Aspartate-beta-hydroxylase Induces Epitope-specific T Cell Responses in Tumors

Abstract

The present invention provides a peptide-based immunotherapy for ASPH-expressing tumors.

Claims

1. A composition comprising a therapeutically effective amount of a purified aspartate-β-hydroxylase (ASPH) peptide, wherein said peptide comprises at least 7 amino acids in length and fewer amino acids than the full length ASPH protein and wherein said peptide comprises an HLA class I or class II-restricted epitope sequence.

2. The composition of claim 1, wherein the peptide comprises the HLA class II restricted sequence of TGYTELVKSLERNWKLI (SEQ ID NO:11).

3. The composition of claim 1, wherein the peptide comprises the HLA class I restricted sequence of YPQSPRARY (SEQ ID NO:26).

4. The composition of claim 1, wherein the peptide is selected from the group consisting of SEQ ID NOs: 1-45.

5. The composition of claim 1, further comprising a pharmaceutical carrier, adjuvant or other co-ingredient.

6. A vaccine comprising the composition of claim 1.

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Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1A and C are histograms, and FIGs. B and D are dot plots. ASPH-specific T cell responses in HCC patients. CD25-depleted or non-depleted, pan T cells purified from the PBMCs of HCC patients (HCC #1-5) were co-cultured with monocyte-derived DCs loaded with recombinant ASPH, AFP or no antigen (control). After 8 days incubation, the T cells were collected and re-stimulated by co-culture with DCs loaded with the same antigen. The response of CD4.sup.+ T cells was evaluated by quantifying the percentage of CD154.sup.+, IFN-γ.sup.+, and IFN-γ.sup.+CD154.sup.+ cells. FIG. 1A: representative flow cytometric analyses; FIG. 1B: summary of the analyses of 5 HCC patients). The response of CD8.sup.+ CTL was evaluated by quantifying the percentage of CD137.sup.+ and IFN-γ.sup.+ cells. FIG. 1C: representative flow cytometric analyses, FIG. 1D: summary of the analyses in 5 HCC patients). The percentages of responsive cells in the ASPH sample are significantly greater than the percentages found in the AFP sample or antigen control, p<0.05 (FIGS. 1B and 1D); the T.sub.reg cell-depleted and non-depleted ASPH samples are significantly different from each other, p<0.05 (FIG. 1B).

[0029] FIG. 2 is a bar graph. The response of healthy donors to predicted ASPH immunoconsensus sequences. PBMCs isolated from healthy donors (HD #1-5) were cultured with the single HLA-DRB1-restricted ASPH peptide (10 μm/ml) enumerated according to the list in Table I. The cells were collected after 2 weeks and the peptide-specific response was quantified by a standard IFN-γ ELISpot assay. Values are expressed as the means±SD of triplicate determination. The dotted lines represent the level of control (0.1% DMSO). *Significantly greater than the control, p<0.05.

[0030] FIG. 3A is a histogram, FIGS. 3B and C are bar graphs, and FIG. 3D is a line graph. Characterization of ASPH peptide-specific T cell responses. PBMCs obtained from patient HD #1 were cultured with the ASPH peptides indicated. After 2 weeks incubation, the cells were collected and re-cultured with the corresponding peptides for 20 hours, followed by analyses. p52-reactive PBMCs were stained and the percentage of IFN-γ.sup.+CD4.sup.+ T cells was determined by flow cytometric analysis (FIG. 3A). The percentage of CD154-expressing CD4.sup.+ T cells assessed by flow cytometric analysis was determined as a measure of the response of cells to the ASPH peptide listed in accordance with Table I (FIG. 3B). The results are the means±SD of triplicate determinations; dotted lines represent the control (0.1% DMSO) values. *Significantly greater than control, p<0.05. PBMCs cultured 2 weeks with the peptides listed were not treated or pretreated with anti-HLA-A,B,C or anti-HLA-DR for 1 hour; the same corresponding peptide (10 μg/ml) was then added and IFN-γ in the culture supernates collected after 5 days was quantified by ELISA (FIG. 3C). Results are the means±SD of triplicate determinations *Significantly less than non-antibody or anti-HLA-A,B,C-treated; p<0.05 Peptide titration assay for the immune response of ASPH peptides-specific cells (FIG. 3D). PBMCs, cultured 2 weeks with the ASPH peptides listed, were collected and reincubated with increasing concentrations of the same peptide. IFN-γ in the culture supernates collected after 5 days was quantified by standard ELISA.

[0031] FIGS. 4A and B are bar graphs. HCC patient respond to ASPH peptides. PBMCs obtained from 7 HCC patients (HCC #6-12) were cultured for 2 weeks with HLA-DRB1-restricted (FIG. 4A) or HLA-class I-restricted (FIG. 4B) ASPH peptide compatible with the patients' HLA background (indicated in parentheses) and enumerated in accordance with Tables I and II. Peptide-specific T cell recognition was evaluated by IFN-γ ELISpot assay. Values are the means±SD of triplicate determination. The dotted lines represent the level of control (0.1% DMSO). *Significantly greater than the control, p<0.05.

