EP1242049A1 - Compositions de vaccins et peptides d'antigene associes a une tumeur de hla-a2 de classe i - Google Patents

Compositions de vaccins et peptides d'antigene associes a une tumeur de hla-a2 de classe i

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Publication number
EP1242049A1
EP1242049A1 EP00986510A EP00986510A EP1242049A1 EP 1242049 A1 EP1242049 A1 EP 1242049A1 EP 00986510 A EP00986510 A EP 00986510A EP 00986510 A EP00986510 A EP 00986510A EP 1242049 A1 EP1242049 A1 EP 1242049A1
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EP
European Patent Office
Prior art keywords
seq
peptide
epitope
hla
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00986510A
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German (de)
English (en)
Other versions
EP1242049A4 (fr
Inventor
John Fikes
Alessandro Sette
John Sidney
Scott Southwood
Esteban Celis
Elissa Keogh
Robert Chesnut
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Epimmune Inc
Original Assignee
Epimmune Inc
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Publication date
Priority claimed from US09/543,608 external-priority patent/US6602510B1/en
Application filed by Epimmune Inc filed Critical Epimmune Inc
Publication of EP1242049A1 publication Critical patent/EP1242049A1/fr
Publication of EP1242049A4 publication Critical patent/EP1242049A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001148—Regulators of development
    • A61K39/00115—Apoptosis related proteins, e.g. survivin or livin
    • A61K39/001151—Apoptosis related proteins, e.g. survivin or livin p53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001182—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001186—MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to the field of biology. In a particular embodiment, it relates to compositions useful to monitor or elicit an immune response to selected tumor-associated antigens.
  • HLA Human Leukocyte Antigen
  • PSA prostate specific antigen
  • CTL cytotoxic T-cell lymphocyte
  • Rosenberg, et al evaluated the safety and mechanism of action of a synthetic HLA-A2 binding peptide derived from the melanoma associated antigen, gplOO, as a cancer vaccine to treat patients with metastatic melanoma (Rosenberg et al, Nature Med., 4:321-327 (1998)).
  • Another aspect of the invention is a vaccine composition
  • a vaccine composition comprising a unit dose of a peptide that comprises less than 50 contiguous amino acids that have 100% identity with a native peptide sequence of CEA, HER2/neu, MAGE2, MAGE3, or p53, the peptide comprising an epitope selected from the group consisting of: VLYGPDAPTV (SEQ ID NO: 1), YLSGANLNV (SEQ ID NO:2), ATVGIMIGV (SEQ ID NO:3), LLPENNVLSPV (SEQ ID NO:4) KLCPVQLWV (SEQ ID NO:5), KLBPVQLWV (SEQ ID NO:6), SLPPPGTRV (SEQ ID NO:7), SMPPPGTRV (SEQ ID NO:8), KLFGSLAFV (SEQ ID NO:9), KVFGSLAFV (SEQ ID NO: 10), VMAGVGSPYV (SEQ ID NO: 11),ALCRWGLLL (SEQ ID
  • LLTFWNPPV (SEQ ID NO: 19), LVFGIELMEV (SEQ ID NO:20), QLVFGIELMEV (SEQ ID NO:21), RLLQETELV (SEQ ID NO:22), VVLGWFGI (SEQ ID NO:23), YLQLVFGIEV (SEQ ID NO:24), and YMIMVKCWMI (SEQ ID NO:25); and a pharmaceutical excipient.
  • the epitope is YLSGANLNV (SEQ ID NO:2), or KLBPVQLWV (SEQ ID NO:6), or SMPPPGTRV (SEQ ID NO:8).
  • the vaccine composition can further comprise an additional epitope, which can be a PanDR binding molecule, and can comprise a liposome, wherein the at least one epitope is on or within the epitope.
  • the pharmaceutical excipient comprises and adjuvant.
  • the vaccine can further comprise an antigen-presenting cell.
  • the epitope can be bound to an HLA molecule on the antigen-resenting cell, whereby when an A2 supertype-restricted cytotoxic T lymphocyte (CTL) is present, a receptor of the CTL binds to a complex of the HLA molecule and the epitope.
  • the antigen-presenting cell can be a dendritic cell.
  • PBMC peripheral blood mononuclear cells
  • donors 431, 397, and 344 were stimulated in vitro.
  • Ficoll-Paque (Pharmacia LKB) purified PBMC were plated at 4 x 10 6 cells/well in a 24-well tissue culture plate (Costar). The peptides were added at a final concentration of 10 ⁇ g/ml and incubated at 37°C for 4 days.
  • Recombinant interleukin-2 was added at a final concentration of 10 ng/ml and the cultures were fed every three days with fresh media and cytokine. Two additional stimulations of the T cells with antigen were performed on approximately days 14 and 28.
  • T cells (3 x 10 5 cells/well) were restimulated with 10 ⁇ g/ml peptide using irradiated (7500 rads) autologous PBMC cells. T cell proliferative responses were determined using a 3 H-thymidine incorporation assay.
  • FIG. 2 depicts that PADRE-specific proliferative responses are induced via peptide vaccination.
  • the proliferation index of multiple wells was calculated as the mean cpm from experimental wells divided by the mean cpm from control wells. PADRE-specific responses were considered positive when the proliferation index exceeded 5.
  • FIG. 3 depicts that splenic DC from ProGP -treated mice present HBV-derived CTL epitopes to a CTL line.
  • l/K b -H-2 bxs transgenic mice (33 ⁇ g/animal, QD, SC for 7 days) were enriched using an anti-CD 1 lc antibody (Miltenyi Biotec).
  • B cells were isolated from normal spleen by magnetic separation after treating cells with biotinylated anti-CD 19 antibody and Strepavidin-coupled beads (Miltenyi Biotec). DC were also generated from bone marrow cells by culture with GM-CSF/IL-4.
  • DC or B cells (1 x 10 5 cells) were incubated with 1 x 10 4 CTL line 1168 and varying concentrations of the HBV Pol 455 peptide in Opti-MEM I medium containing 3 ⁇ g/ml ⁇ 2-microglobulin (Scripps Laboratories). Cells were added to 96-flat bottom well ELISA plates that were pre-coated with an anti-IFN ⁇ capture antibody. After incubation for 18-20 hr at 37 °C, in situ production of IFN ⁇ by stimulated line 1168 was measured using a sandwich ELISA. Data shown is from one experiment. Similar results have been obtained in additional experiments. Studies were performed at Epimmune Inc., San Diego, CA. Figure 4 depicts that splenic DC from ProGP-treated mice induce CTL responses in vivo.
  • Figure 5 presents a schematic of a dendritic cell pulsing and testing procedure.
  • This invention provides a plurality of peptide epitopes that can be used to monitor an immune response to a tumor associated antigen or when one or more peptides are combined to create a cancer vaccine that stimulates the cellular arm of the immune system.
  • vaccines mediate immune responses against tumors in individuals who bear an allele of the HLA-A2 supertype (see Table 5 for a listing of the members of the A2 and other supertypes); such vaccines will generally be referred to as A2 vaccines.
  • An A2 vaccine stimulates the immune system to recognize and kill tumor cells, leading to increased quality of life, and/or disease-free or overall survival rates for patients treated for cancer.
  • an A2 vaccine will be administered to HLA-A2 or HLA-A2 supertype positive individuals with any cancer that expresses at least one of the TAAs from which vaccine epitopes were selected, such as breast, colon or lung cancer.
  • Alternative embodiments of a vaccine are directed at patients who bear additional HLA alleles ⁇ or are not directed to A2 at all.
  • an A2 vaccine improves the standard of care for patients being treated for breast, colon or lung cancer.
  • the peptide epitopes and corresponding nucleic acid compositions of the present invention are useful for stimulating an immune response to TAAs 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 TAAs.
  • the complete sequence of the TAAs 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 TAAs, as will be clear from the disclosure provided below.
  • the peptide epitopes of the invention have been identified in a number of ways, as will be discussed below.
  • 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.
  • 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.
  • binding data results are often expressed in terms of "ICso's.”
  • IC 5 o 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 K D values.
  • Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205, and other publications such Sidney et al, Current Protocols in Immunology 18.3.1 (1998); Sidney, et al, J. Immunol.
  • IC 50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used ⁇ e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC 50 of a given ligand.
  • binding is expressed relative to a reference peptide. Although as a particular assay becomes more, or less, sensitive, the ICso's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change.
  • Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al, Nature 339:392 (1989); Christnick et al, Nature 352:67 (1991); Busch et al, Int. Immunol 2:443 (1990); Hill et al, J.
  • a “computer” or “computer system” generally includes: a processor and related computer programs; at least one information storage/retrieval apparatus such as a hard drive, a disk drive or a tape drive; at least one input apparatus such as a keyboard, a mouse, a touch screen, or a microphone; and display structure, such as a screen or a printer. 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. "Cross-reactive binding" indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • 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.
  • the term "derived" when used to discuss an epitope is a synonym for "prepared.”
  • a derived epitope can be isolated from a natural source, or it can be synthesized in accordance with standard protocols in the art. Synthetic epitopes can comprise artificial amino acids "amino acid mimetics," such as D isomers of natural occurring L amino acids or non-natural amino acids such as cyclohexylalanine.
  • a derived/prepared epitope can be an analog of a native epitope.
  • 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.
  • 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.
  • an epitope can be defined as 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.
  • Epitopes are present in nature, and can be isolated, purified or otherwise prepared/derived by humans. For example, epitopes can be prepared by isolation from a natural source, or they can be synthesized in accordance with standard protocols in the art.
  • Synthetic epitopes can comprise artificial amino acids "amino acid mimetics,” such as D isomers of natural occurring L amino acids or non-natural amino acids such as cyclohexylalanine. Throughout this disclosure, the terms epitope and peptide are often used interchangeably.
  • protein or peptide molecules that comprise an epitope of the invention as well as additional amino acid(s) are still within the bounds of the invention.
  • there is a limitation on the length of a peptide of the invention. The embodiment that is length-limited occurs when the protein/peptide comprising an epitope of the invention comprises a region (i.e., a contiguous series of amino acids) having 100% identity with a native sequence.
  • a region i.e., a contiguous series of amino acids
  • 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.
