Arginase 2 vaccine

Abstract

The present invention relates to novel polypeptides derived from arginase 2 (ARG2), polynucleotides encoding the polypeptides, and compositions comprising the polypeptides or polynucleotides. The present invention also relates to uses of the polypeptides, polynucleotides, or compositions.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel polypeptides derived from Arginase 2 (ARG2), polynucleotides encoding said polypeptides, and compositions comprising said polypeptides or polynucleotides.The present invention also relates to uses of said polypeptides, polynucleotides, and compositions. [Background technology]

[0002] 2. Background of the Invention Arginase is an enzyme that catalyzes the reaction that converts the amino acid L-arginine into L-ornithine and urea. This depletes the microenvironment of arginine, resulting in the suppression of tumor-specific cytotoxic T cell responses. Increased arginase activity has been detected in cancer cells, for example, in patients with breast, lung, colon, or prostate cancer. Mouse macrophages transfected with the rat arginase gene have been shown to promote the growth of co-cultured tumor cells, both in vitro and in vivo. Furthermore, induction of arginase expression by macrophages has been shown to increase tumor angiogenesis via polyamine synthesis. Results from a mouse lung cancer model showed that there was a subpopulation of mature tumor-associated myeloid cells that expressed high levels of arginase. These tumor-associated myeloid cells depleted extracellular L-arginine and inhibited the antigen-specific proliferation of tumor-infiltrating lymphocytes (TILs). Injection of an arginase inhibitor blocked the growth of lung cancer in mice. This demonstrates how induction of arginase expression in tumor cells and tumor-associated myeloid cells can promote tumor growth by suppressing anti-tumor immune responses through its negative effect on TILs.

[0003] MDSCs (myeloid-derived immune suppressor cells) inhibit the activation, proliferation, and cytotoxicity of effector T cells and natural killer cells, while inducing the differentiation and proliferation of Tregs. Both cancer cells and MDSCs can suppress T cells by manipulating L-arginine metabolism via the enzymes nitric oxide synthase (NOS) and arginase. Many tumors show increased expression of arginase and inducible NOS (iNOS), resulting in arginine depletion from the tumor microenvironment. Several studies have highlighted the importance of this altered tumor arginine metabolism in suppressing tumor-specific T cell responses, and it has recently been demonstrated that acute myeloid leukemia (AML) blasts display an arginase-dependent ability to inhibit T cell proliferation and hematopoietic stem cells. Furthermore, arginase and iNOS inhibitors reduce the suppressive activity of AML.

[0004] In mammals, there are two arginase isoenzymes: Arginase 1 and Arginase 2. The two isoenzymes catalyze the same biochemical reaction (and therefore cannot be distinguished by enzyme assays) but differ in cellular expression, regulation, and subcellular localization. Summary of the Invention

[0005] The present inventors have previously identified a 50 amino acid region of Arginase 1 (ARG1) and Arginase 2 (ARG2) that is a "hot spot" for immunogenicity. This region corresponds to positions 161-210 of full-length human ARG1 (SEQ ID NO:20) or positions 180-229 of full-length human ARG2 (SEQ ID NO:19), or the corresponding positions in mouse arginase. This region and peptides derived therefrom are described in WO2018 / 065563.

[0006] The inventors have also identified a specific subset of polypeptides derived from the "hotspot" regions of ARG1 that are particularly effective in stimulating immune responses. These peptides correspond to positions 169-206 of full-length human ARG1, positions 169-200 of full-length human ARG1, or positions 169-210 of full-length human ARG1 (or the corresponding positions in human ARG2 or mouse ARG1). This subset of polypeptides is described in WO 2020 / 064744.

[0007] The inventors further identified that polypeptides derived from a distinct region of human ARG2 are particularly effective in stimulating an immune response. This region spans the C-terminus of the transit peptide of human ARG2 (position 22 of SEQ ID NO: 19). These peptides are described in WO 2020 / 099582 and include an immunogenic peptide called A2L2, which corresponds to positions 2-34 of human ARG2 (SEQ ID NO: 16).

[0008] The present inventors have now identified further immunogenic polypeptides derived from ARG2. Surprisingly, the polypeptides of the invention are not located in the "hotspot" region (i.e., amino acids 180-229 of human ARG2) nor in the A2L2 region (i.e., amino acids 2-34 of ARG2). The polypeptides of the invention inhibit cytotoxic CD8A1 cytotoxicity that is capable of lysing cancer cells (e.g., melanoma cells). + Including T cells, CD8 + or CD4 + It can be advantageously used to stimulate T cells.

[0009] In particular, all immunogenic responses to the immunogenic ARG2 polypeptide observed to date have been CD4 + T cell response. However, CD4 + Epitopes and CD8 + It has been observed that simultaneous activation of epitopes provides strong synergistic protection against tumors. Thus, cytotoxic CD8 + CD8 on T cells etc. +Polypeptides of the invention capable of stimulating T cells are particularly advantageous.

[0010] ARG2-specific T cells generated using the polypeptides of the present invention can specifically recognize and react with activated regulatory T cells (Tregs) with high ARG2 expression, and thus the polypeptides of the present invention have utility in modulating the immunosuppressive tumor microenvironment.

[0011] The present invention provides a polypeptide that is an immunogenic fragment of human arginase 2 (ARG2; SEQ ID NO: 19) comprising or consisting of a sequence of 9 to 19 consecutive amino acids of SEQ ID NO: 19, with the proviso that the fragment does not comprise any one of the following sequences: Amino acids 2 to 34 of SEQ ID NO:19; Amino acids 180 to 229 of SEQ ID NO:19; Amino acids 129 to 141 of SEQ ID NO:19; Amino acids 144 to 156 of SEQ ID NO: 19; or Amino acids 237 to 249 of sequence number 19.

[0012] The polypeptide may comprise or consist of a human leukocyte antigen (HLA) class I restricted epitope. The polypeptide may comprise or consist of an HLA-B8 restricted epitope. The polypeptide may comprise or consist of an HLA-B8 restricted epitope. + Can stimulate T cells. CD8 + The positive T cells may be cytotoxic T cells. + The T cells can be ARG2 specific. + The positive T cells may be ARG2-specific cytotoxic T cells. The polypeptide may comprise or consist of the amino acid sequence: NLIVINPRSV (SEQ ID NO:5).

[0013] The polypeptide may comprise or consist of an HLA class II restricted epitope. +It is capable of stimulating T cells. The polypeptide may comprise or consist of the amino acid sequence: GLLSALDLV (SEQ ID NO: 14).

[0014] The polypeptide may have a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids. The C-terminal amino acid of the polypeptide may be replaced by the corresponding amide.

[0015] The present invention also provides a polynucleotide encoding a polypeptide of the present invention. The polynucleotide may be comprised in a vector.

[0016] The present invention further provides a composition comprising a polypeptide of the present invention and / or a polynucleotide of the present invention. The composition may comprise at least one different polypeptide of the present invention and / or at least one different polynucleotide of the present invention. The composition may comprise at least one pharma- ceutically acceptable diluent, carrier or preservative. The composition may comprise an adjuvant. The adjuvant may be selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, viral dsRNA-based adjuvants, imidazoquinolines, and Montanide ISA adjuvants.

[0017] The present invention also provides a method for treating or preventing a disease or condition in a subject, the method comprising administering to the subject a polypeptide of the invention, a polynucleotide of the invention and / or a composition of the invention.

[0018] The present invention also provides a polypeptide of the invention, a polynucleotide of the invention, and / or a composition of the invention for use in a method of treating or preventing a disease or condition in a subject.

[0019] The present invention further provides the use of a polypeptide of the invention, a polynucleotide of the invention and / or a composition of the invention for the preparation of a medicament for treating or preventing a disease or condition in a subject.

[0020] The disease or condition may be characterized at least in part by inappropriate or excessive immunosuppressive function of ARG2. The excessive immunosuppressive function of ARG2 may be at least in part mediated by activated Treg cells expressing ARG2. The excessive immunosuppressive function of ARG2 may be at least in part mediated by cancer-associated fibroblasts expressing ARG2. The disease or condition may be cancer. The cancer may be melanoma (such as malignant metastatic melanoma), chronic myeloid leukemia (CML), or pancreatic cancer.

[0021] When the disease or condition to be treated is cancer, the polypeptide of the present invention, the polynucleotide of the present invention and / or the composition of the present invention can be used in combination with an additional cancer therapy. The additional cancer therapy can be an immune system checkpoint inhibitor. Preferably, the immune system checkpoint inhibitor is an antibody. More preferably, the immune system checkpoint inhibitor is an anti-PD1 antibody.

[0022] Thus, the present invention provides a method of treating or preventing cancer in a subject, comprising administering to a subject a polypeptide of the invention, a polynucleotide of the invention and / or a composition of the invention, which method further comprises the simultaneous or sequential administration of a further cancer therapy to the subject.

[0023] The present invention also provides a polypeptide of the invention, a polynucleotide of the invention and / or a composition of the invention for use in a method of treating or preventing cancer in a subject, the method comprising administering to the subject a polypeptide of the invention, a polynucleotide of the invention and / or a composition of the invention, the method further comprising simultaneous or sequential administration of a further cancer therapy to the subject.

[0024] The present invention further provides the use of a polypeptide of the invention, a polynucleotide of the invention and / or a composition of the invention for the preparation of a medicament for the treatment or prevention of cancer in a subject, wherein the polypeptide, polynucleotide and / or composition is administered to the subject simultaneously or sequentially with a further cancer therapy.

