[0495] The ability of DC to stimulate immune responses was evaluated in both in vitro and in vivo immune function assays. These assays include the stimulation of CTL hybridomas and CTL cell lines, and the in vivo activation of CTL.

[0496] DC Purification

[0497] Progenipoietin™-mobilized DC were purified from peripheral blood (PB) and spleens of Progenipoietin™-treated C57B1/6 mice to evaluate their ability to present antigen and to elicit cellular immune responses. Briefly, DC were purified from total WBC and spleen using a positive selection strategy employing magnetic beads coated with a CD11c specific antibody (Miltenyi Biotec, Auburn Calif.). For comparison, ex vivo expanded DC were generated by culturing bone marrow cells from untreated C57B1/6 mice with the standard cocktail of GM-CSF and IL-4 (R&D Systems, Minneapolis, Minn.) for a period of 7-8 days (Mayordomo et al., Nature Med. 1:1297-1302 (1995)). Recent studies have revealed that this ex vivo expanded DC population contains effective antigen presenting cells, with the capacity to stimulate anti-tumor immune responses (Celluzzi et al., J. Exp. Med. 83:283-287 (1996)).

[0498] The purities of Progenipoietin™-derived DC (100 μg/day, 10 days, SC) and GM-CSF/IL-4 ex vivo expanded DC were determined by flow cytometry. DC populations were defined as cells expressing both CD11c and MHC Class II molecules. Following purification of DC from magnetic CD11c microbeads, the percentage of double positive PB-derived DC, isolated from Progenipoietin™ treated mice, was enriched from approximately 4% to a range from 48-57% (average yield=4.5×106DC/animal). The percentage of purified splenic DC isolated from Progenipoietin™ treated mice was enriched from a range of 12-17% to a range of 67-77%. The purity of GM-CSF/IL4 ex vivo expanded DC ranged from 31-41% (Wong et al., J. Immunother., 21:32040 (1998)).

[0499] In Vitro Stimulation of CTL Hybridomas and CTL Cell Lines: Presentation of Specific CTL Epitopes

[0500] The ability of Progenipoietin™ generated DC to stimulate a CM cell line was demonstrated in vitro using a viral-derived epitope and a corresponding epitope responsive CTL cell line. Transgenic mice expressing human HLA-A2.1 were treated with Progenipoietin™. Splenic DC isolated from these mice were pulsed with a peptide epitope derived from hepatitis B virus (HBV Pol 455) and then incubated with a CTL cell line that responds to the HBV Pol 455 epitope/HLA-A2.1 complex by producing IFNγ. The capacity of Progenipoietin™-derived splenic DC to present the HBV Pol 455 epitope was greater than that of two positive control populations: GM-CSF and IL-4 expanded DC cultures, or purified splenic B cells. A left shift in the response curve for Progenipoietin™-derived spleen cells versus the other antigen presenting cells revealed that these Progenpoietin™-derived cells required less epitope to stimulate maximal IFNγ release by the responder cell line.

[0501] The ability of ex vivo peptide-pulsed DC to stimulate CTL responses in vivo was also evaluated using the HLA-A2.1 transgenic mouse model. DC derived from Progenipoietin™-treated animals or control DC derived from bone marrow cells after expansion with GM-CSF and IL-4 were pulsed ex vivo with the HBV Pol 455 CTL epitope, washed and injected (IV) into such mice. At seven days post immunization, spleens were removed and splenocytes containing DC and CTL were restimulated twice in vitro in the presence of the HBV Pol 455 peptide. The CTL activity of three independent cultures of restimulated spleen cell cultures was assessed by measuring the ability of the CTL to lyse 51Cr-labeled target cells pulsed with or without peptide. Vigorous CTL responses were generated in animals immunized with the epitope-pulsed Progenipoietin™ derived DC as well as epitope-pulsed GM-CSF/IL-4 DC. In contrast, animals that were immununized with mock-pulsed Progenpoietin™-generated DC (no peptide) exhibited no evidence of CTL induction.

[0502] These data confirm that DC derived from Progenipoietin™ treated mice can be pulsed ex vivo with epitope and used to induce specific CTL responses in vivo. Thus, these data support the principle that Progenpoietin™-derived DC promote CTL responses in a model that manifests human MHC Class I molecules.

[0503] In vivo pharmacology studies in mice have demonstrated no apparent toxicity of reinfusion of pulsed autologous DC into animals.

[0504] Ex vivo Activation of CTL/HTL Responses

[0505] 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, ie., tumor cells.

Example 22

[0506] Alternative Method of Identifying Motif-Bearing Peptides

[0507] 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.

[0508] 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.

[0509] 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, ie., 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.

[0510] 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.

[0511] 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.

5TABLE IPOSITIONPOSITIONPOSITION2 (Primary Anchor)3 (Primary Anchor)C Terminus (Primary Anchor)SUPERMOTIFSA1T, I, L, V, M, SF, W, YA2L, I, V, M, A, T, QI, V, M, A, T, LA3V, S, M, A, T, L, IR, KA24Y, F, W, I, V, L, M, TF, I, Y, W, L, MB7PV, 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, TV, L, I, M, A, TA3L, M, V, I, S, A, T, F, C, G, DK, Y, R, H, F, AA11V, T, M, L, I, S, A, G, N, C, D, FK, R, Y, HA24Y, F, W, MF, L, I, WA*3101M, V, T, A, L, I, SR, KA*3301M, V, A, L, F, I, S, TR, KA*6801A, V, T, M, S, L, IR, KB*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, Y

[0512] Bolded 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.

6TABLE IaPOSITIONPOSITIONPOSITION2 (Primary Anchor)3 (Primary Anchor)C Terminus (Primary Anchor)SUPERMOTIFSA1T, I, L, V, M, SF, W, YA2V, Q, A, TI, V, L, M, A, TA3V, S, M, A, T, L, IR, KA24Y, F, W, I, V, L, M, TF, I, Y, W, L, MB7PV, I, L, F, M, W, Y, AB27R, H, KF, Y, L, W, M, I, 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, SYA2.1V, Q, A, T*V, L, I, M, A, TA3.2L, M, V, I, S, A, T, F, C, G, DK, Y, R, H, F, AA11V, T, M, L, I, S, A, G, N, C, D, FK, R, H, YA24Y, F, WF, L, I, W*If 2 is V, or Q, the C-term is not L

[0513] Bolded 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.

7TABLE IIPOSITION

C-terminusSUPERMOTIFSA11° Anchor1° AnchorT, I, L,F, W, YV, M, SA21° Anchor1° AnchorL, I, V,L, I, V,M, AM, A, TT, QA3preferred1° AnchorY, F, W,Y, F, W,Y, F, W,P, (4/5)1° Anchor(4/5)(4/5)V, S, M, A,(3/5)R, KT, L, IdeleteriousD, E(3/5);D, E, (4/5)P, (5/5)A241° Anchor1° AnchorY, F, W, F, I, Y, I, V, W, L, ML, M, TB7preferredF, W,1° AnchorF, W, YF, W, Y,1° AnchorY(5/5)(4/5)(3/5)V, I, L, F,L, I, V,PM, W, Y, AM, (3/5)deleteriousD, E(3/5);D, E, (3/5)G (4/5)Q, N (4/5)D, E, (4/5)P(5/5);G(4/5);A(3/5);Q, N, (3/5)B271° Anchor1° AnchorR, H, KF, Y, L, W,M, V, AB441° Anchor1° AnchorE, DF, W, Y, L,I, M, V, AB581° Anchor1° AnchorA, T, SF, W, Y, L,I, V, M,AB621° Anchor1° AnchorQ, L, I, F, W, Y, M, V, M, PI, V, L, AMOTIFSA1preferredG, F, Y, W,1° AnchorD, E, A,Y, F, W,P,D, E, Q, N,Y, F, W,1° Anchor9-merS, T, M,YdeleteriousD, E,R, H, K,A,G,A,L, I, VM, P,A1preferredG, R, H, KA, S, T,1° AnchorG, S, T, C,A, S, T, C,L, I, V,D, E,1° Anchor9-merC, L, I,D, E, A, SM,YV, M,deleteriousAR, H, K,D, E,P, Q, N,R, H, K,P, G,G, P,D, E, P,Y, F, W,POSITION

or C-

terminusC-terminusA1peferredY, F, W,1° AnchorD, E, A,A,Y, F, W,P, A, S,G, D, E,P,1° Anchor10-merS, T, MQ, N,Q, N,T, C,YdeleteriousG, P,R, H, K,D, E,R, H, K,Q, N, AR, H, K,R, H, K,AG, L, IY, F,V, M,W,A1preferredY, F, W,S, T, C,1° AnchorA,Y, F, W,P, G,G,Y, F, W,1° Anchor10-merL, I, V M,D, E, A, SYdeleteriousR, H, K,R, H, K,P,G,P, R, H, K,Q, N,D, E,P, Y, F, W,A2.1preferredY, F, W,1° AnchorY, F, W,S, T, C,Y, F, W,A,P1° Anchor9-merL, M, I,V, L, I,V, Q, A, TM, A, T,deleteriousD, E, P,D, E, R,R, K, HD, E, R,K, HK, HA2.1preferredA, Y, F,1° AnchorL, V, I,G,G,F, Y, W, L,1° Anchor10-merW,L, M, I, M,V, I, M,V, L, I,V, Q, M, A, TA, TdeleteriousD, E, P,D, E,R, K, H,P,R, K, H,D, E, R,R, K, H,A,K, H,A3preferredR, H, K,1° AnchorY, F, W,P, R, H,A,Y, F, W,P,1° AnchorL, M, V,K, Y,K, Y, R,I, S,F, W,H, F, AA, T, F,C, GDdeleteriousD, E, P,D, EA11preferredA,1° AnchorY, F, W,Y, FW,A,Y, F, W,Y, FW,P,1° AnchorV, T, L,K, , RY, HM, I,S, A, G,N, C, D, FdeleteriousD, E, P,AG,A24preferredY, F, W,1° AnchorS, T, CY, F, W,Y, F, W,1° Anchor9-merR, H, K,Y, F, W, MF, L, I, WdeleteriousD, E, G,D, E,G,Q, N, P,D, E, R,G,A, Q, N,H, K,A24preferred1° AnchorP,Y, F, W, P,P,1° Anchor10-merY, F, W, MF, L, I, WdeleteriousG, D, EQ, NR, H, KD, EAQ, N,D, E, A,A3101preferredR, H, K,1° AnchorY, F, W,P,Y, F, W,Y, F, W,A, P,1° AnchorM, V, T,R, KA, LI, SdeleteriousD, E, P,D, E,A, D, E,D, E,D, E,D, E,A3301preferred1° AnchorY, F, WA, Y, F,1° AnchorM, V, A,WR, KL, F,I, S, TdeleteriousG, PD, EA6801preferredY, F, W,1° AnchorY, F, W,Y, F, W,P,1° AnchorS, T, C,A, V, T,L, I,R, KM, S, V, ML, IdeleteriousG, P,D, E, G,R, H, K,A,B0702preferredR, H, K,1° AnchorR, H, K,R, H, K,R, H, K,R, H, K,P, A,1° AnchorF, W, Y,PL, M, F,W, Y, A,I, VdeleteriousD, E, Q,D, E, P,D, E,D, E,G, D, E,Q, N,D, E,N, P,B3501preferredF, W, Y,1° AnchorF, W, Y,F, W, Y,1° AnchorL, I, V,PL, M, F,M,W, Y, I,V, A, deleteriousA, G, P,G,G,B51preferredL, I, V,1° AnchorF, W, Y,S, T, C,F, W, Y,G,F, W, Y,1°AnchorM, F, W,PL, I, V,Y,F, W, Y, A, MdeleteriousA, G, P,D, E,G,D, E, Q,G, D, E,D, E, R,N,H, K, S,T, C,B5301preferredL, I, V,1° AnchorF, W, Y,S, T, C,F, W, Y,L, I, V,F, W, Y,1° AnchorM, F, W,PM, F,I, M, F,Y,W, Y,W, Y,A, L, VdeleteriousA, G, P,G,R, H, K,D, E,Q, N,Q, N,B5401preferredF, W, Y,1° AnchorF, W, Y,L, I, V, M,A, L, I,F, W, Y,1° AnchorPL, I, V,V, M,A, P,A, T, I,M,V, L, M, F, W,YdeleteriousG, P, Q,G, D, E,R, H, K,D, E,Q, N, D,D, E,N, D, E,S, T, C,D, EG, 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.

[0514]

8

TABLE III

POSITION

MOTIFS

DR4
preferred
F, M, Y,
M,
T,

I,
V, S, T,
M, H,

M, H

L, I,

C, P, A,

V, W,

L, I, M,

deleterious

W,

R,

W, D, E

DR1
preferred
M, F, L,

P, A, M, Q,

V, M, A,
M,

A, V, M

I, V,

T, S, P,

W, Y

L, I, C,

deleterious

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

G, D, E,
D

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

I, V

I, V,

S, A, C,

W, Y,

T, P, L,

deleterious

C,

G,

G, R, D,
N
G

DR

M, F, L,

V, M, S,

Supermotif

I, V,

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.

[0515]

9

TABLE IV

HLA Class I Standard Peptide Binding Affinity.

STANDARD

BINDING

STANDARD
SEQUENCE
AFFINITY

ALLELE
PEPTIDE
(SEQ ID NO:)
(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
KVFPYALINK
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

[0516]

10

TABLE V

HLA Class II Standard Peptide Binding Affinity.

Bind-

ing

Affin-

Nomen-
Standard
Sequence
ity

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
GRTQDENPVVHFFK
9.1

NIVTPRTPPP

DRB3*0101
DR52a
511
NGQIGNDPNRDIL
470

DRB4*0101
DRw53
717.01
YARFQSQTTLKQKT
58

DRB5*0101
DR2w2β2
553.01
QYIKANSKFIGITE
20

[0517]

11

TABLE VI

HLA-
Allelle-specific HLA-supertype members

supertype
Verifieda
Predictedb

A1
A*0101, A*2501, A*2601, A*2602, A*3201
A*0102, A*2604, A*3601, A*4301, A*8001

A2
A*0201, A*0202, A*0203, A*0204, A*0205,
A*0208, A*0210, A*0211, A*0212, A*0213

A*0206, A*0207, A*0209, A*0214, A*6802, A*6901

A3
A*0301, A*1101, A*3101, A*3301, A*6801
A*0302, A*1102, A*2603, 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*0705, B*1508, B*3501,
B*1511, B*4201, B*5901

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*2702, B*2703, B*2704,
B*2701, B*2707, B*2708, B*3802, B*3903, B*3904,

B*2705, B*2706, B*3801, B*3901, B*3902, B*7301
B*3905, B*4801, B*4802, B*1510, B*1518, B*1503

B44
B*1801, B*1802, B*3701, B*4402, B*4403, B*4404,
B*4101, B*4501, B*4701, 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*5201
B*1301, B*1302, B*1504, 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 pool 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.

[0518]

12

TABLE VII

Prostate A01 Supermotif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
A*0101

PAP
122
11

Kallikrein
147
11

PSA
143
11

Kallikrein
235
9

PSA
231
9
0.0110

PSM
25
8

PSM
25
9

PAP
116
9

PAP
311
9
0.7700

PAP
311
10

PSM
531
11

PSM
643
11

PAP
12
9

PSM
419
8

PSM
13
8

PSM
11
10

PSM
393
10

Kallikrein
241
9

Kallikrein
66
9

PSM
196
10
0.0160

PAP
347
10

PSM
156
9

PAP
201
10

PSA
98
9

PSM
630
10

PSM
453
8

PSM
106
8

PAP
301
10

PSM
137
8

PSM
109
11

PSM
586
10

PAP
80
10

PSM
64
10

PAP
34
9

PSM
480
9

PAP
237
11

PAP
240
8

PSM
560
11

PAP
358
11

PAP
317
9

PAP
317
10

PSM
621
9

PAP
168
10

PSM
703
11

PSM
716
10

PAP
60
8

PAP
216
11

PAP
95
9
0.0980

PAP
170
8

PSM
542
8

PSM
542
11

PSM
557
9

PSM
557
10
0.0260

PSM
727
11

PAP
18
8

PSM
33
9

PSM
33
10

PSA
3
8

Kallikrein
195
8

PSA
191
8

PSM
646
8

PSM
546
11

PSM
639
8

PSM
529
9
0.0025

PAP
204
11

PSM
104
10
0.4800

PAP
196
8

PAP
196
11

PSM
427
8

PSM
680
8

PAP
295
9

PAP
74
11

PSM
168
9
0.0001

PSM
311
9

PSM
516
9

PSM
516
10

Kallikrein
158
8

PSA
154
8

PSM
403
8

Kallikrein
149
9

PSA
145
9

PSM
224
11

PSM
238
9

Kallikrein
221
9

PSA
217
9

Kallikrein
52
8

PSA
48
8

PAP
128
11

PSM
82
9

PAP
270
11

Kallikrein
94
8
0.0260

PSA
90
8
0.0260

Kallikrein
34
10

PSM
347
10
0.0048

PSM
130
10

PSM
416
11

PSM
373
9

PSM
373
11

PSA
69
9

PSA
17
9

PSM
226
9

PSM
226
10

PSM
512
10

PSM
52
10

PSM
200
10

PSM
591
10

PSM
157
8

PSM
199
11

PSM
514
8

PSM
514
11

PAP
193
11

PSM
623
11

PSM
718
8

PSM
324
10

Kallikrein
245
8

PSA
241
8

PSA
16
10

Kallikrein
20
10

PSM
34
8

PSM
34
9

PSA
70
8

PSM
441
9

Kallikrein
178
11

PSM
668
8

PAP
148
8

PAP
148
11

PAP
238
10
12.0000

PAP
194
10

PAP
14
10

PAP
14
11

Kallikrein
179
10

PSA
18
8

PSM
117
11

PAP
315
11

PSM
268
10
0.0082

PAP
70
10
0.6200

PSM
561
10

PAP
359
10

PSM
26
8

PSM
663
8

PAP
114
11

PSA
99
8

PAP
117
8

PSM
69
9

PSM
51
11

PSM
328
10

PSM
153
9

PAP
57
11

PSM
678
9

PSM
678
10

PSA
15
11

Kallikrein
19
11

PAP
147
9
1.2000

PSM
267
11

PAP
212
10

PSM
550
10

PAP
349
8

PSM
290
10

PSM
290
11

PSA
236
10
0.0010

PAP
278
9
0.0031

PAP
54
10

PSM
293
8

Kallikrein
91
11

PAP
276
11

PSM
95
9

PSM
218
11

PSM
91
10

PAP
72
8

PSM
667
9

PAP
69
11

Kallikrein
22
8

Kallikrein
39
9

PSA
84
9

PSA
182
10

PSM
578
8

PSA
87
11

Kallikrein
72
10

PSM
511
11

PSM
527
11

PAP
180
8

PSM
440
10

PSM
662
9

PSM
400
11

PAP
28
10

PSM
414
8

PSM
463
9
11.0000

Kallikrein
89
8

PSM
129
11

PSM
291
9

PSM
291
10

PSM
590
11

PAP
130
9

PSM
142
10

PSM
631
9

PAP
15
9

PAP
15
10

PAP
15
11

PAP
13
8

PAP
13
11

PSA
237
9
0.0017

PSM
615
11

PSM
695
11

PSM
317
11

PSM
348
9
0.0430

PAP
217
10

PSA
67
11

PAP
29
9

PSM
626
8

PSM
361
11

PSM
461
11

PSM
141
11

Kallikrein
150
8

PSA
146
8

PSM
575
11

PAP
145
11

PSM
201
9

PSM
372
10

PSA
68
10

PSM
225
10

PSM
225
11

PSM
690
11

PSM
27
11

PAP
30
8

PSM
592
9

Kallikrein
222
8

PSA
218
8

PSM
603
10

PSM
660
11

PSM
154
8

PSM
154
11

PAP
293
11

Kallikrein
92
10
0.1500

PSA
88
10
0.1500

PAP
129
10

Kallikrein
192
11

PSA
188
11

PSA
1
10

PSM
394
9

PSM
602
11

Kallikrein
74
8

PAP
206
9
0.0046

PSM
497
10

PAP
84
9

PAP
155
10

PSM
228
8

Kallikrein
188
8

PSM
625
9

PSM
537
10

Kallikrein
243
10

PSA
239
10

PSM
371
11

PSM
176
10

PSM
176
11

[0519]

