Immunogenic arginase 2 polypeptides

Abstract

The present invention relates to new polypeptides derived from arginase 2. The present invention also relates to uses of said polypeptides and compositions comprising said polypeptides.

Description

TECHNICAL FIELD

[0001] The present invention relates to new polypeptides derived from arginase 2. The present invention also relates to the use of said polypeptides and of compositions comprising said polypeptides. BACKGROUND

[0002] Arginase is an enzyme that catalyzes the reaction converting the amino acid L-arginine into L-ornithine and urea. This depletes the microenvironment of arginine and leads to the inhibition of tumor-specific cytotoxic T cell responses. For example, elevated arginase activity has been detected in cancer cells of breast cancer, lung cancer, colon cancer or prostate cancer patients. It has been shown that in vitro and in vivo, mouse macrophages transfected with the rat arginase gene promote the proliferation of co-cultured tumor cells. Moreover, induction of arginase expression by macrophages has been shown to increase tumor angiogenesis through polyamine synthesis. Results from a murine lung cancer model have shown that there is a subpopulation of mature tumor-associated myeloid cells that express high levels of arginase. These tumor-associated myeloid cells deplete extracellular L-arginine, which inhibits the antigen-specific proliferation of tumor infiltrating lymphocytes (TILs). In mice, infusion of an arginase inhibitor blocks the growth of lung cancer. This shows how induction of arginase expression in tumor cells and tumor-associated myeloid cells can promote tumor growth by inhibiting the anti-tumor immune response through a negative effect on TILs.

[0003] MDSCs (myeloid-derived suppressor cells) suppress the activation, proliferation and cytotoxicity of effector T cells and natural killer cells, as well as inducing Treg differentiation and expansion. Both tumor cells and MDSCs can suppress T cells by manipulating L-arginine metabolism through the enzymes nitric oxide synthase (NOS) and arginase. Many tumors exhibit increased expression of arginase and inducible NOS (iNOS), leading to depletion of arginine from the tumor microenvironment. Several studies emphasize the importance of this altered tumor arginine metabolism in suppressing tumor-specific T cell responses, and a recent study demonstrated that acute myeloid leukemia (AML) blasts display arginase-dependent ability to suppress T cell proliferation and hematopoietic stem cells. Moreover, arginase and iNOS inhibitors reduce the suppressive activity of AML.

[0004] In mammals, there are two arginase isozymes: arginase 1 and arginase 2. These two isozymes catalyze the same biochemical reaction (and thus cannot be distinguished by enzyme assays), but differ in cellular expression, regulation and subcellular localization. SUMMARY

[0005] The present inventors have previously identified a region of 50 amino acids of arginase 1 and arginase 2 that is an immunogenic "hotspot". This region corresponds to positions 161 to 210 of full-length human arginase 1 (SEQ ID NO: 53) or positions 180 to 229 of full-length human arginase 2 (SEQ ID NO: 51), or the corresponding positions of murine arginase. The region and peptides derived from it are described in WO2018065563. The present inventors have also identified that a particular subset of polypeptides derived from the "hotspot" region of arginase 1 are particularly effective at stimulating an immune response. These peptides correspond to positions 169 to 206 of full-length human arginase 1, positions 169 to 200 of full-length human arginase 1, or positions 169-210 of full-length human arginase 1 (or the corresponding positions of human arginase 2 or murine arginase 1). PCT / EP2019 / 075731 and its prior application GB1815549.9 describe the subset of polypeptides.

[0006] The present inventors have now identified that polypeptides derived from a completely different region of human arginase 2 are particularly effective at stimulating an immune response. Surprisingly, the region spans the C-terminus of the transit peptide of human arginase 2 (position 22 of SEQ ID NO: 51, see schematic in Figure 7 The sequence homology of this region to human arginase 1 is relatively low.

[0007] The polypeptides of the present invention are expected to be particularly effective at stimulating a beneficial immune response against arginase 2 and arginase 2-expressing cells. The development of new immunotherapies for cancer requires a full understanding of the molecules involved in the pathogenesis and the specific proteins recognised by the immune system. In the clinical context, inducing an arginase-specific immune response can support the anti-cancer immune response globally, in addition to killing cancer cells, by inhibiting the immunosuppressive function of arginase-expressing cells, in particular MDSC and tumour-associated macrophages (TAM). Therefore, targeting myeloid dendritic cells (for example by vaccination with the polypeptides of the present invention) will highly cooperate with other anti-cancer immunotherapies, as arginase-expressing cells antagonise the desired effects of other immunotherapies.

[0008] The present invention provides a polypeptide which is an immunogenic fragment of human arginase 2 (SEQ ID NO: 51), comprising or consisting of a sequence of at least 9 contiguous amino acids of SEQ ID NO: 51, which (i) include at least the amino acids at positions 21, 22 and 23 of SEQ ID NO: 51, or (ii) are selected from positions 180 to 229 of SEQ ID NO: 51. The polypeptide can comprise or consist of up to 15, 20, 25, 30, 35, 40, 45 or 50 contiguous amino acids of SEQ ID NO: 51 as defined in (i) or (ii). The polypeptide can comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 59, 58, 57, 54, 55, 56, 2, 3, 19, 20, 21, 60 or 61. The polypeptide has a maximum length of 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids and / or wherein the C-terminal amino acid is replaced by the corresponding amide. The polypeptide can be isolated.

[0009] The present invention also provides a polypeptide which is an immunogenic fragment of murine arginase 2 (SEQ ID NO: 52), comprising or consisting of a sequence of at least 9 contiguous amino acids of SEQ ID NO: 52, which (i) include at least the amino acids at positions 21, 22 and 23 of SEQ ID NO: 52, or (ii) are selected from positions 180 to 229 of SEQ ID NO: 52. The polypeptide can comprise or consist of up to 15, 20, 25, 30, 35, 40, 45 or 50 contiguous amino acids of SEQ ID NO: 52 as defined in (i) or (ii). The polypeptide has a maximum length of 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids and / or wherein the C-terminal amino acid is replaced by the corresponding amide. The polypeptide can be isolated.

[0010] The present invention also provides a composition comprising a polypeptide of the present invention, at least one pharmaceutically acceptable diluent, carrier or preservative, and optionally an adjuvant.

[0011] The present application also provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a polypeptide or composition of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Design of certain polypeptides spanning the "hotspot" regions of human arginase 1 and human arginase 2 is shown. Table X of the specification provides the sequence identifier numbers.

[0013] Figure 2 Identification of ARG2 peptides corresponding to the hotspot regions by peripheral blood mononuclear cells (PBMCs) from healthy donors and melanoma patients (AA914.30) is shown. Spot counts represent the difference between the mean of the wells stimulated with peptide and the mean of the control wells without peptide. 5 x 10 5 cells per well, with peptide and control stimulation performed in triplicate. Responses were analyzed using the Distribution-Free Resampling (DFR) rule.

[0014] Figure 3 Identification of multiple ARG2 library peptides by PBMCs from healthy donors is shown. Spot counts represent the difference between the mean of the wells stimulated with peptide and the mean of the control wells without peptide. 4-4.5 x 10 5 cells per well, with peptide and control stimulation performed in triplicate. Boxes represent the highest, most abundant responses after screening 6 healthy donors.

[0015] Figure 4 Verification of responses to hotspot region peptides is shown. Spot counts represent the difference between the mean of the wells stimulated with peptide and the mean of the control wells without peptide. 5 x 10 5 cells per well, with peptide and control stimulation performed in triplicate. Boxes show peptides with nearly identical sequences to facilitate comparison of responses obtained.

[0016] Figure 5 Verification of responses to ARG2_1, ARG2_5, ARG2_8, ARG2_13, and ARG2_22 is shown. Spot counts represent the difference between the mean of the wells stimulated with peptide and the mean of the control wells without peptide. 3 x 10 5 cells per well, with peptide and control stimulation performed in triplicate. A strong and significant response to ARG2_1 was observed.

[0017] Figure 6A and 6BCD4+ T cell responses to ARG2_1 or no peptide are shown for two healthy donors. PBMC cells were stained with CD4 antibody and analyzed by flow cytometry in an intracellular cytokine release assay with or without ARG2_1 stimulation. As can be seen from the graph, CD4 cells release more IFN-γ (y-axis) or TNF-α (x-axis) when stimulated with the ARG_2 derived peptide.

[0018] Figure 7 A schematic of arginase 2 showing the location of the ARG2_1 fragment is shown.

[0019] Figure 8 Recognition of ARG2 peptides corresponding to the hot spot region by PBMC from healthy donors (BC or Buf-M) and cancer patients (AA, melanoma; UR, renal cell carcinoma) is shown. Spot counts are expressed as the difference between the mean of the wells stimulated with peptide and the mean of the control wells without peptide. 4 x 10 5 cells were seeded per well and peptide and control stimulation was performed in triplicate. Responses were analyzed using the distribution free resampling (DFR) rule. *-p<0.05, **-p<0.01.

[0020] Figure 9 CD8+ T cell responses to ARG2_1 or no peptide are shown for one cancer patient. PBMC cells were stained with CD8 antibody and analyzed by flow cytometry in an intracellular cytokine release assay with or without ARG2-1 stimulation. As can be seen from the graph, CD8+ T cells release more IFN-γ (y-axis) and TNF-α (x-axis) when stimulated with the ARG_2 derived peptide, even though some background release of TNF-α is also observed.

[0021] Figure 10 In vitro ELISPOT responses to ARG2_1 are shown without pre-stimulation but after incubation + / - peptide for 72 hours in ELISPOT plates. 9 x 10 5 cells were seeded per well and ARG2_1 peptide and no ARG2_1 (control) were performed in triplicate. Spot counts are expressed as the difference between the mean of the wells stimulated with peptide and the mean of the control wells without peptide.

[0022] Figure 11 An experimental protocol for in vivo testing of murine Arg2 is shown. 3 mice per group were subcutaneously inoculated with one of 6 peptides. After 7 days, the mice were sacrificed, the spleen was homogenized and PBMC were used for mIFNγ ELISPOT.

[0023] Figure 12 An experimental protocol for in vivo testing of murine Arg2 is shown. 3 mice per group were subcutaneously inoculated with one of 6 peptides. After 7 days, the mice were sacrificed, the spleen was homogenized and PBMC were used for mIFNγ ELISPOT. Figure 11The results of the mIFNγELISPOT experiment were performed afterward. Spot counts are expressed as the difference between the mean of the peptide-stimulated wells and the mean of the control wells without peptide. 8 × 10⁸ spots were seeded per well. 5 Each cell was administered in triplicate, with and without the peptide. Parentheses indicate mice inoculated with the same peptide, and boxes highlight mice with the strongest and most significant responses.

[0024] Figure 13 The results showed that immunization with mARG2_188-196 elicited a strong mIFNγ response to both mARG2_188-196 and mARG2_182-197. 8 × 10⁸ cells were inoculated per well. 5 Cells, peptide and control stimulation were performed three times in parallel.

[0025] Figure 14 This study demonstrates that ARG2-1 is widely recognized by PMBCs from healthy donors and patients with solid tumors or AML. (A) IFNγELISPOT response to the ARG2-1 peptide in PMBCs from healthy donors (n=33), patients with solid tumors (n=19), or patients with AML (n=19). 3–4 × 10⁻⁶ cells per well were seeded. 5 Cells. Peptide and control stimulation were performed in triplicate. Each spot represents one donor and is the number of peptide-specific IFNγ-secreting cells (difference between the mean of the peptide-stimulated wells and the mean of the control wells). (B) Intracellular cytokine staining for IFNγ and TNFα production in healthy donors (HD49 and HD50) and cancer patients with solid tumors stimulated with or without ARG2-1 stimulation (AA27).

[0026] Figure 15 This study demonstrates that the long ARG2 peptide A2F2 induces strong and frequent CD4+ T cell responses in both healthy donors and cancer patients. (A) IFNγELISPOT response to the long peptides A2F1, A2F2, and A2F3 in PBMCs from six healthy donors. 4 × 10⁶ cells per well were seeded. 5 Cells, peptide, and control stimulation were performed in triplicate. Specific spot count (peptide-specific IFNγ-secreting cells) was expressed as the difference in the number of IFNγ spots between the mean of the peptide-stimulated wells and the mean of the control wells. In all three experiments, responses to the peptide were too numerous to count (TNTC) and set at >750 spots. (B) IFNγ ELISPOT response to A2L2 and ARG2-1 in PBMCs from 6 healthy donors. 4 × 10⁶ cells per well were seeded. 5Cells. Peptide and control stimulation were performed in triplicate. Specific spot count (peptide-specific IFNγ-secreting cells) is expressed as the difference in the number of IFNγ spots between the mean of the peptide-stimulated wells and the mean of the control wells. According to the distribution-free resampling rule, *p<0.05 or **p<0.01. (C) IFNγ ELISPOT response to A2L2 peptide in PBMCs from healthy donors (n=30) and solid tumor patients (n=18). 3-4 × 10⁶ cells per well. 5 Cells. Peptide and control stimulation were performed in triplicate. Each spot represents one donor and is also the number of peptide-specific IFNγ-secreting cells (difference between the mean of the wells stimulated with peptide and the mean of the control wells). (D) Representative intracellular cytokine staining for IFNγ and TFNa production in healthy donors (HD48 and HD53) and solid tumor patients (AA27) stimulated with or without A2L2 stimulation. (E) IFNγ EFISPOT response to ARG2-1 and A2L2 in PBMCs from healthy donors (n=26) and solid tumor patients (n=11) to compare the magnitude of the response to the two peptides. 4 × 10⁶ cells per well. 5 Cells were stimulated three times in parallel with both peptide and control stimulation. Specific spot count (peptide-specific IFNγ-secreting cells) was expressed as the difference in the number of IFNγ spots between the mean of peptide-stimulated wells and the mean of control wells. ns:p = 0.7038.

[0027] Figure 16 This illustrates the recognition of ARG2-specific T cells by ARG2-expressing dendritic cells. (A) ARG2-specific T cells were expanded from prostate cancer patients. The specificity of T cell cultures was assessed by staining for intracellular cytokines produced by TNFα and IFNγ in peptide-stimulated cells and non-stimulated controls. Left: Dot plot of ARG2-1 peptide-stimulated and non-stimulated (control) cells. Right: %CD4+ T cells producing IFNγ, TNFα, or both in response to control stimulation (no peptide), ARG2-1 peptide stimulation, or A2L2 peptide stimulation. (B) The specificity of ARG2-specific T cells was assessed by the ELISPOT response to control stimulation (no peptide), ARG2-1 peptide, or A2L2 peptide. 4 × 10-1 cells were seeded per well. 4 Cells. TNTC = Too many to count (over 500 spots). (C) Detection of HLA restriction in ARG2-specific T cells. IFNγELISPOT response of ARG2-specific T cells to ARG2-1 peptide in the presence of different class II inhibitors. (D) IFNγELISPOT response of ARG2-specific T cells to autologous dendritic cells transfected with unrelated control mRNA (mimetic mRNA) or ARG2 mRNA. Effector-to-target ratio was 5:1, with 5 × 10⁶ cells seeded per well.4 Effectors cells. According to the no distribution resampling rule, *p<0.05 or **p<0.01.

