WO2001042267A1 - Declenchement de reponses immunitaires cellulaires a mage2/3 au moyen de compositions de peptides et d'acides nucleiques - Google Patents

Declenchement de reponses immunitaires cellulaires a mage2/3 au moyen de compositions de peptides et d'acides nucleiques Download PDF

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WO2001042267A1
WO2001042267A1 PCT/US2000/033545 US0033545W WO0142267A1 WO 2001042267 A1 WO2001042267 A1 WO 2001042267A1 US 0033545 W US0033545 W US 0033545W WO 0142267 A1 WO0142267 A1 WO 0142267A1
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peptide
epitope
hla
epitopes
peptides
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PCT/US2000/033545
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English (en)
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John Fikes
Alessandro Sette
John Sidney
Scott Southwood
Robert Chesnut
Esteban Celis
Elissa Keogh
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Epimmune Inc.
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Priority to JP2001543564A priority Critical patent/JP2003517310A/ja
Priority to AU20850/01A priority patent/AU2085001A/en
Priority to US10/149,135 priority patent/US20040053822A1/en
Priority to CA002393339A priority patent/CA2393339A1/fr
Priority to EP00984183A priority patent/EP1235841A4/fr
Publication of WO2001042267A1 publication Critical patent/WO2001042267A1/fr

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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
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001186—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
    • C07K14/46—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
    • 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
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464486—MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • CTL cytotoxic T lymphocytes
  • TNF- tumor necrosis factor- ⁇
  • IFN ⁇ interferon- ⁇
  • Tumor-specific helper T lymphocytes are also known to be important for maintaining effective antitumor immunity
  • Their role in antitumor immunity has been demonstrated rn animal models m which these cells not only serve to provide help for induction of CTL and antibody responses, but also provide effector functions, which are mediated by direct cell contact and also by secretion of lymphokines (e , IFN ⁇ and TNF- ⁇ )
  • a fundamental challenge in the development of an efficacious tumor vaccine is immune suppression or tolerance that can occur There is therefore a need to establish vaccine embodiments that elicit immune responses of sufficient breadth and vigor to prevent progression and/or clear the tumor
  • the epitope approach employed in the present invention represents a solution to this challenge, in that it allows the incorporation of various antibody, CTL and HTL epitopes, from discrete regions of a target tumor-associated antigen (TAA), and or regions of other TAAs, in a single vaccine composition
  • TAA tumor-associated antigen
  • Such a composition can simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population
  • MAGE, melanoma antigen genes are a family of related proteins that were first described in 1991 Van der Bruggen and co-workers identified the MAGE gene after isolating CTLs from a patient who demonstrated spontaneous tumor regression These CTLs recognized melanoma cell lines as well as tumor lines from other patient all of whom expressed the same HLA-A1 -restricted gene (van der Bruggen et al , Science 254 1643-1647, 1991, DePlaen et al , Immunogenettcs 40 360-369, 1994)
  • the MAGE genes are expressed in metastatic melanomas (see, e g , Brasseur et al , Int J Cancer 63 375-380, 1995), non-small lung (Weynants et al , Int J Cancer 56 826-829, 1994), gastric (Inoue et al , Gastroenterology 109 1522- 1525, 1995), hepatocellular (Chen et al , Live
  • This invention applies our knowledge of the mechanisms by which antigen is recognized by T cells, for example, to develop epitope-based vaccines directed towards TAAs More specifically, this application communicates our discovery of specific epitope pharmaceutical compositions and methods of use in the prevention and treatment of cancer
  • epitope-based vaccines Upon development of appropriate technology, the use of epitope-based vaccines has several advantages over current vaccmes, particularly when compared to the use of whole antigens in vaccine compositions
  • lmmunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines
  • Such lmmunosuppressive epitopes may, e g , correspond to immunodormnant epitopes rn whole antigens, which may be avoided by selecting peptide epitopes from non-dominant regions (see, e g , Disis et al , J Immunol 156 3151-3158, 1996)
  • An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes (CTL and HTL), and further, to modify the composition of the epitopes, achieving, for example, enhanced lmmunogenicity Accordmgly, the immune response can be modulated, as appropriate, for the target disease Similar engineering of the response is not possible with traditional approaches
  • epitope-based immune-stimulating vaccines Another major benefit of epitope-based immune-stimulating vaccines is their safety The possible pathological side effects caused by infectious agents or whole protein antigens, which might have their own intrinsic biological activity, is eliminated
  • An epitope-based vaccine also provides the ability to dnect and focus an immune response to multiple selected antigens from the same pathogen (a "pathogen” may be an mfectious agent or a tumor associated molecule)
  • pathogen may be an mfectious agent or a tumor associated molecule
  • an epitope-based anti-tumor vaccme also provides the opportunity to combme epitopes derived from multiple tumor-associated molecules This capability can therefore address the problem of tumor-to tumor variability that arises when developing a broadly targeted anti-tumor vaccine for a given tumor type and can also reduce the likelihood of tumor escape due to antigen loss
  • a melanoma in one patient may express a target TAA that differs from a melanoma m another patient
  • Epitopes derived from multiple TAAs can be included m a polyepitopic vaccine that will target both melanomas
  • a need has existed to modulate peptide binding properties, e g , so that peptides that are able to bmd to multiple HLA molecules do so with an affinity that will stimulate an immune response
  • Identification of epitopes restricted by more than one HLA allele at an affinity that correlates with lmmunogenicity is important to provide thorough population coverage, and to allow the ehcitation of responses of sufficient vigor to prevent or clear an infection m a diverse segment of the population Such a response can also target a broad array of epitopes
  • the technology disclosed herein provides for such favored immune responses
  • epitopes for inclusion in vaccme compositions of the invention are selected by a process whereby protein sequences of known antigens are evaluated for the presence of motif or supermotif-bearing epitopes Peptides corresponding to a motif- or supermotif-bea ⁇ ng epitope are then synthesized and tested for the ability to bmd to the HLA molecule that recognizes the selected motif Those peptides that bmd at an intermediate or high affimty t e , an IC 50 (or a K D value) of 500 nM or less for HLA class I molecules or an IC 50 of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response Immunogenic peptide epitopes are selected for inclusion m vaccine compositions
  • Supermotif-bearmg peptides may additionally be tested for the ability to bmd to multiple alleles within the HLA supertype family Moreover, peptide epitopes may be analogued to modify bmding affinity and or the ability to bmd to multiple alleles withm an HLA supertype
  • the mvention also includes embodiments comp ⁇ smg methods for monitoring or evaluatmg an immune response to a TAA m a patient having a known HLA-type
  • Such methods comprise incubating a T lymphocyte sample from the patient with a peptide composition comprising a TAA epitope that has an ammo acid sequence described in, for example, Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI which binds the product of at least one HLA allele present in the patient, and detectmg for the presence of a T lymphocyte that bmds to the peptide
  • a CTL peptide epitope may, for example, be used as a component of a tetrame ⁇ c complex for this type of analysis
  • An alternative modality for defining the peptide epitopes in accordance with the mvention is to recite the physical properties, such as length, primary structure, or charge, which are correlated with binding to a particular allele-specific HLA molecule or group of allele-specific HLA molecules
  • a further modality for defining peptide epitopes is to recite the physical properties of an HLA binding pocket, or properties shared by several allele-specific HLA binding pockets (e g pocket configuration and charge distribution) and reciting that the peptide epitope fits and bmds to the pocket or pockets
  • novel synthetic peptides produced by any of the methods described herem are also part of the mvention HI. BRIEF DESCRIPTION OF THE FIGURES not applicable
  • the peptide epitopes and corresponding nucleic acid compositions of the present mvention are useful for stimulating an immune response to a TAA by stimulating the production of CTL or HTL responses
  • the peptide epitopes which are derived directly or indirectly from native TAA protem ammo acid sequences, are able to bmd to HLA molecules and stimulate an immune response to the TAA
  • the complete sequence of the TAA proteins to be analyzed can be obtained from GenBank.
  • Peptide epitopes and analogs thereof can also be readily determined from sequence mformation that may subsequently be discovered for heretofore unknown variants of particular TAAs, as will be clear from the disclosure provided below.
  • a list of target TAA includes, but is not limited to, the following antigens.
  • the peptide epitopes of the invention have been identified in a number of ways, as will be discussed below. Also discussed in greater detail is that analog peptides have been derived and the bindmg activity for HLA molecules modulated by modifying specific ammo acid residues to create peptide analogs exhibiting altered lmmunogenicity Further, the present invention provides compositions and combmations of compositions that enable epitope-based vaccmes that are capable of mteractmg with HLA molecules encoded by various genetic alleles to provide broader population coverage than prior vaccmes.
  • a “computer” or “computer system” generally mcludes: a processor; at least one information storage/retrieval apparatus such as, for example, a hard drive, a disk drive or a tape drive; at least one input apparatus such as, for example, a keyboard, a mouse, a touch screen, or a microphone, and display structure Additionally, the computer may mclude a communication channel m communication with a network. Such a computer may mclude more or less than what is listed above.
  • a "construct” as used herein generally denotes a composition that does not occur m nature.
  • a construct can be produced by synthetic technologies, e g , recombmant DNA preparation and expression or chemical synthetic techniques for nucleic or amino acids.
  • a construct can also be produced by the addition or affiliation of one material with another such that the result is not found m nature m that form
  • Cross-reactive binding mdicates that a peptide is bound by more than one HLA molecule, a synonym is degenerate bmding
  • a "cryptic epitope” elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when mtact whole protem which comprises the epitope is used as an antigen
  • a "dominant epitope” is an epitope that mduces 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 a set of ammo acid residues which is mvolved in recognition by a particular immunoglobulin, or m the context of T cells, those residues necessary for recognition by T cell receptor pro terns and/or Major Histocompatibility Complex (MHC) receptors
  • MHC Major Histocompatibility Complex
  • 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
  • epitope and peptide are often used mterchangeably It is to be appreciated, however, that isolated or purified protem or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the mvention It is to be appreciated that protem or peptide molecules that comprise an epitope of the mvention as well as additional ammo ac ⁇ d
  • peptide or protem sequences longer than 600 ammo acids are withm the scope of the invention so long as they do not comprise any contiguous sequence of more than 600 ammo acids that have 100% identity with a native peptide sequence, or if longer than 600 amino acids, they are a construct
  • a CTL epitope of the invention be less than 600 residues long in any increment down to eight amino acid residues
  • HLA Human Leukocyte Antigen
  • HLA is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e g , Stites, et al , IMMUNOLOGY, 8TM ED , Lange Publishing, Los Altos, CA, 1994)
  • HLA supertype or family describes sets of HLA molecules grouped on the basis of shared peptide-bmdmg specificities HLA class I molecules that share somewhat similar bmdmg affinity for peptides bearing certain ammo acid motifs are grouped into HLA supertypes
  • HLA superfamily, HLA supertype family, HLA family, and HLA xx-hke molecules are synonyms
  • IC 50 is the concentration of peptide m a bmding assay at which 50% inhibition of bmdmg of a reference peptide is observed Given the conditions in which the assays are run (i e , limiting HLA protems and labeled peptide concentrations), these values approximate K D values Assays for determining bmdmg are described m detail, e g , in PCT publications WO 94/20127 and WO 94/03205 It should be noted that IC 50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e g , HLA preparation, etc ) For example, excessive concentrations of HLA molecules will increase the apparent measured IC 50 of a given ligand
  • bindmg is expressed relative to a reference peptide
  • the ICso's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change
  • the assessment of whether a peptide is a good, intermediate, weak, or negative bmder is generally based on its IC 50 , relative to the IC 50 of a standard peptide
  • Bmdmg may also be determined usmg other assay systems including those usmg live cells (e , Ceppellmi et al , Nature 339 392, 1989, Chnstmck et al , Nature 352 67, 1991, Busch et al , Int Immunol 2 443, 19990, Hill et al , J Immunol 147 189, 1991, del Guercio et al , J Immunol 154 685, 1995), cell free systems usmg detergent lysates (e g , Cerundolo et al , J Immunol 21 2069, 1991), immobilized purified MHC (e g , Hill et al , J Immunol 152, 2890, 1994, Marshall et al , J Immunol 152 4946, 1994), ELISA systems (e g , Reay et al , EMBO J 11 2829, 1992), surface plasmon resonance (e g ,
  • high affinity with respect to HLA class I molecules is defined as bmding with an IC 50 , or K D value, of 50 nM or less
  • intermediate affinity is bmdmg with an IC 50 or K D value of between about 50 and about 500 nM
  • High affinity with respect to bindmg to HLA class II molecules is defined as bmding with an IC 50 or K D value of 100 nM or less
  • intermediate affinity is bmding with an IC 50 or K D value of between about 100 and about 1000 nM
  • identity in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of ammo acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection
  • immunogenic peptide or “peptide epitope” is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bmd an HLA molecule and mduce a CTL and/or HTL response
  • immunogenic peptides of the mvention are capable of bmding to an appropriate HLA molecule and thereafter mducmg a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is de ⁇ ved
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found m its native state
  • isolated peptides m accordance with the mvention preferably do not contain materials normally associated with the peptides m then in situ environment
  • Link refers to any method known m the art for functionally connecting peptides, mcludmg, without limitation, recombmant fusion, covalent bonding, disulfide bondmg, ionic bondmg, hydrogen bondmg, and electrostatic bondmg
  • MHC Major Histocompatibihty Complex
  • HLA complex is a cluster of genes that plays a role m control of the cellular mteractions responsible for physiologic immune responses In humans, the MHC complex is also known as the HLA complex
  • motif refers to the pattern of residues m a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 ammo acids for a class II HLA motif, which is recognized by a particular HLA molecule
  • Peptide motifs are typically different for each protein encoded by each human HLA allele and differ m the pattern of the primary and secondary anchor residues
  • a "non-native" sequence or "construct” refers to a sequence that is not found in nature, i e , is
  • non-naturally occurring sequences include, e g , peptides that are hpidated or otherwise modified, and polyepitopic compositions that contam epitopes that are not contiguous m a native protein sequence
  • a "negative bmdmg residue” or “deleterious residue” is an amino acid which, if present at certain positions (typically not primary anchor positions) m a peptide epitope, results in decreased bmding affinity of the peptide for the peptide 's corresponding HLA molecule
  • peptide is used interchangeably with "ohgopeptide” m 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 ammo acids
  • the preferred CTL-mducmg peptides of the mvention are 13 residues or less m length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues
  • the preferred HTL-mducmg ohgopeptides are less than about 50 residues m length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues
  • “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-adjustmg and buffermg agents, tonicity adjusting agents, wetting agents, preservative, and the like
  • a "primary anchor residue” is an ammo acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule
  • One to three, usually two, primary anchor residues withm a peptide of defined length generally defines a "motif for an immunogenic peptide These residues are understood to fit m close contact with peptide bmdmg grooves of an HLA molecule, with their side chams buried m specific pockets of the bindmg grooves themselves
  • the pnmary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9-res ⁇ due peptide epitope m accordance with the mvention
  • the primary anchor positions for each motif and supermotif are set forth in Table 1
  • analog peptides can be created by altermg the presence or absence of particular residues m these primary anchor positions Such analogs are used to modulate the bmdmg
  • Promiscuous recognition is where a distmct peptide is recognized by the same T cell clone in the context of various HLA molecules Promiscuous recognition or bmdmg is synonymous with cross-reactive bmding
  • a “protective immune response” or “therapeutic immune response” refers to a CTL and or an HTL response to an antigen derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression
  • the immune response may also mclude an antibody response which has been facilitated by the stimulation of helper T cells
  • residue refers to an ammo acid or ammo acid mimetic incorporated into an ohgopeptide by an amide bond or amide bond mimetic
  • a “secondary anchor residue” is an ammo acid at a position other than a primary anchor position m a peptide which may influence peptide bmdmg
  • a secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of ammo acids at one position
  • the secondary anchor residues are said to occur at "secondary anchor positions "
  • a secondary anchor residue can be identified as a residue which is present at a higher frequency among high or intermediate affinity bindmg peptides, or a residue otherwise associated with high or intermediate affinity bmding
  • analog peptides can be created by altering the presence or absence of particular residues m these secondary anchor positions Such analogs are used to finely modulate the bmdmg affinity of a peptide comp ⁇ smg a particular motif or supermotif
  • 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 bindmg specificity shared by HLA molecules encoded by two or more HLA alleles
  • a supermotif-bearmg peptide is recognized with high or intermediate affinity (as defined herem) by two or more HLA molecules
  • “Synthetic peptide” refers to a peptide that is man-made using such methods as chemical synthesis or recombmant DNA technology
  • a “vaccme” is a composition that contams one or more peptides of the mvention
  • vaccines m accordance with the mvention, such as by a cocktail of one or more peptides, one or more epitopes of the invention comprised by a polyepitopic peptide, or nucleic acids that encode such peptides or polypeptides, e , a mimgene that encodes a polyepitopic peptide
  • the "one or more peptides” can mclude any whole umt mteger from 1-150, e g , at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 45, 50,
  • ammo acid residue positions are referred to m a peptide epitope they are numbered m an ammo to carboxyl direction with position one bemg the position closest to the amino terminal end of the epitope, or the peptide or protein of which it may be a part
  • the ammo- 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 smgle letter designations
  • the L-form of an amino acid residue is represented by a capital smgle letter or a capital first letter of a three-letter symbol, and the D-form for those ammo acids havm
  • class I and class II allele-specific HLA bmdmg motifs, or class I or class II supermotifs allows identification of regions withm a protem that have the potential of bindmg particular HLA molecules.
