EP1235841A1

EP1235841A1 - Inducing cellular immune responses to mage2/3 using peptide and nucleic acid compositions

Info

Publication number: EP1235841A1
Application number: EP00984183A
Authority: EP
Inventors: John Fikes; Alessandro Sette; John Sidney; Scott Southwood; Robert Chesnut; Esteban Celis; Elissa Keogh
Original assignee: Epimmune Inc
Current assignee: Epimmune Inc
Priority date: 1999-12-10
Filing date: 2000-12-11
Publication date: 2002-09-04
Also published as: EP1235841A4; CA2393339A1; AU2085001A; JP2003517310A; WO2001042267A1

Abstract

The invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to identify and prepare MAGE2/3 epitopes, and to develop epitope-based vaccines directed towards MAGE2/3-bearing tumors. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of cancer.

Description

INDUCING CELLULAR IMMUNE RESPONSES TO MAGE2/3 USING PEPTIDE AND NUCLEIC

ACID COMPOSITIONS

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

Tumor-specific helper T lymphocytes (HTLs) are also known to be important for maintaining effective antitumor immunity Their role in antitumor immunity has been demonstrated 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 , IFNγ and TNF- α)

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 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 , Liver 19 110-114, 1999), renal (Yamanaka et al , Human Pathol 24 1127-1134, 1998), colorectal (Mori et al , Ann Surg 224 183-188, 1996), and esophageal (Quilhen et al , Anticancer Res 17 387-391, 1997) carcmomas as well as tumors of the head and neck (Lett et al , Acta Otolaryngol 116 633-639, 1996), ovaries (Gillespie et al , Br J Cancer 78 816-821, 1998, Yamada et al , Int J Cancer 64 388-393, 1995), bladder, and osteosarcoma (Sudo et al , J Orthop Res 15 128-132, 1997) Thus, MAGE2/3 are important targets for cancer lmmunotherapy

The information provided m this section is intended to disclose the presently understood state of the art as of the filing date of the present application Information is included m this section which was generated subsequent to the priority date of this application Accordingly, mformation in this section is not intended, m any way, to delmeate the priority date for the mvention

II. SUMMARY OF THE INVENTION

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

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 For example, 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

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) Thus, patient-by-patient variability m the immune response to a particular pathogen may be alleviated by inclusion of epitopes from multiple antigens from the pathogen m a vaccme composition

Furthermore, 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 For example, 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

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

Furthermore, as described herein m greater detail, 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

In a preferred embodiment, 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-beaπng 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 IC₅₀ (or a K_D value) of 500 nM or less for HLA class I molecules or an IC₅₀ of 1000 nM or less for HLA class II molecules, are further evaluated for their ability to induce a CTL or HTL response Immunogenic peptide epitopes are selected for inclusion 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 compπsmg 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 tetrameπc 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 As will be apparent from the discussion below, other methods and embodiments are also contemplated Further, novel synthetic peptides produced by any of the methods described herem are also part of the mvention HI. BRIEF DESCRIPTION OF THE FIGURES not applicable

IV. DETAILED DESCRIPTION OF THE INVENTION 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. MAGE 1, MAGE 2, MAGE 3, MAGE-11, MAGE-A10, BAGE, GAGE, RAGE, MAGE-C1, LAGE-1, CAG-3, DAM, MUCl, MUC2, MUC18, NY-ESO-1, MUM-1, CDK4, BRCA2, NY-LU-1, NY-LU-7, NY-LU-12, CASP8, RAS, KIAA-2-5, SCCs, p53, p73, CEA, Her 2/neu, Melan-A, gplOO, tyrosinase, TRP2, gp75/TRPl, kallikrein, PSM, PAP, PSA, PT1-1, B-catenm, PRAME, Telomerase, FAK, cychn DI protein, NOEY2, EGF-R, SART-1, CAPB, HPVE7, pl5, Folate receptor CDC27, PAGE-1, and PAGE-4.

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.

IV.A. Definitions

The mvention can be better understood with reference to the following definitions, which are listed alphabetically:

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

With regard to a particular ammo acid sequence, 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 In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule Throughout this disclosure epitope and peptide are often used 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 acιd(s) are withm the bounds of the mvention In certain embodiments, there is a limitation on the length of a peptide of the mvention which is not otherwise a construct as defined herem An embodiment that is length-limited occurs when the protein/peptide compπsmg an epitope of the invention compnses a region (l e , a contiguous senes of ammo acids) havmg 100% identity with a native sequence In order to avoid a recited definition of epitope from readmg, e g , on whole natural molecules, the length of any region that has 100% identity with a native peptide sequence is limited Thus, for a peptide compπsmg an epitope of the invention and a region with 100% identity with a native peptide sequence (and which is not otherwise a construct), the region with 100% identity to a native sequence generally has a length of less than or equal to 600 ammo acids, often less than or equal to 500 ammo acids, often less than or equal to 400 amino acids, often less than or equal to 250 ammo acids, often less than or equal to 100 ammo acids, often less than or equal to 85 amino acids, often less than or equal to 75 amino acids, often less than or equal to 65 ammo acids, and often less than or equal to 50 ammo acids In certain embodiments, an "epitope" of the mvention which is not a construct is comprised by a peptide havmg a region with less than 51 ammo acids that has 100% identity to a native peptide sequence, in any mcrement of (50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) down to 5 ammo acids

Certain peptide or protem sequences longer than 600 ammo acids are withm the scope of the invention Such longer sequences are withm the scope of the mvention 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 For any peptide that has five contiguous residues or less that correspond to a native sequence, there is no limitation on the maximal length of that peptide m order to fall withm the scope of the mvention It is presently preferred that a CTL epitope of the invention be less than 600 residues long in any increment down to eight amino acid residues "Human Leukocyte Antigen" or "HLA" is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e g , Stites, et al , IMMUNOLOGY, 8™ ED , Lange Publishing, Los Altos, CA, 1994)

An "HLA supertype or family", as used herein, 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 The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-hke molecules (where xx denotes a particular HLA type), are synonyms

Throughout this disclosure, results are expressed m terms of "IC₅₀'s " IC₅₀ 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 IC₅₀ values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e g , HLA preparation, etc ) For example, excessive concentrations of HLA molecules will increase the apparent measured IC₅₀ of a given ligand

Alternatively, bindmg is expressed relative to a reference peptide Although as a particular assay becomes more, or less, sensitive, the ICso's of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change For example, in an assay run under conditions such that the IC₅₀ of the reference peptide increases 10-fold, the IC₅₀ values of the test peptides will also shift approximately 10-fold Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative bmder is generally based on its IC₅₀, relative to the IC₅₀ 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 , Khilko et al , J Biol Chem 268 15425, 1993), high flux soluble phase assays (Hammer et al , J Exp Med 180 2353, 1994), and measurement of class I MHC stabilization or assembly (e , Ljunggren et al , Nature 346 476, 1990, Schumacher et al , Cell 62 563, 1990, Townsend et al , Cell 62 285, 1990, Parker et al , J Immunol 149 1896, 1992)

As used herem, "high affinity" with respect to HLA class I molecules is defined as bmding with an IC₅₀, or K_D value, of 50 nM or less, "intermediate affinity" is bmdmg with an IC₅₀ 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 IC₅₀ or K_D value of 100 nM or less, "intermediate affinity" is bmding with an IC₅₀ or K_D value of between about 100 and about 1000 nM

The terms "identical" or percent "identity," in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of 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

An "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 Thus, 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 deπved

The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany the material as it is found m its native state Thus, isolated peptides m accordance with the mvention preferably do not contain materials normally associated with the peptides m then in situ environment

"Link" or "join" 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

"Major Histocompatibihty Complex" or "MHC" 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 For a detailed description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^RD ED , Raven Press, New York, 1993 The term "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" Such 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

The term "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 α-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 In one embodiment, for example, the pnmary anchor residues are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9-resιdue peptide epitope m accordance with the mvention The primary anchor positions for each motif and supermotif are set forth in Table 1 For example, 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 affinity of a peptide compπsmg a particular motif or supermotif

"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

The term "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 For example, 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 compπsmg 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 Preferably, 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 As used herem, a "vaccme" is a composition that contams one or more peptides of the mvention There are numerous embodiments of 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, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the mvention The peptides or polypeptides can optionally be modified, such as by hpidation, addition of targetmg or other sequences HLA class I-bmdmg peptides of the invention can be admixed with, or linked to, HLA class II-bιndιng peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes Vaccmes can also comprise peptide-pulsed antigen presentmg cells, e , dendritic cells

The nomenclature used to describe peptide compounds follows the conventional practice wherem the ammo group is presented to the left (the N-terminus) and the carboxyl group to the right (the C- termmus) of each amino acid residue When 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 In the formulae representmg selected specific embodiments of the present mvention, the ammo- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified In the amino acid structure formulae, each residue is generally represented by standard three letter or 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 havmg D-forms is represented by a lower case smgle letter or a lower case three letter symbol Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G The ammo acid sequences of peptides set forth herem are generally designated using the standard smgle letter symbol (A, Alanme, C, Cysteme, D, Aspartic Acid, E, Glutamic Acid, F, Phenylalanine, G, Glycine, H, Histidme, I, Isoleucine, K, Lysine, L, Leucine, M, Methionme, N, Asparagine, P, Prohne, Q, Glutamine, R, Argmme, S, Serine, T, Threonine, V, Valine, W, Tryptophan, and Y, Tyrosme ) In addition to these symbols, "B"ιn the single letter abbreviations used herem designates α-ammo butyric acid

IV.B. Stimulation of CTL and HTL responses

The mechanism by which T cells recognize antigens has been delmeated during the past ten years Based on our understandmg of the immune system we have developed efficacious peptide epitope vaccine compositions that can induce a therapeutic or prophylactic immune response to a TAA m a broad population For an understandmg of the value and efficacy of the claimed compositions, a brief review of immunology-related technology is provided The review is mtended to disclose the presently understood state of the art as of the filing date of the present application Information is mcluded m this section which was generated subsequent to the priority date of this application Accordmgly, mformation m this section is not mtended, m any way, to delmeate the priority date for the mvention A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA- restncted T cells (Buus, S. et al , Cell 47- 1071, 1986; Babbitt, B. P. et al , Nature 317:359, 1985;

Townsend, A. and Bodmer, H , Annu Rev Immunol. 7:601, 1989; Germain, R. N., Annu Rev Immunol

11.403, 1993) Through the study of smgle ammo acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific bindmg to HLA antigen molecules have been identified and are described herein and are set forth in

Tables I, II, and III (see also, e g , Southwood, et al , J Immunol 160 3363, 1998, Rammensee, et al ,

Immunogenetics 41: 178, 1995; Rammensee et al , SYFPEITHI, access via web at . http.//134 2 96.221/scπpts.hlaserver dll/home.htm; Sette, A and Sidney, J. Curr Opin Immunol 10 478, 1998, Engelhard, V. H., Curr Opin Immunol. 6:13, 1994, Sette, A. and Grey, H M , Curr Opin Immunol 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr Biol 6 52, 1994; Ruppert et al , Cell 74:929-937, 1993, Kondo et al , J Immunol 155 4307-4312, 1995; Sidney et al , J Immunol 157-3480-3490, 1996, Sidney et al , Human Immunol 45.79-93, 1996; Sette, A. and Sidney, J Immunogenetics 1999 Nov, 50(3-4) 201-12, Review) Furthermore, x-ray crystallographic analysis of HLA-peptide complexes has revealed pockets withm the peptide bindmg cleft of HLA molecules which accommodate, m an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA bindmg capacity of the peptides m which they are present. (See, e g , Madden, D.R. Annu Rev Immunol. 13:587, 1995, Smith, et al , Immunity 4:203, 1996; Fremont et al , Immunity 8-305, 1998; Stern et al , Structure 2:245, 1994; Jones, E Y. Curr Opin Immunol 9:75, 1997, Brown, J H. et al , Nature 364:33, 1993, Guo, H. C. et al , Proc Natl Acad Sci USA 90.8053, 1993; Guo, H. C et al , Nature 360-364, 1992, Silver, M. L. et al , Nature 360 367, 1992, Matsumura, M et al , Science 257.927, 1992; Madden et al , Cell 70 1035, 1992; Fremont, D H et al , Science 257:919, 1992; Saper, M. A. , Bjorkman, P J. and Wiley, D C, J Mol Biol. 219.277, 1991 ) Accordingly, the definition of 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.

The present mventors have found that the correlation of bmding affinity with lmmunogenicity, which is disclosed herem, is an important factor to be considered when evaluating candidate peptides Thus, by a combmation of motif searches and HLA-peptide bmdmg assays, candidates for epitope-based vaccines have been identified After determining their bmding affinity, additional confirmatory work can be performed to select, amongst these vaccme candidates, epitopes with preferred charactenstics m terms of population coverage, antigenicity, and lmmunogenicity.

Various strategies can be utilized to evaluate lmmunogenicity, mcludmg

1) Evaluation of pnmary T cell cultures from normal individuals (see, e g , Wentworth, P. A. et al , Mol Immunol 32:603, 1995; Cells, E. et al , Proc Natl Acad Sci USA 91:2105, 1994; Tsai, V. et al., J

Immunol 158- 1796, 1997, Kawashima, I. et al , Human Immunol 59: 1, 1998); This procedure mvolves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide m the presence of antigen presentmg cells in vitro over a penod of several weeks T cells specific for the peptide become activated durmg this time and are detected usmg, e g , a ^^Cr-release assay mvolvmg peptide sensitized target cells. 2) Immunization of HLA transgenic mice (see, e g , Wentworth, P A et al , J Immunol 26 97, 1996, Wentworth, P A et al , Int Immunol 8 651, 1996, Alexander, J et al , J Immunol 159 4753, 1997), In this method, peptides m mcomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week Peptide-specific T cells are detected usmg, e g , a

^Cr-release assay mvolvmg peptide sensitized target cells and target cells expressmg endogenously generated antigen

3) Demonstration of recall T cell responses from patients who have been effectively vaccmated or who have a tumor, (see, e g , Rehermann, B et al , J Exp Med 181 1047, 1995, Doolan, D L et al , Immunity 7 97, 1997, Bertoni, R et al , J Clin Invest 100 503, 1997, Threlkeld, S C et al, J Immunol 159 1648, 1997, Diepolder, H M et al , J Virol 71 6011, 1997, Tsang et al , J Natl Cancer lnst 87 982- 990, 1995, Disis et al , J Immunol 156 3151-3158, 1996) In applying this strategy, recall responses are detected by culturing PBL from patients with cancer who have generated an immune response "naturally", or from patients who were vaccmated with tumor antigen vaccines PBL from subjects are cultured in vitro for 1-2 weeks m the presence of test peptide plus antigen presentmg cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells At the end of the culture period, T cell activity is detected using assays for T cell activity including -^Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release

The following describes the peptide epitopes and corresponding nucleic acids of the mvention

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

As mdicated herem, the large degree of HLA polymorphism is an important factor to be taken mto account with the epitope-based approach to vaccme development To address this factor, 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 IC₅₀ 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 IC₅₀ or bmdmg affinity value for class II HLA molecules of 1000 nM or better, (i e , the value is < 1,000 nM) For example, 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 In preferred embodiments, peptides that exhibit cross-reactive bmdmg are then used m cellular screenmg analyses or vaccmes As disclosed herem, higher HLA bmdmg affinity is correlated with greater lmmunogenicity

Greater 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 For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response Moreover, higher 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 Thus, in preferred embodiments of the mvention, high or intermediate affinity bindmg epitopes are particularly useful

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

An affinity threshold associated with lmmunogenicity m the context of HLA class II DR molecules has also been delmeated (see, e g , Southwood et al J Immunology 160 3363-3373,1998, and co-pendmg U S S N 09/009,953 filed 1/21/98) In order to define a biologically significant threshold of DR bmdmg affinity, a database of the bmdmg affinities of 32 DR-restπcted epitopes for their restricting element (t e , the HLA molecule that bmds the motif) was compiled In approximately half of the cases ( 15 of 32 epitopes), DR restriction was associated with high bmdmg affinities, i e binding affinity values of 100 nM or less In the other half of the cases ( 16 of 32), DR restriction was associated with intermediate affinity (binding affinity values m the 100-1000 nM range) In only one of 32 cases was DR restnction associated with an IC₅₀ of 1000 nM or greater Thus, 1000 nM can be defined as an affinity threshold associated with lmmunogenicity m the context of DR molecules

In the case of tumor-associated antigens, many CTL peptide epitopes that have been shown to induce CTL that lyse peptide-pulsed target cells and tumor cell targets endogenously expressing the epitope exhibit bmdmg affinity or IC₅₀ values of 200 nM or less In a study that evaluated the association of bmdmg affimty and lmmunogenicity of such TAA epitopes, 100% (10/10) of the high bmders, 1 e , peptide epitopes bmding at an affinity of 50 nM or less, were immunogenic and 80% (8/10) of them elicited CTLs that specifically recognized tumor cells In the 51 to 200 nM range, very similar figures were obtamed CTL mductions positive for peptide and tumor cells were noted for 86% (6/7) and 71% (5/7) of the peptides, respectively In the 201-500 nM range, most peptides (4/5 wildtype) were positive for mduction of CTL recognizing wildtype peptide, but tumor recognition was not detected

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

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

Such peptide epitopes are identified m the Tables described below

Peptides of the present mvention also comprise epitopes that bind to MHC class II DR molecules. A greater degree of heterogeneity m both size and binding frame position of the motif, relative to the N and C termini of the peptide, exists for class II peptide ligands. This 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. Crystallographic analysis of HLA class II DRB*0101-peptιde complexes showed that the major energy of binding is contributed by peptide residues complexed with complementary pockets on the DRB*0101 molecules An important anchor residue engages the deepest hydrophobic pocket (see, e g , Madden, D R Ann Rev Immunol 13-587, 1995) and is referred to as position 1 (PI). 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

In the past few years evidence has accumulated to demonstrate that a large fraction of HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlappmg peptide bmdmg repertoires, and consensus structures of the mam peptide bmdmg pockets Thus, 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 Tables include a binding affinity ratio listing for some of the peptide epitopes. The ratio may be converted to IC₅₀ by using the following formula: IC₅₀ of the standard peptide/ratio = IC₅₀ of the test peptide (i.e., the peptide epitope). The IC₅₀ values of standard peptides used to determine binding affinities for Class I peptides are shown in Table IV. The IC₅₀ values of standard peptides used to determine binding 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.

