CA2323632A1 - Hla-binding peptides and their uses - Google Patents

Abstract

The present invention provides the means and methods for selecting immunogenic peptides and the immunogenic peptide compositions capable of specifically binding glycoproteins encoded by HLA allele and inducing T cell activation in T cells restricted by the allele. The peptides are useful to elicit an immune response against a desired antigen.

Description

HLA BINDING PEPTIDES AND THEIR USES
BACKGROUND OF THE INVENTION
The present invention relates to compositions and methods for preventing, treating or diagnosing a number of pathological states such as viral diseases and cancers.
In particular, it provides novel peptides capable of binding selected major histocompatibility complex (MHC) molecules and inducing an immune response.
MHC molecules are classified as either Class I or Class II molecules. Class II MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T lymphocytes, B lymphocytes, macrophages, etc.
Class II
MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular immunogenic peptide that is displayed. Class I MHC molecules are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes (CTLs), which then destroy the antigen-bearing cells. CTLs are particularly important in tumor rejection and in fighting viral infections.
The CTL recognizes the antigen in the form of a peptide fragment bound to the MHC class I molecules rather than the intact foreign antigen itself. The antigen must normally be endogenously synthesized by the cell, and a portion of the protein antigen is degraded into small peptide fragments in the cytoplasm. Some of these small peptides translocate into a pre-Golgi compartment and interact with class I heavy chains to facilitate proper folding and association with the subunit ~i2 microglobulin. The peptide-MHC class I complex is then routed to the cell surface for expression and potential recognition by specific CTLs.
Investigations of the crystal structure of the human MHC class I molecule, HLA-A2.1, indicate that a peptide binding groove is created by the folding of the a 1 and a2 domains of the class I heavy chain (Bjorkman et al., Nature 329:506 ( 1987). In these investigations, however, the identity of peptides bound to the groove was not determined.
Buus et al. , Science 242:1065 ( 1988) first described a method for acid elution of bound peptides from MHC. Subsequently, Rammensee and his coworkers (Falk et al . , Nature 351:290 ( 1991 ) have developed an approach to characterize naturally processed peptides bound to class i molecules. Other investigators have successfully achieved direct amino acid sequencing of the more abundant peptides in various HPLC
fractions by conventional automated sequencing of peptides eluted from class I
molecules of the B type (Jardetzky, et a1. , Nature 353 : 326 ( 1991 ) and of the A2.1 type by mass spectrometry (Hunt, et al., Science 225:1261 (1992). A review of the characterization of naturally processed peptides in MHC Class I has been presented by Rotzschke and Falk (Rotzschke and Falk, Immunol. Todav 12:447 (1991).
Sette et al., Proc. Natl. A_cad. Sci. USA 86:3296 (1989) showed that MHC
allele specific motifs could be used to predict MHC binding capacity.
Schaeffer et al., P~roc. Natl. Acad. Sci. USA 86:4649 (1989) showed that MHC binding was related to immunogenicity. Several authors (De Bruijn et al., Eur. J. Immunol., 21:2963-( 1991 ); Pamer et al . , 991 Mature 353 : 852-955 ( 1991 )) have provided preliminary evidence that class I binding motifs can be applied to the identification of potential immunogenic peptides in animal models. Class I motifs specific for a number of human alleles of a given class I isotype have yet to be described. It is desirable that the combined frequencies of these different alleles should be high enough to cover a large fraction or perhaps the majority of the human outbred population.
Despite the developments in the art, the prior art has yet to provide a useful human peptide-based vaccine or therapeutic agent based on this work. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention provides compositions comprising immunogenic peptides having binding motifs for HLA molecules. The immunogenic peptides, which bind to the appropriate MHC allele, comprise conserved residues at certain positions which allow the peptides to bind desire HLA molecules.
Epitopes on a number of immunogenic target proteins can be identified using the peptides of the invention. Examples of suitable antigens include prostate cancer specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C
antigens, Epstein-Ban virus antigens, human immunodeficiency type-1 virus (HIV
1), Kaposi's sarcoma herpes virus (KSHV), human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tuberculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2/neu. The peptides are thus useful in pharmaceutical compositions for both therapeutic and diagnostic applications.
In particular, the invention provides compositions comprising an immunogenic peptide having an HLA binding motif, which immunogenic peptide is a peptide shown in Tables 3-14. Also provided are peptides comprising a conservative substitution of a residue in a peptide shown in Table 3-14. The immunogenic peptide of the invention can be further linked to a second oligopeptide. In some embodiments, the second oligopeptide is a peptide that induces a helper T response.
1~ The invention further provides nucleic acid molecules encoding immunogenic peptides as shown in Tables 3-14, or peptides comprising a conservative substitution of a residue of a peptide shown in Table 3-14. The nucleic acid may further comprise a sequence encoding a second immunogenic peptide or peptide that induces a helper T response.
1.5 The peptides provided here can be used to induce a cytotoxic T cell response either in vivo or in vitro. The methods comprise contacting a cytotoxic T cell with a peptide of the invention.
Definitions The term "peptide" is used interchangeably with "oligopeptide" in the 2~ present specification to designate a series of residues, typically L-amino acids, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of adjacent amino acids. The oligopeptides of the invention are less than about 15 residues in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues .
25 An "immunogenic peptide" is a peptide which comprises an allele-specific motif such that the peptide will bind an MHC molecule and induce a CTL
response.
Immunogenic peptides of the invention are capable of binding to an appropriate HLA
molecule and inducing a cytotoxic T cell response against the antigen from which the immunogenic peptide is derived.
30 Immunogenic peptides are conveniently identified using the algorithms of the invention. The algorithms are mathematical procedures that produce a score which enables the selection of immunogenic peptides. Typically one uses the algorithmic score with a "binding threshold" to enable selection of peptides that have a high probability of binding at a certain affinity and will in turn be immunogenic. The algorithm is based upon either the effects on MHC binding of a particular amino acid at a particular position of a peptide or the effects on binding of a particular substitution in a motif containing peptide.
A "conserved residue" is an amino acid which occurs in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. Typically a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide. At least one to three or more, preferably 1.0 two, conserved residues within a peptide of defined length defines a motif for an immunogenic peptide. These residues are typically in close contact with the peptide binding groove, with their side chains buried in specific pockets of the groove itself.
Typically, an immunogenic peptide will comprise up to three conserved residues, more usually two conserved residues.
As used herein, "negative binding residues" are amino acids which if present at certain positions will result in a peptide being a nonbinder or poor binder and in turn fail to be immunogenic i.e. induce a CTL response.
The term "motif" refers to the pattern of residues in a peptide of defined length, usually about 8 to about 11 amino acids, which is recognized by a particular MHC
~0 allele. The peptide motifs are typically different for each human MHC
allele and differ in the pattern of the highly conserved residues and negative residues.
The binding motif for an allele can be defined with increasing degrees of precision. In one case, all of the conserved residues are present in the correct positions in a peptide and there are no negative residues in positions 1,3 and/or 7.
~5 The phrases "isolated" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. Thus, the peptides of this invention do not contain materials normally associated with their ~;~. environment, e.g., MHC I molecules on antigen presenting cells. Even where a protein has been isolated to a homogenous or dominant band, there 30 are trace contaminants in the range of 5-10 % of native protein which co-purify with the desired protein. Isolated peptides of this invention do not contain such endogenous co-purified protein.

The term "residue" refers to an amino acid or amino acid mimetic incorporated in an oligopeptide by an amide bond or amide bond mimetic.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to the determination of allele-specific peptide 5 motifs for human Class I MHC (sometimes referred to as HLA) allele subtypes, in particular, peptide motifs recognized by HLA alleles.
For HLA-A2.1 alleles a peptide of 9 amino acids preferrably has the following motif: a first conserved residue at the second position from the N-terminus selected from the group consisting of I, V, A and T and a second conserved residue at the C-terminal position selected from the group consisting of V, L, I, A and M. An alternate motif is one in which the first conserved residue at the second position from the N-terminus selected is from the group consisting of L, M, I, V, A and T and the second conserved residue at the C-terminal position selected from the group consisting of A and M. The amino acid at position 1 is preferrably not an amino acid selected from the group consisting of D, and P. The amino acid at position 3 from the N-terminus is not an amino acid selected from the group consisting of D, E, R, K and H. The amino acid at position 6 from the N-terminus is not an amino acid selected from the group consisting of R, K and H . The amino acid at at position 7 from the N-terminus is not an amino acid selected from the group consisting of R, K, H, D and E.
The HLA-A2.1 binding motif for peptide of 10 residues is as follows: a first conserved residue at the second position from the N-terminus selected from the group consisting of L, M, I, V, A, and T, and a second conserved residue at the C-terminal position selected from the group consisting of V, I, L, A and M. The first and second conserved residues are separated by 7 residues. Preferrably, the amino acid at position 1 is not an amino acid selected from the group consisting of D, E and P. The N-terminal residue is not an amino acid selected from the group consisting of D and E.
The residue at position 4 from the N-terminus is not an amino acid selected from the group consisting of A, K, R and H. The amino acid at positon 5 from the N-terminus is not P. The amino acid at position 7 from the N-terminus is not an amino acid selected from the group ~0 consisting of R, K and H. The amino acid at position 8 from the N-terminus is not amino acid selected from the group consisting of D, E, R, K and H. The amino acid at position 9 from the N-terminus is not an amino acid selected from the group consisting of R, K and H.
Te motif for HLA-A3.2 comprises from the N-terminus to C-terminus a first conserved residue of L, M, I, V, S, A, T and F at position 2 and a second conserved residue of K, R or Y at the C-terminal end. Other first conserved residues are C, G or D
and alternatively E. Other second conserved residues are H or F. The first and second conserved residues are preferably separated by 6 to 7 residues.
The motif for HLA-A1 comprises from the N-terminus to the C-terminus a first conserved residue of T, S or M, a second conserved residue of D or E, and a third conserved residue of Y. Other second conserved residues are A, S or T. The first and second conserved residues are adjacent and are preferably separated from the third conserved residue by 6 to 7 residues. A second motif consists of a first conserved residue of E or D and a second conserved residue of Y where the first and second conserved residues are separated by 5 to 6 residues.
The motif for HLA-A 11 comprises from the N-terminus to the C-terminus a first conserved residue of T, V, M, L, I, S, A, G, N, C D, or F at position 2 and a C-terminal conserved residue of K, R, Y or H. The first and second conserved residues are preferably separated by 6 or 7 residues.
The motif for HLA-A24.1 comprises from the N-terminus to the C-terminus a first conserved residue of Y, F or W at position 2 and a C terminal conserved residue of F, I, W, M or L. The first and second conserved residues are preferably separated by 6 to 7 residues.
These motifs are then used to define T cell epitopes from any desired antigen, particularly those associated with human viral diseases, cancers or autoiummune diseases, for which the amino acid sequence of the potential antigen or autoantigen targets is known.
Epitopes on a number of potential target proteins can be identified in this manner. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis B
core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Burr virus antigens, melanoma antigens (e.g., MAGE-1), human immunodeflciency virus (HIV) antigens, human papilloma virus (HPV) antigens, Lassa virus, mycobacterium tubcrculosis (MT), p53, CEA, trypanosome surface antigen (TSA) and Her2lneu.