  • ⁇ Cr-release assay mvolvmg peptide sensitized target cells and target cells expressmg endogenously generated antigen
  • epitope selection encompassmg identification of peptides capable of bmdmg at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bmd at high or intermediate affinity to two or more allele-specific HLA molecules
  • CTL-mducing peptides of mterest for vaccine compositions preferably include those that have an IC 50 or bmding affinity value for class I HLA molecules of 500 nM or better (i e , the value is ⁇ 500 nM) HTL-mducmg peptides preferably mclude those that have an IC 50 or bmdmg affinity value for class II HLA molecules of 1000 nM or better, (i e , the value is ⁇ 1,000 nM)
  • peptide bmdmg is assessed by testmg the capacity of a candidate peptide to bmd to a punfied HLA molecule in vitro Peptides exhibiting high or intermediate affinity are then considered for further analysis Selected peptides are tested on other members of the supertype family
  • peptides that exhibit cross-reactive bmdmg are then used m cellular screenmg analyses or vaccmes As disclosed herem, higher HLA bmdmg
  • lmmunogenicity can be manifested m several different ways lmmunogenicity 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 m 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
  • higher bmdmg affinity peptides lead to more vigorous immunogenic responses As a result, less peptide is required to elicit a similar biological effect if a high or intermediate affinity bmdmg peptide is used
  • high or intermediate affinity bindmg epitopes are particularly useful
  • the bmdmg affimty of peptides for HLA molecules can be determined as described m Example 1, below IV.D. Peptide Epitope Binding Motifs and Supermotifs
  • Peptides of the present mvention also comprise epitopes that bind to MHC class II DR molecules.
  • This mcreased heterogeneity of HLA class II peptide ligands is due to the structure of the bmding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends.
  • PI position 1
  • PI may represent the N-terminal residue of a class II bmdmg peptide epitope, but more typically is flanked towards the N-terminus by one or more residues.
  • Other studies have also pomted to an important role for the peptide residue m the 6 th position towards the C-terminus, relative to PI, for bmding to various DR molecules
  • HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlappmg peptide bmdmg repertoires, and consensus structures of the mam peptide bmdmg pockets
  • peptides of the present mvention are identified by any one of several HLA-specific ammo acid motifs (see, e g , Tables I-III), or if the presence of the motif corresponds to the ability to bmd several allele- specific HLA molecules, a supermotif
  • the HLA molecules that bmd to peptides that possess a particular ammo acid supermotif are collectively referred to as an HLA "supertype"
  • the peptide motifs and supermotifs described below, and summarized m Tables I-III, provide guidance for the identification and use of peptide epitopes m accordance with the mvention.
  • Examples of peptide epitopes bearing a respective supermotif or motif are included in Tables as designated in the description of each motif or supermotif below.
  • the IC 50 values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV.
  • IC 50 values of standard peptides used to determine binding affinities for Class II peptides are shown in Table V.
  • the peptides used as standards for the binding assays described herein are examples of standards; alternative standard peptides can also be used when performing binding studies.
  • the amino acid sequences of MAGE2 and MAGE3 were evaluated for the presence of the designated supermotif or motif, i.e., the amino acid sequences were searched for the presence of the primary anchor residues as set out in Table I (for Class I motifs) or Table III (for Class II motifs) for each respective motif or supermotif.
  • motif- and/or supermotif-bearing amino acid sequences are indicated by position number and length of the epitope with reference to the MAGE2 and MAGE3 sequences and numbering provided below.
  • the "pos” (position) column designates the amino acid position in the MAGE2 or
  • MAGE3 protein sequence that corresponds to the first amino acid residue of the epitope.
  • the "number of amino acids” indicates the number of residues in the epitope sequence and hence the length of the epitope.
  • the first peptide epitope listed in Table VILA is a sequence of 9 residues in length starting at position 154 of the MAGE2 amino acid sequence. Accordingly, the amino acid sequence of the epitope is ASEYLQLVF.
  • Binding data presented in Tables VII-XX is expressed as a relative binding ratio, supra.
  • HLA class I peptide epitope supermotifs and motifs delmeated below are summarized m Table I
  • the HLA class I motifs set out m Table 1(a) are those most particularly relevant to the mvention claimed here
  • Primary and secondary anchor positions are summarized in Table II
  • Allele-specific HLA molecules that comprise HLA class I supertype families are listed m Table VI
  • peptide epitopes may be listed in both a motif and a supermotif Table The relationship of a particular motif and respective supermotif is indicated m the description of the individual motifs
  • the HLA-Al supermotif is characterized by the presence m peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M) primary anchor residue m position 2, and an aromatic (Y, F, or W) primary anchor residue at the C-terminal position of the epitope
  • the corresponding family of HLA molecules that bind to the Al supermotif is comprised of at least A*0101, A*2601, A*2602, A*2501, and A*3201 ( ⁇ ee, e g , D ⁇ B ⁇ no, M et al , J Immunol 151 5930, 1993, DiBnno, M et al , J Immunol 152 620, 1994, Kondo, A et al , Immunogenetics 45 249, 1997)
  • Other allele-specific HLA molecules predicted to be members of the A 1 superfamily are shown in Table VI Peptides bindmg to each of the
  • the corresponding family of HLA molecules (t e , the HLA-A2 supertype that bmds these peptides) is comprised of at least A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207,
  • the HLA-A3 supermotif is characterized by the presence m peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at position 2, and a positively charged residue, R or K, at the C-terminal position of the epitope, e g , m position 9 of 9-mers (see, e g , Sidney et al , Hum Immunol 45 79, 1996) Exemplary members of the corresponding family of HLA molecules (the HLA- A3 supertype) that bind the A3 supermotif mclude at least A*0301, A*l 101, A*3101, A*3301, and A*6801 Other allele-specific HLA molecules predicted to be members of the A3 supertype are shown m Table VI As explained m detail below, peptide bmdmg to each of the individual allele-specific HLA proteins can be modulated by substitutions of ammo acids at the primary and or secondary anchor positions of the peptide, preferably
  • MAGE2 and MAGE3 peptide epitopes that comprise the A3 supermotif are set forth m Tables IX(A) and IX(B), respectively
  • HLA-A24 supermotif The HLA-A24 supermotif is characterized by the presence m peptide ligands of an aromatic (F, W, or Y) or hydrophobic aliphatic (L, I, V, M, or T) residue as a primary anchor m position 2, and Y, F, W, L, I, or M as pnmary anchor at the C-termmal position of the epitope (see, e , Sette and Sidney, Immunogenetics 1999 Nov, 50(3-4) 201-12, Review)
  • the corresponding family of HLA molecules that bind to the A24 supermotif (; e , the A24 supertype) includes at least A*2402, A*3001, and A*2301
  • Other allele-specific HLA molecules predicted to be members of the A24 supertype are shown m Table VI Peptide bmdmg to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choo
  • the HLA-B7 supermotif is characterized by peptides bearmg prolme m position 2 as a primary anchor, and a hydrophobic or aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary anchor at the C-termmal position of the epitope
  • the corresponding family of HLA molecules that bmd the B7 supermotif is comprised of at least twenty six HLA-B protems compnsmg at least B*0702, B*0703, B*0704, B*0705, B*1508, B*3501, B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, B*5602, B*6701, and B*7801 (see, e
  • MAGE2 and MAGE3 peptide epitopes that comprise the B7 supermotif are set forth in Tables XI(A) and XI(B), respectively
  • the HLA-B27 supermotif is characterized by the presence m peptide ligands of a positively charged (R, H, or K) residue as a primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I, A, or V) residue as a primary anchor at the C-terminal position of the epitope (see, e g , Sidney and Sette,
  • Exemplary members of the corresponding family of HLA molecules that bmd to the B27 supermotif include at least B*1401, B*1402, B* 1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301
  • Other allele-specific HLA molecules predicted to be members of the B27 supertype are shown in Table VI
  • Peptide bmding to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosmg respective residues specified for the supermotif
  • Representative MAGE2 and MAGE3 peptide epitopes that comprise the B27 supermotif are set forth m Tables XII(A) and XII(B), respectively
  • the HLA-B44 supermotif is characterized by the presence m peptide ligands of negatively charged (D or E) residues as a primary anchor m position 2, and hydrophobic residues (F, W, Y, L, I, M, V, or A) as a primary anchor at the C-termmal position of the epitope (see, e g , Sidney et al , Immunol Today 17 261, 1996)
  • Exemplary members of the corresponding family of HLA molecules that bmd to the B44 supermotif (i e , the B44 supertype) include at least B*1801, B*1802, B*3701, B*4001, B*4002, B*4006, B*4402, B*4403, and B*4404
  • Peptide bmdmg to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosmg respective residues specified for the supermotif
  • the HLA-B58 supermotif is characterized by the presence m peptide ligands of a small aliphatic residue (A, S, or T) as a primary anchor residue at position 2, and an aromatic or hydrophobic residue (F, W, Y, L, I, V, M, or A) as a primary anchor residue at the C-termmal position of the epitope (see, e g , Sidney and Sette, Immunogenetics 1999 Nov, 50(3-4) 201-12, Review)
  • Exemplary members of the corresponding family of HLA molecules that bmd to the B58 supermotif (i e , the B58 supertype) include at least B* 1516, B*1517, B*5701, B*5702, and B*5801
  • Other allele-specific HLA molecules predicted to be members of the B58 supertype are shown m Table VI Peptide bmdmg to each of the allele-specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions
  • HLA-B62 supermotif The HLA-B62 supermotif is characterized by the presence m peptide ligands of the polar aliphatic residue Q or a hydrophobic aliphatic residue (L, V, M, I, or P) as a prunary anchor m position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A) as a prunary anchor at the C-terminal position of the epitope (see, e g , Sidney and Sette, Immunogenetics 1999 Nov, 50(3-4) 201-12, Review)
  • MAGE2 and MAGE3 peptide epitopes that comprise the B62 supermotif are set forth m Tables XIV(A) and XIV(B), respectively
  • the HLA-Al motif is charactenzed by the presence m peptide ligands of T, S, or M as a primary anchor residue at position 2 and the presence of Y as a primary anchor residue at the C-terminal position of the epitope
  • An alternative allele-specific A 1 motif is characterized by a prunary anchor residue at position 3 rather than position 2
  • This motif is characterized by the presence of D, E, A, or S as a prunary anchor residue m position 3, and a Y as a prunary anchor residue at the C-termmal position of the epitope (see, e g , DiB ⁇ no et al , J Immunol , 152 620, 1994, ondo et al , Immunogenetics 45 249, 1997, and Kubo et al , J Immunol 152 3913, 1994 for reviews of relevant data)
  • Peptide bmdmg to HLA-Al can be modulated by substitutions at
  • peptide epitopes that compnse either Al motif are set forth m Table XV(A and B), MAGE2 and MAGE3, respectively Those epitopes compnsmg T, S, or M at position 2 and Y at the C- terminal position are also included in the listing of HLA-Al supermotif-bea ⁇ ng peptide epitopes listed in Table VII, as these residues are a subset of the A 1 supermotif prunary anchors
  • HLA-A2*0201 motif was determined to be characterized by the presence m peptide ligands of L or M as a prunary anchor residue m position 2, and L or V as a primary anchor residue at the C-terminal position of a 9-res ⁇ due 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-termmal position of a nme ammo acid peptide (see, e g , Hunt et al , Science 255 1261-1263, March 6, 1992, Parker et al , J Immunol 149 3580-3587, 1992)
  • the A*0201 allele-specific motif has also been defined by the present mventors to additionally comprise V, A, T, or Q as a pnmary anchor residue at position 2, and M or T as a pnmary anchor residue at the C-termmal position of
  • A*0201 motif Representative peptide epitopes that comprise an A*0201 motif are set forth in Table VIII(A and B), MAGE2 and MAGE3, respectively.
  • the A*0201 motifs comprising the primary anchor residues V, A, T, or Q at position 2 and L, I, V, A, or T at the C-terminal position are those most particularly relevant to the invention claimed herein.
  • the HLA- A3 motif is characterized by the presence in peptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor residue at position 2, and the presence of K, sY, R, H, F, or A as a primary anchor residue at the C-terminal position of the epitope (see, e.g., DiBrino et al, Proc. Natl. Acad. Sci USA 90: 1508, 1993; and Kubo e/ al, J. Immunol. 152:3913-3924, 1994).
  • Peptide binding to HLA-A3 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • A3 motif Representative peptide epitopes that comprise the A3 motif are set forth in Table XVI(A and B), MAGE2 and MAGE3, respectively. Those peptide epitopes that also comprise the A3 supermotif are also listed in Table IX.