13

TABLE VIII

Prostate A02 Supermotif Peptides with Binding Information

No. of

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

PSM

741
9
0.0002

PSM

741
10

PSM

742
8

PSM

742
9

PSM

735
8

PSM

735
9

PSM

735
11

PSA

59
10
0.0002

PSA

59
11
0.0010
0.0100
0.0140
0.0004
0.0018

Kallikrein

63
11
0.0003
0.0006
0.0450
0.0001
0.0004

PAP

121
9
0.0002

PAP

121
11

PSA

13
9
0.0002

PSA

13
10
0.0002

PAP

3
9

PAP

3
10

PAP

11
9
0.0002

PAP

11
11

PSM

392
8

PAP

299
8

PAP

299
9
0.0520

PSM

711
9
0.0590
6.0000
7.2000
0.0250
0.0009

PAP

122
8

PAP

122
10
0.0044

Kallikrein

147
8
0.0230

PSA

143
8
0.0230

Kallikrein

235
8
0.0009
0.0200
0.0510
0.0001
−0.0001

Kallikrein

235
10
0.0003
0.0050
0.0028
0.0005
−0.0001

PSA

231
8
0.0002

PSA

231
10
0.0008

Kallikrein

9
9
0.0410
0.0038
0.1100
0.0066
−0.0001

Kallikrein

9
10
0.0180
0.2600
0.4000
0.0051
0.0012

PSM

25
10
0.0150

PSM

25
11

PAP

116
8

PSM

302
8

PSM

217
9

PSM

217
10

PSM

217
11

PSA

181
8

PSA

181
9
0.0002

PSM

577
8

PSM

577
11

PSM

13
9
0.0002

PSM

13
11

PAP

227
9
0.0002

PAP

189
9
0.0005

PSM

49
10

PAP

274
10
0.0002

PAP

274
11

PSM

11
11

PSA

44
8
0.0003

PSM

365
8

PSM

365
9
0.0001

PSM

365
10
0.0002

PSM

286
9
0.0042

PSM

635
8

PSM

635
9

PSA

131
9
0.0001

Kallikrein

17
9
0.0001
0.0026
0.0013
0.0020
0.0610

Kallikrein

17
10
0.0014
0.0510
0.0490
0.0035
0.0058

PSM

601
8

PSM

601
11

Kallikrein

41
8
−0.0001
0.0005
0.0011
0.0004
0.0003

PSM

22
8

Kallikrein

198
11
0.0001
0.0003
0.0027
−0.0001
−0.0002

PSA

194
11
0.0013
0.0370
0.0250
0.0002
0.0081

Kallikrein

234
8
−0.0001
−0.0001
−0.0001
−0.0001
−0.0001

Kallikrein

234
9
0.0002
0.0013
0.1100
0.0004
0.0001

Kallikrein

234
11
0.0008
0.0033
0.0120
0.1700
−0.0002

PSA

230
9
0.0001

PSA

230
11
0.0008
0.0130
0.0071
0.0016
0.0023

PSA

180
9
0.0002

PSA

180
10
0.0001

Kallikrein

184
9
−0.0001
0.0006
0.0025
0.0002
0.0012

Kallikrein

184
10
0.0074
0.0710
0.0200
0.0030
0.0071

PSA

62
8
0.0001

PSA

62
9
0.0003

PSA

62
10
0.0001

Kallikrein

66
8
0.0001
0.0006
0.0006
−0.0001
−0.0001

Kallikrein

66
10
0.0001
0.0220
0.0083
0.0002
−0.0001

PAP

372
10
0.0002

Kallikrein

14
8
0.0001
0.0001
0.0001
0.0012
0.0004

PSM

466
8

PSM

466
9
0.0004

PSA

169
11
0.0001

Kallikrein

173
11
0.0002
0.0031
0.0020
0.0009
0.0007

PSM

422
8

PSM

422
11

PSM

710
10
0.0004

PSM

301
9

PSA

130
8
−0.0001
0.0003
−0.0001
−0.0001
0.0001

PSA

130
10
0.0001

PSM

714
11

PSM

156
8

PAP

201
9
0.0002

PSA

171
9
0.0003

PSA

171
11
0.0001

Kallikrein

120
11
0.0022

PSA

116
11
0.0022

PSA

136
8
0.0001

PSA

136
9
0.0003

PSA

136
11
0.0041
0.0180
0.0100
0.0001
0.0009

Kallikrein

3
8
0.0001
−0.0002
−0.0001
−0.0001
0.0006

Kallikrein

3
10
0.0010
0.0180
0.0052
0.0230
0.0051

PSM

173
8

PSM

173
10
0.0004

Kallikrein

182
11
0.0001
0.0018
0.0130
0.0001
0.0170

PSM

191
10
0.0001

PSM

191
11

PSA

98
10
0.0001

PSM

666
9

PSM

666
11

Kallikrein

207
11
0.0001
−0.0001
0.0005
−0.0001
0.0005

PAP

51
8

Kallikrein

85
8
−0.0001
0.0001
−0.0001
−0.0001
0.0002

PSA

81
8
−0.0001
−0.0001
−0.0001
−0.0001
0.0016

PAP

230
9
0.0002

PAP

290
9

PAP

290
10

PAP

290
11

PSA

178
11
0.0001

PAP

108
9

PAP

108
10

PAP

108
11

PSM

114
10

Kallikrein

134
8
−0.0001
−0.0001
−0.0001
−0.0001
0.0024

Kallikrein

134
10
0.0012
0.0230
0.0460
0.0004
0.0017

PAP

301
11

PSM

48
11

PSM

285
8

PSM

285
10
0.0002

PSM

641
10
0.0001

PAP

266
9

PAP

266
10

PSM

397
8

PSM

397
9
0.0002

PSM

109
8

PSM

109
9
0.0028

PSM

586
8

PSM

64
11

PAP

34
8

PAP

237
8

PAP

237
10
0.0008

PAP

240
10
0.0002

PSA

127
8
0.0001

PSA

127
9
0.0001

PSA

127
11
0.0001

PSM

560
10
0.0001

PAP

317
11

PAP

328
8

PAP

76
10

PSM

87
10

PAP

100
8

PAP

100
10

PSM

7
8

PSM

7
9

PSM

542
10
0.0002

PAP

334
9
0.0002

PAP

334
10

PAP

334
11

PSM

522
9
0.0002

PSM

522
10

PSM

727
8

PSM

727
9

PSM

727
10

PSM

351
8

PSM

351
9
0.0002

PSM

351
11

PAP

356
8

PAP

356
9
0.0002

PSM

418
11

PAP

187
8

PAP

187
11

PSM

42
8

PSM

42
9

PSM

42
11

PSM

61
10
0.0160

PSM

670
10
0.0014

PAP

18
9
0.0011

PAP

20
11

PSM

33
11

PAP

92
11

Kallikrein

165
10
0.0410
0.0940
1.1000
0.0068
0.0036

PSA

3
9
0.0150

PSA

3
11
0.0160

PSA

161
10
0.0310

PSM

73
8

PSM

73
11

Kallikrein

195
9
0.0220
0.0019
0.0160
0.0170
0.0006

PSA

191
9
0.0059

PAP

164
8

PAP

164
9

PSM

525
11

PSA

86
11

PSM

333
10
0.0001

PAP

221
8

PAP

221
11

PSM

77
8

PSM

77
10

PSM

737
9

PSM

737
10
0.0001

PAP

326
10

PSA

12
10
0.0005

PSA

12
11
0.1700
0.0220
0.0110
0.0006
0.0017

PSM

391
8

PSM

391
9
0.0002

PSM

24
11

PSM

364
9
0.0001

PSM

364
10
0.0002

PSM

364
11

Kallikrein

16
10
0.0017
0.0520
0.0380
0.0041
0.0057

Kallikrein

16
11
0.0001
0.0004
0.0004
0.0003
0.0003

PSM

282
8

PSM

282
11

PSM

529
10

PSM

385
8

PSM

385
9

PSM

385
10
0.0002

PSM

385
11

PAP

248
11

Kallikrein

225
11
0.0009
0.0014
0.0230
0.0001
0.0004

PSA

221
11
0.0001

PAP

204
10
0.0002

PSM

707
9
0.0210

PSM

104
8

PAP

196
10
0.0340

PSM

427
9
0.0079

PAP

305
11

PSM

680
11

PSM

288
10
0.0340
1.6000
4.7000
0.0015
0.0260

Kallikrein

140
8
−0.0001
0.0003
−0.0001
−0.0001
−0.0001

Kallikrein

140
9
0.0002
0.0092
0.0013
0.0007
−0.0002

Kallikrein

140
11
0.0003
0.0200
0.0450
0.0006
0.0020

PAP

295
8

Kallikrein

200
9
0.0002
0.0007
0.0015
−0.0001
−0.0002

PAP

74
8

PSM

168
8

PSM

168
10
0.0910
1.4000
1.4000
0.0230
0.0013

PSM

508
8

PSM

582
10
0.0024

PSM

582
11

PAP

199
11

PAP

68
8

PSM

85
8

PSM

85
9

PSM

446
11

PSM

224
9

PSM

238
11

Kallikrein

52
9
0.0003

PSA

48
9
0.0003

Kallikrein

52
10
0.0004

PSA

48
10
0.0004

Kallikrein

52
11
0.0002
0.0005
0.0005
0.0014
−0.0001

PSA

48
11
0.0002
0.0005
0.0005
0.0014
−0.0001

PAP

261
8

PAP

261
11

PSM

252
8

PSM

252
10
0.0001

PAP

128
8

PAP

128
9
0.0034

PAP

128
10
0.0016

PSM

345
8

PSM

345
9

PSM

345
11

PSM

82
11

Kallikrein

177
9
0.0020
0.0049
0.0005
0.0009
0.0003

Kallikrein

177
11
0.0290
0.0520
0.1100
0.0088
0.0004

PSM

573
11

PAP

270
8

PAP

378
8

PAP

144
10
0.0002

PAP

144
11

PSA

173
9
0.0001

PSA

173
11
0.0024

PSM

283
10
0.0001

Kallikrein

8
8
0.0001
−0.0002
−0.0001
−0.0001
0.0003

Kallikrein

8
10
0.0013
0.0500
0.0180
0.0180
0.0005

Kallikrein

8
11
0.0009
0.0032
0.0270
0.0100
0.0061

PSM

530
9

PSM

642
9
0.0001

PAP

188
10
0.0002

PSM

130
9
0.0002

PSM

416
8

PSM

373
10
0.0003

PSA

69
8
0.0010

PAP

135
9
1.3000

PAP

135
11

PAP

267
8

PAP

267
9
0.0001

PAP

267
11

PSM

258
11

PSM

226
11

PAP

284
8

PAP

284
9
0.0019

PAP

284
10
0.0610

PSM

96
10

Kallikrein

132
8
0.0001
0.0010
0.0001
−0.0001
0.0002

Kallikrein

132
10
0.0003
0.0084
0.0088
0.0004
0.0005

PSM

52
9

PSM

52
11

Kallikrein

226
10
0.0003
0.0100
0.0031
0.0005
0.0002

Kallikrein

226
11
0.0003
0.0150
0.0007
0.0013
0.0350

PSA

222
10
0.0003
0.0036
0.0030
0.0001
0.0003

PSA

222
11
0.0010
0.0120
0.0096
0.0001
0.0003

PSM

200
9
0.0001

PSM

591
11

PSM

659
10
0.0004

PSM

659
11

PSM

398
8

PSM

66
9
0.0002

PSM

59
9

PSM

723
10
0.0001

PSM

193
8

PSM

193
9
0.0002

PSM

193
10
0.0001

PSM

193
11

Kallikrein

131
8
0.0004
0.0002
0.0017
0.0002
−0.0001

Kallikrein

131
9
0.0047
0.0500
0.0420
0.0021
0.0002

Kallikrein

131
11
0.0002
0.0053
0.1700
0.0011
0.0006

PSM

199
10
0.0002

PSM

187
8

PSM

514
10
0.0140

PAP

282
10
0.0002

PAP

282
11

PSM

304
10
0.0003

PSA

166
9
0.0190

PSA

166
10
0.0370

PAP

234
8

PAP

234
10
0.0040

PAP

234
11

PAP

193
10
0.0026

PSM

343
10
0.0042

PSM

343
11

PAP

251
8

PSM

122
9
0.0002

PSM

122
10
0.0001

PSM

623
10
0.0002

PSM

718
11

PSM

207
8

PSM

207
11

PSM

341
9

PSM

213
8

PSM

213
10

Kallikrein

137
11
0.0001
0.0004
0.0009
0.0012
0.0005

PSA

133
11
0.0014

PSM

324
11

Kallikrein

191
9
0.0035
0.0092
0.1900
0.1600
0.0004

Kallikrein

191
11
0.0010
0.0280
0.0280
0.0160
0.0036

PSA

187
9
0.0020

Kallikrein

245
9
0.0001

PSA

241
9
0.0001

PAP

208
11

PAP

120
10
0.0017

PSM

219
8

PSM

219
9
0.0002

PSM

28
8

PSM

28
11

PSM

83
10
0.0001

PSM

83
11

PSM

110
8

PAP

31
8

PAP

31
9

PAP

31
10
0.0002

PAP

31
11

PAP

8
9
0.0002

PAP

283
9

PAP

283
10

PAP

283
11

PAP

7
8

PAP

7
10
0.0061

PSM

305
9
0.0001

PAP

21
10
0.6000

PAP

21
11

PSM

34
10
0.0058

PSM

428
8

PSM

4
8

PSM

4
9
0.0180

PSM

4
10
0.0006

PSM

4
11

PAP

6
9
0.0120

PAP

6
11

PAP

306
10
0.0017

PAP

306
11

PSM

441
8

PSM

441
10
0.0280
0.7500
1.5000
0.0043
0.0006

Kallikrein

123
8
0.0001

PSA

119
8
0.0001

PSA

119
10
0.0001

PSA

119
11
0.0023
0.0140
0.0150
0.0002
0.0010

Kallikrein

123
10
0.0030
0.0290
0.9200
0.0010
0.0008

Kallikrein

123
11
0.0002
0.0007
0.0180
−0.0001
−0.0001

Kallikrein

178
8
0.0003
0.0073
0.0003
0.0021
−0.0001

Kallikrein

178
10
0.0030
0.0800
0.0280
0.0020
0.0042

PSM

116
8

PAP

136
8

PAP

136
10
0.0074

PAP

136
11

PSM

668
9
0.0110

Kallikrein

121
10
0.0018

PSA

117
10
0.0018

PAP

113
8

PAP

113
9
0.0071

PAP

113
10
0.0037

PAP

113
11

PSM

469
9
0.0780
11.0000
4.8000
0.0340
0.0250

PSM

469
10
0.0046

PSA

167
8

PSA

167
9

Kallikrein

171
8

Kallikrein

171
9

PSM

650
10

PSM

650
11

PSM

442
9

PSM

442
11

PAP

258
10

PAP

258
11

PAP

296
11

PSA

128
8
−0.0001
−0.0001
0.0002
−0.0001
0.0001

PSA

128
10
0.0002

PSA

4
8
0.0003
−0.0001
0.0006
0.0007
0.0001

PSA

4
10
0.0018
0.0450
0.0820
0.0110
0.0910

PSA

4
11
0.0008
0.0014
0:0370
0.0025
0.0062

PSM

268
11

PSA

162
9
0.0003

PSA

162
11
0.0007
0.0087
0.0074
0.0004
0.0021

PSM

574
10

PSM

574
11

PSA

37
8
0.0001

PSA

37
9
0.0003

Kallikrein

217
10
0.0004

PSA

213
10
0.0004

Kallikrein

217
11
0.0007
0.0034
0.0033
0.0049
0.0041

PSA

213
11
0.0007
0.0034
0.0033
0.0049
0.0041

PSM

561
9

PAP

40
11

PSM

473
9
0.0001

Kallikrein

54
8
0.0001

PSA

50
8
0.0001

Kallikrein

54
9
0.0001

PSA

50
9
0.0001

Kallikrein

54
10
0.0001

PSA

50
10
0.0001

Kallikrein

54
11
0.0001

PSA

50
11
0.0001

PSM

26
9
0.0280
0.0030
0.0004
0.1100
0.0003

PSM

26
10
0.0021

Kallikrein

4
9
0.0020
0.0027
0.0085
0.0190
0.0002

PAP

263
9

PSM

174
9

PAP

298
9
0.0037

PAP

298
10
0.0010

Kallikrein

196
8
0.0014
0.0020
0.0018
0.0001
0.0002

PSA

192
8
0.0006
0.0012
0.0033
−0.0001
0.0001

Kallikrein

122
9
0.0610

PSA

118
9
0.0610

PSA

118
11
0.1400

Kallikrein

122
11
0.0044
0.0072
0.2100
0.0019
0.0007

PAP

343
11

PSM

663
9
0.4400
5.7000
5.8000
0.4900
0.0410

PAP

232
10
0.0002

PAP

373
9

PSM

583
9
0.0170

PSM

583
10
0.0140

PSM

583
11

PSM

451
11

PSM

216
10
0.0002

PSM

216
11

PSM

69
10

PSM

257
8

PSM

51
8

PSM

51
10

PAP

119
11

Kallikrein

79
8
0.0002
0.0035
0.0004
−0.0001
0.0004

PSM

3
9
0.0001

PSM

3
10
0.0027

PSM

3
11

PSM

260
9
0.0007

PSM

260
10
0.0002

PSM

57
9
0.0026

PSM

57
11

Kallikrein

102
10
0.0043
0.0260
0.0400
0.0058
0.0020

PSM

357
9

PSM

357
10
0.0001

PSM

153
11

PSM

231
9
0.0001

PSA

125
8
−0.0001
−0.0001
−0.0001
−0.0001
−0.0001

PSA

125
10
0.0002

PSA

125
11
0.0003
0.0028
0.0008
−0.0001
−0.0001

Kallikrein

129
8
0.0001
0.0003
−0.0001
−0.0001
−0.0001

Kallikrein

129
10
0.0011
0.0100
0.0320
0.0006
0.0002

Kallikrein

129
11
0.0002
0.0006
0.0017
−0.0001
0.0001

Kallikrein

146
9
0.0083
0.0210
0.0270
0.0002
0.0035

PSA

142
9
0.0083
0.0210
0.0270
0.0002
0.0035

PSM

273
11

Kallikrein

240
8
0.0001
−0.0001
−0.0001
−0.0001
−0.0001

PAP

49
10
0.0002

PSM

296
10
0.0001

PSM

296
11

PAP

134
8

PAP

134
10
0.0075

PAP

140
9
0.0002

PSM

658
11

PAP

352
8

PAP

352
9
0.0001

PSA

15
8
0.0001

Kallikrein

19
8
0.0001
0.0002
−0.0001
−0.0001
−0.0001

PAP

5
8

PAP

5
10
0.0004

PSM

468
10
0.0008

PSM

468
11

PAP

147
8

PAP

147
10
0.0006

PSM

267
8

Kallikrein

216
8
0.0001

PSA

212
8
0.0001

Kallikrein

216
11
0.0020

PSA

212
11
0.0020

PAP

212
11

PSA

95
8
0.0002

PSM

550
9
0.0002

Kallikrein

99
8
0.0002
0.0008
0.0002
−0.0001
−0.0001

PSM

568
8

PSM

568
9
0.0042

PSM

568
10
0.0005

PAP

365
9

PAP

365
10

PAP

365
11

PSM

619
9

PAP

64
8

PAP

64
10

PSM

166
9

PSM

166
10

PSA

185
8

PSA

185
9

PSA

185
11

PSM

388
8

PSM

388
11

Kallikrein

57
8

PSA

53
8

PSA

53
11

Kallikrein

57
11

Kallikrein

142
9
0.0001

PSA

138
9
0.0001

Kallikrein

142
10
0.0084
0.0220
0.0520
0.0037
0.0005

PSA

138
10
0.0084
0.0220
0.0520
0.0037
0.0005

PSM

293
10

PAP

362
9

Kallikrein

91
10
0.0019
0.0099
0.0680
0.0022
0.0011

PSM

740
10
0.0006

PSM

740
11

PSM

79
8

PAP

276
8

PAP

276
9
0.0002

PAP

276
10

PSM

95
11

PSM

731
8

PSM

731
9
0.0026

PSM

731
11

PSM

218
8

PSM

218
9
0.0001

PSM

218
10
0.0006

PAP

72
10
0.0003

PSM

667
8

PSM

667
10
0.0510
.0.1200
0.1100
0.0003
0.2700

PAP

297
10
0.0002

PAP

297
11

Kallikrein

39
10
0.0004
0.0097
0.0200
0.0005
0.0252

PSA

182
8
−0.0001
−0.0001
0.0001
−0.0001
−0.0001

PSA

182
11
0.0001

PSA

35
10
0.0001

PSA

35
11
0.0001

PSM

578
10
0.0001

PSM

578
11

PSA

87
10
0.0001

Kallikrein

72
9
0.0001
0.0021
0.0011
0.0025
0.0510

PAP

101
9
0.0002

PAP

2
8

PAP

2
10

PAP

2
11

PAP

10
10
0.0002

PSM

673
9
0.0001

PSM

534
10

PAP

273
11

PSA

43
8
−0.0001
−0.0001
0.0003
−0.0001
−0.0001

PSA

43
9
0.0002

Kallikrein

186
8
−0.0001
−0.0001
0.0003
0.0001
−0.0001

Kallikrein

186
11
0.0007
0.0560
0.0016
0.0018
0.0009

PSM

354
8

PSM

354
9
0.0004

PSM

527
9
0.0001

PAP

180
9
0.0006

PAP

180
10
0.0048

PAP

180
11

PSM

440
8

PSM

440
9
0.0001

PSM

440
11

PSM

649
11

PAP

257
8

PAP

257
11

PSA

121
8
0.0004

PSA

121
9
0.0003

PSA

121
11
0.0007

Kallikrein

125
8
−0.0001
0.0005
0.0007
−0.0001
−0.0001

Kallikrein

125
9
−0.0001
−0.0002
0.0009
−0.0001
−0.0002

Kallikrein

125
11
0.0015
0.0043
0.0210
0.0002
0.0006

PSM

662
8

PSM

662
10
0.5100
1.6000
1.3000
0.0930
0.0005

PSM

730
9

PSM

730
10

PSM

181
8

PSM

414
10

PAP

111
8

PAP

111
10
0.0150

PAP

111
11

PSM

463
8

PSM

463
11

PSM

162
8

PAP

287
10
0.0002

PAP

115
8

PAP

115
9
0.0043

PSM

634
9
0.0001

PSM

634
10

Kallikrein

7
9
−0.0001
0.0006
0.0087
0.0006
0.0004

Kallikrein

7
11
0.0029
0.0066
0.0160
0.0100
0.0055

PSM

455
8

PSM

455
10
0.0001

Kallikrein

159
8
0.0001

PSA

155
8
0.0001

PSA

155
9
0.0001

PSM

129
10
0.0001

PSM

613
10

PAP

130
8

PSA

75
8
0.0003
0.0032
0.0028
−0.0001
−0.0001

PSA

75
11
0.0190

PSM

631
10
0.0010

PAP

15
8

Kallikrein

175
9
0.0003
0.0720
0.0180
−0.0001
0.0004

Kallikrein

175
11
0.0390
1.9000
0.6900
0.0005
0.0004

PSM

322
8

Kallikrein

104
8
0.0002
0.0007
0.0002
−0.0001
−0.0001

PSA

100
8
0.0020

PAP

242
8

Kallikrein

170
9
0.0100
0.0840
0.0240
0.0006
0.0031

Kallikrein

170
10
0.0099
0.4000
0.0920
0.0059
0.0008

PAP

13
9
0.0200

PAP

13
10
0.0170

PSM

472
10
0.0002

PSM

615
8

PSM

615
10
0.0001

Kallikrein

35
8

PSA

31
8

PSA

31
9

Kallikrein

71
10

PSM

98
8

PSM

98
11

PSA

203
11
0.0005
0.0150
0.0092
0.0002
0.0035

PAP

106
8

PAP

106
9

PAP

106
11

PSM

431
11

PSM

348
8

PSM

348
11

PSM

338
9
0.0001

PSM

107
9
0.0001

PSM

107
10
0.0002

PSM

107
11

Kallikrein

11
8
0.0004
0.0006
0.0022
0.0003
−0.0001

Kallikrein

11
10
0.0024
0.0760
0.0065
0.0026
0.0035

Kallikrein

11
11
0.0100
0.0010
0.0007
0.0007
0.0005

PAP

217
11

PSA

67
10
0.0001

PAP

29
10
0.0031

PAP

29
11

PSM

626
10

PSM

626
11

PSA

7
8
0.0001

PSA

7
10
0.0001

PSA

7
11
0.0001

PSM

554
8

PSM

554
9
0.0073

PSA

58
11
0.0005
0.0057
0.0085
0.0004
0.0105

PSM

14
8

PSM

14
10

PSM

415
9

PAP

190
8

PAP

171
11

PAP

112
9
0.0650

PAP

112
10
0.0065

PAP

112
11

PAP

222
10
0.0002

PAP

222
11

PSM

461
9
0.0012

PSM

461
10
0.0008

PSA

5
9
0.0016

PSA

5
10
0.0007

PAP

231
8

PAP

231
11

Kallikrein

143
8

PSA

139
8

Kallikrein

143
9

PSA

139
9

PAP

335
8

PAP

335
9

PAP

335
10

PSM

78
9

PAP

275
9

PAP

275
10

PAP

275
11

PSM

339
8

PSM

339
11

PAP

71
11

Kallikrein

150
11
−0.0001
0.0009
0.0025
0.0005
0.1400

PSA

146
11
−0.0001
0.0009
0.0025
0.0005
0.1400

PAP

374
8

PAP

291
8

PAP

291
9

PAP

291
10
0.0020

PSM

575
9

PSM

575
10
0.0005

PAP

145
9
0.0002

PAP

145
10
0.0001

PSM

738
8

PSM

738
9
0.0002

PAP

292
8

PAP

292
9
0.0044

PAP

292
11

PSM

734
8

PSM

734
9

PSM

734
10

PSM

576
8

PSM

576
9
0.0002

PSA

38
8
−0.0001
−0.0001
−0.0001
−0.0001
−0.0001

PSM

12
10
0.0001

Kallikrein

40
9
−0.0001
−0.0001
0.0002
0.0002
0.0004

PSM

447
10
0.0001

PSM

201
8

PSM

358
8

PSM

358
9
0.0002

PSM

372
11

PSA

68
9
0.0003

PSM

225
8

PAP

363