Peptides comprising the epitopes from these antigens are synthesized and then tested for their ability to bind to the appropriate MHC molecules in assays using, for example, purified class I molecules and radioiodonated peptides and/or cells expressing empty class I molecules by, for instance, immunofluorescent staining and flow microfluorometry, peptide-dependent class I assembly assays, and inhibition of CTL
recognition by peptide competition. Those peptides that bind to the class I
molecule are further evaluated for their ability to serve as targets for CTLs derived from infected or immunized individuals, as well as for their capacity to induce primary j~
vitro or in vivo CTL responses that can give rise to CTL populations capable of reacting with virally infected target cells or tumor cells as potential therapeutic agents.
The MHC class I antigens are encoded by the HLA-A, B, and C loci.
HLA-A and B antigens are expressed at the cell surface at approximately equal densities, whereas the expression of HLA-C is significantly lower (perhaps as much as 10-fold lower) . Each of these loci have a number of alleles. The peptide binding motifs of the invention are relatively specific for each allelic subtype.
For peptide-based vaccines, the peptides of the present invention preferably comprise a motif recognized by an MHC I molecule having a wide distribution in the human population. Since the MHC alleles occur at different frequencies within different ethnic groups and races, the choice of target MHC allele may depend upon the target population. Table 1 shows the frequency of various alleles at the HLA-A locus products among different races. For instance, the majority of the Caucasoid population can be covered by peptides which bind to four HLA-A allele subtypes, specifically HLA-A2.1, A1, A3.2, and A24.1. Similarly, the majority of the Asian population is encompassed with the addition of peptides binding to a fifth allele HLA-A11.2.

A Allele/SubtvneN~69)' A(54) A1 10.1(7) 1.8(1) 27.4(138) A2.1 11.5(8) 37.0(20) 39.8( 199) A2.2 10.1 (7) 0 3. 3( 17) A2.3 1.4(1) 5.5(3) 0.8(4) A2.4 - - -A2.5 ~ - - -A3.1 1.4(1) 0 0.2(0) A3.2 5.7(4) 5.5(3) 21.5(108) Al l . l 0 5.5(3) 0 A 11. 2 5.7(4) 31.4( 17) 8.7{44) A 11.3 0 3.7(2) 0 A23 4.3{3) - 3.9(20) A24 2.9(2) 27.7(15) 15.3(77) A24.2 - - -A24.3 - - -A25 1.4(1) - 6.9(35) A26.1 4.3(3) 9.2(5) 5.9(30) A26.2 7.2(5) - 1.0(5) A26V - 3.7(2) -A28.1 10.1(7) - 1.6(8) A28.2 1.4(1) - 7.5(38) A29.1 1.4(1) - 1.4(7) A29.2 10.1(7) 1.8(1) 5.3(27) A30.1 8.6(6) - 4.9(25) A30.2 1.4( 1 ) - 0.2( 1 ) A30.3 7.2(5) - 3.9(20) A31 4.3(3) 7.4(4) 6.9(35) A32 2.8(2) - 7.1{36) Aw33.1 8.6(6) - 2.5(13) Aw33.2 2.8(2) 16.6(9) 1.2(6) Aw34.1 1.4(1) - -Aw34.2 14.5( 10) - 0.8(4) Aw36 5.9(4) - -Table compiled from B. DuPont, Immunobiologv of HLA, Vol. I, Histocompatibility Testing 1987, Springer-Verlag, New York 1989.
' N - negroid; A = Asian; C = Caucasoid. Numbers in parenthesis represent the number of individuals included in the analysis.
The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left {the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G.
The procedures used to identify peptides of the present invention generally follow the methods disclosed in Falk et al., 351:290 (1991), which is incorporated herein by reference. Briefly, the methods involve large-scale isolation of MHC
class I
molecules, typically by immunoprecipitation or affinity chromatography, from the appropriate cell or cell line. Examples of other methods for isolation of the desired MHC
molecule equally well known to the artisan include ion exchange chromatography, lectin chromatography, size exclusion, high performance ligand chromatography, and a combination of all of the above techniques.
In the typical case, immunoprecipitation is used to isolate the desired allele.
A number of protocols can be used, depending upon the specificity of the antibodies used.
For example, allele-specific mAb reagents can be used for the affinity purification of the HLA-A, HLA-B,, and HLA-C molecules. Several mAb reagents for the isolation of HLA-A molecules are available. The monoclonal BB7.2 is suitable for isolating molecules. Affinity columns prepared with these mAbs using standard techniques are successfully used to purify the respective HLA-A allele products.
In addition to allele-specific mAbs, broadly reactive anti-HLA-A, B, C
mAbs, such as W6/32 and B9.12.1, and one anti-HLA-B, C mAb, B1.23.2, could be used in alternative affinity purification protocols as described in previous applications.
The peptides bound to the peptide binding groove of the isolated MHC
molecules are eluted typically using acid treatment. Peptides can also be dissociated from class I molecules by a variety of standard denaturing means, such as heat, pH, detergents, salts, chaotropic agents, or a combination thereof.

Peptide fractions are further separated from the MHC molecules by reversed-phase high performance liquid chromatography (HPLC) and sequenced.
Peptides can be separated by a variety of other standard means well known to the artisan, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectrofocusing, and the like.
Sequencing of the isolated peptides can be performed according to standard techniques such as Edman degradation (Hunkapiller, M. W . , g~,., Methods Enzymol . 2.1., 399 [ 1983]). Other methods suitable for sequencing include mass spectrometry sequencing of individual peptides as previously described (Hunt, et al., Science 225:1261 (1992), 10 which is incorporated herein by reference). Amino acid sequencing of bulk heterogenous peptides (g,,g.,., pooled HPLC fractions) from different class I molecules typically reveals a characteristic sequence motif for each class I allele.
Definition of motifs specific for different class I alleles allows the identification of potential peptide epitopes from an antigenic protein whose amino acid sequence is known. Typically, identification of potential peptide epitopes is initially carried out using a computer to scan the amino acid sequence of a desired antigen for the presence of motifs. The epitopic sequences are then synthesized. The capacity to bind MHC Class molecules is measured in a variety of different ways. One means is a Class I
molecule binding assay as described in the related applications, noted above.
Other alternatives described in the literature include inhibition of antigen presentation (Sette, et al., J. Immyno~. 141:3893 (1991), in vitro assembly assays (Townsend, et al., ~ 62:285 (1990), and FACS based assays using mutated ells, such as RMA.S (Melief, et al., Eur. J.J.
j~y~. 21:2963 (1991)).
Next, peptides that test positive in the MHC class I binding assay are assayed for the ability of the peptides to induce specific CTL responses in vitro. For instance, Antigen-presenting cells that have been incubated with a peptide can be assayed for the ability to induce CTL responses in responder cell populations. Antigen-presenting cells can be normal cells such as peripheral blood mononuclear cells or dendritic cells (Inaba, et al., ). Ex~,"_lVled. 166:182 (1987); Boog, Eur. 1. Immunol. 18:219 [1988]).
Alternatively, mutant mammalian cell lines that are deficient in their ability to load class I molecules with internally processed peptides, such as the mouse cell lines RMA-S (Karre, et al.. , 319:675 (1986); Ljunggren, et al., Fur. J. Immunol.

21:2963-2970 ( 1991 )), and the human somatic T cell hybrid, T-2 (Cerundolo, et al . , Nature 345:449-452 ( 1990)) and which have been transfected with the appropriate human class I genes are conveniently used, when peptide is added to them, to test for the capacity of the peptide to induce in vitro primary CTL responses. Other eukaryotic cell lines which could be used include various insect cell Iines such as mosquito larvae (ATCC
cell lines CCL 125, 126, 1660, 1591, 6585, 6586), silkworm (ATTC CRL 8851), armyworm (ATCC CRL 1711), moth (ATCC CCL 80) and Drosophila cell lines such as a Schneider cell line (see Schneider ~," F4mbr<rol. Exn; ~~hol_ 27:353-365 [1927]).
Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukapheresis of normal donors or patients and used as the responder cell sources of CTL precursors. In one embodiment, the appropriate antigen-presenting cells are incubated with 10-100 ~.M of peptide in serum-free media for 4 hours under appropriate culture conditions. The peptide-loaded antigen-presenting cells are then incubated with the responder cell populations in vitro for 7 to 10 days under optimized culture conditions. Positive CTL activation can be determined by assaying the cultures for the presence of CTLs that kill radiolabeled target cells, both specific peptide-pulsed targets as well as target cells expressing endogenously processed form of the relevant virus or tumor antigen from which the peptide sequence was derived.
Specificity and MHC restriction of the CTL is determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I.
The peptides that test positive in the MHC binding assays and give rise to specific CTL
responses are referred to herein as immunogenic peptides.
The immunogenic peptides can be prepared synthetically, or by recombinant DNA technology or from natural sources such as whole viruses or tumors.
Although the peptide will preferably be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles.
The polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.

Desirably, the peptide will be as small as possible while still maintaining substantially all of the biological activity of the large peptide. When possible, it may be desirable to optimize peptides of the invention to a length of 9 or 10 amino acid residues, commensurate in size with endogenously processed viral peptides or tumor cell peptides that are bound to MHC class I molecules on the cell surface.
Peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.
For instance, the peptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding. By conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for 1.5 another. The substitutions include combinations such as Gly, Ala; Val, Ile, Leu, Met;
Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single amino acid substitutions may also be probed using D-amino acids. Such modifications may be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341-347 (1986), Barany and Merrifield, The Pec~tides, Gross and Meienhofer, eds.
(N.Y., Academic Press), pp. 1-284 (1979); and Stewart and Young, Solid Phase Pec~tide ~, (Rockford, Ill., Pierce), 2d Ed. (1984), incorporated by reference herein.
The peptides can also be modified by extending or decreasing the compound's amino acid sequence, e.g., by the addition or deletion of amino acids. The peptides or analogs of the invention can also be modified by altering the order or composition of certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity.
The non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-a-amino acids, or their D-isomers, but may include non-natural amino acids as well, such as (3-y-8-amino acids, as well as many derivatives of L-a-amino acids.
Typically, a series of peptides with single amino acid substitutions are employed to determine the effect of electrostatic charge, hydrophobicity, etc.
on binding.