[0028] Figure 17 ARG2-specific T cells recognize ARG2-expressing malignant myeloid cells. (A) To identify HLA-matched malignant cells, ARG2-specific T cells were tested for IFNy ELISPOT responses to different relevant cancer cell lines pre-pulsed with ARG2-1 peptide. The same tumor cell lines without peptide stimulation were tested as controls. Effector to target ratio 1 : 1, 1 x 105effectors cells per well. According to the no distribution resampling rule, *p<0.05 or **p<0.01. 4 ARG2-specific T cells recognize ARG2-expressing malignant myeloid cells. (A) To identify HLA-matched malignant cells, ARG2-specific T cells were tested for IFNy ELISPOT responses to different relevant cancer cell lines pre-pulsed with ARG2-1 peptide. The same tumor cell lines without peptide stimulation were tested as controls. Effector to target ratio 1 : 1, 1 x 105effectors cells per well. According to the no distribution resampling rule, *p<0.05 or **p<0.01. 5 ARG2-specific T cells recognize ARG2-expressing malignant myeloid cells. (A) To identify HLA-matched malignant cells, ARG2-specific T cells were tested for IFNy ELISPOT responses to different relevant cancer cell lines pre-pulsed with ARG2-1 peptide. The same tumor cell lines without peptide stimulation were tested as controls. Effector to target ratio 1 : 1, 1 x 105effectors cells per well. According to the no distribution resampling rule, *p<0.05 or **p<0.01. 5effector cells. **p<0.01 and ns = not significant according to the no distribution resampling rule. (H) ARG2 expression in THP-1 cells was assessed by RT-qPCR after 48h stimulation with cytokine cocktail or IFNy. Unstimulated THP-1 cells were used as control. Data are expressed as relative expression to the housekeeping gene RPLPO, mean + SD, n=4. (I) IFNy ELISPOT responses of ARG2-specific T cells to THP-1 cells pre-stimulated with cytokine cocktail (THP-1 + cytokines) or IFNy (THP-1 + IFNy). Effector to target ratio 2.5:1, 5x105effector cells plated per well. Data are expressed as spot forming cells (SFC) per million effector cells, mean + SD, n=4. 4 **p<0.01 and ns = not significant according to the no distribution resampling rule.

[0029] Figure 18 ARG2-specific T cells recognize several ARG2-expressing malignant myeloid cells. (A) ARG2 expression in MONO-MAC-1 (MM1) cells was assessed by RT-qPCR after 48h incubation with cytokine cocktail. Data are expressed as fold change compared to unstimulated MM1 cells, mean + SD, n=4. (B) ARG2 expression in MM1 cells was assessed by RT-qPCR after 48h incubation with cytokine cocktail or IFNy. Data are expressed as fold change compared to unstimulated MM1 cells, mean + SD, n=4. (C) IFNy ELISPOT responses of ARG2-specific T cells to MM1 cells pre-stimulated with cytokine cocktail (MM1 + cytokine cocktail) or IFNy (MM1 + IFNy). Effector to target ratio 2.5:1, 5x105effector cells plated per well. Data are expressed as spot forming cells (SFC) per million effector cells, mean + SD, n=4. 4 **p<0.01 and ns = not significant according to the no distribution resampling rule.

[0030] Figure 19 Recognition of ARG2-expressing cells by ARG2-specific T cells depends on ARG2 expression level in addition to target cell antigen processing devices. (A) IFNy ELISPOT responses of ARG2-specific T cells to unstimulated or pre-stimulated with cytokine cocktail, and mock-transfected or transfected with ARG1 or ARG2 mRNA THP-1 cells. Effector to target ratio 2.5:1, 5x105effector cells plated per well. Data are expressed as spot forming cells (SFC) per million effector cells, mean + SD, n=4. 4(a) Effector cells. **p<0.01 and ns=not significant according to the no-distribution resampling rule. (B) Intracellular staining of TNFα and IFNγ from CD4+ T cells in ARG2-specific T cell cultures when incubated with unstimulated THP-1 cells or THP-1 cells prestimulated with a mixture of cytokines, followed by mock or ARG2 mRNA transfection. Effector-target ratio 2:1, 500,000 effector cells used in each case. (C) ARG2 expression in THP-1 cells assessed by RT-qPCR 48 hours after transfection with ARG2-specific siRNAs. Data are expressed as fold change compared to mock-transfected THP-1 cells, mean +SD, n=4. (D) Intracellular staining of TNFα and IFNγ produced by CD4+ T cells from ARG2-specific T cell cultures during incubation with simulated or siRNA-transfected cells was set up for 48 hours under unstimulated or cytokine mixture-stimulated conditions. The effector-target ratio was 2:1, and 500,000 effector cells were used in each case. (E) ARG2 expression in THP-1 cells was assessed by RT-qPCR after transfection with ARG2-specific siRNAs followed by stimulation with a cytokine mixture for 48 hours. Data are expressed as fold change compared to unstimulated simulated transfected THP-1 cells, mean +SD, n=4.

[0031] Figure 20 (A) Screening of C57BL / 6 mouse spleen cells with mouse IFNγELISPOT using one of six different predicted Arg2 epitopes. 8 × 10⁸ cells per well. 5 Cells, peptide and control stimulation were performed in triplicate. Specific spot count (peptide-specific IFNγ-secreting cells) is expressed as the difference in the number of IFNγ spots between the mean of the peptide-stimulated wells and the mean of the control wells. (B) Mouse IFNγ ELIPOT from spleen cells of C57BL / 6 mice immunized with Arg2 peptide P4 (M1-M5) or control immunization (Ctrl 1-4). 8 × 10⁸ cells per well. 5Cells, peptide and control stimulation were performed in triplicate. Specific spot counts (peptide-specific IFNγ-secreting cells) are expressed as the difference in the number of IFNγ spots between the mean of peptide-stimulated wells and the mean of control wells. (C) Arg2 expression in xenografts of different origins in the C57BL / 6 background. Tumor type was assessed in all 3 xenografts. Data are expressed as relative expression of the housekeeping gene Hprtl, mean + SD, n = 3. (D) Treatment regimens in two separate efficacy studies of Arg2 immunization in LL2-inoculated mice with or without anti-PD-1 therapy. 5 × 10⁵ cells were injected subcutaneously into the right abdomen on day 0. 5 LL2 cells were then treated with: (E) control or Arg2 immunization with Arg2 peptide (n=20 in both groups) or (F) control immunization, Arg2 immunization, anti-PD-1 treatment, or Arg2 immunization + anti-PD-1 treatment (n=10 in all groups). (EF) Mean tumor growth of LL2 tumors in individual efficacy studies. Error bars represent the standard error (SEM) of the mean, ***=p<0.0001. (G) Individual tumor size in the treatment group described in (E). Error bars represent the standard deviation (SD).

[0032] Sequence Overview

[0033] SEQ ID NO: 1 to 38 are the amino acid sequences of the polypeptide derived from human arginase 2.

[0034] SEQ ID NO: 39 and 40 are the corresponding "hot spot" regions of human arginase 2 and arginase 1, respectively.

[0035] SEQ ID NO: 41 to 44 are the amino acid sequences of the polypeptide derived from human arginase 1.

[0036] SEQ ID NO: 45 to 50 are the amino acid sequences of the polypeptide derived from mouse arginase 2.

[0037] SEQ ID NO: 51 is the amino acid sequence of full-length human arginase 2.

[0038] SEQ ID NO: 52 is the amino acid sequence of full-length mouse arginase 2.

[0039] SEQ ID NO: 53 is the amino acid sequence of full-length human arginase 1.

[0040] SEQ ID NO: 54 to 56 are the amino acid sequences of the polypeptide derived from human arginase 2, which correspond to the sequence of the predicted HLA-A2 or A3 epitope in the polypeptide of SEQ ID NO: 2 (Arg2_l).

[0041] SEQ ID NOs: 57-59 are amino acid sequences of additional polypeptides derived from human arginase 2 that include at least one of the epitopes of SEQ ID NOs: 54-56.

[0042] SEQ ID NOs: 60-61 are amino acid sequences of additional polypeptides derived from human arginase 2 that include sequences from the "hot spot" region of SEQ ID NO: 39.

[0043] SEQ ID NO: 62 is a predicted signal sequence of human arginase 2. DETAILED DESCRIPTION

[0044] It should be understood that different applications of the disclosed products and methods can be tailored to specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to be limiting.

[0045] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "a plurality of polypeptides" and the like.

[0046] As used herein, "polypeptide" refers broadly to a compound of two or more subunit amino acids, amino acid analogs or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences, but also longer polypeptides and proteins. As used herein, the term "amino acid" refers to both natural and / or unnatural or synthetic amino acids, both D or L optical isomers, amino acid analogs and peptidomimetics.

[0047] The terms "patient" and "subject" are used interchangeably and generally refer to a human.

[0048] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0049] The present inventors have determined that a region of human arginase 2 spanning the C-terminus of the transit peptide is particularly immunogenic. The C-terminal residue of the transit peptide corresponds to position 22 of SEQ ID NO: 51. Thus, a region spanning the C-terminus of the transit peptide includes at least the amino acids of positions 21, 22 and 23 of SEQ ID NO: 51.

[0050] By "immunogenic" herein is meant that the polypeptide is capable of eliciting an immune response to the arginase 2 protein, preferably when the protein is present in or on a cell expressing the arginase 2 protein. In other words, the polypeptide can be described as being immunogenic to arginase 2. The polypeptide can alternatively be described as being an immunogenic fragment of arginase 2. The immune response is preferably a T cell response, and thus the polypeptide can be described as being an immunogenic fragment of arginase 2 comprising a T cell epitope. The immune response can be detected in at least one individual (or sample extracted from an individual) after administration of the polypeptide to the individual (or the sample).

[0051] Any suitable method, including in vitro methods, can be used to identify a polypeptide as being immunogenic. For example, a polypeptide can be identified as being immunogenic if it has at least one of the following characteristics:

[0052] (i) it is capable of eliciting IFN-γ producing cells in a population of PBLs from healthy subjects and / or cancer patients, by an ELISPOT assay, and / or

[0053] (ii) it is capable of being detected in situ in tumor tissue samples of CTLs reactive with arginase 2; and / or

[0054] (iii) it is capable of inducing the in vitro growth of specific T cells.

[0055] The following Examples section also describes methods suitable for determining whether a polypeptide is immunogenic.

[0056] The polypeptide of the application is an immunogenic fragment of human arginase 2 (SEQ ID NO: 51) comprising or consisting of a sequence of at least 9 contiguous amino acids of SEQ ID NO: 51, which (i) include at least the amino acids at positions 21, 22 and 23 of SEQ ID NO: 51, or (ii) are selected from positions 180 to 229 of SEQ ID NO: 51. The polypeptide can comprise or consist of up to 15, 20, 25, 30, 35, 40, 45 or 50 contiguous amino acids of SEQ ID NO: 51 as defined in (i) or (ii). The polypeptide can comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 59, 58, 57, 54, 55, 56, 2, 3, 19, 20, 21, 60 or 61. The maximum length of the polypeptide is 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids and / or wherein the C-terminal amino acid is replaced by the corresponding amide. The polypeptide can be isolated.

[0057] The polypeptide preferably comprises or consists of at least 9 contiguous amino acids of SEQ ID NO: 51 which include at least the amino acids at positions 21, 22 and 23 of SEQ ID NO: 51, i.e. the sequence KSV. The at least 9 contiguous amino acids of SEQ ID NO: 51 preferably comprise the amino acid sequence of SEQ ID NO: 54 (ILKKSVHSVA), SEQ ID NO: 55 (ILKKSVHSV) or SEQ ID NO: 56 (SILKKSVHSV). A preferred polypeptide of the application can comprise or consist of the amino acid sequence of SEQ ID NO: 54, 55 or 56. Particularly preferred are longer polypeptide fragments of SEQ ID NO: 51 which include these sequences. For example, the application provides a polypeptide of up to 50 contiguous amino acids of SEQ ID NO: 51 in which the contiguous amino acids comprise the amino acid sequence of any one of SEQ ID NO: 54, 55 or 56. Examples of such polypeptides are polypeptides which comprise or consist of the sequence of any one of SEQ ID NO: 2, 3, 57, 58 or 59. Polypeptides which comprise or consist of the sequence of any one of SEQ ID NO: 59, 58 and 57 are preferred. Polypeptides which comprise or consist of the sequence of SEQ ID NO: 59 are particularly preferred.

[0058] In any of the polypeptides described herein, the amino acid sequence can be modified by one, two, three, four or five (i.e. up to five) insertions, deletions or substitutions, provided that the polypeptide having the modified sequence displays the same or enhanced immunogenicity against arginase 2 as compared to the polypeptide having the unmodified sequence. By "the same" it is understood that the polypeptide having the modified sequence does not exhibit a significantly reduced immunogenicity against arginase 2 as compared to the polypeptide having the unmodified sequence. Any comparison of immunogenicity between sequences should be performed using the same assay. Unless otherwise specified, modifications to the polypeptide sequence are preferably conservative amino acid substitutions. A conservative substitution is one in which the replaced amino acid is replaced with another amino acid having a similar chemical structure, similar chemical properties or similar side chain volume. The introduced amino acid has a similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, a conservative substitution can introduce an aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. It is well known in the art that conservative amino acid changes can be selected according to the properties of the 20 main amino acids as defined in Table Al below. If the amino acids have similar polarity, this can be determined with reference to the scale of hydrophilicity of the side chains of the amino acids in Table A2.