To obtain the peptide epitope sequences listed in each of Tables VII-XX, 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.

In the Tables, 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. For example, 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.

MAGE2 Amino Acid Sequence 1 MPLEQRSQHC KPEEGLEARG EALGLVGAQA PATEEQQTAS SSSTLVEVTL GEVPAADSPS 60

PPHSPQGASS FSTTINYTLW RQSDEGSSNQ EEEGPRMFPD LESEFQAAIS RKMVE VHFL 120

LLKYRAREPV TKAE LESVL RNCQDFFPVI FSKASEY QL VFGIEWEW PISHLYI VT 180

CLG SYDGL GDNQVMPKTG IIVLAIIA IEGDCAPEEK IWEELSM EV FEGREDSVFA 240

HPRKLLMQDL VQENYLEYRQ VPGSDPACYE FL GPRALIE TSYVKVLHHT LKIGGEPHIS 300 YPP HERA R EGEE 314

MAGE3 Amino Acid Sequence

1 MPLEQRSQHC KPEEGLEARG EALGLVGAQA PATEEQEAAS SSSTLVEVTL GEVPAAESPD 60

PPQSPQGASS LPTTMNYPL SQSYEDSSNQ EEEGPSTFPD LESEFQAALS RKVAELVHFL 120 LLKYRAREPV TKAEMLGSW GNWQYFFPVI FSKASSSLQL VFGIELMEVD PIGHLYIFAT 180

CLGLSYDGLL GDNQIMPKAG LLIIVLAIIA REGDCAPEEK IWEELSVLEV FEGREDSILG 240

DPKKLLTQHF VQENYLEYRQ VPGSDPACYE FL GPRALVE TSYVKVLHHM VKISGGPHIS 300 YPPLHEWVLR EGEE 314 HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:

The pnmary anchor residues of the 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 In some cases, peptide epitopes may be listed in both a motif and a supermotif Table The relationship of a particular motif and respective supermotif is indicated m the description of the individual motifs

IV.D.1. HLA-Al supermotif

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 (i e , the HLA-Al supertype) is comprised of at least A*0101, A*2601, A*2602, A*2501, and A*3201 (^ee, e g , DιBπno, 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 individual HLA proteins 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 Al supermotif are set forth in Tables VII(A) and VII(B), respectively

IV.D.2. HLA-A2 supermotif

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

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,

A*0209, A*0214, A*6802, and A*6901 Other allele-specific HLA molecules predicted to be members of the A2 superfamily are shown m Table VI As explamed in detail below, bmdmg to each of the individual allele-specific HLA molecules can be modulated by substitutions at the primary anchor and/or secondary anchor positions, preferably choosmg respective residues specified for the supermotif Representative MAGE2 and MAGE3 peptide epitopes that compnse the A2 supermotif are set forth m Tables VIII(A) and VIII(B), respectively The motifs compnsmg the pnmary 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 mvention claimed herein

IV.D.3. HLA-A3 supermotif

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 choosmg respective residues specified for the supermotif

Representative MAGE2 and MAGE3 peptide epitopes that comprise the A3 supermotif are set forth m Tables IX(A) and IX(B), respectively

IV.D.4. 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 choosmg respective residues specified for the supermotif Representative MAGE2 and MAGE3 peptide epitopes that compnse the A24 supermotif are set forth m Tables X(A) and X(B), respectively

IV.D.5. HLA-B7 supermotif

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 (i e , the HLA-B7 supertype) 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, eg , Sidney, et al , J Immunol 154 247, 1995, Barber, et al , Curr Biol 5 179, 1995, Hill, et al , Nature 360 434, 1992, Rammensee, et al, Immunogenetics 41 178, 1995 for reviews of relevant data) Other allele-specific HLA molecules predicted to be members of the B7 supertype are shown m Table VI As explamed m detail below, peptide bmding to each of the individual allele-specific HLA protems can be modulated by substitutions at the primary and/or secondary anchor positions of the peptide, preferably choosmg respective residues specified for the supermotif

Representative MAGE2 and MAGE3 peptide epitopes that comprise the B7 supermotif are set forth in Tables XI(A) and XI(B), respectively

IV.D.6. HLA-B27 supermotif

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,

Immunogenetics 1999 Nov, 50(3-4) 201-12, Review) Exemplary members of the corresponding family of HLA molecules that bmd to the B27 supermotif (i e , the B27 supertype) include at least B*1401, B*1402, B* 1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801, B*3901, B*3902, and B*7301 Other allele-specific HLA molecules predicted to be members of the B27 supertype are shown in Table VI Peptide 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

IV.D.7. HLA-B44 supermotif

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

IV.D.8. HLA-B58 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, preferably choosmg respective residues specified for the supermotif Representative MAGE2 and MAGE3 peptide epitopes that comprise the B58 supermotif are set forth m Tables XIII(A) and XIII(B), respectively

IV.D.9. 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) Exemplary members of the corresponding family of HLA molecules that bmd to the B62 supermotif (; e , the B62 supertype) mclude at least B*1501, B*1502, B* 1513, and B5201 Other allele-specific HLA molecules predicted to be members of the B62 supertype are shown in Table VI Peptide bindmg to each of the allele- specific HLA molecules can be modulated by substitutions at primary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif

Representative MAGE2 and MAGE3 peptide epitopes that comprise the B62 supermotif are set forth m Tables XIV(A) and XIV(B), respectively

IV.D.10. HLA-Al motif

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 , DiBπno 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 pnmary and or secondary anchor positions, preferably choosmg respective residues specified for the motif

Representative 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-beaπng peptide epitopes listed in Table VII, as these residues are a subset of the A 1 supermotif prunary anchors

IV.D.11. HLA-A*0201 motif

An 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-resιdue 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 the epitope (see, e g , Kast et al , J Immunol 152 3904-3912, 1994) Thus, the HLA-A*0201 motif comprises peptide ligands with L, I, V, M, A, T, or Q as primary anchor residues at position 2 and L, I, V, M, A, or T as a primary anchor residue at the C-terminal position of the epitope. The preferred and tolerated residues that characterize the primary anchor positions of the HLA-A*0201 motif are identical to the residues describing the A2 supermotif. (For reviews of relevant data, see, e.g., del Guercio et al, J. Immunol. 154:685-693, 1995; Ruppert et al, Cell 74:929-937, 1993; Sidney et al, Immunol. Today 17:261- 266, 1996; Sette and Sidney, Curr. Opin. in Immunol. 10:478-482, 1998). Secondary anchor residues that characterize the A*0201 motif have additionally been defined (see, e.g., Ruppert et al, Cell 74:929-937, 1993). These are shown in Table II. Peptide binding to HLA-A*0201 molecules can be modulated by substitutions at primary and or secondary anchor positions, preferably choosing respective residues specified for the motif.

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.

IV.D.12. HLA-A3 motif

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.

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.

IV.D.13. HLA-Al 1 motif

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),

MAGE2 and MAGE3, respectively; 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

Representative 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-supermotιf-beanng peptide epitopes, as the primary anchor residues charactenzmg the A24 allele- specific motif comprise a subset of the A24 supermotif primary anchor residues

Motifs Indicative of Class II HTL Inducing Peptide Epitopes

The prunary and secondary anchor residues of the HLA class II peptide epitope supermotifs and motifs delmeated below are summarized m Table III

IV.D.1S. HLA DR-1-4-7 supermotif

Motifs have also been identified for peptides that bmd to three common HLA class II allele- specific HLA molecules HLA DRB1*0401, DRB1*0101, and DRB1*0701 (see, e g , the review by Southwood et al J Immunology 160 3363-3373,1998) Collectively, the common residues from these motifs delmeate the HLA DR-1-4-7 supermotif Peptides that bind to these DR molecules carry a supermotif characterized by a large aromatic or hydrophobic residue (Y, F, W, L, I, V, or M) as a primary anchor residue m position 1, and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as a prunary anchor residue m position 6 of a 9-mer core region Allele-specific secondary effects and secondary anchors for each of these HLA types have also been identified (Southwood et al , supra) These are set forth m Table III Peptide bindmg to HLA- DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by substitutions at pnmary and/or secondary anchor positions, preferably choosing respective residues specified for the supermotif

Potential epitope 9-mer core regions compnsmg the DR-1-4-7 supermotif, wherem position 1 of the supermotif is at position 1 of the nme-residue core, are set forth in Table XIX Respective exemplary peptide epitopes of 15 ammo acid residues m length, each of which comprise a c nme residue core, are also shown m the Table, along with cross-reactive bmdmg data for the exemplary 15-resιdue supermotif-beaπng peptides

IV.D.16. HLA DR3 motifs

Two alternative motifs (t e , submotifs) characterize peptide epitopes that bmd to HLA-DR3 molecules (see, e g , Geluk et al , J Immunol 152 5742, 1994) In the first motif (submotif DR3a) a large, hydrophobic residue (L, I, V, M, F, or Y) is present in anchor position 1 of a 9-mer core, and D is present as an anchor at position 4, towards the carboxyl termmus of the epitope As m other class II motifs, core position 1 may or may not occupy the peptide N-terminal position The alternative DR3 submotif provides for lack of the large, hydrophobic residue at anchor position 1, and/or lack of the negatively charged or amide- like anchor residue at position 4, by the presence of a positive charge at position 6 towards the carboxyl termmus of the epitope Thus, for the alternative allele-specific DR3 motif (submotif DR3b) L, I, V, M, F, Y, A, or Y is present at anchor position 1 , D, N, Q, E, S, or T is present at anchor position 4, and K, R, or H is present at anchor position 6 Peptide bindmg to HLA-DR3 can be modulated by substitutions at pnmary and or secondary anchor positions, preferably choosmg respective residues specified for the motif

Potential 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

Potential peptide epitope 9-mer core regions compnsmg the DR3b submotif and respective exemplary 15-mer peptides compnsmg the DR3 submotif-b epitope are set forth m Table XXb Bmdmg data of exemplary DR3 submotif b-bearmg peptides is also shown

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

IV.E. Enhancing Population Coverage of the Vaccine

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

(Table XXIa) and the combmed population coverage achieved by the A2-, A3-, and B7-supertypes (Table XXIb) The A2-, A3-, and B7 supertypes are each present on the average of over 40% in each of these five major ethnic groups Coverage m excess of 80% is achieved with a combination of these supermotifs These results suggest that effective and non-ethnically biased population coverage is achieved upon use of a limited number of cross-reactive peptides Although the population coverage reached with these three mam peptide specificities is high, coverage can be expanded to reach 95% population coverage and above, and more easily achieve truly multispecific responses upon use of additional supermotif or allele-specific motif bearmg peptides

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 By mcludmg epitopes from the six most frequent supertypes, an average population coverage of 99% is obtamed for five major ethnic groups

IV.F. Immune Response-Stimulating Peptide Analogs

In general, 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,

Bennink, et al , J Exp Med 168 19351939, 1988, Rawle, et al , J Immunol 146 3977-3984, 1991) It has been recognized that lmmunodommance (Benacerraf, et al , Science 175 273-279, 1972) could be explained by either the ability of a given epitope to selectively bmd a particular HLA protem (determinant selection theory) (Vitiello, et al , J Immunol 131 1635, 1983), Rosenthal, et al , Nature 267 156-158, 1977), or to be selectively recognized by the existmg TCR (T cell receptor) specificities (repertoire theory) (Klein, J , IMMUNOLOGY, THE SCIENCE OF SELF/NONSELF DISCRIMINATION, John Wiley & Sons, New York, pp 270- 310, 1982) It has been demonstrated that additional factors, mostly linked to processing events, can also play a key role in dictating, beyond strict lmmunogenicity, which of the many potential determinants will be presented as lmmunodominant (Sercarz, et al , Annu Rev Immunol 11 729-766, 1993)

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

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

Although 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

In brief, 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 Generally, analogs are made for peptides that already bear a motif or supermotif Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II bmdmg peptides are shown in Tables II and III, respectively

For a number of the motifs or supermotifs m accordance with the mvention, 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 For example, m the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of peptides used m the analysis, the incidence of cross-reactivity increased from 22% to 37% (see, e g , Sidney, J et al , Hu Immunol 45 79, 1996) Thus, one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small "neutral" residue such as Ala (that may not influence T cell recognition of the peptide) An enhanced likelihood of cross-reactivity is expected if, together with elimmation of detrimental residues withm a peptide, "preferred" residues associated with high affinity bmdmg to an allele-specific HLA molecule or to multiple HLA molecules withm a superfamily are inserted

To ensure that an 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 For example, a cysteme can be substituted out m favor of α-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 bπdges and sufficiently alter the peptide structurally so as to reduce bmdmg capacity. Substituting α-amino butync acid for cysteme not only alleviates this problem, but actually improves bmdmg and crossbmdmg capability m certain 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) Representative analog peptides are set forth in Tables XXII-XXVII The Table mdicates the length and sequence of the analog peptide as well as the motif or supermotif, if appropriate. The "source" column mdicates the residues substituted at the mdicated position numbers for the respective analog

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

In order to identify supermotif- or motif-bearing epitopes in a target antigen, a native protem sequence, e , a tumor-associated antigen, or sequences from an infectious organism, or a donor tissue for transplantation, is screened using a means for computmg, such as an intellectual calculation or a computer, to determine the presence of a supermotif or motif withm the sequence 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. For example, the target TAA molecules mclude, without limitation, CEA, MAGE, p53 and HER2/neu

It is important that the selection critena utilized for prediction of peptide bmdmg are as accurate as possible, to correlate most efficiently with actual bmdmg. Prediction of peptides that rJmd, for example, to HLA-A*0201, on the basis of the presence of the appropriate primary anchors, is positive at about a 30% rate (see, e g , Ruppert, J et al Cell 74-929, 1993) However, by extensively analyzing peptide-HLA bmdmg data disclosed herem, data in related patent applications, and data m the art, the present inventors have developed a number of allele-specific polynomial algonthms that dramatically mcrease the predictive value over identification on the basis of the presence of primary anchor residues alone These algorithms take mto account not only the presence or absence of pnmary anchors, but also consider the positive or deleterious presence of secondary anchor residues (to account for the impact of different ammo acids at different positions) The algorithms are essentially based on the premise that the overall affimty (or ΔG) of peptide-HLA mteractions can be approximated as a lmear polynomial function of the type ΔG = ai, x a₂, x a₃, x a„ where a , is a coefficient that represents the effect of the presence of a given ammo acid (/) at a given position (i) along the sequence of a peptide of n ammo acids. An important assumption of this method is that the effects at each position are essentially mdependent of each other This assumption is justified by studies that demonstrated that peptides are bound to HLA molecules and recognized by T cells m essentially an extended conformation Derivation of specific algorithm coefficients has been described, for example, m Gulukota, K et al , J Mol Biol 267 1258, 1997

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

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

In utilizing computer screening to identify peptide epitopes, a protein sequence or translated sequence may be analyzed usmg software developed to search for motifs, for example the

