WO2024086298A2 - Hla class i and class ii-restricted t-cell epitopes in pancreatic cancer tissues and uses thereof - Google Patents

Abstract

Provided herein are HLA-class I and HLA-class II restricted and non-restricted epitopes recurrently identified in patients with cancer, that can be presented to T cell receptor (TCR) to induce polyfunctional T cell response, polyepitopes peptides thereof, vaccines thereof, and T cells having a TCR having a binding affinity for the epitopes and polyepitopes peptides, and methods of use thereof. The methods of use include methods of treating cancer.

Description

HLA CLASS I AND CLASS H-RESTRICTED T-CELL EPITOPES IN PANCREATIC CANCER TISSUES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/417,951, filed October 20, 2022. The disclosure of the prior application is considered part of and is herein incorporated by reference in the disclosure of this application in their entirety.

STATEMENT REGARDING GOVERNMENT FUNDING

[0002] This invention was made with government support under Grant Nos. CAI 69702, CAI 97296, awarded by the National Institutes of Health. The government has certain right in the invention.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0003] The present invention relates generally to HL A class I and class Il-restricted T-cell epitopes, and more specifically to pancreatic ductal carcinoma specific HLA class I and class II- restricted T-cell epitopes.

BACKGROUND INFORMATION

[0004] T cell epitopes in pancreatic ductal adenocarcinoma (PDAC) are challenging to identify largely due to lack of knowledge on immunodominant antigens and effective technical approaches. Although in silico epitope prediction from whole-exome sequencing results has been used to predict mutation-associated neoepitopes, such an approach may not predict high-affinity T cellreceptor binding epitopes if the tumors have low tumor mutation burdens (TMB).

SUMMARY OF THE INVENTION

[0005] The present invention is based on the seminal discovery of HLA-class I and/or HLA- class II restricted or non-restricted peptides that can be presented to T cell receptor (TCR), a vaccine thereof, and methods of use thereof such as methods of treating cancer.

[0006] In one embodiment, the present invention provides an isolated peptide having the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2.

[0007] In another embodiment, the invention provides a polyepitope peptide including one or more HLA-class I and/or a HLA-class II restricted or non-restricted epitope, wherein the one or more epitopes are an antigenic fragment of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, 0RMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

[0008] In one aspect, the epitopes have the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2. In various aspects, the epitopes have the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

[0009] In an additional embodiment, the invention provides an isolated T cell including a T cell receptor (TCR) having a binding affinity to an HLA-class I and/or HLA-class II restricted or nonrestricted epitope, wherein the epitope is an antigenic fragment of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, ORMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

[0010] In one aspect, the T cell is an engineered T cell. In another aspect, the epitope has the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2. In various aspects, the epitope has the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

[0011] In a further embodiment, the invention provides a vaccine including one or more HLA- class I and/or HLA-class II restricted or non-restricted epitopes, wherein the epitopes are antigenic fragments of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, ORMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

[0012] In one aspect, the vaccine includes a lipid nanoparticle for presenting the one or more epitopes to antigen presenting immune cells. In another aspect, the one or more presented epitopes have the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2. In various aspects, the epitopes have the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30. [0013] In one embodiment, the present invention provides a method of treating cancer in a subject including administering to the subject one or more of the peptides described herein, one or more of the polyepitope peptides described herein, the T cell described herein, or the vaccine described herein, thereby treating cancer in the subject.

[0014] In one aspect, the HLA-class I and/or HLA-class II restricted or non-restricted peptides or the polyepitope peptides thereof induces polyfunctional T cells in the subject. In some aspects, inducing polyfunctional T cells includes stimulating a T cell response and/or stimulating T cell expression of effector T cell cytokine. In various aspects, the effector T cell cytokines include IFNy, IL-2 and/or TNFa. In other aspects, stimulating a T cell response include stimulating cytotoxic T cell cytokines. In various aspects, the cytotoxic T cell cytokines include IFNy and/or granzyme B from T cells. In another aspect, the cancer is a cancer expressing an epitope having the amino acid sequence of any of SEQ ID NOs:7-32, or of any of the peptides listed in Table 2. In various aspects, the cancer is a cancer expressing an epitope having the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30. In some aspect, the cancer is pancreatic cancer. In various aspects, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). In one aspect, the method further includes administering to the subject an anti-cancer treatment. In some aspects, the anti-cancer treatment is selected from the group consisting of gemcitabine, folfirinox, erlotinib, nab-paclitaxel, liposomal irinotecan, and olaparib.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGURES 1A-1I shows the mass spectrometry analysis of HLA Class I epitopes in PDAC tumor cell lines and tissues. FIGURE 1A shows histograms illustrating the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC cell lines Pancl0.05 (left) and Panc06.03 (right). FIGURE IB shows histograms illustrating the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC tissues. FIGURE 1C is a histogram numbers of HLA Class I epitopes and their associated proteins identified from each individual PDAC tissues. FIGURE ID is a graph illustrating predicted HLA Class I binding affinity of eluted peptides. FIGURE IE is a flow chart illustrating peptide validation. FIGURE IF shows Venn diagrams illustrating peptide overlaps among patients. FIGURE 1G is a graph illustrating T2 cell binding assays of selected HLA-A2 and A29 peptides binding to HLA-A2 expressing T2 cells. FIGURE 1H is a graph illustrating T2 cell binding assays of selected HLA-A2 and A29 peptides binding to HLA-A3 expressing T2 cells. FIGURE II is a graph illustrating T2 cell binding assays of selected HLA-A2 and A29 peptides binding to HLA-A1 expressing T2 cells. MFI: mean fluorescent intensity. Unpaired t test and 1- way ANOVA was used for comparing between samples. *p < 0.05, **p < 0.01.

[0016] FIGURES 2A-2F illustrates mass spectrometry analysis of HLA Class II epitopes in PDAC tumor tissues. FIGURE 2A is a histogram illustrating the numbers of different lengths of peptides affinity purified by anti-HLA Class II antibody from representative human PDAC tissue sample Panl3. FIGURE 2B is a histogram illustrating the numbers of different lengths of peptides affinity purified by anti-HLA Class II antibody from representative human PDAC tissue sample Panl4. FIGURE 2C shows Venn diagrams illustrating the numbers of total HLA class I peptides, HLA class II peptides, and completely overlapped peptides between HLA-I and HLA- II peptides in Panl6 (left), Pan04 (middle) and Panl7 (right). FIGURE 2D is a graph illustrating the ability of selected, HLA class I/II-overlapped peptides in stimulating single cells to express IFN-y, IL-2 and TNF-a in FluoroSpot assays using PBMCs of patient A. FIGURE 2E is a graph illustrating the ability of selected, HLA class I/II-overlapped peptides in stimulating single cells to express IFN-y, IL-2 and TNF-a in FluoroSpot assays using PBMCs of patient B. FIGURE 2F is a graph illustrating the ability of selected, HLA class I/II-overlapped peptides in stimulating single cells to express IFN-y, IL-2 and TNF-a in FluoroSpot assays using PBMCs of patient C. Unpaired t test and 1-way ANOVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0017] FIGURES 3A-3J show additional histograms of the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC tissues. FIGURE 3A is a histogram for PanOl. FIGURE 3B is a histogram for Pan02. FIGURE 3C is a histogram for Pan03. FIGURE 3D is a histogram for Pan04. FIGURE 3E is a histogram for Pan05. FIGURE 3F is a histogram for Pan06. FIGURE 3G is a histogram for Pan07. FIGURE 3H is a histogram for Pan08. FIGURE 31 is a histogram for Pan09. FIGURE 3J is a histogram for PanlO.

[0018] FIGURES 4A-4F show predicted HLA Class I binding affinity of eluted peptides from six additional PDAC tissues using the NetMHC4.0 algorithm. The black dot lines represent the 500 nM threshold of high binding affinity. FIGURE 4A shows predicted HLA Class I binding affinity for Pan04. FIGURE 4B shows predicted HLA Class I binding affinity for Pan05. FIGURE 4C shows predicted HLA Class I binding affinity for Pan06. FIGURE 4D shows predicted HLA Class I binding affinity for Pan07. FIGURE 4E shows predicted HLA Class I binding affinity for Pan09. FIGURE 4F shows predicted HLA Class I binding affinity for PanlO. [0019] FIGURES 5A-5B shows the numbers of HLA class I peptides from two PDAC cell lines and percentages of overlapping with the whole peptide pool of PDAC tissues. FIGURE 5A is a Venn graph for Panl0.05. FIGURE 5B is a Venn graph for Pan06.03.

[0020] FIGURE 6 is a graph showing the numbers of HLA class I peptides defined as strong binders for HLA-A0201and HLA-A0301 of Pan04, Pan06, and Pan07 patients and numbers of overlapping peptides among them.

[0021] FIGURES 7A-7B show the validation of selected HLA class I epitopes identified by mass spectrometry in their ability of stimulating T cell responses. FIGURE 7A is a histography showing ability of the synthetic 9-mer peptides in stimulating the IFN-y expression from T cells in PBMCs from HLA-A2 patients. FIGURE 7B is a histography showing ability of the synthetic 9-mer peptides in stimulating the granzyme B expression from T cells in PBMCs from HLA-A2 patients. Unpaired t test and 1-way AN OVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0022] FIGURES 8A-8B show the validation of selected HLA class I epitopes identified by mass spectrometry in their ability of stimulating T cell responses. FIGURE 8A is a histography showing ability of the synthetic 9-mer peptides in stimulating the IFN-y expression from T cells in PBMCs from HLA- A3 patients. FIGURE 8B is a histography showing ability of the synthetic 9-mer peptides in stimulating the granzyme B expression from T cells in PBMCs from HLA-A3 patients. Unpaired t test and 1-way AN OVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0023] FIGURES 9A-9D illustrate the ability of two representative peptides ELOVL1 and LAMB3 in stimulating the IFN-y and granzyme B expression from T cells in a FluoroSpot assay. FIGURE 9A shows the ability of ELOVL1 peptides to induce IFN-y expression. FIGURE 9B shows the ability of ELOVL1 peptides to induce granzyme B expression. FIGURE 9C shows the ability of LAMB3 peptides to induce IFN-y expression. FIGURE 9D shows the ability of LAMB3 peptides to induce granzyme B expression. Unpaired t test and 1-way ANOVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0024] FIGURE 10 shows Venn diagram illustrating the numbers of HLA class I peptides of Pan06 and Pan04 patients and overlapped peptides between patients.

[0025] FIGURES 11A-11C illustrate the ability of selected 9-mer peptides in stimulating IFN- y expression from T cells in PBMCs from patients with different HLA class I types. FIGURE 11A shows IFN-y expression in A2, Al 1 PBMCs. FIGURE 11B shows IFN-y expression in A29, A33 PBMCs. FIGURE 11C shows IFN-y expression in A2, A29 PBMCs. Unpaired t test and 1-way ANOVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0026] FIGURES 12A-12C illustrate the ability of selected 9-mer peptides in stimulating granzyme B expression from T cells in PBMCs from patients with different HLA class I types. FIGURE 12A shows granzyme B expression in A2, Al 1 PBMCs. FIGURE 12B shows granzyme B expression in A29, A33 PBMCs. FIGURE 12C shows granzyme B expression in A2, A29 PBMCs. Unpaired t test and 1-way ANOVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0027] FIGURES 13A-13D are histograms illustrating the numbers of different lengths of peptides affinity purified by anti-HLA Class II antibody assessed from other human PDAC tissues. FIGURE 13A is for Pan04. FIGURE 13B is for Pan 15. FIGURE 13C is for Panl6. FIGURE 13D is for Panl7.

[0028] FIGURES 14A-14B show the relationship between the input of surgical tissue and the amount protein. FIGURE 14A shows the relationship for extracted protein. FIGURE 14B shows the relationship for identified peptides (FIGURE 14B). The black dot lines represent lOOmg input of surgical tissue.

[0029] FIGURES 15A-15D illustrate the expression patterns of LAMC2 and its correlation with survival. FIGURE 15A shows representative LAMC2 immunohistochemistry (IHC) staining images of human PDAC tumors and matched, para-tumoral normal pancreatic tissues, respectively (n = 20). Positive LAMC2 staining in brown. PanIN3, pancreatic intraepithelial neoplasia-3. Scale bar, 50 pm. FIGURE 15B illustrates the LAMC2 expression level in the HPDE normal pancreatic cell line and multiple PDAC cell lines analyzed by Western blotting. GAPDH was blotted as internal control. FIGURE 15C illustrates LAMC2 mRNA expression profiling data of 183 PDAC tissues and 167 normal samples retrieved from the TCGA database and compared by unpaired t test. ****P <0.0001. FIGURE 15D shows Kaplan-Meier survival curves retrieved from Human Protein Atlas and TCGA compared overall survival between PDAC with high and those with low mRNA expression of LAMC2 (High, n = 37, 5-year survival=17%; Low, n = 139, 5-year survival=71%) by log rank test. LAMC2 expression level is determined by “best expression cut off’ FPKM value. p<0.001.

[0030] FIGURES 16A-16D shows that LAMC2203-211 targeting T cells have a cytotoxic activity on PDAC cells in vitro. FIGURE 16A is a graph illustrating the cytotoxicity of infected T cells (GL121-Jurkat, TCRl-Jurkat and TCR2-Jurkat) measured by CytoTox-Fluor™ Cytotoxicity Assay kit (readout as Dead Cell Luminescence) after co-culture with Pancl0.05 at a ratio of 5:1 for 48 hours in T cell medium. FIGURE 16B is a graph illustrating LAMC2 mRNA expression levels in LAMC2KD cells and shCtr cells measured using RT-qPCR. FIGURE 16C shows immunoblots of Western blot of LAMC2 protein in LAMC2KD cells and shCtr cells. GAPDH was blotted as internal control. FIGURE 16D is a graph showing the cytotoxicity of infected Jurkat cells (GL121-Jurkat, TCRl-Jurkat and TCR2-Jurkat) against the LAMC2KD. Jurkat cells and LAMC2KD or shCtr cells co-cultured at 5: 1 ratio. Data are mean ± SD. *P < 0.05, **P < 0.01; ns, not significant, by unpaired t-test.

[0031] FIGURES 17A-17C illustrates how the adoptive transfer of LAMC2203-211 targeting T cells suppresses tumor growth in mice. FIGURE 17A is a schematic representation of the treatment for subcutaneous mouse model: on Day 0, tumor tissues (cubes ~ 2-3mm in diameter) derived from Pancl0.05 cells were implanted subcutaneously into the flank of NSG mouse. After the surgery, mice were randomized into different treatment groups (9 or 10 mice per group) as indicated. On Day 3. Tumor-bearing mice were treated with either PBS, GL121-jurkat, TCRl- Jurkat or TCR2-Jurkat (5x106 cells/mouse) plus rIL-2 (lOOU/mouse) weekly on days indicated. Tumor size was measured by caliper twice a week until Day 45. FIGURE 17B is a graph illustrating tumor growth curve of the mice. FIGURE 17C shows graphs illustrating orthotopic mouse model tumor growth curve. NSG mice were orthotopically implanted with cubes of PDXs (JH029 and JH072) at ~2mm in diameter. Following tumor implantation, mice were randomized into four treatment groups (n = 5 per group) as indicated. Tumor-bearing mice were treated with either PBS, GL121-Jurkat, TCRl-Jurkat or TCR2-Jurkat (5xl06/mouse) plus rIL-2 (lOOU/mouse) weekly, starting on day 10 post tumor implantation. Tumor size was measured by ultrasound imaging until Day 45. Results are shown as mean ± SEM. Two-way ANOVA was used to assess statistical significance. *p < 0.05, **p < 0.01; ns, not significant. All experiments were repeated at least twice.

[0032] FIGURES 18A-18C illustrates how LAMC2 knockdown reduces tumor growth and abolishes LAMC2203-211 targeting T cell-mediated tumor growth suppression in mice. Mouse model establishment and treatment schema are the same as described in FIGURE 18 A, except that mice were treated weekly for 4 times and tumors were measured until day 40. FIGURE 18A is a graph showing growth curve of shCtr and LAMC2KD tumor in mice that received PBS mocktreatment. FIGURE 18B is a graph showing tumor growth curve of shCtr tumors treated with GL121-Jurkat and TCR2-Jurkat (n = 6 mice per group, two tumors on both flanks per mouse). FIGURE 18C is a graph showing tumor growth of LAMC2KD tumors treated with GL121-Jurkat and TCR2-Jurkat (n = 6 mice per group, two tumors on both flanks per mouse). Two-way ANOVA was used to compare. *p < 0.05, **p < 0.01; ns, not significant. All experiments were repeated at least twice.