[0059] Table A1 - Chemical properties of amino acids

[0060] Ala (A) Aliphatic, Hydrophobic, Neutral Met (M) Hydrophobic, Neutral Cys (C) Polar, Hydrophobic, Neutral Asn (N) Polar, Hydrophilic, Neutral Asp (D) Polar, Hydrophilic, Charge (-) Pro (P) Hydrophobic, Neutral Glu (E) Polar, Hydrophilic, Charge (-) Gln (Q) Polar, Hydrophilic, Neutral Phe (F) Aromatic, Hydrophobic, Neutral Arg (R) Polar, Hydrophilic, Charge (+) Gly (G) Aliphatic, Neutral Ser (S) Polar, Hydrophilic, Neutral His (H) Aromatic, Polar, Hydrophilic, Charge (+) Thr (T) Polar, Hydrophilic, Neutral Ile (I) Aliphatic, Hydrophobic, Neutral Val (V) Aliphatic, Hydrophobic, Neutral Lys (K) Polar, Hydrophilic, Charge (+) Trp (W) Aromatic, Hydrophobic, Neutral Leu (L) Aliphatic, Hydrophobic, Neutral Tyr (Y) Aromatic, Polar, Hydrophobic

[0061] Table A2 - Hydrophilicity Scale

[0062]

[0063]

[0064] In any of the polypeptides disclosed herein, any one or more of the following modifications can be made to improve physicochemical properties (e.g. stability), provided that the polypeptide exhibits the same or increased immunogenicity against arginase 2 compared to the polypeptide with the unmodified sequence:

[0065] a) substitution of the C-terminal amino acid with the corresponding amide (may increase resistance to carboxypeptidases);

[0066] b) substitution of the N-terminal amino acid with the corresponding acylated amino acid (may increase resistance to aminopeptidases);

[0067] c) substitution of one or more amino acids with the corresponding methylated amino acid (may increase proteolytic resistance);

[0068] d) substitution of one or more amino acids with the corresponding D-configuration amino acid (may increase proteolytic resistance);

[0069] Any of the polypeptides disclosed herein can be linked to at least one additional moiety at the N- and / or C-terminus to improve solubility, stability and / or aid manufacture / isolation, provided that the polypeptide exhibits the same or increased immunogenicity against arginase 2 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequence KK, KR or RR can be added at the N- and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0070] The polypeptides disclosed herein can be produced by any suitable method. For example, the polypeptides can be synthesised directly using standard techniques known in the art (e.g. Fmoc solid phase chemistry, Boc solid phase chemistry or by liquid phase peptide synthesis). Alternatively, the polypeptides can be produced by transforming a cell (typically a bacterial cell) with a nucleic acid molecule or vector encoding the polypeptide.

[0071] The present application provides nucleic acid molecules and vectors encoding the polypeptides of the present application. The present application also provides host cells comprising such nucleic acids or vectors.

[0072] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the application can be provided in isolated or substantially isolated form. By substantially isolated it is meant that the polynucleotide can have some, but not all, of the peripheral mediators removed. The polynucleotide can be mixed with a carrier or diluent that does not interfere with its intended use, and it is still considered substantially isolated. A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule, either RNA or DNA, which transcribed in vivo (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences (for example, in an expression vector). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translational stop codon at the 3' (carboxy) terminus. Such nucleic acid sequences can include, but are not limited to, cDNA from a viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0073] Polynucleotides can be synthesized according to methods known in the art, as exemplified in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the application can be provided in the form of expression cassettes, which include control sequences operably linked to the insert sequence, thereby allowing the polypeptides of the application to be expressed in vivo. These expression cassettes are in turn provided within vectors, such as plasmids or recombinant viral vectors. Such expression cassettes can be administered directly to a host subject. Alternatively, vectors including the polynucleotides of the application can be administered to a host subject. Preferably, the polynucleotides are prepared and / or administered using a gene vector. An appropriate vector can be any vector that is capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the application.

[0074] Accordingly, the application includes expression vectors comprising such polynucleotide sequences. Such expression vectors are routinely constructed in the art of molecular biology and can involve, for example, the use of plasmid DNA and appropriate initiation factors, promoters, enhancers and other elements such as, for example, a polyadenylation signal that can be required and positioned in the correct orientation to allow expression of the peptides of the application. Other suitable vectors will be apparent to those skilled in the art. As a further example of this, we refer to Sambrook et al.

[0075] The present application also includes cells modified to express a polypeptide of the present application. Such cells typically include prokaryotic cells, such as bacterial cells, for example, E. coli. Such cells can be cultured using conventional methods to produce a polypeptide of the present application.

[0076] A polypeptide of the present application can be in substantially isolated form. It can be mixed with carriers, preservatives or diluents (discussed below) and / or adjuvants (discussed below) that will not interfere with the intended use of the polypeptide, and still be considered substantially isolated. It can also be in substantially purified form, in which case it typically comprises at least 90%, for example, at least 95%, 98% or 99% of the protein in the preparation.

[0077] Compositions comprising a polypeptide

[0078] In another aspect, the present application provides compositions comprising a polypeptide of the present application. For example, the present application provides a composition comprising one or more polypeptides of the present application and at least one pharmaceutically acceptable carrier, preservative or excipient. The carrier, preservative and excipient must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject to which the composition is administered. Generally, all components and the final composition are sterile and pyrogen-free. The composition can be a pharmaceutical composition. The composition can preferably comprise an adjuvant.

[0079] An adjuvant is any substance that, when mixed into a composition, will increase or otherwise alter the immune response elicited by the composition. In a broad sense, an adjuvant is a substance that promotes an immune response. Adjuvants can also preferably have a depot effect, in that they also cause a slow and sustained release of the active agent from the site of administration. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63.

[0080] The adjuvant can be selected from the group consisting of AlK(S04)2, AlNa(S04)2, AlNH4(S04)2, silicon dioxide, alum, Al(OH)3, Ca3(P04)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1 '2'-dipalmitoyl- sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL + TDM + CWS) in 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharides and various derivatives thereof including lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g. poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis and Brucella members, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21. Various saponin extracts have also been shown to be suitable as adjuvants in immunogenic compositions. Granulocyte-macrophage colony stimulating factor (GM-CSF) can also be used as an adjuvant.

[0081] Preferred adjuvants to be used in the present application include oil / surfactant based adjuvants, such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA based adjuvants, such as adjuvants comprising CpG oligonucleotide sequences. Other preferred adjuvants are viral dsRNA based adjuvants, such as Poly I:C. GM-CSF and Imidazochiniline are also examples of preferred adjuvants.

[0082] Most preferred adjuvant is a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

[0083] It is also described in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986 pp. 61-63) that when the antigen of interest has a low molecular weight or is poorly immunogenic, it is recommended to conjugate to an immunogenic carrier. Thus, the polypeptides of the present application can be conjugated to a carrier. The carrier can be present independently of the adjuvant. The function of the carrier can be, for example, to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life. In addition, the carrier can assist in the presentation of the polypeptide or fragment thereof to T cells. Thus, in the composition, the polypeptide can be associated with a carrier, such as those described below.

[0084] The carrier can be any suitable carrier known to those skilled in the art, such as a protein or antigen presenting cell, such as a dendritic cell (DC). Carrier proteins include key hole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. Alternatively, the carrier protein can be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier can be a dextran such as agarose. The carrier must be physiologically acceptable and safe for humans.

[0085] If the composition includes an excipient, the excipient must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the recipient thereof. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can be present in the excipient. These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, inorganic salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic salts such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, carriers, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0086] Formulation of suitable compositions can be achieved using standard pharmaceutical compounding techniques and methods, all of which are readily available to the reasonable skilled person. These compositions can be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions can be prepared, packaged, or sold in unit- dose or multi-dose containers, for example, in ampoules. Compositions include, but are not limited to, suspensions, solutions, emulsions, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of the composition, the active ingredient is provided in dry (e.g., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before administration of the reconstituted composition. The compositions can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oleaginous suspension or solution. This suspension or solution can be formulated according to known art using suitable dispersing or wetting agents (such as, for example, those mentioned herein) and suspending agents according to known art. Other ingredients can be present in the compositions such as those adjuvants, excipients, and auxiliary agents described herein. Such sterile injectable formulations can be prepared using a non-toxic, parenterally-acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils (e.g., synthetic mono- or diglycerides). Other compositions that can be used include those comprising the active ingredient in microcrystalline form, in liposome formulation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation can include a pharmaceutically acceptable polymeric material or a hydrophobic material, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Alternatively, the active ingredient of the composition can be encapsulated, adsorbed to, or associated with a particulate carrier. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, e.g., polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0087] Methods of use

[0088] The polypeptides or compositions of the present application can be used in a method of treating or preventing a disease or condition in a subject. The polypeptides or compositions of the present application can be used in the manufacture of a medicament for use in a method of treating or preventing a disease or condition in a subject. The method comprises administering the polypeptide or the composition to the subject. A therapeutically or prophylactically effective amount of the polypeptide or the composition can be administered to a subject in need thereof.

[0089] The disease or condition is characterized at least in part by an inappropriate or excessive immunosuppressive function of Arginase 2. The disease or condition can be a cancer, preferably a cancer expressing Arginase 2 and / or associated with an inappropriate or excessive immunosuppressive function of Arginase 2. The cancer can be a kidney cancer, a prostate cancer, a breast cancer, a brain cancer, a head and neck cancer or a small intestine cancer, or can be a colorectal cancer or a gastric cancer, or can be a melanoma, or can be a leukemia, preferably acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL). The cancer can be resistant to additional cancer treatments, in particular it can be resistant to immune system checkpoint inhibitors, such as anti-PDl treatments.

[0090] The method can comprise administering the additional cancer treatment simultaneously or sequentially. The additional cancer treatment can be selected from a cytokine treatment, a T cell treatment, an NK treatment, an immune system checkpoint inhibitor, a chemotherapy, a radiation treatment, an immune stimulatory substance, such as another vaccine, or a gene therapy.

[0091] An immune system checkpoint inhibitor is particularly preferred as the additional cancer treatment. Immunization against Arginase 2 can have a synergistic effect when combined with the inhibition of an immune system checkpoint. Examples of immune system checkpoints include:

[0092] a) the interaction between Indoleamine 2,3-dioxygenase (IDOl) and its substrate;

[0093] b) the interaction between PD1 and PDL1 and / or PD1 and PDL2;

[0094] c) the interaction between CTLA4 and CD86 and / or CTLA4 and CD80;

[0095] d) the interaction between B7-H3 and / or B7-H4 and their respective ligands;

[0096] e) the interaction between HVEM and BTLA;

[0097] f) the interaction between GAL9 and TIM3;

[0098] g) the interaction between MHC class I or II and LAG3; and

[0099] h) the interaction between MHC class I or II and KIR.

[0100] Inhibition of checkpoint (a), (b), (c) is particularly preferred as the additional cancer treatment. The checkpoint inhibitor can be any immunomodulator, such as an antibody, blocking or inhibiting the immune system checkpoint, or it can be an immunotherapeutic composition comprising a component of the immune system checkpoint or an immunogenic fragment of said component, targeted by the immune system to stimulate the checkpoint.

[0101] The additional cancer treatment can be an antibody.

[0102] The antibody may be abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, attumomabpentetate, amatuximab, or anatumomab. mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (= tocilizumab), Atorlimumab, Bapineuzumab, Basiliximab, Ba... Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab Mertansine, Blinatumomab, Blosozomab, Brentuximab-Vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab-Mertansine, Cantuzumab-Lavtansineravtansine), Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enoblituzumab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab, Ensituximab,pegol, enokizumab, enoticumab, ensituximab, epitumomabcituxetan, eprantuzumab, erlizumab, ertumaxomab, etaracizumab, eprantuzumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA 05. Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab-Ozomicin ozogamicin, gevokizumab, girentoximab, glembatumumab vedotin, golimumab, gomiliximab, GS6624, iebalizumab, tiuxetan, icrucumab, iegovomab, imciromab, imgatuzumab, inclacumab, and indatuximab.tositumomab), Narnatumab, Necitumumab, Neflamapimod, Nefluzotumab, Nektumumab, Nelipepimol, Nerbibutant, Nerelimomab, Nerelimonmab, Neratinib, Nersaranib, Nesvacumab, Netrimotag, Netupitant, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, Neulasta, NeulTafenatox, Namilumab, Naptumomabestafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, obbinutuzumab, occaratuzumab, occrelizumab, ocdulimomab, ocfatumumab, oclaratumab, oclokizumab, ocmalizumab, onartuzumab, ocportuzumabmonatox, ocregovomab, orticumab, octelixizumab, ocxelumab, oczanezumab, oczoralizumab, paxicumab gibaximab, palivizumab, panitumumab, panobakucumab, parsatuzumab, pascolizumab, pateclizumab, paltritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab-vedotin, pintumomab, placulumab, polatuzumab-vedotin vedotin), Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140. Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab ab), Rituximab, Robatumumab, Rolledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab-pentetide pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, and tacatuzumab. tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, telimomabAritox), Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (= Tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (= Atlizumab), Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab or Zolimomab aritox).

[0103] Preferred antibodies include Natalizumab, Vedolizumab, Belimumab, Atacicept, Alefacept, Otelixizumab, Teplizumab, Rituximab, Ofatumumab, Ocrelizumab, Epratuzumab, Alemtuzumab, Abatacept, Eculizumab, Omalizumab, Canakinumab, Meplizumab, Reslizumab, Tocilizumab, Ustekinumab, Briakinumab, Etanercept, Inlfliximab, Adalimumab, Certolizumab pegol, Golimumab, Trastuzumab, Gemtuzumab, Ozogamicin, Ibritumomab tiuxetan, Tostitumomab, Cetuximab, Bevacizumab, Panitumumab, Denosumab, Ipilimumab, Brentuximab vedotin.

[0104] Antibodies that can be used in the inventive method include in particular: Daratumumab, Nivolumab, Pembrolizumab, Avelumab, Rituximab, Trastuzumab, Pertuzumab, Alemtuzumab, Cetuximab, Panitumumab, Tositumomab and Ofatumumab. Daratumumab is particularly preferred. Anti-PD1 antibodies, such as Nivolumab and Pembrolizumab, are particularly preferred.

[0105] The additional cancer treatment can be selected from the group consisting of: coenzyme B12 (Actimide), azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, eprubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, revlimid, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine and vinorelbine.

[0106] The polypeptides or compositions of the application can also be used in a method of stimulating arginase 1 -specific T cells (e.g., CD4 and CD8 T cells), comprising contacting the cells with the polypeptides or compositions. The method can be performed in vitro. The cells can be present in a sample taken from a healthy subject or a cancer patient, e.g., a tumor sample.

[0107] The application is further illustrated by the following examples, which should not, however, be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the application in diverse forms thereof.

[0108] Example 1

[0109] Materials and methods

[0110] Patient materials

[0111] In Lymphoprep TM PBMCs from healthy donors were isolated using density gradient separation at STEMCELL Technologies and frozen at -150°C in FBS supplemented with 10% DMSO. PBMCs from cancer patients were isolated from blood samples at least four weeks after the termination of any anticancer treatment. All protocols were approved by the Scientific Ethics Committee of the Danish Capital Region and conducted in accordance with the Declaration of Helsinki. Written informed consent from patients was obtained prior to enrollment in the study.