For instance, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo-oligomers or hetero-oligomers. The number and types of residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions, compared to the affinity of the parent peptide. In any event, such substitutions should employ amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference which might disrupt binding.
Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide. Substitutional variants are those in which at least one residue of a peptide has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 2 when it is desired to finely modulate the characteristics of the peptide.

l b i i i l S
E

Origina u Res st due tut Ala on xemp ary Ser Arg Lys, His Asn Gin Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Lys; Arg Ile Leu; Val Leu Ile; Val Lys Arg; His Met Leu; Ile Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; Phe Tyr Trp; Phe Val Ile; Leu Pro Gly Substantial changes in function (e.g., affinity for MHC molecules or T cell receptors) are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example as a sheet 5 or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c} the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in peptide properties will be those in which (a) hydrophilic residue, e.g. seryl, is substituted for (or by) a hydrophobic residue, e.g.
leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a residue having an electropositive side chain, e.g., lysl, 10 arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (c) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
The peptides may also comprise isosteres of two or more residues in the immunogenic peptide. An isostere as defined here is a sequence of two or more residues 15 that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.
egg, gsnerallv, Spatola, emi t y and Biochemistry yf Amino Acids. tides and pJoteins, Vol. VII (Weinstein ed., 1983).
Modifications of peptides with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide inin vivo.
Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. ~, ~, Verhoef et al., $ur. J. Drub Metab. Pharmacokin. 11:291-302 (1986). Half life of the peptides of the present invention is conveniently determined using a 25 96 human serum (vlv) assay. The protocol is generally as follows. Pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 259b with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6°~b aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled WO 99/45954 PCT/US98/fl5039 (4°C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase IiPLC using stability-specific chromatography conditions.
The peptides of the present invention or analogs thereof which have CTL
stimulating activity may be modified to provide desired attributes other than improved serum half life. For instance, the ability of the peptides to induce CTL
activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Particularly preferred immunogenic peptides/T helper conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e. g. , AIa, Gly, or other neuual spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer.
When IS present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL peptide may be linked to the T helper peptide without a spacer.
The immunogenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide.
The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.
In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes CTL. Lipids have been identified as agents capable of priming CTL inin vivo against viral antigens. For example, palmitic acid residues can be attached to the alpha and epsilon amino groups of a Lys residue and then linked, e.g., via one or more linking residues such as GIy, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The Iipidated peptide can then be injected directly in a micellar form, incorporated into a liposome or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment a particularly effective immunogen comprises palmitic acid attached to alpha and epsilon amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, ~,, coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide. See, Deres et al. , Nature 342:561-564 (1989), incorporated herein by reference. Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime a CTL response to the target antigen. Further, as the induction of neutralizing antibodies can also be primed with P3CSS conjugated to a peptide which displays an appropriate epitope, the two compositions can be combined to more effectively elicit both humoral and cell-mediated responses to infection.
In addition, additional amino acids can be added to the termini of a peptide to provide for ease of linking peptides one to another, for coupling to a carrier support, or larger peptide, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like. Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or N-terminus of the peptide or oligopeptide. Modification at the C terminus in some cases may alter binding characteristics of the peptide. In addition, the peptide or oligopeptide sequences can differ from the natural sequence by being modified by terminal-NH2 acylation, e.g., by alkanoyl (C,-C2o) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications rnay provide sites for linking to a support or other molecule.
The peptides of the invention can be prepared in a wide variety of ways.
Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols.
See, for example, Stewart and Young, Solid Phase Pe~ide ~ n~thesis, 2d. ed., Pierce Chemical Co. ( 1984), supra.
Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes an immunogenic peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., Molecular Clonine. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York ( 1982), which is incorporated herein by reference. Thus, fusion proteins which comprise one or more peptide sequences of the invention can be used to present the appropriate T cell epitope.
As the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., d. Am. Chem. Soc. 103:3185 (1981), modification can be made simply by substituting the appropriate bases) for those encoding the native peptide sequence. The coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop colons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable bacterial hosts. Of course, yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
The peptides of the present invention and pharmaceutical and vaccine compositions thereof are useful for administration to mammals, particularly humans, to treat and/or prevent viral infection and cancer. Examples of diseases which can be treated using the immunogenic peptides of the invention include prostate cancer, hepatitis B, hepatitis C, AIDS, renal carcinoma, cervical carcinoma, lymphoma, CMV and condlyloma acuminatum.
For pharmaceutical compositions, the immunogenic peptides of the invention are administered to an individual already suffering from cancer or infected with the virus of interest. Those in the incubation phase or the acute phase of infection can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate. In therapeutic applications, compositions are administered ~to a patient in an amount sufficient to elicit an effective CTL response to the virus or tumor antigen and to cure or at least partially arrest 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 peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 ~g to about 5000 pug of peptide for a 70 kg patient, followed by boosting dosages of from about 1.0 ~.g to about 1000 ~g of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL
activity in the patient's blood. It must be kept in mind that the peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.
For therapeutic use, administration should begin at the first sign of viral infection or the detection or surgical removal of tumors or shortly after diagnosis in the case of acute infection. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In chronic infection, loading doses followed by boosting doses may be required.
Treatment of an infected individual with the compositions of the invention may hasten resolution of the infection in acutely infected individuals. For those individuals susceptible (or predisposed) to developing chronic infection the compositions are particularly useful in methods for preventing the evolution from acute to chronic infection.
Where the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.
The peptide compositions can also be used for the treatment of chronic infection and to stimulate the immune system to eliminate virus-infected cells in carriers.
It is important to provide an amount of immuno-potentiating peptide in a formulation and mode of administration sufficient to effectively stimulate a cytotoxic T cell response.
Thus, for treatment of chronic infection, a representative dose is in the range of about 1.0 ~g to about 5000 ~cg, preferably about S ~cg to 1000 ~cg for a 70 kg patient per dose.

Immunizing doses followed by boosting doses at established intervals, e.g., from one to four weeks, may be required, possibly for a prolonged period of time to effectively immunize an individual. In the case of chronic infection, administration should continue until at least clinical symptoms or laboratory tests indicate that the viral infection has been 5 eliminated or substantially abated and for a period thereafter.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for 10 parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A
variety of aqueous carriers may be used, e.g., water, buffered water, 0.81 saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be 15 packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, 20 calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The concentration of CTL stimulatory peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.19b, usually at or at least about 2 ~ to as much as 20 °~ to 50 °& or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
The peptides of the invention may also be administered via liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the half life of the peptide composition.
Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e. g. , liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophvs.
Bioeng.
9:467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.
For targeting to the immune cells, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, j~gl ~]j~, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95 % of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25 %-75 % .
For aerosol administration, the immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are 0.01 %-20% by weight, preferably 1 %-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, pahnitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1 °~-20% by weight of the composition, preferably 0.25-5 ~ . The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
In another aspect the present invention is directed to vaccines which contain as an active ingredient an immunogenically effective amount of an immunogenic peptide as described herein. The peptides) may be introduced into a host, including humans, linked to its own carrier or as a homopolymer or heteropolymer of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the virus or tumor cells.
Useful carriers are well known in the art, and include, e.g., thyrogIobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. And, as mentioned above, CTL responses can be primed by conjugating peptides of the invention to lipids, such as P3CSS. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the desired antigen, and the host becomes at least partially immune to later infection, or resistant to developing chronic infection.
Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of viral infection or cancer to elicit an immune response against the antigen and thus enhance the patient's own immune response capabilities. Such an amount is defined to be an "immunogenically effective dose." In this use, the precise amounts again depend on the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. , but generally range from about 1.0 p,g to about 5000 ~,g per 70 kilogram patient, more commonly from about 10 ~g to about 500 ~g mg per 70 kg of body weight.

In some instances it may be desirable to combine the peptide vaccines of the invention with vaccines which induce neutralizing antibody responses to the virus of interest, particularly to viral envelope antigens.
For therapeutic or immunization purposes, nucleic acids encoding one or more of the peptides of the invention can also be admisitered to the patient: A
number of methods are conveniently used to deliver the nucleic acids to the patient. For instance, the nulceic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et. al., Science 247: 1465-1468 (1990) as well as U.S.
Patent Nos.
5,580,859 and 5,589,466. The nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles. The nucleci acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for instance, in WO 96/ 18372; WO 93/24640; Mannino and Gould-Fogerite ( 1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No. 5,279,833; WO 91/06309; and Felgner et al. ( 1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414. The peptides of the invention can also be expressed by attenuated viral hosts, such as vaccinia or fowlpox.
This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g.,U.S. Patent No.
4,722,848, incorporated herein by reference. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)) which is incorporated herein by reference. A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., ~~, vectors and the like, will be apparent to those skilled in the art from the description herein.
A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding multiple epitopes of the invention. To create a DNA sequence encoding the selected CTL epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes are reverse translated. A
human codon usage table is used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences are directly adjoined, creating a continuous polypeptide sequence. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequence that could be reverse translated and included in the minigene sequence include: helper T lymphocyte epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention signal. In addition, MHC
presentation of CTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.
The minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. he ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are included in the vector to ensure expression in the target cells. Several vector elements are required: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance).
Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, 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 immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA
stabilization sequences can also be considered for increasing minigene expression. It has recently been proposed that immunostimulatory sequences (ISSs or CpGs) play a role in the immunogenicity of DNA vaccines. These sequences could be included in the vector, outside the minigene coding sequence, if found to enhance immunogenicity.
In some embodiments, a bicistronic expression vector, to allow production of the minigene-encoded epitopes and a second protein included to enhance or decrease immunogenicity can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e. g. LeIF) or costimulatory molecules.
Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment 5 different than the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction.
In contrast to CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-(3) may be beneficial in certain diseases.
Once an expression vector is selected, the minigene is cloned into the 10 polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis.
Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell 15 bank.
Therapeutic quantities of plasmid DNA are produced by fermentation in E.
coli, followed by purification. Aliquots from the working cell bank are used to inoculate fermentation medium (such as Terrific Broth), and grown to saturation in shaker flasks or a bioreactor according to well known techniques. Plasmid DNA can be purified using 20 standard bioseparation technologies such as solid phase anion-exchange resins supplied by Quiagen. If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile 25 phosphate-buffer saline (PBS). A variety of methods have been described, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and MHC class I presentation of minigene-encoded CTL epitopes. The plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL
chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 labeled and used as target cells for epitope-specific CTL lines. Cytolysis, detected by SICr release, indicates production of MHC presentation of minigene-encoded CTL epitopes.
In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-compiexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested. These effector cells (CTLs) are assayed for cytolysis of peptide-loaded, chromium-51 labeled target cells using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.
Antigenic peptides may be used to elicit CTL exex vivo, as well. The resulting CTL, can be used to treat chronic infections (viral or bacterial) or tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a peptide vaccine approach of therapy. ExEx vivo CTL responses to a particular pathogen (infectious agent or tumor antigen) are induced by incubating in tissue culture the patient's CTL
precursor cells (CTLp) together with a source of antigen-presenting cells (APC) and the appropriate immunogenic peptide. After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (an infected cell or a tumor cell).
The peptides may also find use as diagnostic reagents. For example, a peptide of the invention may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, the peptides may also be used to predict which individuals will be at substantial risk for developing chronic infection.
The following example is offered by way of illustration, not by way of limitation.
S ~~le 1 Class 1 antigen isolation was carried out as described in the related applications, noted above. Naturally processed peptides were then isolated and sequenced as described there. An allele-specific motif and algorithms were determined and quantitative binding assays were carried out.
Using the motifs identified above for various HLA alleles, amino acid sequences from a number of antigens were analyzed for the presence of these motifs.
Tables 3- ** provide the results of these searches.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in 1S the art and are encompassed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference.
Table 3 Se eace Anti en Molecule FTFSPTYKAFLSK HBV POL