[0032] FIGS. 5A and B are histograms. ASPH protein-specific T cell response of a healthy blood donor. Pan T cells purified from the PBMCs of HD #1 were co-cultured with monocyte-derived DCs loaded or not loaded (control) with recombinant ASPH or AFP. After 8 days incubation, the T cells were collected and re-stimulated by culture with a fresh population of DCs loaded with the same antigen. The response of CD4.sup.+ T cells was evaluated by quantifying the percentage of CD154.sup.+, IFN-γ, and IFN-γ.sup.+CD154.sup.+ cells within the total CD4.sup.+ T cell population (FIG. 5A). The response of CD8.sup.+ CTLs was evaluated by quantifying the percentage of CD137.sup.+ and IFN-γ.sup.+ cells within the total CD8.sup.+ T cell population (FIG. 5B). The percentage of cells in the gated area is shown.

[0033] FIG. 6 is a bar graph. Summary and comparison of the responses of the healthy donors (HD) and HCC patients (HCC) to HLA-DRB1-restricted ASPH peptides. The responses (IFN-γ spots/106 PBMCs) of 5 healthy donors (#1-5) and 6 HCC patients (#7-12) to the 15 individual HLA-DRB1-restricted peptides down in FIGS. 2 and 4A, respectively, were combined and averaged; the background, DMSO control was subtracted.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Individual purified HLA class I and II peptides of ASPH induced human peptide specific CD4+ helper and CD8+ cytotoxic T cell responses. Since ASPH is highly expressed on many solid tumors, such as hepatocellular carcinoma, cholangiocarcinoma, pancreatic tumors, breast, prostate, colon, lung and gastric cancers, these peptides are useful for vaccine to induce the host immune response against ASPH expressing cancers. This invention involves an ASPH peptide based prophylactic and therapeutic vaccine to inhibit or reduce ASPH-expressing tumors.

[0035] Prior to the invention, the response of human cytotoxic CD8.sup.+ T lymphocytes (CTL) to ASPH was unknown. The data described herein first demonstrated that full-length ASPH induced significant activation of CTLs, as well as CD4.sup.+ helper T cells, obtained from HCC patients. Next, immunoinformatics tools were used to determine 15 HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple epitopes) contained in ASPH. These ICS were synthesized as peptides and their capacities to bind multiple HLA-DRB1 alleles were determined. Thirty HLA class I-restricted ASPH epitopes were also determined and synthesized. Subsequently, each HLA class I- and class II-restricted peptide was evaluated and demonstrated to be immunogenic for HCC patients. The results of these analyses led to the development of an ASPH epitope-based vaccine for HCC immunotherapy.

[0036] HCC has a poor prognosis due to high recurrence rate and lack of effective systemic therapy. ASPH is a highly conserved transmembrane protein, which is over expressed in HCC and promotes malignant phenotypes. Immunization with ASPH-loaded dendritic cells (DCs) has anti-tumor effects in a murine HCC model. Moreover, ASPH induces a significant increase in antigen-specific CD4+ T cells in cultures of human peripheral blood mononuclear cells (PBMCs) indicating a utility of ASPH-based immunotherapy against HCC.

[0037] Monocyte-derived DCs generated from the PBMCs of HCC patients were loaded with ASPH. Helper CD4.sup.+ T cells and CD8.sup.+ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs. Subsequently T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to determine HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ enzyme-linked immunospot assay.

[0038] ASPH-loaded DCs activated both CD4.sup.+ and CD8.sup.+ T cells contained within the PBMC population derived from HCC patients. Furthermore, the HLA class I- and class II-restricted ASPH peptides were significantly immunogenic in PBMC cultures. Both HLA class I- and class II-restricted peptides derived from ASPH induced T cell activation in HCC. These results indicate the efficacy of an ASPH peptide-based therapeutic vaccine for HCC and other ASPH over-expressing tumors.

[0039] ASPH Peptides

[0040] The ASPH vaccine peptides bind to human HLA molecules and stimulate an ASPH-specific anti-tumor immune response, thereby reducing tumor burden/tumor mass, reducing metastases, and conferring a clinical benefit to the patient suffering from an ASPH-expressing tumor. Methods for measuring binding of peptides to HLA molecules are well known in the art, e.g., Parker et al., 1994, Journal of Immunology 152:163. Methods for measuring T-cell mediated immune responses, e.g., activation of CD8+ cytotoxic T cells and/or helper CD4+ T cells are also well known in the art, e.g., detection of secreted immune activation markers and/or cell surface activation markers in response to immunostimulatory ASPH peptide antigens. Exemplary protocols are described herein.

[0041] Peptide variants that preserve antigenic function are also within the invention. For example, a variant may contain an amino acid residue at a particular position in the sequence which has been substituted by other amino acids or may include an additional residue or residues between two residues of the reference peptide as well as a deletion one or more residues from the reference sequence provided that the peptide retain the function of stimulating an ASPH-specific anti-tumor immune response (e.g., activation of ASPH-specific T cells). Such a peptide variant will have at least about 71%-75% amino acid sequence identity; at least about 76%-79% amino acid sequence identity; at least about 80% amino acid sequence identity, at least about 81% amino acid sequence identity, at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and at least about 99% amino acid sequence identity with a full-length sequence.

[0042] In some examples, the variants contain conservative substitutions. Conservative substitutions are shown in the table below. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound.