[0112] peptides

[0113] The peptides were synthesized using standard methods and dissolved in DMSO to obtain a material concentration of 10 mM. The sequences of the peptides used in these experiments are shown in the section titled "Sequences". The peptides are described by SEQ ID NO, by name, or by referring to the start and end positions of each peptide sequence within the full-length sequence of arginase 2. Each description is used interchangeably. For example, the peptide of SEQ ID NO: 2 can be referred to as Arg2_1, or as Arg2 aa11-30 (giving start position 11 and end position 30). The intended designation in each case will be clear based on the context.

[0114] Enzyme-linked immunospot (ELISPOT) assay

[0115] For in vitro ELISPOT, PBMC from cancer patients and healthy donors were pulsed with 20 mM of arginase 1 -derived peptides (or no peptide as control) and 120 U / ml IL-2 for 7 days in 24-well plates before being used for in vitro ELISPOT assay. Cells were placed in 96-well nitrocellulose ELISPOT plates (MultiScreen MAIP N45; Millipore) pre-coated with IFNy capture antibody (Mabtech). Arginase peptides were added to a final concentration of 5 mM and plates were incubated at 37°C for 14-16 hours. After incubation, cells were washed away and a secondary biotinylated mAb (Mabtech, cat. no. 3420-6-1000) was added for 2 hours at room temperature. Unbound secondary antibody was washed away and streptavidin-conjugated alkaline phosphatase (AP) (Mabtech, cat. no. 3310-10) was added for 1 hour at room temperature. Unbound conjugated enzyme was washed away and the assay was developed by adding BCIP / NBT substrate (Mabtech, cat. no. 3650-10). Developed ELISPOT plates were analyzed on a CTL-Immunospot S6 Ultimate-V analyzer using Immunospot software v5.1. Responses were reported as the difference between the mean number of spots in wells stimulated with arginase 2 peptide and the mean number of spots in wells with no peptide added.

[0116] Intracellular staining

[0117] Intracellular staining TM Intracellular staining was performed on cell cultures after 5 hours of stimulation of PBMC with arginase-derived peptides (or no peptide incubation as control) in the presence of BD GolgiPlug TM II (BD Biosciences) according to the manufacturer's instructions. Antibodies used: IFNy-APC (cat. no. 341117), TNFa-455 BV421 (cat. no. 562783), CD4-FITC (cat. no. 347413), CD8-PerCP (cat. no. 345774), CD3-APC-H7 (cat. no. 560275) (all from BD Biosciences), dead cell stain-FVS510 (564406, BD Biosciences).

[0118] Results

[0119] Screening of arginase 2 peptides based on arginase 1 hot spot

[0120] Based on the previously determined arginase 1 hot spot region of 50 amino acids long at positions 161-210 of arginase 1, four peptides covering the corresponding region (positions 180-229) in arginase 2 were selected for testing. These peptides are Arg2-E17 (aa 180-199), Arg2-E18 (aa 190-209), Arg2-E19 (aa 200-219), and Arg2-E20 (210-229). See Figure 1 for a schematic of arginase 1 and arginase 2. Arg2-E17 could not be synthesized by conventional methods, so the remaining three peptides were used for this experiment.

[0121] To test whether these peptides could be used to identify arginase 2 responses, PBMCs from 12 healthy donors and 1 cancer patient were screened for responses in IFNy ELISPOT. Prior to ELISPOT, PBMCs were stimulated with arginase 2 peptides and low dose IL-2 for 1 week.

[0122] As shown in Figure 2 , the peptides were all recognized by PBMCs from at least one subject. Arg2-E18 exhibited the highest and most consistent responses.

[0123] Peptide pool screening for additional arginase 2 peptides

[0124] The entire arginase 2 protein sequence was divided into overlapping 20 amino acid long peptides (the last peptide was 24 amino acids), resulting in a pool of 34 peptides covering the entire sequence (SEQ ID NO: 1-34). Each peptide in the pool overlaps the first 10 amino acids of the subsequent peptide. Using this arginase 2 peptide pool and the IFNy ELISPOT assay, we next screened PBMCs from 6 healthy donors for spontaneous responses. PBMCs were stimulated with 3-4 adjacent 20-mer arginase 2 pool peptides and low dose IL-2 (120 U / mL) for one week. PBMCs were then set up for the IFNy-ELISPOT assay to screen for responses to each 20-mer peptide, respectively.

[0125] As shown in Figure 3 , the following eight peptides showed the highest and most abundant responses:

[0126] ARG2_1(aal 1-30), ARG2_5(aa51-70), ARG2_8(aa81-100), ARG2_13(aal31-150), ARG2_1 8(181-200), ARG2_20(201-220), ARG2_21(aa211-230) and ARG2_22(221-240).

[0127] Arg2_18, Arg2_20, Arg2_21, and Arg2_22 are all contained within or overlap with the previously identified hotspot region. However, other peptides (including Arg2_1, which has the highest response) originate from different regions of the arginase 2 protein.

[0128] Validation of library filtering (hotspot areas)

[0129] Prior to the IFNγELISPOT assay, PBMCs from two healthy donors were stimulated for one week with a single peptide and a low dose of IL-2 (120 U / mL) to validate the response observed in the library screening.

[0130] ARG2_18, ARG2_19, ARG2_20, and ARG2_21 were all tested in this validation experiment, including ARG2-E18, ARG2-E19, and ARG2-E20, which are included because they overlap with the same region as arginase 2. Arg2-E17 was again excluded because it cannot be synthesized using conventional methods.

[0131] The alignment of the six tested peptides is shown below:

[0132]

[0133] like Figure 4 As shown, responses to each tested peptide were observed, validating the original screening. Overlapping pairs of peptides produced almost identical responses, except for ARG2_21 and ARG2-E20, with a stronger response to ARG2_21 observed in a healthy donor. The strongest and most consistent response was obtained with ARG2_18 in this experiment.

[0134] Validation of library filtering (other areas)

[0135] Prior to the IFNγELISPOTT assay, PBMCs from four healthy donors were stimulated for one week with a single peptide and a low dose of IL-2 (120 U / mL) to validate the response observed in the library screening. In this validation, ARG2_1, ARG2_5, ARG2_13, ARG2_18, and ARG2_22 were tested. Figure 5As shown, strong and significant responses were observed, in particular for Arg2_l.

[0136] Therefore, intracellular cytokine staining was used on the same cells to clarify whether CD4+ or CD8+ responses were present. For two healthy donors, Buf-M-01 and Buf-M-02, 0.2% and 0.1% double positive (DP) CD4+ cells were observed (see Figure 6A and 6B representative figures), indicating CD4+ responses to ARG2_1 in these donors.

[0137] Further screening of cancer patients and healthy donors for responses to ARG2_1

[0138] ARG2-1 was used to screen for responses in 8 melanoma (AA07-AA31), 4 prostate cancer (UR07-27) and 13 healthy donors. PBMC from each were stimulated with single peptide and low dose IL-2 (120 U / mL) for one week prior to IFNy ELISPOT assay.

[0139] As shown in Figure 8 , 15 out of 25 donors tested (60%) showed significant responses, while responses seemed almost equally strong in patients and healthy donors.

[0140] PBMC showing clear responses in IFNy ELIPOT were also used for intracellular cytokine staining. We analyzed CD4 cells from two healthy donors and showed that CD4 cells were specifically reacting to ARG2_1 in both donors. In addition, we found a CD8+ response to ARG2_1 in PBMC from a prostate cancer patient (UR12), see Figure 9 , 0.9% CD8+ DP cells versus 0.5% for the control. Taken together, these ICS results indicate that the ARG2_1 responses detected are T cell mediated and likely co-mediated by CD4 and CD8 T cells.

[0141] Therefore, prediction of HLA-A2 and HLA-A3 epitopes within ARG2_1 was performed using the www.syfpeithi.de server. The following epitopes were predicted to be present within the ARG2_1 sequence:

[0142]

[0143] All 3 predicted epitopes contained the transit peptide border at positions 21, 22 and 23 of SEQ ID NO: 51.

[0144] A response to ARG2_1 was also observed without pre-stimulation (“in vitro ELISPOT”).

[0145] One cancer patient and three healthy donors who showed a strong response in previous experiments (this was in vitro or “indirect” IFNγELISPOT) were also tested in vitro with ELISPOT. This means that PBMCs were not pre-stimulated, but simply incubated with + / -Arg2 peptides for 72 hours prior to the IFNγELISPOT assay. Figure 10 As shown, a response to ARG2_1 was detected. Of particular note is the fact that the specific T-cell response was directly detectable in vitro. With very few exceptions, it is generally not possible to detect tumor-associated antigen-specific T cells in PBMCs directly in vitro by tetramer staining or by ELISPOT without prior in vitro peptide stimulation. This is highly unusual, demonstrating the ability to detect an immune response to a non-viral antigen without prior in vitro stimulation. Therefore, these results highlight the high immunogenicity of the ARG2_1 sequence and its included epitopes. These sequences are therefore excellent targets for immunization, as the data suggest that the immune system can selectively target arginase 2-expressing cells, reducing the immunomodulatory effects of arginase expression and thereby enhancing anti-tumor immune responses. Furthermore, these data suggest that arginase 2-specific T cells (particularly those recognizing ARG2_1 epitopes) play a natural role in the immune system. This implies that immunization against these sequences is unlikely to be toxic to patients.

[0146] discuss

[0147] The presence of arginase 2-expressing cells in cancer contributes to an immunosuppressive tumor microenvironment that prevents the proliferation of tumor-specific effector lymphocytes. Therefore, specifically targeting these arginase 2-expressing cells (which may include tumor cells and other regulatory cells) offers both direct and indirect benefits by reducing immunosuppression, allowing for the activation and proliferation of cancer-specific effector cells. Given that anticancer immunotherapy is often antagonized by immunosuppressive cells, this dual effect of targeting the arginase 2 epitope can be highly synergistic. Considering that these experiments have demonstrated the existence of innate CD4 and CD8 T cell-mediated immunity against arginase 2 (particularly against epitopes within the Arg2_1 sequence), the likelihood of successfully targeting arginase 2 in an immunization setting is high.

[0148] Example 2 - Further Investigation of Human ARG2 Peptides

[0149] Materials and methods

[0150] Patient materials

[0151] In Lymphoprep TMPBMC from healthy donors were isolated by density gradient separation on Ficoll® (Alere) and cryopreserved in FBS (Life Technologies) supplemented with 10% DMSO at -150°C. PBMC from cancer patients were isolated from blood samples at least four weeks after termination of any anti-cancer treatment. PBMC from AML patients were isolated from blood samples from patients in different disease and treatment status, thus including patients under treatment. All protocols were approved by the Scientific Ethics Committee for the Capital Region of Denmark and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from the patients prior to inclusion in the study. PMBC were maintained in X-vivo (Bio Nordika) supplemented with 5% human serum (Sigma-Aldrich).

[0152] Cell culture

[0153] THP-1 were cultured in RPMI (Gibco) supplemented with 10% FBS. Set2 cells were cultured in RPMI containing 20% FBS. OCI-AML-2 cells were cultured in Alpha-MEM (Life Technologies) containing 10% FBS. MONO-MAC-1 cells were cultured in RPMI supplemented with 10% FBS, 1 mM sodium pyruvate (Life Technologies), 2 mM L-glutamine (Life Technologies) and lx non-essential amino acids (Life Technologies). All cell lines were tested and determined mycoplasma negative. Cells were passaged 2-3 times per week.

[0154] Cytokine stimulation with IL-4 (400 U / ml), IL-13 (50 ng / ml), IFNy (100 U / ml) or a mixture of cytokines (400 U / ml IL-4, 100 U / ml GM-CSF and 1000 U / ml TNFα) was performed by seeding 0.5-0.75 x 105 cells / ml culture medium supplemented with the respective cytokines, incubating for 48 hours and then harvesting the cells for various experiments. All cytokines were from Trichem. 6 Cell culture

[0155] Peptides

[0156] A library of 34 20-mer peptides of ARG2 was synthesized by PepScan and dissolved at 10 mM in DMSO for screening of the immune response. In the remaining experiments, ARG2-1 was dissolved at 2 mM in sterile water. The peptides were tested for their ability to stimulate the immune response by measuring the IFNγ production in PBMC from healthy donors. The PBMC were stimulated with the peptides at 2 mM for 48 hours and the supernatant was collected for analysis of the IFNγ production by ELISA (Mabtech). The results are shown in Table 1. Long ARG2 peptides (A2L1, A2L2, A2L3 (SEQ-ID-NO: 58, 59, 57)) were synthesized and dissolved in sterile water at 2 mM. The peptides dissolved in sterile water were filtered through a 0.22 μm filter before use. The purity of the synthesized peptides was >90%. A list of all peptides is provided in Table 1.

[0157] Peptide stimulation and ELISPOT assay

[0158] PBMCs from healthy donors or cancer patients were stimulated in vitro with 10 μM of ARG2-derived peptide to improve assay sensitivity. On day 2, IL-2 was added to a total of 120 U / ml of IL-2 (Novartis). After 7 days, 4–6 × 10⁻⁶ μg / ml of IL-2 was added. 5 PBMCs were placed at the bottom of ELISPOT plates pre-coated with IFNγ-capture antibody (Mabtech). PBMCs from each donor or patient were stimulated three or four times in parallel with the peptide (5 μM ARG2-derived peptide) and control. Cells were incubated in the ELISPOT plates for 14–16 hours in the presence of the antigen, then washed and a second biotinylated antibody (Mabtech) was added. After 2 hours of incubation, the second antibody was washed off for 1 hour before the addition of streptavidin-conjugated alkaline phosphatase (Mabtech). Next, unbound enzyme was washed off, and the assay was developed by adding BCIP / NBT substrate (Mabtech). The developed ELISPOT plates were analyzed using ImmunoSpot software v5.1 on a CTL-Immunospot S6 Ultimate-V analyzer. The response was reported as the difference between the mean number of spots in the wells stimulated with the ARG2-derived peptide and the mean number of spots in the control wells.

[0159] By 1-5×10 4 Effector cells (as shown) and 1-2.5×10 4 Target cells (as shown) were placed at the bottom of the ELISPOT wells to establish the ELISPOT assay, with ARG2-specific T cells (effect cells) and various immune or cancer cells serving as target cells. Peptide pulses of the target cells were performed by incubating the cells with 20 μM peptide for 1 hour, followed by two washes to remove unbound peptides. These cells served as a positive control. Effector cells without target cells served as a negative control. All conditions were performed in duplicate, either three or four times.