GTLPQEHIVLKLK HBV POL

GTLPQEHIVLKIK HBV POL

LWSYVNTNMGLK HBV POL

STTDLEAYFKDCLFKHBV X

LWSYVNVNMGLK HBV NUC

STSSCLHQSAVRK HBV POL

TTVNAHQILPKVLHKHBV X

RTPARVTGGVFLVDKHBV POL

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MYVGDLCGSVF HCV

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KVYLAWVPAHK HIV

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IVASCDKCQLK HIV

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DVKQLTEAVQK HIV

AWIQDNSDIK HIV

WTYQIYQBPFK HIV

VTVYYGVPVWK HIV

LTEDRWNKPQK HIV

ATDIQTKELQK HIV

TKEL K ITK HIV

Sw ence Anti en Molecule WTVQPIVLPEK HIV

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S~ eace Aati en Molwculs 'I

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HSATGFKQSSK Leukemia 3A2 CMI

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Table 6 AA SEQUENCE SOURCE

9 GLNKIVRM1' HIV GAG

9 TLWKAGILY HBV adr 9 ILRGTSFVY HBV adr 9 NTSSSPQPK p53 311 9 NVKIPVAIK c-ERB2 745 TLGFGAYMSK HCV LORE

10 GTRVRAMAfY p53 154 10 EAYSPVSTSK HBV adw 9 QiTKIQNFR HIV POL

9 NTfGLILTR HIV ENV

9 FLWEWASVR HBV adr 9 RTPSPRRRR HBV adr 9 SLARGNQGR HBV adr 10 KTYQGSYGFR p53 101 9 WMCLRRFII HBV ayw 9 WMCLRRFII HBV ayw 8 ETAYFLLK HIV con NUC;XNUCFUS

WO 99/45954 ,~4 PCTNS98/05039 AA SEQUENCE SOURCE

9 ASCVTACPY c-ErbB2 293 9 VMAGVGSPY c-ErbB2 773 9 ASPLDSTFY c-ErbB2 997 9 FSPAFDNLY c-ErbB2 1213 9 RWGLLLALL c-ErbH2 8 9 TYLPTNASL c-ErbH2 63 9 CYGLGMEHL c-ErbB2 342 9 AYSLTLQGL c-ErbB2 440 9 PYVSRLLGI c-ErbB2 780 9 KWMALESIL c-ErbB2 887 9 RFTHQSDVW c-ErbB2 898 9 VWSYGVTVW c-Erb82 905 9 SYGVTVWEL c-ErbH2 907 9 VYMIMVKCW c-ErbB2 951 9 RFRELVSEF c-ErbH2 968 RGTQLFEDNYc-ErbB2 103 10 ESMPNPEGRYc-&bB2 280 10 CMQIAKGMSYc-ErbB2 826 10 PASPLDSTFYc-ErbB2 996 10 FSPAFDNLYYc-ErbB2 1213 10 PSQKTYQGSYp53 98 10 VGSDCTTIHYp53 225 10 LYISAWPDSLc-ErbB2 410 WO 99/45954 ~5 PCT/US98/05039 AA SEQUENCE SOURCE

SYGVTVWELM c-ErbB2 10 VYMIMVKCWM c-ErbH2 10 EYLVPQQGFF c-Erb82 10 RYSEDPTVPL c-ErbB2 !0 EYLVSFGVWI HBV NUC

9 VYNFATCGI LCMV glyco 9 GYCLTKWMI LCMV glyco 9 MFEALPHII LCMV glyco 9 IFALISFLL LCMV glyco 9 LFKTTVNSL LCMV glyco 9 LYTVKYPNL LCMV nucleo 9 PYIACRTSI LCMV nucleo 10 GYCLTKWMIL LCMV giyco 10 AYLVSIFLHL LCMV glyco 9 VWKTWGQYW gp100 152 9 TWGQYWQFL gp100 135 9 RYGSFSVTL gp100 479 9 LMAVVLASL gp100 606 9 HWLRLPRIF gp100 636 AA SEQUENCE SOURCE

YMIMVKCWMI c-ErbH2 10 HFLRNQPLTF gp100 231 9 KLRKPKHKK P. falciparum CSP

9 KILSVFFLA P. falciparum 9 ALFFIIFNK P. falciparum 9 GTGSGVSSK P. falciparum 9 VLYNTEKGR P. falciparum 9 KYKLATSVL P. falciparum 9 PSENERGYY P. falciparum LSAI

9 FLKENKLNK P. falciparum 9 GVSENIFLK P. falciparum 9 ILVNLLIFH P. falciparum 9 KSLYDEHIK P. falcipsrum LSAI

AA SEQUENCE SOURCE

9 LLIFHINGK P. falciparum 9 QSSLPQDNR P. falcipanim 9 QTNFKSLLR P. falciparum 9 RINEEKHEK P. falciparum 9 SLYDEHaQC P. falciparum LSAI

9 VLAEDLYGR P. falcipa~um 9 VLSHNSYEK P. falciparum 6(1 9 FYFILVNLL P. falciparum 9 YYIPHQSSL P. falciparum 9 PSDGKCNLY P. falciparum TRAP

9 LACAGLAYK P. falciparum TRAP

9 LLACAGLAY P. falciparum TRAP

9 LSTNLPYGR P. falciparum TRAP

9 QGINVAFNR P. falciparum TRAP

9 RGDNFAVEK P. falciparum TRAP

9 RSRKRE1LH P. falciparum TRAP

9 SLLSTNLPY P. falciparum TRAP

9 KYLVIVFLI P. falciparum TRAP

9 PYAGEPAPF P. falciparum TRAP

AA SEQUENCE SOURCE

VTCGNGIQVRP. falciparum CSP

10 GTGSGVSSKKP. falcipatum 10 LALFFIIFNKP. falciparum 10 FQDEENIGIYP. falciparum 1.SA1 10 FILVNLLIFHP. falciparum LSAI

10 HVLSHNSYEKP. falciparum 10 KSLYDEHIKKP. falcipatvm 10 ALLACAGLAYP. falciparum TRAP

IO IIRLHSDASKP. falciparum TRAP

10 LLACAGLAYKP. falcipatum TRAP

10 RLHSDASKNKP. falcipatum TRAP

9 ILGFVFTLT-NH2Flu Matru f0 KGILGFVFTL-Flu Matrix P/D

P/D

P/D

11 KQVPLRPMTYK940.03 N-terminal extension 9 KLYEIVAKV A2.1 consensus 9 KLAEYVAKV A2.1 consensus 9 KLAEIVYKV AZ.1 cor>Serotts 9 KVFEYLINK A3.2 consensus 10 KVFPYALINKA3.2 consensus 9 AVFAYAAAK A3.2 consensus 9 ALEPAIAKY Al consensus AA SEQUENCE SOURCE

9 YLEPAIAKY A1 coiuensus 9 ALEPYIAKY Al consensus 9 YLEQYIEKY AI coruensus 9 GTEKLLAKY A1 consensus 9 ATEPAIAKY A1 consensus 9 ATNYPAIQK All consensus 9 ATNVPAIQK All conxnsus 9 ATNAPYIQK All consensus 9 ATNAVYIQK All consensus 9 ATNAAYAQK A11 co~ensus 9 AVNAAYAQK All conxnsus 9 AVNAPYIQK All consensus 9 AVNAVYIQK A11 consensus 9 PTDPKLINY A1 consensus 9 GTDPKLINY A1 consensus 9 YTDPKLINF A1 consensus 9 FTDPKLINY A1 consensus 9 FTDQAVIKY A1 consensus 9 YTDQAVDCF A1 consensus 9 YTDQKLINF AI consensus 9 STNPKPQKK HCV-core 11 STNPKPQKKNKHCV-core 9 SFFPEI'TYIself pcpade of P815 analog; Y2 to F, 9 ATDPNFLLY A 1 consensus 9 ATDKNFLLY A1 consensus 9 ALMEKIYQV A2.1 cortiensus peptide 9 ALSEKIYQV A2.1 consensus peptide 9 AVYDPIIQK A3.2 consensus peptide 9 AVYDKI1QK A3.2 co~ensus peptide 9 AVMNPMIQK All consetuus peptide WO 99/45954 PC'T/US98/05039 AA SEQUENCE SOURCE

9 AVMNEMIQK All consensus peptidc 9 AYMDMVNSF A24 consensus peptide 9 AYIDNVNSF A24 consensus peptide 9 KLAAAAAAK A3.2/AII
poly-A
analog 9 DVFRDPALK Aw68 endogetaus 9 GYKDGNEYI Lm listeriolysin Y

9 RYLRDQQLL HIV env 9 MYRPDAIQL P. Yoelii 10 NYSPNGNTNL P. Yoelii 9 KFNPMKTHI Kd consensus peptide 9 AMIKNLDFI Db consensus 9 AM1KNLYFI Db consetuus analog analog 9 QYDDAVYKL Cw4 consensus 9 IFEANGNLI Flu HA 240248 AA SEQUENCE SOURCE