[0025] The present invention further provides a method for stimulating ARG2-specific T cells, comprising contacting the cells with a polypeptide of the present invention or a composition of the present invention. The cells to be contacted may be present in a sample taken from a healthy subject or a cancer patient. The cells to be contacted may be present in a tumor sample. ARG2-specific T cells are CD8 + Preferably, the ARG2-specific T cells are cytotoxic CD8 + T cells. [Brief description of the drawings]

[0026] [Figure 1] Figure 1 shows the identification of ARG2 peptides eliciting CD4+ or CD8+ T cell responses. (A, left) IFNγ ELISPOT screening of 15 different HLA-A2 responses predicted ARG2-derived peptides in five healthy HLA-A2+ donors. 3x105 cells were seeded per well. Control and peptide stimulations were performed in triplicate. Peptide-specific IFNγ secreting cells are shown as the difference in spots between cells in peptide-stimulated and control wells. (A, right) Representative examples of ELISPOT responses shown in (A). (B and C) Representative intracellular cytokine staining for secreting IFNγ and TNFα in samples from three healthy donors stimulated with control or peptide A2S14 (B) or peptide A2S05 (C). Gating strategy is given in Figure 10. HD = healthy donor. DP = double positive (TNFα+ IFNγ+). [Diagram 2]Figure 2 shows that peptide A2S05 responds in both HLA-A2+ and HLA-A2- donors, and that responses are also detectable ex vivo. (A) IFNγ ELISPOT responses to A2L2 and A2S05 in 17 healthy donors. 3.5x105 cells were plated per well. Control and peptide stimulations were performed in triplicate. Peptide-specific IFNγ secreting cells are shown as the difference in spots between cells in peptide-stimulated and control wells. Each point represents one donor, and the bars represent the mean. (B) IFNγ ELISPOT responses to A2S05 in 30 healthy donors and 13 cancer patients (2 prostate cancer patients, 11 melanoma cancer patients) with known HLA type. Each point represents one donor, and the bars represent the mean. (C) IFNγ ELISPOT responses to 2 HLA-A2+ and 2 HLA-A2- donors. HD = healthy donors. (D) HLA typing data from three healthy donors and one cancer patient (AA01, melanoma) with strong responses to A2S05 shown in (C). HLA types shared between donors are highlighted in yellow. (E, left) Ex vivo ELISPOT responses to A2S05. 9x105 cells were plated per well and control and peptide stimulations were performed in triplicate. *p ≤ 0.05 according to distribution-free resampling method. (E, right) Representative examples of ex vivo ELISPOT responses. [Diagram 3]Figure 3 shows that ARG2-specific CD8+ T cells recognize A2S05 in the context of HLA-B8. (A) ARG2-specific CD8+ T cells were expanded from three healthy donors (HD) and one cancer patient (AA01, melanoma). The specificity of the specific T cell cultures was assessed by intracellular cytokine staining for TNFα and IFNγ. CD107a was included as a marker of cytotoxicity. Bars indicate the percentage of CD8+ T cells expressing CD107a and producing IFNγ, TNFα or both (DP, double positive) in response to control or A2S05 stimulation. (B-D) To identify the HLA restriction of the ARG2-specific T cell cultures, ARG2-specific T cells from two donors (HD78 and HD93) were assessed in an IFNγ ELISPOT using cancer cell lines pre-pulsed with A2S05 peptide. The same cancer cell lines without peptide stimulation were included as controls. 3x104 ARG2-specific T cells were plated together with 1x104 cancer cells (effector:target ratio 3:1). Cell lines were either HLA-A1+ (B), HLA-C7+ (C) or HLA-B8+ (D). T cells were plated alone (-) or T cells were plated together with A2S05 (+pep) as negative and positive controls, respectively. (E) 1Cr release assay showing lysis of 5FM6 and FM6 stimulated with IFNγ for 24 h prior to assay. [Figure 4]FIG. 4 shows that ARG2-specific T cells recognize and react with stimulated regulatory T cells (Tregs). (A) IFNγ ELISPOT response of ARG2-specific T cells against in vitro activated and expanded regulatory T cells (Tregs) or effector T cells (Teffs). 5×105 T cells were plated with 5×103 target cells per well (effector:target ratio 10:1). Bars indicate mean ± SD. Within the bars, background from Tregs and Teffs has been corrected. All conditions were plated in sextuplicate. *p≦0.05 according to distribution-free resampling method. (B-C) RT-qPCR analysis of ARG2 expression (B) or FOXP3 expression (C) in Tregs and Teffs used for ELISPOT in (A). ARG2 or FOXP3 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Teff, bars indicate mean ± SD of technical triplicates. (D) RT-qPCR analysis of ARG2 expression in bulk cultures of Treg, Teff and isolated subsets of activated cells. ARG2 expression was normalized to POL2RA and expressed as relative expression (arbitrary units). Bars indicate mean ± SD of technical triplicates. [Diagram 5] Figure 5 shows ELISPOT responses to ARG2-derived peptides. IFNγ ELISPOT responses of three healthy donors to A2S15 (A), A2S14 (B) or A2S05 (C). 3x105 cells were plated per well. Controls and peptide stimulations were performed in triplicate. TNTC = not measurable number (>500). [Figure 6]Figure 6 shows the results of experiments to characterize FM6 cells. (A) Flow cytometry analysis of HLA-ABC and HLA-B8 expression in the metastatic melanoma cell line FM6. Bars indicate the percentage of HLA-ABC+ or HLA-B8+ cells from the live cell population. (B) Mean fluorescence intensity (MFI) of HLA-B8 in FM6 cells stimulated with IFNγ (100 U / ml) for 24 h and in the HLA-B8+ population of FM6 cells. (C) RT-qPCR analysis of ARG2 expression in FM6. Data are expressed as relative expression (arbitrary units) to the housekeeping gene POL2RA and bars indicate the mean ± SD of technical triplicates. [Figure 7] Figure 7 shows the ELISPOT response of A2S05-specific T cells against HLA-B8+ cell lines. (A) ARG2-specific T cells from two donors (HD81 and AA01) were assessed by IFNγ ELISPOT with the metastatic melanoma cell line FM6 prepulsed with A2S05 peptide (FM6 pep). FM6 without peptide stimulation (F or plkM6) was included as a control. 3x104 ARG2-specific T cells were plated with 1x104 cancer cells (effector:target 3:1). All conditions were set up in triplicate. Bars represent mean ± SD. TNTC = not measurable number (>500). (B) ARG2-specific T cells from HD93 were assessed by IFNγ ELISPOT as above. FM6 cells were prepulsed with an ARG1-derived peptide (FM6 pep(ARG1)) or an A2S05 peptide (FM6 pep(ARG2)). *p≦0.05 (according to a distribution-free resampling method). (C) Validation of the HLA-B8 restriction of A2S05 by IFNγ ELISPOT of four A2S05-specific T cell cultures against three additional HLA-B8+ cell lines: two metastatic melanoma cell lines (FM28 and FM82) and one AML cell line (OCI-M2). The experiment was performed as described in (A). [Figure 8]Figure 8 shows the results of sorting and characterization of regulatory T cells (Treg) and effector T cells (Teff). (A) Flow cytometry plots showing the sorting strategy of Treg and Teff in three donors. Tregs were sorted as CD25highCD127- and Teffs as CD25lowCD127+. The full gating strategy of sorting is shown in Figure 9. (B) Mean fluorescence intensity (MFI) of FOXP3-PE in Treg and Teff sorted from three donors. (C-D) RT-qPCR analysis of IL-10 expression (C) or CTLA4 expression (D) in Treg and Teff. IL-10 or CTLA4 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Teff, bars indicate the mean ± SD of technical triplicates. (E) Representative example of RT-qPCR analysis of ARG1 and ARG2 expression in Treg and Teff isolated from HD93. Data are expressed as relative expression (arbitrary units) to the housekeeping gene POL2RA, and bars indicate the mean ± SD of technical triplicates. [Figure 9] FIG. 9 shows the gating strategy for the selection of regulatory T cells (Treg) or effector T cells (Teff). (A) Gating strategy for the selection of Treg or Teff. Cells were first gated as lymphocytes>singlets>live cells>CD3+>CD4+. A fluorescence minus one (FMO) control for CD127 was used to gate for discriminating CD127+ cells from CD127- cells. Based on this, Tregs were sorted as CD25highCD127- and Teffs as CD25lowCD127+. The Treg and Teff gates aimed to select approximately 5% of CD4+ T cells. (B) CD127-FITC FMO used to gate CD127+. (C) FOXP3-PE FMO used to gate FOXP3+. (D-E) Representative examples of purity assessment of the quality of Treg (D) and Teff (E) sorting. [Figure 10]Figure 10 shows the gating strategy for intracellular cytokine staining for IFNγ and TNFα. Gating strategy for intracellular cytokine staining for IFNγ and TNFα. Cells were first gated as lymphocytes>singlets>live cells>CD3+, then as CD4+ or CD8+. CD107a expression and IFNγ and TNFα secretion were assessed for CD4+ (right) and CD8+ (left) T cells, respectively. Samples without peptide stimulation (control) were used to gate for IFNγ+, TNFα+ and CD107a+. [Figure 11-1]FIG. 11 shows that ARG2-specific T cells recognize and react with stimulated regulatory T cells (Treg). (A-C) IFNγ ELISPOT responses of ARG2-specific CD8+ T cells against in vitro activated and expanded regulatory T cells (Treg) or resting CD4+ T cells (Trest). 5×104 ARG2-specific CD8+ T cells were plated with 5×103 target cells per well (effector:target ratio 10:1). Bars indicate mean IFNγ spot counts ± SD. Within the bars, background from Treg and Trest has been corrected. All conditions were plated in sextuplicate. *p≦0.05 according to distribution-independent resampling method. (D) Flow cytometric assessment of Treg populations after 6 h coculture of activated PBMC with autologous CD8+ T cells (control T cells) or ARG2-specific T cells. Cocultures were set up at a ratio of 3:2 activated PBMC:T cells. Only activated PBMCs were used to set the gate for Tregs, similar to the gate used for sorting Tregs in the ELISPOT experiments in (C). Bars indicate the mean ± SD of triplicate technical replicates of the Treg population when co-cultured with control or ARG2-specific T cells compared to PBMC cultures alone (shown as dots). *p ≤ 0.0194 (unpaired t test). (E) RT-qPCR analysis of ARG2 expression in Tregs and Trests used for ELISPOT in (A-C). Bulk cultures of activated PBMCs from which Tregs and Trests were isolated were also included. ARG2 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Trests. Bars indicate the mean ± SD of technical triplicates. (F) Western blot analysis assessing ARG2 expression in Tregs and Trests sorted from HD93. Cell lines Set2 and UKE-1 were included as ARG2 positive and negative controls, respectively. (GH) IFNγ ELISPOT responses of ARG2-specific CD4+ T cells against in vitro activated and expanded Treg or Trest. 5×104 ARG2-specific CD4+ T cells were plated with 5×103 target cells per well (effector:target ratio 10:1). Bars indicate mean IFNγ spot counts ±SD of six technical replicates.Within bars, background from Treg and Trest has been corrected. All conditions were plated in sextuplicate. *p≦0.05 following a distribution-free resampling method. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12-1] Figure 12 shows the results of sorting and characterization of regulatory T cells (Treg) and resting CD4+ T cells (Trest). (A) Flow cytometry plots showing the sorting strategy of Treg and Trest in four donors. Treg were sorted as CD25highCD127- and Trest as CD25lowCD127+. The overall gating strategy of sorting is shown in Figure 9. (B) Representative example of RT-qPCR analysis of ARG1 and ARG2 expression in Treg and Trest isolated from HD93. Data are expressed as relative expression (arbitrary units) to the housekeeping gene POL2RA, bars show mean ± SD of technical triplicates. (C) RT-qPCR analysis of FOXP3 expression in sorted purified Treg and Trest. FOXP3 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Trest. Bars show mean ± SD of technical triplicates. (D) Representative example of RT-qPCR analysis of ARG2 expression in Treg and Trest isolated from HD93 along with autologous ARG2-specific CD8+ T cells used as effector cells in Fig. 4C. Data are expressed as relative expression (arbitrary units) to the housekeeping gene POL2RA, and bars indicate mean ± SD of technical triplicates. (E) Purity analysis of CD8+ enriched fraction. Percentage of live CD3+ cells pre-sort and post-enrichment of CD8+ MACS enrichment are shown. (F, left) Flow cytometry plots showing the sorting strategy of Treg and Trest in donor AA01. (F, right) Western blot analysis of ARG2 expression in Treg, Trest sorted from AA01. Cell lines Set2 and UKE-1 were included as ARG2 positive and negative controls, respectively. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13-1]Figure 13 shows characterization of sorted purified Tregs. (A) Mean fluorescence intensity (MFI) of FOXP3-PE after intracellular staining of sorted Tregs and Trests from three donors. (B-F) RT-qPCR analysis of IL2RA (B), TNFRSF18 (C), IKZF2 (D), CTLA4 (E), or IL10 (F) expression in Tregs and Trests. Expression of each Treg signature gene was normalized to the housekeeping gene POL2RA and is presented as fold change relative to Trests, with bars representing the mean ± SD of technical triplicates. (G) RT-qPCR analysis of PDCD1 expression in Tregs and Trests. Data are presented as relative expression (arbitrary units) relative to the housekeeping gene POL2RA, with bars representing the mean ± SD of technical triplicates. (H) In vitro Treg suppression assay. (Left) Percentage of undivided CD8+ T cells after 5 days of co-culture with sorted purified Treg (+Treg) or Trest (+Trest). Bars represent the mean of three technical triplicates (shown as dots). **p=0.0069 (unpaired t-test). (Center) Proliferation index of CD8 T cells after 5 days of co-culture with Treg or Trest. Bars represent the mean of three technical triplicates (shown as dots). **p=0.0022 (unpaired t-test). (Right) CFSE staining of CD8+ T cells co-cultured with Treg or Trest for 5 days. Dark grey bars (far right) represent the undivided population. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14-1]FIG. 14 shows the effect of an ARG2-based immunomodulatory vaccine on tumor growth, activation of ARG2-specific CD4+ and CD8+ T cells, and the proinflammatory tumor microenvironment. (A) (A) Summary of the experimental timeline. (B) Mean Pan02 tumor growth in mice receiving control (Ctrl) or ARG2-peptide immunomodulatory vaccine. Mice (n=10 per group) were tumor-inoculated and vaccinated according to the experimental outline in A. Data are presented as mean±SEM. ****p<0.0001 (2-way ANOVA test). (C) IFNγ ELISPOT on splenocytes isolated from mice receiving control vaccine (n=3) or ARG2-based vaccine (n=6) from the experiment shown in B. Briefly, 8×105 splenocytes were plated per well. Control and peptide stimulations were performed in triplicate. Peptide-specific IFNγ-secreting cells were quantified as the difference in the number of spots counted between peptide-stimulated and control wells. Each point represents one mouse, and bars represent mean±SEM. *p=0.0238 (Mann-Whitney test). (D) ARG1- and ARG2-specific IFNγ-secreting cells in splenocytes from Pan02 tumor-bearing mice receiving ARG2-based peptide vaccines were assayed by IFNγ ELISPOT. In this assay, 8×105 splenocytes were plated per well with either ARG1 or ARG2 peptide. Each point represents one mouse, and the bars represent the mean ± SEM. *p=0.0022 (Mann-Whitney test). (E) ARG2-specific IFNγ-secreting cells present in CD4+ and CD8+ sorted T cells isolated from splenocytes of Pan02 tumor-bearing mice treated with ARG2-based peptide vaccines were assayed by IFNγ ELISPOT assay. In this assay, 2.8×105 T cells were plated with 6×105 antigen-presenting cells (spleen cells from naive mice) with or without ARG2 peptide. Each point represents one sample (pooled from 2 mice) and bars represent the mean ± SEM. (F) Randomly selected tumors from Pan02-bearing mice from the experiment in B were harvested on day 31 (each group = 4–6). Tumor RNA was extracted and bulk RNAseq was performed.Differentially expressed genes (FDR<0.05 and absolute log2 fold change>0) in Pan02 tumors from ARG2-inoculated mice compared to control-inoculated mice were identified and represented in Volcano plots. n=282 upregulated genes and n=33 downregulated genes (see also FIG. 18). (G) Immune-related biological processes (gene ontology analysis) associated with significantly upregulated genes in the RNAseq data described in F (see also FIG. 19). (H-J) Bar graphs showing absolute immune infiltration scores (arbitrary units) of all immune populations (H), or immune score ratios of M1 vs. M2 macrophages (I) and CD8 T cells vs. Tregs (J). Immune population scores were generated with the ImmuCC algorithm using bulk tumor RNAseq as described in F. Each point represents one mouse and bars represent mean ± SEM. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15-1]FIG. 15 shows the effect of an immunomodulatory ARG2-based peptide vaccine in a mouse tumor model. (A) Individual tumor growth of Pan02 tumor-bearing mice shown in FIG. 6B. The mean tumor growth of each treatment group is also plotted. One mouse showed complete tumor regression (CR) and is indicated by an arrow. (B) Mean Pan02 tumor growth of mice that received the ARG2-peptide immunomodulatory vaccine or were untreated. Mice (n=8-9 per group) were tumor-inoculated and vaccinated as in the experimental outline in FIG. 6A. Data are shown as mean±SEM. **p=0.0010 (2-way ANOVA test). (C) Representative examples of the percent change in body weight compared to day 0 in Pan02 tumor-bearing mice that were untreated or received the ARG2-based immunomodulatory peptide vaccine. Mice (n=8-9 per group) were treated according to the experimental outline in FIG. 6A. (D) Protein sequence alignment ARG1 (aa162-190) and ARG2 (aa181-209). The ARG2 peptide (aa188-196) used in the study is highlighted in blue, and the corresponding sequence in ARG1 (aa169-177) is highlighted in brown. ARG1_169-177 served as a control for specific recognition in Fig. 6D. (E) C57BL / 6 mice were administered 5x105 MC38 cells and received ARG2-based or control vaccines on days 0 and 7 after tumor inoculation. On day 18, mice were sacrificed and spleens and tumors were harvested. (E, left) Peptide-specific IFNγ-secreting cells (left) identified by IFNγ ELISPOT of splenocytes from MC38 tumor-bearing mice inoculated with control vaccine (n=2) and ARG2 vaccine (n=6). 8x105 splenocytes were plated per well. Control and peptide stimulations were performed in triplicate. Peptide-specific IFNγ-secreting cells are shown as the difference in spots between cells in peptide-stimulated and control wells. Each point represents one mouse and bars represent the mean ± SEM. (E, right) Peptide-specific T cell responses in CD45+ enriched cells derived from MC38 tumors in mice treated with control vaccine (n=2) or ARG2 vaccine (n=2). 5x105 CD45+ cells were plated per well. Control and peptide stimulations were performed in triplicate. Each point represents one mouse and bars represent the mean ± SEM.(F) C57BL / 6 mice were administered 5x105 LL2 cells and vaccinated with control or ARG2-vaccine on days 0 and 5 after tumor inoculation. The study was terminated on day 12 and tumors were harvested. (F, left) Peptide-specific IFNγ-secreting cells identified by IFNγ ELISPOT in splenocytes from LL2 tumor-bearing mice inoculated with control (n=8) and ARG2 (n=8). 8x105 splenocytes were plated per well. Control and peptide stimulations were performed in triplicate. Each point represents one mouse and bars represent mean ± SEM. (F, right) Peptide-specific T cell responses in CD45+ enriched cells from LL2 tumors of mice treated with control (n=4) or ARG2 vaccine (n=3). 5x105 CD45+ cells were plated per well. Control and peptide stimulations were performed in triplicate. Each point represents one mouse and bars represent mean ± SEM. (G) C57BL / 6 mice were administered 5x105 B16-F10 cells and received control or ARG2-vaccine on days 0 and 5 after tumor inoculation. The study was terminated on day 12 and tumors were harvested. (G, left) Peptide-specific IFNγ-secreting cells identified by IFNγ ELISPOT on splenocytes from B16-F10 tumor-bearing mice inoculated with control vaccine (n=3) and ARG2 vaccine (n=3). 8x105 splenocytes were plated per well. Control and peptide stimulations were performed in triplicate. Each point represents one mouse and bars represent mean ± SEM. Each point represents one mouse and bars represent mean ± SEM. (G, right) Peptide-specific T cell responses in CD45+ enriched cells from B16-F10 tumors of mice treated with control vaccine (n=3) or ARG2 vaccine (n=3). 5x105 CD45+ cells were plated per well. Control and peptide stimulations were performed in triplicate. Each point represents one mouse and bars represent the mean ± SEM. Each point represents one mouse and bars represent the mean ± SEM. (H) Gene Ontology (GO) analysis of biological processes was performed using Gene Ontology Resource software (http: / / gebeontology.org / ) by inputting genes significantly upregulated in ARG2-treated mice.The enriched biological processes were first categorized as immune-related or non-immune-related biological processes and are expressed as a percentage of the total number of enriched biological processes (see also FIG. 19). (I) Percentage of immune-related biological processes associated with a positive antitumor response, a negative antitumor response, or an immune response to bacteria that were enriched in mice that received the ARG2-based vaccine compared to mice that received the control vaccine (see also FIG. 19). [Figure 15-2] This is a continuation of Figure 15-1. [Figure 16] Figure 16 shows immune populations calculated by the ImmuCC algorithm in Pan02 tumor-bearing mice receiving a control vaccine or an ARG2-based immunomodulatory vaccine. ImmuCC analysis of immune populations by analysis of RNAseq data from bulk tumor RNA of Pan02 tumor-bearing mice receiving a control vaccine or an ARG2-based immunomodulatory vaccine as described in Figure 6F. Box plots show absolute immune scores for CD8+ T cells (A), CD4+ T cells (B), regulatory T cells (C), natural killer cells (D), dendritic cells (E), B cells (F), macrophages (G), monocytes (H) and granulocytes (I) in Pan02 tumor-bearing mice receiving a control vaccine or an ARG2-based vaccine. Subgroups within the different immune populations are listed in Tables 5 and 6. [Figure 17-1]FIG. 17 shows the results of sorting and characterization of regulatory T cells (Treg) and resting T cells (Trest). (A) Gating strategy for sorting Treg or Trest. Cells were first gated as lymphocytes>singlets>live cells>CD3+>CD4+. Fluorescence minus one (FMO) control for CD127 was used to gate for distinguishing CD127+ cells from CD127- cells. Based on this, Tregs were sorted as CD25highCD127- and Teffs as CD25lowCD127+. Treg and Teff gates were targeted to select approximately 5% of CD4+ T cells. Treg and Teff gates were targeted to select approximately 5% of CD4+ T cells. (B) CD127-FITC FMO used to gate CD127+. (C) FOXP3-PE FMO used to gate FOXP3+. (D-E) Representative examples of purity assessment of sorting quality of Treg (D) and Teff (E). (F-G) Representative examples of FOXP3+ cells in Treg (F) and Trest (G) of HD93. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18-1] FIG. 18 shows captured data regarding gene expression information as shown in FIG. 14F. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 18-3] This is a continuation of Figure 18-2. [Figure 18-4] This is a continuation of Figure 18-3. [Figure 18-5] This is a continuation of Figure 18-4. [Figure 19-1] FIG. 19 shows supplemental data for the Gene Ontology analysis as shown in FIGS. 14G, 15H and 15I. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 19-3] Continuation of Figure 19-2. [Figure 19-4] Continuation of Figure 19-3.