[0160] Intracellular cytokine staining assay

[0161] One week after in vitro stimulation with ARG2-derived peptides, intracellular staining of cell cultures was performed on PBMCs. For assays, BD GolgiPlug... TMARG2-specific T cells were re-stimulated with ARG2-derived peptides (or no peptide incubation as control) in the presence of (added after the first hour of peptide stimulation) 9 x 10 5 PBMC for 5 hours. According to the manufacturer's instructions, stimulated cells were stained with fluorescently tagged antibodies for surface markers (CD3, CD4, CD8) and then permeabilized with Fixation / Permeabilization and Permeabilization Buffer (eBioscience, Cat#00-5123-43). Permeabilized cells were then stained with fluorescently tagged antibodies for IFNy and TNFa. Flow cytometry analysis was performed on a FACSCanto II (BD Biosciences). According to the manufacturer's instructions, the antibodies used: IFNy-APC, TNFa-BV421, CD4-PerCP, CD8-FITC, CD3-APC-H7, CD4-FITC, CD8-PerCP, and dead cell stain-FVS510 (all from BD Biosciences). For intracellular cytokine staining to detect cytokine production by ARG2-specific T cells in response to target cells, 5 x 10 TM PBMC were incubated with 2.5 x 10 5 target cells for 5 hours, with GolgiPlug 5 added after the first hour. TM .

[0162] Establishment of ARG2-specific T cell cultures

[0163] PMBC of prostate cancer patients were initially stimulated by irradiated autologous mature dendritic cells (DCs) loaded with ARG2-1 to establish ARG2-specific T cell cultures. IL-12 (20 U / ml) and IL-7 (40 U / ml) were added the next day. PBMC were re-stimulated every 8 days with autologous DCs loaded with ARG2-1 peptide, followed by the addition of IL-2 (120 U / ml) the next day. After 4 stimulations, ARG2-specific T cells were enriched using an IFNy cell enrichment kit (Miltenyi Biotec). Cells were expanded and ARG2-specific T cells were further enriched using a CD4+ enrichment kit (Miltenyi Biotec).

[0164] Generation of in vitro transcribed mRNA

[0165] The cDNA encoding ARG2 (NM_001172.4) was synthesized and cloned into the HLA-class II targeting plasmid pGEM-sig-DC.LAMP (kindly provided by Dr. K. Thielemans, Vrije Universiteit Brussel, Medical School) using the BamHI restriction site. This pGEM-ARG2-DC-LAMP plasmid was linearized with Spel and then used as DNA template for in vitro transcription (ref Article**.

[0166] Total RNA extraction

[0167] Cells were harvested, washed in PBS and prepared in pellets by centrifugation. Cell pellets were placed on ice or frozen at -80°C until RNA extraction. Total RNA was extracted using the RNAeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions with a final elution in 30 μΐ of RNAse-free water. RNA concentration was measured on a NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA was stored at -80°C.

[0168] RT-qPCR

[0169] Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For each reaction, lOOO ng of RNA was reverse transcribed. For RT-qPCR, cDNA was diluted 1 :5 and RT-qPCR analysis was performed on cDNA using TaqMan Gene Expression Assays on a Roche Lightcycler 480 instrument. RT-qPCR was run in quadruplicate and data were analyzed using the ddCT method and normalized to the expression level of the housekeeping gene RPLPO and to the control sample. For non-amplified low concentration samples, Ct was set to 40. No reverse transcriptase control (cDNA reaction without reverse transcriptase) was set as a control for specific amplification. The list of primers used in this study is as follows:

[0170]

[0171] Electroporation

[0172] For mRNA, DCs or cancer cells were transfected with ARG1-DC-LAMP mRNA, ARG2-DC-LAMP mRNA or control mRNA encoding eGFP using the electroporation parameters described previously. Briefly, cells were washed twice in Opti-MEM medium (Thermo Science) and the final cell density was adjusted to 9-12 x 10 6Cells / ml. 350 μl of cell suspension was pre-incubated on ice for 5 min prior to the addition of 10 μg mRNA. The cell suspension was then rapidly transferred to 2 mm (cancer cells) or 4 mm (DC) gap electroporation cuvettes for electroporation (reference: OM article). After electroporation, cells were rapidly transferred to a dish with preheated medium and incubated in a humidified atmosphere containing 5% CO2 for various experimental analyses. Cells transfected with mRNA were either incubated for 1 h and then set up for an ELISPOT assay, or incubated overnight and then set up for an intracellular cytokine staining assay. Electroporation rate was determined by FACS analysis of GFP-transfected cells 24 hours after transfection.

[0173] siRNA-mediated ARG2 silencing

[0174] A set of three ARG2-targeting siRNA duplexes was obtained from Ambion (ARG2 silencer, selected validated siRNAs, IDs sl571, sl572, sl573). The siRNAs were suspended in nucleate-free water to prepare a 0.1 nmol stock solution and stored at -80°C. For the ARG2 silencing assay, THP-1 cells for electroporation were prepared as described above, and 10 μl of working solution containing 0.02 nmol siRNA was added to each of the three siRNAs before transfection. Immediately after transfection, the cells were transferred to preheated medium and incubated for 1 hour. The transfected cells were then divided into two aliquots, and a cytokine mixture (400 U / ml IL-4, 1,000 U / ml GM-CSF, and 1,000 U / ml TNFα) was added to one half of the cells. Cells were incubated in medium or a medium containing cytokines for 48 hours before establishing the cell line for intracellular cytokine staining assays. Forty-eight hours later, the cells were granulated for RNA to obtain knockout efficiency via RT-qPCR.

[0175] Flow cytometry analysis of HLA-DR expression

[0176] HLA-DR expression analysis was performed on cells stimulated for 48 hours with nothing (no cytokines), IFNy (100 U / ml), or a cocktail of cytokines (400 U / ml IL-4, 1,000 U / ml GM-CSF, and 1,000 U / ml TNFa). Briefly, cells were washed and stained with 7-AAD (cat# 51-68981E, BD Bioscience) and FITC-conjugated mouse anti-human HLA-DR, DP, DQ (cat# 5555551, BD Bioscience) or FITC-conjugated mouse IgGl K isotype Ctrl (FC) (cat# 400109, BD Bioscience) for 30 minutes at 4°C. Cells were analyzed on a FACSCanto II flow cytometer (BD Bioscience) and analyzed using FlowJo software (Tree Star). TM Prior to analysis of cells on the FACSCanto II flow cytometer, excess antibody was washed away. HLA-DR expression levels were expressed as the difference in MFI between MHC class II-stained viable cells and isotype control-stained viable cells.

[0177] Statistical analysis

[0178] ELISPOT responses were analyzed using the distribution free resampling (DFR) method. Statistical analysis of ELISPOT responses was performed using R-studio. Differences in responses to ARG2-1 and A2L2 (specific IFNy-secreting cells) were compared using the Wilcoxon matched-pairs signed-rank test (using Prism 8) with a significance level of 0.05. Statistical analysis of the difference in mean tumor growth between the control and Arg2 vaccinated groups was performed by mixed effects analysis using Prism 8.

[0179] Results

[0180] Spontaneous immune responses to ARG2

[0181] As described in Example 1, ARG2-1 was found to give the highest and most frequent responses in the screened donors. Interestingly, ARG2-1 is part of the transit sequence (aal-22) of ARG2. Signal peptide sequences represent an interesting class of peptide epitopes that, in the context of HLA molecules, largely do not depend on proteosomal degradation or TAP for their processing and presentation. Furthermore, this part of ARG2 has little sequence overlap with the corresponding sequence of ARG1 - see alignment below: ARG1 MSAKS RTIGIIGAPFSKGQPRGGVEEGPTVLRKAGLLE (SEQ ID 63) ARG2 MSLRGSLSRL LQTRVH SILKKSVHSVAVIG APFSQGQKRKGVEHGPAAIREAGLMK (SEQ ID 64)

[0182] Therefore, by IFNy ELISPOT analysis, ARG2-1 was used for screening of the immune response against ARG2 in 33 healthy donors (HD) and 19 patients with solid tumor cancer (11 melanoma, 7 prostate cancer and 1 breast cancer patient). Strong and frequent responses were found in both healthy donors and patients with solid tumor cancer, with about 75% of the screened donors having a significant response (see Figure 14 A. Some of the data points in this figure are also shown in Figure 8 .

[0183] Since ARG2 was reported to play an important role in the immunosuppressive microenvironment observed in patients with acute myeloid leukemia (AML), the potential presence of ARG2-specific T cells in PBMCs of patients diagnosed with AML was also investigated by IFNy ELISPOT. For this, we collected peripheral blood from 9 patients diagnosed with AML. Blood sampling and subsequent PBMC isolation was performed independent of the treatment status, so the included patients represent a broad spectrum of disease and treatment stages. Significant responses were observed in 3 out of 9 patients tested (see Figure 14 A), indicating that ARG2-specific T cells can indeed be present in patients with AML. Intracellular cytokine staining for IFNy and TFNa production in healthy donors and patients with solid tumor cancer mainly showed CD4+ responses against ARG2-1 ( Figure 14 B).

[0184] Characterization of long ARG2 peptide epitopes

[0185] It was previously demonstrated that longer (38-mer) ARG1 peptides are superior in stimulating ARG1 -specific T cells compared to 20-mer and 30-mer ARG1 peptides. To determine an optimal mixed ARG2-derived epitope to stimulate ARG2-specific T cells in individuals independent of the tissue type, longer ARG2 peptide epitopes were therefore also designed based on HLA prediction algorithms (available at www.syfpeithi.de and cbs.dtu.dk) that span a larger sequence portion around ARG2-1. These sequences are shown below, aligned with the predicted signal sequence of human arginase 2 and the 20-mer sequences of ARG2-0, ARG2-1 and ARG2-2.

[0186] SEQ ID NO Sequence Name Length 62 MSLRGSLSRLLQTRVHSILKKS--------------------------- Signal Sequence 22 1 MSLRGSLSRLLQTRVHSILK----------------------------- ARG2-0 20 2 --------------------LQTRVHSILKKSVHSVAVIG---------- ARG2-1 20 3 ---------------------------------------KSVHSVAVIGAPFSQGQKRK ARG2-2 20 Longer ARG2 Sequences 57 -----------SLSRLLQTRVHSILKKSVHSVAVIGAPFS------ A2L3 29 58 ---SLRGSLSRLLQTRVHSILKKSVHSVAVIGA--------- A2L1 30 59 ---SLRGSLSRLLQTRVHSILKKSVHSVAVIGAPFS------ A2L2 33

[0187] To verify whether these long ARG2 peptides can be used to recognize ARG2 responses, PBMCs from 6 healthy donors were stimulated once with each of the three long peptides. Subsequently, PBMCs were used to screen for the immune response in IFNy ELISPOT. As Figure 15As shown in Figure A, all three long peptides recognized an immune response; however, 33-polyA2L2 elicited the strongest and most frequent immune response among the three long peptides in the tested donors. Since ARG2-1 is contained within A2L2, the longer peptide A2L2 was tested to determine whether it more effectively stimulated ARG2-specific T cells. Therefore, six healthy donors were stimulated once with either ARG2-1 or A2L2, followed by an IFNγELIPOT assay. In five of the six donors, the A2L2 immune response was significantly higher than the ARG2-1 response (…). Figure 15 B), although not significant (p = 0.0625), indicates that both peptides can elicit frequent immune responses.

[0188] To characterize the immunogenicity of A2L2, PBMCs from 30 healthy donors and 18 cancer patients (14 melanoma, 3 prostate cancer, and 1 breast cancer) were screened using IFNγELISPOT analysis. Strong and frequent responses were observed in 80% of both healthy donors and cancer patients. Figure 15 C). Intracellular cytokine staining for IFNγ and TNFα production showed a CD4+ response solely to A2L2 stimulation ( Figure 15 D) Similar to what was observed for ARG2-1. In the same donor, the immune response against A2L2 was, on average, higher than that against ARG2-1, although not significantly so (p = 0.7038). Figure 15 E).

[0189] Characterization of ARG2-specific T cells

[0190] To further characterize the immune response against ARG2, an ARG2-specific CD4+ T cell culture was generated. This was achieved by repeatedly stimulating PBMCs isolated from prostate cancer patients with autologous dendritic cells loaded with ARG2 peptides, followed by enrichment and rapid expansion of specific cells. In response to TNFα and IFNγ (… Figure 16 Cytokine staining and IFNγELISPOT (A) Figure 16 In B), T cell cultures showed high specificity for both ARG2 and A2L2. Furthermore, it was found that the addition of an HLA-DR blocker (but not an HLA-DP or HLA-DQ blocker) to IFNγELISPOT inhibited IFNγ production from peptide-stimulated ARG2-specific T cell cultures. Figure 16C). To assess the specificity of the ARG2-specific T cell cultures, the ability of the ARG2-specific T cell cultures to recognize and respond to cells expressing ARG2 intracellularly was tested. To this end, autologous DCs were transfected with mRNA encoding ARG2 fused to the ARG2 DC-LAMP signal sequence, which targets the protein to the lysosomal compartment, thereby directing the protein to class II presentation. A higher reactivity towards ARG2 mRNA transfected DCs was observed compared to mock transfected DCs Figure 16D ). FACS analysis of the transfected cells showed a transfection efficiency of >90%, and mRNA analysis of mock and mRNA transfected DCs showed a strong increase in ARG2 expression 24 hours after transfection (data not shown).

[0191] Having shown reactivity towards ARG2 producing immune cells, the ability of the ARG2-specific T cell cultures to recognize and respond against different cancer cells was investigated using an IFNy ELISPOT assay. Sequencing analysis of the HLA of the donor of the specific T cell cultures allowed us to select three HLA matched (HLA-DR01:01) AML cell lines with low endogenous ARG2 expression (OCI-AML2, THP-1 and MONO-MAC-1, data not shown) and pulse with the ARG2-1 peptide to subsequently use as target cells for the IFNy ELISPOT. Set2, another AML cell line with high endogenous ARG2 expression but HLA not matched to the ARG2-specific T cell cultures was included as a negative control. OCI-AML2, THP-1 and MONO-MAC-1 were efficiently recognized by the ARG2-specific T cells Figure 17 A), while Set2 was not. The HLA-DR restriction of the ARG2-specific T cells was confirmed by the addition of two different HLA-DR specific antibodies, as the addition of the two HLA-DR blockers abrogated the recognition of ARG2-1 pulsed THP-1 cells Figure 17 B).