  • the A3 supermotif primary anchor residues comprise a subset of the A3- and Al 1-allele specific motif primary anchor residues.
  • the HLA-Al 1 motif is characterized by the presence in peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primary anchor residue in position 2, and K, R, Y, or H as a primary anchor residue at the C-terminal position of the epitope (see, e.g., Zhang et al, Proc. Natl. Acad. Sci USA 90:2217-2221, 1993; and Kubo et al, J. Immunol. 152:3913-3924, 1994).
  • Peptide binding to HLA-Al 1 can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the motif.
  • Representative peptide epitopes that comprise the Al 1 motif are set forth in Table XVII(A and B),
  • peptide epitopes comprising the A3 allele-specific motif are also present in this Table because of the extensive overlap between the A3 and Al l motif primary anchor specificities. Further, those peptide epitopes that comprise the A3 supermotif are also listed in Table IX. IV.D.14. HLA-A24 motif
  • the HLA-A24 motif is characterized by the presence m peptide ligands of Y, F, W, or M as a primary anchor residue m position 2, and F, L, I, or W as a prunary anchor residue at the C-terminal position of the epitope (see, e g , Kondo et al , J Immunol 155 4307-4312, 1995, and Kubo et al , J Immunol 152 3913-3924, 1994)
  • Peptide bmdmg to HLA-A24 molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosmg respective residues specified for the motif
  • peptide epitopes that comprise the A24 motif are set forth m Table XVIII(A and B), MAGE2 and MAGE3, respectively These epitopes are also listed in Table X, which sets forth HLA- A24-supermot ⁇ f-beanng peptide epitopes, as the primary anchor residues charactenzmg the A24 allele- specific motif comprise a subset of the A24 supermotif primary anchor residues
  • peptide epitope 9-mer core regions corresponding to a nme residue sequence comprising the DR3a submotif (wherem position 1 of the motif is at position 1 of the nme residue core) are set forth in Table XXa
  • Respective exemplary peptide epitopes of 15 ammo acid residues m length, each of which comprise a nme residue core, are also shown m Table XXa along with bmdmg data of the exemplary DR3 submotif a-bearmg peptides
  • each of the HLA class I or class II peptide epitopes set out m the Tables herem are deemed smgly to be an mventive aspect of this application Further, it is also an inventive aspect of this application that each peptide epitope may be used in combination with any other peptide epitope
  • Vaccmes that have broad population coverage are prefened because they are more commercially viable and generally applicable to the most people Broad population coverage can be obtained usmg the peptides of the mvention (and nucleic acid compositions that encode such peptides) through selecting peptide epitopes that bmd to HLA alleles which, when considered in total, are present m most of the population
  • Table XXI lists the overall frequencies of the HLA class I supertypes in various ethnicities
  • the B44-, A1-, and A24-supertypes are each present, on average, m a range from 25% to 40% m these major ethnic populations (Table XXIa) While less prevalent overall, the B27-, B58-, and B62 supertypes are each present with a frequency >25% m at least one major ethnic group (Table XXIa)
  • Table XXIb summarizes the estimated prevalence of combmations of HLA supertypes that have been identified in five major ethnic groups The mcremental coverage obtamed by the inclusion of Al,- A24-, and B44- supertypes to the A2, A3, and B7 coverage and coverage obtamed with all of the supertypes described herem, is shown The data presented herem, together with the previous definition of the A2-, A3-, and B7- supertypes, mdicates that all antigens, with the possible exception of A29, B8, and B46, can be classified into a total of nme HLA supertypes
  • CTL and HTL responses are not directed agamst all possible epitopes Rather, they are restricted to a few "rmmunodommant" determinants (Zinkernagel, et al , Adv Immunol 27 5159, 1979,
  • T cells to them are eliminated during immunological surveillance and that tolerance is induced
  • CTL responses to tumor epitopes in both normal donors and cancer patient has been detected, which may indicate that tolerance is mcomplete (see, e g , Kawashima et al , Hum Immunol 59 1, 1998, Tsang, J Natl Cancer Inst 87 82-90, 1995, Rongcun J ⁇ / , J Immunol 163 1037, 1999)
  • immune tolerance does not completely eliminate or mactivate CTL precursors capable of recognizing high affinity HLA class I bmdmg peptides
  • peptides with suitable cross-reactivity among all alleles of a superfamily are identified by the screenmg procedures described above, cross-reactivity is not always as complete as possible, and m certain cases procedures to mcrease cross-reactivity of peptides can be useful, moreover, such procedures can also be used to modify other properties of the peptides such as bindmg affinity or peptide stability
  • Havmg established the general rules that govern cross-reactivity of peptides for HLA alleles withm a given motif or supermotif, modification (i e , analoging) of the structure of peptides of particular mterest m order to achieve broader (or otherwise modified) HLA bmdmg capacity can be performed More specifically, peptides which exhibit the broadest cross-reactivity patterns, can be produced m accordance with the teachmgs herem
  • the present concepts related to analog generation are set forth m greater detail m co- pending U S S N 09/226,775 filed 1/6/99
  • the strategy employed utilizes the motifs or supermotifs which correlate with bmdmg to certam HLA molecules
  • the motifs or supermotifs are defined by havmg primary anchors, and m many cases secondary anchors
  • Analog peptides can be created by substituting ammo 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 secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II bmdmg peptides are shown in Tables II and III, respectively
  • residues are defined which are deleterious to bmding to allele-specific HLA molecules or members of HLA supertypes that bind the respective motif or supermotif (Tables II and III) Accordmgly, removal of such residues that are detrimental to bmdmg can be performed in accordance with the present mvention
  • the incidence of cross-reactivity increased from 22% to 37% (see, e g , Sidney, J et al , Hu Immunol 45 79, 1996)
  • one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small "neutral" residue such as Ala (that may not influence T cell
  • the analog peptide when used as a vaccme, actually elicits a CTL response to the native epitope in vivo (or, m the case of class II epitopes, elicits helper T cells that cross-react with the wild type peptides), the analog peptide may be used to immunize T cells in vitro from individuals of the appropriate HLA allele Thereafter, the immunized cells' capacity to mduce lysis of wild type peptide sensitized target cells is evaluated It will be desirable to use as antigen presenting cells, cells that have been either infected, or transfected with the appropriate genes, or, m the case of class II epitopes only, cells that have been pulsed with whole protem antigens, to establish whether endogenously produced antigen is also recognized by the relevant T cells
  • Another embodiment of the mvention is to create analogs of weak bmding peptides, to thereby ensure adequate numbers of cross-reactive cellular bmders Class I bmdmg peptides exhibiting bmdmg affinities of 500-5000 nM, and carrying an acceptable but suboptimal pnmary 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 crossbmdmg activity
  • Another embodiment for generatmg effective peptide analogs mvolves 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 cysteme can be substituted out m favor of ⁇ -amino butyric acid ("B" m the single letter abbreviations for peptide sequences listed herein) Due to its chemical nature, cysteme has the propensity to form disulfide b ⁇ dges and sufficiently alter the peptide structurally so as to reduce bmdmg capacity.
  • a native protem sequence, e a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation
  • a means for computmg such as an intellectual calculation or a computer
  • the mformation obtamed from the analysis of native peptide can be used directly to evaluate the status of the native peptide or may be utilized subsequently to generate the peptide epitope.
  • Computer programs that allow the rapid screenmg of protein sequences for the occurrence of the subject supermotifs or motifs are encompassed by the present mvention; as are programs that permit the generation of analog peptides. These programs are implemented to analyze any identified ammo acid sequence or operate on an unknown sequence and simultaneously determine the sequence and identify motif-bearing epitopes thereof, analogs can be simultaneously determmed as well Generally, the identified sequences will be from a pathogenic organism or a tumor-associated peptide.
  • the target TAA molecules mclude, without limitation, CEA, MAGE, p53 and HER2/neu
  • a protein sequence or translated sequence may be analyzed usmg software developed to search for motifs, for example the
  • Peptides m accordance with the mvention can be prepared synthetically, by recombmant DNA technology or chemical synthesis, or from natural sources such as native tumors or pathogenic organisms Peptide epitopes may be synthesized individually or as polyepitopic peptides Although the peptide will preferably be substantially free of other naturally occurring host cell protems and fragments thereof, in some embodiments the peptides may be synthetically conjugated to native fragments or particles
  • the peptides in accordance with the mvention can be a variety of lengths, and either m their neutral (uncharged) forms or m forms which are salts
  • the peptides m accordance with the mvention are either free of modifications such as glycosylation, side chain oxidation, or phosphorylation, or they contam these modifications, subject to the condition that modifications do not destroy the biological activity of the peptides as descnbed herem
  • HLA class I bmdmg epitopes of the mvention such as can be used in a polyepitopic construct, to a length of about 8 to about 13 ammo acid residues, often 8 to 11, preferably 9 to 10 HLA class II bindmg peptide epitopes of the mvention may be optimized to a length of about 6 to about 30 ammo acids m length, preferably to between about 13 and about 20 residues
  • the peptide epitopes are commensurate m size with endogenously processed pathogen-derived peptides or tumor cell peptides that are bound to the relevant HLA molecules, however, the identification and preparation of peptides that comprise epitopes of the mvention can also be carried out using the techniques described herem
  • epitopes of the mvention can be linked as a polyepitopic peptide, or as a mimgene that encodes a polyepitopic peptide
  • a sequence is generally selected on the basis that it contains the greatest number of epitopes per ammo acid length
  • epitopes can be present in a nested or overlappmg manner, e g a 10 ammo acid long peptide could contam two 9 ammo acid long epitopes and one 10 ammo acid long epitope, upon intracellular processing, each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide
  • This larger, preferably multi-epitopic, peptide can be generated synthetically, recombmantly, or via cleavage from the native source
  • the peptides of the mvention can be prepared in a wide variety of ways
  • the peptides can be synthesized in solution or on a solid support m accordance with conventional techniques Vanous automatic synthesizers are commercially available and can be used in accordance with known protocols (See, for example, Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D ED , Pierce Chemical Co , 1984) Further, individual peptide epitopes can be joined using chemical ligation to produce larger peptides that are still within the bounds of the mvention
  • recombmant DNA technology can be employed wherem a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected mto an appropriate host cell and cultivated under conditions suitable for expression
  • recombmant polypeptides which comprise one or more peptide sequences of the mvention can be used to present the appropriate T cell epitope
  • the nucleotide codmg sequence for peptide epitopes of the preferred lengths contemplated herein can be synthesized by chemical techniques, for example, the phosphot ⁇ ester 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
  • HLA bmdmg peptides Once HLA bmdmg 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 descnbed in PCT publications WO 94/20127 and WO 94/03205 Briefly, peptides compnsmg epitopes from a particular antigen are synthesized and tested for then ability to bmd to the appropriate HLA proteins
  • These assays may mvolve evaluatmg the bindmg of a peptide of the mvention to purified HLA class I molecules m relation to the bmdmg of a radioiodinated reference peptide
  • cells expressing empty class I molecules i e lackmg peptide therem
  • Other assays that may be used to evaluate peptide bmdmg mclude peptide-dependent class I assembly
  • antigen-presenting cells that have been mcubated with a peptide can be assayed for the ability to mduce CTL responses m 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 are deficient m their ability to load class I molecules with internally processed peptides and that have been transfected with the appropriate human class I gene, may be used to test for the capacity of the peptide to induce in vitro prunary CTL responses
  • Peripheral blood mononuclear cells may be used as the responder cell source of CTL precursors
  • the appropnate antigen-presentmg cells are mcubated with peptide, after which the peptide- loaded antigen-presentmg cells are then
  • HTL activation may also be assessed usmg such techniques known to those in the art such as T cell proliferation and secretion of lymphokines, e g IL-2 (see, e g Alexander et al , Immunity 1 751-761, 1994)
  • HLA transgenic mice can be used to determine lmmunogenicity of peptide epitopes
  • transgenic mouse models mcludmg mice with human A2 1, Al 1 (which can additionally be used to analyze HLA-A3 epitopes), and B7 alleles have been characterized and others (e g , transgenic mice for HLA-Al and A24) are bemg developed HLA-DR1 and HLA-DR3 mouse models have also been developed Additional transgenic mouse models with other HLA alleles may be generated as necessary
  • Mice may be immunized with peptides emulsified in Incomplete Freund's Adjuvant and the resultmg T cells tested for then: capacity to recognize peptide-pulsed target cells and target cells transfected with appropnate genes
  • CTL responses may be analyzed usmg cytotoxicity assays described above
  • HTL responses may be analyzed usmg such assays as T cell proliferation or secretion of lymphokmes
  • HLA class I and class II bmdmg peptides as described herem are used as reagents to evaluate an immune response
  • the immune response to be evaluated is mduced by usmg as an immunogen any agent that may result m the production of antigen-specific CTLs or HTLs that recognize and bmd to the peptide ep ⁇ tope(s) to be employed as the reagent
  • the peptide reagent need not be used as the immunogen Assay systems that are used for such an analysis mclude relatively recent technical developments such as tetramers, staming for mtracellular lymphokines and mterferon release assays, or ELISPOT assays
  • a peptide of the invention may be used in a tetramer stammg assay to assess peripheral blood mononuclear cells for the presence of antigen-specific CTLs following exposure to a tumor cell antigen or an immunogen
  • the HLA-tetrame ⁇ c complex is used
  • Peptides of the mvention can also be 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)
  • patient PBMC samples from individuals with cancer may be analyzed for the presence of antigen-specific CTLs or HTLs usmg specific peptides
  • a blood sample contammg mononuclear cells may be evaluated by cultivating the PBMCs and stimulating the cells with a peptide of the mvention After an appropriate cultivation period, the expanded cell population may be analyzed, for example, for CTL or for HTL activity
  • the peptides can also be used as reagents to evaluate the efficacy of a vaccine PBMCs obtamed from a patient vaccmated with an immunogen may be analyzed usmg, for example, either of the methods described above
  • the patient is HLA typed, and peptide epitope reagents that recognize the allele-specific molecules present m that patient are selected for the analysis
  • the lmmunogenicity of the vaccme is indicated by the presence of epitope-specific CTLs and or HTLs in the PBMC sample
  • the peptides of the mvention may also be used to make antibodies, usmg techniques well known in the art (see, e g CURRENT PROTOCOLS IN /MMf/ ⁇ r ⁇ o ⁇ r, Wiley/Greene, NY , and Antibodies A Laboratory Manual, Harlow and Lane, Cold Sprmg Harbor Laboratory Press, 1989), which may be useful as reagents to diagnose or monitor cancer
  • antibodies include those that recognize a peptide m the context of an HLA molecule, i e , antibodies that bmd to a peptide-MHC complex
  • Vaccmes and methods of preparmg vaccmes that contain an lmmunogemcally effective amount of one or more peptides as described herem are further embodiments of the mvention
  • vaccme compositions can mclude, for example, hpopeptides (e g Nitiello, A et al , J Clin Invest 95 341, 1995), peptide compositions encapsulated m poly(DL- lactide-co-glycohde) ("PLG”) microspheres (see, e g , Eld ⁇ dge, 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 contamed m immune stimulating complexes (ISCOMS)
  • Toxm-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc (Needham, Massachusetts) may also be used