"FrNDPATTERNS' program (Devereux, et al Nucl Acids Res 12 387-395, 1984) or MotifSearch 1 4 software program (D Brown, San Diego, CA) to identify potential peptide sequences contammg appropriate HLA bmdmg motifs The identified peptides can be scored usmg customized polynomial algorithms to predict then capacity to bmd specific HLA class I or class II alleles As appreciated by one of ordinary skill in the art, a large array of computer programming software and hardware options are available in the relevant art which can be employed to implement the motifs of the mvention order to evaluate (e g , without limitation, to identify epitopes, identify epitope concentration per peptide length, or to generate analogs) known or unknown peptide sequences

In accordance with the procedures described above, MAGE2/3 peptide epitopes and analogs thereof that are able to bmd HLA supertype groups or allele-specific HLA molecules have been identified (Tables VII-XX, Table XXII-XXXI)

IV.H. Preparation of Peptide Epitopes

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

When possible, it may be desirable to optimize 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 Preferably, 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

In alternative embodiments, epitopes of the mvention can be linked as a polyepitopic peptide, or as a mimgene that encodes a polyepitopic peptide

In another embodiment, it is preferred to identify native peptide regions that contam a high concentration of class I and or class II epitopes Such a sequence is generally selected on the basis that it contains the greatest number of epitopes per ammo acid length It is to be appreciated that 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 For the preferred relatively short size, 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

Alternatively, 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 These procedures are generally known m the art, as described generally m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989) Thus, 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 phosphotπester method of Matteucci, et al , J Am Chem Soc 103 3185 (1981) Peptide analogs can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native peptide sequence, exemplary nucleic acid substitutions are those that encode an ammo acid defined by the motifs/supermotifs herein The codmg sequence can then be provided with appropnate linkers and hgated mto expression vectors commonly available m the art, and the vectors used to transform suitable hosts to produce the desired fusion protem A number of such vectors and suitable host systems are now available For expression of the fusion protems, the codmg sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host For example, promoter sequences compatible with bacterial hosts are provided m plasmids containing convenient restriction sites for insertion of the desired codmg sequence The resulting expression vectors are transformed into suitable bacterial hosts Of course, yeast, msect or mammalian cell hosts may also be used, employmg suitable vectors and control sequences

IV.I. Assays to Detect T-Cell Responses

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 Alternatively, cells expressing empty class I molecules (i e lackmg peptide therem) may be evaluated for peptide bmdmg by immunofluorescent staining and flow microfluoπmetry Other assays that may be used to evaluate peptide bmdmg mclude peptide-dependent class I assembly assays and/or the inhibition of CTL recognition by peptide competition Those peptides that bmd to the class I molecule, typically with an affinity of 500 nM or less, are further evaluated for their ability to serve as targets for CTLs derived from mfected or immunized individuals, as well as for their capacity to mduce primary in vitro or in vivo CTL responses that can give rise to CTL populations capable of reactmg with selected target cells associated with a disease

Analogous assays are used for evaluation of HLA class II bmdmg peptides HLA class II motif- bearmg peptides that are shown to bind, typically at an affinity of 1000 nM or less, are further evaluated for the ability to stimulate HTL responses

Conventional assays utilized to detect T cell responses include proliferation assays, lymphokme secretion assays, direct cytotoxicity assays, and limiting dilution assays For example, 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 Alternatively, 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 (PBMCs) 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 mcubated with the responder cell population under optimized culture conditions Positive CTL activation can be determined by assaymg the culture for the presence of CTLs that kill radio-labeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed forms of the antigen from which the peptide sequence was derived

Additionally, a method has been devised which allows direct quantification of antigen-specific T cells by staining with Fluorescein-labelled HLA tetrameπc complexes (Altman, J D et al , Proc Natl Acad Sci USA 90 10330, 1993, Altman, J O et al , Science 274 94, 1996) Other relatively recent technical developments mclude stammg for mtracellular lymphokines, and interferon-γ release assays or ELISPOT assays Tetramer staming, mtracellular lymphokme stainmg and ELISPOT assays all appear to be at least 10-fold more sensitive than more conventional assays (Lalvani, A et al , J Exp Med 186 859, 1997, Dunbar, P R et al , Curr Biol 8 413, 1998, Murali-Kπshna, K et al , Immunity 8 177, 1998)

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)

Alternatively, immunization of HLA transgenic mice can be used to determine lmmunogenicity of peptide epitopes Several 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 Similarly, HTL responses may be analyzed usmg such assays as T cell proliferation or secretion of lymphokmes

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

In one embodiment of the mvention, 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 epιtope(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 For example, 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-tetrameπc complex is used to directly visualize antigen- specific CTLs (see, e g , Ogg et al , Science 279 2103-2106, 1998, and Altman et al , Science 174 94-96, 1996) and determine the frequency of the antigen-specific CTL population m a sample of peripheral blood mononuclear cells A tetramer reagent usmg a peptide of the mvention may be generated as follows A peptide that bmds to an HLA molecule is refolded in the presence of the conesponding HLA heavy cham and β₂-mιcroglobulιn to generate a tπmolecular complex The complex is biotmylated at the carboxyl terminal end of the heavy cham at a site that was previously engmeered mto the protein Tetramer formation is then mduced by the addition of streptavidin By means of fluorescently labeled streptavidin, the tetramer can be used to stain antigen-specific cells The cells may then be identified, for example, by flow cytometry Such an analysis may be used for diagnostic or prognostic purposes

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) For example, 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/Λrø oσr, 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 Such antibodies mclude those that recognize a peptide m the context of an HLA molecule, i e , antibodies that bmd to a peptide-MHC complex

IV.K. Vaccine Compositions

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 Once appropriately immunogenic epitopes have been defined, they can be sorted and delivered by various means, herem referred to as "vaccine" compositions Such 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 , Eldπdge, 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) (see, e g , Takahashi et al , Nature 344 873-875, 1990, Hu et al , Clin Exp Immunol 113 235-243, 1998), multiple antigen peptide systems (MAPs) (see e g , Tarn, J P , Proc Natl Acad Sci USA 85 5409-5413, 1988, Tam, J P , J Immunol Methods 196 17-32, 1996), peptides formulated as multivalent peptides, peptides for use m ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M E et al , In Concepts in vaccine development, Kaufmann, S H E , ed , p 379, 1996, Chakrabarti, S et al , Nature 320 535, 1986, Hu, S L et al , Nature 320 537, 1986, Kieny, M -P et al , AIDS Bio/Technology 4 790, 1986, Top, F H et al , J Infect Dis 124 148, 1971, Chanda, P K et al , Virology 175 535, 1990), particles of viral or synthetic ongm (e g , Kofler, N et al , J Immunol Methods 192 25, 1996, Eldndge, J H et al , Sem Hematol 30 16, 1993, Falo, L D , Jr et al , Nature Med 7 649, 1995), adjuvants (Wanen, H S , Vogel, F R , and Chedid, L A Annu Rev Immunol 4 369, 1986, Gupta, R K et al , Vaccine 11 293, 1993), liposomes (Reddy, R et al , J Immunol 148 1585, 1992, Rock, K L , Immunol Today 17 131, 1996), or, naked or particle absorbed cDNA (Ulmer, J B et al , Science 259 1745, 1993, Robinson, H L , Hunt, L A , and Webster, R G ,

Vaccine 11 957, 1993, Shiver, J W et al , In Concepts in vaccine development, Kaufmann, S H E , ed , p 423, 1996, Cease, K B , and Berzofsky, J A , Annu Rev Immunol 12 923, 1994 and Eldndge, J H et al , Sem Hematol 30 16, 1993) 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,

5,804,566, 5,739,118, 5,736,524, 5,679,647, WO 98/04720, and m more detail below Examples of 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)

For therapeutic or prophylactic immunization purposes, the peptides of the mvention can also be expressed by viral or bacterial vectors Examples of expression vectors mclude attenuated viral hosts, such as vaccmia or fowlpox As an example of this approach, vaccmia virus is used as a vector to express nucleotide sequences that encode the peptides of the mvention 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) A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e g adeno and adeno- associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled m the art from the description herem

Furthermore, vaccmes in accordance with the invention encompass compositions of one or more of the claimed peptιde(s) The peptιde(s) can be mdividually linked to its own carrier, alternatively, the peptιde(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 Additionally, as disclosed herem, CTL responses can be primed by conjugatmg peptides of the invention to lipids, such as tnpalmitoyl-S-glycerylcystemlyseryl- serme (P₃CSS) Upon immunization with a peptide composition m accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccme by producmg large amounts of CTLs and/or HTLs specific for the desired antigen Consequently, the host becomes at least partially immune to later mfection, or at least partially resistant to developmg an ongoing chronic mfection, or derives at least some therapeutic benefit when the antigen was tumor-associated In some embodiments, it may be desirable to combme the class I peptide components with components that mduce or facilitate neutralizing antibody and or helper T cell responses to the target antigen of mterest A prefened embodiment of such a composition comprises class I and class II epitopes m accordance with the mvention An alternative embodiment of such a composition comprises a class I and/or class II epitope m accordance with the invention, along with a cross-bmdmg HLA class II epitope such as PADRE™ (Epimmune, San Diego, CA) molecule (described, for example, m U S Patent Number

5,736,142)

A vaccme of the mvention can also mclude antigen-presentmg cells (APC), such as dendritic cells

(DC), as a vehicle to present peptides of the invention Vaccme compositions can be created in vitro, following dendntic cell mobilization and harvestmg, whereby loadmg of dendritic cells occurs in vitro For example, 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 may also be used as antigen presenting 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

Preferably, the following pnnciples are utilized when selecting an aπay 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

1 ) 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

2 ) Epitopes are selected that have the requisite bmding affinity established to be correlated with lmmunogenicity for HLA Class I an IC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less

3 ) Sufficient supermotif beaπng-peptides, or a sufficient anay of allele-specific motif- beaπng 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 ) When selectmg epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope When selecting epitopes for infectious disease-related antigens it is preferable to select either native or analoged epitopes

5 ) Of particular relevance 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 When providmg nested epitopes, a general objective is to provide the greatest number of epitopes per sequence Thus, 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 When 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

6 ) If a polyepitopic protem is created, or when creating a πunigene, 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 However, with an artificial polyepitopic peptide, 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 "dommant epitope " A dommant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed

IV.K.1. Minigene Vaccines

A number of different approaches are available which allow simultaneous delivery of multiple epitopes 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

U S S N 09/311,784, Ishio a et al , J Immunol 162 3915-3925, 1999, An, L and Whitton. J L , J Virol

71 2292, 1997, Thomson, S A et al , J Immunol 157 822, 1996, Whitton, J L et al , J Virol 67 348,

1993, Hanke, R et al , Vaccine 16 426, 1998 For example, a multi-epitope DNA plasmid encodmg supermotif- and/or motif-bearmg MAGE2/3 epitopes derived from multiple regions of the MAGE2/3 proteins, the PADRE™ 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 For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, 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 These epitope-encodmg DNA sequences may be directly adjomed, so that when translated, a continuous polypeptide sequence is created To optimize expression and/or lmmunogenicity, additional elements can be incorporated mto the minigene design Examples of ammo acid sequences that can be reverse translated and included m the minigene sequence mclude HLA class I epitopes, HLA class II epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal In addition, HLA presentation of CTL and HTL epitopes may be unproved by mcludmg synthetic (e g poly-alanine) or naturally-occumng flanking sequences adjacent to the CTL or HTL epitopes, these larger peptides compnsmg the epιtope(s) are within the scope of the mvention

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 ampicilhn or kanamycin resistance) Numerous promoters can be used for this purpose, e , the human cytomegalovirus (hCMV) promoter See, e , U S Patent Nos 5,580,859 and 5,589,466 for other suitable promoter sequences

Additional vector modifications may be desired to optimize minigene expression and lmmunogenicity In some cases, 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 The inclusion of mRNA stabilization sequences and sequences for replication m mammalian cells may also be considered for mcreasmg minigene expression

Once an expression vector is selected, 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

In addition, unmunostimulatory sequences (ISSs or CpGs) appear to play a role m the lmmunogenicity of DNA vaccmes These sequences may be mcluded m the vector, outside the minigene codmg sequence, if desired to enhance lmmunogenicity

In some embodiments, 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 Examples of 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 , PADRE™, 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 improving HTL induction In contrast to HTL or CTL induction, specifically decreasmg the immune response by co-expression of lmmunosuppressive molecules (e g TGF- β) may be beneficial in certam diseases

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-Fogeπte, BioTechmques 6(7) 682 (1988), U S Pat No 5,279,833, WO 91/06309, and Feigner, et al , Proc Nat'l Acad Sci USA 84 7413 (1987) In addition, peptides and compounds referred to collectively as protective, interactive, non- condensing compounds (PINC) could also be complexed to punfied plasmid DNA to influence vanables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes For example, the plasmid DNA is introduced into a mammalian cell 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) These cells are then chromιum-51 (⁵¹Cr) labeled and used as target cells for epitope- specific CTL lmes, cyto lysis, detected by ^5lCr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes Expression of HTL epitopes may be evaluated m an analogous manner usmg assays to assess HTL activity

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

Alternatively, the 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 In a further alternative embodiment, 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

IV.K.2. Combinations of CTL Peptides with Helper Peptides

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 For mstance, 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 The use of 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 Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or ammo acid numerics, which are substantially uncharged under physiological conditions The spacers are typically selected from, e , Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar ammo acids It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the ammo or carboxy termmus of the CTL peptide The ammo termmus of either the immunogenic peptide or the T helper peptide may be acylated In certam embodiments, the T helper peptide is one that is recognized by T helper cells present in the majority of the population This can be accomplished by selectmg amino acid sequences that bmd to many, most, or all of the HLA class II molecules These are known as "loosely HLA-restncted" or "promiscuous" T helper sequences Examples of peptides that are promiscuous mclude sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE), Plasmodium falcψarum circumsporozoite (CS) protem at positions 378-398 (DIEKKIAKMEKASSVFNWNS), and Streptococcus 18kD protem at positions 116 (GAVDSILGGVATYGAA) Other examples include peptides bearmg a DR 1-4-7 supermotif, or either of the DR3 motifs

Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, m a loosely HLA-restncted fashion, usmg ammo acid sequences not found m nature (see, e , PCT publication WO 95/07707) These synthetic compounds called Pan-DR-bmding epitopes (e g , PADRE™, Epimmune, Inc , San Diego, CA) are designed to most prefeπably bmd most HLA-DR (human HLA class II) molecules For mstance, a pan-DR-bmdmg epitope peptide having the formula aKXVAAWTLKAAa, where "X" is either cyclohexylalamne, phenylalanine, or tyrosine, and "a" is either D-alanme or L-alamne, has been found to bmd to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type An alternative of a pan-DR bmdmg epitope comprises all "L" natural ammo acids and can be provided m the form of nucleic acids that encode the epitope

HTL peptide epitopes can also be modified to alter their biological properties For example, 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 For example, a T helper peptide can be conjugated to one or more palmitic acid chams at either the ammo or carboxyl termini

IV.K.3. Combinations of CTL Peptides with T Cell Priming Agents In some embodiments it may be desirable to include m the pharmaceutical 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 For example, palmitic acid residues can be attached to the ε-and α- 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 ε- and α- ammo groups of Lys, which is attached via linkage, e g , Ser-Ser, to the ammo terminus of the immunogenic peptide

As another example of hpid priming of CTL responses, E coli hpoproteins, such as tπpalmitoyl-S- glycerylcysteinlyseryl- serine (P₃CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e g , Deres, et al , Nature 342 561, 1989) Peptides of the mvention can be coupled to P₃CSS, for example, and the hpopeptide admmistered to an individual to specifically prime a

CTL response to the target antigen Moreover, because the mduction of neutralizing antibodies can also be primed with P₃CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses

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 However, it is to be noted that modification at the carboxyl termmus of a CTL epitope may, m some cases, alter bmdmg characteristics of the peptide In addition, the peptide or ohgopeptide sequences can differ from the natural sequence by being modified by termιnal-NH₂ acylation, e , by alkanoyl (Cι-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

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

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 Progempoietin™ (Monsanto, St Louis, MO) or GM-CSF/IL-4 After pulsmg the DC with peptides and pπor to reinfusion mto patients, the DC are washed to remove unbound peptides In this embodiment, 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 antigen and the like Optionally, a helper T cell peptide such as a PADRE™ family molecule, can be mcluded to facilitate the CTL response

IV.L. Administration of Vaccines for Therapeutic or Prophylactic Purposes

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

In therapeutic applications, 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

Generally 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 μg and the higher value is about 10,000, 20,000, 30,000, or

50,000 μg Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient This is followed by boosting dosages of between about 1 0 μg to about 50,000 μg of peptide 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

As noted above, 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 peptιde(s), liposomes and the like, can be used, as described herem

When the peptide is contacted in vitro, 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

For pharmaceutical compositions, 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 For therapeutic use, 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 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