8

PAP

363
11

PSA

174
8
0.0001

PSA

174
10
0.0008

PSM

27
8

PSM

27
9
0.1300
19.0000
0.3000
0.1200
0.0028

PAP

30
9
0.0590

PAP

30
10
0.0021

PAP

30
11

Kallikrein

138
10
0.0008
0.0150
0.0110
0.0004
−0.0001

Kallikrein

138
11
−0.0001
0.0007
0.0003
0.0003
0.0006

PSM

115
9
0.0002

PSM

592
10
0.0013

PSM

592
11

PSM

603
9
0.0002

PSM

660
9
0.0001

PSM

660
10
0.0003

Kallikrein

5
8
0.0050
0.0790
0.0200
0.0024
0.0003

Kallikrein

5
11
0.0002
0.0011
0.0048
0.0004
0.0005

PSA

56
8
0.0001

Kallikrein

60
8
0.0002
0.0034
0.0001
0.0001
0.0002

PSA

36
9
0.0001

PSA

36
10
0.0003

Kallikrein

53
8
0.0001

PSA

49
8
0.0001

Kallikrein

53
9
0.0200

PSA

49
9
0.0200

Kallikrein

53
10
0.0001

PSA

49
10
0.0001

Kallikrein

53
11
0.0130

PSA

49
11
0.0130

PAP

262
10
0.0008

PSA

134
10
0.0001

PSA

134
11
0.0021
0.0042
0.0014
0.0001
0.0003

PSM

739
8

PSM

739
11

PSM

253
9

Kallikrein

192
8
−0.0001
0.0003
0.0005
0.0007
0.0007

Kallikrein

192
10
0.0008
0.0180
0.0068
0.0004
0.0030

PSA

188
8
0.0001
0.0002
0.0031
−0.0001
−0.0001

PSM

352
8

PSM

352
10

PSM

352
11

PSA

8
9
0.0110

PSA

8
10
0.0019

PSA

8
11
0.0013
0.0005
0.0009
0.0011
0.0002

PSA

1
8
0.0002

PSA

1
9
0.0008

PSA

1
11
0.0069

PSM

394
11

Kallikrein

246
8
0.0001
0.0021
−0.0001
0.0001
−0.0001

PSA

242
8
0.0001
0.0021
−0.0001
0.0001
−0.0001

Kallikrein

246
11
0.0001
0.0001
0.0002
−0.0001
0.0004

PSA

242
11
0.0001
0.0001
0.0002
−0.0001
0.0004

Kallikrein

135
9
−0.0001
−0.0005
0.0007
0.0008
−0.0002

PSM

602
10
0.0001

PSM

434
8

PSM

434
9
0.0001

Kallikrein

47
8
−0.0001
0.0003
0.0005
0.0001
0.0070

Kallikrein

47
9
−0.0001
0.0004
0.0067
0.0007
0.0310

PAP

226
8

PAP

226
10
0.0002

PSA

10
8
0.0005

PSA

10
9
0.0005

Kallikrein

252
8
0.0002
0.0120
0.1700
0.0002
−0.0001

PSA

248
8
0.0001

PSM

20
8

PSM

20
9
0.0180

PSM

20
10
0.0120

PAP

25
8

PAP

25
11

PAP

138
8

PAP

138
9

PAP

138
11

Kallikrein

38
11

PSA

34
11

PSA

55
9
0.0008

Kallikrein

59
9
0.0003
0.0018
0.0001
0.0160
0.0007

PSM

607
8

PSM

607
10

PSM

700
9
0.0013

PSM

692
10

PSM

179
10
0.0002

PAP

310
9
0.0037

Kallikrein

153
8
−0.0001
0.0009
0.0003
0.0003
0.0120

PSA

149
8
−0.0001
0.0009
0.0003
0.0003
0.0120

PSM
YAVVLRKYA
600
9

PSM
YAYRRGIA
277
8

PSM
YAYRRGIAEA
277
10

PSM
YAYRRGIAEAV
277
11

PSM
YINADSSI
449
8

PAP
YIRKRYRKFL
84
10
0.0002

PAP
YIRSTDVDRT
103
10

PAP
YIRSTDVDRTL
103
11

Kallikrein
YTKVVHYRKWI
243
11
0.0001
−0.0001
0.0004
−0.0001
0.0008

PSA
YTKVVHYRKWI
239
11
0.0001
−0.0001
0.0004
−0.0001
0.0008

PSM
YTLRVDCT
460
8

PSM
YTLRVDCTPL
460
10
0.0015

PSM
YTLRVDCTPLM
460
11

PSM
YVAAFTVQA
733
9

PSM
YVAAFTVQAA
733
10

PSM
YVAAFTVQAAA
733
11

[0520]

14

TABLE IX

Prostate A03 Supermotif with Binding Data

No. of

Posi-
Amino

Protein
tion
Acids
A*0301
A*1101
A*3101
A*3301
A*6801

PSA
59
8

PSA
13
8

PAP
3
8

PSM
392
9

PSM
711
8

Kallikrein
235
11

PSA
231
11

PSM
531
9
0.0086
0.2700

PAP
227
8
0.0003
0.0039

PAP
227
10

PSM
49
11

PAP
274
8
0.0180
0.0700

PAP
274
9
0.1000
1.2000

PSM
11
9

PSM
635
11

Kallikrein
17
8

PSM
393
8

PSM
601
10
0.0026
0.0210

Kallikrein
241
10

Kallikrein
241
11

Kallikrein
198
9

PSA
194
9
0.0006
0.0015

PSA
180
8

PSA
180
11

Kallikrein
184
8

PSM
196
9

PAP
347
9
0.0040
0.0006

Kallikrein
14
11

PSM
710
9
0.0006
0.0002

PSM
301
8

PSM
714
10
0.0003
0.0002

PAP
201
8

PSM
173
9

Kallikrein
182
10

PSM
191
9

PSA
98
8
0.0003
0.0001

PSA
98
11

PSM
9
8

PSM
9
9

PSM
9
11

PSM
630
8

Kallikrein
116
10

PSA
112
10

PSM
453
11

PSM
316
9
0.0032
0.0003

PAP
51
9
0.0001
0.0001

PSA
178
10
0.0007
0.0011

PSM
114
9
0.0006
0.0010

PSM
48
8

PSM
641
9
0.0006
0.0002

PAP
266
8

PSM
397
10

PSM
397
11

PAP
166
8

PAP
80
8

PAP
80
9

PAP
80
11

PSM
64
8

PSM
64
9

PAP
34
10
0.0014
0.0037

PSM
716
8

PAP
95
11

PSM
7
10

PSM
7
11

PAP
170
10
0.0004
0.0140

PAP
170
11

PSM
557
8

PSM
675
10

PSM
61
11

PSM
37
8

PAP
18
11

PAP
20
9
0.0024
0.0004

PSM
646
10
0.0003
0.0007

PSM
506
9

PSM
639
11

PSM
333
9

PSM
333
11

PAP
37
11

PSA
12
9
0.0150
0.0350

PSM
391
10

Kallikrein
16
9

PSM
529
8

PSM
529
11

PAP
248
8

PAP
248
10

PSM
680
9
0.0460
0.0280

PSM
311
10
0.0006
0.1400

PSA
226
10

Kallikrein
158
10

PSM
430
11

PSM
85
10

PSM
403
9

PSM
403
11

PSM
360
11

PSM
345
10

Kallikrein
177
10

PAP
314
9
0.2700
0.5300

PSM
573
8

PSM
347
8

PSM
689
11

PSM
202
9

PSM
530
10

PSM
642
8

PSM
614
10
0.1900
0.1100

PSM
52
8

Kallikrein
25
9
0.0410
0.0190
0.0002
0.0006
0.001

PSA
21
9
0.0410
0.0190
0.0002
0.0006
0.001

PSM
200
8

PSM
200
11

PSM
591
8

PSM
398
9
0.1700
0.0087

PSM
398
10
0.0260
0.0006

PSM
59
8

PSM
723
8

PSM
199
9
0.0740
1.0000

PSM
610
8

PAP
173
8

PSM
491
9
0.4000
2.1000

PSM
491
10
0.3200
0.0810

PSM
655
8

PSM
482
10
0.0044
0.0210

PSA
66
8

PSM
207
9
0.1600
0.1200

PSM
213
11

PSA
187
11

Kallikrein
245
10
0.0450
0.0450

PSA
241
10
0.0450
0.0450

PSM
92
10
0.0031
0.0007

PAP
21
8

PSM
34
11

Kallikrein
105
8

PSA
101
8

Kallikrein
123
9

PAP
243
9
0.0760
0.2000

PAP
243
11

Kallikrein
178
9

PAP
153
11

Kallikrein
121
11

PSM
469
11

PAP
241
11

PAP
244
8

PAP
244
10
0.0520
0.0370

Kallikrein
179
8

PSA
57
8

PSA
57
10
0.1400
0.0830

Kallikrein
61
8

Kallikrein
61
9

PAP
315
8
0.0014
0.0100

PSM
561
11

PAP
40
8
0.0003
0.0002

PSM
473
10

PAP
263
10
0.0560
0.1200

PAP
263
11

PSM
174
8

Kallikrein
196
11

PSA
192
11

Kallikrein
122
10

PSM
663
11

Kallikrein
103
10

PSA
99
10
0.0070
0.0110

PSM
216
8

PSM
51
9

Kallikrein
79
11

PSM
247
9

PSM
57
10

Kallikrein
102
11

PSM
589
10

Kallikrein
70
8

PSM
438
8

PSM
231
10

PSA
125
9
0.0002
0.0002
0.0004
0.0006
0.0001

Kallikrein
129
9

PSM
273
8

PSM
273
9
0.0001
0.0002

Kallikrein
240
11

PAP
49
11

PSM
296
9

PSM
678
11

PSA
95
9
0.2400
0.0370
0.0002
0.0006
0.0001

PSA
95
11

Kallikrein
99
9

PSM
721
9

PSM
721
10
0.0003
0.0002

PSA
236
11

PSM
502
10

PAP
224
11

PSM
91
11

PAP
152
8

PSA
182
9
0.0060
0.0140
0.0028
0.0014
0.0051

PSA
35
9
0.0021
0.0018

PAP
101
11

PAP
2
9
0.1500
0.1200

PAP
273
9
0.0210
0.0600

PAP
273
10
0.0053
0.0250

Kallikrein
24
10
0.0460
0.0670

PSA
20
10
0.0460
0.0670

PSM
354
10
0.3700
0.4300

PSM
527
8

PSM
527
10

PSM
400
8

PAP
28
9
0.0490
0.1100

PSM
181
10

PSM
312
9
0.0006
0.0012

PSM
10
8

PSM
10
10

PSM
455
9

Kallikrein
159
9

Kallikrein
159
11

PSA
155
11

PSM
613
11

PSM
590
9
0.0006
0.0220

Kallikrein
104
9

PSA
100
9
0.0024
0.0470

PAP
242
10
0.4900
2.3000

PSM
472
8

PSM
472
11

PSM
492
8

PSM
492
9
1.0000
2.0000

PAP
245
9
1.1000
0.8000

PAP
245
11

PSA
237
10
0.2800
0.2300

PSA
237
11

PSM
615
9
0.1100
0.0720

Kallikrein
117
9
0.0039
1.2000

PSA
113
9
0.0039
1.2000

PSM
454
10
0.0007
0.0910

PSM
45
11

PSM
317
8

PSM
431
10
0.0005
0.0016

PAP
29
8
0.0017
0.0061

PSM
554
11

PSA
58
9
0.0094
0.0140

Kallikrein
62
8

PSM
404
8

PSM
404
10
0.0007
0.0002

PSM
404
11

PAP
171
9
0.0006
0.0078

PAP
171
10
0.0007
0.0001

PSM
361
10
0.0003
0.0002

PAP
39
9
0.0006
0.0002

PSM
12
8

PSM
201
10

PSM
690
10
0.5400
0.7900

PSM
115
8

PSM
603
8

PSA
56
9
0.0002
0.0005

PSA
56
11

Kallikrein
60
9

Kallikrein
60
10

PSA
36
8

PAP
262
11

PSM
627
11

PSA
188
10
0.0003
0.0120

PAP
38
10

Kallikrein
246
9
0.0072
0.0930
0.5500
0.0490
0.0028

PSA
242
9
0.0072
0.0930
0.5500
0.0490
0.0028

PSM
602
9
0.0390
0.0660

PAP
226
9
0.0006
0.0002

PAP
226
11

PSA
10
11

PAP
25
9
0.0035
0.0150

PSA
55
10
0.0004
0.0001

Kallikrein
59
10

Kallikrein
59
11

PSM
607
11

PSM
692
8

PSM
179
9

PSM
600
11

PAP
84
8

PAP
103
9

PAP
155
9

PSM
471
9
0.0600
0.5400

PSM
537
9

Kallikrein
243
8

PSA
239
8

Kallikrein
243
9
0.0006
0.0580
1.2000
2.8000
1.3000

PSA
239
9
0.0006
0.0580
1.2000
2.8000
1.3000

PSM
371
8

[0521]

15

TABLE X

Prostate A24 Supermotif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
A*2401

PSM
674
8

PSM
60
11

PSM
736
11

PAP
299
8

PAP
299
9

PAP
122
10

PAP
122
11

Kallikrein
147
11

PSA
143
11

Kallikrein
235
9

PSA
231
8

PSA
231
9

PSM
25
8

PSM
25
9

PSM
25
10

PSM
25
11

PAP
116
8

PAP
116
9
0.0150

PSM
13
8

PSM
13
9

PAP
227
9

PAP
189
9

PSM
49
10

PAP
274
10

PAP
274
11

PSM
11
10

PSM
11
11

PSM
365
9

PSM
365
10

PSM
635
8

Kallikrein
17
9

PSM
393
10

PSM
601
11

Kallikrein
241
9

PSM
724
9

PSM
724
10

PSM
448
9
0.0190

Kallikrein
187
9

Kallikrein
187
10

Kallikrein
187
11

PSA
62
8

PSA
62
9

PSA
62
10

Kallikrein
66
9

Kallikrein
66
10

Kallikrein
14
8

PSM
466
8

Kallikrein
173
11

Kallikrein
152
9
0.1700

PSA
148
9
0.1700

PSM
652
8

PSM
652
10

PSM
520
9

PSM
520
11

PSM
184
9

PSM
184
11

PAP
186
9
0.0002

PSM
156
9

PAP
201
10

PSA
136
9

Kallikrein
3
8

PSM
191
10

PSA
98
9
0.0001

PSA
98
10

Kallikrein
207
11

PAP
51
8

PAP
230
9

PAP
290
9

PAP
290
11

PAP
108
10

Kallikrein
134
8

PAP
301
10

PSM
599
9

PSM
233
10

PSM
102
9

PSM
425
10

Kallikrein
164
8

PSA
160
8

Kallikrein
194
8

Kallikrein
194
9

PAP
176
9

PSM
505
8

PSM
505
11

PSM
641
10

PSM
137
8

PSM
397
9

PSM
109
8

PSM
109
9

PSM
109
11

PSM
586
8

PSM
586
10

PAP
80
10

PSM
64
10

PSM
64
11

PAP
34
9

PSM
480
9

PAP
237
8

PAP
237
10

PAP
237
11

PAP
240
8

PAP
240
10

PSA
127
9

PSA
127
11

PSM
560
10

PSM
560
11

PAP
358
11

PAP
317
9

PAP
317
10

PSM
621
9
0.0010

PAP
170
8

PSM
542
8

PSM
542
10

PSM
542
11

PAP
334
9

PAP
334
10

PAP
334
11

PSM
557
9

PSM
557
10

PSM
522
9

PSM
727
11

PSM
351
9

PSM
433
9

PSM
433
10

PSM
276
8

PAP
324
8

PAP
83
10
0.0067

PAP
83
11

PSM
185
8

PSM
185
10

PSM
32
8

PSM
32
10
0.0026

PSM
32
11

PAP
23
9
0.0017

PAP
187
8

PAP
187
11

PSM
42
11

PSM
61
10

PSM
670
10

PAP
18
8

PAP
18
9

PSM
33
9

PSM
33
10

PSM
33
11

PSA
3
8

PSA
3
9

PSM
73
8

PSM
73
11

Kallikrein
195
8

PSA
191
8

PSM
639
8

PSM
737
10

PAP
24
8

PSM
565
8

PSM
565
10
1.1000

PSM
487
8

PSM
487
11

PSM
31
8

PSM
31
9
0.0190

PSM
31
11

PAP
66
8

PAP
66
10

PSM
36
8

PAP
17
8

PAP
17
9
0.0016

PAP
17
10
0.0007

PSM
282
8

PSM
282
11

PSM
529
9

PAP
248
11

PAP
204
10

PAP
204
11

PSM
707
9

PSM
104
10

PAP
196
8

PAP
196
10

PAP
196
11

PSM
427
8

PAP
305
11

PSM
680
8

PSM
288
10

Kallikrein
140
9

PAP
295
9

PAP
74
8

PAP
74
11

PSM
168
9

PSM
508
8

PSM
582
10
0.0002

PSM
85
8

PSM
403
8

Kallikrein
149
9

PSA
145
9

PSM
446
11

PSM
224
11

PSM
238
9

PSM
238
11

Kallikrein
221
9

PSA
217
9

Kallikrein
52
8

PSA
48
8

Kallikrein
52
10

PSA
48
10

PAP
261
8

PAP
261
11

PSM
252
8

PSM
252
10

PAP
128
8

PAP
128
9

PAP
128
10

PAP
128
11

Kallikrein
46
9

Kallikrein
28
11

PSA
24
11

Kallikrein
156
10
0.0001

PSA
152
10
0.0001

Kallikrein
156
11

PSA
152
11

PSM
409
8

PSM
409
9

PSM
409
10
0.0540

PSM
150
8

PSM
271
9

PSM
548
9

PSM
298
8

PSM
298
9

PSM
345
11

PSM
82
9

PSM
82
11

PSM
573
11

PAP
270
8

PAP
270
11

PAP
144
10

PAP
144
11

PSM
112
8

PAP
78
8

Kallikrein
248
10
0.0550

PSA
244
10
0.0550

PSM
130
9

PSM
130
10

PSM
416
11

PSM
373
9

PSM
373
11

PSA
69
8

PSA
69
9

PAP
267
11

PSM
258
11

PSA
17
9

PSM
226
9

PSM
226
10

Kallikrein
132
8

Kallikrein
132
10

PSM
52
10

PSM
52
11

Kallikrein
226
11

PSA
222
11

PSM
200
10

PSM
591
10

PSM
659
10

PSM
659
11

PSM
157
8

PSM
398
8

PAP
131
8

PAP
131
11

PAP
205
9
0.0024

PAP
205
10

PSM
691
10

PSM
708
8

PSM
355
8

PSM
72
9

PSA
190
9
0.0310

PSM
645
9

PSM
545
8

PSM
564
9

PSM
564
11

PSM
193
8

PSM
193
10

Kallikrein
131
9

Kallikrein
131
11

PSM
199
11

PSM
187
8

PSM
514
8

PSM
514
11

PSA
166
10

PAP
234
8

PAP
234
10

PAP
234
11

PAP
193
10

PAP
193
11

PSM
122
9

PSM
122
10

PSM
623
10

PSM
623
11

PSM
718
8

PSM
324
10

Kallikrein
191
11

Kallikrein
245
8

PSA
241
8

Kallikrein
245
9

PSA
241
9

PSM
606
9
12.0000

PSM
606
11

PSM
699
10

PSM
699
11

PSM
417
10

PSM
143
9

PAP
22
10
0.0045

PAP
202
9

PSA
76
11

PAP
19
8

PAP
123
9
0.0033

PAP
123
10
0.0140

PSM
632
8

PSM
632
11

PSA
16
10

Kallikrein
20
10

PAP
7
8

PAP
7
10

PAP
21
11

PSM
34
8

PSM
34
9

PSM
34
10

PSA
70
8

PAP
6
9

PAP
6
11

PAP
306
10

PSM
441
9

PSM
441
10

PSA
119
10

Kallikrein
123
10

Kallikrein
178
11

PSM
668
8

PSM
668
9
0.0075

PAP
113
8

PAP
113
11

PSM
469
9

PSA
128
8

PSA
128
10

PAP
315
11

PSA
4
8

PSM
268
10

PSA
162
11

PAP
70
10
0.0022

PSM
574
10

Kallikrein
217
10

PSA
213
10

PSM
561
9

PSM
561
10

PAP
40
11

PAP
359
10

PSM
473
9

Kallikrein
54
8

PSA
50
8

PSM
26
8

PSM
26
9

PSM
26
10

PAP
263
9

PAP
213
9
0.4400

PAP
213
11

PSA
96
11
0.1200

PAP
318
8

PAP
318
9
2.5000

PSM
551
9

PSM
551
11

PAP
154
11

PSM
74
10
0.2300

PSM
227
8

PSM
227
9
0.4400

PSA
238
8

PSA
238
11

PSM
669
8

PSM
669
11

PSA
118
11

Kallikrein
122
11

PAP
343
11

PSM
663
8

PSM
663
9

PAP
232
10

PAP
117
8

PSM
583
9

PSM
583
11

Kallikrein
1
8

Kallikrein
1
10

PSM
470
8

PSM
89
8

PSM
336
9

PSM
336
11

PSM
638
9
0.0001

PSM
76
8

PSM
69
9

PSM
51
8

PSM
51
11

PSM
260
9

PSM
57
9

Kallikrein
102
10

PSM
328
10

PSM
153
9

PSM
540
10

PSM
178
8

PSM
178
9
0.7700

PSM
178
11

PSM
459
11

PSM
594
11

PAP
157
8

PAP
157
11

PSM
160
10

PSM
685
8

PAP
49
10

PSM
296
10

PSM
296
11

PAP
57
11

PAP
134
8

PAP
140
9

PSM
658
11

PAP
352
8

PSM
678
9

PSM
678
10

PSA
15
11

Kallikrein
19
11

PAP
5
10

PSM
468
10

PAP
147
8

PAP
147
9

PAP
147
10

PSM
267
11

Kallikrein
216
8

PSA
212
8

Kallikrein
216
11

PSA
212
11

PAP
212
10

PSA
95
8

PSM
550
10

Kallikrein
99
8

PAP
54
10

PSM
293
8

Kallikrein
91
10

Kallikrein
91
11

Kallikrein
37
11

PAP
309
10
0.0240

PAP
309
11

PAP
183
9
0.1100

PSM
326
8

PAP
276
8

PAP
276
9

PAP
276
10

PAP
276
11

PSM
95
9

PSM
95
11

PSM
218
9

PSM
218
10

PSM
218
11

PSM
91
10

PAP
72
8

PAP
72
10

PSM
667
9

PSM
667
10

PAP
69
11

PAP
297
10
0.0001

PAP
297
11

Kallikrein
39
9

PSA
84
9

PSA
182
10

PSA
182
11

PSM
578
8

PSM
578
10

PSA
87
10

PSA
87
11

Kallikrein
72
9

Kallikrein
72
10

PSA
54
10
0.0007

Kallikrein
58
10

PAP
355
10
0.0037

PAP
163
10
0.0001

PSM
511
11

PSM
354
9

PSM
527
11

PAP
180
8

PAP
180
9

PSM
440
10

PSM
440
11

PSM
649
11

PAP
257
11

PSA
121
8

Kallikrein
125
8

PSM
662
8

PSM
662
9

PSM
662
10

PSM
181
8

PSM
414
8

PAP
111
10

PSM
463
8

PSM
463
9

PSM
463
11

Kallikrein
89
8

PSM
19
8

PSM
19
10

PAP
88
10
0.0057

PSM
536
11

PSM
401
10

PSM
704
9

PSM
704
10

PSA
91
9
0.0007

PSA
91
11

Kallikrein
95
9

Kallikrein
95
11

PSM
455
8

Kallikrein
159
8

PSA
155
8

PSM
129
10

PSM
129
11

PSM
291
9

PSM
291
10

PSM
613
10

PSM
590
11

PAP
130
8

PAP
130
9

PSM
142
10

PSM
631
9

PAP
15
8

PAP
15
9

PAP
15
10

PAP
15
11

Kallikrein
175
9

Kallikrein
104
8

PSA
100
8

PAP
242
8

Kallikrein
170
9

Kallikrein
170
10

PAP
13
8

PAP
13
9

PAP
13
10

PAP
13
11

PSM
472
10

PSA
237
9

PSM
615
8

PSM
615
11

PSA
203
11

PAP
106
8

PAP
106
9

PSM
431
11

PSM
348
8

PSM
348
9

PSM
338
9

PSM
107
10

PSM
107
11

Kallikrein
11
10

Kallikrein
11
11

PAP
217
10

PSA
67
10

PSA
67
11

PAP
29
9

PSM
626
8

PSA
7
10

PSA
7
11

PSM
554
8

PAP
225
8

PAP
225
11

PSM
420
9

PSM
420
10

Kallikrein
228
9

PSA
224
9
0.0001

PAP
62
9
0.0013

PSM
318
10

PSM
496
11

PAP
96
8

PAP
96
9
0.2600

PAP
279
8

PSM
241
8

PSM
118
10

PSM
118
11

PAP
190
8

PAP
171
11

PAP
112
9

PAP
222
11

PSM
361
11

PSM
461
9

PSM
461
10

PSM
461
11

PAP
231
8

PAP
231
11

Kallikrein
150
8

PSA
146
8

Kallikrein
150
11

PSA
146
11

PAP
291
8

PAP
291
10

PSM
575
9

PSM
575
11

PAP
145
9

PAP
145
10

PAP
145
11

PSM
738
9

PAP
292
9

PSA
9
8

PSA
9
9
0.1100

PSA
9
10
0.3600

PSM
558
8

PSM
558
9

PSM
624
9

PSM
624
10
3.2000

PSM
584
8

PSM
584
10

PSM
523
8

PSA
2
9
2.1000

PSA
2
10
0.0062

PSA
85
8

PAP
41
10
0.0005

PSM
201
9

PSM
372
10

PSA
68
9

PSA
68
10

PSM
225
10

PSM
225
11

PAP
363
11

PSM
690
11

PSM
27
8

PSM
27
9

PSM
27
11

PAP
30
8

PAP
30
11

Kallikrein
138
11

PSM
592
9

Kallikrein
222
8

PSA
218
8

PSM
603
9

PSM
603
10

PSM
660
9

PSM
660
10

PSM
660
11

PSA
56
8

Kallikrein
60
8

Kallikrein
53
9

PSA
49
9

PAP
262
10

PSA
134
11

Kallikrein
192
10

Kallikrein
192
11

PSA
188
11

PSM
352
8

PSM
352
11

PSA
8
9

PSA
8
10

PSA
8
11

PSA
1
10

PSA
1
11

PSM
394
9

Kallikrein
246
8

PSA
242
8

PSM
602
10

PSM
602
11

Kallikrein
73
8

Kallikrein
73
9

PSM
555
11

PAP
302
9
0.0320

Kallikrein
242
8

Kallikrein
242
11

PSM
175
11

PSA
10
8

PSA
10
9

PSM
20
9

PAP
25
11

Kallikrein
74
8

PSM
497
10

PSA
55
9

Kallikrein
59
9

PSM
234
9

PAP
319
8

PAP
319
11

PSM
449
8

PAP
84
9

PAP
84
10

PAP
103
11

PAP
155
10

PSM
537
10

Kallikrein
243
10

PSA
239
10

Kallikrein
243
11

PSA
239
11

PSM
460
10

PSM
460
11

PSM
371
11

PSM
176
10

PSM
176
11

PSM
299
8

PSM
299
11

PAP
330
11

[0522]