[0027] A brief description of the sequence SEQ ID NOs: 1 to 15 are each amino acid sequence of a polypeptide derived from human ARG2. SEQ ID NO:16 is the amino acid sequence of the "hot spot" region of human ARG2. SEQ ID NO:17 is the amino acid sequence of the ARG2 peptide designated A2L2. SEQ ID NO: 18 is the amino acid sequence of the ARG1 peptide designated ARG1_65-73. SEQ ID NO:19 is the amino acid sequence of full-length human ARG2. SEQ ID NO: 20 is the amino acid sequence of full-length human ARG1. SEQ ID NOs:21 and 22 are the amino acid sequences of immunogenic murine ARG2-derived and murine ARG1-derived epitopes, respectively. SEQ ID NO:23 is the mouse ARG1 amino acid sequence comprising SEQ ID NO:22. SEQ ID NO:24 is the mouse ARG2 amino acid sequence comprising SEQ ID NO:21.

[0028] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods may be tailored to particular needs in the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0029] Moreover, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides," and the like.

[0030] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" encompasses short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to any naturally and / or unnaturally occurring or synthetic amino acids, including both the D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0031] The terms "patient" and "subject" are used interchangeably and typically refer to a human.

[0032] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0033] The present inventors have identified additional polypeptides derived from human ARG2 that are particularly highly immunogenic.Unexpectedly, these polypeptides are not present in the regions of human ARG2 that have been shown to be immunogenic in the past.In particular, the polypeptides of the present invention are not present in the "hotspot" regions or A2L2 regions of human ARG2.

[0034] "Immunogenic" in the present specification preferably means that the polypeptide can induce an immune response against the ARG2 protein when the ARG2 protein is present in or on a cell expressing the ARG2 protein. In other words, the polypeptide can be described as being immunogenic against ARG2. Alternatively, the polypeptide can be described as an immunogenic fragment of ARG2. The immune response is preferably a T cell response, and the polypeptide can be described as an immunogenic fragment of ARG2 that contains a T cell epitope. The immune response can be detected in at least one individual (or a sample taken from an individual) after administration of the polypeptide to the individual (or a sample taken from the individual).

[0035] A polypeptide can be identified as immunogenic using any suitable method, including in vitro methods. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics (i) to (iii): (i) capable of inducing IFN-γ producing cells in PBL populations of healthy subjects and / or cancer patients as determined by an ELISPOT assay; and / or (ii) CTLs reactive with Arginase 2 can be detected in situ in a sample of tumor tissue; and / or (iii) It is capable of inducing the in vitro development of specific T cells. Suitable methods for determining whether a polypeptide is immunogenic are also described in the Examples section below.

[0036] The polypeptide of the invention is an immunogenic fragment of human arginase 2 (ARG2; SEQ ID NO: 19) comprising or consisting of a sequence of 9 to 19 consecutive amino acids of SEQ ID NO: 19, however said fragment does not include amino acids 2 to 34 or 180 to 229 of SEQ ID NO: 19. The polypeptide may have a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids and / or the C-terminal amino acid may be replaced by the corresponding amide. The polypeptide may be isolated.

[0037] The polypeptide may comprise or consist of a human leukocyte antigen (HLA) class I restricted epitope. The polypeptide may comprise or consist of an HLA-B8 restricted epitope. The polypeptide is capable of stimulating CD8+ T cells. The CD8+ T cells may be cytotoxic T cells. The CD8+ T cells may be ARG2 specific. An exemplary polypeptide of this type comprises or consists of the amino acid sequence NLIVINPRSV (SEQ ID NO:5; A2S05). The peptide A2S05 is HLA-B8 restricted and capable of stimulating ARG2 specific CD8+ cytotoxic T cells.

[0038] The polypeptide may comprise or consist of an HLA class II restricted epitope. The polypeptide is capable of stimulating CD4+ T cells. An exemplary polypeptide of this type may comprise or consist of the amino acid sequence GLLSALDLV (SEQ ID NO: 14).

[0039] In any of the polypeptides described herein, the amino acid sequence may be modified by one, two, three, four, or five (i.e., up to five) additions, deletions, or substitutions, so long as the polypeptide with the modified sequence exhibits equivalent or increased immunogenicity against ARG2 compared to the polypeptide with the unmodified sequence. By "equivalent," it should be understood that the polypeptide with the modified sequence does not exhibit significantly reduced immunogenicity against ARG2 compared to the polypeptide with the unmodified sequence. Any comparison of immunogenicity between sequences should be performed using the same assay. Unless otherwise stated, the modifications to the polypeptide sequence are preferably conservative amino acid substitutions. In a conservative substitution, an amino acid is replaced with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid it replaces. Alternatively, in a conservative substitution, another amino acid that is aromatic or aliphatic may be introduced in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected according to the properties of the 20 major amino acids defined in Table A1 below. If the amino acids have similar polarity, this can be determined by referring to the hydropathic index for the amino acid side chains in Table A2.

[0040] [Table 1-1]

[0041] [Table 1-2]

[0042] In any of the polypeptides disclosed herein, any one or more of the following modifications may be made to improve physiochemical properties (e.g., stability), provided that the polypeptide exhibits equivalent or increased immunogenicity against ARG2 compared to a polypeptide having an unmodified sequence: a) replacement of the C-terminal amino acid with the corresponding amide (which can increase resistance to carboxypeptidases); b) replacement of the N-terminal amino acid with the corresponding acylated amino acid (which can increase resistance to aminopeptidases); c) replacement of one or more amino acids with corresponding methylated amino acids (which can improve resistance to proteolysis); d) Substitution of one or more amino acids with the corresponding amino acid in the D-configuration, which can improve proteolytic resistance.

[0043] Any of the polypeptides disclosed herein may have at least one additional moiety attached to the N- and / or C-terminus to improve solubility, stability, and / or aid in production / isolation, so long as the polypeptide exhibits equivalent or increased immunogenicity against ARG2 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequences KK, KR, or RR may be added to the N- and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0044] The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides can be directly synthesized using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry, or solution phase peptide synthesis. Alternatively, the polypeptides can be produced by transforming a cell, usually a bacterial cell, with a nucleic acid molecule or vector encoding the polypeptide.

[0045] The invention provides nucleic acid molecules and vectors encoding the polypeptides of the invention. The invention also provides host cells comprising such nucleic acids or vectors.

[0046] 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 analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0047] When the polynucleotide of the present invention is an mRNA, it can be provided as an mRNA vaccine. The mRNA vaccine can be formulated as a lipid nanoparticle composition, for example, a lipid nanoparticle composition comprising an mRNA encoding a polypeptide of the present invention and an ionizable lipid.

[0048] The polynucleotides of the invention may be provided in isolated or substantially isolated form. By substantially isolated, it is meant that there may be substantial, but not complete, isolation of the polypeptide from any surrounding medium. The polynucleotides may be mixed with carriers or diluents that do not interfere with their intended use and still be considered substantially isolated. A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo 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 translation stop codon at the 3' (carboxy) terminus. For the purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA from viruses, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0049] Polynucleotides can be synthesized according to methods well known in the art, as illustrated by examples in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the invention can be provided in the form of expression cassettes, which contain regulatory sequences operably linked to an insert sequence, thereby allowing the expression of the polypeptides of the invention in vivo. These expression cassettes are then usually provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be administered directly to the host subject. Alternatively, a vector containing the polynucleotides of the invention can be administered to the host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptides of the invention.

[0050] Thus, the present invention includes expression vectors comprising such polynucleotide sequences. Such expression vectors are constructed routinely in the art of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as polyadenylation signals, which may be necessary and positioned in the correct orientation to allow expression of the peptides of the invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0051] The present invention also includes cells that have been modified to express a polypeptide of the present invention. Such cells usually include prokaryotic cells, such as bacterial cells, e.g., E. coli. Such cells may be cultured using conventional methods to produce the polypeptide of the present invention.

[0052] The polypeptide of the present invention may be in a substantially isolated form. The polypeptide of the present invention may be mixed with a carrier, preservative, or diluent (discussed below), and / or adjuvant (also discussed below) that does not interfere with the intended use, and still be considered to be substantially isolated. The polypeptide of the present invention may also be in a substantially purified form, in which case the polypeptide of the present invention generally comprises at least 90%, for example at least 95%, 98% or 99% of the protein in the preparation.

[0053] Compositions Comprising Polypeptides In another aspect, the present invention provides compositions comprising a polypeptide of the invention and / or a polynucleotide of the invention. For example, the present invention provides compositions comprising one or more polypeptides of the invention and / or one or more polynucleotides of the invention, and optionally at least one adjuvant, pharma- ceutically acceptable carrier, preservative and / or excipient.

[0054] The composition may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polypeptides of the invention, and optionally at least one adjuvant, pharma- ceutically acceptable carrier, preservative and / or excipient.

[0055] The composition may comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight different polynucleotides of the invention, and optionally at least one adjuvant, pharma- ceutically acceptable carrier, preservative and / or excipient.

[0056] Carriers, preservatives and excipients must be "acceptable" in the sense of being compatible with the other components of the composition and not harmful to the subject to which the composition is administered. Usually, all components and the final composition are sterile and pyrogen-free. The composition may be a pharmaceutical composition. The composition may preferably include an adjuvant.

[0057] An adjuvant is any substance whose incorporation into a composition enhances or otherwise modifies the immune response elicited by the composition. Broadly defined, an adjuvant is a substance that enhances the immune response. The adjuvant may also preferably have a depot effect, in that it also provides 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 ed., 1986) pp. 61-63.

[0058] Adjuvants include AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP11687, also known as nor-MDP), and N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-ol. (1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, also known as MTP-PE), RIBI in 2% squalene / Tween-80 RTM emulsion (MPL+TDM+CWS), lipopolysaccharide and its various derivatives including lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g., poly IC and poly AU acid), Mycobacterium tuberculosis (Mycobacterium tuberculosis), The adjuvant may be selected from the group consisting of wax D from B. tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the Brucella genus, Titermax, ISCOMS, Quil A, ALUN (see US58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrix or GMDP, interleukin 1, interleukin 2, Montanide ISA-51, and QS-21. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Granulocyte-macrophage colony stimulating factor (GM-CSF) may also be used as an adjuvant.