  • an "epitope" of the invention is comprised by a peptide having a region with less than 51 amino acids that has 100% identity to a native peptide sequence, in any increment down to 5 amino acids.
  • peptide or protein sequences longer than 600 amino acids are within the scope of the invention, so long as they do not comprise any contiguous sequence of more than 600 amino acids that have 100% identity with a native peptide sequence.
  • a CTL epitope be less than 600 residues long in any increment down to eight amino acid residues.
  • MHC Histocompatibility Complex
  • HLA supertype or HLA family 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 such HLA supertypes.
  • HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules are synonyms.
  • high affinity with respect to HLA class I molecules is defined as binding with an IC 50 , or K D value, of 50 nM or less; “intermediate affinity” is binding with an IC 50 or K D 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 IC 50 or K D value of 100 nM or less; “intermediate affinity” is binding with an IC 50 or K D value of between about 100 and about 1000 nM.
  • ICso 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 K D values.
  • immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T lymphocyte (CTL) response, or a helper T lymphocyte (HTL) response, to the peptide.
  • CTL cytotoxic T lymphocyte
  • HTL helper T lymphocyte
  • 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.
  • isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • An “isolated” epitope refers to an epitope that does dot include the whole sequence of the antigen or polypeptide from which the epitope was derived. Typically the "isolated” epitope does not have attached thereto additional amino acids that result in a sequence that has 100% identity with a native sequence.
  • the native sequence can be a sequence such as a tumor-associated antigen from which the epitope is derived.
  • MHC Major Histocompatibility Complex
  • HLA human leukocyte antigen
  • motif refers to a pattern of residues in an amino acid sequence of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs often differ in their pattern of the primary and secondary anchor residues.
  • a “native” sequence refers to a sequence found in nature.
  • 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.
  • 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 ⁇ -amino and carboxyl groups of adjacent amino acids.
  • a "PanDR binding peptide” or “PADRETM” molecule is a member of a family of molecules that binds more than one HLA class II DR molecule.
  • the pattern that defines the PADRETM family of molecules can be referred to as an HLA Class II supermotif.
  • a PADRE molecule binds to HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses.
  • HTL human helper T lymphocyte
  • “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition.
  • a “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH- adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.
  • 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, two or three, primary anchor residues within a peptide of defined length generally defines a "motif for an immunogenic peptide.
  • the primary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a peptide epitope in accordance with the invention.
  • the primary anchor positions for each motif and supermotif of HLA Class I and HLA Class II are set forth in Table 2, Table 3 and Table 4.
  • analog peptides can be created by altering the presence or absence of particular residues in these anchor positions. Such analogs are used to modulate the binding affinity of a peptide comprising a particular motif or supermotif.
  • Promiscuous recognition by a TCR is where a distinct peptide is recognized by the various T cell clones in the context of various HLA molecules. Promiscuous binding by an HLA molecule is synonymous with cross-reactive binding.
  • a “protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from an pathogenic antigen (e.g., an antigen from an infectious agent or a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression.
  • the immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • residue refers to an amino acid or amino acid mimetic incorporated into a peptide or protein by an amide bond or amide bond mimetic.
  • 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 HLA-bound peptides than would be expected by random distribution of amino acids at a given position.
  • 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.
  • the secondary anchor residues are said to occur at "secondary anchor positions.”
  • 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.
  • the terminology "fixed peptide" is sometimes used to refer to an analog peptide.
  • 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.
  • a "supermotif is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.
  • a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • Synthetic peptide refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • a "vaccine” is a composition that contains one or more peptides of the invention, see, e.g., Table 6, Table 9 and Table 10.
  • vaccines in accordance with the invention, such as by a cocktail of one or more peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide.
  • the "one or more peptides” can include any whole unit integer from l-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 linked to HLA class II-binding peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes.
  • Vaccines can comprise peptide pulsed antigen presenting cells, e.g., dendritic cells.
  • the nomenclature used to describe peptide/protein 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.
  • 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.
  • the amino- and carboxyl- terminal groups although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified.
  • 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
  • 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.
  • APC Antigen presenting cell
  • CEA Carcinoembryonic antigen
  • CTL Cytotoxic T lymphocyte DC Dendritic cells. DC functioned as potent antigen presenting cells by stimulating cytokine release from CTL lines that were specific for a model peptide derived from hepatitis B virus. In vivo experiments using DC pulsed ex vivo with an HBV peptide epitope have stimulated CTL immune responses in vivo following delivery to naive mice.
  • DLT Dose-limiting toxicity, an adverse event related to therapy.
  • G-CSF Granulocyte colony-stimulating factor
  • GM-CSF Granulocyte-macrophage (monocyte)-colony stimulating factor
  • HBV Hepatitis B virus
  • HER2/neu A tumor associated antigen
  • c-erbB-2 is a synonym.
  • HLA Human leukocyte antigen
  • HLA-DR Human leukocyte antigen class II
  • HTL Helper T Lymphocyte. A synonym for HTC.
  • IFN ⁇ Interferon gamma
  • IL-4 Interleukin-4
  • LU 30 % Cytotoxic activity for 10 6 effector cells required to achieve 30% lysis of a target cell population, at a 100:1 (E:T) ratio.
  • MAb Monoclonal antibody
  • MNC Mononuclear cells
  • PBMC Peripheral blood mononuclear cell
  • ProGP TM ProgenipoietinTM (Searle, St. Louis, MO), a chimeric flt3/G-CSF receptor agonist.
  • TAA Tumor Associated Antigen
  • TNF Tumor necrosis factor
  • WBC White blood cells IV.B. Stimulation of CTL and HTL responses
  • T cells recognize antigens have been elucidated during the past ten years.
  • efficacious peptide epitope compositions that induce a therapeutic or prophylactic immune response to TAA, when administered via various art-accepted modalities.
  • These peptides can also be used diagnostically, e.g., to evaluate the immune response to an antigen.
  • responses can be achieved in significant percentages of a non-genetically biased worldwide population.
  • 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).
  • critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Tables 2, 3, and 4.
  • the A2 supermotif and the allele-specific A2.1 motif due to the substantial population coverage they provide.
  • class I and class II allele-specific HLA binding motifs allows identification of regions within a protein that have the predicted ability to bind particular HLA antigen(s).
  • epitope-based vaccines have been identified.
  • additional work can be performed to select, amongst these vaccine peptides, e.g., epitopes can be selected having optional characteristics in terms of population coverage, antigenicity, and immunogenicity, etc.
  • Peptide-specific T cells are detected using, e.g., a ⁇ Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen. 3) Demonstration of recall T cell responses from individuals exposed to the disease, such as immune individuals who were effectively treated and recovered from disease, and/or from actively ill patients (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.
  • recall responses are detected by culturing PBL from subjects in vitro for 1-2 weeks in the presence of a test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells.
  • APC antigen presenting cells
  • T cell activity is detected using assays for T cell activity including * *Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
  • 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.
  • CTL-inducing peptides of interest for vaccine compositions preferably include those that have an IC 50 or binding affinity value for a class I HLA molecule(s) of 500 nM or better ⁇ i.e., the value is ⁇ 500 nM).
  • HTL- inducing peptides preferably include those that have an IC 50 or binding affinity value for class II HLA molecules of 1000 nM or better, ⁇ i.e., the value is ⁇ 1,000 nM).
  • peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are generally tested on other members of the supertype family.
  • peptides that exhibit cross-reactive binding are then used in cellular screening analyses or vaccines.
  • the relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens was determined for the first time in the art by the present inventors. As disclosed in greater detail herein, higher HLA binding affinity is correlated with greater immunogenicity. Greater immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited.
  • a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response.
  • close to 90% of high binding peptides have been found to elicit a response and thus be "immunogenic," as contrasted with about 50% of the peptides that bind with intermediate affinity.
  • the generated response tended to be more vigorous than the response seen with weaker binding peptides.
  • less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used rather than a lower affinity one.
  • high affinity binding epitopes are used.
  • binding affinity and immunogenicity was analyzed by the present inventors by two different experimental approaches ⁇ see, e.g., Sette, et al, J. Immunol 153:5586- 5592 (1994)).
  • 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.
  • 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.
  • HBV hepatitis B virus
  • an affinity threshold value of approximately 500 nM determines the capacity of a peptide epitope to elicit a CTL response.
  • HLA class II ⁇ i.e., HLA DR
  • HLA DR HLA DR
  • HLA DR restriction was associated with high binding affinities, i.e.
  • 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 IC 5 o of 1000 nM or greater. Thus, 1000 nM is defined as an affinity threshold associated with immunogenicity in the context of DR molecules.
  • All 240 peptides 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 values 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 demonstrated the value of motifs for identification of peptide epitopes to be included in a vaccine.
  • Vaccines of the present invention may also comprise epitopes that bind to MHC class II DR molecules.
  • PI position 1
  • Other studies have also pointed to an important role for the peptide residue in the sixth position towards the C-terminus, relative to PI, for binding to various DR molecules.
  • HLA class I and class II molecules can be classified into a relatively few supertypes, each respective supertype characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets.
  • peptides of the present invention are preferably identified by any one of several HLA-specific amino acid motifs ⁇ see, e.g., Tables 2-4), or if the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens, a supermotif ⁇ again see, e.g., Tables 2-4).
  • the primary anchor residues of the HLA class I peptide epitope supermotifs and motifs are summarized in Table 2.
  • the HLA class I motifs set out in Table 2(a) are particularly relevant to the invention claimed here.
  • Primary and secondary anchor positions for HLA Class I are summarized in Table 3.
  • Allele-specific HLA molecules that are comprised by the various HLA class I supertypes are listed in Table 5.
  • patterns of amino acid residues are present in both a motif and a supermotif. The relationship of a particular motif and any related supermotif is indicated in the description of the individual motifs.
  • HLA-A2 supermotif which when present 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.
  • the corresponding family of HLA molecules ⁇ i.e., the HLA-A2 supertype that binds these peptides) is comprised of at least: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209, A*0214, A*6802, and A*6901.
  • Other allele-specific HLA molecules predicted to be members of the A2 superfamily are shown in Table 5.
  • 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.
  • HLA-A*0201 motif 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, March 6, 1992; Parker et al, J. Immunol.
  • 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).