16

TABLE XI

Prostate B07 Supermotif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
B*0702

PSM
236
11

PSA
14
8

PSA
14
9
0.0007

PAP
4
8

PAP
4
9
0.0210

PAP
4
11

PSM
313
11

PSM
693
8

PSM
693
9
0.0003

PAP
351
9
0.0810

PAP
351
10
0.0054

PSM
230
10
0.0002

PAP
56
8

PSM
677
10
0.0001

PSM
677
11

PSM
266
9
0.0001

PAP
211
8

PAP
211
11

PSM
567
8

PSM
567
10
0.0001

PSM
567
11

PSM
387
8

PSM
387
9
0.0011

PSM
720
9
0.0002

PSA
124
8

PSA
124
9
0.0001

PSA
124
11

Kallikrein
128
8

Kallikrein
128
9

Kallikrein
128
11

Kallikrein
145
9

PSA
141
9

Kallikrein
145
10
0.0002

PSA
141
10
0.0002

Kallikrein
232
10

Kallikrein
232
11

PSA
228
11

PSM
367
8

Kallikrein
82
9

Kallikrein
82
11

Kallikrein
161
11

PSA
157
11

PSM
145
10
0.0001

PSM
705
8

PSM
705
9
0.0013

PSM
705
11

PSA
92
8

PSA
92
10
1.1000

PSA
92
11

Kallikrein
96
8

Kallikrein
96
10

Kallikrein
96
11

PAP
124
8

PAP
124
9
0.0001

PAP
53
11

PSM
330
8

Kallikrein
215
8

PSA
211
8

Kallikrein
215
9
0.0280

PSA
211
9
0.0280

PAP
361
8

PSA
78
8

PSA
78
9
0.0006

PSA
78
11

PSM
295
8

PSM
295
11

PSA
94
8

PSA
94
9
0.0018

Kallikrein
98
8

Kallikrein
98
9

PSM
124
8

PSM
618
8

PSM
618
10
0.0003

PSA
184
8

PSA
184
9
0.1700

PSA
184
10
0.0230

Kallikrein
56
8

PSA
52
8

Kallikrein
56
9
0.0240

PSA
52
9
0.0240

PAP
182
8

PAP
182
10
0.0150

PSM
80
11

PAP
364
10
0.0019

PAP
277
8

PAP
277
9
5.8000

PAP
277
10

PSM
292
8

PSM
292
9
0.0007

PSM
292
11

PAP
141
8

Kallikrein
239
8

Kallikrein
239
9

Kallikrein
239
11

PSM
681
10
0.0007

PSM
681
11

Kallikrein
236
8

Kallikrein
236
11

PSA
232
8

PSA
232
11

PSM
593
8

PSM
593
9
0.0011

PSM
593
10
0.0150

PSM
593
11

PAP
156
9
0.0049

PAP
344
10
0.0360

PSM
248
11

PAP
307
9
0.0029

PSM
289
9
0.0790

PSM
289
11

PAP
223
10
0.0032

Kallikrein
141
8

PSA
137
8

PSM
169
8

PSM
169
9
0.0001

PSM
169
11

PAP
133
9
0.0026

PAP
133
11

PSM
657
8

PSM
314
10
0.0012

PAP
125
8

PAP
125
11

PSM
159
11

PSM
148
10
0.0001

PSM
148
11

PSM
147
8

PSM
147
11

PSM
146
9
0.0001

PAP
308
8

PAP
308
11

PAP
139
8

PAP
139
10
0.2400

Kallikrein
36
8

PSA
32
8

Kallikrein
112
10

Kallikrein
112
11

PSM
684
8

PSM
684
9
0.4700

PSM
684
10
0.7200

PSA
108
10
0.0117

PSA
108
11

PSM
411
8

PSM
411
9
0.7800

PSM
411
11

Kallikrein
167
8

Kallikrein
167
10

PSM
17
9
0.3200

PSM
17
10
5.2000

PSM
17
11

PSA
235
8

PSA
235
9

PSA
235
11

PSM
483
11

PSM
503
10
0.0020

PAP
48
11

PSM
165
10
0.0002

PSM
165
11

PAP
348
9
0.0066

PAP
348
10
0.0002

PSM
501
9
0.0025

PSM
269
9
0.0012

PSM
269
10
0.0001

PSM
269
11

PSM
53
8

PSM
53
9
0.0990

PSM
53
10
0.0200

PSA
163
8

PSA
163
10
0.0006

PSM
467
8

PSM
467
11

Kallikrein
18
8

Kallikrein
18
9

PAP
146
8

PAP
146
9
0.0002

PAP
146
10
0.0011

PAP
146
11

Kallikrein
90
11

PSM
325
9
0.0039

PAP
63
8

PAP
63
11

PSM
272
8

PSM
549
8

PSM
549
11

PSM
119
9
0.0001

PSM
119
10
0.0035

[0523]

17

TABLE XII

Prostate B27 Supermotif with Binding Data

No. of

Protein
Position
Amino Acids

Kallikrein
48
8

PSA
60
9

PSA
60
10

PSA
60
11

Kallikrein
64
10

Kallikrein
64
11

PAP
288
9

PAP
288
11

PSM
111
9

PAP
32
9

PAP
32
10

PAP
32
11

PSM
222
11

Kallikrein
130
9

Kallikrein
130
10

PSM
93
8

PSM
93
11

PAP
9
8

PAP
9
10

PAP
9
11

Kallikrein
185
8

Kallikrein
185
11

PSM
15
9

PSM
15
11

PSM
180
9

PAP
313
8

PSM
597
8

PSM
597
11

PSM
609
8

PSM
654
8

PSM
654
10

PSM
654
11

PSM
683
8

PSM
683
9

PSM
683
10

PSM
683
11

PAP
46
8

PAP
27
9

PAP
27
11

PAP
110
8

PAP
110
11

PSM
563
8

PSM
563
10

PAP
321
9

PAP
321
10

PAP
321
11

Kallikrein
32
9

PSA
28
9

Kallikrein
32
10

Kallikrein
32
11

PSA
28
10

PSA
28
11

Kallikrein
238
9

Kallikrein
238
10

PAP
254
9

PAP
254
10

PAP
254
11

Kallikrein
190
8

Kallikrein
190
10

PSM
672
8

PSM
672
10

PAP
354
10

PAP
354
11

PSM
444
9

PSA
234
9

PSA
234
10

PSA
77
9

PSA
77
10

PSM
186
9

PSM
570
8

PSM
570
10

PSM
209
9

PSM
209
11

PAP
42
9

PAP
158
10

PSM
376
8

PSM
376
11

PSM
198
8

PSM
198
11

PAP
192
11

PSM
490
8

PSM
206
9

PSM
533
9

PSA
42
8

PSA
42
9

PSA
42
10

PAP
250
9

PSM
377
10

PAP
249
10

PSM
346
10

PSM
346
11

PAP
58
10

PSM
70
8

PSM
70
11

PSM
43
10

PAP
85
8

PAP
85
9

PSA
63
8

PSA
63
9

PAP
104
10

PAP
104
11

PSM
55
8

PSM
55
11

PSM
617
9

PSM
617
11

Kallikrein
33
8

PSA
29
8

Kallikrein
33
9

Kallikrein
33
10

Kallikrein
33
11

PSA
29
9

PSA
29
10

PSA
29
11

PSM
406
11

PSM
71
10

PAP
281
8

PSA
165
8

PSA
165
10

PSA
165
11

Kallikrein
68
8

PSM
499
8

PSM
499
11

PAP
272
9

PAP
179
9

PAP
179
10

PAP
179
11

PSM
729
8

PSM
729
9

PSM
729
11

PAP
87
11

PSM
5
8

PSM
5
9

PSM
5
11

PAP
197
9

PAP
197
10

Kallikrein
176
8

Kallikrein
176
10

PAP
181
8

PAP
181
9

PAP
181
11

PSA
172
8

PSA
172
10

PSM
65
9

PSM
65
10

PSM
65
11

PAP
35
8

Kallikrein
67
8

Kallikrein
67
9

PAP
172
10

PSM
481
8

PSM
323
11

PAP
235
9

PAP
235
10

PSM
362
10

PSM
362
11

PSM
604
8

PSM
604
9

PSM
604
11

PSA
120
9

Kallikrein
124
9

PSM
661
8

PSM
661
9

PSM
661
10

PSM
661
11

Kallikrein
111
11

PSA
107
11

Kallikrein
166
9

Kallikrein
166
11

PSM
462
8

PSM
462
9

PSM
462
10

PSM
344
9

PSM
58
8

PSM
58
10

PSM
616
10

PSM
192
9

PSM
192
10

PSM
192
11

PAP
271
10

PSM
622
8

PSM
622
11

PAP
1
8

PAP
1
9

PAP
1
11

PAP
269
9

PSM
544
8

PSM
544
9

PSM
121
8

PSM
121
10

PSM
121
11

PSM
212
8

PSM
212
9

PSM
212
11

PSM
698
8

PSM
698
11

PSM
81
10

PSA
93
9

PSA
93
10

Kallikrein
97
9

Kallikrein
97
10

PSM
54
8

PSM
54
9

PSA
164
9

PSA
164
11

PAP
162
11

PSM
412
8

PSM
412
10

Kallikrein
168
9

Kallikrein
168
11

PSM
18
8

PSM
18
9

PSM
18
10

PSM
18
11

PAP
336
8

PAP
336
9

PAP
77
8

PAP
77
9

PAP
252
11

PSM
303
11

PAP
178
10

PAP
178
11

PSA
186
8

PSA
186
10

PSM
254
8

PSM
254
1

PSM
526
10

Kallikrein
88
9

PAP
43
8

PAP
43
1

PAP
90
8

PAP
86
8

Kallikrein
250
8

PSA
246
8

Kallikrein
250
9

Kallikrein
250
10

PSA
246
9

PSA
246
10

PSM
605
8

PSM
605
10

PSM
280
8

PSM
280
10

PSM
16
8

PSM
16
10

PSM
16
11

PSM
413
9

PSM
413
11

Kallikrein
118
9

PSA
114
9

Kallikrein
44
10

Kallikrein
44
11

PSM
696
10

Kallikrein
93
8

PSA
89
8

Kallikrein
93
9

PSA
89
9

PSA
89
11

Kallikrein
93
11

PSM
722
11

PSM
644
10

PSM
513
9

PSM
513
11

PSM
717
8

PSM
717
9

PAP
207
8

PSA
40
10

PSA
40
11

PSM
439
9

PSM
439
10

PSM
439
11

PAP
256
8

PAP
256
9

PSM
123
8

PSM
123
9

PSM
478
11

PSA
189
10

PSM
498
9

PAP
233
9

PAP
233
11

PSM
538
9

Kallikrein
244
9

PSA
240
9

Kallikrein
244
10

PSA
240
10

PSM
353
10

PSM
395
8

PSM
395
11

PAP
218
9

PAP
218
10

PSM
474
8

PSM
294
9

PSA
183
9

PSA
183
10

PSA
183
11

Kallikrein
55
9

PSA
51
9

Kallikrein
55
10

PSA
51
10

PAP
143
11

Kallikrein
247
11

PSA
243
11

PSM
342
11

PSM
214
9

PSM
636
8

PSM
636
11

PSM
728
8

PSM
728
9

PSM
728
10

PSM
239
8

PSM
239
10

PSM
579
9

PSM
579
10

PSM
100
9

PSM
100
11

PSM
319
9

PSM
319
11

PSM
410
8

PSM
410
9

PSM
410
10

PSM
572
8

PSM
552
8

PSM
552
10

PSM
552
11

PAP
184
8

PAP
184
11

PAP
97
8

PAP
280
9

PAP
89
9

Kallikrein
249
9

PSA
245
9

Kallikrein
249
10

Kallikrein
249
11

PSA
245
10

PSA
245
11

PAP
331
10

PSM
279
8

PSM
279
9

PSM
279
11

[0524]

18

TABLE XIII

Prostate B58 Supermotif with Binding Data

No. of

Protein
Position
Amino Acids

PSM
741
9

PSM
741
10

PSM
742
8

PSM
742
9

PSM
735
8

PSM
735
9

PSA
59
10

PSA
59
11

Kallikrein
63
11

PAP
121
9

PAP
121
11

PSA
13
9

PSA
13
10

PAP
3
9

PAP
3
10

PAP
11
8

PAP
11
9

PAP
11
10

PAP
11
11

PSM
392
8

PSM
392
11

PAP
311
8

PAP
311
9

PAP
311
10

PSM
531
11

PSM
643
8

PSM
643
11

PAP
12
8

PAP
12
9

PAP
12
10

PAP
12
11

PSA
39
11

PSM
419
8

PSM
419
10

PSM
419
11

PSM
13
8

PSM
13
9

PSM
13
11

PAP
227
9

PAP
189
9

PSM
49
10

PAP
274
10

PAP
274
11

PSM
22
8

PSM
22
11

Kallikrein
234
8

Kallikrein
234
9

Kallikrein
234
10

PSA
230
9

PSA
230
10

PSA
180
9

Kallikrein
184
9

PSA
205
9

PSA
205
10

PSM
196
8

PSM
196
10

PAP
347
10

PAP
347
11

Kallikrein
14
8

PSM
466
8

PSM
466
9

PSM
422
8

PSM
710
10

PSM
301
9

PSA
130
8

Kallikrein
212
11

PSA
208
11

PSM
630
10

Kallikrein
116
8

PSA
112
8

Kallikrein
116
9

PSA
112
9

Kallikrein
116
11

PSA
112
11

PSM
453
8

PSM
453
10

PSM
316
8

PSM
316
10

PSM
106
8

PSM
106
10

PSM
106
11

PSM
379
8

Kallikrein
207
11

PAP
51
8

Kallikrein
85
8

PSA
81
8

PAP
230
9

PAP
290
9

PAP
290
10

PAP
290
11

PSM
48
11

PSM
285
8

PSM
285
10

PAP
168
10

PSM
703
9

PSM
703
10

PSM
703
11

PSM
716
9

PSM
716
10

PAP
60
8

PAP
60
11

PAP
216
8

PAP
216
11

PAP
95
8

PAP
95
9

PAP
95
10

PSM
7
9

PAP
170
8

PSM
542
8

PSM
542
10

PSM
542
11

PAP
334
9

PAP
334
10

PAP
334
11

PSM
557
9

PSM
557
10

PAP
356
8

PAP
356
9

PSM
235
8

PSM
418
9

PSM
418
11

PSM
161
9

PSM
633
10

PSM
633
11

PSM
646
8

PSM
506
10

PSM
546
10

PSM
546
11

PSM
164
11

PSM
337
8

PSM
337
10

PSM
639
8

PSM
333
10

PSM
77
8

PSM
737
10

PSA
12
10

PSA
12
11

PSM
391
8

PSM
391
9

PSM
263
10

PSM
221
8

PSM
24
9

PSM
24
10

PSM
24
11

PSM
364
8

PSM
364
9

PSM
364
10

PSM
364
11

Kallikrein
16
10

Kallikrein
16
11

PSM
311
9

PSM
516
8

PSM
516
9

PSM
516
10

Kallikrein
158
8

PSA
154
8

Kallikrein
158
9

PSA
154
9

PSM
321
9

PSM
85
8

PSM
85
9

PSM
403
8

Kallikrein
149
9

PSA
145
9

Kallikrein
94
8

PSA
90
8

PSA
90
10

Kallikrein
94
10

Kallikrein
34
8

Kallikrein
34
9

Kallikrein
34
10

PSA
30
8

PSA
30
9

PSA
30
10

PSM
347
9

PSM
347
10

PSM
553
9

PSM
553
10

PAP
144
10

PAP
144
11

PSM
283
10

Kallikrein
8
10

Kallikrein
8
11

PSM
202
8

PSM
530
8

PSM
642
9

PAP
188
10

PSM
128
11

PSM
512
10

PSM
614
9

PSA
175
9

Kallikrein
132
8

Kallikrein
132
10

PSM
52
9

PSM
52
10

PSM
52
11

Kallikrein
226
10

Kallikrein
226
11

PSA
222
10

PSA
222
11

PSM
66
8

PSM
66
9

PSM
66
10

PSM
59
9

PSM
723
10

PSM
723
11

PAP
173
9

PSM
655
9

PSM
655
10

PSM
500
10

PAP
255
8

PAP
255
9

PAP
255
10

PSM
44
9

PSA
66
11

PSM
240
9

PSM
122
9

PSM
122
10

PSM
623
10

PSM
623
11

PAP
120
10

PSM
219
8

PSM
219
9

PSM
219
10

PSM
28
8

PSM
28
10

PSM
28
11

PSM
83
8

PSM
83
10

PSM
83
11

PSM
110
8

PSM
110
10

PAP
31
8

PAP
31
10

PAP
31
11

PSM
92
9

PSM
587
9

PAP
8
9

PAP
8
11

PAP
148
8

PAP
148
9

PAP
148
11

PAP
238
9

PAP
238
10

PSA
122
10

PSA
122
11

Kallikrein
126
10

Kallikrein
126
11

PAP
194
9

PAP
194
10

PAP
14
8

PAP
14
9

PAP
14
10

PAP
14
11

PAP
241
9

Kallikrein
179
9

Kallikrein
179
10

PSA
18
8

Kallikrein
10
8

Kallikrein
10
9

Kallikrein
10
11

PSA
6
8

PSA
6
9

PSA
6
11

PSM
117
11

PSA
128
8

PSA
128
10

PAP
315
11

PSA
4
8

PSA
4
10

PSA
4
11

PSM
268
10

PSM
268
11

PSA
162
9

PSA
162
11

PAP
70
10

PSM
574
10

PSM
574
11

PAP
298
9

PAP
298
10

PAP
114
8

PAP
114
9

PAP
114
10

PAP
114
11

Kallikrein
103
9

PSA
99
8

PSA
99
9

PAP
232
10

PAP
117
8

PSM
451
10

PSM
216
10

PSM
216
11

Kallikrein
70
11

PSM
438
10

PSM
438
11

PSM
231
9

PSA
125
8

PSA
125
10

PSA
125
11

Kallikrein
129
8

Kallikrein
129
10

Kallikrein
129
11

Kallikrein
146
8

PSA
142
8

Kallikrein
146
9

PSA
142
9

PSM
273
11

Kallikrein
240
8

Kallikrein
240
10

PAP
349
8

PAP
349
9

PAP
349
11

PSM
290
8

PSM
290
10

PSM
290
11

PSM
721
8

PSA
236
8

PSA
236
10

PSM
502
8

PSM
502
11

PSM
694
8

PAP
224
9

PAP
278
8

PAP
278
9

PAP
278
11

PAP
54
10

PSM
740
10

PSM
740
11

PSM
389
10

PSM
389
11

PSM
97
9

Kallikrein
22
8

PAP
2
8

PAP
2
10

PAP
2
11

PAP
10
9

PAP
10
10

PAP
10
11

PSM
673
9

PSM
534
8

PAP
273
8

PAP
273
11

PSA
43
8

PSA
43
9

Kallikrein
186
10

Kallikrein
186
11

PSM
400
11

Kallikrein
169
8

Kallikrein
169
10

Kallikrein
169
11

PAP
105
9

PAP
105
10

PAP
28
8

PAP
28
10

PAP
28
11

PSM
181
8

PSM
414
8

PSM
414
10

PAP
111
10

PAP
111
11

PSM
162
8

PAP
287
10

PAP
115
8

PAP
115
9

PAP
115
10

PSM
312
8

PSM
10
11

PSM
634
9

PSM
634
10

Kallikrein
117
8

PSA
113
8

Kallikrein
117
10

PSA
113
10

PSM
695
11

PSM
454
9

PSM
454
11

PSM
45
8

PAP
61
10

PSM
317
9

PSM
317
11

PSA
203
11

PAP
106
8

PAP
106
9

PAP
106
11

PSM
431
11

PSM
348
8

PSM
348
9

PSM
348
11

PSM
338
9

PSA
58
11

PSM
14
8

PSM
14
10

PSM
141
11

Kallikrein
227
9

Kallikrein
227
10

PSA
223
9

PSA
223
10

Kallikrein
150
8

PSA
146
8

Kallikrein
150
11

PSA
146
11

PAP
291
8

PAP
291
9

PAP
291
10

PSM
734
8

PSM
734
9

PSM
734
10

PSM
576
8

PSM
576
9

PSM
576
10

PSA
38
8

PSM
12
9

PSM
12
10

Kallikrein
40
8

Kallikrein
40
9

PSM
447
10

PSM
154
8

PSM
154
10

PSM
154
11

PSM
627
9

PSM
627
10

PAP
293
8

PAP
293
10

PAP
293
11

Kallikrein
92
9

PSA
88
9

Kallikrein
92
10

PSA
88
10

PAP
129
8

PAP
129
9

PAP
129
10

Kallikrein
174
10

Kallikrein
192
8

Kallikrein
192
10

Kallikrein
192
11

PSA
188
8

PSA
188
11

PSM
352
8

PSM
352
11

PSA
8
9

PSA
8
10

PSA
8
11

PSM
434
8

PSM
434
9

Kallikrein
47
8

Kallikrein
47
9

PAP
226
10

PAP
206
8

PAP
206
9

PSM
497
10

PSM
607
8

PSM
607
10

PSM
700
9

PSM
700
10

PSM
692
9

PSM
692
10

PSM
179
8

PSM
179
10

PAP
310
9

PAP
310
10

PAP
310
11

Kallikrein
153
8

PSA
149
8

PSM
600
8

PSM
600
9

PSM
277
8

PSM
277
10

PSM
277
11

PAP
286
8

PAP
286
11

PSM
228
8

PSM
228
9

Kallikrein
188
8

Kallikrein
188
9

Kallikrein
188
10

Kallikrein
43
11

PSM
612
11

PSM
471
11

PSM
625
8

PSM
625
9

PSM
625
11

PSM
537
10

Kallikrein
243
10

PSA
239
10

Kallikrein
243
11

PSA
239
11

PSM
460
10

PSM
460
11

[0525]