[0059] Preferred adjuvants for use with the present invention include oil / surfactant-based adjuvants, such as Montanide adjuvant (available from Seppic, Belgium), preferably Montanide ISA-51.Other preferred adjuvants are bacterial DNA-based adjuvants, such as adjuvants containing CpG oligonucleotide sequences.Still other preferred adjuvants are viral dsRNA-based adjuvants, such as poly I:C.GM-CSF and imidazoquinoline are also examples of preferred adjuvants.

[0060] The adjuvant is most preferably a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA51 or Montanide ISA720.

[0061] It is also mentioned in Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986) pp. 61-63 that coupling to an immunogenic carrier is recommended when the antigen of interest is of low molecular weight or poorly immunogenic. Thus, the polypeptide of the present invention may be coupled to a carrier. The carrier may exist independent of the adjuvant. The function of the carrier may be to increase the molecular weight of the polypeptide fragment, for example, to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life. Furthermore, the carrier may help present the polypeptide or its fragment to T cells. Thus, in the composition, the polypeptide may be associated with a carrier as described below.

[0062] The carrier can be any suitable carrier known to those skilled in the art, for example, a protein or an antigen-presenting cell, such as a dendritic cell (DC). Carrier proteins include keyhole limpet hemocyanin, serum proteins (such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, etc.), 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 sepharose. The carrier must be physiologically acceptable and safe for humans.

[0063] If the composition contains an excipient, the excipient must be "pharmaceutical acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient thereof. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also 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 that can be administered without undue toxicity. Pharmaceutically acceptable excipients include liquids such as, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts, such as acetates, propionates, malonates, benzoates, and the like. A complete discussion of pharmaceutical acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0064] The formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemistry and techniques, all of which are readily available to those skilled in the art. Such 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 dosage form, for example, in ampoules or in multi-dose containers, optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of the composition, the active ingredient is provided in a dry (e.g., powder or granules) form, for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and can include, in addition to the active ingredient, additional ingredients, such as adjuvants, excipients, and auxiliary substances described herein. Such sterile injectable preparations may be prepared using non-toxic parenterally acceptable diluents or solvents, such as, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic saline, and fixed oils, such as synthetic mono- or diglycerides. Other compositions that are useful include compositions that contain the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. Alternatively, the active ingredient of the composition may be encapsulated, adsorbed to, or associated with particulate carriers. Suitable particulate carriers include carriers derived from polymethylmethacrylate polymers, and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368.Other microparticle systems and polymers can also be used, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0065] How to use The polypeptides, polynucleotides or compositions of the invention may be used in a method of treating or preventing a disease or condition in a subject. The polypeptides, polynucleotides or compositions of the invention may 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 said polypeptide, said polynucleotide or said composition to said subject. Administration may be of a therapeutically or prophylactically effective amount of said polypeptide, said polynucleotide or said composition to a subject in need thereof.

[0066] The disease or condition may be characterized at least in part by inappropriate or excessive immunosuppressive function of ARG2. The disease or condition may be cancer, preferably a cancer that expresses ARG2 and / or is associated with inappropriate or excessive immunosuppressive function of ARG2. The cancer may be kidney cancer, prostate cancer, breast cancer, brain cancer, pancreatic cancer, head and neck cancer, or small intestine cancer, or colorectal cancer or gastric cancer, or may be melanoma, or may be leukemia, such as acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). Preferably, the cancer is melanoma (such as malignant melanoma), CML, or pancreatic cancer. The cancer may be CML characterized by inappropriate or excessive immunosuppressive function of ARG2. The cancer may be melanoma characterized by inappropriate or excessive immunosuppressive function of ARG2. The cancer may be malignant melanoma characterized by inappropriate or excessive immunosuppressive function of ARG2. The cancer may be pancreatic cancer. The excessive immunosuppressive function of ARG2 may be mediated, at least in part, by activated Treg cells characterized by expression of ARG2. The excessive immunosuppressive function of ARG2 may be mediated, at least in part, by cancer-associated fibroblasts (CAFs) characterized by expression of ARG2. The cancer may be resistant to other cancer treatments, particularly to immune system checkpoint inhibitors such as anti-PD1 therapy.

[0067] The method may include simultaneous or sequential administration with a concomitant cancer therapy, which may be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiation therapy, immune stimulants (such as additional vaccines), gene therapy, or antibodies.

[0068] Immune system checkpoint inhibitors are particularly preferred as additional cancer therapy. Vaccine therapy against ARG2 may have a synergistic effect when combined with immune system checkpoint inhibitors. Examples of immune system checkpoints include: a) Interaction of indoleamine 2,3-dioxygenase (IDO1) with its substrates b) Interaction of PD1 with PDL1 and / or with PDL2 c) CTLA4-CD86 and / or CTLA4-CD80 interactions d) Interaction of B7-H3 and / or B7-H4 with their respective ligands e) Interaction of HVEM and BTLA f) GAL9-TIM3 interaction g) Interaction of MHC class I or II with LAG3 h) MHC class I or II interaction with KIR Inhibition of checkpoints (a), (b) and (c) is particularly preferred as an additional cancer therapy.

[0069] A checkpoint inhibitor may be any immunomodulatory agent (such as an antibody) that blocks or inhibits an immune system checkpoint, or it may be an immunotherapeutic composition that includes a component of an immune system checkpoint or an immunogenic fragment of said component, and that stimulates targeting of the checkpoint in the immune system.

[0070] The additional cancer therapy may be an antibody.

[0071] The antibodies are: abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, atinumab, atolizumab (= tocilizumab), atollimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevasimumab, Zumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, con Cizumab, crenezumab, CR6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab situxetan, epratuzumab, erlizumab, ertumaxomab, Taracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fullanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, GS6624,Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Incracumab, Indatuximab avtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Ligelizumab, Lintuzumab, Lirilumab, Roderucizumab, Lorvotuzumab mertansine, Lukatumumab, Rumiriximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimuab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab passudotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narutumumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, obinutuzumab, ocralizumab, ocrelizumab, ozlimomab , ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, plakmab, polatuzumab vedotin, ponezumab, priliximab, pritoxakis Simab, Pritumumab, PRO140, Kirizumab, Racotumomab, Ladolezumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Lorezumab, Romosozumab, Rontalizumab, Rovelizumab, Rupirizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab,Sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab (= tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab (= atlizumab), toralizumab, tositumomab , tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, dillalimumab, or zolimomab alitox.

[0072] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, eculizumab, omalizumab, canakinumab, mepolizumab, reslizumab, tocilizumab, ustec, and serovar 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 numab, briakinumab, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, trastuzumab, gemtuzumab, ozogamicin, ibritumomab, tiuxetan, tositumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab and vedotin.

[0073] Particularly preferred antibodies that can be used in the methods of the present invention include daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab and ofatumumab. Anti-PD1 antibodies such as nivolumab are also particularly preferred. Pembrolizumab is most preferred.

[0074] The concomitant cancer therapy may be selected from the group consisting of actimid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, 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.

[0075] The polypeptides of the compositions of the invention may also be used in a method of stimulating ARG2-specific T cells, such as CD4 and CD8 T cells, comprising contacting cells with said polypeptides or compositions. The method may be performed ex vivo. The method may be performed in vivo. The cells may be present in a sample, such as a tumor sample, taken from a healthy subject or a cancer patient. The cells may be present in a sample, such as a tumor sample, taken from a healthy subject or a cancer patient. The method may be a method of stimulating CD8+ T cells. The CD8+ T cells may be cytotoxic CD8+ T cells. The polypeptides used in the method of stimulating CD8+ T cells may comprise or consist of a human leukocyte antigen (HLA) class I restricted epitope, optionally an HLA-B8 restricted epitope. For example, a polypeptide that may comprise or consist of the amino acid sequence of NLIVINPRSV (SEQ ID NO: 5) may be used in a method of stimulating CD8+ T cells, in particular cytotoxic CD8+ T cells.

[0076] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples, both separately and in any combination thereof, may be material for realizing the invention in diverse forms thereof. EXAMPLES

[0077] Example 1 - Materials and Methods Donor material PBMCs from healthy donors were isolated using density gradient separation on Lymphooprep™ (Alere) and cryopreserved at -150°C in fetal bovine serum (FBS, Life Technologies) supplemented with 10% dimethylsulfide (DMSO). PBMCs from cancer patients with solid tumors were obtained from blood samples a minimum of 4 weeks after administration of therapy. PBMCs were maintained in X-Vivo (BioNordika) supplemented with 5% human serum (Sigma Aldrich). All patient protocols were approved by the Danish Capital Region Scientific Ethics Committee and were carried out in accordance with the provisions of the Declaration of Helsinki. Written informed consent was obtained from patients before study enrollment.

[0078] peptide Peptide sequences were predicted using algorithms available at www.syfpeithi.de_and cbs.dtu.dk. A short peptide list was selected to include only peptides with a score above 24 (SYFPEITHI) or a rank below 1.5 (NetMHC). All peptides were synthesized by Schaefer and were >90% pure. Peptides were dissolved in 100% DMSO to a stock concentration of 10 mM or in sterile water to a concentration of 2 mM. Peptides dissolved in water were filter sterilized before use. All peptides used in this study are shown in Table 1.

[0079] In vitro stimulation and IFNγ ELISPOT PBMCs from healthy donors or cancer patients were subjected to in vitro stimulation with ARG2-derived peptides by plating the cells with 10 μM peptide. The following day, the cells were treated with low dose IL-2 (120 U / ml). 10-14 days after peptide stimulation, the cells were used for IFNγ ELISPOT. ELISPOT plates were coated overnight with 7.5 μg / μl IFNγ capture antibody (MabTech). The following day, the plates were washed and blocked with X-vivo medium, after which 2.5-3.5 × 10 cells were plated for ELISPOT with and without restimulation with 5 μM peptide. 5 PBMCs were plated in wells in triplicate for each condition. The cells were incubated for 14-16 hours, washed, and a biotin-conjugated secondary antibody (Mabtech) was added and then incubated for 2 hours.

[0080] The cells were then washed again and then incubated with streptavidin-conjugated alkaline phosphatase (Mabtech) for 1 h. Finally, BCIP / NBT substrate (Mabtech) was added to develop the spots. The reaction was stopped with tap water. Spots corresponding to IFNγ secretion were quantified by visualization on a CTL ImmunoSpot S6 Ultimate-V analyzer using ImmunoSpot software version 5.1.

[0081] The number of peptide-specific IFNγ-secreting cells was calculated by subtracting the average number of spots in the control wells from the average number of spots in the peptide-stimulated wells. ELISPOT assays using ARG2-specific T cells (effector cells) and various immune cells or cancer cells as target cells were performed using 3 × 10 4 ~5×10 4 effector cells were then added, followed by 5 x 10 3 ~10 4The peptide seeding was performed by incubating the target cells with 20 μM peptide for 1 h followed by washing twice to remove unbound peptide. Effector cells with peptide were used as a positive control, and target cells plated without target cells served as a negative control. In ELISPOT assays with immune cells as target cells, wells containing effector cells only were also included as controls. The average number of spots from replicate wells was subtracted from the number of spots of effector cells plated with the respective target cells.