  • Vaccmes of the mvention mclude nucleic acid-mediated modalities DNA or RNA encodmg one or more of the peptides of the mvention can also be administered to a patient This approach is described, for instance, in Wolff et al , Science 247 1465 (1990) as well as U S Patent Nos 5,580,859, 5,589,466,
  • DNA-based delivery technologies mclude "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic pid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e g , U S Patent No 5,922,687)
  • the peptides of the mvention can also be expressed by viral or bacterial vectors
  • expression vectors mclude attenuated viral hosts, such as vaccmia or fowlpox
  • vaccmia virus is used as a vector to express nucleotide sequences that encode the peptides of the mvention
  • the recombmant vaccmia virus Upon introduction into a host bearing a tumor, the recombmant vaccmia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response
  • Vaccinia vectors and methods useful m immunization protocols are descnbed m, e g , M S Patent No 4,722,848
  • Another vector is BCG (Bacille Calmette Guerm) BCG vectors are described m Stover et al , Nature 351 456-460 (1991)
  • BCG Bacilluent al
  • vaccmes in accordance with the invention encompass compositions of one or more of the claimed pept ⁇ de(s)
  • the pept ⁇ de(s) can be mdividually linked to its own carrier, alternatively, the pept ⁇ de(s) can exist as a homopolymer or heteropolymer of active peptide units
  • Such a polymer has the advantage of mcreased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to mduce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response
  • the composition may be a naturally occurring region of an antigen or may be prepared, e , recombinantly or by chemical synthesis
  • Carners that can be used with vaccmes of the mvention are well known the art, and mclude, e g , thyroglobulm, albumins such as human serum albumin, tetanus toxoid, polyamrno acids such as poly L- lysme, poly L-glutamic acid, influenza, hepatitis B virus core protem, and the like
  • the vaccmes can contam a physiologically tolerable (i e , acceptable) diluent such as water, or salme, preferably phosphate buffered salme
  • the vaccmes also typically mclude an adjuvant Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, alummum hydroxide, or alum are examples of materials well known m the art
  • CTL responses can be primed by conjugatmg peptides of the invention to lipids, such as tnpalmitoyl-S-glycerylc
  • a vaccme of the mvention can also mclude antigen-presentmg cells (APC), such as dendritic cells
  • Vaccme compositions can be created in vitro, following dendntic cell mobilization and harvestmg, whereby loadmg of dendritic cells occurs in vitro
  • dendritic cells are transfected, e , with a minigene m accordance with the mvention, or are pulsed with peptides
  • the dendritic cell can then be administered to a patient to elicit immune responses in vivo
  • Vaccme compositions either DNA- or peptide-based, can also be administered in vivo m combmation with dendritic cell mobilization whereby loadmg of dendritic cells occurs in vivo
  • Antigenic peptides are used to elicit a CTL and or HTL response ex vivo, as well
  • the resulting CTL or HTL cells can be used to treat tumors m patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccme peptide or nucleic acid m accordance with the mvention
  • Ex vivo CTL or HTL responses to a particular tumor-associated antigen are induced by mcubating m tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presentmg cells, such as dendntic cells, and the appropriate immunogenic peptide After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded mto effector cells, the cells are infused back mto the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (an infected cell or a tumor cell) Transfected dendntic cells
  • the vaccine compositions of the invention can also be used in combmation with other treatments used for cancer, mcludmg use m combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like
  • the following pnnciples are utilized when selecting an a ⁇ ay of epitopes for inclusion in a polyepitopic composition for use m a vaccine, or for selectmg discrete epitopes to be mcluded m a vaccme and/or to be encoded by nucleic acids such as a mmigene
  • Exemplary epitopes that may be utilized m a vaccme to treat or prevent cancer are set out in Tables XXIII-XXVII and XXXI It is preferred that each of the following principles are balanced m order to make the selection
  • the multiple epitopes to be incorporated m a given vaccme composition may be, but need not be, contiguous m 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 conelated with tumor clearance For HLA Class I this includes 3-4 epitopes that come from at least one TAA For HLA Class II a similar rationale is employed, again 3-4 epitopes are selected from at least one TAA (see e g , Rosenberg et al , Science 278 1447-1450) Epitopes from one TAA may be used m combmation with epitopes from one or more additional TAAs to produce a vaccme that targets tumors with varying expression patterns of frequently-expressed TAAs as described, e , m Example 15
  • the MAGE2/3 epitopes selected for inclusion are preferably conserved between the two protems
  • Epitopes are selected that have the requisite bmding affinity established to be correlated with lmmunogenicity for HLA Class I an IC 50 of 500 nM or less, or for Class II an IC 50 of 1000 nM or less
  • Sufficient supermotif bea ⁇ ng-peptides, or a sufficient anay of allele-specific motif- bea ⁇ ng peptides, are selected to give broad population coverage For example, it is preferable to have at least 80% population coverage
  • a Monte Carlo analysis a statistical evaluation known m the art, can be employed to assess the breadth, or redundancy of, population coverage 4
  • selectmg epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope
  • selecting epitopes for infectious disease-related antigens it is preferable to select either native or analoged epitopes
  • nested epitopes are epitopes referred to as “nested epitopes " Nested epitopes occur where at least two epitopes overlap m a given peptide sequence
  • a nested peptide sequence can comprise both HLA class I and HLA class II epitopes
  • providmg nested epitopes a general objective is to provide the greatest number of epitopes per sequence
  • an aspect is to avoid providmg a peptide that is any longer than the ammo terminus of the ammo terminal epitope and the carboxyl termmus of the carboxyl terminal epitope m the peptide
  • providmg a multi-epitopic sequence such as a sequence compnsmg nested epitopes, it is generally important to screen the sequence m order to msure that it does not have pathological or other deleterious biological properties
  • a polyepitopic protem is created, or when creating a ⁇ unigene, an objective is to generate the smallest peptide that encompasses the epitopes of mterest This principle is similar, if not the same as that employed when selecting a peptide compnsmg nested epitopes
  • the size minimization objective is balanced agamst the need to integrate any spacer sequences between epitopes in the polyepitopic protem
  • Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present m the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation
  • Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope Of particular concern is a junctional epitope that is a "dom
  • nucleic acids encodmg the peptides of the mvention are a particularly useful embodiment of the mvention
  • Epitopes for inclusion m a minigene are preferably selected accordmg to the guidelines set forth m the previous section
  • a preferred means of administenng nucleic acids encodmg the peptides of the mvention uses mmigene constructs encodmg a peptide comprising one or multiple epitopes of the mvention
  • the use of multi-epitope minigenes is descnbed below and m, e g , co-pending application
  • a multi-epitope DNA plasmid encodmg supermotif- and/or motif-bearmg MAGE2/3 epitopes derived from multiple regions of the MAGE2/3 proteins, the PADRETM universal helper T cell epitope (or multiple HTL epitopes from MAGE2/3), and an endoplasmic reticulum-translocating signal sequence can be engineered
  • a vaccme may also comprise epitopes, m addition to MAGE2/3 epitopes, that are derived from other TAAs
  • the lmmunogenicity of a multi-epitopic minigene can be tested m transgenic mice to evaluate the magnitude of CTL induction responses agamst the epitopes tested Further, the lmmunogenicity of DNA- encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lmes against target cells transfected with the DNA plasmid Thus, these experiments can show that the minigene serves to both 1 ) generate a CTL response and 2 ) that the mduced CTLs recognized cells expressmg the encoded epitopes
  • the ammo 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
  • the minigene sequence may be converted to DNA by assemblmg 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 jomed, for example, usmg T4 DNA hgase
  • This synthetic minigene, encodmg the epitope polypeptide can then be cloned mto a desired expression vector
  • Standard regulatory sequences well known to those of skill m the art are preferably mcluded in the vector to ensure expression m the target cells
  • Several vector elements are desirable a promoter with a down-stream cloning site for minigene insertion, a polyadenylation signal for efficient transcription termination, an E coli ongm of replication, and an E coli selectable marker (e g ampicilh
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occumng introns could be incorporated mto the transcnbed region of the minigene
  • mRNA stabilization sequences and sequences for replication m mammalian cells may also be considered for mcreasmg minigene expression
  • the minigene is cloned mto the polylinker region downstream of the promoter
  • This plasmid is transformed mto an appropriate E coli strain, and DNA is prepared using standard techniques
  • the orientation and DNA sequence of the minigene, as well as all other elements mcluded in the vector, are confirmed usmg restriction mappmg and DNA sequence analysis
  • Bacterial cells harboring the conect plasmid can be stored as a master cell bank and a workmg cell bank
  • unmunostimulatory sequences appear to play a role m the lmmunogenicity of DNA vaccmes
  • ISSs or CpGs unmunostimulatory sequences
  • These sequences may be mcluded m the vector, outside the minigene codmg sequence, if desired to enhance lmmunogenicity
  • a bi-cistromc expression vector which allows production of both the minigene-encoded epitopes and a second protem (included to enhance or decrease lmmunogenicity) can be used.
  • protems or polypeptides that could beneficially enhance the immune response if co- expressed mclude cytokmes (e g , IL-2, IL-12, GM-CSF), cytokme-mducmg molecules (e g , LeIF), costimulatory molecules, or for HTL responses, pan-DR bmding protems (e g , PADRETM, Epimmune, San Diego, CA) Helper (HTL) epitopes can be joined to mtracellular targeting signals and expressed separately from expressed CTL epitopes, this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes If required, this could facilitate more efficient entry of HTL epitopes mto the HLA class II pathway, thereby
  • Therapeutic quantities of plasmid DNA can be produced for example, by fermentation m E coli, followed by purification Ahquots from the workmg cell bank are used to moculate growth medium, and grown to saturation in shaker flasks or a bioreactor accordmg to well known techniques
  • Plasmid DNA can be purified usmg standard bioseparation technologies such as solid phase amon-exchange resms supplied by QIAGEN, Inc (Valencia, California) If required, supercoiled DNA can be isolated from the open circular and lmear forms using gel electrophoresis or other methods
  • Purified plasmid DNA can be prepared for injection usmg a variety of formulations The simplest of these is reconstitution of lyophihzed DNA m sterile phosphate-buffered salme (PBS) This approach, known as "naked DNA," is currently bemg used for intramuscular (IM) admmistration m clmical trials To maximize the lmmunotherapeutic effects of minigene DNA vaccmes, an alternative method for formulating purified plasmid DNA may be desirable A variety of methods have been described, and new techniques may become available Cationic lipids, glycohpids, and fusogemc liposomes can also be used m the formulation (see, e , as descnbed by WO 93/24640, Mannino & Gould-Foge ⁇ te, BioTechmques 6(7) 682 (1988), U S Pat No 5,279,833, WO 91/06309, and Feigner, et al , Pro
  • Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes
  • the plasmid DNA is introduced into a mammalian cell lme that is suitable as a target for standard CTL chromium release assays
  • the transfection method used will be dependent on the final formulation Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection
  • a plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS)
  • FACS fluorescence activated cell sorting
  • These cells are then chrom ⁇ um-51 ( 51 Cr) labeled and used as target cells for epitope- specific CTL lmes, cyto lysis, detected by 5l Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes
  • nucleic acids can be administered usmg ballistic delivery as described, for instance, m U S Patent No 5,204,253 Usmg this technique, particles comprised solely of DNA are administered
  • DNA can be adhered to particles, such as gold particles Mmigenes can also be delivered usmg other bacterial or viral delivery systems well known in the art, e g , an expression construct encodmg epitopes of the mvention can be incorporated mto a viral vector such as vaccmia
  • Vaccme compositions compnsmg the peptides of the present mvention, or analogs thereof, which have lmmunostimulatory activity may be modified to provide desired attributes, such as improved serum half-life, or to enhance lmmunogenicity
  • desired attributes such as improved serum half-life, or to enhance lmmunogenicity
  • the ability of a peptide to mduce CTL activity can be enhanced by linking the peptide to a sequence which contams at least one epitope that is capable of mducmg a T helper cell response
  • T helper epitopes m conjunction with CTL epitopes to enhance lmmunogenicity is illustrated, for example, m the co-pendmg applications U S S N 08/820,360, U S S N 08/197,484, and U S S N 08/464,234
  • a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by
  • pan-DR-bmding epitopes e g , PADRETM, Epimmune, Inc , San Diego, CA
  • HTL peptide epitopes can also be modified to alter their biological properties
  • they can be modified to mclude D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, protems, carbohydrates, and the like to mcrease their biological activity
  • a T helper peptide can be conjugated to one or more palmitic acid chams at either the ammo or carboxyl termini
  • compositions of the invention at least one component which prunes cytotoxic T lymphocytes
  • Lipids have been identified as agents capable of pnming CTL in vivo agamst viral antigens
  • palmitic acid residues can be attached to the ⁇ -and ⁇ - ammo groups of a lysme residue and then linked, e , via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide
  • the hpidated peptide can then be administered either directly m a micelle or particle, incorporated mto a hposome, or emulsified in an adjuvant, e g , mcomplete Freund's adjuvant
  • a prefened immunogenic composition comprises palmitic acid attached to ⁇ - and ⁇ - ammo groups of Lys, which is attached
  • E coli hpoproteins such as t ⁇ palmitoyl-S- glycerylcysteinlyseryl- serine (P 3 CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e g , Deres, et al , Nature 342 561, 1989)
  • Peptides of the mvention can be coupled to P 3 CSS, for example, and the hpopeptide admmistered to an individual to specifically prime a
  • CTL and or HTL peptides can also be modified by the addition of ammo acids to the termini of a peptide to provide for ease of linking peptides one to another, for couplmg to a earner support or larger peptide, for modifying the physical or chemical properties of the peptide or ohgopeptide, or the like
  • Ammo acids such as tyrosine, cysteme, lysme, glutamic or aspartic acid, or the like, can be introduced at the C- or N-termmus of the peptide or ohgopeptide, particularly class I peptides
  • modification at the carboxyl termmus of a CTL epitope may, m some cases, alter bmdmg characteristics of the peptide
  • the peptide or ohgopeptide sequences can differ from the natural sequence by being modified by term ⁇ nal-NH 2 acylation, e , by alkanoyl (C ⁇ -C o) or thioglycolyl acetylation, terminal-carboxyl amidation, e g , ammonia, methylamine, etc In some mstances these modifications may provide sites for linking to a support or other molecule
  • An embodiment of a vaccme composition m accordance with the invention comprises ex vivo admmistration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood
  • a pharmaceutical to facilitate harvestmg of DC can be used, such as ProgempoietinTM (Monsanto, St Louis, MO) or GM-CSF/IL-4
  • the DC are washed to remove unbound peptides
  • a vaccme comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on then- surfaces
  • the DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL response to one or more antigens of interest, e g , a MAGE polypeptide, HER/2neu, p53, CEA, a prostate cancer associated
  • the peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent cancer Vaccine compositions contammg the peptides of the mvention are admmistered to a cancer patient or to an individual susceptible to, or otherwise at risk for, cancer to elicit an immune response agamst TAAs and thus enhance the patient's own immune response capabilities
  • peptide and/or nucleic acid compositions are administered to a patient m an amount sufficient to elicit an effective CTL and/or HTL response to the tumor antigen and to cure or at least partially anest or slow symptoms and/or complications