[0033] FIGURES 19A-19C illustrate the assessment of the expression levels of proteins corresponding to the candidate epitopes in Table 6. FIGURE 19A shows representative images of immunohistochemistry staining of TRAPPCI 1, ORMDL3, MYL12A, ZMYND11 and CTNNBIP1 in human PDAC tumors and matched paratumoral normal pancreatic tissues (n = 5). Scale bar, 50 pm. FIGURE 19B is a graph illustrating protein expression levels in normal human tissues cancer patient according to the Human Protein Atlas (proteinatlas.org). FIGURE 19C is a graph illustrating protein expression levels in cancer patient according to the Human Protein Atlas (proteinatlas . org) .

[0034] FIGURE 20 illustrates a workflow of identifying the LAMC2203-211 epitope specific TCR clonotype. Archived PBMCs were stimulated with the LAMC2203-211 peptides as described in the Method. Following stimulation, CD8+ T cells were positively selected by use of magnetic CD8 Microbeads and subjected to the single-cell V(D)J sequencing. Figure created using BioRender. [0035] FIGURE 21 is a schematic plasmid map of the backbone GL121 lentivirus. TCR with complete a- and [l-chains linked by a P2A element is depicted schematically.

[0036] FIGURE 22 is a graph showing that LAMC2203-211 targeting T cells have cytotoxicity activities on various PDAC cell lines in vitro. Cytotoxicity assay was measured using CellTiter- Glo® Luminescent Cell Viability Assay kit (readout as Live Cell Luminescence), with Panel, Panc7.078, and Pancl0.05 cell lines. Data are mean ± SD. Two-tailed unpaired T test was used for comparison. *p < 0.05, **p < 0.01.

[0037] FIGURE 23 is a schematic representation of the treatment of the orthotopic PDX mouse model related to FIGURE 17.

[0038] FIGURE 24 is a schematic representation of the treatment of the subcutaneously implanted LAMC2KD tumors and shCtr tumors related to FIGURE 18.

[0039] FIGURES 25A-25E illustrate the expression patterns of TMSB10 and its correlation with survival. FIGURE 25A is a graph illustrating TMSB10 peptide inducing polyclonal T-cell activity in HLA matched PBMC. FluoroSpot assays to test ability of selected, HLA class I/II- overlapped peptides in stimulating single cells to express IFN-y, IL-2, and TNF-a. PBMC samples from patients A. Spot forming unit (SFU) is the number of spots per 106 PBMCs. Shown is SFU of each peptide after subtracting that of a negative control peptide; and error bars represent the percentages of deviation. “Unstimulated” indicates the reaction in absence of peptides. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”. Unpaired t-test and 1-way ANOVA were used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001. FIGURE 25B shows representative TMSB10 immunohistochemistry (IHC) staining images of human PDAC tumors and matched paratumoral normal pancreatic tissues, respectively (n = 20). Positive TMSB10 staining in brown. Scale bar, 50 pm. FIGURE 25C is a graph illustrating TMSB10 mRNA expression profiling data of 179 PDAC tissues from the TCGA database and 171 normal samples from GTEx database compared by unpaired t test. ****P <0.0001. FIGURE 25D shows Kaplan-Meier survival curves retrieved from Gene Expression Profiling Interactive Analysis (GEPIA) database compared overall survival between PDAC with high and those with low mRNA expression of TMSB10 (High expression, n = 36, Low expression, n = 142) by log-rank test. TMSB10 expression level is determined by “best expression cut off’ FPKM value. p<0.001. FIGURE 25E shows Kaplan-Meier survival curves illustrating disease free survival.

[0040] FIGURE 26 is a graph illustrating that TMSB 1028-44 targeting T cells have cytotoxicity activities on AsPC-1 cell lines in vitro. The cytotoxicity of infected T cells (GL121- Jurkat, TCRl-Jurkat, TCR2-Jurkat, and TCR4-Jurkat) was measured by CytoTox-Fluor™ Cytotoxicity Assay kit (readout as Dead Cell Luminescence) after co-culture with AsPC-1 at a ratio of 5:1 for 48 hours in T cell medium. Data are mean ± SD. Two-tailed unpaired T-test was used for comparison. *p < 0.05, **p < 0.01.

[0041] FIGURE 27 illustrates the identification of overlapping HLA class I & class II tumor- associated antigen from human PDAC samples. HLA Class I and HLA Class II peptides affinity purified from two patient PDAC tumor specimens were sequenced by Maxquant. Number of total overlapped HLA Class I and HLA Class II peptides between the two patient specimens were indicated above. Six peptide candidates were selected based on their expression level in PDAC tissue and normal pancreatic tissue according Human Protein Atlas.

[0042] FIGURE 28 is a graph illustrating the IFN-y, IL-2 and TNF-a stimulating ability analysis of epitopes shared by HLA class I and class II types in Patient B tumor tissues. TMSB 10 peptide induces polyclonal T-cell activity in HLA matched PBMC. FluoroSpot assays to test ability of selected, HLA class I/II-overlapped peptides in stimulating single cells to express IFN- y, IL-2, and TNF-a. PBMC samples from patients B. Spot forming unit (SFU) is the number of spots per 106 PBMCs. Shown is SFU of each peptide after subtracting that of a negative control peptide; and error bars represent the percentages of deviation. “Unstimulated” indicates the reaction in absence of peptides. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”. Unpaired t-test and 1-way ANOVA were used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0043] FIGURE 29 shows a workflow of identifying the TMSB 1028-44 epitope specific TCR clonotype. Archived PBMCs were stimulated with the TMSB 1028-44 peptides as described in the Method. Following stimulation, CD8+ T cells were positively selected by use of magnetic CD8 Microbeads and subjected to the single-cell V(D)J sequencing. Figure created using BioRender. [0044] FIGURES 30A-30D illustrate the RNA expression levels of TMSB10 among multiple cancer types. FIGURE 30A is a graph illustrating RNA expression levels in normal human tissues (GTEX database). FIGURE 30B is a graph illustrating RNA expression levels in multiple cancer patients (TCGA database) according to the Human Protein Atlas (proteinatlas.org). The RNA summary section shows normal distribution of individual samples across the datasets of multiple RNA-seg analyses visualized with box plots shown as median and 25th and 75th percentiles Points are displayed as outliers if they are above or below 1.5 times the interquartile range. The TMSB10 RNA expression profile across all tumor samples and paired normal tissues on multiple cancer types. FIGURE 30C is a graph illustrating TMSB10 RNA expression levels in normal human tissues and in multiple cancer patients according to the TIMER database (timer.cistrome.org). Each dot represents expression of samples. FIGURE 30D is a graph illustrating screening for TMSB10 expression across various pancreatic cancer cell lines using the Human Protein Atlas (proteinatlas . org) .

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is based on the seminal discovery of HLA-class I and/or HLA- class II restricted or non-restricted peptides that can be presented to T cell receptor (TCR), a vaccine thereof, and methods of use thereof such as methods of treating cancer.

[0046] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0047] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. [0048] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0049] As used herein, the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated. For example, and based on the context, the value can vary up or down by 5-10%. For example, for a value of about 100, means 90 to 110 (or any value between 90 and 110).

[0050] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0052] In one embodiment, the present invention provides an isolated peptide having the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2.

[0053] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to any chain of at least two amino acids, linked by a covalent chemical bound. As used herein polypeptide can refer to the complete amino acid sequence coding for an entire protein or to a portion thereof. A "protein coding sequence" or a sequence that "encodes" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. [0054] The terms "sequence identity" or "percent identity" are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first polypeptide or polynucleotide for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e., overlapping positions) x 100). In some embodiments the length of a reference sequence (e.g., SEQ ID NOs:7-32) aligned for comparison purposes is at least 80% of the length of the comparison sequence, and in some embodiments is at least 90% or 100%. In an embodiment, the two sequences are the same length.

[0055] Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a disclosed sequence and a claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match indicates 100% identity over the length of the reference sequence.

[0056] Polypeptides and polynucleotides that are about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 99.5% or more identical to polypeptides and polynucleotides described herein are embodied within the disclosure.

[0057] For example, a polypeptide can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs:7-32.

[0058] Variants of the disclosed sequences also include peptides, or full-length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties. Illustrative amino acid conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine.

[0059] In another embodiment, the invention provides a polyepitope peptide including one or more HLA-class I and/or a HLA-class II restricted or non-restricted epitope, wherein the one or more epitopes are an antigenic fragment of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, ORMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

[0060] As used herein, the terms “polyepitope peptide”, “multi-epitope peptide” and the like refer to peptide or polypeptide that includes at least two epitopes as describes herein. For example, the polyepitope peptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the epitopes of the invention.

[0061] The term “epitope” refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system. An epitope of a protein such as a tumor antigen preferably comprises a continuous or discontinuous portion of said protein. The terms “epitope”, “antigen peptide”, “antigen epitope”, “immunogenic peptide”, “antigenic fragment” and “MHC binding peptide” can be used interchangeably herein and preferably relate to a representation of an antigen which is capable of eliciting an immune response against the antigen or a cell expressing or comprising and preferably presenting the antigen. An “antigen” according to the invention covers any substance that will elicit an immune response. In particular, an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T-lymphocytes (T cells). According to the present invention, the term “antigen” comprises any molecule which comprises at least one epitope. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction. According to the present invention, any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction is preferably a cellular immune reaction. In the context of the embodiments of the present invention, the antigen is preferably presented by a cell, preferably by an antigen presenting cell which includes a diseased cell, in particular a cancer cell, in the context of MHC molecules, which results in an immune reaction against the antigen. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens include tumor antigens.

[0062] The epitopes described herein are HLA-class I and/or HLA-class II restricted or nonrestricted epitopes.

[0063] The human leukocyte antigen (HLA) system or complex is a complex of genes located on chromosome 6 in humans, and which encode cell-surface proteins responsible for the regulation of the immune system. The HLA system is also known as the human version of the major histocompatibility complex (MHC) found in many animals. HLA genes are highly polymorphic, which means that they have many different alleles, allowing them to fine-tune the adaptive immune system. HLAs corresponding to MHC class I (A, B, and C), all of which are the HLA Classi group, present peptides from inside the cell. These peptides are produced from digested proteins that are broken down in the proteasomes. In general, these particular peptides are small polymers, of about 8-10 amino acids in length. Foreign antigens presented by MHC class I attract T-lymphocytes called killer T-cells (also referred to as CD8-positive or cytotoxic T-cells) that destroy cells. MHC class I proteins associate with p2-microglobulin, which unlike the HLA proteins is encoded by a gene on chromosome 15. HLAs corresponding to MHC class II (DP, DM, DO, DQ, and DR) present antigens from outside of the cell to T-lymphocytes. These particular antigens stimulate the multiplication of T-helper cells (also called CD4-positive T cells), which in turn stimulate antibody-producing B-cells to produce antibodies to that specific antigen. Self-antigens are suppressed by regulatory T cells.

[0064] MHC-restricted antigen recognition, MHC restriction or HLA-restriction, refers to the fact that a T cell can interact with a self-major histocompatibility complex molecule and a foreign peptide bound to it, but will only respond to the antigen when it is bound to a particular MHC molecule. When foreign proteins enter a cell, they are broken into peptides. These peptides or antigens can derive from pathogens such as viruses or intracellular bacteria. Foreign peptides are brought to the surface of the cell and presented to T cells by proteins called the major histocompatibility complex (MHC). During T cell development, T cells go through a selection process in the thymus to ensure that the T cell receptor (TCR) will not recognize MHC molecule presenting self-antigens, i.e., that its affinity is not too high. High affinity means it will be autoreactive, but no affinity means it will not bind strongly enough to the MHC. The selection process results in developed T cells with specific TCRs that might only respond to certain MHC molecules but not others. The fact that the TCR will recognize only some MHC molecules but not others contribute to "MHC restriction". The biological reason of MHC restriction is to prevent supernumerary wandering lymphocytes generation, hence energy saving and economy of cellbuilding materials. T-cells are a type of lymphocyte that is significant in the immune system to activate other immune cells. T-cells will recognize foreign peptides through T-cell receptors (TCRs) on the surface of the T cells, and then perform different roles depending on the type of T cell they are in order to defend the host from the foreign peptide, which may have come from pathogens like bacteria, viruses or parasites. Enforcing the restriction that T cells are activated by peptide antigens only when the antigens are bound to self-MHC molecules, MHC restriction adds another dimension to the specificity of T cell receptors so that an antigen is recognized only as peptide-MHC complexes. MHC restriction in T cells occurs during their development in the thymus, specifically positive selection. Only the thymocytes (developing T cells in the thymus) that are capable of binding, with an appropriate affinity, with the MHC molecules can receive a survival signal and go on to the next level of selection. MHC restriction is significant for T cells to function properly when it leaves the thymus because it allows T cell receptors to bind to MHC and detect cells that are infected by intracellular pathogens, viral proteins and bearing genetic defects.

[0065] In one aspect, the epitopes have the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2. In various aspects, the epitopes have the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

[0066] In an additional embodiment, the invention provides an isolated T cell including a T cell receptor (TCR) having a binding affinity to an HLA-class I and/or HLA-class II restricted or nonrestricted epitope, wherein the epitope is an antigenic fragment of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, ORMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

[0067] T cells are a type of lymphocyte, one of the important white blood cells of the immune system that play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. T cells are bom from hematopoietic stem cells, found in the bone marrow. Developing T cells then migrate to the thymus gland to develop (or mature). T cells derive their name from the thymus. After migration to the thymus, the precursor cells mature into several distinct types of T cells. T cell differentiation also continues after they have left the thymus. Groups of specific, differentiated T cell subtypes have a variety of important functions in controlling and shaping the immune response. One of these functions is immune-mediated cell death, and it is carried out by two major subtypes: CD8+ "killer" and CD4+ "helper" T cells. CD8+ T cells, also known as "killer T cells", are cytotoxic - they are able to directly kill virus-infected cells, as well as cancer cells. CD8+ T cells are also able to use small signaling proteins, known as cytokines, to recruit other types of cells when mounting an immune response. A different population of T cells, the CD4+ T cells, function as "helper cells". Unlike CD8+ killer T cells, the CD4+ helper T (TH) cells function by further activating memory B cells and cytotoxic T cells, which leads to a larger immune response. The specific adaptive immune response regulated by the TH cell depends on its subtype, which is distinguished by the types of cytokines they secrete.

[0068] T-cell receptor (TCR) can be engineered, and used in TCR-engineered T cells, which are a novel option for adoptive cell therapy used for the treatment of several advanced forms of cancer. For example, a TCR can be engineered to have a binding affinity to an antigenic fragment of a protein or gene product.

[0069] The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. A portion, a part or a fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, a part or a fragment of an epitope, peptide or protein is preferably immunologically equivalent to the epitope, peptide or protein it is derived from. In the context of the present invention, a “part” of a structure such as an amino acid sequence preferably comprises, preferably consists of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% of the entire structure or amino acid sequence. A TCR as described herein is for example engineered to have a binding affinity to a continuous element of one of the antigens or epitopes described herein, such as any antigenic epitope derived from protein or gene product encoded by COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, 0RMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, or any of the genes listed in Table 2. Non-limiting examples of such antigenic fragment or epitope include the epitopes having the amino acid sequence of any of SEQ ID NOs:7-32 and any of the peptides listed in Table 2.

[0070] In one aspect, the T cell is an engineered T cell. In another aspect, the epitope has the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2. In various aspects, the epitope has the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

[0071] In a further embodiment, the invention provides a vaccine including one or more HLA- class I and/or HLA-class II restricted or non-restricted epitopes, wherein the epitope are antigenic fragments of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, ORMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

[0072] According to the invention, the term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, in particular a cellular immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell. A vaccine may be used for the prevention or treatment of a disease. The term “individualized cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.

[0073] In one aspect, the vaccine includes a lipid nanoparticle for presenting the one or more epitopes to antigen presenting immune cells.