Where susceptible individuals, e g , individuals who may be diagnosed as being genetically pre- disposed to developmg a particular type of tumor, are identified pnor to diagnosis of cancer, the 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 μg and the higher value is about 10,000, 20,000, 30,000, or 50,000 μg Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient Boostmg dosages of between about 1 0 μg to about 50,000 μg 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 treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts

Thus, for treatment of cancer, a representative dose is m the range disclosed above, namely where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000, 20,000, 30,000, or 50,000 μg, preferably from about 500 μg to about 50,000 μg 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

The pharmaceutical compositions for therapeutic treatment are mtended for parenteral, topical, oral, lntrathecal, or local admmistration Preferably, the pharmaceutical compositions are administered parentally, e g , intravenously, subcutaneously, intradermally, or intramuscularly Thus, 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 A variety of 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 pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjustmg and buffering agents, tonicity adjustmg agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chlonde, sorbitan monolaurate, tπethanolamine oleate, etc

The 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 In these preparations, 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 Thus, 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 Liposomes for use in accordance with the mvention are formed from standard vesicle-forming lipids, which generally mclude neutral and negatively charged phosphohpids and a sterol, such as cholesterol The selection of lipids is generally guided by consideration of, e g , hposome size, acid lability and stability of the liposomes m the blood stream A variety of methods are available for preparmg liposomes, as described in, e g , Szoka, et al , Ann Rev Bwphys Bioeng 9 467 (1980), and U S Patent Nos 4,235,871, 4,501,728, 4,837,028, and 5,019,369

For targetmg cells of the immune system, 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

For solid compositions, conventional 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 For oral admmistration, 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%

For aerosol admmistration, 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, lauπc, 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, as desired, as with, e , lecithin for intranasal delivery

IV.M. HLA EXPRESSION: IMPLICATIONS FOR T CELL-BASED IMMUNOTHERAPY

Disease proeression in cancer and mfectious disease It is well recognized that a dynamic interaction between exists between host and disease, both m the cancer and infectious disease settings In the mfectious disease setting, it is well established that pathogens evolve durmg disease The strains that predominate early in HIV infection are different from the ones that are associated with AIDS and later disease stages (NS versus S strains) It has long been hypothesized that pathogen forms that are effective m establishing mfection may differ from the ones most effective in terms of replication and chromcity Similarly, it is widely recognized that the pathological process by which an individual succumbs to a neoplastic disease is complex Durmg the course of disease, many changes occur m cancer cells The tumor accumulates alterations which are m part related to dysfunctional regulation of growth and differentiation, but also related to maximizing its growth potential, escape from drug treatment and/or the body's lmmunQsurveillance Neoplastic disease results m the accumulation of several different biochemical alterations of cancer cells, as a function of disease progression It also results in significant levels of uitra- and mter- cancer heterogeneity, particularly m the late, metastatic stage

Familiar examples of cellular alterations affecting treatment outcomes mclude the outgrowth of radiation or chemotherapy resistant tumors durmg the course of therapy These examples parallel the emergence of drug resistant viral strains as a result of aggressive chemotherapy, e g , of chronic HB V and HIV mfection, and the cunent resurgence of drug resistant organisms that cause Tuberculosis and Malana It appears that significant heterogeneity of responses is also associated with other approaches to cancer therapy, mcludmg anti-angiogenesis drugs, passive antibody immunotherapy, and active T cell-based lmmunotherapy Thus, m view of such phenomena, epitopes from multiple disease-related antigens can be used in vaccmes and therapeutics thereby counteracting the ability of diseased cells to mutate and escape treatment

The interplay between disease and the immune system

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

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

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

Second, encouragmg results have come from immunotherapy trials

Third, observations made m the course of natural disease conelated the type and composition of T cell infiltrate within tumors with positive clmical outcomes (Couhe PG, et al Antitumor immunity at work m a melanoma patient In Advances in Cancer Research. 213-242, 1999) Finally, tumors commonly have the ability to mutate, thereby changing their immunological recognition For example, the presence of monospecific CTL was also correlated with control of tumor growth, until antigen loss emerged (Riker A, et al , Immune selection after antigen-specific immunotherapy of melanoma Surgery, Aug 126(2) 112-20, 1999, Marchand M, et al , Tumor regressions observed m patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-Al Int J Cancer 80(2) 219-30, Jan 18, 1 99) Similarly, loss of beta 2 microglobulm was detected m 5/13 lmes established from melanoma patients after receivmg immunotherapy at the NCI

(Restifo NP, et al , Loss of functional Beta2 - microglobulm in metastatic melanomas from five patients receiving immunotherapy Journal of the National Cancer Institute, Vol 88 (2), 100-108, Jan 1996) It has long been recognized that HLA class I is frequently altered m various tumor types This has led to a hypothesis that this phenomenon might reflect immune pressure exerted on the tumor by means of class I restricted CTL The extent and degree of alteration in HLA class I expression appears to be reflective of past immune pressures, and may also have prognostic value (van Duinen SG, et al , Level of HLA antigens m locoregional metastases and clmical course of the disease m patients with melanoma Cancer Research

48, 1019-1025, Feb 1988, Moller P, et al , Influence of major histocompatibihty complex class I and II antigens on survival m colorectal carcmoma Cancer Research 51, 729-736, Jan 1991) Taken together, these observations provide a rationale for immunotherapy of cancer and mfectious disease, and suggest that effective strategies need to account for the complex series of pathological changes associated with disease

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 β2-mιcroglobuhn and specific HLA heavy chams, alterations m the regulatory elements controlling over class I expression and loss of entøe chromosome sections There are several reviews on this topic, see, 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 β2-ππcroglobulιn m colorectal cancer Int J Cancer, 51(3) 379-85, May 28,1992 There are three mam types of HLA Class I alteration (complete loss, allele- specific loss and decreased expression) The functional significance of each alteration is discussed separately

Complete loss of HLA expression

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

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, Schπer PI, et al , Relationship between myc oncogene activation and MHC class I expression Adv Cancer Res , 60 181-246, 1993)

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

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

It should also be noted that in the case of heterogeneous HLA expression, both normal HLA- expressmg as well as defective cells are predicted to be susceptible to immune destruction based on "bystander effects " Such effects were demonstrated, e g , in the studies of Rosendahl and colleagues that investigated m vivo mechanisms of action of antibody targeted superantigens (Rosendahl A, et al , Perform and IFN-gamma are involved m the antitumor effects of antibody-targeted superantigens J Immunol

160(11) 5309-13, June 1, 1998) 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 Thus, a practical solution to overcome the potential hurdle of allele-specific loss relies on the induction of multispecific responses Just as the inclusion of multiple disease-associated antigens 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

Decrease in expression (allele-specific or not)

The sensitivity of effector CTL has long been demonstrated (Brower, RC, et al , Minimal requirements for peptide mediated activation of CD8+ CTL Mol Immunol , 31, 1285-93, 1994, Chπustnick, ET, et al Low numbers of MHC class I-peptide complexes required to trigger a T cell response Nature

352 67-70, 1991, Sykulev, Y, et al , Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response Immunity, 4(6) 565-71, June 1996) Even a single peptide/MHC complex can result m tumor cells lysis and release of anti-tumor lymphokines The biological significance of decreased HLA expression and possible tumor escape from immune recognition is not fully known Nevertheless, it has been demonstrated that CTL recognition of as few as one MHC/peptide complex is sufficient to lead to tumor cell lysis

Further, it is commonly observed that expression of HLA can be upregulated by gamma IFN, commonly secreted by effector CTL Additionally, HLA class I expression can be 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

With regard to gamma IFN, 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 47 364-71, 1996) Kaklamakis, et al also suggested that adjuvant immunotherapy with IFN- gamma may be beneficial m the case of HLA class I negative tumors (Kaklamams L, Loss of transporter in antigen processmg 1 transport protem and major histocompatibihty complex class I molecules m metastatic versus prunary breast cancer Cancer Research 55 5191-94, November 1995) It is important to underline that IFN-gamma production is mduced and self-amplified by local mflammation/unmumzation (Halloran, et al. Local T cell responses induce widespread MHC expression J. Immunol 148:3837, 1992), resulting in large increases in MHC expressions even in sites distant from the inflammatory site.

Finally, studies have demonstrated that decreased HLA expression can render tumor cells more susceptible to NK lysis (Ohnmacht, GA, et al, Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma J Cellular Phys 182:332-38, 2000; Liunggren

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 β2m 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). If decreases in HLA expression benefit a tumor because it facilitates CTL escape, but render the tumor susceptible to NK lysis, then a minimal level of HLA expression that allows for resistance to NK activity would be selected for (Garrido F, et al, Implications for immunosurveillance of altered HLA class I phenotypes in human tumours Immunol Today 18(2):89-96, February 1997). Therefore, a therapeutic compositions or methods in accordance with the invention together with a treatment to upregulate HLA expression and/or treatment with high affinity T-cells renders the tumor sensitive to CTL destruction.

Frequency of alterations in HLA expression

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

Immunotherapy in the context of HLA loss A majority of the tumors express HLA class I, with a general tendency for the more severe alterations to be found m later stage and less differentiated tumors This pattern is encouragmg m the context of immunotherapy, especially considermg that 1) the relatively low sensitivity of lmmunohistochemical techniques might underestimate HLA expression m tumors, 2) class I expression can be mduced in tumor cells as a result of local inflammation and lymphokme release, and, 3) class I negative cells are sensitive to lysis by NK cells

Accordingly, 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 For example, 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 concuπently with NK cell- inducing compositions, or these compositions can be admmistered sequentially

In the context of allele-specific HLA loss, 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 Thus, one can simultaneously target multiple HLA specificities and epitopes from multiple disease-related antigens to prevent tumor escape by allele-specific loss as well as disease- related antigen loss In addition, embodiments of the present 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

Moreover, it has been observed that expression of HLA can be upregulated by gamma IFN, which is commonly secreted by effector CTL, and that HLA class I expression can be mduced m vivo by both alpha and beta IFN Thus, embodiments of the mvention can also comprise alpha, beta and/or gamma IFN to facilitate upregualtion of HLA

IV.N. REPRIEVE PERIODS FROM THERAPIES THAT INDUCE SIDE EFFECTS: "Scheduled Treatment Interruptions or Drug Holidays"

Recent evidence has shown that certam patients mfected with a pathogen, whom are initially treated with a therapeutic regimen to reduce pathogen load, have been able to mamtam decreased pathogen load when removed from the therapeutic regimen, I e , dunng a "dmg holiday" (Rosenberg, E , et al , Immune control of HIV-1 after early treatment of acute mfection Nature 407 523-26, Sept 28, 2000) As appreciated by those skilled m the art, many therapeutic regimens for both pathogens and cancer have numerous, often severe, side effects During the drug holiday, the patient's immune system is keeping the disease in check Methods for usmg compositions of the invention are used in the context of drug holidays for cancer and pathogenic mfection

For treatment of an mfection, where therapies are not particularly lmmunosuppressive, 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

For patients with cancer, many therapies are lmmunosuppressive Thus, upon achievement of a remission or identification that the patient is refractory to standard treatment, then upon removal from the lmmunosuppressive therapy, a composition m accordance with the mvention is 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

IV.O. Kits The peptide and nucleic acid compositions of this invenUon can be provided m kit form together with instructions for vaccme admmistration Typically 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 Other kit components that may also be desirable mclude, for example, a sterile syringe, booster dosages, and other desired excipients

IV.P. Overview

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 epιtope(s) of the mvention Upon administration of peptide-based epitope forms, 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 is used to induce an immune response, it is admmistered either as naked DNA, generally m a dose range of approximately l-5mg, or via the ballistic "gene gun" delivery, typically m a dose range of approximately 10- 100 μg The DNA can be delivered m a variety of conformations, e g , lmear, cucular etc. Various viral vectors have also successfully been used that comprise nucleic acids which encode epitopes in accordance with the invention.

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

One composition in accordance with the invention comprises a plurality of peptides. This plurality or cocktail of peptides is generally admixed with one or more pharmaceutically acceptable excipients. The peptide cocktail can comprise multiple copies of the same peptide or can comprise a mixture of peptides. The peptides can be analogs of naturally 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. In a prefened embodiment the peptide cocktail comprises a plurality of different CTL epitopes and at least one HTL epitope. The HTL epitope can be naturally or non-naturally (e.g., PADRE®, Epimmune Inc., San Diego, 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, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200).

An additional embodiment of a composition in accordance with the invention comprises a polypeptide multi-epitope construct, i.e., a polyepitopic peptide. Polyepitopic peptides in accordance with the invention are prepared by use of technologies well-known in the art. By use of these known technologies, epitopes in accordance with the invention are connected one to another. The polyepitopic peptides can be linear or non-linear, e.g., multivalent. These polyepitopic constructs can comprise artificial amino acids, spacing or spacer amino acids, flanking amino acids, or chemical modifications between adjacent epitope units. The polyepitopic construct can be a heteropolymer or a homopolymer. The polyepitopic constructs generally comprise epitopes in a quantity of any whole unit integer between 2-200 (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.). The polyepitopic construct can comprise CTL and/or HTL epitopes. One or more of the epitopes in the construct can be modified, e.g., by addition of a surface active material, e.g. a lipid, or chemically modified, e.g., acetylation, etc. Moreover, bonds in the multiepitopic construct can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc. Alternatively, 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 In this embodiment, 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, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100 percent A further embodiment of a composition m accordance with the invention is an antigen presenting cell that comprises one or more epitopes in accordance with the mvention The antigen presentmg cell can be a "professional" antigen presentmg cell, such as a dendntic cell The antigen presenting cell can comprise the epitope of the mvention by any means known or to be determmed m the art Such means include pulsmg of dendntic cells with one or more individual epitopes or with one or more peptides that comprise multiple epitopes, by nucleic acid admmistration such as ballistic nucleic acid delivery or by other techniques m the art for admmistration of nucleic acids, mcludmg vector-based, e g viral vector, delivery of nucleic acids

Further embodiments of 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 As appreciated by one of ordmary skill m the art, 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

It is to be appreciated that peptide-based forms of the mvention (as well as the nucleic acids that encode them) 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 mvention will be described m greater detail by way of specific examples The following examples are offered for illustrative purposes, and are not intended to limit the invention m any manner Those of skill m the art will readily recognize a variety of non-cntical parameters that can be changed or modified to yield alternative embodiments in accordance with the mvention V. EXAMPLES

The following examples illustrate identification, selection, and use of immunogenic Class I and Class II peptide epitopes for inclusion in vaccme compositions

Example 1 HLA Class I and Class II Binding Assays

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 ¹²⁵I-radιolabeled 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 bmd 10-20% of the total radioactivity All subsequent inhibition and direct bmding assays were performed usmg these HLA concentrations

Smce under these conditions [label]<[HLA] and IC₅₀≥[HLA], the measured IC₅₀ values are reasonable approximations of the true K_D values Peptide inhibitors are typically tested at concentrations rangmg from 120 μg/ml to 1 2 ng/ml, and are tested m two to four completely mdependent experiments To allow companson of the data obtamed m different experiments, a relative bmdmg figure is calculated for each peptide by dividing the IC₅₀ of a positive control for inhibition by the IC₅₀ for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide) For database purposes, and mter- experiment compansons, relative bmdmg values are compiled These values can subsequently be converted back mto IC₅₀ nM values by dividing the IC₅₀ nM of the positive controls for inhibition by the relative bmdmg of the peptide of interest This method of data compilation has proven to be the most accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC Bmdmg assays as outlmed above can be used to analyze supermotif and or motif-bearmg epitopes as, for example, descnbed in Example 2

Example 2 Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes

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 Computer searches and algorthims for identification of supermotif and/or motif-bearing epitopes

The searches performed to identify the motif-bearing peptide sequences in Examples 2 and 5 employed protein sequence data for the tumor-associated antigens MAGE2/3.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs were performed as follows. All translated protein sequences were analyzed using a text string search software program, e g., MotifSearch 1.4 (D. Brown, San Diego) to identify potential peptide sequences containing appropriate HLA binding motifs; alternative programs are readily produced in accordance with information in the art in view of the motif/supermotif disclosure herein. Furthermore, such calculations can be made mentally. Identified A2-, A3-, and DR-supermotif sequences were scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms take into account both extended and refined motifs (that is, to account for the impact of different amino acids at different positions), and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: where a₇, is a coefficient which represents the effect of the presence of a given amino acid (/^') at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue^^' occurs at position / m the peptide, it is assumed to contribute a constant amount j, to the free energy of binding of the peptide inespective of the sequence of the rest of the peptide. This assumption is justified by studies from our laboratories that demonstrated that peptides are bound to MHC and recognized by T cells in essentially an extended conformation (data omitted herein).

The method of derivation of specific algorithm coefficients has been described in Gulukota et al, J. Mol. Biol. 267: 1258-126, 1997; (see also Sidney et al, Human Immunol 45:79-93, 1996; and Southwood et al, J. Immunol 160:3363-3373, 1998). Briefly, for all positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying^^' is calculated relative to the remainder of the group, and used as the estimate of ,. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values 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.