19

TABLE XIV

Prostate B62 Supermotif with Binding Data

No. of

Protein
Position
Amino Acids

PAP
299
8

PAP
299
9

PSM
711
9

PAP
122
8

PAP
122
10

PAP
122
11

Kallikrein
147
8

PSA
143
8

Kallikrein
147
11

PSA
143
11

Kallikrein
235
8

Kallikrein
235
9

PSA
231
8

PSA
231
9

Kallikrein
9
9

Kallikrein
9
10

PSM
25
8

PSM
25
9

PSM
25
10

PSM
25
11

PAP
116
8

PAP
116
9

PSM
236
11

PSA
14
8

PSA
14
9

PAP
4
8

PAP
4
9

PAP
4
11

PSM
313
11

PSM
693
8

PSM
693
9

PSM
302
8

PSM
217
9

PSM
217
10

PSM
217
11

PSA
181
8

PSA
181
11

PSM
577
8

PSM
577
9

PSM
577
11

PSM
11
10

PSM
11
11

PSA
44
8

PSM
365
8

PSM
365
9

PSM
365
10

PSM
286
9

PSM
635
8

PSM
635
9

Kallikrein
17
9

Kallikrein
17
10

PSM
393
10

PSM
601
8

PSM
601
11

Kallikrein
41
8

Kallikrein
241
9

PSA
62
8

PSA
62
9

PSA
62
10

Kallikrein
66
8

Kallikrein
66
9

Kallikrein
66
10

PAP
351
9

PAP
351
10

PSA
169
11

Kallikrein
173
11

PSM
714
11

PSM
156
8

PSM
156
9

PAP
201
9

PAP
201
10

PSA
171
9

PSA
171
11

Kallikrein
120
11

PSA
116
11

PSA
136
8

PSA
136
9

Kallikrein
3
8

Kallikrein
3
10

PSM
173
8

Kallikrein
182
11

PSM
191
10

PSM
191
11

PSA
98
9

PSA
98
10

PSM
230
10

PAP
56
8

PSM
677
10

PSM
677
11

PSM
266
9

PAP
211
8

PAP
211
11

PSM
567
8

PSM
567
10

PSM
567
11

PSM
387
8

PSM
387
9

PSM
720
9

PAP
151
8

PSM
666
9

PSM
666
10

PSM
666
11

PSA
178
11

PAP
108
9

PAP
108
10

Kallikrein
134
8

PAP
301
10

PAP
301
11

PSM
641
10

PSM
137
8

PAP
266
9

PSM
397
9

PSM
109
8

PSM
109
9

PSM
109
11

PSM
586
8

PSM
586
10

PAP
80
10

PSM
64
10

PSM
64
11

PAP
34
8

PAP
34
9

PSM
480
9

PAP
237
8

PAP
237
10

PAP
237
11

PAP
240
8

PAP
240
10

PSA
127
8

PSA
127
9

PSA
127
11

PSM
560
10

PSM
560
11

PAP
358
11

PAP
317
9

PAP
317
10

PAP
317
11

PSM
621
9

PSA
124
8

PSA
124
9

PSA
124
11

Kallikrein
128
8

Kallikrein
128
9

Kallikrein
128
11

Kallikrein
145
9

PSA
141
9

Kallikrein
145
10

PSA
141
10

Kallikrein
232
10

Kallikrein
232
11

PSA
228
11

PSM
367
8

Kallikrein
82
9

Kallikrein
82
11

Kallikrein
161
11

PSA
157
11

PSM
145
10

PAP
76
9

PAP
76
10

PSM
87
10

PAP
100
10

PSM
522
9

PSM
522
10

PSM
727
8

PSM
727
9

PSM
727
10

PSM
727
11

PSM
351
8

PSM
351
9

PAP
187
8

PAP
187
11

PSM
42
8

PSM
42
11

PSM
61
10

PSM
670
10

PAP
18
8

PAP
18
9

PAP
20
11

PSM
33
9

PSM
33
10

PSM
33
11

PAP
92
11

Kallikrein
165
10

PSA
3
8

PSA
3
9

PSA
3
11

PSA
161
10

PSM
73
8

PSM
73
11

Kallikrein
195
8

PSA
191
8

PSM
705
8

PSM
705
9

PSM
705
11

PSA
92
8

PSA
92
10

PSA
92
11

Kallikrein
96
8

Kallikrein
96
10

Kallikrein
96
11

PAP
124
8

PAP
124
9

PAP
53
11

PAP
164
9

PAP
177
8

PAP
177
11

PSM
90
11

PSM
525
11

PSA
86
11

PSM
282
8

PSM
282
11

PSM
529
9

PSM
385
8

PSM
385
9

PSM
385
10

PSM
385
11

PAP
248
11

Kallikrein
225
11

PSA
221
11

PAP
204
10

PAP
204
11

PSM
707
9

PSM
104
8

PSM
104
10

PAP
196
8

PAP
196
10

PAP
196
11

PSM
427
8

PSM
427
9

PAP
305
11

PSM
680
8

PSM
680
11

PSM
288
10

Kallikrein
140
8

Kallikrein
140
9

PAP
295
8

PAP
295
9

PAP
74
8

PAP
74
11

PSM
168
8

PSM
168
9

PSM
168
10

PSM
508
8

PSM
582
10

PSM
582
11

PSM
330
8

Kallikrein
215
8

PSA
211
8

Kallikrein
215
9

PSA
211
9

PAP
361
8

PAP
199
8

PAP
199
11

PAP
68
8

Kallikrein
87
10

PSA
83
10

PSM
446
11

PSM
224
9

PSM
224
11

PSM
238
9

PSM
238
11

Kallikrein
221
9

PSA
217
9

Kallikrein
52
8

PSA
48
8

Kallikrein
52
9

PSA
48
9

Kallikrein
52
10

PSA
48
10

PAP
261
8

PAP
261
11

PSM
252
8

PSM
252
10

PAP
128
8

PAP
128
9

PAP
128
10

PAP
128
11

PSM
345
8

PSM
345
11

PSM
82
9

PSM
82
11

Kallikrein
177
9

Kallikrein
177
11

PSM
573
11

PAP
270
8

PAP
270
11

PSA
78
8

PSA
78
9

PSA
78
11

PSM
295
8

PSM
295
11

PSA
94
8

PSA
94
9

Kallikrein
98
8

Kallikrein
98
9

PSM
124
8

PSM
618
8

PSM
618
10

PSA
184
8

PSA
184
9

PSA
184
10

Kallikrein
56
8

PSA
52
8

Kallikrein
56
9

PSA
52
9

PAP
182
8

PAP
182
10

PSA
173
9

PSA
173
11

PSM
130
9

PSM
130
10

PSM
416
8

PSM
416
11

PSM
373
9

PSM
373
10

PSM
373
11

PSA
69
8

PSA
69
9

PAP
135
9

PAP
267
8

PAP
267
11

PSM
258
11

PSA
17
9

PSM
226
9

PSM
226
10

PSM
226
11

PAP
284
10

PSM
80
11

PAP
364
10

PAP
277
8

PAP
277
9

PAP
277
10

PSM
292
8

PSM
292
9

PSM
292
11

PAP
141
8

PSM
96
8

PSM
96
10

Kallikrein
21
9

PSM
200
9

PSM
200
10

PSM
591
10

PSM
591
11

PSM
659
10

PSM
659
11

PSM
157
8

PSM
398
8

PSM
193
8

PSM
193
9

PSM
193
10

PSM
193
11

Kallikrein
131
8

Kallikrein
131
9

Kallikrein
131
11

PSM
199
10

PSM
199
11

PSM
187
8

PSM
514
8

PSM
514
10

PSM
514
11

PSM
304
10

PSA
166
9

PSA
166
10

PAP
234
8

PAP
234
10

PAP
234
11

PAP
193
10

PAP
193
11

PSM
343
10

Kallikrein
239
8

Kallikrein
239
9

Kallikrein
239
11

PSM
94
10

PAP
251
8

PSM
718
8

PSM
718
11

PSM
207
8

PSM
207
11

PSM
213
8

PSM
213
10

Kallikrein
137
11

PSA
133
11

PSM
324
10

Kallikrein
191
9

Kallikrein
191
11

PSA
187
9

Kallikrein
245
8

PSA
241
8

Kallikrein
245
9

PSA
241
9

PAP
208
11

PSA
16
10

PAP
283
11

Kallikrein
20
10

PAP
7
8

PAP
7
10

PSM
305
9

PAP
21
10

PAP
21
11

PSM
34
8

PSM
34
9

PSM
34
10

PSA
70
8

PSM
428
8

PSM
4
8

PSM
4
9

PSM
4
10

PAP
6
9

PAP
6
11

PAP
306
10

PSM
441
8

PSM
441
9

PSM
441
10

Kallikrein
123
8

PSA
119
8

PSA
119
10

Kallikrein
123
10

Kallikrein
178
8

Kallikrein
178
10

Kallikrein
178
11

PAP
136
8

PAP
136
11

PSM
668
8

PSM
668
9

Kallikrein
121
10

PSA
117
10

PAP
113
8

PAP
113
9

PAP
113
10

PAP
113
11

PSM
469
9

PSM
681
10

PSM
681
11

Kallikrein
236
8

Kallikrein
236
11

PSA
232
8

PSA
232
11

PSM
593
8

PSM
593
9

PSM
593
10

PSM
593
11

PAP
156
9

PAP
344
10

PSM
248
11

PAP
307
9

PSM
289
9

PSM
289
11

PAP
223
10

Kallikrein
141
8

PSA
137
8

PSA
167
8

PSA
167
9

Kallikrein
171
8

Kallikrein
171
9

PSM
650
10

PSM
650
11

PSM
442
8

PSM
442
9

PSM
442
11

PAP
258
10

PAP
258
11

PAP
296
8

PAP
296
11

PSA
37
8

PSA
37
9

Kallikrein
217
10

PSA
213
10

PSM
561
9

PSM
561
10

PAP
40
11

PAP
359
10

PSM
473
9

Kallikrein
54
8

PSA
50
8

Kallikrein
54
10

PSA
50
10

Kallikrein
54
11

PSA
50
11

PSM
26
8

PSM
26
9

PSM
26
10

Kallikrein
4
9

PAP
263
9

Kallikrein
122
9

PSA
118
9

PSA
118
11

Kallikrein
122
11

PAP
343
11

PSM
663
8

PSM
663
9

PSM
169
8

PSM
169
9

PSM
169
11

PSM
583
9

PSM
583
10

PSM
583
11

PSM
69
9

PSM
257
8

PSM
51
8

PSM
51
10

PSM
51
11

PAP
119
11

PSM
3
9

PSM
3
10

PSM
3
11

PSM
260
9

PSM
57
9

PSM
57
11

Kallikrein
102
10

PAP
133
9

PAP
133
11

PSM
657
8

PSM
328
10

PSM
357
9

PSM
357
10

PSM
153
9

PSM
153
11

PAP
49
10

PSM
296
10

PSM
296
11

PAP
57
11

PAP
134
8

PAP
134
10

PAP
140
9

PSM
658
11

PAP
352
8

PAP
352
9

PSM
678
9

PSM
678
10

PSA
15
8

PSA
15
11

Kallikrein
19
8

Kallikrein
19
11

PAP
5
8

PAP
5
10

PSM
468
10

PAP
147
8

PAP
147
9

PAP
147
10

PSM
267
8

PSM
267
11

Kallikrein
216
8

PSA
212
8

Kallikrein
216
11

PSA
212
11

PAP
212
10

PSA
95
8

PSM
550
10

Kallikrein
99
8

PSM
568
9

PSM
568
10

PSM
314
10

PAP
125
8

PAP
125
11

PSM
159
11

PSM
148
10

PSM
148
11

PSM
147
8

PSM
147
11

PSM
146
9

PAP
308
8

PAP
308
11

PAP
365
9

PSM
619
9

PSM
619
11

PAP
64
10

PSM
166
9

PSM
166
10

PSM
166
11

PSA
185
8

PSA
185
9

PSA
185
11

PSM
388
8

PSM
388
11

Kallikrein
57
8

PSA
53
8

PSA
53
11

Kallikrein
57
11

PSM
293
8

PSM
293
10

Kallikrein
91
10

Kallikrein
91
11

PAP
276
8

PAP
276
9

PAP
276
10

PAP
276
11

PSM
95
9

PSM
95
11

PSM
731
9

PSM
731
11

PSM
218
8

PSM
218
9

PSM
218
10

PSM
218
11

PSM
91
10

PAP
72
8

PAP
72
10

PSM
667
8

PSM
667
9

PSM
667
10

PAP
69
11

PAP
297
10

PAP
297
11

PAP
139
8

PAP
139
10

Kallikrein
36
8

PSA
32
8

Kallikrein
39
9

Kallikrein
39
10

PSA
84
9

PSA
182
10

PSA
182
11

PSA
35
10

PSA
35
11

PSM
578
8

PSM
578
10

PSM
578
11

PSA
87
10

PSA
87
11

Kallikrein
72
9

Kallikrein
72
10

PAP
101
9

PSM
511
11

PSM
354
9

PSM
527
9

PSM
527
11

PAP
180
8

PAP
180
9

PAP
180
10

PSM
440
8

PSM
440
9

PSM
440
10

PSM
440
11

PSM
649
11

PAP
257
8

PAP
257
11

PSA
121
8

PSA
121
11

Kallikrein
125
8

Kallikrein
125
11

PSM
662
8

PSM
662
9

PSM
662
10

Kallikrein
112
10

Kallikrein
112
11

PSM
684
8

PSM
684
9

PSM
684
10

PSA
108
10

PSA
108
11

PSM
411
8

PSM
411
9

PSM
411
11

Kallikrein
167
8

Kallikrein
167
10

PSM
17
9

PSM
17
10

PSM
17
11

PSA
235
8

PSA
235
9

PSA
235
11

PSM
730
8

PSM
730
10

PSM
463
8

PSM
463
9

PSM
463
11

Kallikrein
89
8

Kallikrein
7
11

PSM
455
8

PSM
455
10

Kallikrein
159
8

PSA
155
8

PSM
129
10

PSM
129
11

PSM
291
9

PSM
291
10

PSM
613
10

PSM
590
11

PAP
130
8

PAP
130
9

PSM
142
10

PSA
75
11

PSM
631
9

PAP
15
8

PAP
15
9

PAP
15
10

PAP
15
11

Kallikrein
175
9

Kallikrein
175
11

PSM
322
8

Kallikrein
104
8

PSA
100
8

PAP
242
8

Kallikrein
170
9

Kallikrein
170
10

PAP
13
8

PAP
13
9

PAP
13
10

PAP
13
11

PSM
472
10

PSA
237
9

PSM
615
8

PSM
615
11

PSM
483
11

PSM
503
10

PAP
48
11

PSM
165
10

PSM
165
11

PAP
348
9

PAP
348
10

PSM
501
9

Kallikrein
35
8

Kallikrein
35
9

PSA
31
8

PSA
31
9

Kallikrein
71
10

Kallikrein
71
11

PSM
98
8

PSM
98
11

PSM
107
9

PSM
107
10

PSM
107
11

Kallikrein
11
8

Kallikrein
11
10

Kallikrein
11
11

PAP
217
10

PAP
217
11

PSA
67
10

PSA
67
11

PAP
29
9

PAP
29
10

PSM
626
8

PSM
626
10

PSM
626
11

PSA
7
8

PSA
7
10

PSA
7
11

PSM
554
8

PSM
554
9

PSM
415
9

PAP
190
8

PAP
171
11

PAP
112
9

PAP
112
10

PAP
112
11

PAP
222
11

PSM
361
11

PSM
461
9

PSA
68
10

PSM
225
8

PSM
225
10

PSM
225
11

PAP
363
11

PSA
174
8

PSA
174
10

PSM
690
11

PSM
27
8

PSM
27
9

PSM
27
11

PAP
30
8

PAP
30
9

PAP
30
11

Kallikrein
138
10

Kallikrein
138
11

PSM
592
9

PSM
592
10

PSM
592
11

Kallikrein
222
8

PSA
218
8

PSM
603
9

PSM
603
10

PSM
660
9

PSM
660
10

PSM
660
11

Kallikrein
5
8

PSA
56
8

Kallikrein
60
8

PSA
36
9

PSA
36
10

Kallikrein
53
8

PSA
49
8

Kallikrein
53
9

PSA
49
9

Kallikrein
53
11

PSA
49
11

PAP
262
10

PSA
134
10

PSA
134
11

Kallikrein
18
8

Kallikrein
18
9

PAP
146
8

PAP
146
9

PSM
461
10

PSM
461
11

PSA
5
9

PSA
5
10

PAP
231
8

PAP
231
11

PSM
269
9

PSM
269
10

PSM
269
11

PSM
53
8

PSM
53
9

PSM
53
10

PSA
163
8

PSA
163
10

PSM
467
8

PSM
467
11

Kallikrein
143
11

PSA
139
11

PAP
335
8

PAP
335
9

PAP
335
10

PAP
275
9

PAP
275
10

PAP
275
11

PSM
339
8

PAP
71
9

PAP
71
11

PSM
575
9

PSM
575
10

PSM
575
11

PAP
145
9

PAP
145
10

PAP
145
11

PSM
738
9

PAP
292
8

PAP
292
9

PAP
292
11

PSM
201
8

PSM
201
9

PSM
358
8

PSM
358
9

PSM
372
10

PSM
372
11

PSA
68
9

PAP
146
10

PAP
146
11

Kallikrein
90
11

PSM
325
9

PSM
739
8

PSM
739
11

PSM
253
9

PSA
1
8

PSA
1
10

PSA
1
11

PSM
394
9

Kallikrein
246
8

PSA
242
8

PSM
602
10

PSM
602
11

PSA
10
8

PSA
10
9

Kallikrein
252
8

PSA
248
8

PSM
20
8

PSM
20
9

PSM
20
10

PAP
25
8

PAP
25
11

Kallikrein
74
8

PAP
63
8

PAP
63
11

PAP
138
9

PAP
138
11

Kallikrein
38
10

Kallikrein
38
11

PSA
34
11

PSA
55
9

Kallikrein
59
9

PSM
449
8

PAP
84
9

PAP
84
10

PAP
103
11

PAP
155
10

PSM
272
8

PSM
549
8

PSM
549
11

PSM
119
9

PSM
119
10

PSM
733
9

PSM
733
10

PSM
733
11

PSM
371
11

PSM
176
10

PSM
176
11

[0526]

20

TABLE XV

Prostate A01 Motif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
A*0101

PSM
452
9

PSM
220
9

PSM
264
9
0.0099

PSM
701
9
0.0040

PSM
693
8

PAP
311
9
0.7700

PSM
597
11

PSM
196
10
0.0160

PSM
453
8

PSM
106
8

PSM
599
9

PSM
171
9
0.0024

PSM
109
11

PAP
237
11

PAP
240
8

Kallikrein
145
9
0.0011

PSA
141
9
0.0011

PAP
95
9
0.0980

PSM
542
8

PSM
542
11

PSM
557
10
0.0260

PSM
546
11

PSM
565
8

PSM
702
8

PSM
487
8

PSM
529
9
0.0025

PSM
104
10
0.4800

PAP
74
11

PSM
168
9
0.0001

PAP
270
11

Kallikrein
94
8
0.0260

PSA
90
8
0.0260

Kallikrein
34
10

PSM
347
10
0.0048

PSM
112
8

PSM
530
8

PSM
346
11

PSM
450
11

PAP
277
10
0.5700

PAP
205
10
0.0012

PSM
691
10

PSM
66
10
0.0001

PSM
545
8

PAP
322
9
3.4000

PAP
322
10
0.0180

Kallikrein
33
11

Kallikrein
239
11

PAP
272
9
0.0011

PSM
699
11

PSM
105
9

PSM
143
9
0.0010

PAP
81
9
0.7800

PSM
65
11

Kallikrein
178
11

PAP
93
11

Kallikrein
236
8

PSA
232
8
0.0002

PSM
289
11

PSM
442
8

PAP
148
8

PAP
238
10
12.0000

Kallikrein
179
10

PSM
117
11

PAP
315
11

PSM
268
10
0.0082

PAP
70
10
0.6200

PSM
227
8

PSM
169
8

PSM
169
11

PSM
451
10
0.4300

PSM
195
11

PAP
94
10
0.0033

PSM
262
11

PSM
540
10

Kallikrein
233
11

PSA
229
11

PSM
484
11

PAP
147
9
1.2000

PSM
290
10

PSM
290
11

PSA
236
10
0.0010

PAP
278
9
0.0031

Kallikrein
91
11

PAP
309
11

PSM
218
11

PSA
87
11

PSM
363
9
0.0001

PSM
320
8

PAP
332
9
0.0002

PSA
235
11

PSM
463
9
11.0000

PAP
174
11

Kallikrein
93
9
0.0011

PSA
89
9
0.0011

PSM
615
11

Kallikrein
180
9

PSM
317
11

PSM
348
9
0.0430

PSM
349
8

Kallikrein
143
11
0.0190

PSA
139
11
0.0190

PSM
141
11

PSM
558
9
0.0010

PAP
293
11

Kallikrein
92
10
0.1500

PSA
88
10
0.1500

PSM
725
9
0.0010

PAP
206
9
0.0046

PAP
310
10
0.5500

PSM
234
9

PSM
552
8

PSM
272
8

[0527]