  • An amount adequate to accomplish this is defined as "therapeutically effective dose"
  • Amounts effective for this use will depend on, e g , the particular composition admmistered, the manner of admmistration, 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 vaccme compositions of the invention may also be used purely as prophylactic agents
  • the dosage for an initial prophylactic immunization generally occurs m a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 ⁇ g and the higher value is about 10,000, 20,000, 30,000, or
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient This is followed by boosting dosages of between about 1 0 ⁇ g to about 50,000 ⁇ g of peptide admmistered at defined intervals from about four weeks to six months after the initial admmistration of vaccine
  • the lmmunogenicity of the vaccine may be assessed by measurmg the specific activity of CTL and HTL obtamed from a sample of the patient's blood
  • peptides compnsmg CTL and/or HTL epitopes of the mvention mduce immune responses when presented by HLA molecules and contacted with a CTL or HTL specific for an epitope comprised by the peptide
  • the manner m which the peptide is contacted with the CTL or HTL is not critical to the mvention For mstance, the peptide can be contacted with the CTL or HTL either in vivo or in vitro If the contactmg occurs in vivo, the peptide itself can be admmistered to the patient, or other vehicles, e g DNA vectors encodmg one or more peptides, viral vectors encodmg the pept ⁇ de(s), liposomes and the like, can be used, as described herem
  • the vaccmatmg agent can comprise a population of cells, e g , peptide-pulsed dendntic cells, or TAA-specific CTLs, which have been mduced by pulsing antigen- presentmg cells in vitro with the peptide Such a cell population is subsequently admmistered to a patient m a therapeutically effective dose
  • the immunogenic peptides of the mvention, or DNA encoding them are generally admmistered to an individual already diagnosed with cancer
  • the peptides or DNA encodmg them can be admmistered mdividually or as fusions of one or more peptide sequences
  • admmistration should generally begm at the first diagnosis of cancer This is followed by boostmg doses until at least symptoms are substantially abated and for a period thereafter
  • the embodiment of the vaccine composition (1 e , mcludmg, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs) delivered to the patient may vary accordmg to the stage of the disease For example, a vaccine compnsmg TAA-specific CTLs may be more efficacious m killing tumor cells in patients with advanced disease than alternative embodiments
  • the vaccme compositions of the mvention may also be used therapeutically in combination with treatments such as surgery
  • treatments such as surgery
  • An example is a situation m which a patient has undergone surgery to remove a primary tumor and the vaccine is then used to slow or prevent recunence and/or metastasis
  • composition can be targeted to them, thus minimizing the need for admmistration to a larger population
  • the dosage for an initial therapeutic immunization generally occurs m a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 ⁇ g and the higher value is about 10,000, 20,000, 30,000, or 50,000 ⁇ g
  • Dosage values for a human typically range from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient
  • Boostmg dosages of between about 1 0 ⁇ g to about 50,000 ⁇ g of peptide pursuant to a boostmg regimen over weeks to months may be admmistered depending upon the patient's response and condition as determmed by measuring the specific activity of CTL and HTL obtamed from the patient's blood
  • the peptides and compositions of the present invention may be employed m serious disease states, that is, life-threatening or potentially life threatening situations In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides m preferred compositions of the invention, it is possible and may be felt desirable by the
  • a representative dose is m the range disclosed above, namely where the lower value is about 1, 5, 50, 500, or 1,000 ⁇ g and the higher value is about 10,000, 20,000, 30,000, or 50,000 ⁇ g, preferably from about 500 ⁇ g to about 50,000 ⁇ g per 70 kilogram patient
  • Initial doses followed by boostmg doses at established intervals, e g , from four weeks to six months, may be required, possibly for a prolonged period of tune to effectively immunize an individual
  • Admmistration should contmue until at least clmical symptoms or laboratory tests mdicate that the tumor has been eliminated or that the tumor cell burden has been substantially reduced and for a period thereafter
  • the dosages, routes of admmistration, and dose schedules are adjusted m accordance with methodologies known m the art
  • compositions for therapeutic treatment are mtended for parenteral, topical, oral, lntrathecal, or local admmistration
  • the pharmaceutical compositions are administered parentally, e g , intravenously, subcutaneously, intradermally, or intramuscularly
  • the mvention provides compositions for parenteral administration which compnse a solution of the immunogenic peptides dissolved or suspended m an acceptable earner, preferably an aqueous earner
  • aqueous earners may be used, e g , water, buffered water, 0 8% salme, 0 3% glycine, hyaluronic acid and the like
  • These compositions may be stenhzed by conventional, well known sterilization techniques, or may be sterile filtered
  • the resultmg aqueous solutions may be packaged for use as is, or lyophihzed, the lyophilized preparation bemg combined with a sterile solution prior to administration
  • the compositions may contam
  • concentration of peptides of the invention m 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 , m accordance with the particular mode of admmistration selected
  • a human unit dose form of the peptide composition is typically mcluded in a pharmaceutical composition that comprises a human unit dose of an acceptable earner, preferably an aqueous earner, and is admmistered 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 mvention may also be admmistered 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 mcrease the half-life of the peptide composition
  • Liposomes include emulsions, foams, micelles, msoluble monolayers, liquid crystals, phosphohpid dispersions, lamellar layers and the like
  • the peptide to be delivered is incorporated as part of a hposome, alone or m conjunction with a molecule which bmds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bmd to the CD45 antigen, or with other therapeutic or immunogenic compositions
  • liposomes either filled or decorated with a desired peptide of the mvention can be dnected to the site of lymphoid cells, where the liposomes then deliver the peptide compositions Lipo
  • a ligand to be incorporated mto the hposome can include, e g , antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells
  • a hposome suspension contammg a peptide may be admmistered intravenously, locally, topically, etc in a dose which varies accordmg to, inter aha, the manner of admmistration, the peptide bemg delivered, and the stage of the disease bemg treated
  • nontoxic solid earners 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 incorporatmg any of the normally employed excipients, such as those earners previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%
  • the immunogenic peptides are preferably supplied m finely divided form along with a surfactant and propellant Typical percentages of peptides are 0 01%-20% by weight, preferably 1%-10%
  • the surfactant must, of course, be nontoxic, and preferably soluble m the propellant
  • Representative of such agents are the esters or partial esters of fatty acids contammg from 6 to 22 carbon atoms, such as caproic, octanoic, lau ⁇ c, palmitic, stearic, linoleic, hnolenic, olestenc and oleic acids with an aliphatic polyhydnc alcohol or its cyclic anhydride
  • Mixed esters such as mixed or natural glycerides may be employed
  • the surfactant may constitute 0 l%-20% by weight of the composition, preferably 0 25- 5%
  • the balance of the composition is ordinarily propellant
  • a earner can also be mcluded
  • HLA class I antigens m tumors The three mam types of alterations in HLA expression in tumors and then functional significance
  • the level and pattern of expression of HLA class I antigens m tumors has been studied m many different tumor types and alterations have been reported m all types of tumors studied
  • the molecular mechanisms underlining HLA class I alterations have been demonstrated to be quite heterogeneous They include alterations m the TAP/processing pathways, mutations of ⁇ 2-m ⁇ croglobuhn and specific HLA heavy chams, alterations m the regulatory elements controlling over class I expression and loss of ent ⁇ e chromosome sections
  • e g Gamdo F, et al
  • Natural history of HLA expression durmg tumour development Immunol Today 14(10) 491-499, 1993
  • Kaklamams L, et al Loss of HLA class-I alleles, heavy chains and ⁇ 2- ⁇ croglobul ⁇ n m colorectal cancer Int J Cancer, 51(3) 379-85,
  • 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 m advanced melanoma J Cellular Phys 182 332-338, 2000, Liunggren HG, et al , Host resistance dnected selectively against H-2 deficient lymphoma variants Analysis of the mechanism J Exp Med , Dec 1,162(6) 1745-59, 1985, Maio M, et al , Reduction susceptibility to natural killer cell-mediated lysis of human FO-1 melanoma cells after mduction of HLA class I antigen expression by transfection with B2m gene J Clin Invest 88(1) 282-9, July 1991, Sch ⁇ er PI, et al , Relationship between myc oncogene activation and M
  • the bystander effect is understood to be mediated by cytokmes elicited from, e g , CTLs actmg on an HLA-bearmg target cell, whereby the cytokmes are in the environment of other diseased cells that are concomitantly killed
  • Allele-specific loss One of the most common types of alterations in class I molecules is the selective loss of certain alleles m individuals heterozygous for HLA Allele-specific alterations might reflect the tumor adaptation to immune pressure, exerted by an immunodominant response restricted by a single HLA restriction element This type of alteration allows the tumor to retam class I expression and thus escape NK cell recognition, yet still be susceptible to a CTL-based vaccme m accordance with the mvention which comprises epitopes conesponding to the remammg HLA type
  • a practical solution to overcome the potential hurdle of allele-specific loss relies on the induction of multispecific responses Just as the inclusion of multiple disease-associated antigens m a vaccme of the mvention guards against mutations that yield loss of a specific disease antigens, simultaneously targetmg multiple HLA specificities and multiple disease-related antigens prevents disease escape by allele-specific losses
  • HLA class I expression can be upregulated by gamma IFN, commonly secreted by effector CTL Additionally, HLA class I expression can be mduced m vivo by both alpha and beta IFN (Halloran, et al Local T cell responses mduce 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
  • Tones et al (Tones, MJ, et al , Loss of an HLA haplotype m pancreas cancer tissue and its conesponding tumor derived cell lme Tissue Antigens 47 372-81, 1996) note that HLA expression is upregulated by gamma IFN m pancreatic cancer, unless a total loss of haplotype has occurred Similarly, Rees and Mian note that allelic deletion and loss can be restored, at least partially, by cytokines such as IFN-gamma (Rees, R , et al Selective MHC expression m tumours modulates adaptive and innate antitumour responses Cancer Immunol Immunother 48 374-81, 1999) It has also been noted that IFN-gamma treatment results in upregulation of class I molecules m 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
  • 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. Clin. Invest. 88(l):282-9, July 1991 ; Schrier PI, et al, Relationship between myc oncogene activation and MHC class I expression Adv. Cancer Res., 60: 181-246, 1993).
  • 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 m diseased cells
  • various embodiments of the present invention can be selected m view of the fact that there can be a degree of loss of HLA molecules, particularly m the context of neoplastic disease
  • the treatmg physician can assay a patient's tumor to ascertam whether HLA is bemg expressed If a percentage of tumor cells express no class I HLA, then embodiments of the present mvention that comprise methods or compositions that elicit NK cell responses can be employed as noted herem, such
  • NK-mducing methods or composition can comprise a Flt3 ligand or ProGP which facilitate mobilization of dendntic cells, the rationale being that dendntic cells produce large amounts of IL-12 IL-12 can also be admmistered directly m either ammo acid or nucleic acid form It should be noted that compositions in accordance with the invention can be administered concu ⁇ ently with NK cell- inducing compositions, or these compositions can be admmistered sequentially
  • a tumor retams class I expression and may thus escape NK cell recognition, yet still be susceptible to a CTL-based vaccme m accordance with the mvention which comprises epitopes conespondmg to the remammg HLA type
  • the concept here is analogous to embodiments of the mvention that mclude multiple disease antigens to guard against mutations that yield loss of a specific antigen
  • embodiments of the present mvention can be combmed with alternative therapeutic compositions and methods
  • Such alternative compositions and methods compnse, without limitation, radiation, cytotoxic pharmaceuticals, and or compositions/methods that mduce humoral antibody responses
  • embodiments of the mvention can also comprise alpha, beta and/or gamma IFN to facilitate upregualtion of HLA
  • compositions of the mvention are admmistered concunently with the standard therapy Durmg this period, the patient's immune system is directed to induce responses agamst the epitopes comprised by the present mventive compositions Upon removal from the treatment havmg side effects, the patient is primed to respond to the infectious pathogen should the pathogen load begm to mcrease Composition of the invention can be provided during the drug holiday as well
  • compositions m accordance with the mvention are admmistered Accordingly, as the patient's immune system reconstitutes, precious immune resources are simultaneously dnected agamst the cancer Composition of the mvention can also be admmistered concunently with an lmmunosuppressive regimen if desired
  • kits The peptide and nucleic acid compositions of this invenUon can be provided m kit form together with instructions for vaccme admmistration
  • the kit would mclude desired peptide compositions in a contamer, preferably in umt dosage form and instructions for administration
  • An alternative kit would mclude a minigene construct with desired nucleic acids of the mvention m a contamer, preferably m unit dosage form together with instructions for admmistration Lymphokines such as IL-2 or IL-12 may also be mcluded m the kit
  • kits Other kit components that may also be desirable mclude, for example, a sterile syringe, booster dosages, and other desired excipients
  • Epitopes m accordance with the present mvention were successfully used to mduce an immune response Immune responses with these epitopes have been mduced by administermg the epitopes in various forms
  • the epitopes have been admmistered as peptides, as nucleic acids, and as viral vectors comprising nucleic acids that encode the ep ⁇ tope(s) of the mvention
  • immune responses have been mduced by direct loadmg of an epitope onto an empty HLA molecule that is expressed on a cell, and via mternalization of the epitope and processmg via the HLA class I pathway, m either event, the HLA molecule expressmg the epitope was then able to interact with and induce a CTL response
  • Peptides can be delivered directly or usmg such agents as liposomes They can additionally be delivered usmg ballistic delivery, m which the peptides are typically m a crystalline form When DNA
  • compositions in accordance with the invention exist in several forms. Embodiments of each of these composition forms in accordance with the invention have been successfully used to induce an immune response.
  • composition in accordance with the invention comprises a plurality of peptides.
  • This plurality or cocktail of peptides is generally admixed with one or more pharmaceutically acceptable excipients.
  • the peptide cocktail can comprise multiple copies of the same peptide or can comprise a mixture of peptides.
  • the peptides can be analogs of naturally occumng epitopes.
  • the peptides can comprise artificial amino acids and/or chemical modifications such as addition of a surface active molecule, e.g., lipidation; acetylation, glycosylation, biotinylation, phosphorylation etc.
  • the peptides can be CTL or HTL epitopes.
  • the peptide cocktail comprises a plurality of different CTL epitopes and at least one HTL epitope.
  • the HTL epitope can be naturally or non-naturally (e.g., PADRE®, Epimmune Inc., San Diego, CA).
  • the number of distinct epitopes in an embodiment of the invention is generally a whole unit integer from one through two hundred (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 105, 107,
  • composition in accordance with the invention comprises a polypeptide multi-epitope construct, i.e., a polyepitopic peptide.
  • Polyepitopic peptides in accordance with the invention are prepared by use of technologies well-known in the art. By use of these known technologies, epitopes in accordance with the invention are connected one to another.