Selection ofHLA-A2 supertype cross-reactive peptides

The complete protein sequences from MAGE2/3 were scanned, utilizing motif identification software, to identify 8-, 9-, 10-, and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity.

A total of 285 HLA-A2 supermotif-positive sequences were identified within the MAGE2 and/or MAGE3 protein sequences. Of these, 137 of the conesponding peptides were synthesized and tested for the capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule). Nineteen of the peptides bound A*0201 with 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.

Selection ofHLA-A3 supermotif-bearing epitopes

The protein sequences scanned above are also examined for the presence of peptides with the

HLA- A3-supermotif primary anchors using methodology similar to that performed to identify HLA-A2 supermotif-bearing epitopes. 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.

Selection ofHLA-B7 supermotif bearing epitopes

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 IC₅₀ of <500 nM are then tested for binding to other common B7-supertype molecules (B*3501, B*5101, B*5301, and B*5401) to identify those peptides that are capable of binding to three or more of the five B7-supertype alleles tested. Examples of HLA-B7 cross-binding supermotif-bearing peptides identified in accordance with this procedure are provided in Table XXIV.

Selection ofAl and A 24 motif-bearing epitopes

To further increase population coverage, HLA-Al and -A24 motif-bearing epitopes can also be incoφorated 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 IC₅₀ of <500 nM. Examples of peptides identified in accordance with this procedure are provided in Tables XXV and XXVI.

Example 3. Confirmation of lmmunogenicity

Motif analysis and binding studies described in Example 2 identified seventeen potential epitopes for both MAGE2 and MAGE3. Four of the peptide are, however, identical in both MAGE2 and 3, and therefore do not represent distinct epitopes. Peptides were selected for in vitro immunogenicity testing. Testing was performed using the following methodology: Target Cell Lines for Cellular Screening

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 IFNγ (Genzyme) for 48 hours at 37°C before use as targets m the ^5lCr release and in situ IFNγ assays

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC) PBMCs were thawed m RPMI with 30 μg/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) The monocytes were purified by plating 10 x 10⁶ PBMC/well in a 6-well plate After 2 hours at 37°C, the non-adherent cells were removed by gently shaking the plates and aspirating the supernatants The wells were washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells Three ml of complete medium contammg 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 were then added to each well DC were used for CTL mduction cultures following 7 days of culture Induction of CTL with DC and Peptide CD8+ T-cells were isolated by positive selection with

Dynal lmmunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent Typically about 200- 250xl0⁶ PBMC were processed to obtam 24xl0⁶ CD8⁺ T-cells (enough for a 48-well plate culture) Briefly, the PBMCs were thawed m RPMI with 30μg/ml DNAse, washed once with PBS contammg 1% human AB serum and resuspended m PBS/1% AB serum at a concentration of 20xl0⁶cells/ml The magnetic beads were washed 3 tunes with PBS/AB serum, added to the cells (140μl beads/20xl0⁶ 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 lOOxlO⁶ cells/ml (based on the oπgmal cell number) m PBS/AB serum contammg lOOμl/ml detacha-bead® reagent and 30μg/ml DNAse The mixture is mcubated for 1 hour at room temperature with continuous mixmg The beads were washed agam with PBS/AB/DNAse to collect the CD8+ T-cells The DC were collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40μg/ml of peptide at a cell concentration of l-2xl0⁶/ml m the presence of 3 μg/ml β₂- microglobulm for 4 hours at 20°C The DC were then nτadiated (4,200 rads), washed 1 time with medium and counted agam

Setting up induction cultures 0 25 ml cytokine-generated DC (@lxl0⁵ cells/ml) were co-cultured with 0 25ml of CD8+ T-cells (@2xl0⁶ cell/ml) m each well of a 48-well plate m the presence of 10 ng/ml of IL-7 rHuman IL10 was added the next day at a final concentration of 10 ng/ml and rhuman IL2 was added 48 hours later at 10IU/ml

Reshmulation of the induction cultures with peptide-pulsed adherent cells Seven and fourteen days after the prunary mduction the cells were restimulated with peptide-pulsed adherent cells The PBMCS were thawed and washed twice with RPMI and DNAse The cells were resuspended at 5x10⁶ cells/ml and irradiated at ~4200 rads The PBMCs were plated at 2x10⁶ m 0 5ml complete medium per well and mcubated for 2 hours at 37°C The plates were washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with lOμg/ml of peptide m the presence of 3 μg/ml β₂ microglobulm in 0 25ml RPMI/5%AB per well for 2 hours at 37°C Peptide solution from each well was aspnated and the wells were washed once with RPMI Most of the media was aspirated from the mduction cultures (CD8+ cells) and brought to 0 5 ml with fresh media The cells were then transfened to the wells contammg the peptide-pulsed adherent cells Twenty four hours later rhuman IL10 was added at a final concentration of lOng/ml and rhuman IL2 was added the next day and again 2-3 days later at 50IU/ml (Tsai et al , Critical Reviews in Immunology 18(1-2) 65-75, 1998) Seven days later the cultures were assayed for CTL activity in a ^5lCr release assay In some experiments the cultures were assayed for peptide-specific recognition m the in situ IFNγ ELISA at the time of the second restunulation followed by assay of endogenous recognition 7 days later After expansion, activity was measured m both assays for a side by side comparison

Measurement of CTL lytic activity by ^5lCr release. Seven days after the second restimulation, cytotoxicity was determmed in a standard (5hr) ⁵¹Cr release assay by assaying individual wells at a smgle E T Peptide-pulsed targets were prepared by mcubatmg the cells with lOμg/ml peptide overnight at 37°C

Adherent target cells were removed from culture flasks with trypsm-EDTA Target cells were labelled with 200μCι of ⁵¹Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at 37^CC Labelled target cells are resuspended at 10⁶ per ml and diluted 1 10 with K562 cells at a concentration of 3 3xl0^δ/ml (an NK-sensitive erythroblastoma cell lme used to reduce non-specific lysis) Target cells (100 μl) and 1 OOμl of effectors were plated in 96 well round-bottom plates and mcubated for 5 hours at 37°C At that time, 100 μl 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 ^5lCr release sample)/(cpm of the maximal ⁵¹Cr release sample- cpm of the spontaneous ⁵¹Cr release sample)] x 100 Maximum and spontaneous release were determmed by mcubatmg the labelled targets with 1% Tπtion X-100 and media alone, respectively A positive culture was defined as one in which the specific lysis (sample- background) was 10% or higher m the case of individual wells and was 15% or more at the 2 highest E T ratios when expanded cultures were assayed

In situ Measurement of Human γlFN Production as an Indicator of Peptide-specific and Endogenous Recognition

Immulon 2 plates were coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0 IM NaHC0₃, pH8 2) overnight at 4°C The plates were washed with Ca²⁺, Mg²⁺-free PBS/0 05% Tween 20 and blocked with PBS/10% FCS for 2 hours, after which the CTLs (100 μl/well) and targets (100 μl/well) were added to each well, 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% C0₂

Recombmant human IFNγ was added to the standard wells starting at 400 pg or 1200pg/100μl well and the plate mcubated for 2 hours at 37°C The plates were washed and 100 μl of biotinylated mouse anti- human IFNγ monoclonal antibody (4μg/ml m PBS/3%FCS/0 05% Tween 20) were added and mcubated for

2 hours at room temperature After washing agam, 100 μl HRP-streptavidin were added and incubated for

1 hour at room temperature The plates were then washed 6x with wash buffer, lOOμl/well developmg solution (TMB 1 1) were added, and the plates allowed to develop for 5-15 minutes The reaction was stopped with 50 μl well IM H₃P0 and read at OD450 A culture was considered positive if it measured at least 50 pg of IFNγ/well above background and was twice the background level of expression

CTL Expansion Those cultures that demonstrated specific lytic activity agamst peptide-pulsed targets and/or tumor targets were expanded over a two week period with antι-CD3 Briefly, 5xl0⁴ CD8+ cells were added to a T25 flask contammg the following lxlO⁶ inadiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x10⁵ inadiated (8,000 rad) EBV- transformed cells per ml, and OKT3 (antι-CD3) at 30ng per ml m RPMI-1640 contammg 10% (v/v) human AB serum, non-essential ammo acids, sodium pyruvate, 25μM 2-mercaptoethanol, L-glutamme and penicillin/streptomycin rHuman IL2 was added 24 hours later at a final concentration of 200IU/ml and every 3 days thereafter with fresh media at 50lU/ml The cells were split if the cell concentration exceeded lxl0⁶/ml and the cultures were assayed between days 13 and 15 at E T ratios of 30, 10, 3 and 1 1 in the ⁵¹Cr release assay or at lxl0⁶/ml m the in situ IFNγ assay usmg the same targets as before the expansion

lmmunogenicity ofA2 supermotif-bearing peptides

The A2-supermotιf 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 In this analysis, 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

Peptides that were screened m the cellular assay and shown to mduce a response m PBMCs from at least 2 normal donors are shown m Table XXVII CTLs to some of these peptides were also able to recognize endogenously expressed peptide (Table XXVII) Two of these peptide sequences, MAGE3 159 and MAGE3 160, overlap and, while both bmd to 5 allele-specific HLA molecules, MAGE3 160 binds with a higher affinity to 4 of the 5 alleles A IFNγ in situ ELISA of individual CTL cultures mduced with MAGE3 159 showed that cells from five wells recognized the peptide-pulsed targets, and 2 of these wells also recognized the appropriate tumor target Additionally, MAGE3 160 induced a peptide-specific CTL response m 14 of 48 wells and 3 of these wells demonstrated endogenous recognition m the IFNγ assay MAGE3 112, MAGE2 157, and MAGE3 271 have also been identified as epitopes (see, e g , Kawashima et al , Human Immunol 59 1-14, 1998, Visseren, Int J Cancer 73 125, 1997)

Evaluation ofA *03/All lmmunogenicity HLA- A3 supermotif-bearing cross-reactive bmding peptides are also evaluated for lmmunogenicity using methodology analogous for that used to evaluate the lmmunogenicity of the HLA- A2 supermotif peptides Usmg this procedure, peptides that mduce an immune response are identified Examples of such peptides are shown m Table XXIII Evaluation ofB7 lmmunogenicity lmmunogenicity screenmg of the B7-supertype cross-reactive binding peptides identified in Example 2 are evaluated in a manner analogous to the evaluation of A2-and A3-supermotιf-beaπng peptides Using this procedure, peptides that mduce an immune response are identified Examples of such peptides are shown m Table XXIV

Evaluation of lmmunogenicity of Motif/Supermotif-Bearing Peptides

Analogous methodology, as appreciated by one of ordmary skill m the art, is employed to determine lmmunogenicity of peptides beanng HLA class I motifs and/or supermotifs set out herem Usmg such a prodcedure peptides that mduce an immune response are identified, e g , Tables XXV and XXVI

Example 4 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (compnsmg primary and/or secondary residues) 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

Analoguing at Primary Anchor Residues

Peptide engmeermg strategies were implemented to further mcrease the cross-reactivity of the epitopes identified above On the basis of the data disclosed, e , m related and co-pending U S S N

09/226,775, the mam anchors of A2-supermotιf-beaπng 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 (IC₅₀ 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 IC₅₀ 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

Additionally, the selection of analogs for cellular screenmg analysis was further restricted by the capacity of the WT parent peptide to bmd at least weakly, i e , bmd at an IC₅₀ of 5000nM or less, to three of more A2 supertype alleles The rationale for this requuement is that the WT peptides must be present endogenously m sufficient quantity to be biologically relevant Analogued peptides have been shown to have mcreased lmmunogenicity and cross-reactivity by T cells specific for the WT epitope (.see, e , Parkhurst et al , J Immunol 157 2539, 1996, and Pogue et al , Proc Natl Acad Sci USA 92 8166, 1995) In the cellular screening of these peptide analogs, it is important to demonstrate that analog- specific CTLs are also able to recognize the wild-type peptide and, when possible, tumor targets that endogenously express the epitope

Of the 19 MAGE2/3 -derived A*0201 bmding peptides, 14 earned suboptimal anchor residues Analogs of two peptide epitopes were synthesized and tested for bmdmg to HLA-A2 supertype molecules MAGE3 112 analogs exhibited mcreased A*0201 bmding affinity, but the parent peptide bound all 5 A2 supertype HLA molecules and significant improvement was not achieved The MAGE3 220 analog, however, did demonstrate a 3-fold increase in A*0201 bmdmg affinity and unproved cross-reactive bmdmg (Table XXII) In addition, 24 of the 26 weak A*0201 bmdmg peptides also met the criteria for analogumg and can be similarly analyzed for improved bmding properties

Those MAGE2/3 analogs that show improved bmdmg relative to the wildtype peptide are evaluated m the cellular screening analysis as described m Example 3 Usmg this methodology, immunogenic analog peptides are identified (Table XXVII) Using methodology similar to that used to develop HLA-A2 analogs, analogs of HLA-A3 and

HLA-B7 supermotif-bearing epitopes are also generated For example, peptides 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-supermotιf-beanng analog peptides are provided m Table XXIV

Similarly, 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

Analogumg at Secondary Anchor Residues Moreover, 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

For example, 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

Engineered analogs with sufficiently unproved bmdmg capacity or cross-reactivity are tested for lmmunogenicity as above

Other analogumg strategies

Another form of peptide analogumg, unrelated to the anchor positions, mvolves the substitution of a cysteme with α-amino butyric acid Due to its chemical nature, cysteme has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce bmdmg capacity Subtitution of α-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)

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

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

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

Selection of HLA-DR-supermotif-beanng epitopes

To identify HLA class II HTL epitopes, 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 IC₅₀ 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 IC₅₀ value of 1000 nM or less to at least 5 of the 8 alleles tested.

Following the strategy outlined above, 97 DR supermotif-bearing sequences were identified within the MAGE2/3 protein sequences. Of those, 23 scored positive in 2 of the 3 combined DR 147 algorithms. These peptides were synthesized and tested for binding to HLA-DRB 1*0101, DRB1*0401, DRB1*0701 with 13, 3, and 7 peptides binding <1000 nM, respectively. Of the 23 peptides tested for binding to these primary HLA molecules, 7 bound at least 2 of the 3 alleles (Table XXVIII).

These 7 peptides were then tested for binding to secondary DR supertype alleles: DRB5*0101, DRB1*1501, DRB1*1101, DRB1*0802, and DRBl*1302. Three of the peptides bound at least 5 of the 8 alleles tested, and occuned in distinct, non-overlapping regions (Table XXIX).

Selection ofDR3 motif peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations,

DR3 binding capacity is an important criterion in the selection of HTL epitopes. However, data generated previously indicated that DR3 only rarely cross-reacts with other DR alleles (Sidney et al, J. Immunol.

149:2634-2640, 1992; Ge ύs. et al, J. Immunol. 152:5742-5748, 1994; Southwood / al, J. Immunol.

160:3363-3373, 1998). This is not entirely surprising in that the DR3 peptide-binding motif appears to be distinct from the specificity of most other DR alleles. For maximum efficiency in developing vaccine candidates it would be desirable for DR3 motifs to be clustered in proximity with DR supermotif regions. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the distinct binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, the MAGE2/3 protein sequences were analyzed for conserved sequences carrying one of the two DR3 specific binding motifs (Table III) reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Twenty- three motif-positive peptides were identified. The conesponding peptides were then synthesized and tested for the ability to bind DR3 with an affinity of

<1000 nM. Two peptides were identified that met this binding criterion (Table XXX), and thereby qualify as HLA class II high affinity binders.

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 IC₅₀ 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.