21

TABLE XVI

Prostate A03 Motif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
A*0301

PSM
741
10

PSM
742
9

PSM
735
8

PSM
735
9

PSA
59
8

PSA
13
8

PAP
3
8

PAP
3
9

PAP
3
10

PAP
11
8

PAP
11
10

PSM
392
9

PSM
392
11

PSM
608
10

PSM
608
11

PSM
452
9

PSM
232
9
0.0006

PSM
232
11

PSM
674
11

PSM
60
8

PSM
736
8

PSM
220
9

PSM
23
10

PSM
23
11

PSM
264
9

PSM
264
11

PSM
701
9

PSM
701
11

PSM
29
9

PSM
29
11

Kallikrein
199
8

PSA
195
8

PSM
84
10

PSM
84
11

PSM
711
8

Kallikrein
147
8

PSA
143
8

Kallikrein
235
9

Kallikrein
235
11

PSA
231
9
0.0170

PSA
231
11

Kallikrein
9
9

PSM
25
8

PSM
25
9

PAP
116
9

PAP
311
9
0.0002

PAP
311
10

PSM
531
9
0.0086

PSM
643
11

PAP
12
9

PSM
419
8

PSM
13
11

PAP
227
8
0.0003

PAP
227
10

PAP
189
10

PSM
49
8

PSM
49
11

PAP
274
8
0.0180

PAP
274
9
0.1000

PSM
11
9

PSA
44
9

PSM
286
10

PSM
635
9

PSM
635
11

Kallikrein
17
8

PSM
393
8

PSM
393
10

PSM
601
8

PSM
601
10
0.0026

Kallikrein
41
8

Kallikrein
41
9

Kallikrein
241
8

Kallikrein
241
9

Kallikrein
241
10

Kallikrein
241
11

PSM
22
8

PSM
22
11

Kallikrein
198
9

PSA
194
9
0.0006

Kallikrein
234
8

Kallikrein
234
10

PSA
230
10

PSA
180
8

PSA
180
11

Kallikrein
184
8

PSM
196
8

PSM
196
9

PSM
196
10
0.0600

PAP
347
9
0.0040

PAP
347
10

PAP
347
11

Kallikrein
14
11

PSM
466
10

PSM
710
9
0.0006

PSM
301
8

PSM
596
10

PSM
596
11

PSM
465
11

PSA
111
11

PSM
652
11

PSM
520
8

PSM
184
10

PAP
186
8

PSM
134
11

PSM
714
10
0.0003

PSM
714
11

PSM
156
8

PSM
156
9

PAP
201
8

PAP
201
10

PSA
171
11

Kallikrein
120
11

PSA
116
11

PSA
136
8

PSM
173
8

PSM
173
9

Kallikrein
182
10

PSM
191
9

PSA
98
8
0.0003

PSA
98
9

PSA
98
11

PSM
9
8

PSM
9
9

PSM
9
11

PSM
630
8

PSM
630
10

Kallikrein
116
10

PSA
112
10

PSM
453
8

PSM
453
11

PSM
316
9
0.0032

PSM
106
8

PAP
51
9
0.0001

Kallikrein
85
10

PSA
81
10

PAP
290
10

PSA
178
10
0.0007

PAP
108
9

PSM
114
9
0.0006

PSM
114
11

PAP
301
10

PAP
301
11

PSM
48
8

PSM
48
9

PSM
285
11

PAP
371
8

PSM
183
8

PSM
183
11

PAP
150
9

PAP
150
10

Kallikrein
115
11

Kallikrein
84
11

PSA
80
11

PAP
229
8

PSM
102
10

PSM
102
11

PSM
425
11

PAP
176
9

PAP
176
10

PSM
505
10

PSM
171
9

PSM
171
10

PSM
171
11

PSM
486
9

PSM
489
11

PSM
408
11

PSM
641
9
0.0006

PSM
137
8

PAP
266
8

PAP
266
9

PSM
397
10

PSM
397
11

PSM
109
11

PSM
586
10

PAP
166
8

PAP
80
8

PAP
80
9

PAP
80
10

PAP
80
11

PSM
64
8

PSM
64
9

PSM
64
10

PAP
34
9

PAP
34
10
0.0014

PAP
23
11

PSM
383
10

PSM
383
11

PAP
203
8

PSM
103
9

PSM
103
10

PSM
103
11

PSM
426
10

PSM
402
10

PSM
39
11

PSM
675
10

PSM
42
8

PSM
61
11

PSM
37
8

PAP
18
11

PAP
20
9
0.0024

PSM
33
10

PAP
92
8

PSA
106
10

PSA
3
11

PSM
73
10
0.0102

PSM
633
11

PSM
646
8

PSM
646
10
0.0003

PSM
506
9

PSM
546
8

PSM
546
11

PSM
337
9

PSM
337
11

PSM
639
8

PSM
639
11

PSM
333
9

PSM
333
11

PSM
77
8

PAP
37
8

PAP
37
11

PSA
12
9
0.0150

PSM
391
10

PSM
263
10

PSM
221
8

PSM
24
9

PSM
24
10

PSM
364
8

Kallikrein
16
9

PAP
346
10

PAP
346
11

PSM
172
8

PSM
172
9

PSM
172
10

PSM
265
8

PSM
265
10

PAP
45
9

PSM
487
8

PSM
31
9
0.0005

PSM
36
9
0.0007

PAP
17
8

PSM
332
10

PSM
30
8

PSM
30
10

PSM
375
9

PSM
384
9

PSM
384
10

PSM
581
8

PSM
310
11

PAP
260
11

Kallikrein
27
8

PSA
23
8

PSM
529
8

PSM
529
9

PSM
529
11

PSM
385
8

PSM
385
9

PAP
248
8

PAP
248
10

Kallikrein
225
11

PSA
221
11

PAP
204
11

PSM
104
8

PSM
104
9

PSM
104
10

PAP
196
8

PSM
427
9

PAP
305
10

PSM
680
8

PSM
680
9
0.0460

PSM
680
10

PSM
288
8

Kallikrein
140
8

PAP
295
9

PAP
74
11

PSM
168
9
0.0007

PSM
311
10
0.0006

PSA
226
10

PSM
516
9

PSM
516
10

Kallikrein
158
8

PSA
154
8

Kallikrein
158
10

PSM
430
11

PSM
85
9

PSM
85
10

PSM
403
9

PSM
403
11

PSM
360
11

PSM
224
9

PSM
224
11

PAP
261
10

Kallikrein
49
8

PAP
289
11

PAP
44
10

PAP
198
11

PSM
345
10

PSM
82
9

Kallikrein
177
9

Kallikrein
177
10

Kallikrein
177
11

PAP
314
9
0.2700

PSM
573
8

PAP
270
11

Kallikrein
94
8
0.0890

PSA
90
8
0.0890

Kallikrein
34
8

Kallikrein
34
10

PSA
30
10

PSM
347
8

PSM
347
10
0.0005

PSA
173
9

PSM
689
9

PSM
689
11

Kallikrein
8
10

PSM
202
8

PSM
202
9

PSM
530
8

PSM
530
10

PSM
642
8

PAP
188
11

PSM
676
9

PSM
676
11

PSM
386
8

PSM
386
11

PAP
50
10

PSA
11
10

PSM
297
8

PSM
130
10

PSM
416
8

PSM
416
11

PSM
373
11

PSA
69
9

PSA
69
10

PAP
135
10

PAP
267
8

PSM
226
9

PSM
226
10

PSM
226
11

PSM
512
10

PSM
614
10
0.1900

PSA
175
10

PSM
52
8

PSM
52
9

PSM
52
10

Kallikrein
226
10

PSA
222
10

Kallikrein
25
9
0.0410

PSA
21
9
0.0410

Kallikrein
25
10

PSA
21
10

PSM
200
8

PSM
200
10

PSM
200
11

PSM
591
8

PSM
591
10

PSM
591
11

PSM
157
8

PSM
398
9
0.1700

PSM
398
10
0.0260

PSM
66
8

PSM
66
10

PSM
59
8

PSM
59
9

PSM
723
8

PSM
723
11

PAP
185
9
0.0006

PAP
91
8

PAP
91
9

PSM
72
11

PSA
190
8

PSM
645
9

PSM
645
11

PSM
545
8

PSM
545
9

PAP
36
8

PAP
36
9

PSM
564
8

PSM
564
9

PSM
564
10

PAP
322
9
0.0002

PAP
322
10
0.0057

PAP
322
11

PSM
223
10

PSM
193
11

PSM
199
9
0.0740

PSM
199
11

PSM
610
8

PSM
610
9
0.1800

PSM
514
8

PSM
514
11

PAP
282
8

PSM
304
10

PSA
166
8

PAP
193
11

PAP
173
8

PAP
173
10

PSM
491
9
0.4000

PSM
491
10
0.3200

PSM
655
8

PSM
482
10
0.0044

PSA
66
8

PSA
66
9
0.0025

PSM
623
11

PSM
207
9
0.1600

PSM
207
11

PSM
213
8

PSM
213
10

PSM
213
11

Kallikrein
137
11

PSA
133
11

PSM
324
10

Kallikrein
191
9

PSA
187
9

PSA
187
11

Kallikrein
245
10
0.0450

PSA
241
10
0.0450

PSM
219
10
0.0004

PSM
28
10

PSM
83
8

PSM
83
11

PSM
110
10

PSM
92
10
0.0031

PSM
587
9

PAP
8
11

PSM
21
9

Kallikrein
197
10

PSA
193
10

PSM
62
10

PSM
62
11

PAP
26
8

PAP
26
11

PSM
105
8

PSM
105
9

PAP
300
11

PSM
417
10

Kallikrein
80
10

PSM
143
9

PAP
22
11

PAP
202
9

PSA
76
11

PAP
19
10

PSM
632
8

PAP
81
8

PAP
81
9
0.0002

PAP
81
10
0.0003

PAP
81
11

PSM
35
8

PSM
35
10
0.0007

PAP
16
8

PAP
16
9

PSM
374
10

PSM
528
8

PSM
528
9
0.0006

PSM
528
10

PAP
191
8

PSM
679
8

PSM
679
9

PSM
679
10

PSM
679
11

Kallikrein
139
9

PSA
71
8

PSM
515
10

PSM
515
11

PSM
305
9
0.0006

PAP
21
8

PSM
34
9

PSM
34
11

PSA
70
8

PSA
70
9

PSM
428
8

PSM
4
8

PSM
4
10
0.0005

Kallikrein
105
8

PSA
101
8

PAP
306
9
0.0010

PSM
441
8

PSM
441
9

Kallikrein
123
8

PSA
119
8

Kallikrein
123
9

PAP
243
8

PAP
243
9
0.0760

PAP
243
11

Kallikrein
178
8

Kallikrein
178
9

Kallikrein
178
10

Kallikrein
178
11

PSM
116
9
0.0006

PAP
136
9

PAP
153
11

PSM
668
8

Kallikrein
121
10

PSA
117
10

Kallikrein
121
11

PAP
113
9
0.0005

PAP
113
10
0.0005

PSM
469
11

PAP
148
8

PAP
148
11

PAP
238
10
0.0005

PSA
122
10

PAP
194
10

PAP
14
10

PAP
14
11

PAP
241
10
0.0003

PAP
241
11

PAP
244
8

PAP
244
10
0.0520

Kallikrein
179
8

Kallikrein
179
9

Kallikrein
179
10

Kallikrein
10
8

PSA
6
8

PSA
6
9

PSM
117
8

PSM
117
11

PSA
57
8

PSA
57
10
0.1400

Kallikrein
61
8

Kallikrein
61
9

PAP
315
8
0.0014

PAP
315
11

PSA
4
10

PSA
4
11

PSM
268
10
0.0005

PSM
268
11

PAP
70
9

PAP
70
10
0.0150

PSA
37
8

PSM
561
10

PSM
561
11

PAP
40
8
0.0003

PSM
473
10

Kallikrein
54
10

PSA
50
10

Kallikrein
54
11

PSA
50
11

PSM
26
8

PAP
263
8

PAP
263
10
0.0560

PAP
263
11

PSM
174
8

Kallikrein
183
9

PSA
135
9

PSM
569
9

Kallikrein
196
11

PSA
192
11

Kallikrein
122
9

PSA
118
9

Kallikrein
122
10

PSM
663
8

PSM
663
11

PAP
114
8

PAP
114
9

PAP
114
11

Kallikrein
103
10

PSA
99
8

PSA
99
10
0.0070

PAP
117
8

PSM
451
10

PSM
216
8

PSM
195
9

PSM
195
10

PSM
195
11

PSM
519
9

Kallikrein
181
8

Kallikrein
181
11

PSM
665
9

PSM
665
10

PSM
665
11

PSA
177
8

PSA
177
11

PSM
336
8

PSM
336
10

PSM
638
8

PSM
638
9
0.0005

PAP
220
8

PSM
76
9

PSM
262
11

PAP
304
8

PAP
304
11

PSM
69
9

PSM
257
8

PSM
51
9

PSM
51
10

PSM
51
11

Kallikrein
79
11

PSM
3
9
0.0006

PSM
3
11

PSM
247
9

PSM
57
10

PSM
57
11

Kallikrein
102
11

PSM
589
10

Kallikrein
70
8

Kallikrein
70
9

PSM
438
8

PSM
438
11

PAP
34
11

PSM
480
9

PAP
237
11

PAP
240
8

PAP
240
11

PSM
560
11

PAP
317
9

PAP
317
10

PSM
621
9
0.0005

PAP
328
10

PAP
168
10

PSM
703
9

PSM
703
11

PSM
716
8

PSM
716
9

PAP
60
8

PAP
95
9
0.0002

PAP
95
11

PSM
7
9

PSM
7
10

PSM
7
11

PAP
170
8

PAP
170
10
0.0004

PAP
170
11

PSM
542
8

PSM
542
11

PSM
557
8

PSM
557
9

PSM
557
10
0.0006

PSM
522
10

PSM
727
9

PSM
727
10

PSM
727
11

PSM
235
8

PSM
418
9

PSM
595
11

PSM
713
11

PSM
653
10

PSM
629
9

PSM
629
11

PSM
185
9

PSM
32
8

PSM
32
11

PSM
524
8

PSM
524
11

PAP
23
10

PSM
328
10

PSM
357
9

PSM
153
9

PSM
153
11

PSM
231
10

PSA
125
9
0.0002

Kallikrein
129
9

Kallikrein
146
8

PSA
142
8

Kallikrein
146
9

PSA
142
9

PSM
273
8

PSM
273
9
0.0001

Kallikrein
240
9

Kallikrein
240
10

Kallikrein
240
11

Kallikrein
233
9

Kallikrein
233
11

PSA
229
11

PSM
484
8

PSM
484
11

PSM
682
8

PSM
682
11

PSM
368
10

PSM
368
11

PSM
315
10

PSM
594
8

PAP
157
8

PSM
685
8

PSM
685
9

PAP
345
11

PSM
331
11

PSM
706
8

PSM
270
8

PSM
270
9

PSM
270
10

PSM
270
11

PAP
49
11

PSM
296
9

PAP
57
11

PAP
134
11

PSM
678
9

PSM
678
10

PSM
678
11

PAP
5
8

PSM
468
8

PAP
147
9
0.0005

PSM
267
8

PSM
267
11

PAP
212
8

PAP
212
10

PSA
95
9
0.2400

PSA
95
11

PSM
550
10
0.0004

Kallikrein
99
9

Kallikrein
99
10

PSM
568
10
0.0005

PAP
349
8

PAP
349
9

PSM
290
10

PSM
290
11

PSM
721
9

PSM
721
10
0.0003

PSA
236
9

PSA
236
10
0.0079

PSA
236
11

PSM
502
10

PSM
694
8

PAP
224
11

PAP
278
9
0.0002

PAP
278
11

PSM
293
8

PSM
293
10

Kallikrein
91
8

Kallikrein
91
11

PSM
740
11

PAP
200
9
0.0006

PAP
200
11

PSM
167
10

PAP
276
11

PSM
95
9

PSM
731
11

PSM
218
11

PSM
91
11

PAP
72
8

PAP
152
8

PSM
667
8

PSM
667
9

PAP
69
10

PAP
69
11

PSM
389
8

Kallikrein
109
11

Kallikrein
39
10

Kallikrein
39
11

PSA
84
9

PSA
84
11

PSA
182
9
0.0060

PSA
182
10

PSA
35
9
0.0021

PSA
35
10

PSM
578
8

PSM
578
11

PSA
87
8

PSA
87
11

Kallikrein
72
10

PAP
101
11

PAP
2
8

PAP
2
9
0.1500

PAP
2
10

PAP
2
11

PAP
10
9

PAP
10
11

PAP
273
8

PAP
273
9
0.0210

PAP
273
10
0.0053

PSA
43
10
0.0110

Kallikrein
186
10

PSM
190
10
0.0021

PSM
598
8

PSM
598
9
0.0024

PSM
598
10

PSM
598
11

PSA
105
11

PAP
163
11

PSM
363
8

PSM
363
9

PSM
580
9

PSM
255
10

PSM
210
8

PSM
210
11

PSM
320
8

PSM
445
8

PSM
511
11

Kallikrein
24
10
0.0460

PSA
20
10
0.0460

Kallikrein
24
11

PSA
20
11

PSM
354
10
0.3700

PSM
527
8

PSM
527
9
0.0032

PSM
527
10

PSM
527
11

PAP
180
8

PAP
180
10
0.0005

PSM
440
9
0.0012

PSM
440
10
0.0220

PSA
121
11

PSM
662
9

PSM
400
8

Kallikrein
169
9

PAP
28
9
0.0490

PAP
28
10

PSM
181
10

PSM
414
10

PAP
111
11

PSM
463
9

Kallikrein
89
8

Kallikrein
89
10

PAP
115
8

PAP
115
10

PSM
312
9
0.0006

PSM
10
8

PSM
10
10

PSM
634
10

PAP
312
8

PAP
312
9

PAP
312
11

PAP
350
8

PSM
155
9

PSM
155
10

PSM
229
8

PSM
628
8

PSM
628
10

PSM
401
11

PSM
704
8

PSM
704
10

PSM
390
11

PSM
197
8

PSM
197
9

PSM
197
11

PAP
195
9

PAP
294
10

PSM
507
8

PSM
517
8

PSM
517
9

PSM
517
11

PSM
532
8

Kallikrein
155
11

PSA
151
11

PSM
547
10

Kallikrein
7
11

PSM
455
9

Kallikrein
159
9

Kallikrein
159
11

PSA
155
11

PSM
129
11

PSM
291
9

PSM
291
10
0.0940

PSM
613
11

PSM
590
9
0.0006

PSM
590
11

PSM
142
10

PSM
631
9

PAP
15
9

PAP
15
10

Kallikrein
175
11

Kallikrein
104
9

PSA
100
9
0.0024

PAP
242
9
0.0006

PAP
242
10
0.4900

Kallikrein
170
8

Kallikrein
110
10

PAP
13
8

PAP
13
11

PSM
472
8

PSM
472
11

PSM
492
8

PSM
492
9
1.0000

PAP
245
9
1.1000

PAP
245
11

PSA
237
8

PSA
237
9
0.6800

PSA
237
10
0.2800

PSA
237
11

PSM
615
9
0.1100

PSM
615
11

Kallikrein
117
9
0.0039

PSA
113
9
0.0039

PSM
695
11

PSM
454
10
0.0007

PSM
45
11

PSM
317
8

PSM
317
11

PAP
106
11

PAP
369
10

PSM
431
10
0.0005

PSM
348
9
0.0016

PSM
338
8

PSM
338
10

PAP
217
11

PSA
67
8

PSA
67
11

PAP
29
8
0.0017

PAP
29
9

PSM
626
8

PSM
626
10

PSA
7
8

PSM
554
11

PSA
58
9
0.0094

Kallikrein
62
8

PSM
14
10

PSM
8
8

PSM
8
9

PSM
8
10

PAP
107
10

PAP
52
8

Kallikrein
15
10

PSM
334
8

PSM
334
10
0.0007

Kallikrein
86
9

Kallikrein
86
11

PSA
82
9
0.0002

PSA
82
11

PSM
415
9

PAP
190
9

PSM
404
8

PSM
404
10
0.0007

PSM
404
11

PAP
171
9
0.0006

PAP
171
10
0.0007

PAP
112
10
0.0005

PAP
112
11

PSM
361
10
0.0003

PSM
361
11

PSM
461
11

PSA
5
9

PSA
5
10

PAP
39
9
0.0006

PSM
141
11

Kallikrein
227
9

PSA
223
9

PAP
291
9

PSM
575
11

PAP
145
11

PAP
292
8

PSM
734
8

PSM
734
9

PSM
734
10

PSM
576
10

PSM
12
8

Kallikrein
40
9

Kallikrein
40
10

PSA
179
9

PSA
45
8

PSM
464
8

PSM
719
11

PAP
109
8

PSM
523
9

PSM
382
11

PSA
85
8

PSA
85
10

PSM
208
8

PSM
208
10

Kallikrein
26
8

PSA
22
8

Kallikrein
26
9

PSA
22
9

PSM
287
9

PSM
329
9

PSM
201
9

PSM
201
10

PSM
358
8

PSA
68
10

PSA
68
11

PSM
225
8

PSM
225
10

PSM
225
11

PSA
174
8

PSA
174
11

PSM
690
8

PSM
690
10
0.5400

PSM
690
11

PSM
27
11

PAP
30
8

Kallikrein
138
10

PSM
115
8

PSM
115
10

PSM
592
9

PSM
592
10
0.0005

PSM
603
8

PSM
603
10

PSM
660
11

PSA
56
9
0.0002

PSA
56
11

Kallikrein
60
9

Kallikrein
60
10

PSA
36
8

PSA
36
9

Kallikrein
53
11

PSA
49
11

PAP
262
9
0.0019

PAP
262
11

PSA
134
10

PSM
154
8

PSM
154
10

PSM
154
11

PSM
627
9

PSM
627
11

PAP
293
11

Kallikrein
92
10
0.0003

PSA
88
10
0.0003

Kallikrein
192
8

PSA
188
8

PSA
188
10
0.0003

PAP
38
10

PSM
394
9

Kallikrein
246
9
0.0072

PSA
242
9
0.0072

PSM
602
9
0.0390

PSM
602
11

Kallikrein
47
10

PAP
226
9
0.0006

PAP
226
11

Kallikrein
2
8

PSM
41
9

PSM
725
9

PSM
725
11

Kallikrein
229
11

PSA
225
11

Kallikrein
157
9

PSA
153
9

Kallikrein
157
11

PSA
10
11

Kallikrein
252
8

PSA
248
8

PSM
20
10
0.0026

PAP
25
8

PAP
25
9
0.0035

Kallikrein
74
8

PAP
206
9
0.0002

PAP
368
11

PSM
497
10

PSA
55
10
0.0004

Kallikrein
59
10

Kallikrein
59
11

PSM
607
11

PSM
700
10

PSM
692
8

PSM
692
9

PSM
692
10

PSM
179
8

PSM
179
9

PAP
310
10
0.0003

PAP
310
11

PSM
600
8

PSM
600
9

PSM
600
11

PSM
277
8

PSM
277
10

PAP
214
8

PSM
709
10

PSM
300
9
0.0006

PSA
97
9

PSA
97
10

PAP
210
10

PSM
566
8

PSM
113
10
0.0005

PSM
234
9

PAP
319
8

PAP
325
8

PAP
247
9
0.0006

PAP
247
11

PSM
205
9
0.0006

PSM
205
11

PAP
84
8

PAP
84
9

PAP
103
9

PAP
155
9

PAP
155
10

PSM
228
8

PSM
228
9

Kallikrein
188
8

PSM
471
9
0.0600

PSM
625
9

PSM
625
11

PSM
537
9

PSM
537
10

Kallikrein
243
8

PSA
239
8

Kallikrein
243
9
0.0006

PSA
239
9
0.0006

PSM
733
9

PSM
733
10

PSM
733
11

PSM
371
8

PSM
176
10

PSM
176
11

[0528]