  • the polyepitopic peptides can be linear or non-linear, e.g., multivalent.
  • These polyepitopic constructs can comprise artificial amino acids, spacing or spacer amino acids, flanking amino acids, or chemical modifications between adjacent epitope units.
  • the polyepitopic construct can be a heteropolymer or a homopolymer.
  • the polyepitopic constructs generally comprise epitopes in a quantity of any whole unit integer between 2-200 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, etc.).
  • 2-200 e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the polyepitopic construct can comprise CTL and/or HTL epitopes.
  • One or more of the epitopes in the construct can be modified, e.g., by addition of a surface active material, e.g. a lipid, or chemically modified, e.g., acetylation, etc.
  • bonds in the multiepitopic construct can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.
  • a composition in accordance with the mvention comprises construct which comprises a series, sequence, stretch, etc , of ammo acids that have homology to ( i e , conesponds to or is contiguous with) to a native sequence
  • This stretch of ammo acids comprises at least one subsequence of ammo acids that, if cleaved or isolated from the longer series of ammo acids, functions as an HLA class I or HLA class II epitope m accordance with the mvention
  • the peptide sequence is modified, so as to become a construct as defined herem, by use of any number of techniques known or to be provided m the art
  • the polyepitopic constructs can contain homology to a native sequence in any whole unit mteger mcrement from 70-100%, e , 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
  • compositions m accordance with the mvention comprise nucleic acids that encode one or more peptides of the mvention, or nucleic acids which encode a polyepitopic peptide m accordance with the invention
  • vanous nucleic acids compositions will encode the same peptide due to the redundancy of the genetic code
  • Each of these nucleic acid compositions falls withm the scope of the present mvention
  • This embodiment of the mvention comprises DNA or RNA, and in certain embodiments a combmation of DNA and RNA It is to be appreciated that any composition compnsmg nucleic acids that will encode a peptide in accordance with the invention or any other peptide based composition in accordance with the mvention, falls withm the scope of this mvention
  • peptide-based forms of the mvention can comprise analogs of epitopes of the invention generated usmg prmiciples already known, or to be known, m the art Principles related to analogmg are now known m the art, and are disclosed herem, moreover, analogmg principles (heterochtic analogmg) are disclosed m co-pendmg application serial number U S S N 09/226,775 filed 6 January 1999 Generally the compositions of the mvention are isolated or purified
  • the folio wing example of peptide bmding to HLA molecules demonstrates quantification of bmding affinities of HLA class I and class II peptides Bmdmg assays can be performed with peptides that are either motif-bearmg or not motif-bearmg
  • HLA class I and class II bmding assays usmg purified HLA molecules were performed in accordance with disclosed protocols (e g , PCT publications WO 94/20127 and WO 94/03205, Sidney et al , Current Protocols in Immunology 18 3 1 (1998), Sidney, et al , J Immunol 154 247 (1995), Sette, et al , Mol Immunol 31 813 (1994)) Bnefly, purified MHC molecules (5 to 500nM) were mcubated with various unlabeled peptide inhibitors and 1-lOnM 125 I-rad ⁇ olabeled probe peptides as described Following incubation, MHC-peptide complexes were separated from free peptide by gel filtration and the fraction of peptide bound was determmed Typically, m preliminary experiments, each MHC preparation was titered m the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bm
  • Vaccme compositions of the invention may mclude multiple epitopes that comprise multiple HLA supermotifs or motifs to achieve broad population coverage
  • This example illustrates the identification of supermotif- and motif-bearmg epitopes for the inclusion m such a vaccme composition
  • Calculation of population coverage is performed using the strategy described below
  • the ARB values conesponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
  • HLA-A*0201 is considered a prototype A2 supertype molecule.
  • IC50 values ⁇ 500 nM.
  • the 19 A*0201 -binding peptides were subsequently tested for the capacity to bind to additional
  • A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). As shown in Table XXII, 17 of the 19 peptides were found to be A2-supertype cross-reactive binders, binding at least three of the five A2- supertype alleles tested.
  • HLA- A3-supermotif primary anchors using methodology similar to that performed to identify HLA-A2 supermotif-bearing epitopes. Peptides conesponding to the supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the two most prevalent A3-supertype alleles. The peptides that are found to bind one of the two alleles with binding affinities of ⁇ 500 nM are then tested for binding cross-reactivity to the other common A3-supertype alleles (A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA- A3 -supertype molecules tested. Examples of HLA-A3 cross-binding supermotif-bearing peptides identified in accordance with this procedure are provided in Table XXIII.
  • HLA-B7-supermotif- bearing sequences The same target antigen protein sequences are also analyzed to identify HLA-B7-supermotif- bearing sequences.
  • the conesponding peptides are then synthesized and tested for binding to HLA-
  • B*0702 the most common B7-supertype allele (i.e., the prototype B7 supertype allele).
  • Those peptides that bind B*0702 with IC 50 of ⁇ 500 nM are then tested for binding to other common B7-supertype molecules (B*3501, B*5101, B*5301, and B*5401) to identify those peptides that are capable of binding to three or more of the five B7-supertype alleles tested.
  • Examples of HLA-B7 cross-binding supermotif-bearing peptides identified in accordance with this procedure are provided in Table XXIV.
  • HLA-Al and -A24 motif-bearing epitopes can also be inco ⁇ orated into potential vaccine constructs.
  • An analysis of the protein sequence data from the target antigen utilized above is also performed to identify HLA-Al- and A24-motif-containing conserved sequences.
  • the conesponding peptide sequence are then synthesized and tested for binding to the appropriate allele-specific HLA molecule, HLA-Al or HLA-24.
  • Peptides are identified that bind to the allele-specific HLA molecules at an IC 50 of ⁇ 500 nM. Examples of peptides identified in accordance with this procedure are provided in Tables XXV and XXVI.
  • the 221A2 1 cell lme produced by transfemng the HLA-A2 1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell lme 721 221, was used as the peptide-loaded target to measure activity of HLA-A2 1-restncted CTL
  • the HLA-typed melanoma cell lines (624mel and 888mel) were obtamed from Y Kawakami and S Rosenberg, National Cancer Institute, Bethesda, MD
  • the cell lines were mamtamed m RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential ammo acids and 10% (v/v) heat mactivated FCS
  • the melanoma cells were treated with lOOU/ml IFN ⁇ (Genzyme) for 48 hours at 37°C before use as targets m the 5l Cr release and in situ IFN ⁇ assays
  • PBMCs were thawed m RPMI with 30 ⁇ g/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential ammo acids, sodium pyruvate, L-glutamme and penicillin/strpetomycin)
  • complete medium RPMI-1640 plus 5% AB human serum, non-essential ammo acids, sodium pyruvate, L-glutamme and penicillin/strpetomycin
  • the monocytes were purified by plating 10 x 10 6 PBMC/well in a 6-well plate After 2 hours at 37°C, the non-adherent cells were removed by gently shaking the plates and aspirating the supernatants
  • the wells were washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells
  • Dynal lmmunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent Typically about 200- 250xl0 6 PBMC were processed to obtam 24xl0 6 CD8 + T-cells (enough for a 48-well plate culture) Briefly, the PBMCs were thawed m RPMI with 30 ⁇ g/ml DNAse, washed once with PBS contammg 1% human AB serum and resuspended m PBS/1% AB serum at a concentration of 20xl0 6 cells/ml The magnetic beads were washed 3 tunes with PBS/AB serum, added to the cells (140 ⁇ l beads/20xl0 6 cells) and mcubated for 1 hour at 4°C with contmuous mixing The beads and cells were washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at lOOxlO 6 cells/ml
  • Adherent target cells were removed from culture flasks with trypsm-EDTA Target cells were labelled with 200 ⁇ C ⁇ of 51 Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37 C C
  • Labelled target cells are resuspended at 10 6 per ml and diluted 1 10 with K562 cells at a concentration of 3 3xl0 ⁇ /ml (an NK-sensitive erythroblastoma cell lme used to reduce non-specific lysis)
  • Target cells 100 ⁇ l
  • 1 OO ⁇ l of effectors were plated in 96 well round-bottom plates and mcubated for 5 hours at 37°C At that time, 100 ⁇ l of supernatant were collected from each well and percent lysis was determmed according to the formula [(cpm of the test sample- cpm of the spontaneous 5l Cr release sample)/(cpm of the maximal 51 Cr release sample- cpm of the spontaneous 51 Cr release sample)] x 100 Maximum and spontaneous release were determmed
  • Immulon 2 plates were coated with mouse anti-human IFN ⁇ monoclonal antibody (4 ⁇ g/ml 0 IM NaHC0 3 , pH8 2) overnight at 4°C The plates were washed with Ca 2+ , Mg 2+ -free PBS/0 05% Tween 20 and blocked with PBS/10% FCS for 2 hours, after which the CTLs (100 ⁇ l/well) and targets (100 ⁇ l/well) were added to each well, leavmg empty wells for the standards and blanks (which received media only)
  • the target cells either peptide-pulsed or endogenous targets, were used at a concentration of lxl 0 e cells/ml
  • the plates were mcubated for 48 hours at 37°C with 5% C0 2
  • Recombmant human IFN ⁇ was added to the standard wells starting at 400 pg or 1200pg/100 ⁇ l well and the plate mcubated for 2 hours at 37°C The plates were washed and 100 ⁇ l of biotinylated mouse anti- human IFN ⁇ monoclonal antibody (4 ⁇ g/ml m PBS/3%FCS/0 05% Tween 20) were added and mcubated for
  • the A2-supermot ⁇ f cross-reactive bindmg peptides that were selected for further evaluation were tested m the cellular assay for the ability to mduce peptide-specific CTL in normal individuals
  • a peptide was considered to be an epitope if it mduced peptide-specific CTLs m at least 2 donors (unless otherwise noted) and if those CTLs also recognized the endogenously expressed peptide
  • HLA motifs and supermotifs are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herem Moreover, the definition of HLA motifs and supermotifs also allows one to engmeer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analogued, or "fixed” to confer upon the peptide certam characteristics, e g greater cross-reactivity withm the group of HLA molecules that comprise a supertype, and/or greater bmdmg affinity for some or all of those HLA molecules Examples of analog peptides that exhibit modulated bmding affinity are set forth in this example and provided m Tables XXII through XXVII
  • the mam anchors of A2-supermot ⁇ f-bea ⁇ ng peptides are altered, for example, to introduce a prefened L, I, V, or M at position 2, and I or V at the C-terminus
  • Peptides that exhibit at least weak A*0201 bmdmg (IC 50 of 5000 nM or less), and carrying suboptimal anchor residues at either position 2, the C-termmal position, or both, can be fixed by introducing canonical substitutions (L at position 2 and V at the C- terminus)
  • Those analogued peptides that show at least a three- fold mcrease m A*0201 bindmg and bmd with an IC 50 of 500 nM, or less were then tested for A2 cross-reactive bmdmg along with their wild-type (WT) counterparts
  • Analogued peptides that bmd at least three of the five A2 supertype alleles were then selected for cellular screening analysis
  • HLA-B7 supermotif-bearing epitopes are also generated.
  • peptides bmdmg at least weakly to 3/5 of the A3-supertype molecules can be engineered at prunary anchor residues to possess a prefened residue (V, S, M, or A) at position 2
  • the analog peptides are then tested for the ability to bmd A*03 and A* 11 (prototype A3 supertype alleles)
  • Those peptides that demonstrate ⁇ 500 nM bmdmg capacity are then tested for A3-supertype cross-reactivity
  • Examples of HLA-A3 supermotif analog peptides are provided m Table XXIII
  • B7 supermotif-bearing peptides can, for example, be engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position (see, e g Sidney et al (J Immunol 157 3480-3490, 1996) Analoged peptides are then tested for cross-reactive bindmg to B7 supertype alleles Examples of B7-supermot ⁇ f-beanng analog peptides are provided m Table XXIV
  • HLA-Al and HLA-A24 motif-bearing peptides can be engmeered at primary anchor residues to improvde bmding to the allele-specific HLA molecule or to improve cross-reactive bmding
  • Examples of analoged HLA-Al and HLA-A24 motif-bearmg peptides are provided m Tables XXV and XXVI Analoged peptides that exhibit unproved bmdmg and/or or cross-reactivity are evaluated for lmmunogenicity usmg methodology similar to that described for the analysis of HLA-A2 supermotif- bearing peptides Usmg such a procedure, peptides that mduce an immune response are identified
  • HLA supermotifs are of value m engineenng highly cross-reactive peptides and/or peptides that bmd HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties Examples of such analoged peptides are provided in Table XXIV
  • the bmdmg capacity of a B7 supermotif-bearing peptide representing a discreet single ammo acid substitution at position 1 can be analyzed
  • a peptide can, for example, be analogued to substitute L with F at position 1 and subsequently be evaluated for mcreased bmdmg affinity/ and or increased cross-reactivity This procedure will identify analogued peptides with modulated bmdmg affinity
  • cysteme has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce bmdmg capacity
  • Subtitution of ⁇ -amino butyric acid for cysteme not only alleviates this problem, but has been shown to improve bmdmg and crossbmdmg capabilities m some mstances (see, e g , the review by Sette et al , In Persistent Viral Infections. Eds R Ahmed and I Chen, John Wiley & Sons, England, 1999)
  • Example 5 Identification of peptide epitope sequences with HLA-DR bindmg motifs Peptide epitopes bearmg an HLA class II supermotif or motif may also be identified as outlmed below using methodology similar to that described m Examples 1-3
  • the MAGE2/3 protem sequences were analyzed for the presence of sequences bearmg an HLA-DR-motif or supermotif Specifically, 15-mer sequences were selected compnsmg a DR- supermotif, further compnsmg a 9-mer core, and three-residue N- and C-terminal flanking regions (15 ammo acids total)
  • Protocols for predictmg peptide binding to DR molecules have been developed (Southwood et al , J Immunol 160 3363-3373, 1998) These protocols, specific for individual DR molecules, allow the scoring, and rankmg, of 9-mer core regions Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (I e , at position 1 and position 6) withm a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors Usmg allele specific selection tables (see, e , Southwood et al , ibid ), it has been found that these protocols efficiently select peptide sequences with a high probability of bmdmg a particular DR molecule Additionally, it has been found that performing these protocols in tandem, specifically those for DRl, DR4w4, and DR7, can efficiently select DR cross-reactive peptides
  • the MAGE2/3-denved peptides identified above were tested for then bmding capacity for various common HLA-DR molecules All peptides were initially tested for bmdmg to the DR molecules in the prunary panel DRl, DR4w4, and DR7 Peptides binding at least 2 of these 3 DR molecules with an IC 50 value of 1000 nM or less, were then tested for bmdmg to DR5 *0101 , DRB 1*1501, DRB 1*1101, DRB 1*0802, and DRB1*1302. Peptides were considered to be cross-reactive DR supertype binders if they bound at an IC 50 value of 1000 nM or less to at least 5 of the 8 alleles tested.
  • HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations.