[0074] Adjuvants are often used to modify or augment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus providing increased immunity to a particular disease. Adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called pathogen-associated molecular patterns, which include liposomes, lipopolysaccharide, molecular cages for antigens, components of bacterial cell walls, and endocytosed nucleic acids such as RNA, double-stranded RNA, single-stranded DNA, and unmethylated CpG dinucleotide-containing DNA. Because immune systems have evolved to recognize these specific antigenic moieties, the presence of an adjuvant in conjunction with the vaccine can greatly increase the innate immune response to the antigen by augmenting the activities of dendritic cells, lymphocytes, and macrophages by mimicking a natural infection.

[0075] The vaccine described herein can be formulated with a lipid nanoparticle as an adjuvant to enhance the presentation of the antigens to antigen presenting cells, and therefore to increase the immune response induce by the antigens.

[0076] Almost all cell types can present antigens in some way. They are found in a variety of tissue types. Professional antigen-presenting cells, including macrophages, B cells and dendritic cells, present foreign antigens to helper T cells, while virus-infected cells (or cancer cells) can present antigens originating inside the cell to cytotoxic T cells. In addition to the MHC family of proteins, antigen presentation relies on other specialized signaling molecules on the surfaces of both APCs and T cells. Antigen-presenting cells are vital for effective adaptive immune response, as the functioning of both cytotoxic and helper T cells is dependent on APCs. Antigen presentation allows for specificity of adaptive immunity and can contribute to immune responses against both intracellular and extracellular pathogens. It is also involved in defense against tumors. Some cancer therapies involve the creation of artificial APCs to prime the adaptive immune system to target malignant cells.

[0077] In another aspect, the one or more presented epitopes have the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2. In various aspects, the epitopes have the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30. [0078] In one embodiment, the present invention provides a method of treating cancer in a subject including administering to the subject one or more of the peptides described herein, one or more of the polyepitope peptides described herein, the T cell described herein, or the vaccine described herein, thereby treating cancer in the subject.

[0079] The term “subject” as used herein refers to any individual or patient to which the subject methods are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0080] The term "treatment" is used interchangeably herein with the term "therapeutic method" and refers to both 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic conditions or disorder, and 2) and prophylactic/ preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventive measures).

[0081] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., treating cancer). The effective amount can be determined as described herein.

[0082] The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration.

[0083] By “pharmaceutical composition” it is meant that the peptides, polyepitope peptides, vaccine or T cell described herein are formulated with a “pharmaceutically acceptable” carrier, diluent or excipient that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof, nor to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, for example Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3 -pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Examples of carrier include, but are not limited to, liposome, nanoparticles, ointment, micelles, microsphere, microparticle, cream, emulsion, and gel. Examples of excipient include, but are not limited to, anti-adherents such as magnesium stearate, binders such as saccharides and their derivatives (sucrose, lactose, starches, cellulose, sugar alcohols and the like) protein like gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate and parabens. Examples of diluent include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil and dimethyl sulfoxide (DMSO).

[0084] In one aspect, the HLA-class I and/or HLA-class II restricted or non-restricted peptides or the polyepitope peptides thereof induces polyfunctional T cells in the subject.

[0085] The immune system is a system of biological structures and processes within an organism that protects against disease. This system is a diffuse, complex network of interacting cells, cell products, and cell-forming tissues that protects the body from pathogens and other foreign substances, destroys infected and malignant cells, and removes cellular debris: the system includes the thymus, spleen, lymph nodes and lymph tissue, stem cells, white blood cells, antibodies, and lymphokines. B cells or B lymphocytes are a type of lymphocyte in the humoral immunity of the adaptive immune system and are important for immune surveillance. T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. There are two major subtypes of T cells: the killer T cell and the helper T cell. In addition, there are suppressor T cells which have a role in modulating immune response. Killer T cells only recognize antigens coupled to Class I MHC molecules, while helper T cells only recognize antigens coupled to Class II MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cell. A third minor subtype are the

T cells that recognize intact antigens that are not bound to MHC receptors. In contrast, the B cell antigen-specific receptor is an antibody molecule on the B cell surface and recognizes whole pathogens without any need for antigen processing. Each lineage of B cell expresses a different antibody, so the complete set of B cell antigen receptors represent all the antibodies that the body can manufacture.

[0086] A “cellular immune response”, a “cellular response”, a “cellular response against an antigen” or a similar term is meant to include a cellular response directed to cells characterized by presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T-lymphocytes which act as either “helpers” or “killers”. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as cancer cells, preventing the production of more diseased cells. In preferred embodiments, the present invention involves the stimulation of an anti-tumor CTL response against tumor cells expressing one or more tumor expressed antigens and preferably presenting such tumor expressed antigens with class I MHC.

[0087] The terms “immunoreactive cell” “immune cells” or “immune effector cells” in the context of the present invention relate to a cell which exerts effector functions during an immune reaction. An “immunoreactive cell” preferably is capable of binding an antigen or a cell characterized by presentation of an antigen, or an antigen peptide derived from an antigen and mediating an immune response. For example, such cells secrete cytokines and/or chemokines, secrete antibodies, recognize cancerous cells, and optionally eliminate such cells. For example, immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

[0088] In some aspects, inducing polyfunctional T cells includes stimulating a T cell response and/or stimulating T cell expression of effector T cell cytokine. [0089] In various aspects, the effector T cell cytokines include IFNy, IL-2 and/or TNFa.

[0090] Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses. IFNs belong to the large class of proteins known as cytokines, molecules used for communication between cells to trigger the protective defenses of the immune system that help eradicate pathogens. Examples of IFNs include IFN-a, IFN-ff IFN-c, IFN-K and IFN-y.

[0091] The tumor necrosis factor (TNF) superfamily refers to a superfamily of cytokines that can cause cell death. All TNF superfamily members form homotrimeric (or heterotrimeric in the case of LT-alpha/beta) complexes that are recognized by their specific receptors. Examples of TNF super family members include TNF, TNF-[3, lymphotoxin-alpha, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L and TRAIL.

[0092] Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that are expressed and secreted by white blood cells (leukocytes) as well as some other body cells. The human genome encodes more than 50 interleukins and related proteins. The function of the immune system primarily depends on interleukins, and rare deficiencies of a number of them have been described, all featuring autoimmune diseases or immune deficiency. The majority of interleukins are synthesized by CD4 helper T-lymphocyte, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, and hematopoietic cells.

[0093] T lymphocytes regulate the growth and differentiation of T cells and certain B cells through the release of secreted protein factors, which include interleukin 2 (IL2). IL2 is a lymphokine that induces the proliferation of responsive T cells. In addition, it acts on some B cells, via receptor-specific binding, as a growth factor and antibody production stimulant. The protein is secreted as a single glycosylated polypeptide, and cleavage of a signal sequence is required for its activity.

[0094] In other aspects, stimulating a T cell response include stimulating cytotoxic T cell cytokines.

[0095] In various aspects, the cytotoxic T cell cytokines include IFNy and/or granzyme B. [0096] Granzyme B (GrB) is one of the serine protease granzymes most commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells along with the pore forming protein perforin to mediate apoptosis in target cells. Granzyme B has also been found to be produced by a wide range of non-cytotoxic cells ranging from basophils and mast cells to smooth muscle cells. The secondary functions of granzyme B are also numerous. Granzyme B has shown to be involved in inducing inflammation by stimulating cytokine release and is also involved in extracellular matrix remodeling.

[0097] In another aspect, the cancer is a cancer expressing an epitope having the amino acid sequence of any of SEQ ID NOs:7-32, or of any of the peptides listed in Table 2. In various aspects, the cancer is a cancer expressing an epitope having the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

[0098] The epitopes described herein are epitopes that were recurrently found presented by antigen presenting cells of patients with cancer. They are expected to be potent at inducing T cells response in any patient having a cancer whose cell express said epitope.

[0099] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. In 2015, about 90.5 million people had cancer, about 14.1 million new cases occur a year and it caused about 8.8 million deaths (15.7% of deaths). The most common types of cancer in males are lung cancer, prostate cancer, colorectal cancer and stomach cancer. In females, the most common types are breast cancer, colorectal cancer, lung cancer and cervical cancer. The term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 100 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure environmental pollutant, tobacco and or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof. As used herein, “neoplasm” or “tumor” including grammatical variations thereof, means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, pancreatic, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

[0100] Exemplary cancers described by the national cancer institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom's; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non- Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland' Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma (Osteosarcoma Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom's Macro globulinemia; and Wilms' Tumor.

[0101] In some aspect, the cancer is pancreatic cancer.

[0102] In various aspects, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). [0103] In one aspect, the method further includes administering to the subject an anti-cancer treatment.

[0104] In some aspects administration can be in combination with one or more additional therapeutic agents. The phrases “combination therapy”, “combined with” and the like refer to the use of more than one medication or treatment simultaneously to increase the response. The compositions of the present invention might for example be used in combination with other drugs or treatment in use to treat cancer. Specifically, the administration of peptides, polyepitope peptides, vaccine or T cell to a subject can be in combination with any other relevant anti-cancer therapy. Such therapies can be administered prior to, simultaneously with, or following administration of the composition of the present invention. [0105] The term “anti-cancer therapy” or “anti-cancer treatment” as used herein is meant to refer to any treatment that can be used to treat cancer, such as surgery, radiotherapy, chemotherapy, immunotherapy, and checkpoint inhibitor therapy.

[0106] Examples of chemotherapy include treatment with a chemotherapeutic, cytotoxic or antineoplastic agents including, but not limited to, (i) anti-microtubules agents comprising vinca alkaloids (vinblastine, vincristine, vinflunine, vindesine, and vinorelbine), taxanes (cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel), epothilones (ixabepilone), and podophyllotoxin (etoposide and teniposide); (ii) antimetabolite agents comprising anti-folates (aminopterin, methotrexate, pemetrexed, pralatrexate, and raltitrexed), and deoxynucleoside analogues (azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, doxifhiridine, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, mercaptopurine, nelarabine, pentostatin, tegafur, and thioguanine); (iii) topoisomerase inhibitors comprising Topoisomerase I inhibitors (belotecan, camptothecin, cositecan, gimatecan, exatecan, irinotecan, lurtotecan, silatecan, topotecan, and rubitecan) and Topoisomerase II inhibitors (aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicinm, merbarone, mitoxantrone, novobiocin, pirarubicin, teniposide, valrubicin, and zorubicin); (iv) alkylating agents comprising nitrogen mustards (bendamustine, busulfan, chlorambucil, cyclophosphamide, estramustine phosphate, ifosamide, mechlorethamine, melphalan, prednimustine, trofosfamide, and uramustine), nitrosoureas (carmustine (BCNU), fotemustine, lomustine (CCNU), N-Nitroso-N-methylurea (MNU), nimustine, ranimustine semustine (MeCCNU), and streptozotocin), platinum-based (cisplatin, carboplatin, dicycloplatin, nedaplatin, oxaliplatin and satraplatin), aziridines (carboquone, thiotepa, mytomycin, diaziquone (AZQ), triaziquone and triethylenemelamine), alkyl sulfonates (busulfan , mannosulfan, and 1WEST\289674921.1 MINO1240 treosulfan), non-classical alkylating agents (hydrazines, procarbazine, triazenes, hexamethylmelamine, altretamine, mitobronitol, and pipobroman), tetrazines (dacarbazine, mitozolomide and temozolomide); (v) anthracyclines agents comprising doxorubicin and daunorubicin. Derivatives of these compounds include epirubicin and idarubicin; pirarubicin, aclarubicin, and mitoxantrone, bleomycins, mitomycin C, mitoxantrone, and actinomycin; (vi) enzyme inhibitors agents comprising FI inhibitor (Tipifarnib), CDK inhibitors (Abemaciclib, Alvocidib, Palbociclib, Ribociclib, and Seliciclib), PrI inhibitor (Bortezomib, Carfilzomib, and Ixazomib), Phi inhibitor (Anagrelide), IMPDI inhibitor (Tiazofurin), LI inhibitor (Masoprocol), PARP inhibitor (Niraparib, Olaparib, Rucaparib), HDAC inhibitor (Belinostat, Panobinostat, Romidepsin, Vorinostat), and PIKI inhibitor (Idelalisib); (vii) receptor antagonist agent comprising ERA receptor antagonist (Atrasentan), Retinoid X receptor antagonist (Bexarotene), Sex steroid receptor antagonist (Testolactone); (viii) ungrouped agent comprising Amsacrine, Trabectedin, Retinoids (Alitretinoin Tretinoin) Arsenic trioxide, Asparagine depleters (Asparaginase/Pegaspargase), Celecoxib, Demecolcine Elesclomol, Elsamitrucin, Etoglucid, Lonidamine, Lucanthone, Mitoguazone, Mitotane, Oblimersen, Omacetaxine mepesuccinate, and Eribulin.

[0107] Examples of immunotherapy include treatment with antibodies including, but not limited to, alemtuzumab, Avastin (bevacizumab), Bexxar (tositumomab), CDP 870, and CEA- Scan (arcitumomab), denosumab, Erbitux (cetuximab), Herceptin (trastuzumab), Humira (adalimumab), IMC-IIF 8, LeukoScan (sulesomab), MabCampath (alemtuzumab), Mab Thera (Rituximab), matuzumab, Mylotarg (gemtuzumab oxogamicin), natalizumab, NeutroSpec (Technetium (99mTc) fanolesomab), panitumamab, Panorex (Edrecolomab), ProstaScint (Indium- Ill labeled Capromab Pendetide), Raptiva (efalizumab), Remicade (infliximab), ReoPro (abciximab), rituximab, Simulect (basiliximab), Synagis (palivizumab), TheraCIM hR3, tocilizumab, Tysabri (natalizumab), Verluma (nofetumomab), Xolair (omalizumab), Zenapax (dacliximab), Zevalin (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), Gilotrif (afatinib), Lynparza (olaparib), Perjeta (pertuzumab), Otdivo (nivolumab), Bosulif (bosutinib), Cabometyx (cabozantinib), trastuzumab -dkst (Ogivri), Sutent (sunitinib malate), Adcetris (brentuximab vedotin), Alecensa (alectinib), Calquence (acalabrutinib), Yescarta (ciloleucel), Verzenio (abemaciclib), Keytruda (pembrolizumab), Aliqopa (copanlisib), Nerlynx (neratinib), Imfinzi (durvalumab), Darzalex (daratumumab), Tecentriq (atezolizumab), and Tarceva (erlotinib).

[0108] “Checkpoint inhibitor therapy” is a form of cancer treatment that uses immune checkpoints which affect immune system functioning. Immune checkpoints can be stimulatory or inhibitory. Tumors can use these checkpoints to protect themselves from immune system attacks. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function. Checkpoint proteins include programmed cell death 1 protein (PDCD1, PD-1; also known as CD279) and its ligand, PD-1 ligand 1 (PD-L1, CD274), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), A2AR (Adenosine A2A receptor), B7-H3 (or CD276), B7-H4 (or VTCN1), BTLA (B and T Lymphocyte Attenuator, or CD272), IDO (Indoleamine 2, 3 -dioxygenase), KIR (Killercell Immunoglobulin-like Receptor), LAG3 (Lymphocyte Activation Gene-3), TIM-3 (T- cell Immunoglobulin domain and Mucin domain 3), and VISTA (V-domain Ig suppressor of T cell activation). Immunotherapy also includes the use of adoptive transfer of genetically engineered T cells, modified to recognize and eliminate cancer cells specifically. For example, T cells can be genetically modified to stably express on their surface chimeric antigen receptors (CAR). CAR are synthetic proteins comprising of a signaling endodomain, consisting of an intracellular domain of the CD3-zeta chain, a transmembrane domain, and an extracellular domain consisting of the antigen recognition fragment of a monoclonal antibody which gives the receptor its specificity for tumor associated antigen (e.g., an scFv, or single chain variable region fragment). Upon interaction with the target cancer cell expressing the scFv’s cognate antigen, the chimeric antigen receptor triggers an intracellular signaling leading to T-cell activation and to a cytotoxic immune response against tumor cells. Such therapies have been shown to be efficient against relapsed/refractory disease. Additionally, CAR-T cells can be engineered to include co- stimulatory receptor that enhance the T-cell-mediated cytotoxic activity. Furthermore, CAR-T cells can be engineered to produce and deliver protein or agent of interest in the tumor microenvironment.