In summary, 3 DR supertype cross-reactive binding peptides were identified from the MAGE2/3 protein sequences. Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides may be analogued to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9- mer core sequence is an optimal residue for DR3 binding, and substitution for that residue may improve DR

3 binding. Example 6 lmmunogenicity of HTL epitopes

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

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

Where frequency data was not available at the level of DNA typing, conespondence to the serologically defined antigen frequencies was assumed To obtam total potential supertype population coverage no linkage disequihbnum was assumed, and only alleles confirmed to belong to each of the supertypes were mcluded (minimal estimates) Estimates of total potential coverage achieved by mter-loci combinations were made by addmg to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e , total=A+B*(l-A)) Confirmed members of the A3-lιke supertype are A3, Al 1, A31, A*3301, and A*6801 Although the A3-lιke supertype may also include A34, A66, and A*7401, these alleles were not included m overall frequency calculations Likewise, confirmed members of the A2-lιke supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901 Finally, the B7-lιke supertype-confirmed alleles are B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602) Population coverage achieved by combmmg the A2-, A3- and B7-supertypes is approximately

86% m five major ethnic groups (see Table XXI) Coverage may be extended by mcludmg peptides bearmg the Al and A24 motifs On average, Al is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chmese, Japanese, and Hispanic) Together, these alleles are represented with an average frequency of 39% m these same ethnic populations The total coverage across the major ethmcities when Al and A24 are combmed with the coverage of the A2-, A3- and B7-supertype alleles is >95% An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearmg epitopes Example 8 Recognition Of Generation Of Endogenous Processed Antigens After Pπming

This example determines that CTL mduced by native or analogued peptide epitopes identified and selected as described m Examples 1-6 recognize endogenously synthesized, i e , native antigens, usmg a transgenic mouse model Effector cells isolated from transgenic mice that are immunized with peptide epitopes (as descnbed, e g , in Wentworth et al , Mol Immunol 32 603, 1995), for example HLA-A2 supermotif- bearing epitopes, are re-stimulated in vitro usmg peptide-coated stimulator cells Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated An additional six days later, these cell lines are tested for cytotoxic activity on ^5lCr labeled Jurkat- A2 1/K target cells m the absence or presence of peptide, and also tested on ⁵¹Cr labeled target cells bearmg the endogenously synthesized antigen, i e cells that are stably transfected with TAA expression vectors

The result will demonstrate that CTL lmes obtained from animals pruned with peptide epitope recognize endogenously synthesized antigen The choice of transgenic mouse model to be used for such an analysis depends upon the epιtope(s) that is being evaluated In addition to HLA-A*0201/K^b transgenic mice, several other 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

Example 9 Activity Of CTL-HTL Coniugated Epitopes In Transgenic Mice

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 XXXI The peptides may be hpidated, if desired

Immunization procedures Immunization of transgenic mice is performed as described (Alexander et al , J Immunol 159 4753-4761, 1997) For example, A2/K^b 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) In vitro CTL activation One week after pruning, spleen cells (30xlO^δ cells/flask) are co-cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (lOxlO⁶ cells/flask) in 10 ml of culture medιum/T25 flask After six days, effector cells are harvested and assayed for cytotoxic activity

Assay for cytotoxic activity Target cells (1 0 to 1 5xl0⁶) are incubated at 37°C in the presence of 200 μl of^5lCr After 60 mmutes, cells are washed three tunes and resuspended m medium Peptide is added where req red at a concentration of 1 μg/ml For the assay, 10^{4 51}Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96- well plates After a 6 hour mcubation penod at 37°C, a 0 1 ml aliquot of supernatant is removed from each well and radioactivity is determmed in a Micromedic automatic gamma counter The percent specific lysis is determined by the formula percent specific release = 100 x (experimental release - spontaneous release)/(maxιmum release - spontaneous release) To facilitate comparison between separate CTL assays run under the same conditions, % ^5lCr release data is expressed as lytic units/10⁶ cells One lytic unit is arbitranly defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells m a 6 hour ⁵¹Cr release assay To obtam specific lytic units/ 10⁶, the lytic units/ 10⁶ obtamed in the absence of peptide is subtracted from the lytic units/ 10⁶ obtained m the presence of peptide For example, if 30% ^5lCr release is obtamed at the effector (E) target (T) ratio of 50 1 (I e , 5xl0⁵ effector cells for 10,000 targets) m the absence of peptide and 5 1 (l e , 5xl0⁴ effector cells for 10,000 targets) m the presence of peptide, the specific lytic umts would be [(l/50,000)-( 1/500,000)] x 10⁶ = 18 LU

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 In accordance with these procedures it is found that a CTL response is mduced, and concomitantly that an HTL response is mduced upon admmistration of such compositions

Example 10 Selection of CTL and HTL epitopes for inclusion in a cancer vaccme

This example illustrates the procedure for the selection of peptide epitopes for vaccme compositions of the mvention 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 ρeptιde(s), or may be smgle and or polyepitopic peptides

The following principles are utilized when selectmg an anay of epitopes for inclusion in a vaccme composition Each of the following principles is balanced m order to make the selection

Epitopes are selected which, upon administration, mimic immune responses that have been observed to be conelated with tumor clearance For example, 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.

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

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

Example 11. Construction of Minigene Multi-Epitope DNA Plasmids

This example provides general guidance for the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of CTL and/or HTL epitopes or epitope analogs as described herein. Expression plasmids have been constructed and evaluated as described, for example, in co-pending U.S.S.N. 09/311,784 filed 5/13/99.

A minigene expression plasmid may include multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-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. Similarly, HLA class II epitopes are selected from multiple tumor antigens to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

This example illustrates the methods to be used for construction of such a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art. 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-puπfied The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence The final multiepitope mmigene is assembled by extending the overlapping oligonucleotides m three sets of reactions usmg PCR A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed usmg the following conditions 95°C for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pan) for 30 sec, and 72°C for 1 mm

For the first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended Oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined m 100 μl reactions contammg Pfu polymerase buffer (lx= 10 mM KCL, 10 mM (NH₄)₂S0₄, 20 mM Tπs-chloride, pH 8 75, 2 mM MgS0₄, 0 1% Triton X-100, 100 μg/ml BSA), 0 25 mM each dNTP, and 2 5 U of Pfu polymerase The full-length dimer products are gel-purified, and two reactions 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 the full length product for 25 additional cycles The full-length product is gel-puπfied and cloned mto pCR-blunt (Invitrogen) and individual clones are screened by sequencmg

Example 12 The plasmid construct and the degree to which it induces lmmunogenicity

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

Alternatively, plasmid constructs can be evaluated in vitro by testing for epitope presentation by APC following transduction or transfection of the APC with an epitope-expressmg nucleic acid construct Such a study determmes "antigemcity" and allows the use of human APC 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 determining the concentration of peptide necessary to obtamed equivalent levels of lysis or lymphokme release (see, e g , Kageyama et al , J Immunol 154 567-576, 1995)

To assess the capacity of the mmigene construct (e g , a pMin mmigene construct generated as decπbed in U S S N 09/311,784) to mduce CTLs in vivo, HLA-Al 1/K^b transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a 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 ^5lCr 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. It is, therefore, found that 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.

To assess the capacity of a class II epitope encodmg mmigene to mduce HTLs in vivo, I- A^b restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA As a means of comparmg the level of HTLs mduced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i e HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded m the minigene). The HTL response is measured using a ³H-thymιdιne 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)

For example, the efficacy of the DNA mmigene may be evaluated m transgenic mice In this example, A2.1/K^b transgenic mice are immunized IM with 100 μg of the DNA mmigene encodmg the immunogenic peptides After an incubation period (rangmg from 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinant vaccmia virus expressmg the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccmia without the mmigene sequence, or with DNA encodmg the mmigene, but without the vaccmia boost After an additional mcubation penod of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay Additionally, splenocytes are stimulated in vitro with the A2 -restricted peptide epitopes encoded m the mmigene and recombmant vaccmia, then assayed for peptide-specific activity in an IFN-γ 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 by HLA- A3 and HLA-B7 motif or supermotif epitopes Example 13 Peptide Composition for Prophylactic Uses

Vaccme compositions of the present invention are used to prevent cancer in persons who are at risk for developing a tumor For example, a polyepitopic peptide epitope composition (or a nucleic acid 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 μg, generally 100-5,000 μg, for a 70 kg patient The initial admmistration of vaccme is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample Additional booster doses are admmistered as required The composition is found to be both safe and efficacious as a prophylaxis against cancer

Alternatively, the polyepitopic peptide composition can be admmistered as a nucleic acid m accordance with methodologies known m the art and disclosed herem

Example 14 Polyepitopic Vaccine Compositions Derived from Native TAA Sequences

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 As noted herein, epitope motifs may be nested or overlapping (i e , frame shifted relative to one another) For example, with frame shifted overlappmg epitopes, two 9-mer epitopes and one 10-mer epitope can be present m a 10 ammo acid peptide Such a vaccme composition is admmistered for therapeutic or prophylactic purposes

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

Related to this embodiment, computer programs can be denved in accordance with principles m the art, which identify in a target sequence, the greatest number of epitopes per sequence length

Example 15 Polyepitopic Vaccine Compositions Directed To Multiple Tumors

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 For example, a set of TAA epitopes can be selected that allows the targeting of most common epithelial tumors (see, e g , Kawashima et al , Hum Immunol 59 1-14, 1998) Such a composition includes epitopes from CEA, HER-2/neu, and MAGE2/3, all of which are expressed to appreciable degrees (20-60%) 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 dendntic cells which have been loaded with the peptide epitopes in vitro

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 For example, approximately 50% of breast tumors express CEA, 20% express MAGE3, and 30% express HER-2/neu Thus, the use of a smgle antigen for immunotherapy would offer only limited patient coverage The combmation of the three TAAs, however, 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 In the following example, peptides m accordance with the mvention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen

In this example, highly sensitive human leukocyte antigen tetramenc complexes ("tetramers") are used for a cross-sectional analysis of, for example, tumor-associated antigen HLA-A*0201-specιfic 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 . N Engl J Med 337 1267, 1997) Briefly, punfied HLA heavy cham (A*0201 m this example) and β2-mιcroglobulm 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, β2-mιcroglobuhn, 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. Louis, Missouri), adenosine 5 'tnphosphate and magnesium. Streptavidin-phycoerythπn conjugate is added ιn a l.4 molar ratio, and the tetramenc product is concentrated to 1 mg/ml The resultmg product is refened to as tetramer-phycoerythπn

For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes and resuspended m 50 μl of cold phosphate-buffered saline Tn-color analysis is performed with the tetramer-phycoerythπn, along with antι-CD8-Tπcolor, and antι-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.

Example 17 Use of Peptide Epitopes to Evaluate Recall Responses

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

For example, 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 μg/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 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help durmg the first week of stimulation

In the microculture format, 4 x 10⁵ PBMC are stimulated with peptide m 8 replicate cultures m 96- well round bottom plate m 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well On day 7 the cultures are transfened mto a 96- well flat-bottom plate and restimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad) autologous feeder cells The cultures are tested for cytotoxic activity on day 14. A positive CTL response requues two or more of the eight replicate cultures to display greater than 10% specific ⁵¹Cr 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)

Cytotoxicity assays are performed m the following manner 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 μM, and labeled with 100 μCi of ⁵¹Cr (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 ⁵¹Cr 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 Percent cytotoxicity is determmed from the formula 100 x [(experimental release-spontaneous release)/maxιmum 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 results of such an analysis mdicate the extent to which HLA-restncted CTL populations have been stimulated by previous exposure to the TAA or TAA vaccme

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 5xl0⁵ cells/well and are stimulated with 10 μg/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 μCi ³H-thymιdιne 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 ³H-thymιdιne incorporation Antigen- specific T cell proliferation is calculated as the ratio of ³H-thymιdιne incoφoration m the presence of antigen divided by the ³H-thymιdιne incoφoration m the absence of antigen

Example 18 Induction Of Specific CTL Response In Humans

A human clmical trial for an immunogenic composition compπsing 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 μg of peptide composition,

Group II 3 subjects are injected with placebo and 6 subjects are mjected with 50 μg peptide composition,

Group III 3 subjects are mjected with placebo and 6 subjects are injected with 500 μg of peptide composition

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

The endpomts measured m this study relate to the safety and tolerabihty of the peptide composition as well as its lmmunogenicity Cellular immune responses to the peptide composition are an mdex of the intrinsic activity of the peptide composition, and can therefore be viewed as a measure of biological efficacy The following summarize the clmical and laboratory data that relate to safety and efficacy endpomts

Safety The mcidence of adverse events is monitored m the placebo and drug treatment group and assessed m terms of degree and reversibility

Evaluation of Vaccme Efficacy For evaluation of vaccme efficacy, subjects are bled before and after injection Penpheral blood mononuclear cells are isolated from fresh hepaπnized blood by Ficoll- Hypaque density gradient centnfugation, ahquoted m freezing media and stored frozen Samples are assayed for CTL and HTL activity

The vaccme is found to be both safe and efficacious

Example 19 Therapeutic Use m Cancer Patients

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

There are three patient groupmgs 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

Example 20 Induction of CTL Responses Usmg a Pnme Boost Protocol

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

For example, 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 μg) 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 5-10⁷ to 5xl0⁹ 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 For evaluation of vaccme efficacy, patient blood samples will be obtamed before immunization as well as at mtervals following admmistration of the initial vaccme and booster doses of the vaccme Penpheral blood mononuclear cells are isolated from fresh hepanmzed blood by Ficoll-Hypaque density gradient centnfugation, ahquoted m freezing media and stored frozen Samples are assayed for CTL and HTL activity

Analysis of the results will mdicate that a magnitude of response sufficient to achieve protective immunity agamst cancer is generated

Example 21 Admmistration of Vaccme Compositions Using Dendntic Cells

Vaccmes compnsmg peptide epitopes of the mvention may be admmistered usmg antigen- presentmg cells (APCs), or "professional" APCs such as dendntic cells (DC) In this example, the peptide- pulsed DC are admmistered to a patient to stimulate a CTL response in vivo In this method, 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 For example, a cocktail of epitope-bearmg peptides is admmistered ex vivo to PBMC, or isolated

DC therefrom, from the patient's blood A pharmaceutical to facilitate harvestmg of DC can be used, such as Progenipoietin™ (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

As appreciated clinically, and readily determmed by one of skill based on clmical outcomes, 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 10⁶ dendntic cells per patient are typically admmistered, larger number of dendntic cells, such as 10⁷ or 10⁸ can also be provided Such cell populations typically contam between 50-90% dendntic cells

In some embodiments, peptide-loaded PBMC are injected mto patients without purification of the DC For example, PBMC contammg DC generated after treatment with an agent such as Progempoietin™ are mjected mto patients without purification of the DC The total number of PBMC that are admmistered often ranges from 10⁸ to 10¹⁰ Generally, 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 Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 10⁶ DC, then the patient will be injected with a total of 2 5 x 10⁸ peptide-loaded PBMC The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill m the art

Ex vivo activation of CTL HTL responses Alternatively, 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. Alternative Method of Identifying Motif-Bearing Peptides

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

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

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

TABLE I

Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table. TABLE la

*If 2 is V, or Q, the C-term is not L

Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.

SF 1199173 vl

TABLE II

POSITION

¥ C-terminus

SUPERMOTIFS

Al 1 ° Anchor 1° Anchor T,I,L, V,M,S F,W,Y

A2 1° Anchor 1° Anchor

CO L,I,V,M, , L,I,V,M,A,T c m T,Q co A3 preferred 1° Anchor Y,F,W, (4/5) Y,F, , Y,F,W, (4/5) P, (4/5) 1 "Anchor

V,S,M,A,7; (3/5) R,K

L,l m deleterious D,E (3/5); P, (5/: 5) D,E, (4/5) co 1° Anchor 1° Anchor m A24 Y,F,W,I, V, F,l,Y, tV,L,M m L,M, T

B7 prefened F,W,Y (5/5) 1 "Anchor F,W,Y (4/5) F.W.Y, 1 "Anchor

7i c L,IN,M, (3/5) P (3/5) V,l,L,F,M, W ,A m deleterious D,E (3/5); P(5/5); D,E, (3/5) G, (4/5) Q,N, (4/5) G(4/5); A(3/5); D,E, (4/5) Q,N, (3/5)

1° Anchor 1" Anchor

B27 R,H,K F,Y,L, ^, ,

1° Anchor 1° Anchor

B44 E,D F,W,Y,L,I,M,V,A

1° Anchor 1 ° Anchor

B58 A,T,S F,W,Y,L,I, V,M,A

1 ° Anchor 1° Anchor

B62 Q,L,/, V,M, F,W,Y,M,I, V,L,A P

POSITION

0 C-terminus

MOTIFS

Al preferred G.F.Y.W, l°Anchor D.E.A, Y.F.W, P, D,E,Q,N, Y,F,W, 1 "Anchor

9-mer S,T,M, Y deleterious D,E, R.H.K.L. V A, G, A, M,P,

CO c m co

Al preferred G,R,H,K A,S,T,C,L,I 1 "Anchor G,S,T,C, A,S,T,C, L,I,V,M, D,E, PAnchor

9-mer V,M, O,E ,S Y m deletenous A R,H,K,D,E, D,E, P,Q,N, R,H,K, PA G,P, co P,Y,F,W, m m

POSITION s @ C-terminus or C-terminus

Al pefened Y,F,W, 1 "Anchor D,E,A,Q,N, A, Y.F.W.Q.N, P,A,S,T,C, G,D,E, P, 1 "Anchor

10-mer S,T,M Y deleterious G,P, R,H,K,G,L,I D,E, R,H,K, Q,N,A R,H,K,Y,F, R,H,K, V,M, W,

CO m m

73 A2.1 preferred Y.F.W, l°Anchor Y,F,W, S,T,C, Y,F,W, A, 1 "Anchor

C 9-mer L,M,I. V,Q, VJL,I,M,A,T m A,T deleterious D,E,P, D,E,R,K,H R,K,H D,E,R,K,H