[0192] THP-1 cell line is a monocyte cell line derived from peripheral blood of AML patients. THP-1 cells are reported to maintain a certain plasticity whose function depends on the presence of specific cytokines in their surrounding environment. IL-4 and IL-13 are reported to be the main inducers of ARG1, but their effect on ARG2 is not known. In addition, THP-1 cells are reported to acquire DC-like characteristics when stimulated for 48 hours with a mixture of cytokines (referred to herein as "cytokine cocktail") of IL-4, GM-CSF and TFN alpha. Therefore, THP-1 cells were stimulated with IL-4, IL-13 or the cytokine cocktail to test whether ARG2 expression in THP-1 cells would be increased. When stimulated with the cytokine cocktail, more than 2-fold induction of ARG2 expression was found, whereas IL-4 and IL-13 had no major effect on ARG2 expression levels Figure 17 C). It was next investigated whether the increase in ARG2 expression after cytokine cocktail stimulation would be able to elicit an immune response from ARG2 specific T cells. Indeed, the cytokine cocktail was found to lead to recognition of stimulated THP-1 cells in IFNy ELISPOTs Figure 17 D), and TNFa and IFNy production detected by intracellular cytokine staining Figure 17 E). After cytokine treatment of THP-1 cells, only ARG2 expression was increased, while ARG1 expression remained unchanged Figure 17 F). The response to cytokine stimulated THP-1 cells could be blocked by HLA-DR specific antibodies Figure 17 G).

[0193] Notably, cytokine cocktail stimulated THP-1 cells changed morphology with more colony formation, small protrusions and acquired adhesion compared to unstimulated cells (data not shown). Importantly, the cytokine cocktail did not upregulate HLA-DR expression (data not shown). In contrast, treatment of THP-1 cells with IFNy increased HLA-DR expression on the cell surface (not shown), but did not increase ARG2 expression Figure 17 H), and IFNy stimulated THP-1 cells could not be recognized by ARG2 specific T cells in the IFNy ELISPOT assay Figure 18 I).

[0194] MONO-MAC-1 is an AML cell line that, like THP-1 cells, still has the capacity to differentiate or be influenced by cytokine stimulation. Similar to the observations with THP-1 cells, it is possible to increase ARG2 expression in MONO-MAC-1 cells by cytokines Figure 18a). Stimulation of MONO-MAC-1 cells with a cytokine cocktail did not increase HLA-DR expression compared to unstimulated cells in MONO-MAC-1 cells (not shown). Furthermore, MONO-MAC-1 cells stimulated with IFNy did not upregulate ARG2 expression Figure 18 B), and in IFNy ELISPOTs, only cytokine-treated MONO-MAC-1 cells were recognized by ARG2-specific T cells Figure 19 C). MONO-MAC-1 stimulated with a cytokine cocktail also changed morphology, similar to what was observed for THP-1 cells (not shown).

[0195] To further test the concept of ARG2 expression-dependent T cell recognition, THP-1 cells were transfected with ARG2 mRNA using the ARG2-DC-LAMP construct. The DC-LAMP sequence was reported to be specific for mature DCs, but THP-1 cells can differentiate into DC-like cells, so the construct was equally suitable for transfection of THP-1 cells. Indeed, ARG2-specific T cell cultures were observed to react to THP-1 cells transfected with ARG2-DC-LAMP mRNA Figure 7 A). Furthermore, reactivity against cells transfected with ARG2-DC-LAMP mRNA was significantly higher compared to cells transfected with ARG1-DC-LAMP mRNA Figure 19 A), emphasizing the specificity of ARG2-specific T cells. Furthermore, stimulation with cytokines for 48 hours prior to transfection increased the immune response Figure 19 A). Intracellular cytokine staining for TNFa and IFNy production showed similar trends Figure 19 B). After 24 hours of transfection, electroporation efficiency was assessed by FACS analysis of GFP-expressing cells and showed efficient transfection (>99% GFP+ cells) (data not shown). In line with this, ARG2 and ARG1 expression was observed to be substantially multiplied compared to mock cells after 24 hours of transfection with ARG2-DC-LAMP mRNA or ARG1-DC-LAMP mRNA, respectively (data not shown). ARG2 expression levels in mRNA-transfected THP-1 cells were high compared to endogenous ARG2 expression levels in Set2 cells (data not shown).

[0196] Secondly, we further demonstrated that T cell recognition and activation are dependent on ARG2 expression using siRNA-mediated knockdown of ARG2. Transfection of THP-1 cells with a pool of three ARG2-specific siRNAs resulted in efficient ARG2 KD Figure 19C). TNFa and IFNy production was detected by intracellular cytokine staining after 48h of siRNA transfection and cytokine stimulation. We observed a decrease in TNFa and IFNy production by ARG2 specific T cells on siRNA+cytokine cells compared to mock+cytokine cells, even though we failed to completely eliminate production of Figure 19 D). RT-qpr of ARG2 expression in cells showed ARG2 KD in siRNA+cytokine cells, but ARG2 expression levels were slightly higher than the levels obtained with siRNA-KD only SEQ ID E).

[0197] Example 3 - Proof of principle of the vaccine in a mouse model

[0198] To prove the therapeutic potential of a vaccine against arginase 2, a mouse model was developed. Murine arginase 2 has 85% sequence homology with human arginase 2, so it was not possible to simply use the human sequence. Using an epitope prediction server, possible epitopes in murine arginase 2 of C57 mice (H-2Kb, H2DB) were investigated.

[0199] Epitopes predicted to bind to H2Kb and H2Db were found in two clusters, around aa85 and aa182. These are shown in the table below, aligned to illustrate the overlap.

[0200] Sequence Name Start Position End Position VVYPRSVGL--------- 45 mArg2 P1 VVYPRSVGLANQELAEVV 85 93 46 ​ mArg2 P2 85 102 47 --------------- PNIVYIGL mArg2 P3 191 198 48 WIKPCLSPPNIVYIGL mArg2 P4 182 197 49 ---------- LSPPNIVYI-- mArg2 P5 188 196 50 IQYFSMREI mArg2 P6 213 221

[0201] Each predicted epitope was predicted to bind to either H2-Kb or H2-Db, but mArg2_P2 brings together two predicted epitopes - theoretically - able to bind to both H2-Kb and H2-Db. mArg2_P6 was predicted to bind to both H2-Kb and H2-Db.

[0202] These peptides were used for immunization screening (see experimental scheme in Figure 11 ), in which three C57 mice were subcutaneously inoculated with one of the six peptides. One week later the mice were sacrificed and the spleens were homogenized to obtain PBMCs. These were used to set up IFNy ELISPOTs to screen for responses against the peptide used for immunization and other predicted epitope peptides with overlapping sequences.

[0203] As shown in Figure 12 , mice inoculated with mArg2 P5 (aa188-196) gave the strongest, most significant response. Further immunization experiments were performed with this peptide. As shown in Figure 13As shown, mice vaccinated with mArg2 P5 responded to both mArg2 P5 (aal88-196) and P4 (aal82-197), but not to mArg2 P3 (asl91-198) or only weakly. This indicates that the main epitope is located in aal88-196. However, in general, the experiments demonstrate that vaccination with arginase 2 peptides is able to stimulate an immune response in vivo, verifying the therapeutic potential of arginase 2 vaccines as a general principle.

[0204] Example 4 - Further demonstration of vaccine principle in mouse model - Lewis lung cancer

[0205] Materials and methods

[0206] Peptide design - see Example 3.

[0207] Cell culture

[0208] The tumor-derived cell line Lewis-Lung (LL2) was cultured in DMEM medium supplemented with penicillin, streptomycin and 10% PBS. Cells were passaged 2-3 times per week by detachment from flasks with 0.25% trypsin-EDTA (Gibco).

[0209] Animal experiments

[0210] Animal experiments were performed in the animal facilities at the Department of Oncology, Herlev Hospital. All experiments with mice were reviewed and approved by the Danish Animal Experimentation Council. Daily care and breeding of C57BL / 6 mice was performed by the animal facility’s animal caretakers. For therapeutic vaccination studies, C57BL / 6 mice were purchased from Taconic.

[0211] Tumor injections

[0212] LL2 cells (5 x 10 5 ) were resuspended in 100 ul serum-free medium and injected subcutaneously in the right flank of female C57BL / 6 mice. Tumor volume was measured with electronic calipers, and the endpoint of the tumor study was either when the tumors reached the threshold size of 800 mm 3 or due to formation of ulcerations on the tumors.

[0213] Peptide vaccines and murine ELISPOT

[0214] Murine Arg2 peptides (P1-P6) were purchased from PepScan or Synthesized and reportedly soluble peptides were dissolved in ultrapure water or DMSO at 2 mM or 10 mM, respectively. The dissolved peptides were then emulsified with Montanide adjuvant (50 μl / mouse) (Seppic Inc.) to obtain the optimal dose of 100 μg total peptide in a total volume of 100 μl. The emulsified peptide vaccine was subcutaneously injected into the base, tail, or flank of 12–16 week old C57BL / 6 mice using a 27G needle. Control mice were given a total volume of 100 μl of water and Montanide emulsion. For therapeutic vaccine studies in tumor-inoculated mice, vaccination was performed on day 0 and day 7 post-tumor inoculation via the flank of the tail and left flank. For epitope screening and validation experiments, a single dose of vaccine was injected into the right flank of mice. Mice were sacrificed one week later, and spleens were reclaimed. The spleens were crushed and passed through a 70 μM filter, and red blood cells were lysed with red blood cell lysis buffer (Qiagen). Cells were washed four times and counted, then set at 8 × 10⁸ cells per well. 6 Cells were subjected to mouse IFNγELISPOT analysis.

[0215] Treatment with PD-1 blocking antibodies

[0216] The anti-mouse PD-1 (CD279) monoclonal antibody was purchased from BioXCell (clone: ​​RMP1-14). For efficacy studies, mice were intraperitoneally injected with 250 μg of PD-1 blocking antibody in 200 μl PBS. Starting from day 4 post-LL2 vaccination, mice were treated with anti-PD-1 three times weekly.

[0217] Statistical analysis

[0218] ELISPOT responses were analyzed using a distributionless resampling (DFR) method (described in Moodie et al. (reference)). Statistical analysis of ELISPOT responses was performed using R-studio. Differences in responses to ARG2-1 and A2L2 (specific IFNγ-secreting cells) were compared using a Wilcoxon paired signed-rank test (Prism 8) at a significance level of 0.05. Statistical analysis of the difference in mean tumor growth between the control and Arg2-inoculated groups was performed using a mixed-effects analysis with Prism 8.

[0219] result

[0220] Example 1 and 2 show ARG2 as a target for specific T cells in vitro, where both immune cells and cancer cells expressing ARG2 are specifically recognized by ARG2 specific T cells. To test the potential functional effects of ARG2 specific T cells in vivo, Example 3 identified relevant murine ARG2 peptide epitopes, which were further evaluated. C57BL / 6 mice were screened for immune responses by subcutaneous immunization of mice in peptide-montanide emulsion. Each of 6 candidate peptides was inoculated in groups of 3 mice, and 7 days later the mice were sacrificed, spleen cells were isolated and analyzed in an in vitro mIFNy ELISPOT assay. A strong immune response was observed in all 3 mice inoculated with P4 (see Figure 12 , and the same data is shown in Figure 20 A). This was subsequently confirmed in a similar experiment with more mice per group Figure 20 B).

[0221] To identify the most relevant tumor model, Arg2 expression was evaluated in different groups of transplanted tumors derived from C57BL / 6. We found the most consistent high expression of Arg2 in tumors formed by Lewis lung (LL2) tumor cells Figure 20 C). C57BL / 6 mice were challenged with LL2 tumor cells and then immunized twice (day 0 and day 7 after tumor inoculation - see treatment schedule in Figure 2 D), resulting in reduced tumor growth in Arg2 inoculated mice (P4) Figure 20 E). Mice were sacrificed on day 12 after tumor inoculation due to tumor ulceration. To test whether the Arg2 vaccine modifies the TME in a way that could induce the effects of anti-PDl antibodies, we combined the vaccine against mARG2 (188-197) (denoted P4) with anti-PDl in the LL2 model. As expected, single treatment with anti-PDl antibodies did not affect tumor growth in this model. However, the combination of anti-PDl with the ARG2 vaccine had an additive effect Figure 20 F and 20G). Mice were sacrificed on day 12 after tumor inoculation due to tumor ulceration.

[0222] Summary and discussion of Examples 1-4

[0223] These examples show that ARG2 is a target for specific T cells and, thus, specific ARG2-specific effector T cells can be developed as a possible new approach to target ARG2-expressing immunosuppressive cells. First, these examples identified naturally occurring peripheral ARG2-specific T cells in cancer patients and healthy donors by screening a peptide library covering the entire ARG2 sequence. Interestingly, it was found that ARG2 contains multiple epitopes that are frequently recognized by peripheral T cells. The frequent T cell response against ARG2 emphasizes the high immunogenicity of ARG2 and supports the possibility to enhance ARG2-specific immune responses in ARG2-expressing cancer patients, such as prostate cancer or AML patients. Moreover, the strong immune response in healthy individuals indicates that ARG2-specific T cells are a natural component of the immune system and can be important for immune homeostasis. Furthermore, specific CD4+ T cells were isolated and expanded that reacted to peptides from the apparently most immunogenic region of ARG2. The results show that ARG2-specific T cells do recognize and react to ARG2-expressing myeloid cells.

[0224] Generally, tumors are now classified into different categories according to the immune infiltration; (i) lack of immune infiltrates (in so-called "cold" tumors); (ii) immune infiltrates excluded from contact with malignant cells (in so-called "excluded" tumors); or (iii) rich tumor infiltrates (in so-called "hot" tumors) that are controlled by strong immunosuppressive mechanisms. An important therapeutic strategy is the clinical combination treatment of "cold" and "excluded" tumors into "hot" tumors, since the latter are usually associated with improved disease outcomes with immunotherapy, in particular with checkpoint blockade. An important feature of arginases is its expression in "excluded" type tumors, due to arginase-expressing immunosuppressive immune cells in these tumors. ARG1 is well described to be upregulated in M2-like macrophages in response to Th2 cytokines, such as IL-4 and IL-13 (in addition to IL-10 and TGF-β). In contrast, the regulation of ARG2 is only very limited described, but interestingly, it has been suggested that Toll-like receptor ligands such as lipopolysaccharide and oligodeoxynucleotides containing a high number of non-methylated cytosine guanine motifs (CpG) induce ARG2 expression in murine macrophages.