[0109] In some aspects, the anti-cancer treatment is selected from the group consisting of gemcitabine, folfirinox, erlotinib, nab-paclitaxel, liposomal irinotecan, and olaparib.

[0110] Presented below are examples discussing the peptides and multiepitope peptides described herein, contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES EXAMPLE 1

MATERIAL AND METHODS

[0111] Human cancer cell lines and primary human tissues [0112] Pane 10.05 cells, W6/32 cells, and IVA12 cells, T2 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained by following the protocols suggested by ATCC. Pane 06.03 cells had been kept in the laboratory since the establishment of the cell lines. Pane 10.05 and Pane 06.03 are the two cell lines that the irradiated, allogeneic GM- CSF secreting whole cell vaccine (GV AX) derived from. T2-A1 and T2-A3 were genetically modified from T2 cells, a human B and T lymphoblast hybrid expressing only the HLA-A2 allele, to express the HLA-A1 and HLA-A3 allele. Human pancreatic ductal adenocarcinoma (PDAC) resection specimens were obtained from the patients who underwent surgery at the Johns Hopkins Hospital under the Johns Hopkins Medical Institution (JHMI) Institutional Review Board (IRB) approved protocol (IRB00244430) which allows the access of de-identified tumor specimens and peripheral blood mononuclear cells (PBMCs) archived from clinical trial participants who consented for using their specimens for research. HLA types, which were the results of PCR tests performed at the clinical laboratory at the Johns Hopkins Medicine, were also part of the archived database associated with the above IRB protocol. Information on biospecimens including PDAC tumor tissues and PBMC was summarized in Table 1.

[0113] Preparation of antibody-conjugated affinity purification columns

[0114] The antibody-conjugated affinity purification columns were prepared by using a modified protocol. Briefly, W6/32 cells were cultured for pan-HLA-I (A, B, C) antibody and IVA12 cells for pan-HLA-II (DR, DP, DQ) antibody, respectively, and the supernatant was collected from the cultures. There is no crossreactivity of this IVA12 antibody towards HLA-I molecules The supernatants which were diluted in the Pierce Protein A or Protein G binding buffer (Thermo Scientific, Waltham, MA) were applied to the columns packed with the Pierce Protein A Plus Agarose for HLA-I antibodies or Protein G Plus Agarose for HLA-II antibodies (Thermo Scientific), respectively. After washing the columns with the binding buffer and subsequently with the 0.2M sodium borate buffer (pH 9), agarose beads were cross-linked with dimethyl pimelimidate (Thermo Scientific) at the final concentration of 20mM in the sodium borate buffer. After the agarose beads were rotated for 2 hours(h) in the 2.5X beads volumes of 200mM ethanolamine (pH 8), they were washed with the binding buffer and stored in the phosphate- buffered saline at 4°C.

[0115] Purification of HLA bound peptides [0116] The procedures for purification of HLA bound peptides were modified from those used in the following published studies. In brief, the tissues weighted between 100 mg and 1000 mg (Table 1) were immediately frozen in liquid nitrogen after surgical resection and stored at -80°C until the experiment. In preliminary experiments, specimens below 100 mg yielded a suboptimal number of unique peptides while more than 100 mg did not yield higher numbers of peptides (FIGURES 14A and 14B). In contrast, more than 1000 mg yielded peptides that did not peak at 9 mer. The tissue samples used in this study weigh between 100 mg and 680 mg. They were grounded using a mortar and a pestle and incubated in 1-2 ml Pierce IP lysis buffer (Thermo Scientific) containing a Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) at 4 °C with constant agitation for 2 h at 4 °C. To lyse cultured cancer cells, 10⁸ cells were pelleted and lysed in 1ml lysis buffer for 1 h. After 30 min centrifugation at 16000g, the supernatant was incubated with unconjugated Protein A beads at 4 °C for 1 h to block the non-specific binding. Then, the supernatants were incubated with the pan-HLA-I antibody-conjugated Protein A beads at the 1/1 Oth lysate volume overnight. After centrifugation, the beads were washed; and then HLA- I bound peptides were eluted as described below. If HLA-II bound peptides were to be purified, after the protein lysate was incubated with the HLA-I antibody conjugated beads to remove the HLA-I bound peptides, the flow-through was used for the isolation of HLA-II bound peptides with the pan-HLA-II antibody-conjugated Protein G beads followed by washing as described above. For peptide elution, The HLA antibody-conjugated beads were washed with Buffer A containing 150 mM NaCl, 20 mM Tris-HCl at a 10X beads volume, Buffer B containing 400 mM NaCl, 20 mM Tris- HC1 at a 10X beads volume, Buffer A at a 10X beads volume again, and 20 mM Tris- HCl (pH 8) at a 7X beads volume twice at 4 °C. HLA molecules were eluted at room temperature by 500 pl of 0.1 N acetic acid (pH 3) for 15 minutes. Eluted peptides were loaded to the Sep-Pak C18 3cc/200mg Vac Cartridge (Waters, Milford, MA) which was first activated with 1 mL of methanol and washed with 1 mL 0.1% Trifluoroacetic acid (TFA) three times. The flow-through was repeatedly loaded to the cartridges two more times. The cartridges were washed twice with 300ul of 0.1% TFA. After washing, the peptides were eluted for three times with 400 ul, 300 ul, and 300ul of 80% Acetonitrile in 0.1% TFA, respectively, into a clean 1.5 mL Eppendorf tube. The eluted samples were dried thoroughly at 30°C using vacuum centrifugation and then stored at - 80°C. [0117] LC-MS/MS analysis of HLA peptides

[0118] Above samples were rehydrated in 20 ul of 2% acetonitrile, 0.1% formic acid and placed in an EasyLC autosampler and nanoLC system coupled to an Orbitrap Lumos mass spectrometer (Thermo Fisher). 10 ul of the sample was injected onto a trap column at 5ul/min and then eluted into the mass spectrometer at 300 nl/min over a 90-minute gradient from 2% acetonitrile to 90% acetonitrile in 0.1% formic acid. An in house made nanoLC column (75um ID x 250mm packed with ReproSil-Pur 120 C18-AQ 3um particles (Dr. Maisch, Germany)) was used to separate the peptides. The mass spectrometer was operated at a resolution of 120,000 for MS and 30,000 for MS2. The peptides were fragmented with an isolation window of 1.6 Daltons and collision energy of 30% NCE via higher-energy C-trap dissociation (HCD). As many peptides as possible in a 3- second cycle having a charge of 2-6 were fragmented before doing the next MS precursor scan and precursors which had been previously fragmented were dynamically excluded for 15 seconds. The AGC target for MS was set to 4e5 ions with a maximum injection time of 50 milliseconds and MS2 was set to le5 ions and 100 milliseconds maximum. The precursor masses were subjected to calibration on the fly using the Easy-IC fluoranthene lock mass system.

[0119] MS data analysis of HLA peptides

[0120] Andromeda of the MaxQuant computational platform, a peptide search engine integrated into the MaxQuant environment (Max Planck Institute of Biochemistry, Munich, Germany) was used to search the peak lists against the UniProt databases (Human 93,609 entries, Feb 2018). The settings were used as suggested in the literature. Briefly, the second peptide identification option in Andromeda was enabled. Enzyme specificity was set as unspecific. A false discovery rate of 0.01 was minimally required. The initially allowed mass deviation of the precursor ion was set to 6 p.p.m. The maximum fragment mass deviation was set to 20 p.p.m. 10 out of the 12 processed PDAC samples were chosen after excluding Pan03 and Pan08, which may have non-specific HLA-binding peptides as the lengths of the peptides from these samples did not peak unimodally (FIGURES 3A-3J). the peptide sequences that were considered to be reverse sequences or contaminants were filtered out by MaxQuant. NetMHC-4.0 and NetMHCIIpan-4.0 (Department of Health Technology, Lyngby, Denmark) were used to predict binding affinities of peptides. [0121] FluoroSpot assay

[0122] The peptides were synthesized and purified to > 95% purity by Peptide 2.0 (Chantilly, VA) according to the sequences identified by MaxQuant. Peptides were stocked in 100% DMSO and diluted in the cell culture medium to yield a final peptide concentration at 10 ng/ml. Archived, cryopreserved PBMCs were recovered and immediately subjected to the FluoroSpot assay. 2xl0⁵ PBMCs per well were plated into a 96-well FluoroSpot assay plate from the Human IFN-y /Granzyme B FluoroSpotPLUS kit or the Human IFN-y/IL-2/TNF-a FluoroSpotPLUS kit (Mabtech, Cincinnati, OH). Peptides of interest at a concentration of 2 pg/ml were incubated with PBMCs according to the manufacturer’s instruction. Positive controls were PBMCs stimulated with anti-CD3/anti-CD28 antibodies (Mabtech, Cincinnati, OH) or the CEF peptides (Immunospot, Cleveland, OH). Negative controls were PBMC without stimulation. Cytokines produced by PBMCs following peptide stimulation were captured by their specific antibodies conjugated by different fluorescences. Plates were read by an AID iSpot Spectrum reader (Autoimmun Diagnostika GmbH), and the results were processed by the software provided by the manufacturer. Spots that expressed IFN-y or Granzyme B were counted separately. Spots expressing one, two, or all three cytokines among IFN-y, IL-2, and TNF-a were counted, respectively.

[0123] Peptide/MHC Binding Assay

[0124] The CEF peptides were used as positive controls (Bio-Synthesis, Lewisville, TX) including CEF1 (GILGFVFTL, SEQ ID NO:1) and CEF20 (NLVPMVATV, SEQ ID NO:2) for HLA-A2, CEF24 (VSDGGPNLY, SEQ ID NOG) and CEF25 (CTELKLSDY, SEQ ID NO:4) for HLA-A1, and CEF4 (RVLSFIKGTK, SEQ ID NOG) and CEF26 (ILRGSVAHK, SEQ ID NO:6) for HLA-A3. T2 cells are mono-allelic with HLA-A2. T2-A1, T2-A3, and T2-A11 cells are bi- allelic as they intrinsically express a low amount of HLA-A2 in addition to HLA-A1 , A3, and Al 1 , respectively. T2 cells expressing the HLA molecule of interest were resuspended in serum-free AimV medium (Fisher Scientific, Waltham, MA) to a concentration of 10⁶ cells/ml and pulsed with P-2 microglobulin (final concentration at 3ug/ml, Sigma- Aldrich, St. Louis, MO) and peptide (final concentration at 50ug/ml) at room temperature overnight. Cell surface MHC molecules stabilized by the peptide binding were quantified by a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA) with anti-HLA-A2, Al, or A3 mouse monoclonal antibodies (One Lambda, West Hills, CA) as primary antibodies, respectively, and a rabbit anti-mouse FITC-conjugated IgG secondary antibody (Dako, Santa Clara, CA). Dead cells were excluded by being stained with the Live Dead Aqua Dead Cell Kit (Invitrogen). Flow cytometry results were analyzed using the CytExpert software (Beckman Coulter) and were presented as an increase in mean fluorescence intensity (MFI) of cells that were bound with the tested peptide compared to cells without peptide.

[0125] Comparison of the peptide sequences with DNA sequencing

[0126] The Novor software (Rapid Novor Inc, Kitchener, Canada) was used for de novo peptide sequencing, database searching, characterizing unspecific PTMs, and detecting peptide variant sequences according to the user’s manual. After the sequences of peptides were obtained by MS, they were uploaded to the MS-Homology portal (University of California, San Francisco, prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=mshomology). The number of amino acid differences allowed was set to be one. The protein identities of the resulted peptide sequences were obtained. The Mutalyzer software (Leiden University Medical Center, Leiden, Netherlands, mutalyzer.nl/) was used to identify the amino acid changes by comparing the resulted peptide sequences to the wide-type protein sequences. Finally, the peptide variant sequences were compared with the translated protein sequences from the mutated nucleotide sequences according to WES.

[0127] Statistical analyses

[0128] All statistical analyses and most of graphs were performed using GraphPad Prism software (GraphPad Software). Venn’s diagrams were drawn with VENNY2.1 (CNB-CSIC, Madrid, Spain). The mean fluorescent intensities or mean values of spot forming units (SFU) in the FluoroSpot assay were compared by Welch’s t-test for two-group comparisons and by oneway ANOVA for multiple group comparisons. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”. Because SFUs of negative control peptides vary significantly among different samples, the comparison was made between stimulated and unstimulated peptide/samples. A p- value of less than 0.05 was considered statistically significant. EXAMPLE 2

RESULTS

[0129] HLA class I (HLA-I) and HLA class II (HLA-II)-restricted peptides, respectively, were isolated from tissues of human PDAC, a low-TMB tumor, by using the pan-HLA-I or pan-HLA- II affinity purification column and identified T cell epitopes by peptidome analysis with mass spectrometry (MS). Bioinformatics analysis identified 553 and 1709 HLA-I bound peptides from two human PDAC cell lines, Pancl0.05 and Panc6.03, respectively (FIGURE 1A), and similar numbers of HLA-I bound peptides from 12 surgically resected human PDAC tissues (Table 1; Table 2; FIGURES 1B-C; FIGURES 3A-3J). The numbers of peptides with different lengths peaked at 9 amino-acid, an anticipated length of HLA-I bound peptides. 9-mer peptides from 8 PDAC specimens whose HLA types were available were filtered and their binding affinity to their corresponding HLA-I types was predicted by using NetMHC-4.0. However, the results suggested that the predicting algorithm may have missed many HLA-binding peptides (FIGURE ID; FIGURES 3A-3J). Interestingly, it was found that eluted epitopes were shared among different PDACs as well as PDAC cell lines (Table 3; FIGURES 5A-5B and 6). For further validation, eight shared peptides were chosen (FIGURE IE), which were among predicted high-affinity binding peptides shared by multiple patients (FIGURE IF).

[0130] Surprisingly, T cell response as demonstrated by the expression of either IFN-y or granzyme B or both was significantly stimulated by these peptides not only in the PBMC from at least one of two patients whose tumors were used for identifying these peptides, but also in those from HLA type-unmatched patients (FIGURES 7A-7B, 8A-8B and 9A-9D). Thus, five peptides shared by multiple HLA-A2 PDACs and seven peptides shared by multiple HLA-A29 PDACs were selected (FIGURE 10) and examined their binding to HLA molecules in the T2-b inding assay (FIGURE 1G). The results showed that the peptides have a specific binding to HLA-A2 and A29, respectively; however, five HLA-A2 peptides also bound to HLA- A3 (p<0.01, FIGURE 1H) and seven HLA-A29 peptides bound to HLA-Al(p<0.05, FIGURE II). Note that T2 cells used were not reported to bind A2 peptides and A29 peptides, respectively. These peptides are not predicted to bind HLA-A1 or A3 according to NetMHC (Table 4), either. Moreover, the A29 peptides and the A2 peptides were able to stimulate T cells from an HLA-(A2, Al 1) patient and an HLA-(A29, A33) patient, respectively (FIGURES 11A-11C and 12A-12C). [0131] After the use the pan-HLA class I affinity purification column to bind the HLA-I peptides in the lysate of PDAC tissues, the flow-through for HLA-II peptide isolation was subjected to the pan-HLA-II affinity purification column. The numbers of HLA-II peptides with different lengths peaked at 14-16 amino acids (FIGURES 2A-2B; FIGURES 13A-13D). It was found that HLA-II and HLA-I epitopes purified separately from the same PDAC specimens frequently contained overlapped peptide sequences (FIGURE 2C). Next, 30 peptides shared between two PDACs were selected and eluted from both HLA-I and HLA-II affinity purification columns, respectively, and according to similar criteria in FIGURE IE, they were narrowed down to 6 peptides which binding to HLA-II could be predicted by NetMHCIIpan-4.0 and NetMHC-4.0 according to the patients’ known HLA-II types. 12 to 21-mer peptides were then synthesized according to the core MHC binding sequences that were predicted by NetMHCIIpan (Table 5) and examined the ability of these synthetic peptides in stimulating the IFN-y, TNF-a, and IL-2 expression from T cells in HL A- type unmatched PBMCs (FIGURES 2D-2F). The results showed that some peptides could stimulate the expression of all three cytokines, suggesting that polyfunctional T cells are induced by these peptides.