  • 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 less preferred tolerated residues that characterize the primary anchor positions of the HLA-A*0201 motif are identical to the residues describing the A2 supermotif.
  • HLA class II peptide epitope supermotifs and motifs are summarized in Table 4. Also see, U.S. Patent 5,736,142, and a co-pending application entitled Alteration Of Immune Responses Using Pan DR Binding Peptides, U.S.S.N. 09/310,462, filed 12 May 1999.
  • CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few "immunodominant" determinants (Zinkemagel, 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).
  • dominance and subdominance are relevant to immunotherapy of both infectious diseases and malignancies.
  • recruitment of subdominant epitopes can be important for successful clearance of the infection, especially if dominant CTL or HTL specificities have been inactivated by functional tolerance, suppression, mutation of viruses and other mechanisms (Franco, et al, Curr. Opin. Immunol. 7:524-531, 1995).
  • CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.
  • TAA tumor associated antigens
  • TIL tumor infiltrating lymphocytes
  • CTL CTL bound in the 50-500 nM range.
  • T cells to dominant epitopes may have been clonally deleted, and selecting subdominant epitopes may allow existing T cells to be recruited, which will then lead to a therapeutic or prophylactic response.
  • the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones.
  • peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screening procedures described above, cross-reactivity is not always as complete as possible, and in certain cases procedures to increase cross-reactivity of peptides can be useful; moreover, such procedures can also be used to modify other properties of the peptides such as binding affinity or peptide stability. Having established the general rules that govern cross-reactivity of peptides for HLA alleles within a given motif or supermotif, modification ⁇ i.e., analoging) of the structure of peptides of particular interest in order to achieve broader (or otherwise modified) HLA binding capacity can be performed.
  • peptides that 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.S.N. 09/226,775 filed 6 January 1999.
  • the analoging strategy utilizes the motifs or supermotifs that correlate with binding to certain HLA molecules.
  • Analog peptides can be created by substituting amino acid residues at primary anchor, secondary anchor, or at primary and secondary anchor positions.
  • analogs are made for peptides that already bear a motif or supermotif.
  • preferred primary and secondary anchor residues of supermotifs and motifs for HLA class I and HLA class II binding peptides are shown in Tables 3 and 4, respectively.
  • 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 3 and 4).
  • one strategy to improve the cross-reactivity of peptides withm 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)
  • a small "neutral" residue such as Ala (that may not influence T cell recognition of the peptide)
  • An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, "preferred" residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted
  • the analog peptide when used as a vaccme, 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 induce T cells in vitro from individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to lyse wild type peptide sensitized target cells is evaluated. Alternatively, evaluation of the cells' activity can be evaluated by monitoring IFN release Each of these cell monitoring strategies evaluate the recognition of the APC by the CTL.
  • antigen presenting cells cells that have been either infected, or transfected with the approp ⁇ ate genes, or, (generally only for class II epitopes, due to the different peptide processing pathway for HLA class II), cells that have been pulsed with whole protein antigens, to establish whether endogenously produced antigen is also recognized by the T cells mduced by the analog peptide
  • peptide/protein-pulsed dendritic cells can be used to present whole protein antigens for both HLA class I and class II
  • Another embodiment of the invention is to create analogs of weak bmding peptides, to thereby ensure adequate numbers of cellular binders.
  • Class I binding peptides exhibiting binding affinities of 500-5000 nM, and carrymg an acceptable but suboptunal primary anchor residue at one or both positions can be "fixed” by substituting preferred anchor residues m accordance with the respective supertype The analog peptides can then be tested for binding and/or cross-bindmg capacity.
  • Another embodiment of the invention is to create analogs of peptides that are already cross-reactive bmders and are vaccine candidates, but which bmd weakly to one or more alleles of a supertype
  • the cross-reactive binder carries a suboptunal residue (less preferred or deleterious) at a primary or secondary anchor position
  • the peptide can be analoged by substituting out a delete ⁇ ous residue and replacing it with a preferred or less preferred one, or by substituting out a less preferred reside and replacmg it with a preferred one
  • the analog peptide can then be tested for cross-binding capacity.
  • 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
  • a cysteine (C) can be substituted out in favor of ⁇ -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.
  • amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like.
  • Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide, particularly class I peptides. 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.
  • the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH 2 acylation, e.g., by alkanoyl (C C 2 o) or thioglycolyl acetylation, teiminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.
  • Peptides in accordance with the invention can be prepared synthetically, by recombinant
  • 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.
  • 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 can contain modifications such as glycosylation, side chain oxidation, or phosphorylation, generally subject to the condition that modifications do not destroy the biological activity of the peptides.
  • the peptides of the invention can be prepared in a wide variety of ways.
  • 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).
  • individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
  • 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.
  • recombinant polypeptides which comprise one or more peptide sequences of the invention, can be used to present the appropriate T cell epitope.
  • 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.
  • 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.
  • 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.
  • yeast, insect or mammalian cell hosts may also be used, employing suitable vectors and control sequences. It is generally preferable that the peptide epitope be as small as possible while still maintaining substantially all of the immunologic activity of the native protein.
  • HLA class I binding peptide epitopes of the invention may be desirable to optimize HLA class I binding peptide epitopes of the invention to a length of about 8 to about 13 amino acid residues, preferably 9 to 10. It is to be appreciated that one or more epitopes in this size range can be comprised by a longer peptide (see the Definition Section for the term "epitope" for further discussion of peptide length).
  • HLA class II binding epitopes are preferably optimized to a length of about 6 to about 30 amino acids in length, preferably to between about 13 and about 20 residues.
  • the epitopes are commensurate in size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules.
  • the identification and preparation of peptides of various lengths can be carried out using the techniques described herein.
  • An alternative preferred embodiment of the invention comprises administration of peptides of the invention linked as a polyepitopic peptide, or as a minigene that encodes a polyepitopic peptide.
  • Another preferred embodiment is obtained by identifying 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 frame-shifted 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.
  • a larger, preferably multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source.
  • HLA binding peptides 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, e.g., 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 relevant HLA proteins. These assays may involve evaluation of peptide binding 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.
  • cell surface HLA molecules that lack any bound peptide 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 an HLA 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 pathology.
  • HLA class II binding peptides are used for evaluation of HLA class II binding peptides.
  • HLA class II motif-bearing peptides that are shown to bind are further evaluated for the ability to stimulate HTL responses.
  • T cell responses include proliferation assays, lymphokine secretion assays, direct cytotoxicity assays, and limiting dilution assays.
  • 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.
  • mutant, non-human mammalian cell lines that have been transfected with a human class I MHC gene, and that are deficient in their ability to load class I molecules with internally processed peptides, are used to evaluate the capacity of the peptide to induce in vitro primary CTL responses.
  • PBMCs Peripheral blood mononuclear cells
  • 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 lyse radio-labeled target cells, either specific peptide-pulsed targets or target cells that express endogenously processed antigen from which the specific peptide was derived.
  • the presence of epitope-specific CTLs can be determined by IFN ⁇ in situ ELISA.
  • HTL activation may also be assessed using techniques known to those in the art, such as T cell proliferation or lymphokine secretion ⁇ see, e.g. Alexander et al, Immunity 1 :751-761, 1994).
  • immunization of HLA transgenic mice can be used to determine immunogenicity of peptide epitopes.
  • transgenic mouse strains e.g., mice with human A2.1, Al l (which can additionally be used to analyze HLA-A3 epitopes), and B7 alleles have been characterized.
  • Other transgenic mice strains e.g., transgenic mice for HLA-A1 and A24
  • HLA-DR1 and HLA-DR3 mouse models have been developed. In accordance with principles in the art, additional transgenic mouse models with other HLA alleles are generated as necessary.
  • mice can be immunized with peptides emulsified in Incomplete Freund's Adjuvant; thereafter any resulting T cells can be tested for their capacity to recognize target cells that have been peptide-pulsed or transfected with genes encoding the peptide of interest.
  • CTL responses can be analyzed using cytotoxicity assays described above.
  • HTL responses can be analyzed using, e.g., T cell proliferation or lymphokine secretion assays.
  • HLA class I and class II binding peptides can be used as reagents to evaluate an immune response.
  • the evaluated immune response can be induced by any immunogen.
  • the immunogen may result in the production of antigen-specific CTLs or HTLs that recognize the peptide e ⁇ itope(s) employed as the reagent.
  • a peptide of the invention may or may not be used as the immunogen.
  • Assay systems that can be used for such analyses include tetramer-based protocols, staining for intracellular lymphokines, interferon release assays, or ELISPOT assays.
  • a peptide of the invention can be used in a tetramer staining assay to assess peripheral blood mononuclear cells for the presence of any antigen-specific CTLs.
  • the HLA-tetrameric complex is used to directly visualize antigen-specific CTLs and thereby determine the frequency of such antigen-specific CTLs in a sample of peripheral blood mononuclear cells ⁇ see, e.g., Ogg et al, Science 279:2103-2106, 1998; and Altaian et al, Science 174:94- 96, 1996).
  • a tetramer reagent comprising 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 ⁇ 2 -microglobulin to generate a trimolecular complex. The complex is biotinylated at the carboxyl terminal end of the HLA heavy chain, at a site that was previously engineered into the protein. Tetramer formation is then induced by adding streptavidin. When fluorescently labeled streptavidin is used, the tetrameric complex is used to stain antigen-specific cells. The labeled cells are then readily identified, e.g., by flow cytometry. Such procedures are used for diagnostic or prognostic purposes; the cells identified by the procedure can be used for therapeutic purposes.
  • 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.
  • a PBMC sample from an individual expressing a disease- associated antigen e.g. a tumor-associated antigen such as CEA, p53, MAGE2/3,HER2neu, or an organism associated with neoplasia such as HPV or HS V
  • a blood sample containing mononuclear cells may 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 may be analyzed, for example, for CTL or for HTL activity.
  • the peptides can be used to evaluate the efficacy of a vaccine.
  • PBMCs obtained from a patient vaccinated with an immunogen may be analyzed by methods such as those described herein.
  • the patient is HLA typed, and peptide epitopes that are bound by the HLA molecule(s) present in that patient are selected for analysis.
  • the immunogenicity of the vaccine is indicated by the presence of CTLs and/or HTLs directed to epitopes present in the vaccine.