  • DR3 binding capacity is an important criterion in the selection of HTL epitopes.
  • DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles.
  • DR3 motifs For maximum efficiency in developing vaccine candidates it would be desirable for DR3 motifs to be clustered in proximity with DR supermotif regions.
  • peptides shown to be candidates may also be assayed for their DR3 binding capacity.
  • peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.
  • the 2 DR3 binding peptides were then tested for binding to the DR supertype alleles (Table XXXI). Both DR3 binding peptides bound DRB1*1302 with an IC 50 of 269 nM, but neither was a DR supertype cross-reactive binder. Conversely, the DR supertype cross-reactive binding peptides were also tested for DR3 binding capacity, with no measurable DR3 binding observed.
  • 3 DR supertype cross-reactive binding peptides were identified from the MAGE2/3 protein sequences.
  • the class II motif-bearing peptides may be analogued to improve affinity or cross-reactivity.
  • aspartic acid at position 4 of the 9- mer core sequence is an optimal residue for DR3 binding, and substitution for that residue may improve DR
  • This example determines immunogenic DR supermotif- and DR3 motif-bearmg epitopes among those identified using the methodology in Example 5 lmmunogenicity of HTL epitopes are evaluated m a manner analogous to the determination of lmmunogenicity of CTL epitopes by assessing the ability to stimulate HTL responses and/or by usmg appropnate transgenic mouse models lmmunogenicity is determmed by screenmg for I ) in vitro primary mduction usmg normal PBMC or 2 ) recall responses from cancer patient PBMCs
  • Example 7 Calculation of phenotypic frequencies of HLA-supertypes in various ethnic backgrounds to determine breadth of population coverage
  • This example illustrates the assessment of the breadth of population coverage of a vaccme composition comprised of multiple epitopes compnsmg multiple supermotifs and/or motifs
  • transgenic mouse model to be used for such an analysis depends upon the ep ⁇ tope(s) that is being evaluated.
  • transgenic mouse models including mice with human Al l, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e , transgenic mice for HLA-Al and A24) are bemg developed HLA-DR 1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes
  • This example illustrates the mduction of CTLs and HTLs m transgenic mice by use of a tumor associated antigen CTL/HTL peptide conjugate whereby the vaccme composition comprises peptides to be admmistered to a cancer patient
  • the peptide composition can comprise multiple CTL and/or HTL epitopes and further, can comprise epitopes selected from multiple- tumor associated antigens
  • the epitopes are identified using methodology as described m Examples 1-6
  • This analysis demonstrates the enhanced lmmunogenicity that can be achieved by inclusion of one or more HTL epitopes m a vaccme composition
  • Such a peptide composition can compnse an HTL epitope conjugated to a prefened CTL epitope containing, for example, at least one CTL epitope selected from Tables XXVII and XXIII-XXVI, or other analogs of that epitope
  • the HTL epitope is, for example, selected from Table
  • mice which are transgenic for the human HLA A2 1 allele and are useful for the assessment of the lmmunogenicity of HLA-A*0201 motif- or HLA- A2 supermotif-bearing epitopes, are pruned subcutaneously (base of the tail) with 0 1 ml of peptide conjugate formulated m salme, or DMSO/salme Seven days after priming, splenocytes obtamed from these animals are reshmulated with syngemc irradiated LPS-activated lymphoblasts coated with peptide
  • the target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA- A2 1/K b chimeric gene (e , Vitiello et al , J Exp Med 173 1007, 1991)
  • HLA- A2 1/K b chimeric gene e , Vitiello et al , J Exp Med 173 1007, 1991
  • spleen cells (30xlO ⁇ cells/flask) are co-cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (lOxlO 6 cells/flask) in 10 ml of culture med ⁇ um/T25 flask After six days, effector cells are harvested and assayed for cytotoxic activity
  • Target cells (1 0 to 1 5xl0 6 ) are incubated at 37°C in the presence of 200 ⁇ l of 5l Cr After 60 mmutes, cells are washed three tunes and resuspended m medium Peptide is added where req red at a concentration of 1 ⁇ g/ml
  • 10 4 51 Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 ⁇ l) in U-bottom 96- well plates
  • a 0 1 ml aliquot of supernatant is removed from each well and radioactivity is determmed in a Micromedic automatic gamma counter
  • % 5l Cr release data is expressed as lytic
  • the results are analyzed to assess the magnitude of the CTL responses of animals mjected with the immunogenic CTL/HTL conjugate vaccme preparation
  • the magnitude and frequency of response can also be compared to the CTL response achieved usmg the CTL epitopes by themselves Analyses similar to this may be performed to evaluate the lmmunogenicity of peptide conjugates contammg multiple CTL epitopes and/or multiple HTL epitopes.
  • the peptides m the composition can be m the form of a nucleic acid sequence, either single or one or more sequences (l e , minigene) that encodes ⁇ ept ⁇ de(s), or may be smgle and or polyepitopic peptides
  • Epitopes are selected which, upon administration, mimic immune responses that have been observed to be conelated with tumor clearance
  • a vaccine can mclude 3-4 epitopes that come from at least one TAA
  • Epitopes from one TAA can be used in combination with epitopes from one or more additional TAAs to produce a vaccme that targets tumors with varying expression patterns of frequently-expressed TAAs as described, e g , m Example 15
  • Epitopes are preferably selected that have a bmdmg affinity (IC50) of 500 nM or less, often 200 nM or less, for an HLA class I molecule, or for a class II molecule, 1000 nM or less Sufficient supermotif bearing peptides, or a sufficient anay of allele-specific motif bearing peptides, are selected to give broad population coverage. For example, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage. When selecting epitopes from cancer-related antigens it is often prefened to select analogs because the patient may have developed tolerance to the native epitope.
  • IC50 bmdmg affinity
  • junctional epitope is a potential HLA binding epitope, as predicted, e.g., by motif analysis. Junctional epitopes are generally to be avoided because the recipient may bind to an HLA molecule and generate an immune response to that epitope, which is not present in a native protein sequence.
  • CTL epitopes for inclusion in vaccine compositions are, for example, selected from those listed in Tables XXVII and XXIII-XXVI. Examples of HTL epitopes that can be included in vaccine compositions are provided in Table XXXI.
  • a vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response that results in tumor cell killing and reduction of tumor size or mass.
  • Minigene plasmids may, of course, contain various configurations of CTL and/or HTL epitopes or epitope analogs as described herein.
  • Expression plasmids have been constructed and evaluated as described, for example, in co-pending U.S.S.N. 09/311,784 filed 5/13/99.
  • a minigene expression plasmid may include multiple CTL and HTL peptide epitopes.
  • HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-Al and -A24 motif- bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes.
  • Prefened epitopes are identified, for example, in Tables XXIII-XXVII and XXXI.
  • HLA class I supermotif or motif-bearing peptide epitopes derived from multiple TAAs are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage.
  • HLA class II epitopes are selected from multiple tumor antigens to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct.
  • the selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
  • the mmigene DNA plasmid contams a consensus Kozak sequence and a consensus murme kappa Ig-hght chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herem
  • the sequence encodes an open readmg frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3 1 Myc-His vector
  • Overlappmg oligonucleotides for example eight oligonucleotides, averagmg approximately 70 nucleotides m length with 15 nucleotide overlaps, are synthesized and HPLC-pu ⁇ fied
  • the oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence,
  • the full-length dimer products are gel-purified, and two reactions contammg the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles Half of the two reactions are then mixed, and 5 cycles of annealing and extension earned out before flanking primers are added to amplify
  • Example 12 The plasmid construct and the degree to which it induces lmmunogenicity
  • Example 11 The degree to which the plasmid construct prepared using the methodology outlined m Example 11 is able to mduce lmmunogenicity is evaluated through in vivo injections mto mice and subsequent in vitro assessment of CTL and HTL activity, which are analysed usmg cytotoxicity and proliferation assays, respectively, as detailed e g , m U S S N 09/311 ,784 filed 5/ 13/99 and Alexander et al , Immunity 1 751- 761, 1994
  • plasmid constructs can be evaluated in vitro by testing for epitope presentation by APC following transduction or transfection of the APC with an epitope-expressmg nucleic acid construct
  • the assay determines the ability of the epitope to be presented by the APC m a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface Quantitation can be performed by directly measurmg the amount of peptide eluted from the APC (see, e g , Sijts et al , J Immunol 156 683-692, 1996, Demotz et al , Nature 342 682-684, 1989), or the number of peptide-HLA class I complexes can be estimated by measurmg the amount of lysis or lymphokme release mduced by mfected or transfected target cells, and then
  • HLA-Al 1/K b transgenic mice are immunized intramuscularly with 100 ⁇ g of naked cDNA.
  • a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a smgle polypeptide as they would be encoded by the mmigene.
  • Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded m the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a 5l Cr release assay
  • the results mdicate the magnitude of the CTL response directed agamst the A3 -restricted epitope, thus indicating the in vivo lmmunogenicity of the minigene vaccme and polyepitopic vaccme.
  • the mmigene elicits immune responses directed toward the HLA- A3 supermotif peptide epitopes as does the polyepitopic peptide vaccine
  • a similar analysis is also performed usmg other HLA-A2 and HLA-B7 transgenic mouse models to assess CTL mduction by HLA-A2 and HLA-B7 motif or supermotif epitopes.
  • I- A b restricted mice are immunized intramuscularly with 100 ⁇ g of plasmid DNA
  • a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant.
  • CD4+ T cells i e HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded m the minigene).
  • the HTL response is measured using a 3 H-thym ⁇ d ⁇ ne incorporation proliferation assay, (see, e g , Alexander et al Immunity 1:751-761, 1994). The results mdicate the magnitude of the HTL response, thus demonstrating the in vivo lmmunogenicity of the mmigene.
  • DNA mimgenes constructed as described Example 11, may also be evaluated as a vaccme m combmation with a boostmg agent usmg a prime boost protocol
  • the boostmg agent may consist of recombmant protem (e g , Barnett et al , Aids Res and Human Retroviruses 14, Supplement 3 S299-S309, 1998) or recombmant vaccmia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e g , Hanke et al , Vaccine 16:439-445, 1998; Sedegah et al , Proc Natl Acad Sci USA 95.7648-53, 1998, Hanke and McMichael, Immunol Letters 66 177-181, 1999, and Robmson et al , Nature Med 5:526-34, 1999)
  • the efficacy of the DNA mmigene may be evaluated m transgenic mice
  • A2.1/K b transgenic mice are immunized IM with 100 ⁇ g of the DNA mmigene encodmg the immunogenic peptides
  • the mice are boosted IP with 10 7 pfu/mouse of a recombinant vaccmia virus expressmg the same sequence encoded by the DNA minigene.
  • mice are immunized with 100 ⁇ g of DNA or recombinant vaccmia without the mmigene sequence, or with DNA encodmg the mmigene, but without the vaccmia boost
  • splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay
  • splenocytes are stimulated in vitro with the A2 -restricted peptide epitopes encoded m the mmigene and recombmant vaccmia, then assayed for peptide-specific activity in an IFN- ⁇ ELISA It is found that the mmigene utilized m a prune-boost mode elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone
  • Such an analysis is also performed usmg other HLA-Al 1 and HLA-B7 transgenic mouse models to assess CTL mduction
  • Vaccme compositions of the present invention are used to prevent cancer in persons who are at risk for developing a tumor
  • a polyepitopic peptide epitope composition (or a nucleic acid compnsmg the same) contammg multiple CTL and HTL epitopes such as those selected m Examples 9 and/or 10, which are also selected to target greater than 80% of the population, is admmistered to an individual at nsk for a cancer, e g , melanoma
  • the composition is provided as a single polypeptide that encompasses multiple epitopes
  • the vaccme is admmistered m an aqueous earner comprised of Freunds Incomplete Adjuvant
  • the dose of peptide for the initial immunization is from about 1 to about 50,000 ⁇ g, generally 100-5,000 ⁇ g, for a 70 kg patient
  • the initial admmistration of vaccme is followed by booster dosages at 4 weeks followed by evaluation of the magnitude
  • polyepitopic peptide composition can be admmistered as a nucleic acid m accordance with methodologies known m the art and disclosed herem
  • a native TAA polyprotein sequence is screened, preferably usmg computer algonthms defined for each class I and or class II supermotif or motif, to identify "relatively short” regions of the polyprotem that comprise multiple epitopes and is preferably less m length than an entire native antigen
  • This relatively short sequence that contains multiple distmct, even overlappmg, epitopes is selected and used to generate a mmigene construct
  • the construct is engineered to express the peptide, which conesponds to the native protein sequence
  • the "relatively short” peptide is generally less than 1 ,000, 500, or 250 ammo acids m length, often less than 100 amino acids m length, preferably less than 75 ammo acids m length, and more preferably less than 50 ammo acids in length
  • the protem sequence of the vaccme composition is selected because it has maximal number of epitopes contamed withm the sequence, i e , it has a high concentration of epitopes
  • the vaccme composition will preferably mclude, for example, three CTL epitopes and at least one HTL epitope from TAAs
  • This polyepitopic native sequence is admmistered either as a peptide or as a nucleic acid sequence which encodes the peptide Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes compnse substitutions that alter the cross-reactivity and/or bmding affimty properties of the polyepitopic peptide
  • the embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processmg will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-mducing vaccme compositions Additionally such an embodiment provides for the possibility of motif-bearmg epitopes for an HLA makeup that is presently unknown Furthermore, this embodiment (absent analogs) directs the immune response to multiple peptide sequences that are actually present in native TAAs thus avoidmg the need to evaluate any junctional epitopes Lastly, the embodiment provides an economy of scale when producmg nucleic acid vaccme compositions
  • the MAGE2/3 peptide epitopes of the present mvention are used m conjunction with peptide epitopes from other target tumor antigens to create a vaccine composition that is useful for the treatment of various types of tumors
  • a set of TAA epitopes can be selected that allows the targeting of most common epithelial tumors (see, e g , Kawashima et al , Hum Immunol 59 1-14, 1998)
  • Such a composition includes epitopes from CEA, HER-2/neu, and MAGE2/3, all of which are expressed to appreciable degrees (20-60%) m frequently found tumors such as lung, breast, and gastrointestinal tumors
  • the composition can be provided as a smgle polypeptide that incorporates the multiple epitopes from the various TAAs, or can be admmistered as a composition compnsmg one or more discrete epitopes Alternatively, the vaccme can be administered as a mmigene construct or as dendn
  • Targeting multiple tumor antigens is also important to provide coverage of a large fraction of tumors of any particular type
  • a single TAA is rarely expressed m the majority of tumors of a given type
  • approximately 50% of breast tumors express CEA, 20% express MAGE3, and 30% express HER-2/neu
  • the use of a smgle antigen for immunotherapy would offer only limited patient coverage
  • the combmation of the three TAAs would address approximately 70% of breast tumors
  • a vaccme composition compnsmg epitopes from multiple tumor antigens also reduces the potential for escape mutants due to loss of expression of an individual tumor antigen
  • Example 16 Use of peptides to evaluate an immune response
  • Peptides of the mvention may be used to analyze an immune response for the presence of specific CTL or HTL populations dnected to a TAA Such an analysis may be performed usmg multunenc complexes as described, e g , by Ogg et al, Science 279 2103-2106, 1998 and Greten et al , Proc Natl Acad Sci USA 95 7568-7573, 1998
  • peptides m accordance with the mvention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen
  • tetramers highly sensitive human leukocyte antigen tetramenc complexes
  • tetramers highly sensitive human leukocyte antigen tetramenc complexes
  • tetramers are used for a cross-sectional analysis of, for example, tumor-associated antigen HLA-A*0201-spec ⁇ fic CTL frequencies from HLA A*0201 -positive individuals at different stages of disease or following immunization usmg a TAA peptide contammg an A*0201 motif Tetramenc complexes are synthesized as described (Musey et al .