[0132] As illustrated in FIGURES 1A-1I, mass spectrometry analysis of HLA Class I epitopes was performed in PDAC tumor cell lines and tissues. MaxQuant was used to identify the peptide sequences with a false discovery rate (FDR) of 1%. The histograms in FIGURE 1A show the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC cell lines, Pane 10.05 and Panc06.03. These peptides correspond to 363 and 1238 unique proteins, respectively. The representative histograms in FIGURE IB show the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC tissues. The numbers of HLA Class I epitopes and their associated proteins identified from each individual PDAC tissues were identified (FIGURE 1C). From the 10 PDACs, a total of 14632 peptides and 11849 unique peptides, corresponding to 6086 non-redundant proteins, were identified. The numbers of eluted peptides from different PDAC specimens varied between 296 and 3270 (1331 on average). These peptides correspond to 123 to 2041 proteins (782 on average), respectively. [0133] Predicted HLA Class I binding affinity of eluted peptides from representative PDAC tissues, Panl2 and Panl 1 were obtained using the NetMHC4.0 algorithm (FIGURE ID). The dot lines represent the 500 nM threshold of high binding affinity. Note that 339 eluted peptides and 219 eluted peptides from the Panl2 PDAC specimen (81.7% and 52.8% of the total of 415 9-mer peptides, respectively) showed a low predicted binding affinity to the patient’s class I HLA types, HLA-A*2902 and HLA-A*3301, respectively. Similarly, 343 eluted peptides and 319 eluted peptides from the Panl l PDAC specimen (73.3% and 68.2% of the total of 468 9-mer peptides, respectively) showed a low predicted binding affinity to HLA-A*0101 and HLA-A*2902, respectively. A similar degree of net MHC in missing the peptides with high binding affinity was previously reported.

[0134] As shown in FIGURE IE, several criteria were followed for selecting peptides for validation: 1) those that are shared by multiple patients; 2) those whose predicted HLA binding affinity ranks among the top 0.5% of all peptides, which is the recommended threshold for the selection of peptides by NetMHC); 3) those whose corresponding proteins are overexpressed in tumor epithelia of PDAC compared to normal pancreas according to the Human Protein Atlas (www.proteinatlas.org). These criteria were used with a consideration of developing therapeutic agents. Eight peptides that met the selection criteria include four HLA-A2 peptides (COL6A3, ELOVL1, LAMC2, RASAL2) and four HLA-A3 peptides (DYNLRB1, ICE1, LAMB3, MYH9) (FIGURE 6). Numbers of HLA class I peptides from representative PDAC samples including Pan04, Pan06, and Pan07 and those of completely overlapped peptides among all three or any two of three PDAC samples were indicated (FIGURE IF, left). Numbers of peptides considered as strong binders (ranks among the top 0.5%) for HLA-A0201 (FIGURE IF, upper right) and HLA- A0301 (FIGURE IF, lower right) in Pan06 and Pan07, respectively, and those of overlapped peptides between Pan06 and Pan07 were also indicated. The sources of eight selected peptides were indicated.

[0135] FIGURES 1G-1I show T2 cell binding assays of selected HLA-A2 and A29 peptides binding to HLA-A2 expressing T2 cells (FIGURE 1G), HLA-A3 expressing T2 cells (FIGURE 1H), and HLA-A1 expressing T2 cells (FIGURE II). Twelve peptides that consisted of five peptides (ORMDL3, MYL12A, LAMC2, WDR82, TRRAP) shared by multiple HLA-A2 PDACs and seven peptides (TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1) shared by multiple HLA-A29 PDACs were selected according to the criteria listed in FIGURE IE (FIGURE 10). Controls indicate negative control peptides. In FIGURE 10, the numbers of HLA class I peptides of Pan06 and Pan04 patients and overlapped peptides between patients were indicated (left). Peptide numbers of Pan09, Panl l, and Panl2 patients, and overlapping peptide numbers among patients (right). Note that both HLA-I and HLA-II peptide sequences were compared with the DNA WES results available from 4 PDACs and no peptide sequence matched to the nucleotide sequence variants including single nucleotide polymorphism (SNP). It is possible that HLA-bound peptide identification may have missed the mutations-associated neoepitopes. However, such a result is consistent with the known rareness of the genomic mutation-associated neoepitopes in PDAC.

[0136] Mass spectrometry analysis of HLA Class II epitopes was performed in PDAC tumor tissues. MaxQuant was used to identify the peptide sequences. The MS analysis of eluted HLA-II peptides showed an average of 490 peptide sequences (ranging between 249 and 689), corresponding to an average of 116 proteins (ranging between 62 andl42) from six PDAC tissue samples. FIGURES 2A and 2B histograms illustrating the numbers of different lengths of peptides affinity purified by anti-HLA Class II antibody from two representative human PDAC tissue samples, Panl3 and Panl4. Numbers of total HLA class I peptides, HLA class II peptides, and completely overlapped peptides between HLA-I and HLA-II peptides were indicated in FIGURE 2C. Three representative PDAC samples were used. The ability of selected, HLA class I/II- overlapped peptides in stimulating single cells to express IFN-y, IL-2 and TNF-a in FluoroSpot assays was assessed and is illustrated in FIGURES 2D-2F. PBMC samples from three representative patients were shown. Spot forming unit (SFU) is the number of spots per 10⁶ PBMCs. Shown is SFU of each peptide after subtracting that of a negative control peptide; and error bars represent the percentages of deviation. “Unstimulated” indicates the reaction in absence of peptides. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”.

[0137] The HLA-I bound peptides were isolated from 12 surgically resected human PDAC tissue samples by using the same pan-HLA-I affinity purification column. The peptide-length distribution histograms peaked at 9-mer in all 12 PDAC specimens (FIGURE IB and FIGURES 3A-3J). As shown in FIGURE 1C, the numbers of eluted peptides from different PDAC specimens varied between 296 and 3270 (1331 on average). These peptides correspond to 123 to 2041 proteins (782 on average), respectively.

[0138] Whether eluted peptides could be predicted to have a high affinity binding to HLA molecules was examined. The eluted peptides from 8 PDAC specimens were filtered to include only 9-mer peptides and their binding affinity to their corresponding HLA-I types was predicted by using NetMHC-4.0 (FIGURE ID and FIGURES 4A-4F). HLA typing information was available with these 8 out of the 12 processed specimens. The cutoff for the low predicted binding affinity was set as 500 nM (indicated by block dot lines). Note that 339 eluted peptides and 219 eluted peptides from the Panl2 PDAC specimen (81.7% and 52.8% of the total of 415 9-mer peptides, respectively) showed a low predicted binding affinity to the patient’s class I HLA types, HLA-A*2902 and HLA-A*3301, respectively. Similarly, 343 eluted peptides and 319 eluted peptides from the Panl l PDAC specimen (73.3% and 68.2% of the total of 468 9-mer peptides, respectively) showed a low predicted binding affinity to HLA-A*0101 and HLA-A*2902, respectively.

[0139] As seen in FIGURES 5A-5B, 153 peptides (27.7%) from Pane 10.05 cells and 569 peptides (33.3%) from Pane 06.03 cells were also found in the peptides eluted from the 10 PDAC tissues.

[0140] Whether eluted epitopes were shared among different PDACs were examined in 10 out of the 12 processed PDAC samples after excluding Pan03 and Pan08, which may have non-specific HLA-binding peptides as the lengths of the peptides from these samples did not peak unimodally (see FIGURES 3A-3J and FIGURE 6).

[0141] To validate selected HLA class I epitopes identified by mass spectrometry, their ability of stimulating T cell responses was assessed. Ability of the synthetic 9-mer peptides in stimulating the IFN-y and granzyme B expression from T cells in PBMCs from HLA-A2 patients including Pan06 (HLA-A*0201, A*0301) and Pan04 (HLA-A*0201, A*1101) in a FluoroSpot assay was shown in the histograms at FIGURES 7A and 7B. Pre vaccine: PBMCs collected before the patients received the GM-CSF-secreting, allogeneic pancreatic tumor whole cell vaccine (PDAC GV AX vaccine) therapy. Post vaccine: PBMCs collected after the patients received the PDAC GV AX vaccine therapy. For this study, use PBMCs from the patients who received the GV AX vaccine was not intended. However, many patients at JHMI received the GV AX vaccine. The PBMC samples were always archived before and after the PDAC patients received the first treatment of GV AX through the past clinical trials and available for other research under the JHMI IRB general banking protocol. More specifically, the PBMC samples used were obtained from the HLA-A2 (Pan06 and Pan04) and A3 patients (Pan06) whose tumors were used for identifying these peptides. Similarly, the tumor specimens archived under the JHMI IRB-approved general banking protocol and used in this study happened to be obtained from patients who underwent the surgical resection following the treatment of GV AX. However, it is not anticipated that the treatment of GV AX, which is made of irradiated, allogenic whole tumor cells, would have an impact on the identification of epitopes; however, archived biospecimen repositories of clinical trials would provide PBMCs to compare peripheral T cell response at different time points. It is anticipated that the treatment of GV AX, which expresses many epitopes that were identified in the PDAC tissues (FIGURES 5A-5B), would enhance the peripheral T cell response to these epitopes. The results showed that T cell response as demonstrated by the expression of either IFN-y or granzyme B or both was significantly stimulated by each of the eight selected peptides in the PBMC from at least one of two patients whose tumors were used for identifying these peptides. Note that T cell response was also stimulated by the peptides in the PBMC samples from other HLA type-matched patients. As anticipated, T cell response was observed in the PBMC collected before receiving the GV AX vaccine. However, T cell response was more likely observed in the PBMC collected after receiving the GV AX vaccine, suggesting this whole cell vaccine expresses at least some of the shared antigens and thus was able to induce the proliferation of T cells specific for those antigens. Nevertheless, T cell response in some of the peptides was decreased in the PBMC collected after receiving the GV AX vaccine, suggesting the GV AX vaccine treatment did not adequately present every one of these 8 epitopes. Spot forming unit (SFU) is the number of spots per 10^A6 PBMCs.

[0142] Ability of the synthetic 9-mer peptides in stimulating the IFN-y and granzyme B expression from T cells in PBMCs from HLA- A3 patients in a FluoroSpot assay was shown in the histograms at FIGURES 8A and 8B. Pre vaccine: PBMCs collected before the patients including Pan06 (HLA-A*0201, A*0301) and Pan07 (HLA-A*0301) received the PDAC GV AX vaccine therapy. Post vaccine: PBMCs collected after the patients (Pan06 and Pan07) received the PDAC GV AX vaccine therapy. Spot forming unit (SFU) is the number of spots per 10^A6 PBMCs. [0143] As shown in FIGURES 9A-9D, the ability of two representative peptides ELOVL1 and LAMB3 in stimulating the IFN-y and granzyme B expression from T cells was assessed in a FluoroSpot assay. PBMCs from the patient (designated “original”: Pan06 (HLA-A*0201, A*0301)) where the peptides were eluted from and those from other patients (designated “other”: Panl8 (HLA-A*0201, A*0301); Pan08 (HLA-A*0201, A*2501); Panl9 (HLA-A*0301, A*2902)) were tested. Pre vaccine: PBMCs collected before the patients received the PDAC GV AX vaccine therapy. Post vaccine: PBMCs collected after the patients received the PDAC GV AX vaccine therapy. Spot forming unit (SFU) is the number of spots per 10^A6 PBMCs.

[0144] The validation of selected HLA class I epitopes identified by mass spectrometry in their ability of binding to unmatched HLA class I molecules and stimulating T cell responses in unmatched PBMC was assessed. Ability of selected 9-mer peptides in stimulating the IFN-y expression from T cells in PBMCs from patients including Pan04 (HLA-A*0201, A*1101), Pan09 (HLA-A*2902, A*3301), and Pan20 (HLA-A*0201, A*2902) with HLA class I types indicated, respectively, in a FluoroSpot assay, was shown in the histograms at FIGURES 11A-11C. The PBMC samples were archived before and after the PDAC patients received the first treatment of GV AX through the past clinical trials and available for other research under the JHMI IRB general banking protocol. MFI: mean fluorescent intensity. Pre vaccine: PBMCs collected before the patients received the PDAC GV AX vaccine therapy. Post vaccine: PBMCs collected after the patients received the PDAC GV AX vaccine therapy. Spot forming unit (SFU) is the number of spots per 10^A6 PBMCs. Shown is SFU of each peptide after subtracting that of a negative control peptide; and error bars represent the percentages of deviation. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”.

[0145] Ability of selected 9-mer peptides in stimulating the granzyme B expression from T cells in PBMCs from patients with HLA class I types indicated, respectively, in a FluoroSpot assay, was assessed as shown in FIGURES 12A-12C. MFI: mean fluorescent intensity. Pre vaccine: PBMCs collected before the patients received the PDAC GV AX vaccine therapy. Post vaccine: PBMCs collected after the patients received the PDAC GV AX vaccine therapy. Spot forming unit (SFU) is the number of spots per 10^A6 PBMCs. Shown is SFU of each peptide after subtracting that of a negative control peptide; and error bars represent the percentages of deviation. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”. Unpaired t test and 1-way AN OVA was used for comparing between stimulated and unstimulated peptide/samples. *p < 0.05, **p < 0.01, ***p < 0.001.

[0146] Note that the HLA-A29 peptides were able to stimulate T cells from both Pan04 (HLA- A*0201, A*1101), a non-HLA-A29 patient, and Pan20 (HLA-A*0201, A*2902), an HLA-A29 patient; and the HLA-A2 peptides were able to stimulate T cells from Pan09 (HLA-A*2902, A*3301), a non-HLA-A2 patient. Thus, our results suggested that above identified HLA-A2 and - A29 epitopes are able to bind unmatched HLA molecules and stimulate the T cell response in HLA-unmatched PBMC samples.

[0147] As shown in FIGURES 13A-13D, the numbers of different lengths of peptides affinity purified by anti-HLA Class II antibody were assessed from other human PDAC tissues.

[0148] FIGURES 14A-14B show the relationship between the input of surgical tissue and the amount of extracted protein (FIGURE 14A) and between the input of surgical tissue and the number of identified peptides (FIGURE 14B). The black dot lines represent lOOmg input of surgical tissue.

[0149] Table 1. Summary of Biospecimen Information.

PanOl Tumor tissue 500 657 NA NA NA NA NA NA NA NA NA

Pan02 Tumor tissue 500 3271 NA NA NA NA NA NA NA NA NA

Pan03 Tumor tissue 100 296 NA NA NA NA NA NA NA NA NA

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA- HLA- HLA-

Pan04 320 1679 689

PBMC A*0201 A*1101 B*5201 B*5201 C*1202 C*1202 DRB 1 *0405 DRBl* 1103

HLA- HLA- HLA- HLA- HLA- HLA-

Pan05 Tumor tissue 480 732 NA NA NA

A*2402 A*3101 B* 1501 B*5101 C*0303 C*1402

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA-

Pan06 610 1909 NA NA NA

PBMC A*0201 A*0301 B* 1402 B*1501 C*0303 C*0802

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA-

Pan07 310 979 NA NA NA

PBMC A*0301 A*0301 B*0702 B*3501 C*0401 C*0702

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA-

Pan08 500 516 NA NA NA

PBMC A*0201 A*2501 B*3801 B*5101 C*0701 C*1203

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA-

Pan09 320 1791 NA NA NA

PBMC A*2902 A*3301 B* 1402 B*4403 C*0802 C*1601

HLA- HLA- HLA- HLA- HLA- HLA-

Pan 10 Tumor tissue 550 1717 NA NA NA

A*0101 A*2402 B*0702 B*5101 C*0102 C*0702

HLA- HLA- HLA- HLA- HLA- HLA-

Panl l Tumor tissue 320 1054 NA NA NA

A*0101 A*2902 B*3502 B*4403 C*0401 C*1601

HLA- HLA- HLA- HLA- HLA- HLA-

Pan 12 Tumor tissue 600 843 NA NA NA

A*2902 A*3301 B*0702 B*6501 C*0701 C*0802

HLA- HLA- HLA- HLA- HLA- HLA- HLA- HLA-

Pan 13 Tumor tissue 590 NA 665

A*1101 A*2601 B*4901 B*5701 C*0501 C*0601 DRB 1 *0801 DRBl* 1303

HLA- HLA- HLA- HLA- HLA- HLA- HLA- HLA-

Pan 14 Tumor tissue 672 NA 450

A*0201 A*2402 B*4502 B*6201 C*0401 C*1201 DRBl *0103 DRB 1*0701

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA- HLA- HLA-

Pan 15 550 109 249

PBMC A*0101 A*2301 B*0802 B*1303 C*1001 C*1801 DRBl *1101 DRB 1*0801

Tumor tissue, HLA- HLA- HLA- HLA- HLA- HLA- HLA- HLA-

Pan 16 678 1236 444

PBMC A*0201 A*2402 B*3501 B*3701 C*0401 C*0602 DRB1 *1418 DRB 1*0304

HLA- HLA- HLA- HLA- HLA- HLA- HLA- HLA-

Pan 17 Tumor tissue 845 184 508

A*0101 A*0201 B*0801 B*1301 C*0601 C*0701 DRB 1 *0401 DRBl* 1703

HLA- HLA- HLA- HLA- HLA- HLA-

Pan 18 PBMC NA NA NA NA NA

A*0201 A*0301 B*4001 B*4403 C*0304 C*1601

HLA- HLA- HLA- HLA- HLA- HLA-

Pan 19 PBMC NA NA NA NA NA

A*0301 A*2902 B*4901 B*5501 C*0303 C*0701

HLA- HLA- HLA- HLA- HLA- HLA-

Pan20 PBMC NA NA NA NA NA

A*0201 A*2902 B*4402 B*4403 C*0501 C*1601 [0150] Table 2. PDAC Peptidome.