  • the peptides of the invention may also be used to make antibodies, using techniques well known in the art (see, e.g. CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY; and Antibodies A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor Laboratory Press, 1989). Such antibodies are useful as reagents to determine the presence of disease-associated antigens. Antibodies in this category include those that recognize a peptide when bound by an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex.
  • Vaccines that contain an immunologically effective amount of one or more peptides of the invention are a further embodiment of the invention.
  • the peptides can be delivered by various means or formulations, all collectively referred to as "vaccine" compositions.
  • Such vaccine compositions, and/or modes of administration can include, for example, naked cDNA in cationic lipid formulations; lipopeptides (e.g.,Vitiello, A. et al., J. Clin. Invest.
  • naked cDNA or peptides encapsulated e.g., 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.
  • ISCOMS immune stimulating complexes
  • MAPs multiple antigen peptide systems
  • MAPs multiple antigen peptide systems
  • viral, bacterial, or, fungal delivery vectors See e.g., Tam, J. P., Proc. Natl Acad. Sci. U.S.A. 85:5409-5413, 1988; Tarn, J.P., J. Immunol. Methods 196:17-32, 1996
  • viral, bacterial, or, fungal delivery vectors Perkus, M. E. et al, In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996
  • Vaccines of the invention comprise nucleic acid mediated modalities.
  • DNA or RNA encoding one or more of the peptides of the invention can be administered to a patient.
  • This approach is described, for instance, in Wolff et. al, Science 247:1465 (1990) as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and, WO 98/04720.
  • 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. Patent No. 5,922,687).
  • peptide vaccines of the invention can be expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox.
  • vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention.
  • the recombinant vaccinia virus Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848.
  • 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 vims vectors, alpha vims vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, are apparent to those skilled in the art from the description herein.
  • vaccines in accordance with the invention can comprise one or more peptides of the invention.
  • 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 probability for immunological reaction and, where different peptide epitopes are used to make up the polymer, the ability to induce antibodies and/or T cells that react with different antigenic determinants of the antigen targeted for an immune response.
  • the composition may be a naturally occurring region of an antigen or can be prepared, e.g. , recombinantly or by chemical synthesis.
  • 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 vims proteins, hepatitis B vims core protein, and the like.
  • the vaccines can contain a physiologically tolerable diluent such as water, or a saline solution, preferably phosphate buffered saline.
  • the vaccines also 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-glyceryl-cysteinyl-seryl-serine (P 3 CSS).
  • P 3 CSS tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine
  • a peptide composition in accordance with the invention Upon immunization with a peptide composition in accordance with the invention, via injection (e.g., SC, ID, IM), aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing antibodies, CTLs and or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to subsequent exposure to the TAA, or at least partially resistant to further development of TAA-bearing cells and thereby derives a prophylactic or therapeutic benefit.
  • injection e.g., SC, ID, IM
  • aerosol e.g., oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes
  • the immune system of the host responds to the vaccine by producing antibodies, CTLs and or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to subsequent exposure to the TAA,
  • components that induce T cell responses are combined with components that induce antibody 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.
  • a composition comprises a class I and/or class II epitope in accordance with the invention, along with a PADRETM molecule (Epimmune, San Diego, CA).
  • Vaccine of the invention can comprise antigen presenting cells, such as dendritic cells, as a vehicle to present peptides of the invention.
  • dendritic cells are transfected, e.g., with a minigene construct in accordance with the invention, in order to elicit immune responses. Minigenes are discussed in greater detail in a following section.
  • Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro.
  • the vaccine compositions of the invention may also be used in combination with antiviral drugs such as interferon- , or immune adjuvants such as IL-12, GM-CSF, etc.
  • antiviral drugs such as interferon-
  • immune adjuvants such as IL-12, GM-CSF, etc.
  • the following principles are utilized when selecting epitope(s) for inclusion in a vaccine, either peptide-based or nucleic acid-based formulations.
  • Exemplary epitopes that may be utilized in a vaccine to treat or prevent TAA-associated disease are set out in Table 6. Each of the following principles can be balanced in order to make the selection.
  • the epitopes may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with prevention or clearance of TAA-expressing tumors. For HLA Class I, this generally includes 3-4 epitopes derived from at least one TAA. 2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less. For HLA Class I it is presently preferred to select a peptide having an IC 50 of 200 nM or less, as this is believed to better correlate not only to induction of an immune response, but to in vitro tumor cell killing as well.
  • Supermotif bearing-peptides or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. In general, 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 of population coverage.
  • Nested epitopes occur where at least two epitopes overlap in a given peptide sequence.
  • a peptide comprising "transcendent nested epitopes” is a peptide that has both HLA class I and HLA class II epitopes in it.
  • 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.
  • 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
  • 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.
  • nucleic acids encoding multiple epitopes are a useful embodiment of the invention; discrete peptide epitopes or polyepitopic peptides can be encoded.
  • the epitopes to be included in a minigene are preferably selected according to the guidelines set forth in the previous section.
  • HLA class I epitopes HLA class II epitopes
  • a ubiquitination signal sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.
  • ER endoplasmic reticulum
  • multi-epitope minigenes are also described in, e.g., co-pending application U.S.S.N. 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.
  • a multi-epitope DNA plasmid encoding nine dominant HLA-A*0201- and Al 1-restricted CTL epitopes derived from the polymerase, envelope, and core proteins of HBV and human immunodeficiency vims (HIV), a PADRETM universal helper T cell (HTL) epitope, and an endoplasmic reticulum-translocating signal sequence has been engineered. Immunization of HLA transgenic mice with this plasmid construct resulted in strong CTL induction responses against the nine CTL epitopes tested.
  • 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.
  • additional elements can be incorporated into the minigene design such as spacer amino acid residues between epitopes.
  • 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 e ⁇ itope(s) are within the scope of the invention.
  • 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.
  • 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.
  • a promoter with a downstream 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. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • Optimized peptide expression and immunogenicity can be achieved by certain modifications to a minigene construct.
  • introns facilitate efficient gene expression, thus one or more synthetic or naturally-occurring introns can 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.
  • the minigene is cloned into the polylinker region downstream of the promoter.
  • This plasmid is transformed into an appropriate bacterial 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 cell banks.
  • immunostimulatory sequences appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence to enhance immunogenicity.
  • a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (e.g., one that modulates immunogenicity) can be used.
  • proteins or polypeptides that, if co-expressed with epitopes, can enhance an immune response include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or pan-DR binding proteins (PADRETM, Epimmune, San Diego, CA).
  • Helper T cell (HTL) epitopes such as PADRE molecules can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes.
  • HTL epitopes This can be done in order to direct HTL epitopes to a cell compartment different than that of the CTL epitopes, one that provides for more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction.
  • immunosuppressive molecules ⁇ e.g. TGF- ⁇
  • TGF- ⁇ immunosuppressive molecules
  • 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 are grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA is purified using standard bioseparation technologies such as solid phase anion-exchange resins available, e.g., from QIAGEN, Inc. (Valencia, California). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as "naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene vaccines, alternative methods of formulating purified plasmid DNA may be used. A variety of such 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., WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988);
  • peptides and compounds referred to collectively as protective, interactive, non-condensing compounds can also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • Target cell sensitization can be used as a functional assay of the expression and HLA class I presentation of minigene-encoded epitopes.
  • the plasmid DNA is introduced into a mammalian cell line that is a suitable 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).
  • FACS fluorescence activated cell sorting
  • the transfected cells are then chromium-51 ( 51 Cr) labeled and used as targets for epitope- specific CTLs.
  • Cytolysis of the target cells indicates both the production 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.
  • 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).
  • splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTLs, standard assays are conducted to determine if there is cytolysis of pep tide-loaded, Cr-labeled target cells. Once again, lysis of target cells that were exposed to epitopes corresponding to those in the minigene, demonstrates DNA vaccine function and induction of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic mice in an analogous manner.
  • the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment for ballistic delivery, DNA can be adhered to particles, such as gold particles.
  • Combinations of CTL Peptides with Helper Peptides Vaccine compositions comprising CTL peptides of the present invention can be modified to provide desired attributes, such as improved serum half-life, broadened population coverage or enhanced immunogenicity.
  • the ability of a peptide to induce CTL activity can be enhanced by linking the CTL peptide to a sequence which contains at least one HTL epitope.
  • T helper epitopes in conjunction with CTL epitopes to enhance immunogenicity is illustrated, for example, in co-pending applications U.S.S.N. 08/820,360, U.S.S.N. 08/197,484, and U.S.S.N. 08/464,234.
  • CTL epitope/HTL epitope conjugates are linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, e.g., 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 optional 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, commonly three to 13, more frequently three to six residues.
  • the CTL peptide epitope may be linked to the T helper peptide epitope, directly or via a spacer, at either it's amino or carboxyl terminus.
  • the amino terminus of either the CTL peptide or the HTL peptide can be acylated.
  • 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.
  • amino acid sequences that are promiscuous include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:26), Plasmodiumfalciparum CS protein at positions 378-398 (DIEK__OAKMEKASSVFNVVNS; SEQ ID NO:27), and Streptococcus 18kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO:28).
  • antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:26), Plasmodiumfalciparum CS protein at positions 378-398 (DIEK__OAKMEKASSVFNVVNS; SEQ ID NO:27), and Streptococcus 18kD protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO:28).
  • Other examples include peptides bearing
  • Synthetic compounds fall within the family of molecules called Pan-DR-binding epitopes (e.g., PADRETM, Epimmune Inc., San Diego, CA).
  • PADRETM peptides are designed to bind multiple HLA-DR (human HLA class II) molecules.
  • pan-DR-binding epitope peptide having the formula: aKXVAAZTLKAAa, where "X” is either cyclohexylalanine, phenylalanine, or tyrosine; “Z” is either tryptophan, tyrosine, histidine or asparagine; and “a” is either D-alanine or L-alanine (SEQ ID NO:29), has been found to bind to numerous allele-specific HLA-DR molecules. Accordingly, these molecules stimulate a T helper lymphocyte response from most individuals, regardless of their HLA type.
  • Certain pan-DR binding epitopes comprise all "L” natural amino acids; these molecules can be provided as peptides or in the form of nucleic acids that encode the peptide.