TABLE-US-00001 Exemplary substitutions Original Preferred residue Exemplary substitutions substitutions Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Norleucine Leu

[0043] The polypeptides of the invention can be either synthesized in vitro or expressed recombinantly from polynucleotide sequences. The polypeptides of the invention can be readily synthesized in vitro using polypeptide chemistry. For example, polypeptide synthesis can be carried out in a stepwise manner on a solid phase support using an automated polypeptide synthesizer, such as a Rainin Symphony Peptide Synthesizer, Advanced Chemtech Peptide Synthesizer, Argonaut Parallel Synthesis System, or an Applied Biosystems Peptide Synthesizer. The peptide synthesizer instrument combines the Fmoc chemistry with HOBt/HBTU/DIEA activation to perform solid-phase peptide synthesis.

[0044] “Percent (%) nucleic acid sequence identity” with respect to nucleic acid sequences is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining % nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0045] The following materials and methods were used to generate the date described herein.

[0046] Recombinant ASPH and ASPH Peptides

[0047] Full-length human ASPH (GenBank Accession No. 583325; Version: 583325.1 hereby incorporated by reference) was cloned into the EcoRI site of the pcDNA vector (Invitrogen, Carlsbad, Calif.). Recombinant protein was produced in a Baculovirus system (Invitrogen) according to the manufacturer's instruction..sup.11, 12

TABLE-US-00002 Human ASPH amino acid sequence SEQ ID NO 46 MAQRKNAKSSGNSSSSGSGSGSTSAGSSSPGARRETKHGGHKNGRKGGLSG TSFFTWFMVIALLGVWTSVAVVWFDLVDYEEVLGKLGIYDADGDGDFDVDD AKVLLGLKERSTSEPAVPPEEAEPHTEPEEQVPVEAEPQNIEDEAKEQIQS LLHEMVHAEHVEGEDLQQEDGPTGEPQQEDDEFLMATDVDDRFETLEPEVS HEETEHSYHVEETVSQDCNQDMEEMMSEQENPDSSEPVVEDERLHHDTDDV TYQVYEEQAVYEPLENEGIEITEVTAPPEDNPVEDSQVIVEEVSIFPVEEQ QEVPPETNRKTDDPEQKAKVKKKKPKLLNKFDKTIKAELDAAEKLRKRGKI EEAVNAFKELVRKYPQSPRARYGKAQCEDDLAEKRRSNEVLRGAIETYQEV ASLPDVPADLLKLSLKRRSDRQQFLGHMRGSLLTLQRLVQLFPNDTSLKND LGVGYLLIGDNDNAKKVYEEVLSVTPNDGFAKVHYGFILKAQNKIAESIPY LKEGIESGDPGTDDGRFYFHLGDAMQRVGNKEAYKWYELGHKRGHFASVWQ RSLYNVNGLKAQPWWTPKETGYTELVKSLERNWKLIRDEGLAVMDKAKGLF LPEDENLREKGDWSQFTLWQQGRRNENACKGAPKTCTLLEKFPETTGCRRG QIKYSIMHPGTHVWPHTGPTNCRLRMHLGLVIPKEGCKIRCANETRTWEEG KVLIFDDSFEHEVWQDASSFRLIFIVDVWHPELTPQQRRSLPAI
His motif is underlined; conserved sequences within the catalytic domain are designated by bold type. Additional information regarding ASPH is found in U.S. Pat. No. 6,783,758, hereby incorporated by reference.