22

TABLE XVII

Prostate All Motif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
A*1101

PSA

59
8

PSA

13
8

PAP

3
8

PSM

392
9

PSM

608
10

PSM

608
11

PSM

452
9

PSM

232
9
0.0051

PSM

232
11

PSM

674
11

PSM

220
9

PSM

264
9

PSM

701
9

Kallikrein

199
8

PSA

195
8

PSM

84
11

PSM

711
8

Kallikrein

235
9

Kallikrein

235
11

PSA

231
9
0.0013

PSA

231
11

PSM

274
8

PSM

588
11

PAP

311
9
0.0550

PSM

531
9
0.2700

PAP

227
8
0.0039

PAP

227
10

PAP

189
10

PSM

49
8

PSM

49
11

PAP

274
8
0.0700

PAP

274
9
1.2000

PSM

11
9

PSA

44
9

PSM

286
10

PSM

635
11

Kallikrein

17
8

PSM

393
8

PSM

601
10
0.0210

Kallikrein

41
9

Kallikrein

241
8

Kallikrein

241
9

Kallikrein

241
10

Kallikrein

241
11

Kallikrein

198
9

PSA

194
9
0.0015

Kallikrein

234
10

PSA

230
10

PSA

180
8

PSA

180
11

Kallikrein

184
8

PSM

196
9

PSM

196
10
0.0490

PAP

347
9
0.0006

Kallikrein

14
11

PSM

466
10

PSM

710
9
0.0002

PSM

301
8

PSM

596
10

PSM

596
11

PSM

465
11

PSA

111
11

PSM

652
11

PSM

520
8

PSM

184
10

PAP

186
8

PSM

714
10
0.0002

PAP

201
8

PAP

201
10

PSM

173
9

Kallikrein

182
10

PSM

191
9

PSA

98
8
0.0001

PSA

98
11

PSM

9
8

PSM

9
9

PSM

9
11

PSM

630
8

Kallikrein

116
10

PSA

112
10

PSM

453
8

PSM

453
11

PSM

316
9
0.0003

PSM

106
8

PAP

51
9
0.0001

Kallikrein

85
10

PSA

81
10

PSA

178
10
0.0011

PSM

114
9
0.0010

PSM

114
11

PAP

301
10

PSM

48
8

PSM

48
9

PSM

285
11

PAP

371
8

PSM

183
8

PSM

183
11

PAP

150
10

Kallikrein

115
11

Kallikrein

84
11

PSA

80
11

PAP

229
8

PSM

102
11

PAP

176
9

PAP

176
10

PSM

505
10

PSM

171
9

PSM

171
11

PSM

486
9

PSM

489
11

PSM

641
9
0.0002

PAP

266
8

PSM

397
10

PSM

397
11

PSM

109
11

PAP

166
8

PAP

80
8

PAP

80
9

PAP

80
10

PAP

80
11

PSM

64
8

PSM

64
9

PAP

34
10
0.0037

PAP

34
11

PAP

237
11

PAP

240
8

PAP

240
11

PAP

317
9

PAP

328
10

PSM

68
8

PSM

437
9

PSM

716
8

PAP

95
9
0.0002

PAP

95
11

PSM

7
10

PSM

7
11

PAP

170
10
0.0140

PAP

170
11

PSM

542
8

PSM

542
11

PSM

557
8

PSM

557
10
0.0002

PSM

235
8

PSM

595
11

PSM

713
11

PSM

653
10

PSM

629
9

PSM

185
9

PSM

524
11

PAP

23
11

PAP

203
8

PSM

103
10

PSM

103
11

PSM

402
10

PSM

675
10

PSM

61
11

PSM

37
8

PAP

18
11

PAP

20
9
0.0004

PAP

92
8

PSA

106
10

PSM

73
10
0.0036

PSM

646
10
0.0007

PSM

506
9

PSM

546
8

PSM

546
11

PSM

337
9

PSM

337
11

PSM

639
11

PSM

333
9

PSM

333
11

PAP

37
8

PAP

37
11

PSA

12
9
0.0350

PSM

391
10

PSM

263
10

PSM

221
8

PSM

364
8

Kallikrein

16
9

PAP

346
10

PSM

172
8

PSM

172
10

PSM

265
8

PSM

487
8

PSM

36
9
0.0014

PSM

332
10

PSM

310
11

PAP

260
11

Kallikrein

27
8

PSA

23
8

PSM

529
8

PSM

529
9

PSM

529
11

PAP

248
8

PAP

248
10

PAP

204
11

PSM

104
9

PSM

104
10

PAP

305
10

PSM

680
8

PSM

680
9
0.0280

PSM

680
10

PSM

288
8

PAP

295
9

PAP

74
11

PSM

168
9
0.0002

PSM

518
10

PSM

335
9

PSM

335
11

PSM

311
10
0.1400

PSA

226
10

Kallikrein

158
10

PSM

430
11

PSM

85
10

PSM

403
9

PSM

403
11

PSM

360
11

PSM

224
11

PAP

261
10

Kallikrein

49
8

PAP

198
11

PSM

345
10

Kallikrein

177
10

PAP

314
9
0.5300

PSM

573
8

PAP

270
11

PSM

475
8

PSM

56
11

Kallikrein

94
8
0.0006

PSA

90
8
0.0006

Kallikrein

34
10

PSM

347
8

PSM

347
10
0.0002

PSM

689
9

PSM

689
11

PSM

202
9

PSM

530
8

PSM

530
10

PSM

642
8

PAP

188
11

PSM

676
9

PSM

386
11

PAP

50
10

PSA

11
10

PSM

297
8

PSA

69
10

PAP

135
10

PSM

226
9

PSM

450
11

PSM

194
11

PSM

614
10
0.1100

PSA

175
10

PSM

52
8

Kallikrein

25
9
0.0190

PSA

21
9
0.0190

Kallikrein

25
10

PSA

21
10

PSM

200
8

PSM

200
11

PSM

591
8

PSM

591
10

PSM

398
9
0.0087

PSM

398
10
0.0006

PSM

66
10

PSM

59
8

PSM

723
8

PSM

723
11

PAP

185
9
0.0004

PAP

91
8

PAP

91
9

PSM

72
11

PSA

190
8

PSM

645
11

PSM

545
8

PSM

545
9

PAP

36
8

PAP

36
9

PSM

564
8

PSM

564
9

PSM

564
10

PAP

322
9
0.0002

PAP

322
10
0.0890

PAP

322
11

PSM

199
9
1.0000

PSM

610
8

PSM

610
9
0.1200

PAP

282
8

PSA

166
8

PSM

215
9

PSM

637
9

Kallikrein

69
9

Kallikrein

69
10

PSM

539
11

PAP

173
8

PAP

173
10

PSM

491
9
2.1000

PSM

491
10
0.0810

PSM

655
8

PSM

482
10
0.0210

PSA

66
8

PSA

66
9
0.0014

PSM

207
9
0.1200

PSM

213
11

PSA

187
11

Kallikrein

245
10
0.0450

PSA

241
10
0.0450

PSM

219
10
0.0002

PSM

110
10

PSM

92
10
0.0007

Kallikrein

197
10

PSA

193
10

PSM

62
10

PSM

62
11

PAP

26
8

PAP

26
11

PSM

105
8

PSM

105
9

PAP

300
11

Kallikrein

80
10

PSM

143
9

PAP

202
9

PAP

19
10

PAP

81
8

PAP

81
9
0.0002

PAP

81
10
0.0002

PAP

81
11

PSM

35
10
0.3700

PSM

528
9
0.0002

PSM

528
10

PAP

191
8

PSM

679
9

PSM

679
10

PSM

679
11

PSA

71
8

PAP

21
8

PSM

34
11

PSA

70
9

Kallikrein

105
8

PSA

101
8

PAP

306
9
0.0002

PSM

441
9

Kallikrein

123
9

PAP

243
8

PAP

243
9
0.2000

PAP

243
11

Kallikrein

178
9

Kallikrein

178
11

PSM

116
9
0.0003

PAP

136
9

PAP

153
11

Kallikrein

121
11

PSM

469
11

PAP

93
11

PAP

148
8

PAP

238
10
0.0004

PAP

241
10
0.0002

PAP

241
11

PAP

244
8

PAP

244
10
0.0370

Kallikrein

179
8

Kallikrein

179
10

PSM

117
8

PSM

117
11

PSA

57
8

PSA

57
10
0.0830

Kallikrein

61
8

Kallikrein

61
9

PAP

315
8
0.0100

PAP

315
11

PSM

268
10
0.0002

PAP

70
9

PAP

70
10
0.0024

PSM

561
11

PAP

40
8
0.0002

PSM

473
10

PAP

263
8

PAP

263
10
0.1200

PAP

263
11

PSM

174
8

Kallikrein

183
9

Kallikrein

196
11

PSA

192
11

Kallikrein

122
10

PSM

663
11

PSM

664
10

Kallikrein

103
10

PSA

99
10
0.0110

PSM

451
10

PSM

216
8

PSM

195
10

PSM

195
11

PSM

519
9

Kallikrein

181
8

Kallikrein

181
11

PSM

665
9

PSA

177
8

PSA

177
11

PSM

336
8

PSM

336
10

PSM

638
8

PSM

262
11

PAP

304
11

PSM

51
9

Kallikrein

79
11

PSM

247
9

PSM

57
10

Kallikrein

102
11

PSM

589
10

Kallikrein

70
8

Kallikrein

70
9

PSM

438
8

PSM

231
10

PSA

125
9
0.0002

Kallikrein

129
9

Kallikrein
PALGTTCY
146
8

PSA
PALGTTCY
142
8

PSM
PANEYAYR
273
8

PSM
PANEYAYRR
273
9
0.0002

Kallikrein
PAVYTKVVH
240
9

Kallikrein
PAVYTKVVHY
240
10

Kallikrein
PAVYTKVVHYR
240
11

Kallikrein
PCALPEKPAVY
233
11

PSA
PCALPERPSLY
229
11

PSM
PDEGFEGK
484
8

PSM
PDEGFEGKSLY
484
11

PSM
PDRPFYRH
682
8

PSM
PDRPFYRHVIY
682
11

PSM
PDRYVILGGH
368
10

PSM
PDRYVILGGHR
368
11

PSM
PDSSWRGSLK
315
10

PSM
PFYRHVIY
685
8

PAP
PGCSPSCPLER
345
11

PSM
PGFTGNFSTQK
331
11

PSM
PGYPANEY
270
8

PSM
PGYPANEYAY
270
10

PSM
PGYPANEYAYR
270
11

PAP
PIDTFPTDPIK
49
11

PSM
PIGYYDAQK
296
9

PAP
PILLWQPlPVH
134
11

PSM
PLGLPDRPFY
678
10

PSM
PLGLPDRPFYR
678
11

PSM
PLMYSLVH
468
8

PAP
PLSEDQLLY
147
9
0.0001

PSM
PLTPGYPANEY
267
11

PAP
PLYCESVH
212
8

PSA
PLYDMSLLK
95
9
0.0370

PSA
PLYDMSLLKNR
95
11

PSM
PLYHSVYETY
550
10
0.0002

Kallikrein
PLYNMSLLK
99
9

Kallikrein
PLYNMSLLKH
99
10

PSM
PNKTHPNY
120
8

PSM
PSIPVHPIGY
290
10

PSM
PSIPVHIGYY
290
11

PSM
PSKAWGEVK
721
9

PSM
PSKAWGEVKR
721
10
0.0002

PSA
PSLYTKVVH
236
9

PSA
PSLYTKVVHY
236
10
0.0003

PSA
PSLYTKVVHYR
236
11

PSM
PSPEFSGMPR
502
10

PAP
PSWATEDTMTK
224
11

PAP

278
9
0.0002

PSM

293
8

Kallikrein

91
8

Kallikrein

91
11

PAP

200
9
0.0008

PAP

200
11

PSM

167
10

PAP

276
11

PSM

218
11

PSM

91
11

PAP

72
8

PAP

152
8

PAP

69
10

PAP

69
11

PSM

389
8

Kallikrein

109
11

Kallikrein

39
11

PSA

84
11

PSA

182
9
0.0140

PSA

35
9
0.0018

PSA

87
8

PSA

87
11

PAP

101
11

PAP

2
9
0.1200

PAP

273
8

PAP

273
9
0.0600

PAP

273
10
0.0250

PSA

43
10
0.0310

PSM

190
10
0.0002

PSM

598
8

PSM

598
9
0.0190

PSM

598
10

PSA

105
11

PAP

163
11

PSM

363
8

PSM

363
9

PSM

320
8

Kallikrein

24
10
0.0670

PSA

20
10
0.0670

Kallikrein

24
11

PSA

20
11

PSM

354
10
0.4300

PSM

527
8

PSM

527
10

PSM

527
11

PSM

440
10
0.0005

PAP

332
9
0.0002

PSA

64
10

PSA

64
11

PSM

400
8

Kallikrein

169
9

PAP

28
9
0.1100

PSM

181
10

PSM

463
9

Kallikrein

89
10

PSM

312
9
0.0012

PSM

10
8

PSM

10
10

PAP

312
8

PAP

312
11

PSM

628
10

PSM

401
11

PSM

390
11

PSM

197
8

PSM

197
9

PSM

197
11

PAP

294
10

PSM

507
8

PSM

517
11

PSM

532
8

PSM

547
10

PSM

455
9

Kallikrein

159
9

Kallikrein

159
11

PSA

155
11

PSM

291
9

PSM

291
10
1.4000

PSM

613
11

PSM

590
9
0.0220

PSM

590
11

PSM

142
10

Kallikrein

104
9

PSA

100
9
0.0470

PAP

242
9
0.0002

PAP

242
10
2.3000

Kallikrein

170
8

Kallikrein

110
10

PSM

472
8

PSM

472
11

PSM

492
8

PSM

492
9
2.0000

PAP

245
9
0.8000

PAP

245
11

PSA

237
8

PSA

237
9
0.0140

PSA

237
10
0.2300

PSA

237
11

PSM

615
9
0.0720

PSM

615
11

Kallikrein

180
9

PSA

176
9

PSM

46
10

PSM

46
11

Kallikrein

117
9
1.2000

PSA

113
9
1.2000

PSM

454
10
0.0910

PSM

45
11

PSM

317
8

PSM

317
11

PAP

369
10

PSM

431
10
0.0016

PSM

348
9
0.0083

PSM

338
8

PSM

338
10

PSA

67
8

PAP

29
8
0.0061

PSM

554
11

PSA

58
9
0.0140

Kallikrein

62
8

PSM

8
9

PSM

8
10

PAP

52
8

Kallikrein

15
10

PSM

334
8

PSM

334
10
0.0002

Kallikrein

86
9

PSA

82
9
0.0002

PAP

190
9

PSM

404
8

PSM

404
10
0.0002

PSM

404
11

PAP

171
9
0.0078

PAP

171
10
0.0001

PSM

361
10
0.0002

PSM

361
11

PSM

461
11

PAP

39
9
0.0002

PSM

349
8

PSM

50
10

PSM

543
10

PSM

543
11

PSM

141
11

PAP

145
11

PSM

12
8

Kallikrein

40
10

PSA

179
9

PSA

45
8

PSM

464
8

PSM

719
11

PSA

85
10

PSM

208
8

Kallikrein

26
8

PSA

22
8

Kallikrein

26
9

PSA

22
9

PSM

287
9

PSM

201
10

PSA

68
11

PSM

225
10

PSA

174
11

PSM

690
8

PSM

690
10
0.7900

PSM

690
11

PSM

115
8

PSM

115
10

PSM

592
9

PSM

603
8

PSM

603
10

PSA

56
9
0.0005

PSA

56
11

Kallikrein

60
9

Kallikrein

60
10

PSA

36
8

PAP

262
9
0.0030

PAP

262
11

PAP

264
9

PAP

264
10

PSM

177
11

PSM

627
11

PAP

293
11

Kallikrein

92
10
0.0015

PSA

88
10
0.0015

PSA

188
10
0.0120

PAP

38
10

Kallikrein

246
9
0.0930

PSA

242
9
0.0930

PSM

602
9
0.0660

PSM

602
11

Kallikrein

47
10

PAP

226
9
0.0002

PAP

226
11

PSM

725
9

Kallikrein

229
11

PSA

225
11

Kallikrein

157
11

PSA

10
11

PAP

25
9
0.0150

PSM

246
10

PAP

206
9
0.0002

PAP

368
11

PSA

55
10
0.0001

Kallikrein

59
10

Kallikrein

59
11

PSM

607
11

PSM

700
10

PSM

692
8

PSM

692
9

PSM

179
9

PAP

310
10
0.0002

PSM

600
8

PSM

600
11

PSM

709
10

PSM

300
9
0.0002

PSA

97
9

PAP

210
10

PSM

566
8

PSM

113
10
0.0016

PSM

234
9

PAP

325
8

PAP

247
9
0.0002

PAP

247
11

PSM

205
9
0.0002

PSM

205
11

PAP

84
8

PAP

103
9

PAP

155
9

PSM

75
8

PAP

303
8

Kallikrein

101
8

PSM

356
8

PSM

471
9
0.5400

PSM

537
9

Kallikrein

243
8

PSA

239
8

Kallikrein

243
9
0.0580

PSA

239
9
0.0580

PSM

371
8

[0529]

23

TABLE XVIII

Prostate A24 Motif Peptides with Binding Data

No. of

Protein
Position
Amino Acids
A*2401

PSM
674
8

PSM
60
11

PSM
736
11

PAP
116
8

PAP
116
9
0.0150

PSM
724
9

PSM
448
9
0.0190

Kallikrein
187
9

Kallikrein
187
11

Kallikrein
152
9
0.1700

PSA
148
9
0.1700

PSM
652
8

PSM
652
10

PSM
520
9

PSM
520
11

PSM
184
11

PAP
186
9
0.0002

PSM
191
10

PSA
98
9
0.0001

PSA
98
10

PSM
102
9

PSM
425
10

Kallikrein
164
8

PSA
160
8

Kallikrein
194
8

Kallikrein
194
9

PSM
505
8

PSM
505
11

PSM
621
9
0.0010

PSM
433
9

PSM
433
10

PSM
276
8

PAP
83
10
0.0067

PAP
83
11

PSM
185
10

PSM
32
8

PSM
32
10
0.0026

PSM
32
11

PAP
23
9
0.0017

Kallikrein
195
8

PSA
191
8

PAP
24
8

PSM
565
10
1.1000

PSM
487
11

PSM
31
8

PSM
31
8
0.0190

PSM
31
11

PAP
66
8

PSM
36
8

PAP
17
8

PAP
17
9
0.0016

PAP
17
10
0.0007

PAP
74
8

PSM
508
8

PSM
582
10
0.0002

Kallikrein
46
9

Kallikrein
28
11

PSA
24
11

Kallikrein
156
10
0.0001

PSA
152
10
0.0001

Kallikrein
156
11

PSA
152
11

PSM
409
8

PSM
409
9

PSM
409
10
0.0540

PSM
150
8

PSM
298
8

PSM
298
9

PAP
270
8

PAP
78
8

Kallikrein
248
10
0.0550

PSA
244
10
0.0550

PAP
131
8

PAP
131
11

PAP
205
9
0.0024

PSM
708
8

PSM
355
8

PSM
72
9

PSA
190
9
0.0310

PSM
645
9

PSM
564
11

PSM
606
9
12.0000

PSM
699
10

PSM
417
10

PAP
22
10
0.0045

PSA
76
11

PAP
19
8

PAP
123
9
0.0033

PAP
123
10
0.0140

PSM
632
8

PSM
632
11

PSM
668
8

PSM
668
9
0.0075

PAP
113
8

PAP
113
11

PSM
469
9

PAP
213
9
0.4400

PAP
213
11

PSA
96
11
0.1200

PAP
318
9
2.5000

PSM
551
11

PAP
154
11

PSM
74
10
0.2300

PSM
227
9
0.4400

PSA
238
11

PSM
669
8

PSM
669
11

PSM
663
8

PSM
663
9

Kallikrein
1
8

Kallikrein
1
10

PSM
470
8

PSM
89
8

PSM
336
11

PSM
638
9
0.0001

PSM
76
8

PSM
57
9

Kallikrein
102
10

PSM
178
8

PSM
178
9
0.7700

PSM
178
11

PSM
459
11

PSM
594
11

PAP
157
8

PAP
157
11

Kallikrein
37
11

PAP
309
10
0.0240

PAP
183
9
0.1100

PSM
326
8

PAP
297
10
0.0001

PAP
297
11

PSA
54
10
0.0007

Kallikrein
58
10

PAP
355
10
0.0037

PAP
163
10
0.0001

PSM
662
9

PSM
662
10

PSM
10
0

PSM
19
10

PSM
536
11

PSM
401
10

PSM
704
9

PSM
704
10

PSA
91
11

Kallikrein
95
11

PAP
225
11

PSM
420
9

PSM
420
10

Kallikrein
228
9

PSA
224
9
0.0001

PAP
62
9
0.0013

PSM
496
11

PAP
96
9
0.2600

PSM
241
8

PSM
118
11

PAP
231
8

PAP
231
11

PSA
9
8

PSA
9
9
0.1100

PSA
9
10
0.3600

PSM
558
8

PSM
624
9

PSM
624
10
3.2000

PSM
584
8

PSM
584
10

PSM
523
8

PSA
2
9
2.1000

PSA
2
10
0.0062

PSA
85
8

PAP
41
10
0.0005

PSA
134
11

Kallikrein
73
8

Kallikrein
73
9

PSM
555
11

Kallikrein
242
11

PSM
175
11

PAP
319
8

PSM
299
8

[0530]

24

TABLE XIX

Prostate DR Supermotif Peptides

Protein
Position

PAP
1

Kallikrein
1

PSA
1

Kallikrein
2

PSA
2

PSA
3

PAP
124

PSA
16

PAP
6

PAP
14

PSM
611

PSM
287

PSM
426

PAP
360

PSA
198

PSA
63

PAP
35

PAP
302

Kallikrein
12

PSA
17

PAP
7

Kallikrein
188

Kallikrein
157

PSA
153

PSM
289

PSA
134

Kallikrein
20

PSA
183

PAP
218

Kallikrein
222

PSA
218

PAP
164

PSM
469

PSM
488

PSM
523

PSA
174

Kallikrein
6

PSM
570

PSM
669

PSM
686

PAP
30

PAP
113

PSM
456

PAP
293

Kallikrein
166

PSA
162

PSM
105

PSM
192

PSM
447

PSM
719

PSM
525

PSM
279

PAP
359

PAP
26

PAP
70

PAP
21

PSA
6

PAP
167

PSM
164

PSM
549

PSM
642

PSM
394

PSM
175

PSM
268

PSM
33

PSM
253

PSA
213

Kallikrein
217

PAP
263

PSM
493

PSM
209

PSM
585

PSM
138

PSM
259

PSM
214

PSM
333

PSA
214

Kallikrein
218

PAP
364

PAP
202

Kallikrein
90

PSA
86

PSA
45

PSM
449

PSM
227

PSA
51

Kallikrein
55

PAP
131

PSM
248

PSA
118

Kallikrein
122

PSM
399

PAP
340

PAP
102

Kallikrein
81

PSA
97

Kallikrein
101

PSA
55

Kallikrein
59

PSA
77

PSM
556

PSM
115

PAP
53

PSM
300

PSM
73

PAP
138

PAP
280

Kallikrein
229

PSA
225

PSM
614

PSM
62

PSM
410

PSM
75

PSM
226

Kallikrein
242

PAP
258

PSM
344

PSM
574

PSM
113

PSM
65

PAP
303

PSM
309

PAP
25

PSM
41

PSM
38

Kallikrein
179

PAP
184

PSA
175

PAP
286

PAP
24

PAP
156

PSM
671

PSA
120

Kallikrein
124

PAP
310

PSM
292

PAP
226

PSA
170

Kallikrein
174

PSM
653

Kallikrein
226

PSA
222

PAP
238

PSM
664

PAP
241

PAP
197

PAP
244

PSM
177

PSM
572

PSM
512

PAP
117

Kallikrein
106

PSA
102

PAP
120

Kallikrein
4

PSM
473

PAP
97

PAP
223

PAP
307

Kailikrein
223

PSA
219

Kallikrein
105

PAP
136

PSM
592

PSM
143

PSM
462

PSM
234

Kallikrein
236

PSA
232

Kallikrein
165

PAP
129

PSA
96

Kallikrein
100

PAP
137

PAP
143

PSA
167

PAP
8

PAP
344

PAP
368

PSM
622

PSM
169

PSA
188

Kallikrein
171

PSM
21

PSM
329

PAP
342

PAP
262

PSM
734

PSM
100

Kallikrein
75

PAP
104

PSA
57

Kallikrein
61

PSM
676

PSM
381

PSM
583

PSM
691

Kallikrein
253

PSA
249

PSM
530

PSM
20

PSA
238

PSM
733

PAP
50

Kallikrein
92

PSM
158

Kallikrein
192

PSA
117

Kallikrein
121

Kallikrein
10

PAP
210

Kallikrein
178

PAP
16

PSM
659

PSA
34

PSA
22

Kallikrein
26

PSM
442

PAP
109

PSM
434

PSM
110

PSA
70

PSM
629

PSA
10

PSM
383

PSA
132

Kallikrein
136

Kallikrein
196

Kallikrein
18

PSM
337

PSM
418

PSM
464

PSA
8

PSM
546

PSM
356

PSM
144

PAP
148

PSM
627

PSM
737

PSM
579

Kallikrein
43

PSM
450

PAP
330

PSM
587

PSA
88

PSM
297

PSA
71

PSM
639

Kallikrein
141

PSM
663

PSA
137

Kallikrein
21

PSM
161

PSM
157

PAP
132

PSA
11

PSA
4

Kallikrein
138

Kallikrein
5

PSM
103

PSM
5

PAP
135

PAP
141

PSM
603

PSM
712

PAP
213

PSM
569

PSM
154

PSM
497

PAP
283

PAP
306

PAP
343

PSM
690

Kallikrein
252

PSA
248

[0531]