[0082] Ex vivo ELISPOT PBMCs were thawed and allowed to rest overnight. The next day, 9 × 10 5 Cells were plated at 1000 x g for 24 h. Control and peptide stimulations were performed in at least triplicate. The remainder of the protocol was performed as described above.

[0083] cell line K562, K562-A1, FM6, FM28, and FM82 cell lines were maintained in RMPI-1640 (Gibco) supplemented with 10% FBS. OCI-M2 was maintained in Iscove's MDM (Gibco) containing 20% ​​FBS. Cells were subcultured every 2–3 days. Adherent cells (FM6, FM28, and FM82) were subcultured after dissociation from flasks with 0.25% trypsin (Gibco). All cell lines were confirmed to be mycoplasma negative.

[0084] Intracellular cytokine staining PBMCs were stimulated in vitro with peptides as described above. 10-14 days after stimulation, these cells were used for intracellular cytokine staining. Cells were incubated with peptide or no peptide control and CD107a-PE (BD) for 1 h, followed by the addition of GolgiPlug (BD). After a 4-h incubation period, cells were washed and stained for extracellular markers CD3-APC / H7, CD4-FITC, and CD8-PerCP, and dead cells were stained with FVS-510. Cells were then permeabilized using Fixation / Permeabilization buffer (Invitrogen) before staining with IFNγ-APC and TNFα-BV421. Data were acquired using a BD Canto II flow cytometer and analyzed using FlowJo. The gating strategy is shown in Figure 10 and the antibodies used in this study are listed in Table 2.

[0085] [Table 2]

[0086] Flow cytometry-based analysis of HLA expression Pan-HLA (HLA-ABC) and HLA-B8 expression levels of the cell lines were analyzed by staining with HLA-ABC-FITC (BD) and HLA-B8-PE (Miltenyi Biotec), respectively. + Cells and HLA-B8 + Cells were gated. Data was acquired using a BD Canto II flow cytometer and analyzed using FlowJo. Antibodies used in this study are listed in Table 2 above.

[0087] Generation of ARG2-specific T cell cultures ARG2-specific T cell cultures were obtained by stimulating PBMCs with ARG2-S05 peptide followed by low-dose IL-2 (120 UL) the next day. On day 12 after peptide stimulation, cells were restimulated with peptide and IFNγ-secreting cells were isolated using magnetic beads. These cells were then expanded using a rapid expansion protocol including feeder cells, CD3 antibody, and anti-dose IL-2 (3000-6000 U / mL). On days 16-17, the specificity of the cultures was determined by stimulating with ARG2-S05 peptide and measuring cytokine release by ICS.

[0088] Activation, expansion, and isolation of regulatory T cells (Treg) and effector T cells (Teff) or resting T cells (Trest) PBMCs were thawed and stimulated with CD3 / CD28 Dynabeads (Gibco) according to the manufacturer's instructions in X-vivo supplemented with 10% FBS, 1% sodium pyruvate (Gibco), and 1% non-essential amino acids (Gibco). 300 U / mL IL-2 was added on days 2, 5, and 7 after bead activation. On day 8, beads were removed and cells were plated in medium without IL-2. On day 9, cells were stained with CD3-APC / H7, CD4-PerCP, CD25-Pe-Cy / 7, and dead cell stain FVS-510. Some cells were used as fluorescence minus one (FMO) control for CD127, and the remaining cells were stained with CD127-FITC. From the CD127-FITC stained cells, a portion was fixed, permeabilized, and stained for FOXP3. From the live population, Tregs were identified as CD3 + CD4 + CD25 high CD127 - and Teff or Trest were selected as CD3 + CD4 + CD25 low CD127 + Sorting was performed using BD FACSMelody™ and data were visualized using BD FACSChorus software. The gating strategy and specific sorting gates are shown in Figures 8, 9 and 17, and the antibodies used are listed in Table 2.

[0089] Total RNA extraction For RNA extraction, cells were harvested, washed with PBS, and pelleted. Cell pellets were stored at -80°C until RNA isolation. Total RNA isolation was performed using the RNEasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions and eluted in 30 μL of RNase-free water. RNA concentration was determined using a NanoDrop2000 spectrophotometer (Thermo Scientific). RNA was stored at -80°C.

[0090] cDNA synthesis and RT-qPCR cDNA was synthesized using the High Capacity cDNA Transcription Kit (Applied Biosystems) with random primers and 400-1000 ng of RNA as input. cDNA was diluted 1:2-1:4 prior to RT-qPCR analysis. RT-qPCR analysis was performed using TaqMan Gene Expression Assay on a Roche Lightcycler 480 instrument. Assays were performed in triplicate and results were analyzed as previously described (Bookout et al., Curr. Protoc. Mol. Biol. 73, 15.8.1-15.8.28 (2006)). For low-expressing samples that had no amplification during the assay, the Ct value was set at 40. A no-reverse transcriptase control was also included in the primer validation analysis. The primers used in this study are listed in Table 3 below.

[0091] [Table 3]

[0092] Preparation of cell lysates for Western blotting Set2, UKE-1 and sorted Treg and Trest cells were washed twice with sterile PBS, pelleted and stored at -80°C. Cell pellets were resuspended in ice-cold RIPA lysis buffer (Thermo Scientific) supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Scientific) at a 1:100 dilution. Cell lysates were placed under continuous stirring at 4°C for 15 min and then centrifuged at 16800×g for 15 min at 4°C. The supernatants were then transferred to new Eppendorf tubes and used for protein concentration determination using the BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol.

[0093] Western blot analysis of ARG2 expression in selected Treg and Trest A volume of cell lysate corresponding to 20 μg total protein was mixed with distilled water, Bolt™ Sample Reducing Agent (diluted 1:10) and Bolt™ LDS Sample Buffer (diluted 1:4, Invitrogen) to a total sample volume of 50 μL. Samples were incubated at 99°C for 10 min to aid denaturation and then separated on Bolt™ 4-12% Bis-Tris Plus gels (Invitrogen) at 200 V for 20 min using a PowerPac HV (BioRad) and Bolt™ MES SDS Running Buffer (Invitrogen). A BioRad Precision Plus Protein Dual Color ladder was utilized to allow quantification of protein size. Gels were transferred to an iBlot 2 PVDF Ministack (Invitrogen) and electroblotting was performed using an iBlot Gel 2 Transfer device (Invitrogen) following the manufacturer's guidelines. The membrane was cut into two pieces so that ARG2 and vinculin proteins could be stained separately. Both parts of the membrane were blocked for 1 h in TBST buffer (TBS buffer (Thermo Scientific) supplemented with 0.1% Tween-20 (Sigma Aldrich)) supplemented with 5% nonfat dry milk. Then, one half of the membrane was incubated overnight at 4°C with ARG2-specific primary antibody diluted 1:1,000 in blocking buffer, and the other half of the membrane was kept in TBST overnight. The next day, the unstained part of the membrane was incubated for 1 h with vinculin-specific antibody diluted 1:100,000 in blocking buffer. Afterwards, the membrane was washed three times for 5 min with TBST. The ARG2-stained and vinculin-stained membranes were then incubated for 1 h with anti-rabbit or anti-mouse secondary antibodies, respectively, diluted 1:2,000 in blocking buffer.After three washes, the membrane was developed with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) for 5 minutes and visualized on a Gel Doc™ XR System (BioRad) using ImageLab software (V.5.2.1). The antibodies used are listed in Table 2 above.

[0094] Cytotoxicity assay As previously described by Andersen et al. J. Immunol. 163, 3812-3818 (1999), conventional 51 Cr release assay was performed to identify ARG2-specific CD8 + The cytotoxicity of T cells was evaluated. Briefly, target cells were treated with 100 μCi of radioactive 51 After labeling with Cr for 1 hour, the cells were washed twice to remove extracellular excess 51 Cr was removed. Effector and target cells were plated at various effector:target (E:T) ratios and incubated for 4 hours. 100 μL of supernatant was then collected and 51 Cr release was determined using a 2470 Automatic γ Counter (Perkin Elmer). 51 Cr release was determined in separate wells by adding 100 μL of 10% Triton-X to target cells. Spontaneous target cell lysis was determined in other wells by incubating target cells with medium alone. Assays were set up with technical duplicates at all E:T ratios, and maximum and minimum releasing wells were set up with sextuplicate technical replicates.

[0095] In vitro Treg suppression assay Eight days after the start of stimulation, the CD8 positive fraction of activated PBMC used for Treg / Trest selection was isolated using magnetic bead separation (Miltenyi Biotec). The purity of the sorted cells was assessed by flow cytometry after staining the cells with CD3-APC / H7, CD8-FITC and FVS-510. The following day, CD8 T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) dye (Sigma Aldrich) at a concentration of 5 μM. Then, 1.5 × 10 5 0.3 x 10 CFSE-labeled CD8 T cells in a round-bottom 96-well plate 5 The cells were co-cultured with selected and purified Treg or Trest cells from 1000 ng / mL of IgG. One day after the initiation of co-culture, the cells were stimulated with human T activator anti-CD3 / CD28 Dyna beads (Gibco) at a ratio of 1:25 and 300 U / mL IL-2 was added. The proliferation status of CD8 T cells was analyzed by flow cytometry on days 0 and 5 after the initiation of co-culture. Cells were stained with CD4-PerCP to distinguish Treg / Trest cells from CFSE-labeled CD8 T cells. Triplicate technical replicates were set up for each analysis time point. Data were acquired using a BD Canto II flow cytometer and analyzed using FCS Express V.7 and FlowJo V.10. The antibodies used are listed in Table 2 above.

[0096] Treg / Trest population analysis in co-cultures of activated PBMCs and effector T cells On the 9th day after the start of stimulation, 7.5 × 10 5 Activated PBMCs alone or 5 x 10 5 of ARG2-specific CD8 T cells or 5 × 10 5ARG2-specific CD8 T cells were incubated with either 0.1% Treg or 0.1% autologous control CD8 T cells. Control CD8 T cells were isolated from activated PBMCs as described above. Cells were cultured in 48-well plates for 6 hours and then stained using the same procedure as for Treg and Trest sorting described above. In addition, a similar gating strategy was utilized based on samples containing only activated PBMCs (Figure 9). The Treg and Trest population sizes in samples containing ARG2-specific CD8 T cells or control CD8 T cells were compared to samples containing only activated PBMCs to determine the percent reduction of the two populations. Triplicate technical replicates were included for both conditions with added T cells, and sextuple technical replicates were included for the activated PBMCs alone condition.