  • HTL peptide epitopes can be modified to alter their biological properties.
  • HTL peptide epitopes can be modified in the same manner as CTL peptides. For instance, they may be modified to include D-amino acids or be conjugated to other molecules such as lipids, proteins, sugars and the like. Peptides comprising D-amino acids generally have increased resistance to proteases, and thus have an extended serum half- life.
  • peptides of the invention can be conjugated to other molecules such as lipids, proteins or sugars, or any other synthetic compounds, to increase their biological activity.
  • a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or the carboxyl termini.
  • lipids have been identified as agents capable of facilitating the priming in vitro CTL response against viral antigens.
  • palmitic acid residues can be attached to the ⁇ -and ⁇ - amino groups of a lysine residue and then linked to an immunogenic peptide.
  • One or more linking moieties can be used such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like.
  • the lipidated peptide can then be administered 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 ⁇ - and ⁇ - amino groups of Lys via a linking moiety, e.g., Ser-Ser, added to the amino terminus of an immunogenic peptide.
  • E. coli lipoproteins such as tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P 3 CSS) can be used to prime CTL when covalently attached to an appropriate peptide.
  • P 3 CSS tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine
  • peptides of the invention can be coupled to P 3 CSS, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen.
  • two such compositions can be combined to elicit both humoral and cell-mediated responses.
  • 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 ProgenipoietinTM (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.
  • a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes in HLA molecules on their surfaces.
  • the DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to one or more antigens of interest, e.g., tumor associated antigens (TAA) such as HER2/neu, p53, MAGE 2, MAGE3, and/or carcinoembryonic antigen (CEA).
  • TAA tumor associated antigens
  • CEA carcinoembryonic antigen
  • TAA helper T cell
  • PADRE helper T cell
  • a vaccine in accordance with the invention comprising epitopes from HER2/neu, p53, MAGE 2, MAGE3, and carcinoembryonic antigen (CEA) is used to treat minimal or residual disease in patients with malignancies such as breast, colon or lung cancer; any malignancies that bear any of these TAAs can also be treated with the vaccine.
  • a TAA vaccine can be used following debulking procedures such as surgery, radiation therapy or chemotherapy, whereupon the vaccine provides the benefit of increasing disease free survival and overall survival in the recipients.
  • a vaccine of the invention is a product that treats a majority of patients across a number of different tumor types.
  • a vaccine comprising a plurality of epitopes, preferably supermotif-bearing epitopes, offers such an advantage.
  • peptides of the present invention are useful for administration to mammals, particularly humans, to treat and/or prevent disease.
  • vaccine compositions (peptide or nucleic acid) of the invention are administered to a patient who has a malignancy associated with expression of one or more TAAs, or to an individual susceptible to, or otherwise at risk for developing TAA-related disease. Upon administration an immune response is elicited against the TAAs, thereby enhancing the patient's own immune response capabilities.
  • peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective immune response to the TAA-expressing cells and to thereby cure, arrest or slow symptoms and/or complications.
  • 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.
  • the vaccine compositions of the invention can be used purely as prophylactic agents.
  • 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 ⁇ g of peptide and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g of peptide.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g of peptide per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 ⁇ g to about 50,000 ⁇ g 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.
  • peptides comprising CTL and or HTL epitopes of the invention induce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide.
  • the manner in which the peptide is contacted with the CTL or HTL is not critical to the invention.
  • the peptide can be contacted with the CTL or HTL either in vitro or in vivo.
  • peptide can be administered directly, or in other forms/vehicles, e.g., DNA vectors encoding one or more peptides, viral vectors encoding the peptide(s), liposomes, antigen presenting cells such as dendritic cells, and the like, as described herein.
  • the peptides or polypeptides can be administered directly.
  • the peptide/polypeptides can be administered indirectly presented on APCs, or as DNA encoding them.
  • the peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences.
  • administration should generally begin at the first diagnosis of TAA- related disease. This is followed by boosting doses at least until symptoms are substantially abated and for a period thereafter. In chronic disease states, loading doses followed by boosting doses may be required.
  • 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 ⁇ g of peptide and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g of peptide.
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g of peptide per 70 kilogram patient.
  • Boosting dosages of between about 1.0 ⁇ g to about 50,000 ⁇ g of peptide, administered pursuant to a boosting regimen over weeks to months, can be administered depending upon the patient's response and condition. Patient response can be determined by measuring the specific activity of CTL and HTL obtained from the patient's blood.
  • peptides and compositions of the present invention are used in serious disease states. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be desirable to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
  • a representative dose is in the range disclosed above, namely where the lower value is about 1, 5, 50, 500, or 1,000 ⁇ g of peptide and the higher value is about 10,000; 20,000; 30,000; or 50,000 ⁇ g of peptide, preferably from about 500 ⁇ g to about 50,000 ⁇ g of peptide per 70 kilogram patient.
  • administration should continue until at least clinical symptoms or laboratory tests indicate that the disease has been eliminated or substantially abated, and for a follow-up period thereafter.
  • the dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
  • compositions for therapeutic treatment are intended for parenteral, topical, oral, intrathecal, or local administration.
  • the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • 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.
  • compositions may contain pharmaceutically acceptable auxiliary substances or pharmaceutical excipients as may be 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.
  • pharmaceutically acceptable auxiliary substances or pharmaceutical excipients as may be 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.
  • 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.
  • a human unit dose form of the peptide composition is typically included in a pharmaceutical composition that also 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, 17 th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985).
  • the peptides of the invention can 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.
  • 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.
  • 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.
  • a ligand can be incorporated into the liposome, 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.
  • 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.
  • 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, often at a concentration of 25%-75%.
  • 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, often 1%-10%.
  • the surfactant must, of course, be pharmaceutically acceptable, 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, although an atomizer may be used in which no propellant is necessary and other percentages are adjusted accordingly.
  • a carrier can also be included, e.g., lecithin for intranasal delivery.
  • Antigenic peptides of the invention have been used to elicit a CTL and or HTL response ex vivo, as well.
  • the resulting CTLs or HTLs can be used to treat chronic infections, or tumors in patients that do not respond to other conventional forms of therapy, or who do not respond to a therapeutic peptide or nucleic acid vaccine in accordance with the invention.
  • Ex vivo CTL or HTL responses to a particular 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 (APC), such as dendritic cells, and the appropriate immunogenic peptide.
  • APC antigen-presenting cells
  • the cells 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).
  • CTL destroy
  • HTL facilitate destruction
  • 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.
  • 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 chrome HBV and HIV infection, and the current resurgence of dmg 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 lmmunotherapy, and active T cell- based lmmunotherapy 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
  • HLA class I antigens in tumors 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 ⁇ 2-microglobulin and specific HLA heavy chains, alterations in the regulatory elements controlling over class I expression and loss of entire chromosome sections.
  • HLA class I While the complete absence of class I expression will eliminate CTL recognition of those tumor cells, the loss of HLA class I will also render the tumor cells extraordinary sensitive to lysis from NK cells (Ohnmacht, GA, et al, Heterogeneity in expression of human leukocyte antigens and melanoma- associated antigens in advanced melanoma J Cellular Phys 182:332-338, 2000; Liunggren HG, et al, Host resistance directed selectively against H-2 deficient lymphoma variants: Analysis of the mechanism J. Exp.
  • 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.
  • 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.
  • 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.
  • a practical solution to overcome the potential hurdle of allele-specific loss relies on the induction of multispecific responses.
  • HLA class I expression 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.
  • Torres et al Torres, MJ, 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.
  • 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).
  • 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).
  • IFN-gamma production is induced and self-amplified by local inflammation immunization (Halloran, et al. Local T cell responses induce widespread MHC expression/. Immunol 148:3837, 1992), resulting in large increases in MHC expressions even in sites distant from the inflammatory site.
  • HLA expression can render tumor cells more susceptible to NK lysis (Ohnmacht, GA, et al, Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma J Cellular Phys 182:332-38, 2000; Liunggren HG, et al, Host resistance directed selectively against H-2 deficient lymphoma variants: Analysis of the mechanism/. Exp. Med., 162(6): 1745-59, December 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 ⁇ 2m gene J.
  • 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.
  • HLA class I 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 mduced in tumor cells as a result of local inflammation and lymphokine release, and, 3) class I negative cells are sensitive to lysis by NK cells
  • 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 m the context of neoplastic disease
  • the treating physician can assay a patient's tumor to ascertain whether HLA is bemg 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
  • NK-inducing methods or composition can comprise a Flt3 ligand or ProGP which facilitate mobilization of dendritic cells, the rationale bemg that dendritic cells produce large amounts of IL-12 IL- 12 can also be administered directly in either ammo acid or nucleic acid form
  • compositions in accordance with the invention can be administered concurrently with NK cell-inducing compositions, or these compositions can be administered sequentially
  • 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
  • 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
  • embodiments of the present mvention 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
  • embodiments of the mvention can also comprise alpha, beta and/or gamma IFN to facilitate upregualtion of HLA
  • compositions of the invention are administered concurrently with the standard therapy Du ⁇ ng this pe ⁇ od, the patient's immune system is directed to mduce responses against the epitopes comprised by the present inventive compositions Upon removal from the treatment havmg side effects, the patient is pruned 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
  • compositions in accordance with the mvention are administered concurrently with an immunosuppressive regimen if desired
  • kits can be provided m kit form together with instructions for vaccine administration
  • the kit would include desired compos ⁇ t ⁇ on(s) of the mvention in a container, preferably m unit dosage form and instructions for administration
  • a kit would mclude an APC, such as a dendritic cell, previously exposed to and now presenting peptides of the mvention in a container, preferably m umt dosage form together with instructions for administration
  • An alternative kit would mclude a minigene construct with desired nucleic acids of the mvention in a container, preferably in unit dosage form together with instructions for administration. Lymphokmes such as IL-2 or IL-12 may also be included m the kit
  • Other kit components that may also be desirable mclude, for example, a sterile sy ⁇ nge, booster dosages, and other desired excipients
  • TAAs are set forth in Table 12 Peptides were evaluated based upon MHC binding motifs, on the capacity to bind MHC molecules, and the ability to activate tumor-reactive CTL in vitro using lymphocyte cultures from normal individuals. This approach has several advantages. First, it does not require the isolation of patient-derived cells such as CTL or tumor cells. Secondly, the identification of epitopes that stimulate CTL in normal individuals permits the identification of a broad range of epitopes, including subdominant as well as dominant epitopes.