TABLE-US-00003 Human ASPH cDNA sequence (GENBANK Accession No. S83325; codon encoding initiating methionine is underlined). SEQ ID NO: 47 1 cggaccgtgc aatggcccag cgtaagaatg ccaagagcag cggcaacagc agcagcagcg 61 gctccggcag cggtagcacg agtgcgggca gcagcagccc cggggcccgg agagagacaa 121 agcatggagg acacaagaat gggaggaaag gcggactctc gggaacttca ttcttcacgt 181 ggtttatggt gattgcattg ctgggcgtct ggacatctgt agctgtcgtt tggtttgatc 241 ttgttgacta tgaggaagtt ctaggaaaac taggaatcta tgatgctgat ggtgatggag 301 attttgatgt ggatgatgcc aaagttttat taggacttaa agagagatct acttcagagc 361 cagcagtccc gccagaagag gctgagccac acactgagcc cgaggagcag gttcctgtgg 421 aggcagaacc ccagaatatc gaagatgaag caaaagaaca aattcagtcc cttctccatg 481 aaatggtaca cgcagaacat gttgagggag aagacttgca acaagaagat ggacccacag 541 gagaaccaca acaagaggat gatgagtttc ttatggcgac tgatgtagat gatagatttg 601 agaccctgga acctgaagta tctcatgaag aaaccgagca tagttaccac gtggaagaga 661 cagtttcaca agactgtaat caggatatgg aagagatgat gtctgagcag gaaaatccag 721 attccagtga accagtagta gaagatgaaa gattgcacca tgatacagat gatgtaacat 781 accaagtcta tgaggaacaa gcagtatatg aacctctaga aaatgaaggg atagaaatca 841 cagaagtaac tgctccccct gaggataatc ctgtagaaga ttcacaggta attgtagaag 901 aagtaagcat ttttcctgtg gaagaacagc aggaagtacc accagaaaca aatagaaaaa 961 cagatgatcc agaacaaaaa gcaaaagtta agaaaaagaa gcctaaactt ttaaataaat 1021 ttgataagac tattaaagct gaacttgatg ctgcagaaaa actccgtaaa aggggaaaaa 1081 ttgaggaagc agtgaatgca tttaaagaac tagtacgcaa ataccctcag agtccacgag 1141 caagatatgg gaaggcgcag tgtgaggatg atttggctga gaagaggaga agtaatgagg 1201 tgctacgtgg agccatcgag acctaccaag aggtggccag cctacctgat gtccctgcag 1261 acctgctgaa gctgagtttg aagcgtcgct cagacaggca acaatttcta ggtcatatga 1321 gaggttccct gcttaccctg cagagattag ttcaactatt tcccaatgat acttccttaa 1381 aaaatgacct tggcgtggga tacctcttga taggagataa tgacaatgca aagaaagttt 1441 atgaagaggt gctgagtgtg acacctaatg atggctttgc taaagtccat tatggcttca 1501 tcctgaaggc acagaacaaa attgctgaga gcatcccata tttaaaggaa ggaatagaat 1561 ccggagatcc tggcactgat gatgggagat tttatttcca cctgggggat gccatgcaga 1621 gggttgggaa caaagaggca tataagtggt atgagcttgg gcacaagaga ggacactttg 1681 catctgtctg gcaacgctca ctctacaatg tgaatggact gaaagcacag ccttggtgga 1741 ccccaaaaga aacgggctac acagagttag taaagtcttt agaaagaaac tggaagttaa 1801 tccgagatga aggccttgca gtgatggata aagccaaagg tctcttcctg cctgaggatg 1861 aaaacctgag ggaaaaaggg gactggagcc agttcacgct gtggcagcaa ggaagaagaa 1921 atgaaaatgc ctgcaaagga gctcctaaaa cctgtacctt actagaaaag ttccccgaga 1981 caacaggatg cagaagagga cagatcaaat attccatcat gcaccccggg actcacgtgt 2041 ggccgcacac agggcccaca aactgcaggc tccgaatgca cctgggcttg gtgattccca 2101 aggaaggctg caagattcga tgtgccaacg agaccaggac ctgggaggaa ggcaaggtgc 2161 tcatctttga tgactccttt gagcacgagg tatggcagga tgcctcatct ttccggctga 2221 tattcatcgt ggatgtgtgg catccggaac tgacaccaca gcagagacgc agccttccag 2281 caatttagca tgaattcatg caagcttggg aaactctgga gaga

[0048] ASPH ICS were determined..sup.13, 14 The entire protein was parsed into overlapping 9-mer frames and each frame was evaluated using EpiMatrix for its potential to bind a panel of eight common HLA-DRB1 alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501), which represent >95% of the MHC diversity in the human population..sup.15 HLA-DRB1-restricted ICS were constructed using EpiAssembler, an algorithm that maximizes epitope density by assembling potentially immunogenic 9-mers into 18-25 amino acid stretches..sup.16 Additionally, all parsed 9-mers were scored for their potential to bind a panel of six common class I “supertype” alleles; A*0101, A*0201, A*0301, A*2402, B*0702, and B*4403, which cover over 95% of the human population..sup.17 Based upon these predictions, the peptide sequences were synthesized using FMOC chemistry and purified >85% by HPLC (21′ Century Biochemicals, Marlboro, Mass.); each was dissolved in 100% DMSO (100 μg/μl) and stored at −80° C. The amino acid sequences of the HLA-DRB1-restricted peptides and HLA class I-restricted peptides are shown in Table I and Table II, respectively.

[0049] Healthy Blood Donors and HCC Patients

[0050] Blood samples were obtained from 12 HCC patients (HCC #1-12) and 5 healthy blood donors (HD #1-5). The peripheral blood mononuclear cells (PBMCs) were purified from whole blood of the HCC patients using known methods..sup.11 HLA typing on DNA extracted from each sample was performed using known methods.

[0051] Used, de-identified whole-blood leukocyte reduction filters (blood filters; Sepacell RZ-2000, Baxter Healthcare Corporation, Irvine Calif.), served as the source of PBMCs derived from blood donated with informed consent by healthy volunteers. The PBMCs were recovered by back-flushing the filters according to the methods of Meyer et al. and purified by centrifugation on Ficoll-Paque Plus (1.077; Pharmacia, Uppsala, Sweden) gradient using known methods..sup.18, 19

[0052] Generation of DCs

[0053] DCs were generated using known methods..sup.11, 20 Monocytes were isolated from PBMCs using anti-CD14 microbeads (Miltenyi Biotec, Auburn, Calif.) and cultured for 5 days in X-VIVO 15 medium (Lonza, Walkerville, Md.) supplemented with human GM-CSF (R&D Systems, Minneapolis, Minn.) and IL-4 (R&D Systems). ASPH (1 μg/ml) was added on day 5. On the next day, TNF-α (R&D Systems) was added to stimulate DC maturation and the cells were incubated for another 48 hours. DCs incubated with α-fetoprotein (AFP; Zynaxis Cell Science, Malvern, Pa.) or alone served as the control. Mature, epitope-expressing DCs were collected at the end of the incubation period and used in the experiments described.