[0151] Table 2 provides a shortened list of peptides that were found in 3 or more patients, with redundant peptides having been removed.

[0152] Table 3 Number of PDACs that share the epitopes and number of epitopes that are shared.

[0136] Table 4. Selected HLA-A2 peptides and A3 peptides.

[0137] Table 5. Six synthesized HLA class II peptides.

[0138] This study is the first one to examine HLA class I and class II restricted peptidomes in human PDAC. Previously, similar studies in few other malignant diseases were successfully conducted and reported. This study is also one of the few using MS to identify HLA class II epitopes. Therefore, this study has opened a new direction for the investigation of T cell epitopes and for the development of T cell epitope-based immunotherapy such as vaccine and TCR-T cell therapy in immune “desert” tumors, specifically PDAC.

EXAMPLE 3

LAMC2 TCR ANTI-TUMOR STUDY

[0139] Tumor antigens serve as crucial targets for T cell-based therapy to induce tumor-specific rejection. However, identifying pancreatic ductal adenocarcinoma (PDAC) specific T cell epitopes have been challenging. Using advanced mass spectrometry (MS) analysis, cancer-associated, class I MHC-bound epitopes shared by multiple PDAC patients with different HLA-A types were previously identified. Here, one of the epitopes, LAMC2203-211, a naturally occurring nonmutated epitope, on the protein LAMC2 was investigated. Following stimulation with the LAMC2203-211 peptides, T cell receptors (TCRs) were cloned and transduced in the Jurkat human T cell line using lentiviral vector. It was found Jurkat cells expressing LAMC2203-211 specific TCRs elicit potent, LAMC2-specific, in vitro cytotoxic effects on PDAC cells. Furthermore, mice that harbored either subcutaneously or orthotopically implanted tumors originated from both HLA-A allele matched and unmatched PDAC patients showed tumor growth suppression in a LAMC2-dependent manner following the infusion of LAMC2-targeting T cells. A LAMC2-specific TCR-based T cell therapy strategy likely suitable for many PDAC patients was therefore developed. This is the first study to adopt MS analysis to identify natural CD8+ T cell epitopes in PDAC that could potentially serve as targets for PDAC immunotherapeutic strategies.

[0140] Materials and Methods

[0141] Cell lines and. cell culture

[0142] Human PDAC cell lines (HPDE, Panc-1, AsPC-1) and human T cell line Jurkat were purchased from ATCC (Manassas, VA). Panc6.03, Pancl0.05, Panc9.05 and Panc7.078 are primary pancreatic cancer cell lines that were established from surgically resected PDAC specimen in accordance with the Johns Hopkins Medical Institution Institutional Review Board (JHMI IRB)- approved protocols and authenticated by DNA and gene expression profding and previously described (35). Human peripheral blood mononuclear cells (PBMC) were obtained from patients under Johns Hopkins Medical Institution (JHMI) Institutional Review Board (IRB) approved protocol (IRB00244430). HPDE, Panc-1, AsPC-1, Panc6.03, Pancl0.05, Panc9.05 and Panc7.078 were cultured in RPMI 1640 media (Life Technologies), 10% fetal bovine serum (Atlas Biologicals), 1% 1-glutamine (Life Technologies), 1% Non-essential Amino Acids (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). PBMCs and Jurkat cells were cultured in RPMI- 1640 Medium (Life Technologies) supplemented with 10% fetal bovine serum (Atlas Biologicals), 1% 1-glutamine (Life Technologies), and 1% penicillin/streptomycin (Life Technologies). All cells were maintained at 37°C in a humidified incubator with 5% CO2.

[0143] Immunohistochemistry (IHC)

[0144] Tumor tissues for human correlative IHC staining were obtained from specimens collected from 20 patients who underwent surgery at the Johns Hopkins Hospital under the JHMI IRB approved protocol (IRB00244430). Formalin- fixed paraffin-embedded (FFPE) tissues were sectioned at 5-pm and subjected to heat-induced antigen retrieval. IHC staining was performed using Dako Catalyzed Signal Amplification system as previously described (36). The following antibodies were used: anti-LAMC2 (Atlas Antibodies, AMAb91098), anti-TRAPPCl l(Biorbyt, orbl86301), anti-ZMYND 11 (Thermo Fisher Scientific, PA540960), anti-CTNNBIPl (MyBioSource, MBS2527764), anti-ORMDL3(Millipore Sigma, ABN417) and anti-MYL12A (Santa Cruz Biotechnology, sc-28329 HRP). All slides were scanned and analyzed using Image Analysis Software (Aperio Technologies).

[0145] Immunoblotting

[0146] Total proteins were extracted using Radioimmunoprecipitation assay (RIP A) lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and separated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred onto 0.45pm polyvinylidene difluoride (PVDF) membranes. The transferred membranes were then blocked with 5% non-fat milk and subsequently incubated overnight at 4 °C with anti-glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, 5174S) (1:1000) and anti-LAMC2 primary antibodies (Atlas Antibodies, AMAb91098) (1:1000), followed by incubation with corresponding secondary antibodies (Invitrogen, 656140; Cell Signaling Technology, #7076). The immunoreactive proteins on the membranes were visualized using a ChemiDoc XRS System.

[0147] T cell cultures with the LAMC2203-211 peptides

[0148] Archived PBMCs collected by the JHMI IRB approved protocol (IRB00244430) were stimulated in 24-well cell-culture plates at the concentration of 5 x 10⁶ cells per well with the LAMC2203-211 peptides (10 pg/ml) in the presence of IL-7 (20 ng/ml; Peprotech). On day 3, low- dose Recombinant interleukin-2 (rIL-2) (20 U/ml; Amgen) was added. Half-medium change was performed with fresh medium supplemented with rIL-2(20 U/ml) and IL-7(20 ng/ml) every 3 days. PBMCs cultured only in the presence of the cytokines but not peptides were used to provide a baseline TCR repertoire profile for comparison. After 21 days, CD8+ T cells were sorted through magnetic-activated cell sorting (MACS) using the Human CD8+ T Cell Isolation Kit, Human (Miltenyl Biotec) following the manufacturer’s instruction and processed following the lOx Genomics Chromium Single Cell Protocol. [0149] Single Cell TCR sequencing using the lOx Chromium platform

[0150] The single-cell RNA sequencing libraries were prepared following the protocol provided by the 10^x genomics Chromium Single Cell Immune Profiling Solution. Cellular suspensions were loaded on a Chromium Single Cell Controller instrument (lOx Genomics) to generate single-cell Gel Bead-In Emulsions (GEMs). After reverse transcription of mRNA, droplets were broken, and barcoded cDNA was purified with DynaBeads. Barcoded, full-length V(D)J segments were enriched from amplified cDNA with primers specific for the human TCR constant regions. V(D)J region-enriched libraries were size selected with SPRI beads (avg. size 600 bp) and sequenced on an Illumina HiSeq 2500 instrument. The Cell Ranger Software Suite (version 2.1.0) was used for V(D)J sequence assembly

[0151] TCR reconstitution in Jurkat cells

[0152] Lentiviral transfer plasmids were synthesized by OBiO Technology. Variable regions of TCRa- and [3- chains of TCR1 and TCR2 were linked by a P2A peptide element to yield the transgene cassette 5'-TCR[3-P2A-TCRa-3'. The transgene cassette was synthesized and integrated into the GFP-tagged retrovirus vector GL121 (Figure S3). The P2A linker peptide results in higher expression and functionality of human TCR(37). To enhance TCR surface expression, the constant regions of both TCR chain genes were exchanged by their mouse counter parts(38). All constructs were verified by sequence analysis.

[0153] Lentivirus transfer plasmids carrying TCR1 and TCR2 were co-transfected into HEK293T cells with packaging plasmid pCMV-dR8.91 and envelope plasmid pCMV-VSV-G to produce lentivirus particles. Lipofectamine 2000 transfection reagent (Invitrogen, 11668027) was added according to manufacturer’s instruction. The supernatants containing the relevant lentivirus were harvested 48 h and 72 h post transfection.

[0154] To establish TCR1 -expressing, TCR2-expressing, and GL121 backbone lentivirus- infected cell lines (TCR 1 -Jurkat, TCR2 -Jurkat, and GL121-Jurkat, respectively), Human Jurkat T cells (ATCC, Clone E6-1) were infected by respective recombinant lentiviruses in the presence of polybrene (1:500) (Sigma-Aldrich, TR1003). The medium was changed to the normal culture medium 24 h post infection. [0155] Generate Stable LAMC2 knockdown cells with shRNA

[0156] Bacterial glycerol stock of LAMC2-shRNA (TRCN0000083390) and control shRNA (SHC002) was purchased through Sigma-Aldrich. The expanded plasmids were used to produce lentivirus following the same procedure as described above. To stablish stable LAMC2 knock down Pancl0.05 (LAMC2KD ^_,“^,“^,Pancl0.05) and control Pancl0.05 cell lines (shCtr Pancl0.05), the resultant lentivirus was transduced into Pancl0.05 cells grown to -70% confluency. The medium was changed to the normal culture medium 24 h post infection. For selection, puromycin was added to the culture media at a final concentration of 1 pg/ml.

[0157] In vitro Jurkat Cytotoxicity assay

[0158] 5xl0³ Pancl0.05 tumor cells and 2.5xl0⁴ TCR- transduced Jurkat cells were cultured at a ratio of 1 :5 in Tumor cell medium in an opaque-walled, flat-bottomed, 96-well plate for 48 hours. Jurkat cells cultured alone were measured for the baseline death level of Jurkat cells. The effector: target ratios were tested at 5 : 1 and 10:1, respectively; and the ratio of 5 : 1 was chosen as the optimal condition. Cell death was measured using CytoTox-Fluor™ Cytotoxicity Assay kit (Promega) according to the manufacturer’s instructions. Tumor cell death was calculated by subtracting the death of the co-cultured cells with the baseline death of Jurkat cells alone.

[0159] Mouse models and in vivo antitumor experiments

[0160] Mice: female NOD/LtSzPrkdcscidIL2rytmlWjl (NSG) mice (6-8 weeks) were purchased from Harlan Laboratories and maintained in accordance with the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC) guidelines. Mice considered to have reached a “survival endpoint,” including hunched posture, lethargy, dehydration, and rough hair coat, were euthanized. The IACUC mouse protocol was maintained by IACUC.

[0161] Subcutaneous model: In this model, 2x106 Pancl0.05 cells were inoculated subcutaneously into both flank of NSG mice. After 3-5 weeks, the subcutaneous tumors were harvested and cut into -2 mm3 pieces and then implanted subcutaneously into 8-10 weeks old NSG mice. Mice were randomized into different treatment groups. Three days later, tumor-bearing NSG mice were randomly assigned to the treatment groups as indicated. Mice were treated with 1x106 Jurkat cells as indicated weekly by tail vein, and rIL-2 (100U) was administered concurrently during each treatment. Tumor size was measured twice a week using caliper. Tumor measurements were taken by researcher blinded to the group assignment. The long(L) and short(S) axes of each tumor were measured on harvested tumors with calipers. Tumor volume(V) was calculated as V = (L x S2)/2.

[0162] Orthotopic model: JH029 and JH072 were PDXs that derived from patients with pancreatic ductal adenocarcinoma from our institution. Small pieces of PDXs were inoculated subcutaneously into both flank of NSG mice. After 5-8 weeks, the subcutaneous tumors were harvested and cut into ~2 mm³ pieces and then implanted orthotopically into the pancreases of 8- 10 weeks old NSG mice. Eight days later, tumor-bearing NSG mice were randomly assigned to the treatment groups as indicated. Jurkat cells as indicated were injected once a week by tail vein as described above for the subcutaneous model. Tumor size was measured twice a week with by a Vevo750 small animal ultrasound. Tumor volume(V) was calculated as V = (L x S2)/2.

[0163] Results

[0164] Characterization of potential T cell antigens in PDACs and. selection of LAMC2 as a target for T cell therapy development

[0165] HLA class I restricted peptides were recently isolated from tissues of human PDAC by using the pan-HLA class I affinity purification column and identified T cell epitopes by peptidome analysis with mass spectrometry. Through peptidome analysis, multiple T cell epitopes that are shared by multiple patients with unique HLA-A alleles were identified. Six epitopes were then selected from the shared epitope pool that also induced T cell response in HLA-type matched and unmatched patient PBMC (as manifested by the production of IFN-y, granzyme B or both) for further investigation.

[0166] Using immunohistochemistry, the expression of corresponding proteins of these six peptides in human PDAC tumors and adjacent non-tumoral normal tissues was analyzed (FIGURES 15A and 19A). The LAMC2203-211 epitope (Table 6), which consists of amino acid sequences 203 to 211 of the LAMC2 protein, was discovered in two patient’s PDAC tissue specimens, one patient with HLA-A2, A3 and the other with HLA-A2, Al l. LAMC2 was found to be highly expressed on invasive PDAC tumor cells but was undetectable in normal pancreas and para-tumoral tissue (which includes stroma cells surrounding the tissue cells) in all 20 PDAC samples tested, making it an ideal target for therapeutic intervention (FIGURE 15A). In contrast, TRAPPCI 1, ORMDL3, and MYL12A were found to be expressed in both tumors and adjacent non-tumoral tissues, whereas ZMYND11 and CTNNBIP1 were not detected in either tumors or adjacent non-tumoral normal tissues (FIGURE 19A).

[0167] Table 6. List of candidate HLA-Class I epitopes

Epitope Sequence HLA-A type

TRAPPCI 1191-199 FYEHAQTYY A29

ZMYND 11348-356 DELELHQRF A29

CTNNBIP 136-44 SEEEFLRTY A29

ORMDL3 59-67 GMYIFLHTV A2

MYLI2A121-129 YLRELLTTM A2

LAMC2₂O3-211 SVHKITSTF A2

[0168] Next, LAMC2 expression was evaluated in various PDAC cell lines. It was found that LAMC2 expression was low in the HPDE normal pancreatic ductal epithelial cell line and high in most of PDAC cell lines (FIGURE 15B). The Cancer Genome Atlas (TCGA) data also revealed a significantly increase in the expression of LAMC2 in PDAC tumor tissues compared to normal pancreatic tissue (FIGURE 15C). In addition, Kaplan-Meier survival analysis of PDAC patients based on the Human Protein Atlas (proteinatlas.org) indicated that those with a high LAMC2 expression had significantly worse 5-year survival (17%) than those with a low LAMC2 expression (71%) (pO.OOl) (FIGURE 15D). The combination of these results suggested that LAMC2 might be a potential antigenic target for PDAC. Note that LAMC2 protein is expressed at relatively low levels in thyroid gland, salivary gland, and skeletal muscle, and is only moderately expressed in nasopharynx, bronchus, colon, urinary bladder, fallopian tube, endometrium, breast, appendix, tonsil (FIGURE 19B). Nevertheless, LAMC2 is not expressed in vital organs, such as liver, kidney, and brain. Thus, it is anticipated that TCR T cell therapies targeting LAMC2 will be safe. Moreover, LAMC2 is overexpressed in essentially all PDACs and in more than 90% of many other types of cancer (FIGURE 19C), thereby supporting LAMC2 as a specific target for the TCR T cell therapies. Thus, we have chosen to clone LAMC2203-211 epitope specific-TCRs to develop TCR T cell therapies.