9 SYIPSAEKI P. bergaii 9 KYQAVTTTL Tumour P198 MYPHFMPTNLMCMV pp89 9 AYPNVSAKI Lm listeriolysin 9 AYTGGKINI Lm listeriolysin 9 RAKWNNTLK HIV env 370 9 MAVFIHNFK HIV po1909 9 TAGILELLK HPV 6b El 9 RAALLGKFK HPV 6b El 9 CATMCRHYK HPV 6b El 9 TAACSHEGK Flu HA-1 9 NANANSAVK P. fal csp 9 GAFKVPGVK LCMV glyco 9 NMLES1LQC LCMV m~c 9 WMILAAELK LCMV glyco 9 EMNLPGRWK H1V pot 107 9 GSTHVS~YPKHBV POL 398 FUS

9 ASQIYAGIK HIV pol 438 9 ASCDKCQLK HIV po1769 9 MSLAADLEK LCMV nuc VSSKNLMEK Mel. tyro 25~

AA SEQUENCE SOURCE

9 LSTNLPYGK P. fal ssp2 9 STDHIPILY A1 Nat.
Processed 9 STAPPAHGV Breast mucin 9 LMAVVLASL yp100 9 WSQKRSFVY gp100 9 PLDCVLYRY gp100 PSSVGSRSEY gp100 9 YTAVVPLVY Hu ) chain Table 7 AA SEQUENCE SOURCE

El 11 El 358 El 14 El 77 El 101 9 TTRQTVIEH HPV 6b/I I
El 341 9 KLIEPLSLY HPV 6b/I I
El 254 El 462 9 fTVYAEPPK CEA 316 El 396 El 339 El 439 10 P'fISPLNTSYCEA 240 El 254 AA SEQUENCE SOURCE

!0 ATPGPAYSGRCEA 89 IO 10 QFLRHQNIEFHPV 6b/I I
El 445 10 TFTFPNPFPFHPV 6b/11 El 586 9 RVDCTPLMY Prost.Ca PSM

9 LLSLYGIHK Prost.Ca PAP

9 SIVLPFDCR Prost.Ca PSM

IS 9 KSLYESWTK Prost.Ca PSM

9 SMKHPQEMK Prost.Ca PSM

9 SLYESWTKK Prost.Ca PSM

9 YSLVHNLTK Prost.Ca PSM

9 HLTELYFEK Prost.Ca PAP

ZO 9 RATQIPSYK Prost.Ca PAP

9 ASGRARYTK Prost.Ca PSM
53l 9 SLYGIHKQK Prost.Ca PAP

9 RDYAWLRK Prost.Ca PSM

9 SSHDLMLLR Prost.Ca PSA

25 9 GAAPLILSR Prost.Ca PSA

9 KIVIARYGK Prost.Ca PSM

9 RAAPLLLAR Prost.Ca PAP

9 VVLRKYADK Prost.Ca PSM

9 GLPDRPFYR Prost.Ca PSM

3O 9 WLDRSVLAK Prost.Ca PAP

9 KVFRGNKVK Prost.Ca PSM

9 IVRSFGTLK Prost.Ca PSM

9 KIYSISMKH Prost.Ce PSM

9 RSVLAKELK Prost.Cs PAP

35 9 STNEVTRIY Prost.Ca PSM

9 GFFLLGFLF Prosc.Ca PSM
3l AA SEQUENCE SOURCE

9 LYSDPADYFProst.Ca PSM

9 KYADKIYSIProst.Ca PSM

9 NYARTEDFFProst.Ca PSM

9 AYINADSSIProst.Ca PSM

9 AFTFSP'fYKHBV POL 655 10 NADSSIEGNYProst.Ca PSM

10 GLDSVELAHYProst.Ca PSM

I S 10 RATQIPSYKKProst.Ca PAP

10 LGFLFGWFIICProst.Ca PSM

10 SSIEGNYTLRProst.Ca PSM

10 KSLYESWTKKProst.Ca PSM

10 SLLSLYGIHKProst.Ca PAP

10 FLYNFTQIPHProst.Ca PSM

10 VIYAPSSHNKProst.Ca PSM

10 AVVLRKYADKProst.Ca PSM

10 KSPDEGFEGKProst.Ca PSM

10 IVRSFGTLKKProst.Ca PSM

25 10 RIYNVIGTLRProst.Ca PSM

10 LSLYGIHKQKProst.Ca PAP

10 MSLLKNRFLRProst.Ca PSA

10 ISMKHPQEMKProst.Ca PSM

10 RAVCGGVLVHProst.Ca PSA

10 GSAPPDSSWRProst.Ca PSM

10 SIPVHPIGYYProst.Ca PSM

10 CSGKIVIARYProst.Ca PSM

10 ETYELVEKFYProst.Ca PSM

10 RLLQERGVAYProst.Ca PSM

3S 10 FYDPMFKYHLProst.Ca PSM

10 TYSVSFDSLFProst.Ca PSM

AA SEQUENCE SOURCE

10 LYNFTQIPHLProst.Ca PSM

10 GWRPRRTILFProst.Ca PSM

9 FIFHKFQTK HTLV-I tax 9 FLTNVPYKR NTLV-I tax lU 9 TTWDPIDGR HTLV-I tax 9 SALQFLIPR HTLV-I tax 9 LSFPDPGLR HTLV-I tax 9 QSSSFIFHK HTLV-1 tax 9 GLCSARLHR HTLV-1 tax I S 9 RLPSFPTQR HTLV-I tax 9 AMRKYSPFR HTLV-1 tax 9 ISGGLCSAR HTLV-I tax 9 ALFTAQEAK HPV 16 El 9 GVSFSELVR HPV 16 El 9 KAAMLAKFK HPV 16 El 9 LTNILNVLK HPV 16 El 9 LVRPFKSNK HPV 16 El ZS 9 MSFLTALKR HPV 16 El 9 NSNASAFLK HPV 16 El 9 QMSMSQWIK HPV 16 El 9 SMSQW1KYR HPV 16 El 9 TAAALYWYK HPV 16 El 9 WLLLVRYK HPV 16 El 35 9 CATMCKHYR HPV 18 El 9 FTfFLGALK HPV IH E1 $7 AA SEQUENCE SOURCE

9 GVLILALLR HPV 18 El 9 LILALLRYK HPV 18 El S 9 NMSQWiRFR HPV 18 El 9 WTYFDTYMR HPV 18 El IO 9 fIKNFDIPK GCDFP-13 9 TLWKAGILY HHV pol 150 11 HTLWKAGiLYKHBV POL 149 9 WMNSTGFTK HCV consensus 9 RVLED(iVNYHCV consensus 9 RLLAPTfAY HCV consensus 9 GVLAALAAY HCV contcnsus 3S 9 RVCEKMALY HCV consensus WO 99/45954 8g PCT/US98/05039 PEPTIDE AA UENCE