[0225] ARG2 has recently also been described to be induced in neuroblastoma cells by IL1 β and TNFα. Importantly, in the present study we further show that a cocktail of cytokines, namely IL4, GM-CSF and TNFα, induce ARG2 in malignant myeloid cells. Thus, ARG2 seems to be induced by environments not only in the exclusionary tumor but also in more "intermediate" to "hot" tumors. Therefore, it is not surprising that ARG1 and ARG2 are found in the tumor microenvironment (TME) to be expressed by different cells and in different tumor types. Thus, ARG1 is mainly expressed by MDSCs and TAMs, while ARG2 has been described to be expressed by various solid tumor cells, AML blasts and CAFs. Therefore, the combination of ARG1 and ARG2 for immunization can be advantageous to target different immunosuppressive arginase expressing cells in the TME, thus benefiting more patients. Moreover, activated M1 macrophages have been well described to appear in response to propagating inflammation by Th1 cytokines, such as IFNy. Importantly, many stromal cells are not terminally differentiated cells and can revert to immune competent cells under proinflammatory stimuli. Activating arginase specific T cells by e.g. immunization indeed leads to Thl inflammatory signals at the tumor site. Other types of anti-regulatory T cells, such as IDO- and PD-L1 specific proinflammatory T cells, are known to exist and Thl -inflammatory signals, such as IFNy, have been reported to spontaneously lead to expansion of such IDO- and PD-L1 specific T cells, suggesting a potential synergistic effect of arginase with IDO- or PDL1 based vaccines. In this case, ARG1 / ARG2 immunization can induce Thl inflammation at the tumor site, where otherwise arginase expressing cells prevent lymphocyte infiltration. In turn, this effect will induce IDO and / or PD-L1, thus allowing for further targeting by anti-Tregs recognizing epitopes derived from these targets. Thus, a combination of epitopes from different anti-Treg target antigens can be an additive in the immunization approach. Likewise, the combination of an ARG2 immunomodulatory vaccine activating ARG2 specific T cells and checkpoint blockade antibodies should increase the number of patients responding to treatment compared to checkpoint blockade alone, which is only effective in inflamed tumors. Therefore, arginase expressing cells prevent effector lymphocyte proliferation at the tumor site and are thus an important reason for the lack of anti-PDl treatment effect in many cancer patients. In the present study we find that ARG2 is indeed expressed in the well described PD-L1 anti-tumor model Lewis lung. We find that activation of ARG2 specific T cells by immunization inhibits the growth of Lewis lung cells, most importantly in synergy with anti-PDl. Thus, the combination with immunomodulatory ARG2 immunization can indeed sensitize resistant Lewis lung cells to anti-PDl treatment.

[0226] In summary, these examples show that ARG2-specific T cells exist as a natural component of the immune system and can easily tip the balance away from immunosuppression in cancer. Therapeutic immunization against ARG2 should promote the generation of an inflammatory TME, which would favor a cancer-specific immune response against cancer cells. Thus, ARG2-based vaccines are likely to synergize with other immunotherapies, particularly checkpoint inhibitors. The most immunogenic peptides from human ARG2 used in this example are effective in stimulating ARG2-specific T cell responses, which can be critical for rebalancing the microenvironment and should increase the potency of T cell enhancing drugs, such as checkpoint blockers, compared to single pathway therapies or current cancer vaccines that only target cancer cells.

[0227] Sequence

[0228] The start and stop positions indicate positions within the full-length human arginase 2 (SEQ ID NO: 51), unless otherwise noted.

[0229] Table X

[0230]

[0231]

[0232]

[0233] * indicates sequence from human arginase 1. The start and stop positions are positions in human arginase 1 (SEQ ID NO: 53).

[0234] # indicates sequence from murine arginase 2. The start and stop positions are positions in murine arginase 2 (SEQ ID NO: 52), although the numbering is also matched to human arginase 2 since the murine and human proteins are of the same length.

[0235] Full-length human arginase 2 (NP_001163.1) (SEQ ID NO: 51)

[0236]

[0237] Regions identified as immunogenic hotspots based on arginase 1 homology are indicated in bold and underlined. The transport peptide boundary is identified as the center of previously unrecognized hotspots with bold and italicized "KSV".

[0238] Full-length murine arginase 2 (NP_033835.1) (SEQ ID NO: 52)

[0239]

[0240]

[0241] Full length human Arginase 1 (NP_000036.2) (SEQ ID NO: 53)

[0242]