[0054] Induction of Epitope-Specific T Cells

[0055] Epitope-specific T cells were induced according to known methods..sup.21 Briefly, 2.5×10.sup.5 PBMCs/200 μl X-VIVO 15 medium supplemented with 1 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 U/ml recombinant human IL-2 (R&D Systems) in round-bottom 96-well plates were cultured for 2 weeks with 10 μg/ml individual peptide. When mature DCs were used as antigen-presenting cells, the T cells were isolated from PBMCs by negative selection using the Pan T Cell Isolation Kit II (Miltenyi Biotec). Regulatory T.sub.(reg) cells were removed by the addition of anti-CD25 microbeads (Miltenyi Biotec) where indicated. T.sub.reg cell-depleted or non-depleted T lymphocytes (2.4×10.sup.6) were co-cultured for 8 days with 4×10.sup.4 mature DCs loaded with relevant antigen in 24-well plates..sup.11

[0056] Blocking of T Cell Response

[0057] To demonstrate the contribution of HLA molecules to ASPH peptide-dependent T cell activation, the cells were incubated with antibodies specific for HLA class I (clone W6/32; BioLegend, San Diego, Calif.) or HLA-DR (clone L432; BioLegend) (15 μg/ml) for 1 hour at 37° C. prior to analyses.

[0058] Flow Cytometric Analysis

[0059] Flow cytometric analysis was conducted..sup.11 Intracellular cytokine staining was performed to evaluate T cell activation. Conjugated mouse monoclonal antibodies specific for the following determinants were used: CD4 (clone OKT4; BioLegend, San Diego, Calif.), CD8a (clone RPA-T8; BioLegend), CD137 (clone 4B4-1; BD Biosciences, San Diego, Calif.), CD154 (clone TRAP1; BD Biosciences), and IFN-γ (clone B27; BD Biosciences). Appropriate isotype controls were included in each analysis.

[0060] Enzyme-Linked Immunospot Assay

[0061] Human IFN-γ enzyme-linked immunospot (ELISpot) assays were performed as we described previously using a kit purchased from eBioscience (San Diego, Calif.) to determine T cell immune-reactivity..sup.21 Cells (5×10.sup.4/well) were added to ELISpot plates (Millipore, Bedford, Mass.) pre-coated with anti-IFN-γ capture antibody and incubated with peptides (10 μg/ml) for 20 hours. Subsequently, the plates were washed and incubated sequentially with biotinylated IFN-γ detection antibody then avidin-HRP. The plates were developed by adding substrate, 3-amino-9-ethyl carbazole, and the number of spots/well was quantified using a CTL-immunospot S5 UV Analyzer (Cellular Technology Limited, Shaker Heights, Ohio).

[0062] Enzyme-Linked Immunosorbent Assay

[0063] Enzyme-linked immunosorbent assays (ELISA) were performed to quantify IFN-γ in cell culture supernatants using a human IFN-γ ELISA kit (eBioscience)..sup.11

[0064] Statistical Analysis

[0065] Data are expressed as mean±SD. Differences between groups were assessed by the χ.sup.2-test, Fisher's exact test or the Mann-Whitney U test. A p value <0.05 was considered statistically significant. Data analyses were performed using StatView (version 5.0; SAS Institute Inc., Cary, N.C.).

[0066] Immunogenic Response of HCC Patients to ASPH

[0067] Experiments were undertaken to determine the capacity of ASPH to activate CD8.sup.+ CTLs, as well as helper CD4.sup.+ T cells, contained in the PBMC populations derived from HCC patients. The experimental procedure for evaluating ASPH-specific T cell activation was optimized using PBMCs derived from a healthy donor..sup.11 Pan T cells were co-cultured with autologous, ASPH-pulsed DCs; T cells co-incubated with AFP-pulsed or non-pulsed DCs served as controls. After 8 days culture, the T cells were collected and re-incubated with freshly prepared DCs pre-loaded with the same antigen; antigen-specific T cell activation was evaluated by flow cytometric analysis. In addition to IFN-γ, the expression of CD154 and CD137 was used as a surrogate marker for antigen-specific activation of CD4.sup.+ T cells and CD8.sup.+ CTLs, respectively..sup.22, 23 These experiments revealed significant increases in the activation of both CD4.sup.+ T cells and CTLs in response to ASPH compared to AFP or no antigen (FIGS. 5A and B). Moreover, they support the premise that immunization with ASPH activates protein-specific CD4.sup.+ and CD8.sup.+ T cells in HCC patients.