1235.01 10 AVFDRKSDAK

S 26.0149 9 CALRFTSAR

26.0153 9 SSAGPCALR

F104.02 9 SLTPPHSAK

F105.01 9 AIF SSMTK

F105.02 9 G>F SSMTK

F105.03 9 AAF SSMTK

F105.04 9 AIA SSMTK

FI05.05 9 AIFASSMTK

F105.06 9 AIF ASMTK

F105.07 9 AIF SAMTK

IS F105.08 9 AIF SSATK

F105.09 9 AIF SSMAK

FI05.10 9 AIF SSMTA

F105.11 9 FIF SSMTK

F105.12 9 SIF SSMTK

F105.14 9 ANF SSMTK

F105.16 9 AIF CSMTK

FI05.17 9 AIF SSMTR

F105.19 9 AIF SSMTY

F105.20 9 AIL SSMTR

25 FI05.21 9 AIF RSMTR

F105.24 10 PAIF SSMTK

F105.25 10 AIF SSMTKI

27.0103 9 AIILH K

27.0104 9 YGFRLGFLH

27.0108 9 SSCMGGMNR

27.0235 10 TCTYSPALNK

27.0239 10 NSSCMGGMNR

27.0240 10 SSCMGGMNRR

27.0250 10 KSKKG STSR

3S 27.0232 10 TSRHKKLMFK

28.0062 8 FMFSP'fYK

28.0063 8 FVFSPTYK

8.0066 8 MXXK

PEPTIDE AA SE UENCE

28.0322 9 SMICSWRR

28.0323 9 SV1CSWRR

28.0324 9 KVGNFTGLK

28.0325 9 KVGNFTGLR

28.0326 9 VVFFS FSR

28.0327 9 SVNRPIDWK

28.0328 9 TLWKAGILK

28.0329 9 TLWKAGILR

28.0330 9 TMWKAGILY

1~ 28.0331 9 TVWKAGIL.Y

28.0332 9 RMYLHTLWK

28.0333 9 RVYLHTLWK

28.0334 9 AMTFSPTYK

28.0335 9 AVTFSPTYK

I S 28.0336 9 SVVRRAFPR

28.0337 9 SWRRAFPK

28.0338 9 ISEYRHYXY

28.0339 9 GTGXNGWFY

28.0340 9 ASXHLTELY

28.0341 9 ASXDKX LK

28.0371 9 RVXEKMALY

28.0372 9 XTGWFMVEA

28.0374 9 HISXLTFGR

28.0375 9 AVXTRGVAK

28.0377 9 HLIFXHSKK

28.0378 9 HTMLXMXXK

28.0381 9 RLKAD~IC

28.0383 9 TLFXASDAK

28.0384 9 ALLRYKXGK

30 28.0387 9 ATMXRHYKR

28.0388 9 XATMXRHYK

28.0390 9 ATMXKHYRR

28.0391 9 LLAXAGLAY

28.0392 9 LAXAGLAYK

35 28.0393 9 SIVLPFDXR

28.0394 9 MXWWAGIK

2 .0628 10 MFTFS

PEPTIDE AA S UENCE

28.0629 10 VFTFSPTYK

28.0630 10 TMWKAGILYK

28.0631 10 TVWKAGILYK

28.0632 10 VMGGVFLVDK

S 28.0633 10 VVGGVFLVDK

28.0635 10 SVLPETTVVR

28.0638 10 HTLWKAGI1..K

28.0640 10 HMLWKAGILY

28.0395 9 SADtSWRR

IO 28.0644 10 GTFNSVVLSR

28.0645 10 YMFDVVLGAK

28.0646 10 MMWYWGPSLK

28.0647 10 MMWYWGPSLR

28.0665 10 IVGG WEXEK

I S 28.0667 10 IILEXVYXK

28.0668 10 SIPHAAXHK

28.0670 10 IVXPIXS K

28.0671 10 LIRXLRX K

28.0672 10 XTYSPALNK

ZO 28.0675 10 TVXAGGXAR

28.0676 10 HISXLTFGR

28.0677 10 XVNXS FLR

28.0678 10 LIFXHSKKK

28.0679 10 FVLGGXRHK

ZS 28.0713 10 TSADCSWRR

28.0714 10 HLIFXHSKKK

28.0715 10 LLIRXWX K

28.0716 10 GIVXPIXS K

28.0717 10 LLIRXLRX K

3O 28.0718 10 SLE RSLHXK

28.0720 10 RIVGGWEXEK

28.0721 10 DIILEXVYXK

28.0722 10 XVYXK LLR

28.0723 10 RAVXGGVLVH

3S 28.0725 10 LTAAHXIRNK

28.0728 10 KAAXWNAGIK

28.07 0 W P R

PEPT'lDE AA SE UENCE
_", j , 28.0731 10 l LLGiWGXSGK

28.0732 10 TTLFXASDAK

28.0734 10 RTVXAGGXAR

28.0736 10 GT RXEKXSK

S 28.0737 !0 LV NANPDXK

28.0738 10 VTXGNGI VR

28.0739 10 DXATMXRHYK

28.0740 10 GLAXH LXAR

28.0741 10 ALLAXAGLAY

1~ 28.0742 10 LLAXAGLAYK

28.0743 10 XVARXPSGVK

28.0745 10 LVEIXTEMEK

28.0746 10 LLNWXM IAK

28.0824 11 HMLWKAGILYK

15 28.0825 11 HVLWKAGILYK

28.0826 II SMLPETTVVRR

28.0827 11 SVLPETTWRR

28.0828 11 GMDNSVVLSRK

28.0829 11 GVDNSVVLSRK

28.0830 11 GTFNSWLSRK

28.0369 9 GLAXH LXA

1259.02 9 DTVDTVLEK

1259.10 9 PVTIGECPK

1259.14 10 FTAVGKEFNK

25 1259.16 11 RTLDFHDSNVK

1259.21 I KTRPII SPLTK

1239.26 11 GTHPSSSAGLK

1259.28 11 ILWILDRLFFK

1259.29 9 WILDRLFFK

1259.30 11 CIYRRFKYGLK

1259.31 9 KSMREEYRK

1239.33 9 YI MCTELIC

1259.37 10 MVMELVRMIK

1239.38 9 VMELVRMIK

1259.41 11 LIRPNENPAHK

26.0023 8 VSFGVWIR

6.0024 8 V I

WO 99/45954 PC'T/US98/05039 PEPTIDE AA S UENCE

26.0026 8 ASFCGSPY

26.0035 9 TSPYELSLY

26.0036 9 TSIPFLHEY

26.0041 9 FNDPGPGTY

S 26.0045 9 YVDLGALRY

26.0051 9 DADRSFIEY

26.0055 9 NMDKAVKLY

26.0056 9 TTDNFYRNY

26.0058 9 HSAEAL KY

IO 26.0059 9 LTAGLDFAY

26.0061 9 LTYKYN FY

26.0062 9 CSNDKSLVY

26.0063 9 RSARASSRY

26.0065 9 ASADKPYSY

I S 26.0067 9 STTAGPNEY

26.0069 9 LSGNGHFHY

26.0073 9 NTFV ANLY

26.0074 9 GTATYLPPY

26.0081 9 RLDAFR TY

2~ 26.0082 9 KAEVHTFYY

26.0083 9 VAEGDTVIY

26.0084 9 LTEIDIRDY

26.0085 9 HTEFE VY

26.0086 9 VSDGGPNLY

2S 26.0092 9 IIED YNRY

26.0093 9 FLD WVVTEY

26.0095 9 FVEDPNGKY

26.0096 9 ISDESYRVY

26.0156 9 YLAEADLSY

26.0197 9 ALLAVGATK

26.0198 9 ALNFPG K

26.0199 9 AVGATKVPR

26.0203 9 FSVSVS LR

26.0204 9 GTATLRLVK

3S 26.0205 9 GVSR LRTK

26.0207 9 LIYRRRLMK

6.0211 LVL

PEPTIDE AA UENCE

26.0212 9 SSHWLRLPR

26.0214 9 TMEVTVYHR

26.0216 9 VLASLIYRR

26.0217 9 VSC GGLPK

26.0218 9 WLASLIYR

26.0227 9 GT CALTRR

26.0251 9 FTIPYWDWR

26.0252 9 GTPEGPLRR

26.0253 9 KSYLE ASR

26.0255 9 LVSLLCRHK
' 26.0256 9 MVPF1PLYR

26.0258 9 TSAGHFPR

26.0259 9 SIFE WLRR

26.0260 9 SLLCRHKRK

1$ 26.0261 9 SSW IVCSR

26.0267 10 NM 1GGVLTY

26.0273 10 RMA NFAMRY

26.0274 10 FTV SLSGY

26.0275 10 TSPYELSLY

20 26.0276 10 SSNAILSLSY

26.0280 10 TS PWWPADY

26.0284 10 VSDVSIIIPY

26.0285 10 ASDA SANKY

26.0286 10 FTETNLAGEY

25 26.0287 10 WDGFEPNGY

26.0291 10 FNDPGPGTYY

26.0296 10 FLD WWTEYY

26.0299 10 AAEFATETAY

26.0309 10 NAEVVLN LY

3~ 26.0311 10 FVDGDSLPEY

26.0316 10 PSEDA VAVY

26.0317 10 MSDNIRTGLY

26.0318 10 ESELREILNY

26.0319 10 CMESVRNGTY

35 26.0320 10 KTENG1TRLY

26.0321 ! LTEIDIRDYY

.0 97 10 L V MAW

WO 99/45954 94 PCT/US9$/05039 PEPTIDE AA SE UENCE

26.0424 10 AWLASL1YR

I
26.0425 10 GALLAVGATK

26.0426 10 GTATLRLVKR

26.0427 10 HTMEVTVYHR

26.0428 10 IALNFPGS K

26.0432 10 LRALDGGNK

26.0433 10 VPLDCVLYR

26.0434 10 SLIYRRRLMK

26.0435 10 SSSHWLRLPR

26.0438 10 TVSC LPK

26.0442 10 WLASLIYRR

26.0466 10 YVKVLHHTLK

26.0473 10 LIGCWYCRRR

26.0474 10 LLIGCWYCRR

1 s 26.0485 10 SSMHNALHIY

26.0504 10 CVSSKNLMEK

26.0510 10 FSSW IVCSR

26.0511 10 GLVSLLCRHK

26.0518 10 YMVPFIPLYR

26.0535 11 GVWIRTPPAYR

26.0339 11 RLVVDFS FSR

26.0545 11 TLPETT'VVRRR

26.0549 11 LLPIFFCLWVY

ZS 26.0550 11 RAFPHCLAFSY

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Table 10 AA SEQUENCE SOURCE

9 YIFATCLGL MAGE 3 l69 ~ 75 9 RLWHYPCTV HCV Env2 bl4 9 RLWHYPCTI HCV Env2 9 FLLLADARI HCV Env2 9 GVWPLLLLL HCV Env2 15 9 GMWPLLLLL HCV Env2 9 LMAVVLASL gp100 606 10 YMIMVKCWMI c-ErbB2 952 25 9 AILSVSSFL P. falciparum 9 GLIMVLSFL P. falcipe<um CSP

9 VLLGGVGLV P. falciparum 9 GLLGNVSTV P. falcipanrm 9 LLGNVSTVL P. falciparum 30 9 VLAGLLGNV P. falcipuum AA SEQUENCE SOURCE

9 KILSVFFLA P. falciparum 9 FL1FFDLFL P. falciparum TRAP

9 LIFFDLFLV P. falciparum TRAP

9 FMKAVCVEV P. falci~tum TRAP

9 LLMDCSGSI P. falciparum TRAP

10 1LSVSSFLFV P. falciparum 10 VLLGGVGLVL P. falciparum 10 GLLGNVSTVL P. falciparum 10 FLIFFDLFLV P. falciparum TRAP

1~ 10 GLALLACAGL P. falciparum TRAP

9 TLMSAMTNL Prost.Ca PAP Il2 9 LLLARAASL Prost.Ca 9 ALDVYNGLL Prost.Ca 10 SLSLGFLFLL Ptrost.Ca 10 FLPSDFFPSV(CONH2)HHc 18-27 10 FLPSDFFPSV-NH2HBc 18-27 g ~,gVpTLT.~ Flu Matrix l0 KG1LGFVFTL-NH2F1u Matrix I FLPSDFFPSVR HBc t8-28 t 9 FLPSDFFPS HBc 18-26 25 9 GILGICVFTI. Flu Matrix arulog 9 FLSKQYLNL HBV polymerase g KLQCVpLHV PSA 166-174 P/D

WO 99/45954 PC'T/US98/05039 AA SEQUENCE SOURCE

P/D

PID

9 IU.YEIVAKV A2.1 consensus 9 1CLAEYVAKV A2.1 consensus S 9 KLAEIVYKV A2.1 consensus 9 TLTSCNTSV HIV gp 120 env. RE
vans. 197 9 ALMEKIYQV A2.1 coruensus peptide 9 ALSEKIYQV A2.1 consensus peptide 9 FLMSYFPSV 941.01 9-mcr analog 1~ 9 FLPSYFPSV 941.01 9-mer analog 10 FLMSDYFPSV 941.01 M2 analog 9 FLYCYFALV Chiron consensus 9 FMYCYFALV Chiron consensus 10 SLVGFGILCV Chiron consensus 1S 10 SLMGCGLFWV Chiron consensus 8 GLLGPLLV HBVadr-ENV

9 AMAKAAAAI A2.1 poly-A

9 FLPSYFPSA analog of 994.02:
chiron comb 9 FAPSYFPSV analog of 994.02:
chiron comb 9 FLPSYFPSS analog of 994.02:
chiron comb 9 FSPSYFPSV analog of 994.02:
chiron comb ?.S I1 EIWEELSVMEV MAGE-1 lI5 AA SEQUENCE SOURCE

8 TLG1YSPI HPV, analog of 1088.01 8 TLGIVXPI HPV, analog of 1088.01 1l VLLDYQGMLPV HBV env 11 CILLLCLIFLL HBV env 9 FLGGSPVCL HBV env 11 TVIEYLVSFGV HBV core 11 TVLEYLVSFGV HBV core 10 FLLAQFTSAI HBV pol 9 GLYSSTVPI HBV pol 1 s 9 GLYSSTAPI HBV pot 9 GLDVLTAKV HIV foam VIN.