[0097] Animal testing Animal experiments were performed in the animal facility of the Oncology Department of Heref, Copenhagen University Hospital. Experiments were approved by the Danish Ethics Committee on Experimental Animal Welfare (Dyreforsogstilsynet). C57BL / 6 female mice were bred in-house on a C57BL / 6JBomTac background. Routine care was provided by the animal caretaker at the animal facility.

[0098] Peptide vaccination Mouse ARG1 peptide (mARG1_169-177, ISAKDIVYI (SEQ ID NO: 22)) was synthesized by Schafer and dissolved in DMSO to a stock concentration of 10 mM. Mouse ARG2 peptide (mARG2_188-196, LSPPNIVYI (SEQ ID NO: 21)) was synthesized by PepScan or Schafer and dissolved in sterile water to a stock concentration of 2 mM. ARG2 peptides were filtered through a sterile 0.22 μm filter before use. All synthetic peptides were >90% pure. All peptides used in this study are listed in Table 1. ARG2 peptide (100 μg) was suspended in a volume of 50 μL of sterile water and emulsified 1:1 with Montanide ISA 51 (Seppic). The control vaccine contained 50 μL of sterile water and emulsified 1:1 with Montanide. Mice were inoculated subcutaneously at the base of the tail with 100 μL of control or peptide emulsion. In tumor studies, mice were vaccinated on days 1 and 17 (Pan02), 0 and 7 (MC38), or 0 and 5 (B16-F10 and LL2) after tumor inoculation. In the remaining studies, mice were vaccinated 1 to 3 times with a minimum of 1 week between inoculations.

[0099] Tumor Testing Pan02, MC38, B16-F10 and LL2 cell lines were thawed one week before inoculation and cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin and streptavidin (P / S) (Life Technologies).

[0100] Female C57BL / 6 mice were given 5 × 10 subcutaneous injections in the right flank area. 5 Pan02, MC38, B16-F10 or LL2 cells were inoculated into each mouse. Tumor volumes were measured with digital calipers and expressed as (length × width). 2 The mean tumor volume was calculated as 0.01% (n = 1) / 2. The study endpoint was defined as a tumor volume greater than 1000 mm3 or the presence of tumor ulceration. Investigators performing tumor measurements were blinded to treatment groups.

[0101] Mouse IFNγ ELISPOT Mice were sacrificed at the endpoint in the tumor study and 1 week after the last vaccination in the remaining studies. Spleens were harvested, disrupted through a 70 μm filter, and red blood cells were lysed with Red Blood Cell Lysis Solution (Qiagen). 5 10 splenocytes were plated onto IFNγ ELISPOT plates. IFNγ ELISPOT was performed as previously described. Tumors were harvested, cut into small pieces, and enzymatically digested in RPMI medium containing 2.1 mg / ml collagenase type 1 (Worthington), 75 μg / ml DNase I (Worthington), 5 mM CaCl2, and 1% P / S. Cells were filtered through a 70 μm cell strainer, and red blood cells were lysed as described above. CD45+ tumor-infiltrating lymphocytes (TILs) were isolated from tumor single-cell suspensions using CD45 microbeads (for MC38 and LL2 tumors) (Miltenyi Biotec) or CD45 (TIL) microbeads (for B16-F10 tumors) (Miltenyi Biotec). Isolated CD45+ cells were rested overnight. 5 × 10 per well were cultured at 10 × 10 5 CD45+ cells were plated onto IFNγ ELISPOT plates. To determine the phenotype of ARG2 responses, CD8+ and CD4+ T cells were isolated from the spleens of vaccinated mice using CD8a (Ly-2) and CD4+ (L3T4) microbeads (Miltenyi Biotec), respectively, according to the manufacturer's instructions. Isolated T cells were rested overnight. 2.8 × 10 5 6 × 10 CD8+ or CD4+ T cells from naive mice (used as antigen-presenting cells) were 5 The cells were plated onto IFNγ ELISPOT plates along with 1000 cells of splenocytes. Peptide-specific responses were reported as the difference in mean spot number between peptide-stimulated and non-stimulated wells.

[0102] Tumor RNA extraction and RNA sequencing Tumor fragments (≦30 mg) were stored in RNAlater (Invitrogen) at −80°C. Tumors were homogenized in a TissueLyser (Qiagen) and RNA was extracted using the RNEasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration was measured with a Nanodrop 2000 spectrophotometer. Isolated RNA was stored at −80°C. RNAseq was performed on tumors from 4 untreated and 6 vaccinated mice as previously described in Fjaestad et al., Oncogene 41, 1364-1375 (2022). Briefly, 500 ng of purified RNA (RIN score >7) was enriched for polyadenylated mRNA using oligo-dT magnetic beads (Illumina), followed by fragmentation and cDNA synthesis using random priming (NEBNext). cDNA was prepared for Illumina sequencing by adaptor ligation, followed by PCR indexing and size selection. The concentration of the cDNA libraries was determined by KAPA Library Quantification Kits (Roche) and sequenced using the Novaseq 6000 Illumina sequencing platform. RNAseq data are available in the GEO repository (GSE212500). Read alignment and quantification were performed as previously described by Fjaestad et al. Briefly, sequenced DNA was aligned to the GRCm39 reference genome assembly using STAR-v2.7.8, and reads were quantified using featureCounts and ENSEMBL genes and transcripts version 104. Differential gene expression was analyzed with DESeq2. Volcano plots were generated using the EnhancedVolcano package (v1.8.0) based on differential gene expression analysis. The Gene Ontology Resource (http: / / geneontology.org / ) was used to evaluate the biological processes associated with the differentially upregulated genes and then classify the most specific sub-processes.

[0103] ImmuCC We characterized and quantified 25 immune cell subtypes using the computational framework of the CIBERSORT analysis tool and the developed ImmuCC signature matrix (non-tissue specific), suitable for deconvolution of bulk RNA-Seq data from mice. The ImmuCC signature matrix used consisted of 511 genes, of which 510 were mapped from our bulk RNA-Seq data (1 gene was missing). For deconvolution of bulk RNA-Seq samples with CIBERSORT, the median of the ratio-normalized data from DEseq2 was used to create an input mixture matrix. Furthermore, the analysis included both CIBERSORT-Relative and CIBERSORT-Absolute modes. CIBERSORT-Relative represents immune cell fractions relative to total immune abundance and is therefore suitable for intra-sample comparisons, while CIBERSORT-Absolute generates a score that quantifies the abundance of each cell type, making it suitable for both intra-sample comparisons between cell types and between samples of the same cell type. CIBERSORT output was generated with 1000 sorts and the quantile normalization parameter disabled. In this study, two population schemes were defined (compact and expanded, Tables 5 and 6) to aggregate a portion of the 25 immune subpopulations. The total absolute score of the integrated subpopulations was calculated as the sum of the subpopulations. Relative fractions were recalculated based on the new total immune abundance for each scheme.

[0104] statistical analysis Statistical analysis of ELISPOT responses was performed in R studio using distribution-free resampling (DFR) as described by Moodie et al. (Methods Mol. Biol. 792, 185-196 (2012)). Descriptive statistics from proliferation assays and changes in Treg population size from Treg / Trest population analysis were statistically analyzed using unpaired t-tests. To determine statistical significance of tumor growth curves, two-way ANOVA with comparisons between multiple time points was performed. Statistical significance of differences in peptide-specific IFNγ-secreting cells and ImmuCC-related analysis between treatment groups was assessed with Mann-Whitney U test and unpaired two-tailed t-test, respectively. Statistical analysis was performed in GraphPad Prism (version 9).

[0105] Example 2 – Natural CD8 against ARG2 + T cell response The present inventors have demonstrated that ARG2-specific CD8 + T cells were examined. Using an in silico HLA prediction algorithm (available at syfpeithi.de and cbs.dtu.dk), a library of 15 short ARG2-derived peptides (9-mers and 10-mers) predicted to bind strongly to HLA-A2 was generated, as shown in Table 4 below.

[0106] [Table 4]

[0107] These 15 peptides (A2S01 to A2S15) were isolated from HLA-A2 positive (HLA-A2 + Peripheral blood mononuclear cells (PBMCs) from five healthy donors (HDs) who were confirmed to be resistant to IFNγ were screened. PBMCs were stimulated in vitro with each peptide and restimulated in an IFNγ ELISPOT assay after 12-14 days of culture.

[0108] Peptides A2S05 (SEQ ID NO:5), A2S14 (SEQ ID NO:14) and A2S15 (SEQ ID NO:15) were identified as strong candidates, each eliciting significant responses in PBMCs from three or more donors (Figure 1A). However, repeated IFNγ ELISPOT in HD did not show a significant response, thus failing to validate the response seen with A2S15 (Figure 5A). On the other hand, A2S14 elicited a strong IFNγ ELISPOT response in a validation set-up (Figure 5B). Further characterization of the response by intracellular cytokine staining (ICS) for TNFα and IFNγ revealed that CD4 + A2S05 was also verified to induce a strong IFNγ ELISPOT response (Figure 5C), which was amplified by ICS-mediated CD8 T cell responses (Figure 1B). + Thus, A2S05 was associated with CD8 T cells (Fig. 1C). + It was identified as the ARG2 peptide that induces a T cell response.

[0109] Next, 17 HDs were screened for responses to A2S05 and the previously described highly immunogenic ARG2-derived peptide A2L2 (SEQ ID NO: 17). Strong and frequent responses to A2L2 were observed. However, only a few responses were observed to A2S05 in this particular assay (Figure 2A). The HDs used in this assay were not HLA typed. It was therefore considered possible that the screened donors had HLA types that were incompatible with the peptide A2S05. Therefore, an additional 38 donors (13 cancer patients with solid tumors and 25 healthy donors) with known HLA-A2 status (positive or negative) were screened for responses to A2S05. Most donors were selected on the basis of being HLA-A2+ (n=29), but some HLA-A2 donors (n=9) were included as controls. Again, responses to A2S05 were less common, with statistically significant responses seen in only 10 of 38 donors (Figure 2B). Interestingly, responses to A2S05 were not associated with HLA-A2 + and HLA-A 2This was observed in both donors (Figure 2C), indicating that A2S05 is not HLA-A2 restricted as suggested by the in silico HLA prediction algorithm.

[0110] Complete HLA typing of the four donors who showed strong A2S05 responses was performed, and all four donors were found to have HLA-A1, HLA-B8, and HLA-C7 (Figure 2D). Importantly, some donors who showed strong in vitro responses to A2S05 also showed strong and significant ex vivo responses in PBMCs (Figure 2E).

[0111] Example 3 – ARG2-specific CD8 + Characterization of T cells ARG2-specific CD8 + To further characterize the T cells, A2S05-specific T cell cultures were established from four donors. Briefly, donor PBMCs were stimulated in vitro with A2S05. After 12 days, peptide-specific T cells were restimulated and isolated using an IFNγ capture kit. IFNγ-producing cells were expanded using a rapid expansion protocol and specificity for IFNγ and TNFα was examined by ICS on days 16-17 of expansion. High specificity CD8 + Cultures were established (Figure 3A).