  • HLA heavy cham A*0201 m this example
  • ⁇ 2-m ⁇ croglobulm are synthesized by means of a prokaryotic expression system
  • the heavy cham is modified by deletion of the transmembrane-cytoso c tail and COOH-terminal addition of a sequence contammg a BirA enzymatic biohnylation site
  • the heavy cham, ⁇ 2-m ⁇ croglobuhn, and peptide are refolded by dilution
  • the 45-kD refolded product is isolated by fast protem liquid chromatography and then biotinylated by BirA m the presence of biotm (Sigma, St.
  • PBMCs For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes and resuspended m 50 ⁇ l of cold phosphate-buffered saline Tn-color analysis is performed with the tetramer-phycoeryth ⁇ n, along with ant ⁇ -CD8-T ⁇ color, and ant ⁇ -CD38
  • the PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples Controls for the tetramers mclude both A*0201 -negative individuals and A*0201 -positive unmfected donors.
  • the percentage of cells stained with the tetramer is then determmed by flow cytometry.
  • the results mdicate the number of cells m the PBMC sample that contam epitope-restncted CTLs, thereby readily indicating the extent of immune response to the TAA epitope, and thus the stage of tumor progression or exposure to a vaccme that elicits a protective or therapeutic response.
  • the peptide epitopes of the mvention are used as reagents to evaluate T cell responses, such as acute or recall responses, m patients. Such an analysis may be performed on patients who are m remission, have a tumor, or who have been vaccmated with a TAA vaccme
  • the class I restricted CTL response of persons who have been vaccinated may be analyzed
  • the vaccme may be any TAA vaccme PBMC are collected from vaccmated mdividuals and HLA typed Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples denved from mdividuals who bear that HLA type
  • PBMC from vaccinated mdividuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, MO), washed three tunes m HBSS (GIBCO Laboratories), resuspended in RPMI- 1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50 ⁇ g/ml), and Hepes (lOmM) contammg 10% heat- inactivated human AB serum (complete RPMI) and plated using microculture formats
  • a synthetic peptide compnsmg an epitope of the invention is added at 10 ⁇ g/ml to each well and HBV core 128-140 epitope is added at 1 ⁇ g/ml to each well as a source of T cell help durmg the first week of stimulation
  • a positive CTL response requues two or more of the eight replicate cultures to display greater than 10% specific 51 Cr release, based on comparison with unmfected control subjects as previously described (Rehermann, et al , Nature Med 2:1104,1108, 1996; Rehermann et al , J Clin Invest. 97 1655-1665, 1996, and Rehermann et al J Clin Invest 98 1432-1440, 1996).
  • Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibihty and Immunogenetics (ASHI, Boston, MA) or established from the pool of patients as described (Guilhot, et al J Virol 66 2670-2678, 1992)
  • Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell lme that are mcubated overnight with the synthetic peptide epitope of the mvention at 10 ⁇ M, and labeled with 100 ⁇ Ci of 51 Cr (Amersham Corp , Arlmgton Heights, IL) for 1 hour after which they are washed four tunes with HBSS
  • Cytolytic activity is determmed m a standard 4 hour, split- well 51 Cr release assay usmg U- bottomed 96 well plates contammg 3,000 targets/well Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50 1 on day 14
  • E/T effector/target
  • Percent cytotoxicity is determmed from the formula 100 x [(experimental release-spontaneous release)/max ⁇ mum release-spontaneous release)]
  • Maximum release is determined by lysis of targets by detergent (2% Triton X-100, Sigma Chemical Co , St Louis, MO) Spontaneous release is ⁇ 25% of maximum release for all experiments
  • the class II restricted HTL responses may also be analyzed
  • Purified PBMC are cultured m a 96- well flat bottom plate at a density of 1 5xl0 5 cells/well and are stimulated with 10 ⁇ g/ml synthetic peptide, whole antigen, or PHA Cells are routmely plated replicates of 4-6 wells for each condition After seven days of culture, the medium is removed and replaced with fresh medium contammg lOU/ml IL-2 Two days later, 1 ⁇ Ci 3 H-thym ⁇ d ⁇ ne is added to each well and mcubation is continued for an additional 18 hours Cellular DNA is then harvested on glass fiber mats and analyzed for 3 H-thym ⁇ d ⁇ ne incorporation
  • Antigen- specific T cell proliferation is calculated as the ratio of 3 H-thym ⁇ d ⁇ ne inco ⁇ oration m the presence of antigen divided by the 3 H-thym ⁇ d ⁇ ne inco ⁇ oration m the absence of antigen
  • a human clmical trial for an immunogenic composition comp ⁇ sing CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study Such a trial is designed, for example, as follows
  • a total of about 27 subjects are enrolled and divided into 3 groups Group I 3 subjects are mjected with placebo and 6 subjects are injected with 5 ⁇ g of peptide composition,
  • Group II 3 subjects are injected with placebo and 6 subjects are mjected with 50 ⁇ g peptide composition
  • Group III 3 subjects are mjected with placebo and 6 subjects are injected with 500 ⁇ g of peptide composition
  • the vaccme is found to be both safe and efficacious
  • Evaluation of vaccme compositions are performed to validate the efficacy of the CTL-HTL peptide compositions m cancer patients
  • the mam objectives of the trials are to determine an effective dose and regimen for mducmg CTLs m cancer patients, to establish the safety of mducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clmical picture of cancer patients, as manifested by a reduction in tumor cell numbers
  • Such a study is designed, for example, as follows
  • the studies are performed m multiple centers
  • the trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a smgle dose followed six weeks later by a single booster shot of the same dose
  • the dosages are 50, 500 and 5,000 micrograms per injection
  • Drug-associated adverse effects (severity and reversibility) are recorded
  • the first group is mjected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively.
  • the patients within each group range m age from 21-65, mclude both males and females (unless the tumor is sex-specific, e g , breast or prostate cancer), and represent diverse ethnic backgrounds
  • a prune boost protocol similar m its underlying principle to that used to evaluate the efficacy of a DNA vaccme m transgenic mice, which was descnbed m Example 12, may also be used for the admmistration of the vaccme to humans
  • Such a vaccme regimen may mclude an initial administration of, for example, naked DNA followed by a boost using recombinant virus encodmg the vaccme, or recombmant protein/polypeptide or a peptide mixture admmistered m an adjuvant
  • the initial immunization may be performed usmg an expression vector, such as that constructed in Example 11, m the form of naked nucleic acid admmistered IM (or SC or ID) m the amounts of 0 5-5 mg at multiple sites
  • the nucleic acid (0 1 to 1000 ⁇ g) can also be admmistered using a gene gun Following an mcubation penod of 3-4 weeks, a booster dose is then admmistered
  • the booster can be recombinant fowlpox virus admmistered at a dose of 5-10 7 to 5xl0 9 pfu
  • An alternative recombinant virus such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protem or a mixture of the peptides can be admmistered
  • patient blood samples will be obtamed before immunization as
  • Vaccmes compnsmg peptide epitopes of the mvention may be admmistered usmg antigen- presentmg cells (APCs), or "professional" APCs such as dendntic cells (DC)
  • APCs antigen- presentmg cells
  • DC dendntic cells
  • the peptide- pulsed DC are admmistered to a patient to stimulate a CTL response in vivo
  • dendntic cells are isolated, expanded, and pulsed with a vaccme compnsmg peptide CTL and HTL epitopes of the invention
  • the dendntic cells are infused back mto the patient to elicit CTL and HTL responses in vivo
  • the mduced CTL and HTL then destroy (CTL) or facilitate destruction (HTL) of the specific target tumor cells that bear the proteins from which the epitopes m the vaccme are derived
  • CTL destroy
  • HTL facilitate destruction
  • DC therefrom from the patient's blood
  • a pharmaceutical to facilitate harvestmg of DC can be used, such as ProgenipoietinTM (Monsanto, St Louis, MO) or GM-CSF/IL-4 After pulsmg the DC with peptides and prior to reinfusion mto patients, the DC are washed to remove unbound peptides
  • the number of dendntic cells remfused mto the patient can vary (see, e g , Nature Med 4 328, 1998, Nature Med 2 52, 1996 and Prostate 32 272, 1997) Although 2-50 x 10 6 dendntic cells per patient are typically admmistered, larger number of dendntic cells, such as 10 7 or 10 8 can also be provided Such cell populations typically contam between 50-90% dendntic cells
  • peptide-loaded PBMC are injected mto patients without purification of the DC
  • PBMC contammg DC generated after treatment with an agent such as ProgempoietinTM are mjected mto patients without purification of the DC
  • the total number of PBMC that are admmistered often ranges from 10 8 to 10 10
  • the cell doses mjected mto patients is based on the percentage of DC m the blood of each patient, as determmed, for example, by lmmunofluorescence analysis with specific anti-DC antibodies
  • ProgenipoietinTM mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 10 6 DC
  • the patient will be injected with a total of 2 5 x 10 8 peptide-loaded PBMC
  • the percent DC mobilized by an agent such as ProgenipoietinTM is typically estimated to be between 2-10%, but can vary as appreciated
  • ex vivo CTL or HTL responses to a particular tumor-associated antigen can be mduced by mcubatmg m tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presentmg cells (APC), such as dendntic cells, and the appropriate immunogenic peptides After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded mto effector cells, the cells are infused back mto the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i e , tumor cells Example 22.
  • CTL destroy
  • HTL facilitate destruction
  • Another way of identifying motif-bearing peptides is to elute them from cells bearing defined MHC molecules.
  • EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can then be infected with a pathogenic organism or transfected with nucleic acids that express the tumor antigen of interest. Thereafter, peptides produced by endogenous antigen processing of peptides produced consequent to infection (or as a result of transfection) will bind to HLA molecules within the cell and be transported and displayed on the cell surface.
  • the peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al, J. Immunol. 152:3913, 1994). Because, as disclosed herein, the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides conelated with the particular HLA molecule expressed on the cell. Alternatively, cell lines that do not express any endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele.
  • These cells may then be used as described, i.e., they may be infected with a pathogenic organism or transfected with nucleic acid encoding an antigen of interest to isolate peptides conesponding to the pathogen or antigen of interest that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that conespond to binding to the single HLA allele that is expressed in the cell.
  • 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.
  • Secondary anchor specificities are designated for each position independently.
  • Table IV HLA Class I Standard Peptide Binding Affinity.
  • 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*l 102, A*2603, A*3302, A*3303, A*3401,
  • Verified alleles include alleles whose specificity has been determined by pool sequencing analysis, peptide binding assays, or by analysis m 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.
  • VKVLHHTLKIGGEPH MAGE2.284

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Abstract

L'invention utilise nos connaissances des mécanismes par lesquels l'antigène est reconnu par les lymphocytes T pour identifier et préparer des épitopes MAGE2/3, et pour développer des vaccins à base d'épitopes, ciblés sur des tumeurs porteuses de MAGE2/3. Plus spécialement, l'invention concerne des compositions pharmaceutiques et leurs procédés d'utilisation dans la prévention et le traitement du cancer.
PCT/US2000/033545 1999-12-10 2000-12-11 Declenchement de reponses immunitaires cellulaires a mage2/3 au moyen de compositions de peptides et d'acides nucleiques WO2001042267A1 (fr)

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AU20850/01A AU2085001A (en) 1999-12-10 2000-12-11 Inducing cellular immune responses to mage2/3 using peptide and nucleic acid compositions
US10/149,135 US20040053822A1 (en) 2000-12-11 2000-12-11 Inducing cellular immune responses to mage2/3 using peptide and nucleic acid compositions
CA002393339A CA2393339A1 (fr) 1999-12-10 2000-12-11 Declenchement de reponses immunitaires cellulaires a mage2/3 au moyen de compositions de peptides et d'acides nucleiques
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US8822182B2 (en) 2001-05-18 2014-09-02 Mayo Foundation For Medical Education And Research Chimeric antigen-specific T cell-activating polypeptides
FR2837837A1 (fr) * 2002-03-28 2003-10-03 Roussy Inst Gustave Epitopes peptidiques communs a des antigenes d'une meme famille multigenique
WO2003083124A2 (fr) * 2002-03-28 2003-10-09 Institut Gustave Roussy Epitopes peptidiques communs a des antigenes d'une meme famille multigenique
WO2003083124A3 (fr) * 2002-03-28 2004-04-01 Roussy Inst Gustave Epitopes peptidiques communs a des antigenes d'une meme famille multigenique
US7419669B2 (en) 2002-03-28 2008-09-02 Institut Gustave Roussy Peptide epitopes common to antigens of the same multigene family
US7488793B2 (en) * 2003-09-22 2009-02-10 Ludwig Institute For Cancer Research Isolated peptide which binds to HLA-Cw*07 and uses thereof
US8263560B2 (en) 2005-04-01 2012-09-11 University Of Maryland Baltimore HPV 16 peptide vaccine for head and neck cancer
WO2010086294A2 (fr) 2009-01-28 2010-08-05 Epimmune Inc. Polypeptides de liaison de pan-dr et leurs utilisations
CN102459322A (zh) * 2009-06-09 2012-05-16 瓦克松生物技术公司 用于免疫治疗的hla-b*0702共有表位的鉴定、优化与应用
WO2010143010A1 (fr) * 2009-06-09 2010-12-16 Vaxon Biotech Identification, optimisation et utilisation d'épitopes hla-b*0702 partagés pour l'immunothérapie
CN102459322B (zh) * 2009-06-09 2015-11-25 瓦克松生物技术公司 用于免疫治疗的hla-b*0702共有表位的鉴定、优化与应用
US10024868B2 (en) 2009-06-09 2018-07-17 Vaxon Biotech Identification, optimization and use of shared HLA-B*0702 epitopes for immunotherapy
US10934338B2 (en) 2015-03-27 2021-03-02 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11407810B2 (en) 2015-03-27 2022-08-09 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11434273B2 (en) 2015-03-27 2022-09-06 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11873329B2 (en) 2015-03-27 2024-01-16 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors
US11897934B2 (en) 2015-03-27 2024-02-13 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against various tumors

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JP2003517310A (ja) 2003-05-27
CA2393339A1 (fr) 2001-06-14
EP1235841A1 (fr) 2002-09-04
EP1235841A4 (fr) 2006-04-12
AU2085001A (en) 2001-06-18

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