A2.1 prefened A,Y,F,W, 1 "Anchor L,V,I,M, G, G, F,Y,W,L, 1 "Anchor 10-mer ,M,I, V,Q, V,I,M, WJ. .M.A.T

A.T deleterious D,E,P, D,E, R,K,H,A, P, R,K,H, D,E,R,K, R,K,H, H,

POSITION s Θ g c- or terminus

C-terminus

A3 prefened R,H,K, 1 "Anchor Y,F,W, P,R,H,K,Y, A, Y,F,W, P, 1 "Anchor L,M,V,I,S, F,W, K,Y,R,H,F,A A,T,F,C,G D deleterious D,E,P, D,E

CO c Al l preferred 1 "Anchor Y,F,W, Y,FW, A, Y,F,W, Y,FW, 1 "Anchor

V,T,L,M,I, K„RY,H

CO S,A,G,N,C,

D,F m deleterious D,E,P, G,

CO

I m A24 preferred Y,F,W,R,H,K, 1 "Anchor s,τ,c Y.F.W, Y,F,W, 1° Anchor m 9-mer Y,F,W,M F,L,I,W deleterious D,E,G, D,E, G, Q,N,P, D,E,R,H,K, G, A.Q.N,

A24 preferred 1 "Anchor P, Y,F,W,P, P, 1 "Anchor ro 10-mer Y,F,W F,L,I,W deleterious G,D,E Q,N R,H,K D,E Q,N, D,E,A,

A3101 preferred R,H,K, l°Anchor Y.F.W, P, Y,F,W, Y,F,W, A,P, 1 "Anchor

M,V,T^4,L, R,K

IS deleterious D,E,P, D,E, A,D,E, D,E, D,E, D.E,

POSITION

Θ I B Θ C- or terminus C-terminus

A3301 preferred 1 "Anchor Y,F,W A,Y,F,W 1 "Anchor

M,V,A,L,F, R,K

I.S deleterious G,P D,E

CO A6801 preferred Y,F,W,S,T,C, 1 "Anchor Y,F,W,L,I, Y,F,W, P, 1 "Anchor

C m A,V M,S, V,M R,K co L.I deleterious G,P, D,E,G, R,H,K, A, m

CO

I m m B0702 preferred R,H,K,F,W,Y, 1 "Anchor R,H,K, R,H,K, R,H,K, R,H,K, P,A, 1 "Anchor

P LMF,W,Y,A,

73 IV c m deleterious D,E,Q,N,P, D,E,P, D,E, D,E, G,D,E, Q,N, D,E, ro

B3501 preferred F,W,Y,L,I,V,M, 1 "Anchor F,W,Y, F.W.Y, 1 "Anchor P L.M.F.W.Y,/,

V.A deleterious A,G,P, G, G,

POSITION

0 @ I @ 1 C- or terminus

C-terminus

B51 preferred L,I/V,M,F,W,Y, l"Anchor F,W,Y, s,τ,c, F.W.Y, G, F,W,Y, 1 "Anchor

P L, V,F,0',

Y.A.M deleterious A,G,P,D,E,R,H,K, D,E, G, D,E,Q,N, G,D,E, S,T,C,

CO

C CD B5301 preferred L,I,V,M,F,W,Y, 1 "Anchor F,W,Y, S.T.C, F.W.Y, L,I/V,M,F, F,W,Y, 1 "Anchor CO P W,Y, I,M,F,W,Y,

A,L, V m deleterious A,G,P,Q,N, G, R,H,K,Q,N, D,E,

CO

I m m

B5401 preferred F,W,Y, 1 "Anchor F,W,Y,L,I,V L,I,V,M, A.L.I.V.M, F,W,Y,A,P, 1 "Anchor

73 P M, A,T,I,V,L, c M,F, W,Y m ro deleterious G,P,Q,N,D,E, G,D,E,S,T,C, R,H,K,D,E, D,E, Q,N,D,G,E, D,E,

Italicized residues indicate less preferred or "tolerated" residues.

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

Secondary anchor specificities are designated for each position independently.

SF 1199181 vl

Table III

POSITION

MOTIFS 1° anchor 1 1° anchor 6 @

DR4 preferred F, M, Y,L, I, M, V, S, T, C, P, A, M, H, M, H V, W, L, I, M, deleterious w, R, W, D, E

DRl preferred M, F, L, I, V, P, A, M, Q, V, M, A, T, S, P, M, A, V, M W, Y,

CO L, /, C c

CD deleterious C C, H F, D C, W, D G, D, E, D CO

DR7 preferred M, F, L, I, V, M, W, A, I, V, M, S, A, C, M, ι. v W, Y, T, P, L, m deleterious c, G, G, R, D, N G co m m DR Supermotif M, F, L, I, V, V, M, S, T, A, C,

W, Y, P, L, I,

73 c m ro

DR3 MOTIFS 1 ° anchor 1 1° anchor 4] |l° anchor 6 motif a L, I, V, M, F, preferred Y, D motif b L, I, V, M, F, D, N, Q, E, preferred A, Y, S, T K, R, H

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

Table IV: HLA Class I Standard Peptide Binding Affinity.

SF l 199173 vl Table V. HLA Class II Standard Peptide Binding Affinity.

1199173 vl

Table VI

Allelle-specific HLA-supertype members

HLA-supertype Verified³ Predicted"

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

A2 A*0201, A*0202, A*0203, A*0204, A*0205, A*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,

A*3402, A*6601, A*6602, A*7401

A24 A*2301, A*2402, A*3001 A*2403, A*2404, A*3002, A*3003

B7 B*0702, B*0703, B*0704, B*0705, B* 1508, B*3501, B*3502, B*3503, B*1511, B*4201, B*5901

B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101, B*5102, co B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502, B*5601, c

CD B*5602, B*6701, B*7801 CO B27 B* 1401 , B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*2701, B*2707, B*2708, B*3802, B*3903, B*3904, B*3801, B*3901, B*3902, B*7301 B*3905, B*4801, B*4802, B* 1510, B* 1518, B*1503

B44 B* 1801, B*1802, B*3701, B*4402, B*4403, B*4404, B*4001 , B*4002, B*4101, B*4501, B*4701, B*4901, B*5001 m B*4006 co B58 B*5701 , B*5702, B*5801, B*5802, B* 1516, B* 1517

I B62 B*1501, B*1502, B*1513, B*5201 B*1301 , B*1302, B*1504, B*1505, B*1506, B*1507, m m B* 1515, B* 1520, B* 1521, B* 1512, B* 1514, B* 1510

73

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

SF l l 66636 vl

Table VII A Mage 2 API Supermotif Peptides with Binding Data

_- -* _ c Ξ _ _- ° — Ξ

Table VII B Mage 3 API Supermotif Peptides with Binding Data

Table VIII A Mage 2 AP2 Supermotif with Binding Data

Table VIII A

Mage 2 A02 Supermotif with Binding Data

« c oe .

Table VIII A Mage 2 AP2 Supermotif with Binding Data

Table VIII A Mage 2 A02 Supermotif with Binding Data

^βe i; _β oo β'

Table VIII B

Mage 3 A02 Supermotif with Binding Data

Table VIII B Mage 3 A02 Supermotif with Binding Data

Table VIII B Mage 3 AP2 Supermotif with Binding Data

K C - — oc _ __ _ oc c OC J>X ? _

— — ~~ — N - — " " - - - n «M

Table IX A Mage 2 AP3 Supermotif with Binding Data

Table IX B Mage 3 AP3 Supermotif with Binding Data

Table X A Mage 2 A24 Supermotif Peptides with Binding Data

« _ oo oc _ oc _ oo θ' θe ».

Table X A Mage 2 A24 Supermotif Peptides with Binding Data

Table X B Mage 3 A24 Supermotif Peptides with Binding Data

— __ ϋ

Table X B Mage 3 A24 Supermotif Peptides with Binding Data

Table XI A Mage 2 BP7 Supermotif Peptides with Binding Data

Table XI B Mage 3 BP7 Supermotif Peptides with Binding Data

Table XII A Mage 2 B27 Supermotif Peptides

Table XII B Mage 3 B27 Supermotif Peptides

Table XIII A

Mage 2 B58 Supermotif Peptides

Table XIII A Mage 2 B58 Supermotif Peptides

Table XIII B Mage 3 B58 Supermotif Peptides

&■ — g> — _ .

Table XIII B Mage 3 B58 Supermotif Peptides

Table XIV A Mage 2 B62 Supermotif Peptides

Ξ Ξ

Table XIV A Mage 2 B62 Supermotif Peptides

Table XIV B Mage 3 B62 Supermotif Peptides

Table XIV B Mage 3 B62 Supermotif Peptiαes

, _ ~ _ ft _- — oc — x „ x ft * _ ft CC !

Table XV A Mage 2 API Motif Peptides with Binding Data

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Table XV B Mage 3 API Motif Peptides with Binding Data

Table XVI A Mage 2 AP3 Motif Peptides with Binding Data

— ft

>n

Table XVI A Mage 2 AQ3 Motif Peptides with Binding Data

Table XVI A Mage 2 AP3 Motif Peptides with Binding Data

* * - X - - « _ * ; x ftr * « θ θ_' -

Table XVI A Mage 2 AP3 Motif Peptides with Binding Data

Table XVI B Mage 3 AP3 Motif Peptides with Binding Data

Table XVI B Mage 3 A03 Motif Peptides with Binding Data

Table XVII A Mage 2 All Motif Peptides with Binding Data

ft Ξ Ξ ft oC ft .— _ CC QC 0C — — OC OC — — αC N Ξ Ξ ft —

τ - ». - ft ft κ £

Table XVII A Mage 2 All Motif Peptides with Binding Data

o CN _— cc

Table XVII A Mage 2 All Motif Peptides with Binding Data

Table XVII B Mage 3 All Motif Peptides with Binding Data

Table XVIII A Mage 2 A24 Motif Peptides with Binding Data

Table XVIII B Mage 3 A24 Motif Peptides with Binding Data

* x ft ~ oo oe X ft ft —

Table XIX A Mage 2 DR Super Motif Peptides with Binding Data ft ft ft ft

x 3 >^• ' έ < ^

n =_ ≤ >

— > ^ — —

Ξ < Ξ Ξ Table XIX A Mage 2 DR Super Motif Peptides with Binding Data

: a. < ^■

>

> = — > -.-;>

£ — _^ — — Table XIX B Mage 3 DR Super Motif Peptides with Binding Data

Table XIX B Mage 3 DR Super Motif Peptides with Binding Data

Table XXa B o z _wo-_N- _»„_* Ma^ge 3 DR 3 a Motif Peptides with Binding Data

Ϊ

Q

ϊ

C O O — = 000

C

Table XXa B

Mage 3 DR 3a Motif Peptides with Binding Data o z a 00 = 000 = 0

i a

lableXXb A Mage 2 DR 3b Motif Peptides with Binding Data

Table XXb A Mage 2 DR 3b Motif Peptides with Binding Data

Table XXb B Mage 3 DR 3b Motif Peptides with Binding Data

ϊ

Ξ

a

a.

i

O o

r y c

- < _S

-- ; > : _.

- « fi

- -j as ^c. < C

Table XXb B Mage 3 DR 3b Motif Peptides with Binding Data

TABLE XXI. Population coverage with combined HLA Supertypes

PHENOTYPIC FREQUENCY

Caucasian North Japanese Chinese Hispanic Average

HLA-SUPERTYPES American

Black a. Individual Supertypes

A2 45.8 39.0 42.4 45.9 43.0 43.2

A3 37.5 42.1 45.8 52.7 43.1 44.2

B7 43.2 55.1 57.1 43.0 49.3 49.5

Al 47.1 16.1 21.8 14.7 26.3 25.2

A24 23.9 38.9 58.6 40.1 38.3 40.0

B44 43.0 21.2 42.9 39.1 39.0 37.0

B27 28.4 26.1 13.3 13.9 35.3 23.4

B62 12.6 4.8 36.5 25.4 11.1 18.1

B58 10.0 25.1 1.6 9.0 5.9 10.3 b. Combined 1 Supertypes

A2, A3, B7 84.3 86.8 89.5 89.8 86.8 87.4

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

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

B2 ', B62, B58

SF 1199173 vl

Table XXII. Crossbinding data A2 supermotif peptides

A*0201 A*0202 A*0203 A*0206 A*6802 No. A2 Alleles

Source AA Sequence nM nM nM nM nM Crossbound

MAGE2.112 9 KMVELVHFL 38 15 9.1 49 364 5

MAGE2.112 10 KMVELVHFLL 23 39 127 9.0 2667 4

CO MAGE2.112 11 KMVELVHFLLL 5.0 45 63 109 7692 4 c

CD MAGE2.153 9 KASEYLQLV 152 116 17 185 4878 4 CO MAGE2.157 10 YLQLVFGIEV 50 165 345 370 9302 4

MAGE2.160 10 LVFGIEVVEV 357 21 44 29 8.0 5

MAGE2.220 9 KIWEELSML 167 642 175 29 ~ 3 m MAGE2.271 9 FLWGPRALI 238 96 137 1542 95 4 co MAGE2.277 10 ALIETSYVKV 500 729 125 1947 3077 2 m MAGE2/3.44 10 TLVEVTLGEV 67 39 4.3 218 33 5 m

MAGE3.112 9 KVAELVHFL 68 29 14 168 17 5

73 MAGE3.112 10 KVAELVHFLL 54 36 217 206 11 5 c MAGE3.159 11 QLVFGIELMEV 7.9 74 217 185 267 5 m MAGE3.160 10 LVFGIELMEV 29 20 7.7 29 14 5

MAGE3.174 11 HLYIFATCLGL 56 741 769 — 4494 1

MAGE3.176 9 YIFATCLGL 185 45 37 1028 222 4

MAGE3.195 11 IMPKAGLLIIV 20 226 15 176 — 4

MAGE3.220 9 KIWEELSVL 333 391 2381 308 — 3

MAGE3.271 9 FLWGPRALV 31 43 14 336 40 5

— indicates binding affinity =10,000nM.

Table XXIII. HLA-A3 Supermotif-bearing Peptides

No. of A3 Published Published

A*0301 A* 1 101 A*3101 A*3301 A*6801 CTL CTL

AA Sequence Source Alleles CTL CTL nM nM nM nM nM Wildtype Tumor Crossbound Wildtype Tumor

10 LLGDNQIMPK MAGE1/3.189 500 375 - - 372 3

9 SVFSTTINK MAGE2.69.V2K9 20 8.2 3333 9667 5.7 3

9 SVFSTTINR MAGE2.69.V2R9 58 6.3 62 88 6.7 5

9 SSFSTTINK MAGE2.69 69 3.0 2195 - 26 3

1 1 FSTTINYTLWR MAGE2.71 1000 353 257 3919 163 3

CO c 10 STTINYTLWK MAGE2.72 126 9.2 - - 258 3

CD CO 9 TTΓNYTLWR MAGE2.73 204 1 1 237 171 17 5 7/7 2/5

9 TVINYTLWR MAGE2.73.V2 262 77 720 433 15 4

9 TVΓNYTLWK MAGE2.73.V2K9 306 97 9000 - 62 3 m 8 LVHFLLLK MAGE2/3.1 16 379 40 - - 400 3 co m 9 LVHFLLLKK MAGE2/3.1 16.K9 21 4.3 - ~ 381 3 m 9 SMLEVFEGR MAGE2.226 5500 273 37 9.0 1818 3

73 9 SMLEVFEGK MAGE2.226 116 3.8 120 387 2581 4 c 8 SVFAHPRK MAGE2.237 78 74 1385 - 182 3 m

9 AVIETSYVK MAGE2.277.V2 393 63 - - 31 3

9 AVIETSYVR MAGE2.277.V2R9 ~ 171 129 1 160 15 3

9 ALIETSYVK MAGE2.277 136 32 900 - 286 3

9 IVYPPLHER MAGE2.299.V2 1 17 375 95 32 14 5

9 IVYPPLHEK MAGE2.299.V2K9 42 103 857 2990 42 3

9 ISYPPLHER MAGE2.299 324 214 23 36 81 5

9 LVHFLLLKY MAGE2/3.116 297 500 - 8788 8000 2

9 LVHFLLLKR MAGE2/3.1 16.R9 440 375 237 94 27 5

9 YFFPVIFSK MAGE3.138 5000 462 316 207 571 3

9 YVFPVIFSK MAGE3.138.V2 24 3.0 2769 784 1.7 3

9 YVFPVIFSR MAGE3.138.V2R9 36 2.6 6.0 13 0.50 5

- indicates binding affinity >10,000nM.