[0068] Using the same experimental approach, ASPH-specific T cell activation was evaluated in

[0069] PBMCs derived from five HCC patients (HCC #1-5) characterized in Table 3. As shown in FIGS. 1A and B, incubation with ASPH-pulsed DCs resulted in significant increases in CD4.sup.+CD154.sup.+ and CD4.sup.+IFN-γ.sup.+ T cells compared to incubation with AFP-pulsed or non-pulsed DCs. CD4.sup.+ T cells expressing both IFN-γ and CD154 were also expanded significantly. In addition, the percentage of CD8.sup.+ CTLs expressing CD137 and IFN-γ was significantly higher in co-cultures that contained ASPH-pulsed DCs compared to co-cultures that contained AFP-pulsed or non-pulsed DCs (FIGS. 1C and D). T.sub.reg cell depletion using anti-CD25 microbeads prior to co-culture enhanced ASPH-specific T cell activation..sup.20 These results, which demonstrate the ASPH-specific activation of CD4.sup.+ and CD8.sup.+ T cells among PBMCs derived from HCC patients, support the use of purified ASPH peptides in HCC immunotherapy for ASPH-overexpressing tumors.

[0070] Prediction and Validation of HLA-DRB1-Restricted ASPH Peptide Sequences

[0071] The demonstrated immunogenicity of purified ASPH peptides affords the development of an ASPH epitope-based anti-tumor vaccine. Both MHC class II-restricted (CD4.sup.+) and MHC class I-restricted (CD8.sup.+) T cells are preferably used for sustained anti-tumor immunity..sup.24, 25 Initially, HLA-DRB1-restricted ICS composed of multiple epitopes and covering full-length human ASPH were determined and constructed using bioinformatics tools..sup.13, 14 Fifteen promiscuous ISC, each capable of binding multiple HLA-DRB1 alleles representing >95% of the human population, were synthesized (Table I)..sup.15

[0072] The validity of these predicted ASPH epitopes (ICS) was documented by demonstrating their ability to induce the response of naïve T cells contained within PBMC populations derived from 5 healthy blood donors (HD #1-5). The PBMCs obtained from each donor were transferred to multiple wells in a 96-well round bottom plate and cultured with a single, ASPH peptide sequence. After 2 weeks, the capacity of each sequence to induce a peptide-specific response was evaluated by IFN-γ ELISpot assay. As shown in FIG. 2, each peptide sequence induced a statistically significant increase in IFN-γ producing cells, albeit, the response to any single sequence varied among donors. While these ICS are promiscuous and bind multiple HLA-DRB1 alleles, it is likely that differences in the donors' MHC backgrounds contribute to this varied response. Notably, however, all the ASPH peptides exhibited immunogenicity in at least one healthy blood donor, and p322 (ID #4, Table I) was immunogenic for all 5 donors. p581 (ID #11, Table I) also emerged as a preferred peptide.

[0073] To quantify and characterize the IFN-γ-producing cells, the PBMCs derived from HD #1 following induction by p52 (ID #1, Table I) were stained and evaluated by flow cytometric analysis. As predicted, essentially all HLA-DRB1-restricted, p52-reactive, IFN-γ-producing cells expressed CD4 (FIG. 3A). In addition, CD154 expression was assessed as a measure of CD4.sup.+ T cells activation following incubation with each ASPH peptide (FIG. 3B). The pattern of CD154 expression was consistent with the IFN-γ ELISpot data presented above. HLA blocking experiments confirmed the dependence of ASPH peptide-induced IFN-γ production on HLA-DRB1 expression. IFN-γ production by the ASPH peptide-responsive cells was significantly abrogated by the presence of anti-HLA-DR, but not anti-HLA-class I, antibody (FIG. 3C). Titration experiments revealed dose-dependent increases in IFN-γ production following ASPH peptide stimulation (FIG. 3D). Together, these results substantiate the HLA-DR-restricted, dose-dependent CD4.sup.+ T cell responses induced by the ASPH peptides. IL-4 production was not observed following incubation with any of the ASPH peptide sequences indicating that these sequences induced Th1-like CD4.sup.+ T cell responses.

[0074] ASPH Peptide-Specific T Cell Activation in HCC Patients

[0075] Based upon the results demonstrating the HLA-DRB1-restricted, ASPH ICS-specific T cell response of healthy blood donors, the response of CD4.sup.+ T cells derived from seven HCC patients (HCC #6-12) to the same peptides was evaluated. Each HLA class II-restricted peptide sequence induced a statistically significant increase in IFN-γ producing cells, though the response to individual peptides varied among patients (FIG. 4A). None of the peptides elicited a significant response in HCC patient #6. All the predicted ASPH peptides exhibited immunogenicity in at least one HCC patients, however, a pattern similar to that found in the healthy blood donors shown above.

[0076] CD8.sup.+, as well as CD4.sup.+, T cells are required for anti-tumor immunity. As such, HLA class I-restricted ASPH-derived epitopes were also determined and 30 HLA-A- and HLA-B-restricted peptides were synthesized (Table II). The response of CD8.sup.+ CTLs derived from the HCC patients to the peptides compatible with their HLA class I alleles was subsequently evaluated. Histoincompatibility precluded including HCC patient #8 in this analysis. All the HLA class I-restricted peptides induced significant increases in IFN-γ producing cells albeit, as in the case of those restricted by HLA-DRB1, the response to individual peptides varied substantially among patients (FIG. 4B). HCC patient #6, for example, failed to exhibit a significant response to any peptide among a panel of 11 HLA-A*01/*03- and 5 HLA-B*07-restricted peptides. HCC patient #12, on the other hand, responded to 85% of the peptides compatible with his HLA background. ASPH 371 was determined to be a preferred class I restricted peptide.