25

TABLE XXa

Prostate DR 3a Submotif Peptides

Protein
Position

PAP
124

PSM
669

PSM
186

PAP
331

PSM
405

PAP
167

PSM
394

PAP
263

PAP
298

PAP
364

PSM
227

PSM
700

Kallikrein
81

Kallikrein
111

PSA
77

PAP
53

PSM
131

PAP
325

PSM
65

Kallikrein
179

PSA
175

PAP
24

PAP
318

PSM
4

PAP
97

PSM
441

PSM
462

PSM
366

PSM
583

PAP
172

PAP
148

PSM
627

PSM
450

PSM
663

Kallikrein
160

PSA
156

PSM
103

PAP
213

PSM
130

PAP
92

[0532]

26

TABLE XXbP

Prostate DR 3b Submotif Peptides

Protein

Position

PSM
IQSQWKEFG
96

PSM
FDIESKVDP
713

PSM
YSISMKHPQ
612

PSM
INCSGKIVI
194

PAP
YCESVHNFT
214

PSM
LERDMKINC
188

PSM
YAPSSHNKY
692

PSM
VIGTLRGAV
358

PAP
IMYSAHDTT
284

PAP
LGMEQHYEL
73

PSM
FLDELKAEN
61

PSM
AWGEVKRQI
724

PAP
LNESYKHEQ
93

PAP
LAKELKFVT
31

PSM
LPFDCRDYA
593

PSA
VCAQVHPQK
179

PSM
AVATARRPR
11

PAP
MTTNSHQGT
373

PSM
AEENSRLLQ
435

PSM
LTKELKSPD
477

[0533]

27

TABLE XXI

Population coverage with combined HLA Supertypes

PHENOTYPIC FREQUENCY

North

Cauca-
American
Japa-
Chi-
His-
Aver-

HLA-SUPERTYPES
sian
Black
nese
nese
panic
age

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

[0534]

28

TABLE XXII

Prostate Antigen Peptides

Antigen

Binding affinity

≦ 200 nM
Sequence

PSA.117
LMLLRLSEPA

PSA.118
MLLRLSEPAEL

PSA.118
MILLRLSEPA

PSA.143
ALGTTCYA

PSA.161
FLTPKKLQCV

PSA.166
KLQCVDLHV

PAP.6
LLLARAASLSL

PAP.21
LLFFWLDRSV

PAP.30
VLAKELKFV

PAP.92
FLNESYKHEQV

PAP.112
TLMSAMTNL

PAP.135
ILLWQPIPV

PAP.284
IMYSAHDTTV

PAP.299
ALDVYNGLL

PSM.26
LVLAGGFFL

PSM.27
VLAGGFFLL

PSM.168
GMPEGDLVYV

PSM.288
GLPSIPVHPI

PSM.441
LLQERGVAYI

PSM.469
LMYSLVHNL

PSM.662
RMMNDQLMFL

PSM.663
MMNDQLMFL

PSM.667
QLMFLERAFI

PSM.711
ALFDIESKV

HuK2.165
FLRPRSLQCV

HuK2.175
SLHLLSNDMCA

Binding affinity

> 200 nM
Sequence

PSM.4
LLHETDSAV

PSM.25
ALVLAGGFFL

PSM.427
GLLGSTEWA

PSM.514
KLGSGNDFEV

[0535]

29

TABLE XXIIIA

A2 supermotif cross-reactive binding data

A2

Cross-

A*0201
A*0202
A*0203
A*0206
A*6802
Reac-

Peptide
AA
Sequence
Source
nM
nM
nM
nM
nM
tivity

20.0044
9
LLLARAASL
PAP.6
208
13
29
425
—
4

63.0136
11
LLLARAASLSL
PAP.6
8.1
3.1
5.3
80
143
5

60.0201
9
LLLARAASV
PAP.6.V9
18
215
6.7
95
—
4

20.0203
10
LLARAASLSL
PAP.7
500
5.2
63
9250
5714
3

63.0031
10
LLARAASLSV
PAP.7.V10
109
10
21
378
121
4

63.0137
11
AASLSLGFLFL
PAP.11
227
23
53
95
—
4

1419.51
10
SLSLGFLFLL
PAP.13
40
13
403
21
8560
4

1419.52
10
SLSLGFLFLV
PAP.13.V10
1.8
3.9
17
42
355
5

1419.50
9
SLSLGFLFV
PAP.13.V9
77
25
21
93
—
4

60.0203
9
FLFLLFFWV
PAP.18.V9
42
307
625
308
90
4

63.0138
11
FLLFFWLDRSV
PAP.20
14
17
2.8
285
364
5

1097.09
10
LLFFWLDRSV
PAP.21
28
0.60
1.6
231
—
4

1418.23
10
LTFFWLDRSV
PAP.21.T2
118
11
9.6
43
16
5

63.0139
11
LLFFWLDRSVL
PAP.21
65
2.9
2.7
822
4444
3

63.0033
10
SLLAKELKFV
PAP.29.L2
64
5.7
3.8
38
6667
4

1097.171
9
VLAKELKFV
PAP.30
96
3.6
6.7
168
—
4

63.0142
11
VLAKELKFVTL
PAP.30
6.9
8.1
21
25
—
4

63.0034
10
VLAKELKFVV
PAP.30.V10
31
12
189
86
2286
4

1419.55
11
FLNESYKHEQV
PAP.92
29
1.4
5.6
381
6154
4

1177.01
9
TLMSAMTNL
PAP.112
43
0.80
2.9
285
296
5

20.0312
10
TLMSAMTNLA
PAP.112
385
3.6
37
3700
6667
3

63.0037
10
TLMSAMTNLV
PAP.112.V10
63
3.9
12
43
242
5

1419.56
9
TLMSAMTNV
PAP.112.V9
10
2.4
3.6
54
62
5

1419.58
10
LLALFPPEGV
PAP.120.L2
5.0
0.70
1.6
148
163
5

1419.59
10
LVALFPPEGV
PAP.120.V2
156
17
4.8
463
28
5

1419.6
10
ALFPPEGVSI
PAP.122
278
11
133
2643
—
3

1419.61
10
ALFPPEGVSV
PAP.122.V10
15
1.0
18
119
4444
4

63.0041
10
GVSIWNPILV
PAP.128.V10
250
94
23
451
2286
4

60.0207
9
GVSIWNPIV
PAP.128.V9
455
269
909
308
—
3

63.0042
10
PLLLWQPIPV
PAP.134.L2
238
47
19
336
3333
4

1044.04
9
ILLWQPIPV
PAP.135
3.3
39
1.8
71
1702
4

1418.25
9
ITLWQPIPV
PAP.135.T2
34
1720
6.2
26
32
4

1419.69
10
LLWQPIPVHV
PAP.136.V10
25
1.8
17
287
60
5

1166.11
10
GLHGQDLFGI
PAP.196
26
0.90
2.5
315
—
4

1419.62
10
GLHGQDLFGV
PAP.196.V10
12
2.3
3.1
18
—
4

63.0048
10
KLRELSELSV
PAP.234.V10
263
9.1
7.1
49
1818
4

1097.05
10
IMYSAHDTTV
PAP.284
217
1.5
14
411
—
4

1389.06
10
ILYSAHDTTV
PAP.284.L2
385
1.0
15
1480
5714
3

60.0213
9
TVSGLQMAV
PAP.292.V9
294
12
122
195
5.7
5

1177.02
9
ALDVYNGLL
PAP.299
73
29
256
3083
—
3

1419.64
10
LLPPYASCHV
PAP.306.V10
88
15
16
98
5260
4

— indicates binding affinity > 10,000 nM.

[0536]

30

TABLE XXIIIB

A2 supermotif cross-reactive binding data

A2

Cross-

A*0201
A*0202
A*0203
A*0206
A*6802
Reac-

Peptide
AA
Sequence
Source
nM
nM
nM
nM
nM
tivity

1126.10
9
VLAGGFFLL
PSM.27
39
0.20
33
31
2857
4

1389.20
9
VLAGGFFLV
PSM.27.V9
26
0.40
5.0
57
216
5

1129.04
10
GMPEGDLVYV
PSM.168
55
3.1
7.1
161
6154
4

1389.22
10
GLPEGDLVYV
PSM.168.L2
42
2.0
2.1
112
964
4

1418.29
10
GTPEGDLVYV
PSM.168.T2
313
134
53
40
571
4

1129.10
10
GLPSIPVHPI
PSM.288
147
2.7
2.1
2467
308
4

1389.24
10
GLPSIPVHPV
PSM.288.V10
55
0.70
0.60
308
121
5

1129.01
10
LLQERGVAYI
PSM.441
179
5.7
6.7
861
—
3

1126.14
9
LMYSLVHNL
PSM.469
64
0.40
2.1
109
320
5

1126.06
10
RMMNDQLMFL
PSM.662
9.8
2.7
7.7
40
—
4

1126.01
9
MMNDQLMFL
PSM.663
11
0.80
1.7
7.6
195
5

1126.16
10
QLMFLERAFI
PSM.667
98
36
91
—
30
4

1129.08
9
ALFDIESKV
PSM.711
85
0.70
1.4
148
8889
4

1418.30
9
ATFDIESKV
PSM.711.T2
238
27
44
82
258
5

[0537]

31

TABLE XXIIIC

A2 supermotif cross-reactive binding data

A2

Cross-

Alternate
A*0201
A*0202
A*0203
A*0206
A*6802
Reac-

Peptide
AA
Sequence
Source
Source
nM
nM
nM
nM
nM
tivity

1419.25
11
VVFLTLSVTWI
PSA.1

385
159
63
2846
—
3

63.0185
11
VVFLTLSVTWV
PSA.1.V11

89
88
71
336
—
4

63.0186
11
FLTLSVTWIGV
PSA.3.V11

6.8
3.0
18
65
114
5

60.0216
9
FLTLSVTWV
PSA.3.V9

53
8.4
8.3
49
—
4

60.0217
9
TLSVTWIGV
PSA.5.V9

26
4.9
40
712
229
4

1419.10
11
VLVHPQWVLTA
PSA.49
HuK2.53
294
7.7
101
2056
—
3

1419.11
11
VLVHPQWVLTV
PSA.49.V11
HuK2.53.V11
11
1.5
16
31
8889
4

63.0109
11
DLMLLRLSEPV
PSA.116.V11
HuK2.120.V11
50
57
29
148
2759
4

63.0014
10
LMLLRLSEPA
PSA.117
HuK2.121
200
17
67
925
5000
3

1418.43
10
LMLLRLSEPV
PSA.117.V10
HuK2.121.V10
114
67
29
25
6154
4

1419.02
9
MLLRLSEPA
PS A.118
HuK2.122
195
745
145
49
—
3

1389.10
9
MLLRLSEPV
PSA.118.V9
HuK2.122.V9
36
36
46
638
421
4

1389.12
11
MLLRLSEPAEV
PSA.118.V11

294
331
115
1762
4444
3

1419.01
8
ALGTTCYA
PSA.143
HuK2.147
15
19
13
561
—
3

1389.14
8
ALGTTCYV
PSA.143.V8
HuK2.147.V8
74
6.4
12
264
—
4

1098.02
10
FLTPKKLQCV
PSA.161

52
8.3
13
755
—
3

990.01
9
KLQCVDLHV
PSA.166

79
205
91
6167
—
3

63.0058
10
KLQCVDLHVV
PSA.166.V10

13
84
9.1
500
—
4

60.0220
9
KVTKFMLCV
PSA.187.V9

69
518
53
128
—
3

1419.17
11
PLVCNGVLQGV
PSA.212.V11
HuK2.216.V11
27
127
19
255
4314
4

1418.55
10
LVCNGVLQGV
PSA.213.V10
HuK2.217.V10
10
2.9
12
5.6
3.5
5

[0538]

32

TABLE XXIIID

A2 supermotif cross-reactive binding data

A2

Cross-

Alternate
A*0201
A*0202
A*0203
A*0206
A*6802
Reac-

Peptide
AA
Sequence
Source
Source
nM
nM
nM
nM
nM
tivity

1418.13
9
LLLSIALSV
HuK2.4.L2

88
176
147
189
—
4

1418.57
11
ILLSVGCTGAV
HuK2.8.L2

36
33
36
308
—
4

1418.59
11
ITLSVGCTGAV
HuK2.8.T2

294
134
40
206
121
5

1419.05
10
ALSVGCTGAV
HuK2.9

53
75
17
542
—
3

1418.15
9
ALSVGCTGV
HuK2.9.V9

24
17
9.1
264
—
4

1418.35
10
SVGCTGAVPV
HuK2.11.V10

104
287
154
552
216
4

1419.10
11
VLVHPQWVLTA
HuK2.53
PSA.49
294
7.7
101
2056
—
3

1419.11
11
VLVHPQWVLTV
HuK2.53.V11
PSA.49.V11
11
1.6
16
31
9378
4

63.0109
11
DLMLLRLSEPV
HuK2.120.V11
PSA.116.V11
50
57
29
148
2759
4

63.0014
10
LMLLRLSEPA
HuK2.121
PSA.117
200
17
67
925
5000
3

1418.43
10
LMLLRLSEPV
HuK2.121.V10
PSA.117.V10
114
67
29
25
6154
4

1419.02
9
MLLRLSEPA
HuK2.122
PSA.118
195
745
145
49
—
3

1389.10
9
MLLRLSEPV
HuK2.122.V9
PSA.118.V9
36
36
46
638
421
4

1419.01
8
ALGTTCYA
HuK2.147
PSA.143
15
19
13
561
—
3

1389.14
8
ALGTTCYV
HuK2.147.V8
PSA.143.V8
74
6.4
12
264
—
4

1419.07
10
FLRPRSLQCV
HuK2.165

186
4.8
4.2
—
—
3

60.0191
9
SLQCVSLHL
HuK2.170

500
51
417
6167
2581
3

1419.66
10
SLQCVSLHLL
HuK2.170

263
4.9
71
446
5000
4

1418.52
10
SLQCVSLHLV
HuK2.170.V10

13
6.3
2.8
5.2
205
5

1418.19
9
SLQCVSLHV
HuK2.170.V9

56
165
48
4111
1600
3

1419.14
11
SLHLLSNDMCA
HuK2.175

71
4.8
71
—
—
3

1418.66
11
SLHLLSNDMCV
HuK2.175.V11

8.6
0.80
10
2313
2162
3

1419.15
11
HLLSNDMCARA
HuK2.177

417
391
250
374
—
4

1418.67
11
HLLSNDMCARV
HuK2.177.V11

26
1.3
5.3
37
860
4

1418.20
9
HLLSNDMCV
HuK2.177.V9

119
102
278
176
—
4

1418.53
10
LLSNDMCARV
HuK2.178.V10

5.3
0.70
4.3
10
1702
4

1418.71
11
KVTEFMLCAGV
HuK2.191.V11

56
10
26
29
143
5

1418.21
9
KVTEFMLCV
HuK2.191.V9

53
27
31
34
6667
4

1418.22
9
FMLCAGLWV
HuK2.195.V9

29
12
91
51
—
4

1419.17
11
PLVCNGVLQGV
HuK2.216.V11
PSA.212.V11
27
127
19
255
4314
4

1418.55
10
LVCNGVLQGV
HuK2.217.V10
PSA.213.V11
10
2:9
12
5.6
3.5
5

[0539]

33

TABLE XXIVA

Immunogenicity of A2 cross-reactive binding peptides and peptide analogs

Cross-

Reac-
A2

Peptide

A*0201
A*0202
A*0203
A*0206
A*6802
tivity
pep-
A2
A2

ID
AA
Sequence
Source
nM
nM
nM
nM
nM
(<200nM)
tide
native
in vivo

1419.51
10
SLSLGFLFLL
PAP.13
40
13
403
21
8560
3

1419.52
10
SLSLGFLFLV
PAP.13.V10
1.8
3.9
17
42
355
4

1097.09
10
LLFFWLDRSV
PAP.21
28
0.60
1.6
231
—
3
3/3

0/3

1418.23
10
LTFFWLDRSV
PAP.21.T2
118
11
9.6
43
16
5
3/3
2/3

1097.17
9
VLAKELKFV
PAP.30
96
3.6
6.7
168
—
4
1/3

0/3

1177.01
9
TLMSAMTNL
PAP.112
43
0.80
2.9
285
296
3
2/2

3/3

1419.58
10
LLALFPPEGV
PAP.120.L2
5.0
0.72
1.6
146
164
5

1419.61
10
ALFPPEGVSV
PAP.122.V10
15
1.0
18
120
4387
4
1/3
1/3

1044.04
9
ILLWQPIPV
PAP.135
3.3
39
1.8
71
8511
4
5/5

1/6

1418.25
9
ITLWQPIPV
PAP.135.T2
34
1723
6.2
26
32
4
3/3
2/3

1419.69
10
LLWQPIPVHV
PAP.136.V10
25
1.8
17
287
60
4

1166.11
10
GLHGQDLFGI
PAP.196
26
0.9
2.5
315
—
3

1419.62
10
GLHGQDLFGV
PAP.196.V10
12
2.3
3.2
18
—
4

1097.05
10
IMYSAHDTTV
PAP .284
217
1.5
14
411
—
2
3/3

0/3

1419.64
10
LLPPYASCHV
PAP.306.V10
88
15
16
98
5260
4

[0540]

34

TABLE XXIVB

Immunogenicity of A2 cross-reactive binding peptide and peptide analogs

Cross-

Reac-
A2

Peptide

A*0201
A*0202
A*0203
A*0206
A*6802
tivity
pep-
A2
A2

ID
AA
Sequence
Source
nM
nM
nM
nM
nM
(<200nM)
tide
native
in vivo

1126.10
9
VLAGGFFLL
PSM.27
39
0.20
33
31
—
4
1/2

3/3

1389.20
9
VLAGQFFLV
PSM.27.V9
26
0.40
5.0
57
216
4
1/2
1/2

1129.04
10
GMPEGDLVYV
PSM.168
55
3.1
7.1
161
—
4
0/1

1/3

1129.10
10
GLPSIPVHPI
PSM.288
147
2.7
2.1
2467
1538
3
2/4

0/3

1389.24
10
GLPSIPVHPV
PSM.288.V10
55
0.70
0.60
308
121
4
4/4
3/4

1129.01
10
LLQERGVAYI
PSM.441
179
5.7
6.7
861
—
3
3/3

1126.14
9
LMYSLVHNL
PSM.469
64
0.40
2.1
109
1600
4
3/3

3/3

1126.06
10
RMMNDQLMFL
PSM.662
9.8
2.7
7.7
40
—
4
1/1

20/22

1126.01
9
MMNDQLMFL
PSM.663
11
0.80
1.7
7.6
976
4
2/2

3/3

1129.08
9
ALFDffiSKV
PSM.711
85
0.70
1.4
148
—
4
2/2

3/3

[0541]

35

TABLE XXIVC

Immunogenicity of A2 cross-reactive binding peptides and peptide analogs

Cross-

A*
A*
A*
A*
A*
Reac-
A2

A2

Peptide

Alternate
0201
0202
0203
0206
6802
tivity
pep-
A2
in

ID
AA
Sequence
Source
Source
nM
nM
nM
nM
nM
(<200nM)
tide
native
vivo

1419.27
11
FLTLSVTWIGV
PSA.3.V11

6.8
3.0
18
65
113
5
3/3
3/3

1419.11
11
VLVHPQWVLTV
PSA49.V11
HuK2.53.V11
11
1.6
16
31
9378
4

1419.13
11
DLMLLRLSEPV
PSA.116.V11
HuK2.120.V11
50
57
29
148
2759
4

1419.02
9
MLLRLSEPA
PSA.118
HuK2.122
195
745
145
49
—
3

1389.10
9
MLLRLSEPV
PSA.118.V9
HuK2.122.V9
36
36
46
638
421
3
3/3
1/3

1419.01
8
ALGTTCYA
PSA.143
PSA.143
15
19
13
562
—
3

1389.14
8
ALGTTCYV
PSA.143.V8
HuK2.147.V8
74
6.4
12
264
—
3
2/3
1/3

1098.02
10
FLTPKKLQCV
PSA.161

52
8.3
13
755
—
3
3/4

0/6

990.01
9
KLQCVDLHV
PSA.166

79
205
91
6167
—
2
1/2

1/3

1419.24
10
KLQCVDLHVV
PSA.166.V10

13
84
9.5
502
—
3
1/2
1/2

1419.17
11
PLVCNGVLQGV
PSA.212.V11
HuK2.216.V11
27
127
19
255
4314
3

[0542]

36

TABLE XXIVD

Immunogenicity of A2 cross-reactive binding peptides and peptide analogs

Cross-

A*
A*
A*
A*
A*
Reac-
A2

A2

Peptide

Alternate
0201
0202
0203
0206
6802
tivity
pep-
A2
in

ID
AA
Sequence
Source
Source
nM
nM
nM
nM
nM
(<200nM)
tide
native
vivo

1418.13
9
LLLSIALSV
HuK2.4.L2

88
176
147
189
—
4
2/2
2/2

1419.05
10
ALSVGCTGAV
HuK2.9

53
75
17
542
—
3

1419.11
11
VLVHPQWVLTV
HuK2.53.V11
PSA49.V11
11
1.6
16
31
9378
4
2/2
2/2

1419.13
11
DLMLLRLSEPV
HuK2.120.V11
PSA.116.V11
50
57
29
148
2759
4
2/2
2/2

1419.02
9
MLLRLSEPA
HuK2.122
PSA.118
195
745
145
49
—
3

1389.10
9
MLLRLSEPV
HuK2.122.V9
PSA.118.V9
36
36
46
638
421
3

1419.01
8
ALGTTCYA
HuK2.147
PSA.143
15
19
13
562
—
3
1/2

1389.14
8
ALGTTCYV
HuK2.147.V8
PSA.143.V8
74
6.4
12
264
—
3

1419.07
10
FLRPRSLQCV
HuK2.165

186
4.8
4
—
—
3
1/3

1419.14
11
SLHLLSNDMCA
HuK2.175

72
4.8
73
—
—
3
1/3

1419.17
11
PLVCNGVLQGV
HuK2.216.V11
PSA.212.V11
27
127
19
255
4314
3
2/2
2/2

[0543]

37

TABLE XXV

DR supermotif and DR3 motif-bearing peptides

cross-reactive binding peptides

DR supermotif
DR3

Antigen
Motif+
Algorithm+*
Motif+

PAP
67
39/15
21

PSM
45
25/7
4

PSA
108
54/20
31

HuK2
45
21/6
4

Total
265
139/48
60

*Number scoring positive in the combined DR1, DR4w4 and DR7 algorithms (≧1/≧2)

Inducing cellular immune responses to prostate cancer antigens using peptide and nucleic acid compositions

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

PCT Information