  • a vaccine comprises epitopes (as one or more peptides or as nucleic acids encoding them) from among these four, or any other, TAAs. Accordingly, this vaccine induces CTL responses against several major cancer types.
  • CEA is a 180 kD cell surface and secreted glycoprotein produced by a number of different tumors at high levels of expression, particularly colon cancer. This antigen is present.in normal physiology associated with fetal tissue (see, e.g., Ruddon, R., Cancer Biology, 3 rd ed., p 126 (1995); and copending USSN 09/458,302, filed 10 December 1999)). The abnormally high expression on cancer cells makes CEA an important target for immunotherapy.
  • MAGE, melanoma antigens are a family of related proteins whose expression is normally limited to testis and placenta, but are also expressed by melanomas and a variety of other carcinomas. These proteins are known to be recognized by cytotoxic T cells (see, e.g., copending USSN 09/458,298, filed 10 December 1999).
  • HER2/neu is a 185 kD transmembrane protein that is similar to the EGF receptor.
  • HER2/neu is a tyrosine kinase capable of autophosphorylation. Over-expression of HER2/neu is co ⁇ elated with oncogenic transformation. It is expressed primarily in breast, ovarian and gastric cancers (see, e.g., copending USSN 09/458,299, filed 10 December 1999).
  • a fourth TAA targeted, p53 is normally a tumor suppressor gene but can be mutated.
  • p53 has been observed in colon, lung, prostate and osteosarcomas as well as other tumors (see, e.g., copending USSN 09/458,297, filed 10 December 1999).
  • p53 peptides in a vaccine of the invention are derived from non-mutated sequences that are common between all cancer patients.
  • Other TAAs that can be included in a vaccine composition are associated with prostate cancer (see, e.g., copending Provisional Application USSN 60/171312, filed 21 December 1999).
  • Table 7 below delineates the tumor antigen expression in breast, colon and lung.
  • Table 8 shows the incidence, 5-year survival rates, and the estimated number of deaths per year for these tumors in the U.S for each type of cancer in Table 7. In terms of estimated new cases, estimated deaths and 5 year survival rates each of these tumor types has a large unmet need. Globally, the incidence of these tumors is significantly greater
  • Protein sequences from the four targeted tumor antigens were analyzed, to identify 8-, 9-, 10-, and 11-mer sequences containing the HLA-A2 supertype binding motif.
  • This motif [leucine (L), isoleucine (I), valine (V), methionine (M), alanine (A), threonine (T), or glutamine (Q) at position 2, and leucine (L), isoleucine (I), valine (V), methionine (M), alanine (A), or threonine (T) at the C-terminus; see Table 2] is the predominant factor in determining peptide binding to the HLA molecules within the A2 supertype (see, e.g., del Guercio et al, J.
  • HLA-A2 peptide motifs were tested directly for binding to human class I HLA molecules, since a subset of motif-bearing peptides bind with a biologically significant affinity, data depicted in Table 6.
  • An affinity threshold ⁇ 500 nM to the HLA-A2 molecule was previously shown to define the capacity of a peptide epitope to elicit a CTL response (Sette et al, J. Immunol 153:5586-5592 (1994)).
  • a competitive inhibition assay using purified HLA molecules was used to quantify peptide binding.
  • Motif-bearing peptides were initially tested for binding to HLA-A*0201, the prototype member of the HLA-A2 supertype.
  • A*0201-binding peptides found to bind at least one additional A2 supertype member were selected for further testing. Analogs of the native sequences for the CEA and p53 were evaluated to identify additional CTL peptide epitopes, as described below.
  • HLA-A2 is a species restricted molecule
  • the binding and functional activities of the A2 vaccine epitopes were measured in vitro using human molecules and cells.
  • CTL epitopes were identified that demonstrated high or intermediate HLA-A2 binding affinity (IC 50 of ⁇ 500 nM). These epitopes also bound to at least one additional member of the HLA-A2 supertype family with an IC 50 ⁇ 500 nM.
  • Each epitope stimulated the in vitro induction of a specific human CTL that recognized and lysed peptide-pulsed target cells and tumor cell lines expressing the relevant TAA.
  • a PADRE molecule is optionally included in the vaccine to promote the induction of long lasting CTL responses (Alexander et al, Immunologic Research, In Press.).
  • Class I HLA peptides can be modified, or "analoged” by substitution of amino acids at a given position to increase their HLA binding affinity and or supertype cross-reactivity (see, e.g., Table 2, and Zitvogel et al, J Exp Med 183:87-97 (1996); Sette, et al, J. Immunol. 153:5586-5592 (1994)).
  • the amino acids at position 2 and the C terminus of a peptide are the primary contact or "anchor" residues that interact with the HLA-A2 binding pocket.
  • anchor residues were modified by substitution with a presently preferred or less prefe ⁇ ed anchor residue, at position 2 and/or at the C-terminus.
  • Another type of modification utilized involved the substitution of ⁇ -amino butyric acid (B) for endogenous cysteine (C) residues to avoid the potential complication of disulfide bridge formation during product development.
  • peptides can also be analoged by modification of a secondary anchor residue.
  • a peptide can be analoged by removal of a deleterious residue in favor of an acceptable or prefe ⁇ ed one; an acceptable residue can be exchanged for a different acceptable residue or a prefe ⁇ ed residue, or a prefe ⁇ ed residue can be exchanged for another prefe ⁇ ed one.
  • peptide sequences were modified using one or more of the strategies described above.
  • the peptides were tested for HLA-A2 supertype binding using the molecular binding assay.
  • Supertype-binding data for analog peptides are shown in Table 6.
  • the peptides of the invention were also evaluated for their potential to stimulate CTL precursor responses to the TAA-derived peptide ⁇ in vitro primary CTL induction) and CTL recognition of tumor cells expressing the target TAA peptide epitope (recognition of endogenous targets). These criteria provided evidence that the peptides are functional epitopes.
  • Peripheral blood monocytic cell-derived (or bone-ma ⁇ ow-derived) human DC generated in vitro using GM-CSF and IL-4 and pulsed with a peptide of interest, were used as antigen presenting cells (APCs) in primary CTL induction cultures.
  • the peptide pulsed DC were incubated with CD8 T cells (positively selected from normal donor lymphocytes using magnetic beads) which served as the source of CTL precursors.
  • CD8 T cells positively selected from normal donor lymphocytes using magnetic beads
  • One week after stimulation with peptide primary cultures were tested for epitope-specific CTL activity using either a standard chromium-release assay which measures cytotoxicity or a sandwich ELISA-based interferon gamma (IFN ⁇ ) production assay.
  • IFN ⁇ sandwich ELISA-based interferon gamma
  • T cell cultures testing positive for recognition of peptide-pulsed targets were expanded and evaluated for their ability to recognize human tumor cells that endogenously express the TAA.
  • the chromium-release and IFN ⁇ production assays were used for these evaluations, with tumor cell lines serving as the targets.
  • Tumor cell lines lacking expression of either the TAA or the HLA- A2.1 molecule served as the negative control for non-specific activity.
  • CTL cultures were generated which recognized tumor cells in a peptide-specific and HLA-A2 -restricted manner (Table 6).
  • a prefe ⁇ ed embodiment of a vaccine includes a molecule from the PADRETM family of universal T helper cell epitopes (HTL) that target most DR molecules in a manner designed to stimulate helper T cells.
  • HTL universal T helper cell epitopes
  • PADRE molecules are the peptides, aKFVAAWTLKAAa, aKYVAAWTLKAAa, aKFVAAYTLKAAa, aKXVAAYTLKAAa, aKYVAAYTLKAAa, aKFVAAHTLKAAa, aKXVAAHTLKAAa, aKYVAAHTLKAAa, aKFVAANTLKAAa, aKXVAANTLKAAa, aKYVAANTLKAAa, AKXVAAWTLKAAA (SEQ ID NO:30), AKFVAAWTLKAAA (SEQ ID NO:31), AKYVAAWTLKAAA (SEQ ID NO:32), AKFVAAYTLKAAA (SEQ ID NO:33), AKXVAAYTLKAAA (SEQ ID NO:34), AKYVAAYTLKAAA (SEQ ID NO:35), AKFVAAHTLKAAA (SEQ ID NO:36), AKXVAAHTLKAAA
  • the PADRE peptide is amidated.
  • a particularly prefe ⁇ ed amidated embodiment of a PADRE molecule is conventionally written aKXVAAWTLKAAa-NH 2 .
  • PADRE is predicted to be present in >95% of all humans. Therefore, this PADRE molecule is anticipated to induce an HTL response in virtually all patients, despite the extensive polymorphism of HLA-DR molecules in the human population.
  • PADRE has been specifically engineered for optimal immunogenicity for human T cells.
  • Representative data from in vitro primary immunizations of normal human T cells with TT 830-843 antigen and the PADRE molecule aKXVAAWTLKAAa-NH 2 are shown in Figure 1.
  • Peripheral blood mononuclear cells (PBMC) from three normal donors were stimulated with the peptides in vitro. Following the third round of stimulation, it was observed that PADRE generated significant primary T cell responses for all three donors as measured in a standard T cell proliferation assay. With the PADRE peptide, the 10,000 cpm proliferation level was generally reached with 10 to 100 ng/ml of antigen.
  • TT 830-843 antigen generated responses for only 2 out of 3 of the individuals tested. Responses approaching the 10,000 cpm range were reached with about 10,000 ng/ml of antigen. In this respect, it was noted that PADRE was, on a molar basis, about 100-fold more potent than TT 830-843 antigen for activation of T cell responses.
  • PADRETM peptide components of the vaccine bind with broad specificity to multiple allelic forms of HLA-DR molecules.
  • PADRETM peptide component(s) bind with high affinity (IC 50 ⁇ IOOO nM), i.e., at a level of affinity co ⁇ elated with being immunogenic for HLA Class II restricted T cells.
  • the in vivo administration of PADRETM peptide(s) stimulates the proliferation of HTL in normal humans as well as patient populations.