9 RILGAVAKV HIV form VIN.

9 LLFGYPVYV HTLV, tax lI-19 9 ALFGYPVYV tax 11-19, SAAS

9 LLFGAPVYV tax I1-19, SAAS

9 LLFGYAVYV tax 11-19, SAAS

9 LLFGYPYAV tax 11-19.
SAAS

25 9 ILTV11.GVL MARTI32-40 10 ILSVSSFLFV Plus. falcip.
CSA-A

9 GLIMVLSFL Plas. falcip.
CSA-A

AA SEQUENCE SOURCE

9 IMVLSFLFL Plas. falcip.
CSA-A

10 FLIFFDLFLV Plas. falcip.
TRAP-A

9 FMKAVCVEV Plas. falcip.
TRAP-A

9 1MPGQEAGL gp100 9 GLGQVPLiV gp100 9 LMAVVLASL gp100 9 RLMKQDFSV gp100 9 HLAVIGALL gp100 9 LLAVGATKV gp100 lO 9 MLGTHTMEV gp100 10 LLDGTATLRL &Pl~

10 VLYRYGSFSV gp100 10 VLPSPACQLV gp100 10 SLADTNSLAV gp100 IS 10 VLMAVVLASL gp100 10 LMAVVLASLI gp100 10 RLDCWRGGQV gp100 10 AMLGTHTMEV gp100 10 ALDGGNKHFL gp100 Z.O 9 YLEPGPVTA gp100 9 KTWGQYWQV gp100 9 ITDQVPFSV gp100 25 9 YLEPGPVTA gp100 10 LLDGTATLRL Bp100 10 VLYRYGSPSV gp100 10 ALDGGNKHFL Sp100 9 GILTVIL.GV MART131-39 3O 9 YMNGTMSQV Human Tyroainase 9 MLLAVLYBL Human Tyrosinase 9 LLWSFQTSA Human Tyrosinase AA SEQUENCE SOURCE

9 YLTLAKHTI Human Tyrosinase 9 FLPWHRLFL Human Tyrosinase 9 FLLRWEQEI Human Tyrosinase 9 RIWSWLLGA Human Tyrosinase 9 LLGAAMVGA Human Tyrosinase 9 AMVGAVLTA Human Tyrosinase 9 VLTALLAGL Human Tyrosinase 9 ALLAGLVSL Human Tyrosinase 9 LLAGLVSLL Human Tyrosinase 1 O 10 BLLWSFQTSA Human Tyrosinase 10 WMHYYVSMDA Human Tyrosinase 10 FLPWHRLFLL Human Tyrosinase 10 WLLGAAMVGA Human Tyrosinase 10 AMVGAVLTAL Human Tyrosinase 15 10 VLTALLAGLV Human Tyrosinase 10 TALLAGLVSL Human Tyrosinase 10 ALLAGLVSLL Human Tyrosinase 9 NLTDALLQV P. falciparum 9 SAWENVKNV P. falciparum 2O 10 FLIFFDLFLV P. falciparum 9 NLNDNAIHL P. falciparum 10 YLLMDCSGSI P. falciparum 9 TLQDVSLEV conaols Table I1 AA SEQUENCE SOURCE

El El El El IO 9 GIYDALFDI PSMAg El EI

10 NLLDGNPMSIHPV 6b/I
I EI

EI

10 TL1KCPPLLVHPV 6b11I
El ZO 10 MVFELANSIVPSMAg El 10 WLCAGALVLAPSMAg ~

AA SEQUENCE SOURCE

to tax tax tax tax tax lO 9 LLLVLCLQL GCDFP-15 IS

El EI

EI

EI

EI

EI

El AA SEQUENCE SOURCE

El El Ei B E I

El EI

10 FIQGAVISFVHPVl8 El El El El 9 GLACHQLCA HER2/neu 9 1LDEAYVMA HER2/neu 9 SIISAVVGI HER2lneu 2,O 9 WLGWFGI HER2/neu 9 YMIMVKCWM HER2/neu 10 ALCRWGLLLAHER2/neu 10 QLFEDNYALAHER2/neu AA SEQUENCE SOURCE

consensus 1 p 9 AIIDPLIYA MSH 291 1 S 9 SLYNTVATL HIV p17/SB

Table 12 PEPTIDE NO. PEPTIDE LENGTHS UENCE

1237.01 9 FLWG~ALV

1237.02 9 FLWGPNALV

1237.03 9 FLWGPHALV

1237.04 9 FLWGPKALV

1237.05 9 FLWGPFALV

26.0158 9 AVIGALLAV

26.0172 9 LLHLAVIGA

1~ 26.0186 9 SLADTNSLA

26.0192 9 VMGTTLAEM

26.0240 9 LLAVLYCLL

26.0383 10 FLRN PLTFA

26.0390 10 HLAVIGALLA

15 26.0395 10 LAVIGALLAV

26.0418 10 TLAEMSTPEA

26.0423 10 YLAEADLSYT

26.0497 10 MLLAVLYCLL

1183.10 10 VLYRYGSFSV

2~ 27.0007 9 ILSSLGLPV

27.0012 9 LLFLGWFL

27.0019 9 GLYGA YDV

27.0022 9 FVVALIPLV

27.0023 9 GLMTAVYLV

25 27.0027 9 ALVLLMLPV

27.0028 9 ILLSIARV V

27.0029 9 SLYFGGICV

27,0030 9 LIPCMDW

27.0031 ~~ 9 V STY(.~L

27.0032 . AIHNWHAI

27,0034 9 GLHGVGVSV

27.0035 9 GLVDFVKHI

27.0036 9 LLFRFMRPL

27.0038 9 LMLPGMNGI

35 27.0043 9 TVLRFVPPL

27,0044 9 MLGNAPSVV
' 27,0050 9 YLDLALMSV

7. PPV

PEPTIDE NO. PEPTIDE LENGTHSE UENCE

27.0082 9 FLLPDA SI

27.0083 9 MTYAAPLFV

27.0088 9 LLPLGYPFV

27,ppgg 9 GLYYLTTEV

S 27.0090 9 MALLRLPLV

27.0091 9 RLPLVLPAV

27.0093 9 RMFAANLGV

27.0095 9 RLLDDTPEV

27.0096 9 YLYVHSPAL

1~ 27.0100 4 GLYLS IAV

27.0101 9 YLS IAVLL

27.0102 9 SLAGFVRML

27.0137 10 ATYDKGILTV

27.0146 10 KIFMLVTAVV

IS 27.0151 10 FLLADERVRV

27.0153 10 MLATDLSLRV

27.0154 10 VGWEV

27.0161 10 FLMPVEDVFI

27.0165 10 RMSRVTTFTV

2O 27.0168 10 LALVLLMLPV

27.0169 10 ALVLLMLPVV

27.0170 10 GIVSGILLSI

27.0171 10 SLYFGGICV1 27.0173 10 LIPCMDVVL

2S 27.0181 10 LLFRFMRPLI

27.0183 10 VLLEDGGVEV

27.0184 10 AMPAYNWMTV

27.0186 10 GLAGTVLRFV

27.0188 10 VLIAFGRFPI

27.0189 10 FLTCDANLAV

27.0197 l0 AIAWGAWGEV

27.0204 10 LLLETSVVEAI

27.0217 IO RMPEAAPPVA

27.0223 10 WMAE1T1.GRV

3S 27.0226 10 AMALLRLPLV

27.0229 10 FMSLAGFVRM

27.0 I i L V YV

PEPTIDE PEPTIDE LENGTHSE UENCE
NO.