[0169] Jurkat cells expressing LAMC2203-211- specific TCR can effectively eliminate LAMC2 + human pancreatic cancer cells in vitro [0170] To identify LAMC2203 -211 -specific TCRs, PBMCs from donors whose tumors were used to identify the LAMC2203-211 epitope were stimulated with a synthesized LAMC2203-211 peptide (FIGURE 20). Top seven most expanded TCR clones in LAMC2203-211 peptide stimulated CD8+ T cells comparing to TCR repertoire in unstimulated CD8+ T cells were identified (Table 7). Two most expanded TCRs (designated TCR1 and TCR2) were selected to validate their specificity for the LAMC2203-211 epitope. Subsequently, we transduced the two TCR constructs (FIGURE 21; Table 8) into human Jurkat cells by lentiviral infection, establishing TCRl-Jurkat and TCR2-Jurkat cell lines. The backbone lentiviral vector, GL121, was also transduced into Jurkat cells to establish the control GL 121 -Jurkat cell line. Jurkat cells expressing the LAMC2203 -211 -specific TCRs or the backbone lentiviral vector, both expressing GFP, were sorted by flow cytometer.

[0171] Table 7. Highly expanded TCR clones following the LAMC2203-211 peptide stimulation.

Clone Folds of Expansion*

TCR1 16

TCR2 13

TCR3 13

TCR4 13

TCR5 11

TCR6 11

TCR7 10

*Fold of expansion is calculated as the ratio between the number of cells from the same TCR clone in the T cell culture with peptide stimulation and that without peptide stimulation.

[0172] Table 8 : TCR sequences identified and used in this study.

TCR Sequence

TRA TGTGCTTCCATGGAATATGGAAACAAACTGGTCTTT;

TRB TGCGCCAGCAGCCACGGGACTGCCGACCTCAATGAGCAGTTCTTC

TRA TGTGCAGAGATCATCCCTACCGGCACTGCCAGTAAACTCACCTTT;

TRB TGTGCCAGCAGCTTCCCCCGGGACAATCCACAGCCTACGCAGTATTTT

TRA TGTGCAGCTCCCAGGAGGTGGAACAATGCCAGACTCATGTTT;

TRA TGTGCCCCGTCGTTATCTGGTTCTGCAAGGCAACTGACCTTT;

TRB TGTGCCAGCAGCAACACTACCATAGCGGGGGGGGGAGAGACCCAGTACTTC

TRA TGTGCAGGAGGGAAGCCGTTT;

TRB TGTGCCAGCAGTCTCGGACTAAACTACGAGCAGTACTTC

TRA TGTGCAATGAGCGGTAGTGGAGGTAGCAACTATAAACTGACATTT;

TRB TGCAGTGCCTCACCGACGCAGTATTTT;

TRB TGCGCCAGCAGCCAGGGTGGGGAGCAGTTCTTC

TRA TGTGCAATGAGCTACAACCAGGCAGGAACTGCTCTGATCTTT;

TRB TGTGCCAGCAGAGGGACAGGGGGCAACTACGAGCAGTACTTC

TRA TGTGCAGGAGTTCCCGACAGCAGTGCTTCCAAGATAATCTTT;

TRA TGTGCTACGGATGGAGCCGACAAGCTCATCTTT;

TRB TGTGCCAGTAGTATATCGGTGGACTCTGGAAACACCATATATTTT

[0173] To assess the efficacy of LAMC2203 -211 -targeting Jurkat cells against PDAC cells, the TCR1, TCR2, or GL121 -infected Jurkat cells were co-cultured with the human Pancl0.05 PDAC cells (HLA-A1, A 19) and the cytotoxic activity of the infected Jurkat cells was examined. Cytotoxic activities were measured using the CytoTox-Fluor™ Cytotoxicity Assay kit and reported as dead-cell luminescence. Following co-incubation with Pancl0.05 cells, both LAMC2 TCR1 and TCR2-infected Jurkat cells showed a significantly higher killing compared to the control GL 121 -Jurkat cells (FIGURE 16A).

[0174] To further investigate TCR1 and TCR2-Jurkat cell’s epitope-specificity, a stable LAMC2KD -Pane 10.05 cell line was established using lentivirus-carried shRNA to knock down LAMC2 expression and a shCtr Pancl0.05 cell line through infection of lentivirus carrying the non-mammalian control shRNA (designated LAMC2KD cells and shCtr cells, respectively). LAMC2 knockdown efficiency was confirmed by RT-PCR and Western blot (FIGURES 16B and 16C). The above-described co-culture was then repeated with the LAMC2KD -Pancl0.05 cells. This time, no significant differences in the cytotoxic activities between the LAMC2203-211- targeting Jurkat cells and GL121-Jurkat cells were observed (FIGURE 16D). Altogether, these results indicate LAMC2 TCR1 and TCR2 are specific for LAMC2. Furthermore, it was found that TCR2-Jurkat cells, as representative of LAMC2203-211- targeting Jurkat cell can effectively kill other human PDAC cells, namely Panc-1 (HLA-A2, Al l) and Panc7.078(HLA-A2) cells (FIGURE 22). This finding suggests the LAMC2203-211 epitope may be presented by varying alleles of HLA class I molecule, and the LAMC2203-211- targeting Jurkat cells can induce targetspecific elimination against tumor cells expressing this epitope in most of patients.

[0175] Adoptive transfer of LAMC2203-211-targeting T cells suppress tumor growth in in vivo pancreatic carcinoma models

[0176] Next, the in vivo antitumor effect of LAMC2203-211 targeting TCR1 and TCR2- infected Jurkat cells in PDAC was assessed. First, a xenograft mouse model of PDAC was established by implanting Panel 0.05 derived tumor subcutaneously into theNSG mice. Three days after tumor implantation, mice were adoptively transferred with either the TCR1 -Jurkat, TCR2- Jurkat, or control GL121-Jurkat cells via weekly intravenous injections. (FIGURE 17A). rIL-2 was administered together with the T cells. The results showed that LAMC2203 -211-targeting TCR1 and TCR2-infected Jurkat T cells, but not the GL121-Jurkat T cells, displayed antitumor activity (FIGURE 17B). The capacity of Jurkat cells that target LAMC2203-211, which is predicted to bind HLA-A2, to control tumor growth in two PDAC patient-derived tumor xenograft (PDX) mouse models, JH029 (HLA-A2, A31) and JHO72(HLA-A1, A68) was also tested (FIGURES 23 and 24). In line with the previous findings, JH029 and JH072 both showed significantly slower rate of growth in mice following adoptive transfer of either TCR1 or TCR2 Jurkat cells compared to GL121-Jurkat cells (FIGURE 17C). Taken together, these results demonstrates that both HLA-matched and unmatched PDACs can be targeted in vivo by LAMC2203 -211-targeting T cells.

[0177] Adoptive transfer of LAMC2203-211-targeting T cells do not suppress growth of LAMC2-deficient tumor [0178] The specificity of the antitumor activity of LAMC2203 -211 -targeting T cells in vivo was then further evaluated. The LAMC2KD Pancl0.05 cells or Pancl0.05 cells infected with lentivirus expressing the non-mammalian shRNA control were used to implant the tumors (LAMC2KD tumor and shCtr tumor, respectively) subcutaneously. Following the same treatment schema as above described (FIGURE 17), mice began receiving weekly injection (four weeks total) of the TCR2-Jurkat cells as representative LAMC2203 -211 -targeting T cells and the GL121- Jurkat cells or PBS as controls three days post-tumor implantation.

[0179] In agreement with previous studies on LAMC2(34), it was found that LAMC2KD tumors displayed a slower tumor growth compared to shCtr tumors in the PBS-treated group (pO.OOl) (FIGURE 18A). This finding further suggests that LAMC2 may play an important tumor-promoting role in PDAC. In line with the above findings (FIGURE 17), it was found that TCR2-Jurkat cells significantly decreased the growth of shCtr-tumors compared to GL121-Jukat cells (p<0.05) (FIGURE 18B). In contrast, the effect of TCR2-Jurkat cells in tumor growth suppression was not observed with the LAMC2KD tumors (FIGURE 18C). Taken together, these results suggest that LAMC2203 -211 -targeting T cells can suppress PDAC tumor growth in a LAMC2-dependent manner.

[0180] The toxicities of LAMC2203 -211 -targeting T cells were also monitored and none was observed until the number of LAMC2203-211-Jurkat cell infusion was increased to four weekly treatments (FIGURE 18). Treatment-related toxicities including decreased mobility and hunched back ere observed. Such toxicities were not limited to LAMC2 TCR-infected Jurkat cells or GL 121-Jurkat cells. However, after the Jurkat cells were filtered with a strainer before infusion, the toxicity associated with the 4th Jurkat cells infusion in the repeated experiments was no longer observed.

EXAMPLE 4

CHARACTERIZATION OF POTENTIAL T CELL ANTIGENS IN PDACS AND SELECTION OF TMSB10 AS A TARGET FOR T CELL THERAPY DEVELOPMENT

[0181] HLA class I and class Il-restricted peptides from tissues of human PDAC were successfully isolated and T cell epitopes identified by peptidome analysis with mass spectrometry. Notably, the peptidome analysis revealed a substantial overlap between HLA-I and HLA-II epitopes derived from the same patient samples. The HLA-I and HLA-II overlapped peptides shared between two PDAC samples and found 30 such peptides were examined (FIGURE 27). Subsequently, those peptides were narrowed down to 6 peptides whose corresponding proteins are overexpressed in tumor epithelia of PDAC compared to normal pancreas according to the Human Protein Atlas (proteinatlas.org) (Table 9). These six epitopes were selected for further investigation in this study. The HLA-binding affinity of these peptides was predicted using NetMHC4.0, it was found that TMSB10 has the highest binding affinity to HLA-II compared to the other five epitopes (FGA, IGHG, H1F2, VIM, and HBD). Based on core MHC binding sequences predicted by NetMHC II pan, 12-21-mer peptides were synthesized (Table 9) and the ability of these synthetic peptides to stimulate the expression of IFN-y, TNF-a, and IL-2 in T cells from HLA-type unmatched PBMCs was examined. Cytokine Fluorospot assay showed that TMSB 1028-44 peptide, which consists of amino acid sequences 28 to 44 of the Thymosin Beta 10 protein (TMSB 10), can stimulate the expression of all three cytokines, suggesting that this peptide can induce polyfunctional T cells (FIGURE 25 A and FIGURE 28).

[0182] Table 9. List of candidate epitopes shared by HLA class I and class II types

„ . . G „ene PDAC Normal , . T. i .ssue Pred .ic. te .d . b.in. ding

Peptide sequence names expressi .on expressi .on affinity (uM) ' ' (DRB1 0113-restricted)

ADSGEGDFLAEGGGVR FGA Low All 1443.6

NSGALTSGVHTFPAVLQS IGHG NA NA 914.2

SGPPVSELITKAVAASKER H1F2 Medium Lymphoid tissue 73.1

TLPTKETIEQEKRSEIS TMSB10 Medium Blood 6807.9

TVETRDGQVINETSQHHDDLEVIM Medium Liver 2331.4

VVAGVANALAHK HBD Low Bone marrow 356.5

[0183] Assessing TMSB1028-44 as a potential PDAC immunotherapeutic target

[0184] TMSB 1028-44 peptide was then further investigated as a potential target for PDAC immunotherapy. The expression of TMSB 10 protein levels in human PDAC tumors and adjacent non-tumoral normal tissues was analyzed by using immunohistochemistry. TMSB 10 was found to be highly expressed in tumor tissues but was undetectable in normal pancreas, making it an ideal target for therapeutic intervention (FIGURE 25B). The Cancer Genome Atlas (TCGA) data also revealed a significant increase in the mRNA expression of TMSB10 in PDAC tumor tissues compared to normal pancreatic tissue from GTEx database (FIGURE 25C). In addition, Kaplan- Meier survival analysis of PDAC patients based on the Gene Expression Profiling Interactive Analysis (GEPIA) database (gepia.cancer-pku.cn) demonstrated that those with a high TMSB10 expression had significantly worse overall survival (OS) and disease-free survival (DFS) than those with a low TMSB10 expression (FIGURES 25D and 25E, both p<0.05). The combination of these results suggested that TMSB10 might be a potential antigenic target for PDAC. Note that TMSB10 mRNA is expressed at relatively low levels in vital organs, such as the liver, cerebellum, pituitary gland, thyroid gland, and kidney (FIGURES 30A and 30B). Thus, it is anticipated that TCR T cell therapies targeting TMSB10 will be safe. In addition, the TMSB10 mRNA expression is relatively low in the normal pancreas, which indicates targeting TMSB10 will minimize harm to healthy pancreas tissues. Moreover, TMSB10 is overexpressed in essentially all PDACs and in nearly 60% of 33 types of cancer in the TCGA database (FIGURE 30C), thereby supporting TMSB10 as a specific target for the TCR T cell therapies. Thus, we have chosen to clone TMSB 1028-44 epitope-specific TCRs to develop TCR T cell therapies.

[0185] Identification of TMSB1028-44-specific CD4 and CD8 TCRs by single-cell TCR sequencing.

[0186] To identify TMSB1028-44-specific CD4 and CD8 TCRs, peripheral blood mononuclear cells (PBMCs) from donors whose tumors were used to identify the TMSB 1028-44 epitope were stimulated with a synthesized TMSB 1028-44 peptide (FIGURE 29). Droplet-based scTCR-seq libraries from the two patients’ isolated CD8+ and CD4+ T cells were generated, which were isolated from the patient’s PBMC stimulated with TMSB 1028-44 peptide and unstimulated PBMC.

[0187] The expanded CD8 TCR clones in TMSB 1028-44 peptide stimulated CD8+ T cells were identified by comparing to TCR repertoire in unstimulated CD8+ T cells. In addition, expanded CD4 TCR clones from CD4 TCR repertoire were also identified using the same method. Based on the change of matched clonotype frequency in stimulated compared to unstimulated samples, the two most expanded CD8 TCRs (designated TCR1 and TCR2) and CD4 TCRs (designated TCR3 and TCR4) were selected to validate their specificity for the TMSB1028-44 epitope (Table 10). [0188] Table 10: Highly expanded TCR clones following the TMSBIO28-44 peptide stimulation.

TCR TCRV CDR3 Se uence Clonot e Count- Clonot e Count-

*Fold of expansion is calculated as the ratio between the number of cells from the same TCR clone in the T cell culture with peptide stimulation and that without peptide stimulation.

[0189] Jurkat cells expressing TMSB 1028-44- specific TCR can effectively eliminate TMSB 10+ human pancreatic cancer cells in vitro

[0190] Subsequently, the four TCR constructs were transduced into human Jurkat cells by lentiviral infection, establishing CD8 TCR- Jurkat (TCR1 and TCR2) and CD4 TCR-Jurkat (TCR3 and TCR4) cell lines. The backbone lentiviral vector, GL 121, was also transduced into Jurkat cells to establish the control GL 121 -Jurkat cell line. Jurkat cells expressing the TMSB 1028-44 -specific TCRs or the backbone lentiviral vector, both expressing GFP, were sorted by flow cytometer.

[0191] To select the target cancer cell line, TMSB 10 expression was screened for across various pancreatic cancer cell lines using the PHA database and found that AsPC-1 has the second highest TMSB10 expression among 46 types of human PDAC cell lines. Therefore, AsPC-1 was chosen for further study (FIGURE 30D).

[0192] To assess the efficacy of TMSB1028-44-targeting Jurkat cells against PDAC cells, the CD8 TCR (TCR1 and TCR2) or GL121 -infected Jurkat cells were co-cultured with the human AsPC-1 PDAC cells (HLA-A1, A2; HLA-DRB1-4, DPB1-13) and examined the cytotoxic activity of the infected Jurkat cells. Cytotoxic activities were measured using the CytoTox-Fluor™ Cytotoxicity Assay kit and reported as dead-cell luminescence. Following co-incubation with AsPC-1 cells, both TMSB 10 CD8 TCR (TCR1 and TCR2) -infected Jurkat cells demonstrated a significantly higher killing compared to the control GL 121 -Jurkat cells (FIGURE 26).