[0077] Immunogenic ASPH Epitopes in HCC Patients

[0078] The aim of cancer immunotherapy is to induce an effective immune response that specifically targets tumor cells..sup.26 Success is predicated upon the existence of a tumor-associated antigen (TAA), which is uniquely over expressed in tumor tissue and elicits a specific immune response in the tumor-bearing host. To date, several HCC-related TAAs have been identified: AFP, glypican-3, and NY-ESO-1..sup.27-30 While T cell responses specific for these TAAs are often observed, however, they are not sufficiently robust to affect clinical outcomes. Although the factors that contribute to these unsatisfactory outcomes are complicated and require further explorations, one factor seems to be a diminished CD4.sup.+ helper T cell response. Both activated CD4.sup.+ T cells and CD8.sup.+ CTLs are required to maintain a sustained anti-tumor response..sup.24, 25

[0079] ASPH was found here to elicit the antigen-specific activation of CD4.sup.+ and CD8.sup.+ T cells among human PBMCs indicating that ASPH possesses both HLA class I- and class II-restricted epitopes making it useful for HCC immunotherapy. Such an approach utilizes an epitope-based vaccine for HCC, which consists of a combination of purified peptides (epitopes) capable of eliciting the robust responses of CD4.sup.+ and CD8.sup.+ T cells. Toward this end, immunoinformatics tools were used to identify 30 ASPH-derived HLA class I-restricted and 15 class II-restricted epitopes. These epitopes induced both CD8.sup.+ CTL and CD4.sup.+ T cell responses by six out of seven HCC patients indicating that these chosen ASPH peptides are useful for HCC immunotherapy. Notably, one patient (HCC #6) failed to respond to any of the epitopes compatible with her HLA background suggesting that she would not be a good candidate. Such a response profile of an individual patient provides basis for personalized treatment regimen, e.g., pre-screening patients prior to vaccination to determine which of the peptides (SEQIDNOS 1-45) are most suitable for a combination of peptides in the vaccine for a specific patient.

[0080] In addition to selecting the ideal TAA to target, strategies to optimize anti-tumor immunity incorporate approaches to prevent the escape of cancer cells from immune recognition. Mechanisms that underlie cancer cell escape, e.g., cell surface inhibitory molecules and immunoregulatory cells including T.sub.reg and myeloid-derived suppressor cells have been reported..sup.31-34 The findings demonstrated an enhanced T cell response to ASPH following CD25.sup.+ T.sub.reg cell depletion. Moreover, residual tumor cells still expressed cell surface ASPH in a mouse model following immunization with ASPH-loaded DCs implying the existence of tumor cells that escape immunotherapy..sup.12 Thus, a combination therapy strategy using ASPH-based immunotherapy in combination with other approaches (e.g., antagonizing T.sub.reg cell function) circumvent immune escape and achieve a successful clinical outcome..sup.35 Low doses of cyclophosphamide, for example, reportedly decrease the number and inhibitory activity of T.sub.reg cells by suppressing the expression of important functional markers: forkhead box P3 and glucocorticoid-induced TNF-receptor-related protein..sup.36 In a tumor-bearing mouse model, cyclophosphamide treatment suppressed T.sub.reg cell function and augmented the immune response and tumor eradication..sup.37 Similarly, sorafenib (a multi-targeted anti-angiogenic tyrosine kinase inhibitor used to treat patients with advanced HCC) reduced T.sub.reg cell function and enhanced anti-tumor immunity in mice bearing established orthotropic HCC..sup.38 Optionally, these agents are used as an adjunct therapy to the vaccine peptides.

[0081] Other treatment options such as transarterial embolization or local ablation therapy are useful to augment ASPH-based HCC immunotherapy. Transarterial embolization, for example, not only induced tumor necrosis directly, but also expanded the T cell-mediated immune response..sup.39 Radiofrequency HCC ablation induced peptide-specific CTLs..sup.40 SPH-based immunotherapy can be combined with additional approaches to treat HCC and augment the possible clinical effect of peptide epitope based therapy.

[0082] The results indicate that T.sub.reg cells comprise a small component of the T cell population activated by the entire ASPH protein sequence. A major advantage of peptide-based, versus whole protein-based, vaccines is that T.sub.reg cell epitopes can readily be identified and omitted.

[0083] The data reported herein demonstrated that ASPH elicits antigen-specific CD4.sup.+ T cell and CD8.sup.+ CTL activation, and that ASPH-derived HLA class I- and class II-restricted purified peptides activate CD4.sup.+ T cells and CD8.sup.+ CTLs derived from HCC patients. The average responses of the healthy donors and the HCC patients to the HLA-DRB1-restricted ASPH peptides (shown in FIG. 2 and FIG. 4A respectively) are summarized and compared in FIG. 6. In the case of healthy donors (HD) naïve T cells represent the vast majority of induced antigen-specific T-cell. However, in HCC patients it may be a combination of both naïve cells and previously sensitized cells. Considering the immunogenicity of ASPH, its tissue specificity and association with tumor progression, ASPH peptide-based vaccination represents an improved HCC treatment and treatment of other tumor types characterized by aberrant, e.g., overexpression of ASPH. Overexpression of ASPH refers to expression level of ASPH of the tissues or cells from a subject is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more than the expression level of ASPH of the tissues or cells from a healthy subject or a subject without cancer.