27.0268 11 GILGFVFTLTV

27.0269 11 VLDVGDAYFSV

27.0271 11 KIWEELSMLEV

27.0272 11 STLVEVTLGEV

$ 27.0273 11 GLAP HLIRV

27.0274 11 HLIRVEGNLRV

27.0005 9 YLLALRYLA
~

27.0013 9 GLYR WALA

27.0017 9 LLW DPVPA

27.0040 9 ALLSDWLPA

27.0045 9 WLL1DTSNA

27.0046 9 MLASTLTDA

27.0081 9 YLSEGDMAA

27.0094 9 LLACAVIHA

I S 2?.0144 10 LLCCSGVATA

27.0191 10 LLATVFKLTA

27.0192 10 KLTADGVLTA

27.0195 10 GLGGLGLFFA

28.0064 8 TLG1VXPI

28.0065 8 ALGTTXYA

28.0293 9 FLLTRILTV

28.0294 9 ALMPLYACV

28.0293 9 LLA FTSAV

28.0296 9 LLPFV WFV

28.0297 9 FLLA FTSV

28.0298 9 KLHLYSHPV

28.0299 9 KLFLYSHPI

28.0300 9 LLSSNLSWV

28.0301 9 FLLSLGIHV

28.0302 9 MMWYWGPSV

28.0303 9 VL AGFFLV

28.0304 9 PLLPIFFCV

28.0305 9 FLLPIFFCL

28.0306 9 VLLDY MV

28.0307 9 YMDDVVLGV

28.0308 9 YMFDVVLGA

X8.0309 9 GL ~WSPOV

PEPTIDE NO. PEPTIDE SE UENCE
LENGTH

28.0342 9 YMIMVKXWM

28.0343 9 YIFATXLGL

28.0345 9 SLHXKPEEA

28.0346 9 ALGLVXV A

S 28.0348 9 LLMDXSGSI

28.0349 9 FAFRDLXIV

28.0352 9 GTLG1VXP1 28.0353 9 TLGIVXPIX

28.0354 9 LLWFHISXL

1 ~ 28.0355 9 KLTPLXVTL

28.0356 9 ALVEDITEM

28.0357 9 LTFGWXFKL

28.0359 9 KL XVDLHV

28.0360 9 FMKAVXVEV

1 S 28.0361 9 LL YXLYL

28.0362 9 XLYLHI SL

28.0363 9 SLAXSWGMV

28.0364 9 ILYAHI XL

28.0365 9 KLLSKLLXV

2~ 28.0366 9 PLLPff FXL

28.0367 9 TLIKXPPLL

28.0368 9 ALMPLYAXI

28.0370 9 XILESLFRA

28.0609 10 FLLA FTSAV

2S 28.0610 10 YLHTLWKAGV

28.0611 10 YLFTLWKAGI

28.0612 10 YLLTLWKAGI

28.0613 10 LLFY GMLPV

28.0614 10 LLLY MLPV

3O 28.0615 10 LLVL AGFFV

28.0616 10 ILLLCLIFLV

28.0650 10 ALXRWGLLL

28.0651 10 KLPDLXTEL

28.0652 10 HLY X, 3S 28.0653 10 XILESLFRA

28.0654 10 KL XVDLHV

28.065 10 YIFA X

PEPTIDE NO. PEPTIDE LENGTHSE HENCE

F111.01 9 SLYNTVATL

Fi11.02 9 ALYNTVATL

F111.04 9 SLANTVATL

F111.06 9 SLFNAVATL

S F111.07 9 SLFNLLATL

F111.10 9 SLFNTIAVL

FI11.11 9 SLFNAVAVL

F111.09 9 SLFNTIVVL

Fi11.12 9 SLFNAtAVL

1~ F111.13 9 SLFNTVAVL

F111.14 9 SLFNTVCVI

F111.15 9 SLHNTVATL

F111.17 9 SLHNTVAVL

F111.18 9 SLYATVATL

IS F111.19 9 SLYNAVATL

F111.21 9 SLYNTAATL

F111.22 9 SLYNTIAVL

F111.23 9 SLYNTSATL

F111.25 9 SLYNTVAVL

2~ F111.26 9 SLYNTVATA

F111.27 9 SLYNAIATL

F111.28 9 SLYNLVAVL

Fi 11.29 9 SLFNLLAVL

F111.32 9 SLFNTVVTL

25 F111.34 9 SLYNTVAAL

1039.031 9 MMWYWGPSL

1211.40 10 SLLNATAIAV

FAFRDLCIV

TLGIVCPIC

Table 13 A SEQUENCE SOURCE

A

9 QPRGRRQPI HCV Core 9 SPRGSRPSW HCV Core 9 DPRRRSRNL HCV Core 9 LPGCSFSIF HCV Core WO 99/45954 12g PCT/US98/05039 A SEQUENCE SOURCE

A

A SEQUENCE SOURCE
A

9 HPSDGKCNL P. falciparum S

9 RPRGDNFAV P. falciparum S

9 QPRPRGDNF P. falciparum S

9 LPNDKSDRY P. falciparum S

10 LPRRGPRLGV HCV Core 10 APLGGAARAL HCV Core 10 LPGCSFSIFL HCV Core A SEQUENCE SOURCE

A

A SEQUENCE SOURCE
A

10 IPYSPLSPKV P. falciparum S

10 TPYAGEPAPF P. falciparum S

' A SEQUENCE SOURCE
A

10 ' FPDLESEFQA MAGE2/3 98 9 EPLSLYAHI HPV 6b/11 El 9 PPLLVTSNI HPV 6b/11 9 SPRLDAIKL HPV 6b/11 9 TPKKNCIAI HPV 6b/11 9 FPFDRNGNA HPV 6b/11 10 CPPLLVTSNI HPV 6b/11 10 FPFDRNGNAV HPV 6b/11 WO 99/45954 PCTlUS98/05039 A SEQUENCE SOURCE

A

8 HPvhAGPI HIV con.

GAG

8 GPGvRyPL HIV con.

NEF

8 SPIETVPV HIV con.

POL

8 NPYNTPVF HIV con.

POL

8 LPIQKETW H1V con.

POL

A SEQUENCE SOURCE

A

8 VPRRKaKi HIV con.

POL

8 VpLQLPPI HIV con.

REV

8 VPLAMKLI P. falciparum 8 LPYGRTNL P. falciparum 8 RPRGDNFA P. falciparum 8 IPQQEPNI P. falciparum 8 TPFAGEPA P. falciparum 9 SPINTIAEA HPV 6b EI

9 SPRLDAIKL HPV 6b/11 9 EPLSLYAHI HPV 6b/11 z 9 EPPKIQSGV HPV 6b/11 9 IPFLTKFKL HPV 6b El 9 TPKKNCIAI HPV 6b/11 9 PPLLVTSNI HPV 6b/11 A SEQUENCE SOURCE
A

9 FPFDRNGNA HPV 6b/11 9 GPTLIGANA gp100 74 9 LPDGQVIWV gp100 97 9 VPLAHSSSA gp100 198 9 QPLTFALQL gp100 236 9 DPSGYLAEA gp100 246 9 EPGPVTAQV gp100 282 9 MPTAESTGM gp100 366 9 TPAEV SIV V gp 100 401 9 LPKEACMEI gp100 520 9 LPSPACQLV gp100 545 9 VPLIVGILL gp100 596 9 LPHSSSHWL gp100 630 A SEQUENCE SOURCE
A

9 CPIGENSPL gp100 647 9 SPLLSGQQV gp100 653 9 APLGPQFPF Tyrosinase ~ 9 IPIGTYGQM Tyrosinase 9 TPMFNDINI Tyrosinase 9 LPWHRLFLL Tyrosinase 9 IPYWDWRDA Tyrosinase ' 2 9 SPASFFSSW Tyrosinase 9 LPSSADVEF Tyrosinase 9 SPLTGIADA Tyrosinase 9 DPIFLLHHA Tyrosinase 9 IPLYRNGDF Tyrosinase 9 LPSIPVHPI Prost.Ca PSM

9 IPVHPIGYY Prost.Ca PSM

9 RPFYRHVIY Prost. Ca PSM

9 TPKHNMKAF Prost.Ca PSM

9 FPGIYDALF Prost. Ca PSM

9 RPRWLCAGA Prost.Ca PSM

9 DPLTPGYPA Prost.Ca PSM

WO 99!45954 PCT/US98/05039 A SEQUENCE SOURCE
A

9 RPRRTILFA Prost. Ca PSM

9 LPFDCRDYA Prost. Ca PSM

10 QPIPVHTVPL Prost. Ca PAP

10 HPYKDFIATL Prost.Ca PAP

10 LPGCSPSCPL Prost.Ca PAP

10 LPSWATEDTM Prost.Ca PAP

10 VPLSEDQLLY Prost.Ca PAP

10 FPHPLYDMSL Prost. Ca PSA

10 RPGDDSSHDL Prost. Ca PSA

10 HPQKVTKFML Prost.Ca PSA

10 LPFDCRDYAV Prost.Ca PSM

10 YPNKTHPNYI Prost. Ca PSM

10 SPEFSGMPRI Prost.Ca PSM

10 RPRWLCAGAL Prost.Ca PSM

10 TPKHNMKAFL Prost.Ca PSM

10 RPFYRHVIYA Prost.Ca PSM

9 SPREGPLPA HER2/neu 9 KPDLSYMPI HER2/neu 9 HPPPAFSPA HER2/neu A SEQUENCE SOURCE

A

9 GPLPAARPA HER2/neu 9 APQPHPPPA HER2/neu 9 EPLTPSGAM HER2/neu 9 LPTHDPSPL HER2/neu 9 DPLNNTTPV HER2/neu 9 SPLTSIISA HER2/neu 9 SPKANKEIL HER2/neu 9 LPTNASLSF HER2/neu 9 CPSGVKPDL HER2/neu 9 SPLAPSEGA HER2/neu 9 MPNQAQMRI HER2/neu 9 LPAARPAGA HER2/neu 9 LPQPPICTI HER2/neu 9 SPAFDNLYY HER2/neu A SEQUENCE SOURCE

A

9 TPTAENPEY HER2/neu 9 LPSETDGYV HER2/neu 10 LPTNASLSFL HER2/neu b5 10 CPAEQRASPL HER2Jneu 10 KPCARVCYGL HER2/neu 10 APQPHPPPAF HER2/neu 10 SPGGLRELQL HER2/neu 10 SPLTSIISAV HER2/neu 10 MPNQAQMRIL HER2/neu 70b 10 SPYVSRLLGI HER2/neu 10 HPPPAFSPAF HER2/neu 10 SPREGPLPAA HER2/neu 10 NPHQALLHTA HER2/neu 10 MPYGCLLDHV HER2/neu A SEQUENCE SOURCE

A

10 GPASPLDSTF HER2/neu 9 LPTTLFQPV HTLV-I tax zl 9 IPPSFLQAM HTLV-I tax 9 FPGFGQSLL HTLV-I tax 9 WPLLPHVIF HTLV-I tax 9 SPPITWPLL HTLV-I tax 9 VPYKRIEEL HTLV-I tax 9 RPQNLYTLW HTLV-I tax 9 CPKDGQPSL HTLV-I tax A SEQUENCE SOURCE

A

10 WPYLHNRLVV HPV16 El A SEQUENCE SOURCE
A

9 HPSDGKCNL Pf SSP2 206 9 RPRGDNFAV Pf SSP2 305 9 QPRPRGDNF Pf SSP2 303 10 TPYAGEPAPF Pf SSP2 539 Table 14 PEPTIDE AA SE UENCE

25.0129 9 LPPLERLTL

26.0445 10 EPGPVTA VV

26.0448 10 LPRIFCSCPI

26.0449 10 LPSPAC LVL

26.0455 10 VPLAHSSSAF

26.0458 10 VPRN DWLGV

26.0476 10 APPAYEKLSA

26.0478 10 MPREDAHFIY

26.0519 10 APAFLPWHRL

26.0522 IO GPNCTERRLL

26.0523 10 IPLYRNGDFF

26.0529 10 TPRLPSSADV

19.0101 9 TPAEVSIVV

26.0554 ll APFT CGYPAL

26.0561 11 NPADDPSRG

26.0564 11 RPPNAPILSTL

26.0566 11 SPFLLA FTSA

26.0567 11 SPHHTALR AI

26.0568 11 TPARVTGGVF

Claims (7)

WHAT IS CLAIMED IS:

1. A composition comprising an immunogenic peptide having an HLA
binding motif, which immunogenic peptide is a peptide shown in Tables 3-14 or a peptide comprising a conservative substitution of a residue in a peptide shown in Table 3-14.

2. The composition of claim 1, wherein the immunogenic peptide is linked to a second oligopeptide.

3. The composition of claim 2, wherein the second oligopeptide is a peptide that induces a helper T response.

4. A composition comprising a nucleic acid molecule encoding an immunogenic peptide as shown in Tables 3-14, or a peptide comprising a conservative substitution of a residue of a peptide shown in Table 3-14.

5. The composition of claim 4, wherein the nucleic acid further comprises a sequence encoding a second immunogenic peptide.

6. The composition of claim 4, wherein the nucleic acid further comprises a sequence encoding an oligopeptide that induces a helper T response.

7. A method of inducing a cytotoxic T cell response comprising contacting a cytotoxic T cell with a peptide of claim 1.