[0243] Regions identified as immunogenic hotspots are shown in bold and underlined. SEQUENCE LISTING <110> IO BIOLOGICAL TECHNOLOGIES LTD <120> Immunogenic Arginase 2 polypeptide <130> N414657WO <150> GB1818576.9 <151> 14 November 2018 <160> 64 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> PRT <213> Homo sapiens <400> 1 Met Ser Leu Arg Gly Ser Leu Ser Arg Leu Leu Gln Thr Arg Val His 1 5 10 15 Ser Ile Leu Lys 20 <210> 2 <211> 20 <212> PRT <213> Homo sapiens <400> 2 Leu Gln Thr Arg Val His Ser Ile Leu Lys Lys Ser Val His Ser Val 1 5 10 15 Ala Val Ile Gly 20 <210> 3 <211> 20 <212> PRT <213> Homo sapiens <400> 3 Lys Ser Val His Ser Val Ala Val Ile Gly Ala Pro Phe Ser Gln Gly 1 5 10 15 Gln Lys Arg Lys 20 <210> 4 <211> 20 <212> PRT <213> Homo sapiens <400> 4 Ala Pro Phe Ser Gln Gly Gln Lys Arg Lys Gly Val Glu His Gly Pro 1 5 10 15 Ala Ala Ile Arg 20 <210> 5 <211> 20 <212> PRT <213> Homo sapiens <400> 5 Gly Val Glu His Gly Pro Ala Ala Ile Arg Glu Ala Gly Leu Met Lys 1 5 10 15 Arg Leu Ser Ser 20 <210> 6 <211> 20 <212> PRT <213> Homo sapiens <400> 6 Glu Ala Gly Leu Met Lys Arg Leu Ser Ser Leu Gly Cys His Leu Lys 1 5 10 15 Asp Phe Gly Asp 20 <210> 7 <211> 20 <212> PRT <213> Homo sapiens <400> 7 Leu Gly Cys His Leu Lys Asp Phe Gly Asp Leu Ser Phe Thr Pro Val 1 5 10 15 Pro Lys Asp Asp 20 <210> 8 <211> 20 <212> PRT <213> Homo sapiens <400> 8 Leu Ser Phe Thr Pro Val Pro Lys Asp Asp Leu Tyr Asn Asn Leu Ile 1 5 10 15 Val Asn Pro Arg 20 <210> 9 <211> 20 <212> PRT <213> Homo sapiens <400> 9 Leu Tyr Asn Asn Leu Ile Val Asn Pro Arg Ser Val Gly Leu Ala Asn 1 5 10 15 Gln Glu Leu Ala 20 <210> 10 <211> 20 <212> PRT <213> Homo sapiens <400> 10 Ser Val Gly Leu Ala Asn Gln Glu Leu Ala Glu Val Val Ser Arg Ala 1 5 10 15 Val Ser Asp Gly 20 <210> 11 <211> 20 <212> PRT <213> Homo sapiens <400> 11 Glu Val Val Ser Arg Ala Val Ser Asp Gly Tyr Ser Cys Val Thr Leu 1 5 10 15 Gly Gly Asp His 20 <210> 12 <211> 20 <212> PRT <213> Homo sapiens <400> 12 Tyr Ser Cys Val Thr Leu Gly Gly Asp His Ser Leu Ala Ile Gly Thr 1 5 10 15 Ile Ser Gly His 20 <210> 13 <211> 20 <212> PRT <213> Homo sapiens <400> 13 Ser Leu Ala Ile Gly Thr Ile Ser Gly His Ala Arg His Cys Pro Asp 1 5 10 15 Leu Cys Val Val 20 <210> 14 <211> 20 <212> PRT <213> Homo sapiens <400> 14 Ala Arg His Cys Pro Asp Leu Cys Val Val Trp Val Asp Ala His Ala 1 5 10 15 Asp Ile Asn Thr 20 <210> 15 <211> 20 <212> PRT <213> Homo sapiens <400> 15 Trp Val Asp Ala His Ala Asp Ile Asn Thr Pro Leu Thr Thr Ser Ser 1 5 10 15 Gly Asn Leu His 20 <210> 16 <211> 20 <212> PRT <213> Homo sapiens <400> 16 Pro Leu Thr Thr Ser Ser Gly Asn Leu His Gly Gln Pro Val Ser Phe 1 5 10 15 Leu Leu Arg Glu 20 <210> 17 <211> 20 <212> PRT <213> Homo sapiens <400> 17 Gly Gln Pro Val Ser Phe Leu Leu Arg Glu Leu Gln Asp Lys Val Pro 1 5 10 15 Gln Leu Pro Gly 20 <210> 18 <211> 20 <212> PRT <213> Homo sapiens <400> 18 Leu Gin Asp Lys Val Pro Gin Leu Pro Gly Phe Ser Trp lie Lys Pro 1 5 10 15 Cys lie Ser Ser 20 <210> 19 <211 > 20 <212> PRT <213> Homo sapiens <400> 19 Phe Ser Trp lie Lys Pro Cys lie Ser Ser Ala Ser lie Val Tyr lie 1 5 10 15 Gly Leu Arg Asp 20 <210> 20 <211 > 20 <212> PRT <213> Homo sapiens <400> 20 Ala Ser lie Val Tyr lie Gly Leu Arg Asp Val Asp Pro Pro Glu His 1 5 10 15 Phe lie Leu Lys 20 <210> 21 <211 > 20 <212> PRT <213> Homo sapiens <400> 21 Val Asp Pro Pro Glu His Phe lie Leu Lys Asn Tyr Asp lie Gin Tyr 1 5 10 15 Phe Ser Met Arg 20 <210> 22 <211 > 20 <212> PRT <213> Homo sapiens<213> Homo sapiens <400> 22 Asn Tyr Asp Ile Gln Tyr Phe Ser Met Arg Asp Ile Asp Arg Leu Gly 1 5 10 15 Ile Gln Lys Val 20 <210> 23 <211> 20 <212> PRT <213> Homo sapiens <400> 23 Asp Ile Asp Arg Leu Gly Ile Gln Lys Val Met Glu Arg Thr Phe Asp 1 5 10 15 Leu Leu Ile Gly 20 <210> 24 <211> 20 <212> PRT <213> Homo sapiens <400> 24 Met Glu Arg Thr Phe Asp Leu Leu Ile Gly Lys Arg Gln Arg Pro Ile 1 5 10 15 His Leu Ser Phe 20 <210> 25 <211> 20 <212> PRT <213> Homo sapiens <400> 25 Lys Arg Gln Arg Pro Ile His Leu Ser Phe Asp Ile Asp Ala Phe Asp 1 5 10 15 Pro Thr Leu Ala 20 <210> 26 <211> 20 <212> PRT <213> Homo sapiens <400> 26 Asp Ile Asp Ala Phe Asp Pro Thr Leu Ala Pro Ala Thr Gly Thr Pro 1 5 10 15 Val Val Gly Gly 20 <210> 27 <211> 20 <212> PRT <213> Homo sapiens <400> 27 Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu Gly 1 5 10 15 Met Tyr Ile Ala 20 <210> 28 <211> 20 <212> PRT <213> Homo sapiens <400> 28 Leu Thr Tyr Arg Glu Gly Met Tyr Ile Ala Glu Glu Ile His Asn Thr 1 5 10 15 Gly Leu Leu Ser 20 <210> 29 <211> 20 <212> PRT <213> Homo sapiens <400> 29 Glu Glu Ile His Asn Thr Gly Leu Leu Ser Ala Leu Asp Leu Val Glu 1 5 10 15 Val Asn Pro Gln 20 <210> 30 <211> 20 <212> PRT <213> Homo sapiens <400> 30 Ala Leu Asp Leu Val Glu Val Asn Pro Gln Leu Ala Thr Ser Glu Glu 1 5 10 15 Glu Ala Lys Thr 20 <210> 31 <211> 20 <212> PRT <213> Homo sapiens <400> 31 Leu Ala Thr Ser Glu Glu Glu Ala Lys Thr Thr Ala Asn Leu Ala Val 1 5 10 15 Asp Val Ile Ala 20 <210> 32 <211> 20 <212> PRT <213> Homo sapiens <400> 32 Thr Ala Asn Leu Ala Val Asp Val Ile Ala Ser Ser Phe Gly Gln Thr 1 5 10 15 Arg Glu Gly Gly 20 <210> 33 <211> 20 <212> PRT <213> Homo sapiens <400> 33 Ser Ser Phe Gly Gln Thr Arg Glu Gly Gly His Ile Val Tyr Asp Gln 1 5 10 15 Leu Pro Thr Pro 20 <210> 34 <211> 24 <212> PRT <213> Homo sapiens <400> 34 His Ile Val Tyr Asp Gln Leu Pro Thr Pro Ser Ser Pro Asp Glu Ser 1 5 10 15 Glu Asn Gln Ala Arg Val Arg Ile 20 <210> 35 <211> 20 <212> PRT <213> Homo sapiens <400> 35 Gly Phe Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser Ile Val Tyr 1 5 10 15 Ile Gly Leu Arg 20 <210> 36 <211> 20 <212> PRT <213> Homo sapiens <400> 36 Ser Ala Ser Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Pro Glu 1 5 10 15 His Phe Ile Leu 20 <210> 37 <211> 20 <212> PRT <213> Homo sapiens <400> 37 Asp Val Asp Pro Pro Glu His Phe Ile Leu Lys Asn Tyr Asp Ile Gln Tyr Phe Ser Met Arg Asp Ile Asp Arg Leu Gly Ile Gln Lys 1 5 10 15 Tyr Phe Ser Met 20 <210> 38 <211> 20 <212> PRT <213> Homo sapiens <400> 38 Lys Asn Tyr Asp Ile Gln Tyr Phe Ser Met Arg Asp Ile Asp Arg Leu Gly Ile Gln Lys 1 5 10 15 Gly Ile Gln Lys 20 <210> 39 <211> 50 <212> PRT <213> Homo sapiens <400> 39 Gly Phe Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser Ile Val Tyr 1 5 10 15 Ile Gly Leu Arg Asp Val Asp Pro Pro Glu His Phe Ile Leu Lys Asn Tyr Asp Ile Gln Tyr Phe Ser Met Arg Asp Ile Asp Arg Leu Gly Ile Gln Lys 20 25 30 Tyr Phe Ser Met 35 40 45 Gln Lys 50 <210> 40 <211> 50 <212> PRT <213> Homo sapiens <400> 40 Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr 1 5 10 15 Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr 20 25 30 Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile 35 40 45 Gly Lys 50 <210> 41 <211> 20 <212> PRT <213> Homo sapiens <400> 41 Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr 1 5 10 15 Ile Gly Leu Arg 20 <210> 42 <211> 20 <212> PRT <213> Homo sapiens <400> 42 Ala Lys Asp Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Gly Glu 1 5 10 15 His Tyr Ile Leu 20 <210> 43 <211> 20 <212> PRT <213> Homo sapiens <400> 43 Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr Leu Gly Ile Lys 1 5 10 15 Tyr Phe Ser Met 20 <210> 44 <211> 20 <212> PRT <213> Homo sapiens <400> 44 Lys Thr Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu 1 5 10 15 Gly Ile Gly Lys 20 <210> 45 <211> 9 <212> PRT <213> Mus musculus <400> 45 Val Val Tyr Pro Arg Ser Val Gly Leu 1 5 <210> 46 <211> 18 <212> PRT <213> Mus musculus <400> 46 Val Val Tyr Pro Arg Ser Val Gly Leu Ala Asn Gln Glu Leu Ala Glu 1 5 10 15 Val Val <210> 47 <211> 8 <212> PRT <213> Mus musculus <400> 47 Pro Asn Ile Val Tyr Ile Gly Leu 1 5 <210> 48 <211> 16 <212> PRT <213> Mus musculus <400> 48 Trp lie Lys Pro Cys Leu Ser Pro Pro Asn lie Val Tyr lie Gly Leu 1 5 10 15 <210> 49 <211> 9 <212> PRT <213> Mus musculus <400> 49 Leu Ser Pro Pro Asn lie Val Tyr lie 1 5 <210> 50 <211> 9 <212> PRT <213> Mus musculus <400> 50 lie Gin Tyr Phe Ser Met Arg Glu lie 1 5 <210> 51 <211> 354 <212> PRT <213> Homo sapiens <400> 51 Met Ser Leu Arg Gly Ser Leu Ser Arg Leu Leu Gin Thr Arg Val His 1 5 10 15 Ser lie Leu Lys Lys Ser Val His Ser Val Ala Val lie Gly Ala Pro 20 25 30 Phe Ser Gin Gly Gin Lys Arg Lys Gly Val Glu His Gly Pro Ala Ala 35 40 45 Ile Arg Glu Ala Gly Leu Met Lys Arg Leu Ser Ser Leu Gly Cys His 50 55 60 Leu Lys Asp Phe Gly Asp Leu Ser Phe Thr Pro Val Pro Lys Asp Asp 65 70 75 80 Leu Tyr Asn Asn Leu Ile Val Asn Pro Arg Ser Val Gly Leu Ala Asn 85 90 95 Gln Glu Leu Ala Glu Val Val Ser Arg Ala Val Ser Asp Gly Tyr Ser 100 105 110 Cys Val Thr Leu Gly Gly Asp His Ser Leu Ala Ile Gly Thr Ile Ser 115 120 125 Gly His Ala Arg His Cys Pro Asp Leu Cys Val Val Trp Val Asp Ala 130 135 140 His Ala Asp Ile Asn Thr Pro Leu Thr Thr Ser Ser Gly Asn Leu His 145 150 155 160 Gly Gln Pro Val Ser Phe Leu Leu Arg Glu Leu Gln Asp Lys Val Pro 165 170 175 Gln Leu Pro Gly Phe Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser 180 185 190 Ile Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Pro Glu His Phe Ile 195 200 205 Leu Lys Asn Tyr Asp He Gin Tyr Phe Ser Met Arg Asp He Asp Arg 210 215 220 Leu Gly He Gin Lys Val Met Glu Arg Thr Phe Asp Leu Leu He Gly 225 230 235 240 Lys Arg Gin Arg Pro He His Leu Ser Phe Asp He Asp Ala Phe Asp 245 250 255 Pro Thr Leu Ala Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr 260 265 270 Tyr Arg Glu Gly Met Tyr He Ala Glu Glu He His Asn Thr Gly Leu 275 280 285 Leu Ser Ala Leu Asp Leu Val Glu Val Asn Pro Gin Leu Ala Thr Ser 290 295 300 Glu Glu Glu Ala Lys Thr Thr Ala Asn Leu Ala Val Asp Val He Ala 305 310 315 320 Ser Ser Phe Gly Gin Thr Arg Glu Gly Gly His He Val Tyr Asp Gin 325 330 335 Leu Pro Thr Pro Ser Ser Pro Asp Glu Ser Glu Asn Gin Ala Arg Val 340 345 350 Arg He <210> 52 <211> 354 <212> PRT <213> Mus musculus <400> 52 Met Phe Leu Arg Ser Ser Ala Ser Arg Leu Leu His Gly Gin He Pro 1 5 10 15 Cys Val Leu Thr Arg Ser Val His Ser Val Ala He Val Gly Ala Pro 20 25 30 Phe Ser Arg Gly Gin Lys Lys Leu Gly Val Glu Tyr Gly Pro Ala Ala 35 40 45 He Arg Glu Ala Gly Leu Leu Lys Arg Leu Ser Arg Leu Gly Cys His 50 55 60 Leu Lys Asp Phe Gly Asp Leu Ser Phe Thr Asn Val Pro Gin Asp Asp 65 70 75 80 Pro Tyr Asn Asn Leu Val Val Tyr Pro Arg Ser Val Gly Leu Ala Asn 85 90 95 Gln Glu Leu Ala Glu Val Val Ser Arg Ala Val Ser Gly Gly Tyr Ser 100 105 110 Cys Val Thr Met Gly Gly Asp His Ser Leu Ala He Gly Thr He He 115 120 125 Gly His Ala Arg His Arg Pro Asp Leu Cys Val He Trp Val Asp Ala 130 135 140 His Ala Asp Ile Asn Thr Pro Leu Thr Thr Val Ser Gly Asn Ile His 145 150 155 160 Gly Gln Pro Leu Ser Phe Leu Ile Lys Glu Leu Gln Asp Lys Val Pro 165 170 175 Gln Leu Pro Gly Phe Ser Trp Ile Lys Pro Cys Leu Ser Pro Pro Asn 180 185 190 Ile Val Tyr Ile Gly Leu Arg Asp Val Glu Pro Pro Glu His Phe Ile 195 200 205 Leu Lys Asn Tyr Asp Ile Gln Tyr Phe Ser Met Arg Glu Ile Asp Arg 210 215 220 Leu Gly Ile Gln Lys Val Met Glu Gln Thr Phe Asp Arg Leu Ile Gly 225 230 235 240 Lys Arg Gln Arg Pro Ile His Leu Ser Phe Asp Ile Asp Ala Phe Asp 245 250 255 Pro Lys Leu Ala Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr 260 265 270 Tyr Arg Glu Gly Val Tyr Ile Thr Glu Glu Ile His Asn Thr Gly Leu 275 280 285 Leu Ser Ala Leu Asp Leu Val Glu Val Asn Pro His Leu Ala Thr Ser 290 295 300 Glu Glu Glu Ala Lys Ala Thr Ala Arg Leu Ala Val Asp Val Ile Ala 305 310 315 320 Ser Ser Phe Gly Gln Thr Arg Glu Gly Gly His Ile Val Tyr Asp His 325 330 335 Leu Pro Thr Pro Ser Ser Pro His Glu Ser Glu Asn Glu Glu Cys Val 340 345 350 Arg Ile <210> 53 <211> 322 <212> PRT <213> Homo sapiens <400> 53 Met Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser 1 5 10 15 Lys Gly Gln Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg 20 25 30 Lys Ala Gly Leu Leu Glu Lys Leu Lys Glu Gln Glu Cys Asp Val Lys 35 40 45 Asp Tyr Gly Asp Leu Pro Phe Ala Asp Ile Pro Asn Asp Ser Pro Phe 50 55 60 Gln Ile Val Lys Asn Pro Arg Ser Val Gly Lys Ala Ser Glu Gln Leu 65 70 75 80 Ala Gly Lys Val Ala Glu Val Lys Lys Asn Gly Arg Ile Ser Leu Val 85 90 95 Leu Gly Gly Asp His Ser Leu Ala Ile Gly Ser Ile Ser Gly His Ala 100 105 110 Arg Val His Pro Asp Leu Gly Val Ile Trp Val Asp Ala His Thr Asp 115 120 125 Ile Asn Thr Pro Leu Thr Thr Thr Ser Gly Asn Leu His Gly Gln Pro 130 135 140 Val Ser Phe Leu Leu Lys Glu Leu Lys Gly Lys Ile Pro Asp Val Pro 145 150 155 160 Gly Phe Ser Trp Val Thr Pro Cys Ile Ser Ala Lys Asp Ile Val Tyr 165 170 175 Ile Gly Leu Arg Asp Val Asp Pro Gly Glu His Tyr Ile Leu Lys Thr 180 185 190 Leu Gly Ile Lys Tyr Phe Ser Met Thr Glu Val Asp Arg Leu Gly Ile 195 200 205 Gly Lys Val Met Glu Glu Thr Leu Ser Tyr Leu Leu Gly Arg Lys Lys 210 215 220 Arg Pro Ile His Leu Ser Phe Asp Val Asp Gly Leu Asp Pro Ser Phe 225 230 235 240​​​​​​​​​​ Thr Pro Ala Thr Gly Thr Pro Val Val Gly Gly Leu Thr Tyr Arg Glu 245 250 255 Gly Leu Tyr Ile Thr Glu Glu Ile Tyr Lys Thr Gly Leu Leu Ser Gly 260 265 270 Leu Asp Ile Met Glu Val Asn Pro Ser Leu Gly Lys Thr Pro Glu Glu 275 280 285 Val Thr Arg Thr Val Asn Thr Ala Val Ala Ile Thr Leu Ala Cys Phe 290 295 300 Gly Leu Ala Arg Glu Gly Asn His Lys Pro Ile Asp Tyr Leu Asn Pro 305 310 315 320 Pro Lys <210> 54 <211> 10 <212> PRT <213> Homo sapiens <400> 54 Ile Leu Lys Lys Ser Val His Ser Val Ala 1 5 10 <210> 55 <211> 9 <212> PRT <213> Homo sapiens <400> 55 Ile Leu Lys Lys Ser Val His Ser Val 1 5 <210> 56 <211> 10 <212> PRT <213> Homo sapiens <400> 56 Ser Ile Leu Lys Lys Ser Val His Ser Val 1 5 10 <210> 57 <211> 29 <212> PRT <213> Homo sapiens <400> 57 Ser Leu Ser Arg Leu Leu Gln Thr Arg Val His Ser Ile Leu Lys Lys 1 5 10 15 Ser Val His Ser Val Ala Val Ile Gly Ala Pro Phe Ser 20 25 <210> 58 <211> 30 <212> PRT <213> Homo sapiens <400> 58 Ser Leu Arg Gly Ser Leu Ser Arg Leu Leu Gln Thr Arg Val His Ser 1 5 10 15 Ile Leu Lys Lys Ser Val His Ser Val Ala Val Ile Gly Ala 20 25 30 <210> 59 <211> 33 <212> PRT <213> Homo sapiens <400> 59 Ser Leu Arg Gly Ser Leu Ser Arg Leu Leu Gln Thr Arg Val His Ser 1 5 10 15 Ile Leu Lys Lys Ser Val His Ser Val Ala Val Ile Gly Ala Pro Phe 20 25 30 Ser <210> 60 <211> 29 <212> PRT <213> Homo sapiens <400> 60 Phe Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser Ile Val Tyr Ile 1 5 10 15 Gly Leu Arg Asp Val Asp Pro Pro Glu His Phe Ile Leu 20 25 <210> 61 <211> 32 <212> PRT <213> Homo sapiens <400> 61 Leu Pro Gly Phe Ser Trp Ile Lys Pro Cys Ile Ser Ser Ala Ser Ile 1 5 10 15 Val Tyr Ile Gly Leu Arg Asp Val Asp Pro Pro Glu His Phe Ile Leu 20 25 30 <210> 62 <211> 22 <212> PRT <213> Homo sapiens <400> 62 Met Ser Leu Arg Gly Ser Leu Ser Arg Leu Leu Gln Thr Arg Val His 1 5 10 15 Ser Ile Leu Lys Lys Ser 20 <210> 63 <211> 56 <212> PRT <213> Homo sapiens<213> Homo sapiens <400> 63 Met Ser Leu Arg Gly Ser Leu Ser Arg Leu Leu Gin Thr Arg Val His 1 5 10 15 Ser Ile Leu Lys Lys Ser Val His Ser Val Ala Val Ile Gly Ala Pro 20 25 30 Phe Ser Gin Gly Gin Lys Arg Lys Gly Val Glu His Gly Pro Ala Ala 35 40 45 Ile Arg Gin Ala Gly Leu Met Lys 50 55 <210> 64 <211> 38 <212> PRT <213> Homo sapiens <400> 64 Met Ser Ala Lys Ser Arg Thr Ile Gly Ile Ile Gly Ala Pro Phe Ser 1 5 10 15 Lys Gly Gin Pro Arg Gly Gly Val Glu Glu Gly Pro Thr Val Leu Arg 20 25 30 Lys Ala Gly Leu Leu Glu 35

Claims

1. A polypeptide which is an immunogenic fragment of SEQ ID NO: 51 consisting of the amino acid sequence of any one of SEQ ID NO: 59, 58, 57 or 2.

2. The polypeptide according to claim 1, wherein the C-terminal amino acid is replaced by the corresponding amide.

3. A composition comprising the polypeptide of claim 1 or 2 and an adjuvant.

4. The composition according to claim 3, comprising at least one pharmaceutically acceptable diluent, carrier or preservative.

5. The composition according to claim 3 or 4, wherein the adjuvant is selected from the group consisting of a bacterial DNA-based adjuvant, an oil / surfactant-based adjuvant, a viral dsRNA-based adjuvant and imidazoquinolines.

6. The composition of claim 5, wherein, The oil / surfactant-based adjuvant is Montanide ISA adjuvant.

7. Use of the polypeptide of claim 1 or 2 or the composition of any one of claims 3 to 6 in the manufacture of a medicament for treating a disease or condition in a subject, wherein the disease or condition is a cancer selected from prostate cancer, melanoma and acute myeloid leukemia.

8. An in vitro method of stimulating T cells specific for Arginase 2, wherein the method comprises contacting the cells with the polypeptide of claim 1 or 2 or the composition of any one of claims 3 to 6.

9. The method according to claim 8, wherein the cells are present in a sample taken from a healthy subject or a cancer patient.

10. The method according to claim 9, wherein the sample taken from a cancer patient is a tumor sample.

11. A nucleic acid encoding the polypeptide of claim 1 or 2.

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