Table XXIII. HLA-A3 Supermotif-bearing Peptides

No. of A3 Published Published

A*0301 A*1101 A*3101 A*3301 A*6801 CTL CTL

9 SVLEVFEGR MAGE3.226 43 106 44 93 9 SVLEVFEGK MAGE3.226.K9 83 6.7 129 460 186

CO c

CD CO

m co

I m m

73 c m

— indicates binding affinity >10,000nM.

Table XXIV. HLA-B7 Supermotif-Bearing Peptides

No. of B7 _CTL

B*0702 B*3501 B*5101 B*5301 B*5401 CTL

AA Sequence Source Alleles nM nM nM nM nM Tumor Crossbound

9 FPIGHLYII MAGE3.170.F119 3.4 77 5.0 7.2 0.60 5

10 FPIGHLYIFA MAGE3.170.F1 39 51 56 179 0.40 5

10 FPIGHLYIFI MAGE3.170.F1110 63 139 5.7 8.5 2.9 5

9 MPKAGLLI1 MAGE3.196 932 5143 393 90 248 3

9 MPVAGLLII MAGE3.196.V3 86 66 1.2 2.3 1 12 5

CO 10 MP AGLLIIV MAGE3.196 1774 ~ 393 - 12 2

CO 8 MPKAGLLI MAGE3.196 42 - 12 358 313 4 CO

H 10 MPKAGLLIII MAGE3.196.I10 324 2400 62 176 102 4 H C 8 FPKAGLLI MAGE3.196.F1 I8 31 ~ 8.2 775 46 3 H m 10 FPKAGLLIII MAGE3.196.F1 I 10 204 2667 65 846 21 3

CO

I 10 FP AGLLIIV MAGE3.196.F1 220 878 190 4650 1.1 3 m m 11 FPRALVETSYI MAGE3.274.F1I1 1 7.2 5539 1 17 620 59 3

H

11 FPRALVETSYV MAGE3.274.F1 4.2 4235 204 - 10 3

*J

C

I- 9 FPH1SYPPI MAGE3.296.F1 I9 2.9 360 18 233 1.4 5 m ro σ>

— indicates binding affinity >10,000nM.

Table XXV. HLA-A1 Motif-Bearing Peptides

Published Published

A*0101

AA Sequence Source CTL CTL nM

Wildtype Tumor

10 ASSFSTTINY MAGE2.68 147

10 ATSFSTTI Y MAGE2.68.T2 455

10 ASDFSTTINY MAGE2.68.D3 25

9 STFSTTΓNY MAGE2.69.T2 490

1 1 VVEVVPISHLY MAGE2.166 125

8 VTDLGLSY MAGE2.179.D3 2.7

10 LTQDLVQENY MAGE2.246.T2 58

9 MQDLVQENY MAGE2.247 17

9 MTDLVQENY MAGE2.247.T2 0.80

10 ASSLPTTMNY MAGE3.68 9.6

10 ATSLPTTMNY MAGE3.68.T2 208

10 ASDLPTTMNY MAGE3.68.D3 2.6

9 SSLPTTMNY MAGE3.69 676

9 STLPTTMNY MAGE3.69.T2 58

1 1 TMNYPLWSQSY MAGE3.74 301

9 GTVVGNWQY MAGE3.137.T2 36

1 1 LMEVDPIGHLY MAGE3.166 3.3

9 EVDPIGHLY MAGE3.168 1.4

9 ETDPIGHLY MAGE3.168.T2 0.70

8 ATCLGLSY MAGE3.179 227

10 LTQHFVQENY MAGE3.246 96

10 LTDHFVQENY MAGE3.246.D3 2.3

9 ITGGPHISY MAGE3.293.T2 36

1) Tuting et al., Journal of Immunology 160(3): 1 139, 1998

Table XXVIa. HLA-A24 Motif-Bearing Peptides

Published Published

A*2402

AA Sequence Source CTL CTL nM

Wildtype Tumor

1 1 SFSTTINYTLW MAGE2.70 429

9 MYPDLESEF MAGE2.97.Y2 52

1 1 IFSKASEYLQL MAGE2.150 126

9 EYLQLVFGI MAGE2.156 3.4

9 EYLQLVFGF MAGE2.156.F9 4.0

10 LYILVTCLGF MAGE2.175.F10 18

9 VMPKTGLLI MAGE2.195 52

10 VMPKTGLLII MAGE2.195 207

8 LWGPRALI MAGE2.272 100

10 SYVKVLHHTL MAGE2.282 75

10 SYVKVLHHTF MAGE2.282.F10 34

9 TYPDLESEF MAGE3.97.Y2 218

9 NWQYFFPVI MAGE3.142 23

10 NYQYFFPVIF MAGE3.142.Y2 23

8 QYFFPVIF MAGE3.144 100

1 1 IFSKASSSLQL MAGE3.150 132

10 LYIFATCLGF MAGE3.175.F10 10

9 IMPKAGLLI MAGE3.195 29

10 IMPKAGLLII MAGE3.195 240

1 1 IWEELSVLEVF MAGE3.221 462

8 SYPPLHEW MAGE3.300 286

10 SYPPLHEWVL MAGE3.300 20

10 SYPPLHEWVF MAGE3.300.F10 5.5

3) Tahara et al., Clinical Cancer Research 5(8):2236, 1999

4) Tanaka et al., Cancer Research 57(20):4465, 1997

Table XXVI B A24 Motif-bearing Peptides

Peptide AA Seαuence Source A^*2401 nM

52.0072 8 LWGPF ALI MAG E2.272 100

52.0073 8 QYFFPVIF MAG E3.144 100

52.0078 8 SYPPLHEW MAGE3.300 285.7

52.0102 10 SYPPLHEWVL MAGE3.300 20.3

52.0166 11 SFSTTINYTLW MAGE2.70 428.6

52.0167 11 IFSKASEYLQL MAG E2.150 126.3

52.017 11 IFSKASSSLQL MAG E3.150 131.9

52.0172 11 IWEELSVLEVF MAGE3.221 461.5

57.006 9 MYPDLESEF MAGE2.97.Y2 52.2

57.0061 9 KYVELVHFF MAGE2.1 12.Y2F9 7.1

57.0062 9 IYSKASEYF MAGE2.150.Y2F9 14.6

57.0063 9 EYLQLVFGF MAGE2.156.F9 4

57.0064 9 VYPKTGLLF MAGE2.195.Y2F9 5.5

57.0065 9 TYPDLESEF MAGE3.97.Y2 218.2

57.0066 9 NYQYFFPVF MAGE3.142.Y2F9 3.4

57.0067 9 IYSKASSSF MAGE3.150.Y2F9 375

57.0068 9 IYPKAGLLF MAGE3.195.Y2F9 9.2

57.0084 10 SYSTTINYTF MAGE2.70.Y2F10 14.8

57.0085 10 LYILVTCLGF MAGE2.175.F10 17.6

57.0086 10 VYPKTGLLIF MAGE2.195.Y2F10 2.9

57.0087 10 EYLWGPRALF MAGE2.270.Y2F10 10

57.0088 10 SYVKVLHHTF MAGE2.282.F10 34.3

57.009 10 NYQYFFPVIF MAGE3.142.Y2 22.6

57.0092 10 LYIFATCLGF MAGE3.175.F10 10

57.0093 10 lYPKAGLLIF MAGE3.195.Y2F10 1.2

57.0095 10 SYPPLHEWVF MAGE3.300.F10 5.5

Table XXVIIa. lmmunogenicity of A2 supermotif peptides

A*0201 A*0202 A*0203 A*0206 A*6802 No. A2 Alleles CTL CTL

Source AA Sequence nM nM nM nM nM Crossbound Wild-type Tumor

MAGE2.1 12 9 KMVELVHFL 9.8 25 17 123 2353 4 1/1 0/1

MAGE2.112 10 KMVELVHFLL 23 39 127 9.0 2667 4 1/1 0/1

MAGE2.112 11 KMVELVHFLLL 5.0 45 63 109 7692 4 1/1 0/1

MAGE2.153 9 KASEYLQLV 152 116 17 185 4878 4 2/4 0/2

MAGE2.157 10 YLQLVFGIEV 50 165 345 370 9302 4 3/3 1/3

MAGE2.160 10 LVFGIEVVEV 357 20 43 28 8.0 5 4/4 0/3 O MAGE3.112 9 KVAELVHFL 68 29 14 168 17 5 3/4 3/4 c CD MAGE3.112 10 KVAELVHFLL 54 36 217 206 11 5 0/1 0/1 O MAGE3.159 11 QLVFGIELMEV 7.9 74 217 185 267 5 3/3 1/3²

MAGE3.160 10 LVFGIELMEV 29 20 7.7 28 14 5 4/4 1/4²

3 m MAGE3.195 11 IMPKAGLLIIV 20 226 14 176 4 3/4 0/3 co MAGE3.220 9 KIWEELSVL 357 391 2381 308 — 3 3/4 0/3

I m MAGE3.271 9 FLWGPRALV 31 43 14 336 40 5 4/4 2/4 m

73 1) Indicates the number of donors positive over the total number of donors tested.

C 2) A positive result was seen after the second restim. m 3) ~ indicates binding affinity =10,000nM. ro σ>

Table XXVIII. DR supertype primary binding

DRl 47 DRl 47

DRl DR4w4 DR7 Algo Sequence Source Cross- nM nM nM Sum binding

2 LGEVPAADSPSPPHS MAGE2.50 — — — 0

3 ESEFQAAISR MVEL MAGE2.102 4.2 281 49 3

2 GIEVVEVVPISHLYI MAGE2.163 595 6429 278 2

2 DGLLGDNQVMPKTGL MAGE2.187 ~ ~ - 0

2 NQVMPKTGLLI1VLA MAGE2.193 2632 - - 0

2 TGLLIIVLAIIAIE MAGE2.198 417 1216 862 2

2 TGLLIIVLAIIAIEG MAGE2.199 6250 — — 0

2 GLLIIVLAIIAIEGD MAGE2.200 500 — ~ 1

3 LLIIVLAIIAIEGDC MAGE2.201 581 3750 1923 1

2 LI1VLAIIAIEGDCA MAGE2.202 417 8824 2083 1

2 EPHISYPPLHERALR MAGE2.296 — — — 0

3 ALGLVGAQAPATEEQ MAGE2/3.22 152 - - 1

2 ESEFQAALSRKVAEL MAGE3.102 2.6 763 34 3

2 NWQYFFPVIFSKASS MAGE3.142 46 409 446 3

3 PV1FSKASSSLQLVF MAGE3.148 98 1875 281 2

3 LQLVFGIELMEVDPI MAGE3.158 200 ~ 258 2

3 GHLYIFATCLGLSYD MAGE3.173 455 4091 - 1

2 DGLLGDNQIMPKAGL MAGE3.187 - - - 0

2 NQIMPKAGLLIIVLA MAGE3.193 1 14 - - 1

2 KAGLLIIVLAIIARE MAGE3.198 1 163 - - 0

2 AGLLIIVLAIIAREG MAGE3.199 l l l l -- >9615 0

3 LLIIVLAIIAREGDC MAGE3.201 1923 — — 0

2 GPHISYPPLHEWVLR MAGE3.296 2273 — — 0

— indicates binding affinity =10,000nM

Table XXIX. DR supertype crossbinding

DRl 47 Broad

DRl DR4 4 DR7 DR2w2βl DR2w2B2 DR6wl9 DR5wl l DR8w2

Peptide Sequence Source Cross- Binding nM nM nM nM nM nM nM nM binding (5/8)

co m m

73

C m

Table XXX. DR3 binding

DR3

Sequence Source nM

GPRMFPDLESEFQAA MAGE2.94 3371

FPDLESEFQAAISRK MAGE2.98 -

EFQAAISRKMVELVH MAGE2.104 -

QLVFGIEVVEVVPIS MAGE2.159 -

CLGLSYDGLLGDNQV MAGE2.181 2143

YDGLLGDNQVMPKTG MAGE2.186 -

LAIIAIEGDCAPEEK MAGE2.206 —

IIAIEGDCAPEEKIW MAGE2.208 4546

EEKIWEELSMLEVFE MAGE2.218 —

RKLLMQDLVQENYLE MAGE2.243 2000

MQDLVQENYLEYRQV MAGE2.247 1500

VKVLHHTLKIGGEPH MAGE2.284 —

TLKIGGEPHISYPPL MAGE2.290 —

FPDLESEFQAALSRK MAGE3.98 -

EFQAALSRKVAELVH MAGE3.104 -

QLVFGIELMEVDPIG MAGE3.159 -

IELMEVDPIGHLYIF MAGE3.164 167

CLGLSYDGLLGDNQI MAGE3.181 -

YDGLLGDNQIMPKAG MAGE3.186 -

LAIIAREGDCAPEEK MAGE3.206 —

EEKIWEELSVLEVFE MAGE3.218 —

EDSILGDPKKLLTQH MAGE3.235 448

TQHFVQENYLEYRQV MAGE3.247 1071

— indicates binding affinity =10,000nM

Claims

WHAT IS CLAIMED IS

1. An isolated prepared MAGE2/3 epitope consisting of a sequence selected from the group consisting of the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI.

2. A composition of claim 1, wherein the epitope is admixed or joined to a CTL epitope.

3. A composition of claim 2, wherein the CTL epitope is selected from the group set out in claim 1.

4. A composition of claim 1, wherein the epitope is admixed or joined to an HTL epitope.

5. A composition of claim 4, wherein the HTL epitope is selected from the group set out in claim 1.

6. A composition of claim 4, wherein the HTL epitope is a pan-DR binding molecule.

7. A composition of claim 1, comprising at least three epitopes selected from the group set out in claim 1.

8. A composition of claim 1, further comprising a liposome, wherein the epitope is on or within the liposome.

9. A composition of claim 1 , wherein the epitope is joined to a lip id.

10. A composition of claim 1, wherein the epitope is joined to a linker.

11. A composition of claim 1 , wherein the epitope is bound to an HLA heavy chain, β2-microglobulin, and strepavidin complex, whereby a tetramer is formed.

12. A composition of claim 1, further comprising an antigen presenting cell, wherein the epitope is on or within the antigen presenting cell.

13. A composition of claim 12, wherein the epitope is bound to an HLA molecule on the antigen presenting cell, whereby when a cytotoxic lymphocyte (CTL) is present that is restricted to the HLA molecule, a receptor of the CTL binds to a complex of the HLA molecule and the epitope.

14 A clonal cytotoxic T lymphocyte (CTL), wherem the CTL is cultured in vitro and bmds to a complex of an epitope selected from the group set out m Tables XXIII, XXIV, XXV, XXVI, and XXVII, bound to an HLA molecule

15 A peptide compπsmg at least a first and a second epitope, wherein the first epitope is selected from the group consisting of the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI, wherem the peptide comprise less than 50 contiguous ammo acids that have 100% identity with a native peptide sequence

16 A composition of claim 15, wherem the first and the second epitope are selected from the group of claim 14

17 A composition of claim 16, further comprising a third epitope selected from the group of claim 15

18 A composition of claim 15, wherem the peptide is a heteropolymer

19 A composition of claim 15, wherein the peptide is a homopolymer

20 A composition of claim 15, wherem the second epitope is a CTL epitope

21 A composition of claim 20, wherem the CTL epitope is from a tumor associated antigen that is not MAGE2/3

22 A composition of claim 15, wherem the second epitope is a PanDR bmdmg molecule

23 A composition of claim 1, wherem the first epitope is linked to an a linker sequence

24 A vaccme composition compnsmg a unit dose of a peptide that comprises less than 50 contiguous ammo acids that have 100% identity with a native peptide sequence of MAGE2/3, the peptide compπsmg at least a first epitope selected from the group consistmg of the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI, and, a pharmaceutical excipient

25 A vaccme composition m accordance with claim 24, further compnsmg a second epitope

26. A vaccine composition of claim 24, wherein the second epitope is a PanDR binding molecule.

27. A vaccine composition of claim 24, wherein the pharmaceutical excipient comprises an adjuvant.

28. An isolated nucleic acid encoding a peptide comprising an epitope consisting of a sequence selected from the group consisting of the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI.

29. An isolated nucleic acid encoding a peptide comprising at least a first and a second epitope, wherein the first epitope is selected from the group consisting of the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI; and wherein the peptide comprises less than 50 contiguous amino acids that have 100% identity with a native peptide sequence.

30. An isolated nucleic acid of claim 29, wherein the peptide comprises at least two epitopes selected from the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI.

31. An isolated nucleic acid of claim 30, wherein the peptide comprises at least three epitopes selected from the sequences set out in Tables XXIII, XXIV, XXV, XXVI, XXVII, and XXXI.

32. An isolated nucleic acid of claim 29, wherein the second peptide is a CTL epitope.

33. An isolated nucleic acid of claim 32, wherein the CTL is from a tumor- associated antigen that is not MAGE2/3.

34. An isolated nucleic acid of claim 20, wherein the second peptide is an HTL epitope.