[0193] To further investigate CD8 T cells combined with CD4 T cells enhanced the tumor cell killing ability TCR1 and TCR2-Jurkat cells co-cultured with TCR3 and TCR4-Jurkat cells were used to test the cytotoxic activity of the combination group. Interestingly, when TMSB 10 TCR1 and TCR2-infected Jurkat cells combined with TCR4-infected Jurkat cells, significantly higher killing ability was observed compared to the control GL 121 -Jurkat cells or single CD8 TCR-Jurkat cells (FIGURE 26). Altogether, these results showed that the combined action of TMSB10 CD8 TCR and CD4 TCR T-cells can effectively kill more tumor cells and lead to enhanced anti-tumor immunity.

[0194] Materials and Methods

[0195] Cell lines and. cell culture

[0196] Human PDAC cell lines (AsPC-1) and human T cell line Jurkat were purchased from ATCC (Manassas, VA). Human peripheral blood mononuclear cells (PBMC) were obtained from patients under Johns Hopkins Medical Institution (JHMI) Institutional Review Board (IRB) approved protocol (IRB00244430). AsPC-1 was cultured in RPMI 1640 media (Life Technologies), 10% fetal bovine serum (Atlas Biologicals), 1% 1-glutamine (Life Technologies), 1% Non-essential Amino Acids (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). PBMCs and Jurkat cells were cultured in RPMI-1640 Medium (Life Technologies) supplemented with 10% fetal bovine serum (Atlas Biologicals), 1% 1-glutamine (Life Technologies), and 1% penicillin/streptomycin (Life Technologies). All cells were maintained at 37°C in a humidified incubator with 5% CO2.

[0197] Preparation of antibody-conjugated affinity purification columns

[0198] The antibody-conjugated affinity purification columns were prepared following a modified protocol. Briefly, W6/32 cells were cultured to produce the pan-HLA-I (A, B, C) antibody, and IVA12 cells were cultured for the pan-HLA-II (DR, DP, DQ) antibody. The culture supernatant was collected, there is no cross reactivity of this IVA 12 antibody towards HLA-I molecules. These supernatants were then appropriately diluted with Pierce Protein A or Protein G binding buffer (Thermo Scientific) and loaded onto columns packed with Pierce Protein A Plus Agarose for HLA-I antibodies or Protein G Plus Agarose for HLA-II antibodies (Thermo Scientific), respectively. After washing the columns with the binding buffer and subsequently with the 0.2M sodium borate buffer (pH 9), agarose beads were cross-linked using dimethyl pimelimidate (Thermo Scientific) at the final concentration of 20mM in the sodium borate buffer. Following rotation of the agarose beads for 2 hours in 2.5X bead volumes of 200mM ethanolamine (pH 8), they were washed with the binding buffer and stored in phosphate-buffered saline at 4°C. [0199] Purification ofHLA bound peptides

[0200] The procedures for purification of HLA bound peptides were modified from those used in our previous stud y. In brief, the tissues after surgical resection were immediately frozen in liquid nitrogen and stored at -80°C until the experiment. The tissue samples and cell samples were lysate by Pierce IP lysis buffer (Thermo Scientific) containing a Complete Protease Inhibitor Cocktail (Roche) at 4 °C. The supernatant were sequence incubated with unconjugated Protein A beads and the pan-HLA-I antibody-conjugated Protein A beads. After that, HLA-I bound peptides were eluted. Then, the HLA-II bound peptides were also purified as described as below, after the protein lysate was incubated with the HLA-I antibody conjugated beads to remove the HLA-I bound peptides, the flow-through was used for the isolation of HLA-II bound peptides with the pan-HLA-II antibody-conjugated Protein G beads followed by washing as described above. For peptide elution, The HLA antibody-conjugated beads were washed with washing buffer. HLA molecules were eluted at room temperature by 500 pl of 0.1 N acetic acid (pH 3) for 15 minutes. Eluted peptides were loaded to the Sep-Pak C18 3cc/200mg Vac Cartridge (Waters, Milford, MA). The flow-through was repeatedly loaded to the cartridges two more times. The cartridges were washed twice with 300ul of 0.1% TFA. After washing, the peptides were eluted for three times with 400 ul, 300 ul, and 300ul of 80% Acetonitrile in 0.1% TFA, respectively, into a clean 1.5 mL Eppendorf tube. The eluted samples were dried thoroughly at 30°C using vacuum centrifugation and then stored at -80°C.

[0201] LC-MS/MS analysis ofHLA peptides

[0202] Above samples were rehydrated in 20 ul of 2% acetonitrile, 0.1% formic acid and placed in an EasyLC autosampler and nanoLC system coupled to an Orbitrap Lumos mass spectrometer (Thermo Fisher). 10 ul of the sample was injected onto a trap column at 5ul/min and then eluted into the mass spectrometer at 300 nl/min over a 90-minute gradient from 2% acetonitrile to 90% acetonitrile in 0.1% formic acid. An in house made nanoLC column (75um ID x 250mm packed with ReproSiLPur 120 C18-AQ 3um particles was used to separate the peptides. The mass spectrometer was operated at a resolution of 120,000 for MS and 30,000 for MS2. The peptides were fragmented with an isolation window of 1.6 Daltons and collision energy of 30% NCE via higher-energy C-trap dissociation (HCD). As many peptides as possible in a 3-second cycle having a charge of 2-6 were fragmented before doing the next MS precursor scan and precursors which had been previously fragmented were dynamically excluded for 15 seconds. The AGC target for MS was set to 4e5 ions with a maximum injection time of 50 milliseconds and MS2 was set to le5 ions and 100 milliseconds maximum. The precursor masses were subjected to calibration on the fly using the Easy-IC fluoranthene lock mass system.

[0203] MS data analysis of HLA peptides

[0204] Andromeda of the MaxQuant computational platform, a peptide search engine integrated into the MaxQuant environment (Max Planck Institute of Biochemistry, Munich, Germany) was used to search the peak lists against the UniProt databases (Human 93,609 entries, Feb 2018). The settings used were as suggested in the previous study. Briefly, the second peptide identification option in Andromeda was enabled. Enzyme specificity was set as unspecific. A false discovery rate of 0.01 was minimally required. The initially allowed mass deviation of the precursor ion was set to 6 p.p.m. The maximum fragment mass deviation was set to 20 p.p.m. The peptide sequences that were considered to be reverse sequences or contaminants by MaxQuant were filtered out. NetMHC-4.0 and NetMHCIIpan-4.0 (Department of Health Technology, Lyngby, Denmark) were used to predict binding affinities of peptides.

[0205] FluoroSpot assay

[0206] The peptides were synthesized and purified to > 95% purity by Peptide 2.0 (Chantilly, VA) according to the sequences identified by MaxQuant. Peptides were stocked in 100% DMSO and diluted in the cell culture medium to yield a final peptide concentration at 10 ng/ml. Archived, cryopreserved PBMCs were recovered and immediately subjected to the FluoroSpot assay. 2x105 PBMCs per well were plated into a 96-well FluoroSpot assay plate from the Human IFN-y /Granzyme B FluoroSpotPLUS kit or the Human IFN-y/IL-2/TNF-a FluoroSpotPLUS kit (Mabtech, Cincinnati, OH). Peptides of interest at a concentration of 2 pg/ml were incubated with PBMCs according to the manufacturer’s instruction. Positive controls were PBMCs stimulated with anti-CD3/anti-CD28 antibodies (Mabtech, Cincinnati, OH) or the CEF peptides (Immunospot, Cleveland, OH). Negative controls were PBMC without stimulation. Cytokines produced by PBMCs following peptide stimulation were captured by their specific antibodies conjugated by different fluorescences. Plates were read by an AID iSpot Spectrum reader (Autoimmun Diagnostika GmbH) at the Johns Hopkins University Immune Monitoring Core. The results were processed by the software provided by the manufacturer. Spots that expressed IFN-y or Granzyme B were counted separately. Spots expressing one, two, or all three cytokines among IFN-y, IL-2, and TNF-a were counted, respectively.

[0207] Immunohistochemistry (IHC)

[0208] Tumor tissues for human correlative IHC staining were obtained from specimens collected from patients who underwent surgery at the Johns Hopkins Hospital under the JHMI IRB approved protocol (IRB00244430). Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 5-pm and subjected to heat-induced antigen retrieval. IHC staining was performed using Dako Catalyzed Signal Amplification system as previously described. Anti-TMABIO (Thermo Fisher Scientific, PA5-116041) was used in our study. All slides were scanned and analyzed using Image Analysis Software (Aperio Technologies).

[0209] T cell cultures with the TMSB1028-44 peptides

[0210] Archived PBMCs collected by the JHMI IRB approved protocol (IRB00244430) were stimulated in 24-well cell-culture plates at the concentration of 5 x 106 cells per well with the TMSB1028-44 peptides (10 pg/ml) in the presence of IL-7 (20 ng/ml; Peprotech). On day 3, low- dose rIL-2 (20 U/ml; Amgen) was added. Half-medium change was performed with fresh medium supplemented with rIL-2(20 U/ml) and IL-7(20 ng/ml) every 3 days. PBMCs cultured only in the presence of the cytokines but not peptides were used to provide a baseline TCR repertoire profile for comparison. After 21 days, CD8+ and CD4+ T cells were sorted through magnetic-activated cell sorting (MACS) and processed following the lOx Genomics Chromium Single Cell Protocol. [0211] Single Cell TCR sequencing using the lOx Chromium platform

[0212] The single-cell RNA sequencing libraries were prepared following the protocol provided by the 10^x genomics Chromium Single Cell Immune Profiling Solution. Cellular suspensions were loaded on a Chromium Single Cell Controller instrument (lOx Genomics) to generate single-cell Gel Bead-In Emulsions (GEMs). After reverse transcription of mRNA, droplets were broken, and barcoded cDNA was purified with DynaBeads. Barcoded, full-length V(D)J segments were enriched from amplified cDNA with primers specific for the human TCR constant regions. V(D)J region-enriched libraries were size selected with SPRI beads (avg. size 600 bp) and sequenced on an Illumina HiSeq 2500 instrument. The Cell Ranger Software Suite (version 2.1.0) was used for V(D)J sequence assembly [0213] TCR reconstitution in Jurkat cells

[0214] Lentiviral transfer plasmids were synthesized by OBiO Technology. Variable regions of TCRa- and [3- chains of CD8-TCR and CD4-TCR were linked by a P2A peptide element to yield the transgene cassette 5'-TCR[3-P2A-TCRa-3'. The transgene cassette was synthesized and integrated into the GFP-tagged retrovirus vector GL121. The P2A linker peptide results in higher expression and functionality of human TCR . To enhance TCR surface expression, the constant regions of both TCR chain genes were exchanged by their mouse counter parts . All constructs were verified by sequence analysis. Lentivirus transfer plasmids carrying TCR1 , TCR2, TCR3 and TCR4 were co-transfected into HEK293T cells with packaging plasmid pCMV-dR8.91 and envelope plasmid pCMV-VSV-G to produce lentivirus particles. Lipofectamine 2000 transfection reagent (Invitrogen, 11668027) was added according to manufacturer’s instruction. The supernatants containing the relevant lentivirus were harvested 48 h and 72 h post transfection. To establish TCR1 -expressing, TCR2-expressing, TCR3 -expressing, TCR4-expressing, and GL121 backbone lentivirus-infected cell lines (TCR1 -Jurkat, TCR2-Jurkat, TCR3-Jurkat, TCR4-Jurkat, and GL121-Jurkat, respectively), Human Jurkat T-cells (ATCC, Clone E6-1) were infected by respective recombinant lentiviruses in the presence of polybrene (1:500) (Sigma- Aldrich, TRI 003). The medium was changed to the normal culture medium 24 h post infection.

[0215] In vitro Jurkat Cytotoxicity assay

[0216] In total, 5X103 AsPC-1 tumor cells and 2.5x104 Transfected Jurkat cells were cultured in at a ratio of 1 :5 in an opaque-walled flat-bottomed 96-well plate for 48 hours in T cell medium. For the group with two effector cell types, we added 1.25 xl04 cells for each cell types. Cell viability was tested using CytoTox-Fluor™ Cytotoxicity Assay kit (Promega) according to the manufacturer’s instructions.

[0217] Statistical analyses

[0218] All statistical analyses and most of graphs were performed using GraphPad Prism software (GraphPad Software). Venn’s diagrams were drawn with VENNY2.1 (CNB-CSIC, Madrid, Spain). The mean fluorescent intensities or mean values of spot forming units (SFU) in the FluoroSpot assay were compared by Welch’s t-test for two-group comparisons and by oneway ANOVA for multiple group comparisons. If the SFU of a peptide in a sample is less than that of the negative control peptide, it is set as zero; and such a result would be considered “unstimulated”. Because SFUs of negative control peptides vary significantly among different samples, the comparison was made between stimulated and unstimulated peptide/samples. A p- value of less than 0.05 was considered statistically significant.

[0219] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. An isolated peptide having the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2.

2. A polyepitope peptide comprising one or more HLA-class I and/or a HLA-class II restricted or non-restricted epitopes, wherein the one or more epitopes are an antigenic fragment of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, 0RMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

3. The polyepitope peptide of claim 2, wherein the one or more epitopes have the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2.

4. The polyepitope peptide of claim 3, comprising an epitope having the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

5. An isolated T cell comprising a T cell receptor (TCR) having a binding affinity to an HLA- class I and/or HLA-class II restricted or non-restricted epitope, wherein the epitope is an antigenic fragment of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, 0RMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

6. The isolated T cell of claim 5, wherein the T cell is an engineered T cell.

7. The isolated T cell of claim 5, wherein the epitope has the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2.

8. The isolated T cell of claim 7, wherein the epitope has the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

9. A vaccine comprising one or more HLA-class I and/or HLA-class II restricted or nonrestricted epitopes, wherein the epitopes are antigenic fragments of a protein or gene product encoded by a gene selected from the group consisting of COL6A3, ELOVL1, LAMC2, RASAL2, DYNLRB1, ICE1, LAMB3, MYH9, 0RMDL3, MYL12A, LAMC2, WDR82, TRRAP, TFIP11, ACBD3, CKS2, IGF1, TRAPPCI 1, ZMYND11, CTNNBIP1, TMSB10, and any of the genes listed in Table 2.

10. The vaccine of claim 9, comprising a lipid nanoparticle for presenting the one or more epitopes to antigen presenting immune cells.

11. The vaccine of claim 10, wherein the one or more presented epitopes have the amino acid sequence of any of SEQ ID NOs:7-32 or of any of the peptides listed in Table 2.

12. The vaccine of claim 11, wherein the one or more epitopes have the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.

13. A method of treating cancer in a subject comprising administering to the subject one or more of the peptides of claim 1, one or more of the polyepitope peptides of any of claims 2-4, the T cell of any of claim 5-8, or the vaccine of any of claims 9-12, thereby treating cancer in the subject.

14. The method of claim 13, wherein the HLA-class I and/or HLA-class II restricted or nonrestricted peptides or the polyepitope peptides thereof induces polyfunctional T cells in the subj ect.

15. The method of claim 13, wherein inducing polyfunctional T cells comprises stimulating a T cell response and/or stimulating T cell expression of effector T cell cytokine.

16. The method of claim 15, wherein the effector T cell cytokines comprise IFNy, IL-2 and/or TNFa.

17. The method of claim 15, wherein stimulating a T cell response comprise stimulating cytotoxic T cell cytokines.

18. The method of claim 17, wherein the cytotoxic T cell cytokines comprise IFNy and/or granzyme B.

19. The method of claim 13, wherein the cancer is pancreatic cancer.

20. The method of claim 19, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

21. The method of claim 13, further comprising administering to the subject an anti-cancer treatment.

22. The method of claim 21, wherein the anti-cancer treatment is selected from the group consisting of gemcitabine, folfirinox, erlotinib, nab-paclitaxel, liposomal irinotecan, and olaparib.

23. The method of claim 13, wherein the cancer is a cancer expressing an epitope having the amino acid sequence of any of SEQ ID NOs:7-32, or of any of the peptides listed in Table 2.

24. The method of claim 13, wherein the cancer is a cancer expressing an epitope having the amino acid sequence of SEQ ID NO: 17 and/or the amino acid sequence of SEQ ID NO:30.