TABLE-US-00004 TABLE I Predicted, HLA-DRB1-restricted ASPH immunogenic consensus sequences Peptide Peptide AA SEQ ID ID# name position AA sequence NO 1 p52 52-71 TSFFTWFMVIALLGVWTSVA 1 2 p103 103-117 AKVLLGLKERSTSEP 2 3 p148 148-166 KEQIQSLLHEMVHAEHVEG 3 4 p322 322-337 QKAKVKKKKPKLLNKF 4 5 p415 415-432 PADLLKLSLKRRSDRQQF 5 6 p427 427-444 SDRQQFLGHMRGSLLTLQ 6 7 p437 437-452 RGSLLTLQRLVQLFPN 7 8 p443 443-458 LQRLVQLFPNDTSLKN 8 9 p492 492-509 VHYGFILKAQNKIAESIP 9 10 p557 557-576 ASVWQRSLYNVNGLKAQPWW 10 11 p581 581-597 TGYTELVKSLERNWKLI 11 12 p588 588-607 KSLERNWKLIRDEGLAVMDK 12 13 p725 725-738 HEVWQDASSFRLIF 13 14 p731 731-747 ASSFRLIFIVDVWHPEL 14 15 p740 740-758 VDVWHPELTPQQRRSLPAI 15 ASPH, aspartate-β-hydroxylase; AA, amino acid

TABLE-US-00005 TABLE II Predicted, HLA class I-restricted ASPH peptide sequences SEQ ID Peptide ID# Peptide name AA position AA sequence Restricting HLA allele NO 1 ASPH48 48-56 GLSGTSFFT A*0201 16 2 ASPH53 53-61 SFFTWFMVI A*2402 17 3 ASPH58 58-66 FMVIALLGV A*0201 18 4 ASPH62 62-70 ALLGVWTSV A*0201 19 5 ASPH72 72-80 VVWFDLVDY A*0101, A*0301, B*0702 20 6 ASPH79 79-87 DYEEVLGKL A*2402 21 7 ASPH81 81-89 EEVLGKLGI B*4403 22 8 ASPH252 252-260 TDDVTYQVY A*0101, B*4403 23 9 ASPH258 258-266 QVYEEQAVY A*0101, A0301 24 10 ASPH261 261-269 EEQAVYEPL B*4403 25 11 ASPH371 371-379 YPQSPRARY A*0101, B*0702 26 12 ASPH374 374-382 SPRARYGKA B*0702 27 13 ASPH406 406-414 QEVASLPDV B*4403 28 14 ASPH411 411-419 LPDVPADLL B*0702 29 15 ASPH475 475-483 KVYEEVLSV A*0201 30 16 ASPH478 478-486 EEVLSVTPN B*4403 31 17 ASPH484 484-492 TPNDGFAKV B*0702 32 18 ASPH488 488-496 GFAKVHYGF A*2402 33 19 ASPH491 491-499 KVHYGFILK A*0301 34 20 ASPH503 503-511 KIAESIPYL A*0201 35 21 ASPH521 521-529 GTDDGRFYF A*0101 36 22 ASPH537 537-545 RVGNKEAYK A*0301 37 23 ASPH557 557-565 ASVWQRSLY A*0101, A*0301, B*4403 38 24 ASPH563 563-571 SLYNVNGLK A*0301 39 25 ASPH582 582-590 GYTELVKSL A*2402 40 26 ASPH611 611-619 LFLPEDENL A*2402 41 27 ASPH681 681-689 GPTNCRLRM B*0702 42 28 ASPH693 693-701 LVIPKEGCK A*0301 43 29 ASPH701 701-709 KIRCANETR A*0301 44 30 ASPH711 711-719 WEEGKVLIF B*4403 45 ASPH, aspartate-β-hydroxylase; AA, amino acid

TABLE-US-00006 TABLE 3 HCC patient characteristics Maximum HLA alleles Age tumor Risk ID A B C DR (years) Gender Histology size (cm) Multiplicity factor HCC #1 ND 67 Male W 1.0 Single Alcohol HCC #2 ND 58 Male M 5.5 Multiple HCV HCC #3 ND 84 Male W 8.0 Single Alcohol HCC #4 ND 72 Male W 15.0 Multiple None HCC #5 ND 53 Male W 2.0 Single HCV HCC #6 *01/*03 *07/*08 *07/—  *02/*06 70 Female W 8.0 Single HCV HCC #7 *01/*02 *49/*53 *04/*07 *05/*06 82 Male ND 6.5 Single None HCC #8 *25/*68 *18/*35 *04/*12 *06/*07 69 Male ND 4.8 Single None HCC #9 *02/*24 *35/*53 *04/—  *05/*06 57 Male ND 1.6 Single None HCC #10 *02/*33 *44/*65 *04/*08 *02/*09 64 Female M 9.4 Single None HCC #11 *02/*29 *51/—  *04/—  *05/*06 70 Female W 5.8 Multiple HCV HCC #12 *03/*23 *07/*44 *04/*07 *02/*05 70 Male M 2.7 Single HCV ND, not determined: W, well differentiated; M, moderately differentiated; HCV, hepatitis C virus

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