  • a vaccine in accordance with the invention comprises epitope-bearing peptides of the invention delivered via dendritic cells (DC). Accordingly, DC were 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. DC Purification
  • ProGP-mobilized DC were purified from peripheral blood (PB) and spleens of ProGP-treated C57B1/6 mice to evaluate their ability to present antigen and to elicit cellular immune responses.
  • DC were purified from total WBC and spleen using a positive selection strategy employing magnetic beads coated with a CD1 lc specific antibody (Miltenyi Biotec, Auburn CA).
  • ex vivo expanded DC were generated by culturing bone ma ⁇ ow cells from untreated C57B1 6 mice with the standard cocktail of GM-CSF and IL-4 (R&D Systems, Minneapolis, MN) for a period of 7-8 days (Mayordomo et al, Nature Med. 1 :1297-1302 (1995)).
  • the percentage of purified splenic DC isolated from ProGP treated mice was enriched from a range of 12-17% to a range of 67-77%o.
  • the purity of GM-CSF/IL-4 ex vivo expanded DC ranged from 31-41% (Wong et al, J. Immunother., 21:32040 (1998)).
  • ProGP generated DC to stimulate a CTL cell line was demonstrated in vitro using a viral-derived epitope and a co ⁇ esponding epitope responsive CTL cell line.
  • Transgenic mice expressing human HLA-A2.1 were treated with ProGP.
  • Splenic DC isolated from these mice were pulsed with a peptide epitope derived from hepatitis B vims (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 IFN ⁇ .
  • ProGP-derived splenic DC The capacity of ProGP-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 (Figure 3).
  • the left shift in the response curve for ProGP-derived spleen cells versus the other antigen presenting cells reveal that these ProGP-derived cells require less epitope to stimulate maximal IFN ⁇ release by the responder cell line.
  • Peptide-pulsed ProGP-Derived DC Promote In Vivo CTL responses
  • the ability of e vivo peptide-pulsed DC to stimulate CTL responses in vivo was also evaluated using the HLA-A2.1 transgenic mouse model.
  • DC derived from ProGP-treated animals or control DC derived from bone ma ⁇ ow 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.
  • spleens were removed and splenocytes containing DC and CTL were restimulated twice in vitro in the presence of the HBV Pol 455 peptide.
  • each peptide of the invention is prepared by chemical synthesis and is isolated as a solid by lyophilization. Peptides are manufactured in compliance with Good Manufacturing
  • Bulk peptides of the invention following identity and release testing, are formulated as an aqueous or non-aqueous solution, sterile filtered, and aseptically filled into sterile, depyrogenated vials.
  • Sterile rubber stoppers are inserted and overseals applied to the vials.
  • the vialed formulations undergo
  • Dendritic Cell Isolation, Pulsing, Testing and Administration A presently prefe ⁇ ed procedure for vaccination is set forth herein.
  • patients are treated with ProGP to expand and mobilize DC into the circulation.
  • proGP to expand and mobilize DC into the circulation.
  • leukapheresis approximately 15L process, possibly repeated once if required to collect sufficient mononuclear cells.
  • the mononuclear cell product is admixed with peptides of the invention by injection through micropore filters (this admixing protocol is not needed if sterile peptides are used).
  • the cell product vaccine embodiment is resuspended in cryopreservative solution (final 10% DMSO) and, for those protocols involving multiple vaccination boosts, divided into aliquots.
  • cryopreservative solution final 10% DMSO
  • the pulsed mononuclear cell product(s) are frozen and stored according to accepted procedures for hematopoietic stem cells.
  • Vaccination is performed by injection or intravenous infusion of thawed cell product after the hematologic effects of ProGP in the patient have dissipated ⁇ i.e., the hemogram has returned to baseline).
  • Figure 5 provides a flow chart of ex vivo pulsing of DC with peptides, washing of DC, DC testing, and cryopreservation. A more detailed description of the process is provided in the following Examples.
  • Patients are treated with ProGP daily by subcutaneous injection (dose and schedule determined in accordance with standard medical procedures). On the evening before leukapheresis, patients are assessed by an apheresis physician or nurse/technologist for adequacy of intravenous access for large- bore apheresis catheters. If peripheral venous access is deemed inadequate to maintain rapid blood flow for apheresis, then central venous catheters (inguinal, subclavian or internal jugular sites) can be inserted by appropriate medical/surgical personnel.
  • leukapheresis On the day of predicted peak DC mobilization, leukapheresis (approximately 3 blood volumes or 15L) is performed, for example, on a Cobe Spectra or Fenwal CS3000 (flow rate >35mL/min) to obtain mononuclear cells.
  • the number of DC in the leukapheresis product is estimated by flow cytometric counting of mononuclear cells possessing the immunophenotypes lin- /HLA-DR+/CD1 lc+ and lin-/HLA-DR+/CD 123+ in a lmL sample aseptically withdrawn from the apheresis product.
  • CBC/differential The numbers of granulocytes and lymphocytes in the leukapheresis product are counted by automated cytometry (CBC/differential). CBC/differential is performed immediately after the leukapheresis procedure and every other day for ten days to monitor resolution of the hematologic effects of the hematopoietin treatment and apheresis.
  • Plasma is removed from the leukapheresis product by centrifugation and expression of supernatant.
  • the cells from the centrifugation pellet are resuspended in OptiMEM medium with 1% Human Serum Albumin (HSA) at a cell density of 10 7 DC/ml in up to 100 ml.
  • HSA Human Serum Albumin
  • the peptide(s) of the invention are administered directly into the DC culture bag through an injection port, using aseptic technique. After mixing, e.g., by repeated squeezing and inversion, the cell suspension is incubated for four hours at ambient temperature. Cryopreservative solution is prepared by dissolving 50 mL pharmaceutical grade dimethylsulfoxide (DMSO) in 200 mL Plasmalyte ® . After the pulsing period, the cell suspension is washed by centrifugation and resuspension in an equal volume of phosphate buffered saline solution.
  • DMSO dimethylsulfoxide
  • the washing procedure is repeated a defined number of times, e.g., until studies validate that peptides have been removed.
  • Samples of one milliliter each are removed for viability testing and microbiological testing.
  • the cells are then prepared for freezing by centrifugation and resuspension in an equal volume of cryopreservative solution (final 10% DMSO).
  • the cell suspension in cryopreservative is then divided into six equal aliquots, transfe ⁇ ed to 50 ml freezing bags (Fenwal) and frozen at controlled rate of l°C/min for storage in liquid nitrogen until needed for vaccination procedure.
  • Antigen presenting cells, long-term stimulated T cells co ⁇ esponding to peptides of the invention, or T cell hybridomas, are used to determine the optimal procedure for incubating the peptide reagents of a vaccine with human cells.
  • Pulsing studies are done using one or more of the following cell sources: purified DC from ProGP treated HLA- A2.1 transgenic mice; human tumor cell lines that express HLA-A2; peripheral blood mononuclear cells from normal human volunteers; peripheral blood mononuclear cells from ProGP treated patients; and/or DC obtained from normal human HLA-A2 volunteers following the ex vivo culture of their peripheral blood mononuclear cells with GM-CSF and IL- 4.
  • Evaluated conditions include, e.g.:
  • a vaccine of the invention Following pulsing with the peptide reagents, DC from the patient are washed several times to remove excess peptides prior to infusing the cells back into the patient. In this embodiment of a vaccine of the invention, the washing procedure removes unbound peptides. Accordingly, there is no, or negligible, systemic exposure of the patient to the peptides.
  • Alternative vaccines of the invention involve direct administration of peptides of the invention to a patient, administration of a multiepitopic polypeptide comprising one or more peptides of the invention, administration of the peptides in a form of nucleic acids which encode them, e.g. , by use of minigene constructs.
  • Residual peptides are detected using a mass spectrometer set-up to monitor the protonated molecular ions of each peptide as they elute from the HPLC column.
  • the peptides are quantified by comparing the area response ratio of analyte and internal standard to that obtained for standards in a calibration curve.
  • peptide reagents may be formulated using 0.1% trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • the number of DC in the leukapheresis product is estimated by flow cytometric counting of mononuclear cells possessing the immunopheno types lin7HLA-DR + /CDl lc + and lin " /HLA-DR + /CD123 + in a 1 ml sample aseptically withdrawn from the apheresis product.
  • Lin " cells excludes monocytes, T- lymphocytes, B-lymphocytes, and granulocytes, by using a cocktail of antibodies to lineage markers CD3, CD14, DC16, CD19, CD20, CD56.
  • Viability of mononuclear cells is assessed after pulsing and washing, prior to suspension in cryopreservative, by trypan blue dye exclusion. In general, if the cell product contains more than 50% tiypan blue-positive cells, the product is not administered to a patient. Microbiological Testing
  • the cell suspension in cryopreservative is examined for microbial contamination by gram stain and routine clinical bacterial and fungal culture/sensitivity. If tests are positive for bacterial or fungal contamination, implicit evidence of significant contamination, the product is not infused. If, e.g., a gram stain is negative, the product may be infused for the first vaccination while awaiting results of culture/sensitivity. Antibiotic therapy based on culture results is instituted at the discretion of the treating physician if the patient shows appropriate signs of infection that could be clinically attributable to the infused contaminant.
  • Example 17 Patient Vaccination
  • an aliquot of frozen pulsed dendritic cell product is removed from a liquid nitrogen freezer and kept frozen in an insulated vessel containing liquid nitrogen during transport to the infusion site.
  • the product is thawed by immersion with gentle agitation in a water bath at 37°C.
  • the cell suspension is infused through intravenous line by gravity or by syringe pump.
  • the vaccine is administered by injection, e.g., subcutaneously, intradermally, or intramuscularly.
  • the patient's vital signs are monitored before infusion/injection and at 5 minute intervals during an infusion, then at 15 minute intervals for 1 hour after infusion/injection.
  • a vaccine in accordance with the invention comprises eight peptide epitopes bearing the HLA-A2 supermotif. Collectively, these eight epitopes are derived from the tumor associated antigens (TAAs) HER2/neu, p53, MAGE 2, MAGE3, and carcinoembryonic antigen (CEA), and stimulate CTL responses to these TAAs. (see Table 9) These eight peptides, which are also presented in Table 6, bear an HLA-A2 supermotif.
  • a ninth peptide an HTL epitope that enhances CTL responses such as a pan-DR-binding peptide (PADRETM, Epimmune, San Diego, CA), is included.
  • PDARETM pan-DR-binding peptide
  • HLA-A2 peptide components of the A2 vaccine bind to multiple HLA-A2 superfamily molecules with high or intermediate affinity (IC 50 ⁇ 500 nM).
  • HLA-A2-specific analog and native peptide components of the A2 vaccine stimulate CTL from the peripheral blood of normal human volunteers. These CTL recognize native peptides that have been pulsed onto HLA-A2 expressing APCs, as well as endogenous peptides presented by HLA-matched tumor cell lines.
  • the A2 vaccine is effective in stimulating the cellular arm of the immune system to mediate immune responses against tumors. It is to be appreciated that vaccines comprising peptides bearing other motifs, or nucleic acids encoding such peptides, are also used in accordance with the principles set forth herein, and are within the scope of the present invention.
  • an A2 vaccine comprises DC pulsed ex vivo with the nine peptides.
  • This embodiment of a vaccine can be used with progenipoietin (ProGP)-mobilized DC.
  • ProGP progenipoietin
  • An A2 vaccine comprises a cocktail of 12 peptides, 10 of which stimulate CTL responses to the tumor associated antigens (TAA) HER2/neu, p53, MAGE 2/3, and carcinoembryonic antigen (CEA).
  • TAA tumor associated antigens
  • CEA carcinoembryonic antigen
  • This embodiment of an A2 Vaccine is used in combination with an emulsion-based adjuvant such as Montanide® ISA51 or ISA720 (Seppic, Paris, France) or an Incomplete Freund's Adjuvant, preferably administered by injection.
  • an emulsion-based adjuvant such as Montanide® ISA51 or ISA720 (Seppic, Paris, France) or an Incomplete Freund's Adjuvant, preferably administered by injection.
  • an emulsion-based adjuvant such as Montanide® ISA51 or ISA720 (Seppic, Paris, France) or an Incomplete Freund's Adjuvant, preferably administered by injection.
  • the eight HLA-A2 CTL peptide components of this vaccine embodiment bind to multiple HLA-A2 superfamily molecules with high or intermediate affinity (IC 50 ⁇ 500 nM).
  • the HLA-A2-specific analog and native peptide components of the present vaccine stimulate CTL from patient's blood. These CTL recognize native peptides that were pulsed onto HLA-A2 expressing APCs, as well as endogenous peptides presented by HLA-matched tumor cell lines.
  • PADRETM peptide components of the A2 vaccine bind with high affinity and broad specificity to multiple allelic forms of HLA-DR molecules (IC 50 ⁇ IOOO nM).
  • the in vivo administration of PADRE peptide stimulates the proliferation of HTL in normal humans as well as patient populations.
  • this vaccine embodiment is effective in stimulating the cellular arm of the immune system to mediate immune responses against tumors.
  • 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.
  • 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.
  • V,S,M,A,7 (3/5) R,K L,I deleterious D,E (3/5); P, (5/5) D,E, (4/5)
  • A.L. V deleterious A,G,P,Q,N, G, R.H.K.Q.N, D,E,
  • B5401 preferred F,W,Y, 1 "Anchor F.W.Y.L.I.V L,I,V,M, A,L,I,V,M, F,W,Y,A,P, l"Anchor P M, A,T,I,V,L,
  • Secondary anchor specificities are designated for each position independently.
  • A2 A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0208, A*0210, A*0211, A*0212, A*0213 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
  • 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.
  • Predicted alleles are alleles whose specificity is predicted on the basis of B and F pocket structure to overlap with the supertype specificity.
  • the peptide designations are derived from the target antigen (e.g. CEA) and the numeral relates to the first amino acid in the protein (e.g. 691). Analogs are noted by the amino acid inserted by substitution and the peptide position substituted (e.g. V9).
  • HLA binding was measured by a competitive binding assay where lower values indicate greater binding affinity.
  • TAA Tumor Associated Antigens and Genes
  • CAG-3 Wang R-Rosenberg S, J.Immunology, 161 :3591-3596, 1998)
  • CDK4 Wood T., Beach D, Science 269:1281, 1995
  • Her 2/neu (Disis M., Cheever M, Cancer Res 54: 1071 , 1994)
  • TRP2 Wang R., Rosenberg S.A, J.Ex.Med 184:2207, 1996) gp75/TRPl (Wang R., Rosenberg S.A, J.Ex.Med 183:1131, 1996)
  • NOEY2 (Yu Y., Bat RC, PNAS, 96(1):214-219, 1999)
  • Kallikrein 2 (DarsomUrology, 49:857-862, 1997)

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Abstract

Compositions ou compositions de vaccins comprenant au moins un peptide possédant moins de 600 d'acides aminés contigus présentant une identité de 100 % par rapport à une séquence native de CEA, HER 2/neu, MAGE2, MAGE3, ou p53, ce peptide possédant, de plus, au moins un déterminant antigénique sélectionné dans la table T6.
EP00986510A 1999-12-13 2000-12-13 Compositions de vaccins et peptides d'antigene associes a une tumeur de hla-a2 de classe i Withdrawn EP1242049A4 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US543608 1983-10-20
US17044899P 1999-12-13 1999-12-13
US170448P 1999-12-13
US09/543,608 US6602510B1 (en) 2000-04-05 2000-04-05 HLA class I A2 tumor associated antigen peptides and vaccine compositions
US58320000A 2000-05-30 2000-05-30
US583200 2000-05-30
PCT/US2000/034318 WO2001041741A1 (fr) 1999-12-13 2000-12-13 Compositions de vaccins et peptides d'antigene associes a une tumeur de hla-a2 de classe i

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EP1242049A1 true EP1242049A1 (fr) 2002-09-25
EP1242049A4 EP1242049A4 (fr) 2005-01-05

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JP (1) JP2003516344A (fr)
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WO (1) WO2001041741A1 (fr)

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JP2004517609A (ja) * 2000-09-01 2004-06-17 エピミューン インコーポレイテッド Ｈｌａ−ａ２．１結合ペプチドおよびそれらの用途
EP1903056A3 (fr) * 2002-12-10 2008-05-07 Idm Pharma, Inc. Peptides se liant au HLA-A1, -A2, -A3, -A24, -B7 and -B44 et comprenant des épitopes d'antigène associé à une tumeur, et compositions les comprenant
CA2511775A1 (fr) * 2002-12-10 2004-06-24 Epimmune Inc. Peptides diriges contre des antigenes associes aux tumeurs hla-a1, a2 -a3,-a24,-b7, et -b44 et compositions
CA2505870A1 (fr) * 2002-12-11 2004-06-24 Soren Mouritsen Epitopes uniques de ciblage
CA2522812C (fr) * 2003-04-18 2012-08-21 Idm Pharma, Inc. Peptides antigenes hla-a2 associes a une tumeur et compositions
JP5015601B2 (ja) 2003-10-21 2012-08-29 セダーズ−シナイ メディカル センター 中枢神経系の癌を含む癌の処置のための系および方法
US8129184B2 (en) 2006-09-26 2012-03-06 Cedars-Sinai Medical Center Cancer stem cell antigen vaccines and methods
WO2008039974A2 (fr) 2006-09-28 2008-04-03 Cedars-Sinai Medical Center Vaccins contre le cancer et méthodes de vaccination
WO2009059011A2 (fr) 2007-11-01 2009-05-07 Mayo Foundation For Medical Education And Research Peptides de liaison hla-dr et leurs utilisations
WO2010028066A2 (fr) 2008-09-02 2010-03-11 Cedars-Sinai Medical Center Épitopes cd133
WO2010086294A2 (fr) 2009-01-28 2010-08-05 Epimmune Inc. Polypeptides de liaison de pan-dr et leurs utilisations
RU2600798C2 (ru) * 2009-04-01 2016-10-27 Юниверсити Оф Майами Композиции вакцин и способы их применения
ES2618573T3 (es) 2009-05-07 2017-06-21 ImmunoCellular Therapeutics,Ltd Epítopos de CD133
CN103961698A (zh) * 2013-02-05 2014-08-06 日东电工株式会社 经皮或粘膜给予用疫苗组合物
IN2014CH00396A (fr) * 2013-02-05 2015-04-03 Nitto Denko Corp
EP2956544B1 (fr) 2013-02-14 2017-11-01 Immunocellular Therapeutics Ltd. Vaccins contre le cancer et méthodes de vaccination
US9701729B2 (en) * 2013-03-08 2017-07-11 Taiho Pharmaceutical Co., Ltd. Peptide having 5 linked CTL epitopes
JP6692294B2 (ja) 2013-07-31 2020-05-13 バイオベンチャーズ・リミテッド・ライアビリティ・カンパニーＢｉｏＶｅｎｔｕｒｅｓ， ＬＬＣ 腫瘍関連糖鎖抗原を標的として癌を治療及び予防するための組成物及び方法
SG11201603165TA (en) 2013-10-21 2016-05-30 Taiho Pharmaceutical Co Ltd Novel Peptide Having 4 Linked CTL Epitopes
GB201504502D0 (en) * 2015-03-17 2015-04-29 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against pancreatic cancer and other cancers
SG10202111399YA (en) 2015-12-22 2021-11-29 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against breast cancer and other cancers
GB201522667D0 (en) 2015-12-22 2016-02-03 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against breast cancer and other cancers
GB201604458D0 (en) * 2016-03-16 2016-04-27 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against cancers
WO2018200586A1 (fr) 2017-04-26 2018-11-01 Eureka Therapeutics, Inc. Constructions reconnaissant spécifiquement le glypicane 3 et utilisations de ces dernieres

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KR960700739A (ko) * 1993-03-05 1996-02-24 카린 이스텀 Hla-a2. 1 결합 펩티드 및 그의 용도(hla-a2. 1 binding peptides and their uses)
WO1998033888A1 (fr) * 1997-01-31 1998-08-06 Epimmune, Inc. Cellules a peptides ou a antigenes charges de peptides

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See also references of WO0141741A1 *

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WO2001041741A1 (fr) 2001-06-14
AU2273701A (en) 2001-06-18
CA2393730A1 (fr) 2001-06-14
WO2001041741A9 (fr) 2002-05-30
EP1242049A4 (fr) 2005-01-05

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