[0112] Using established cultures, the HLA restriction of A2S05 was determined. IFNγ ELISPOT was performed using A2S05-specific T cells from HD78 and HD93 co-cultured with various target cell lines pulsed with A2S05 peptide. These results suggest that HLA-A1 + and HLA-C7 + There was no reactivity to cell lines (Figures 3B and 3C), but HLA-B8 + The recognition and reactivity to the metastatic melanoma cell line FM6 was demonstrated (Figure 3D). FM6 was identified as a HLA-B8 +The remaining two ARG2-specific CD8 + T cell cultures were confirmed to be reactive to FM6 (Figure 7A) and all four ARG2-specific CD8 + T cell cultures were further enriched for three HLA-B8 + The specificity of the ARG2-specific CD8 T cell cultures was confirmed by IFNγ ELISPOT assay, which showed no reactivity to a peptide with the corresponding ARG1 sequence (ARG1_65-73) (Figure 7B).

[0113] Next, ARG2-specific CD8 + To determine whether T cells could lyse FM6 cells, standard 51 Indeed, ARG2-specific CD8 T cells lysed FM6 cells in a concentration-dependent manner (Figure 3E), thus confirming the existence of ARG2-specific CD8 T cells. + The cytotoxic potential of T cells was demonstrated: stimulation with IFNγ enhanced the mean fluorescence intensity (MFI) of HLA-B8 expression (Figure 6) and led to a small increase in the lysis of FM6 cells (Figure 3E).

[0114] Therefore, the HLA-B8 restriction of the A2S05 peptide was confirmed by the metastatic melanoma cell line FM6 and three other HLA-B +The peptide was confirmed by its specific recognition against cancer cell lines and cytolytic activity. This was surprising, given that this peptide was predicted to bind to HLA-A2. Interestingly, it has been suggested that the HLA-B8 haplotype may play a protective role against melanoma, based on the finding that the frequency of HLA-B8 is significantly decreased in advanced melanoma patients compared to healthy donors (Fensterle et al., BMC Med. 4, 1-6 (2006)). Furthermore, in chronic myeloid leukemia (CML), HLA-B8 expression is associated with a reduced incidence of CML (Posthuma et al., Blood 93, 3863-3865 (1999)). Upregulation of ARG2 has also been observed in Tregs and malignant melanoma cells in metastatic melanoma (Lowe et al., JCI Insight 4, (2019) and Yu et al., J. Cell. Physiol. 235, 9997-10011 (2020)), and an immunosuppressive role of ARG2 in acute myeloid leukemia has been described (Mussai et al., Blood 122, 749-758 (2013)). Without being bound by theory, the HLA-B8 restriction of class I ARG2 peptides suggests that ARG2-specific T cells play a role in immune surveillance in melanoma and CML.

[0115] Example 4 – ARG2-specific T cells specifically recognize activated regulatory T cells In a recent study, Lowe et al. showed that activated Tregs from peripheral blood express activated effector CD4 +We found that activated Tregs showed higher ARG2 expression compared to T cells (Teff), demonstrating ARG2-dependent suppression of T cell proliferation by Tregs (Lowe et al., JCI Insight 4, (2019)). It was hypothesized that if activated Tregs expressed higher ARG2 levels than activated Teffs, they could be preferentially recognized by ARG2-specific T cells. To test this hypothesis, PBMCs were activated using CD3 / CD28 beads and IL-2 was added on days 2, 5, and 7 based on the experiments described by Lowe et al. On day 9 after activation, purified Tregs and Teffs were purified using FACS (Figures 8A and 9). The purified Tregs or Teffs were then plated as target cells for autologous ARG2-specific T cells in an IFNγ ELISPOT assay. Significantly higher responses to Tregs than to Teffs were observed in all three donors (Figure 4A), and RT-qPCR analysis showed 3-7 fold higher ARG2 expression in Tregs than in Teffs (Figure 4B), with minimal levels of ARG1 expression (Figure 8E), thus indicating ARG2-dependent recognition. The Treg phenotype was confirmed by preferential expression of Treg signature genes, including FOXP3 (Figure 4C), IL-10, and CTLA-4 (Figures 8C-D) in Tregs. Finally, ARG2 expression levels in Tregs and Teffs were compared to those in bulk cultures of activated cells from which the two subsets were isolated, revealing that the bulk cultures expressed comparable levels of ARG2 to Teffs (Figure 4D).

[0116] Thus, the ability of ARG2-specific T cells to recognize and react with Tregs was shown to highlight their immunomodulatory function. The ability of ARG2-specific T cells to target regulatory cells indicates that ARG2-specific T cells are anti-Tregs characterized by their ability to "control the regulators." The preferential expression of ARG2 in activated Tregs, compared with activated bulk cultures or activated Teffs, suggests that ARG2 induction serves as an active mechanism for the augmentation of the immunosuppressive capacity of Tregs. Thus, without being bound by theory, it can be reasonably predicted that targeting of activated Tregs by ARG2-specific T cells will have an important immunomodulatory capacity by removing the immunosuppression exerted by activated Tregs with high ARG2 expression. Many tumors are characterized by such abundance of Tregs, and therefore, targeting of activated immunosuppressive Tregs by ARG2-specific anti-Tregs may alleviate Treg-mediated immunosuppression of tumor-infiltrating T cells (TILs).

[0117] Example 5 – ARG2-specific T cells specifically recognize activated regulatory T cells In a recent study, Lowe et al. showed that activated Tregs from peripheral blood express activated effector CD4 +We found that Tregs showed higher ARG2 expression compared to T cells (Teff), demonstrating ARG2-dependent suppression of T cell proliferation by Tregs (Lowe et al., JCI Insight 4, (2019)). It was hypothesized that if activated Tregs expressed higher ARG2 levels than other T cells, they could be preferentially recognized by ARG2-specific T cells. To test this hypothesis, PBMCs were activated using CD3 / CD28 beads and IL-2 was added on days 2, 5, and 7 based on the experiments described by Lowe et al. On day 9 after activation, purified Tregs and resting T cells (Trest) were purified using FACS (Figures 12A and 13). Purified Tregs or Trests were then plated as target cells for autologous ARG2-specific T cells in an IFNγ ELISPOT assay. Significantly higher responses to Tregs than Trests were observed in all three donors (Figures 11A-C). Furthermore, co-culture of activated PBMCs with ARG2-specific T cells increased autologous CD8 + Compared to activated PBMCs co-cultured with T cells, this resulted in a significant reduction in the Treg population (Figure 11D), indicating differential cytotoxic activity of ARG2-specific T cells against Tregs. Importantly, RT-qPCR analysis showed 3-7 fold higher ARG2 expression in Tregs than Treats (Figure 11E), with minimal levels of ARG1 expression (Figure 12B), indicating ARG2-dependent recognition of Tregs. Preferential ARG2 expression at the protein level in Tregs was confirmed by Western blot analysis (Figures 11F and 12F). ARG2 expression levels in Tregs and Trests were also compared to those in bulk cultures of activated PBMCs from which these two T cell subsets were isolated, and it was found that the bulk cultures expressed ARG2 at levels comparable to Trests (Figure 11E). Furthermore, ARG2 expression in ARG2-specific T cells was comparable to that of Trests (Figure 12D). Finally, in an IFNγ ELISPOT assay, autologous ARG2-specific CD4 + Treg or Trest were plated as target cells for T cells. Interestingly, ARG2-specific CD4 +T cells also showed significantly higher responses to Treg than to Trest (Figures 11F-G).

[0118] To confirm the immunosuppressive phenotype of isolated ARG2-expressing Tregs, a portion of activated PBMCs was stained intracellularly for FOXP3. FOXP3 expression was assessed between cells sorted as Tregs and Trests, showing higher FOXP3 expression in Tregs compared to Trests (Figure 13A). This was also confirmed by RT-qPCR analysis of FOXP3 expression in Tregs and Trests (Figure 12C). To further validate the sorting and purification strategy, the expression of several other Treg signature genes was evaluated by RT-qPCR, and IL2RA (CD25), TNFRSF18 (GITR), IKZF2 (HELIOS), CTLA4, and IL10 were found to be preferentially expressed in Tregs (Figure 13B-F). Interestingly, Tregs also preferentially express PDCD1 (PD-1) over Trests (Figure 13G). Finally, the functional phenotype of sorted and purified Tregs was confirmed by proliferation assays. For this purpose, autologous CD8 + A highly pure population of T cells was isolated (Figure 12E). Anti-CD3 / CD28 and IL-2 stimulated CD8 T cells co-cultured with Trest or Tregs. + When T cell proliferation was assessed over a 5-day period, Tregs were significantly more abundant than Trests in CD8 + It was found to significantly suppress T cell proliferation (Figure 13H).

[0119] Example 6 - ARG2-specific T cells induced by peptide vaccination immunomodulate and inhibit tumor growth in a mouse model of pancreatic cancer The present inventors previously identified a highly immunogenic murine ARG2-derived epitope (mARG2_188-196, LSPPNIVYI) that induced a strong and frequent T cell response after a single vaccination. Vaccination with an immunogenic ARG2-derived peptide was shown to delay tumor growth in a mouse model of lung cancer. To confirm the antitumor effect of ARG2-derived peptide vaccination in another tumor model and to investigate the immune regulatory function of ARG2-specific T cells, a syngeneic mouse model, Pan02, was used. Pan02 is a model of pancreatic ductal adenocarcinoma (PDAC) that is suitable for studying ARG2-based vaccines because ARG2 expression has previously been shown to correlate with poor prognosis in PDAC patients, and because high Arg2 expression has been demonstrated in Pan02 tumors at tumor end-stage and at tumor endpoint.

[0120] C57BL / 6 mice were administered Pan02 cells, and on day 11, when tumors were palpable, mice with similar mean tumor volumes were assigned to different treatment groups. On days 11 and 17 after tumor inoculation, mice received either the ARG2-based vaccine or a control vaccine as previously described (Figure 14A). Interestingly, a significant inhibition of tumor growth was observed in ARG2-inoculated mice, and in fact, tumor growth stopped after day 20 (Figure 14B). Moreover, one mouse in the ARG2-inoculated group showed complete tumor regression (Figure 15A). These results were confirmed in an independent study (Figure 15B). The safety of the vaccine was also demonstrated by the absence of body weight loss in the ARG2-inoculated group (Figure 15C).

[0121] Six ARG2-inoculated mice and three control-inoculated mice were randomly selected for ELISPOT analysis 31 days after tumor inoculation. Here, strong responses were observed in all ARG2-inoculated mice, and none in the control-inoculated mice (Figure 14C). The observed immune response was specific to ARG2, since no cross-reactivity was observed against a peptide derived from the corresponding region of ARG1 (mARG1_169-177, ISAKDIVYI) despite the partial sequence similarity (Figures 14D and 15D). Next, pairs of ARG2-inoculated mice were randomly selected, and pooled splenocyte samples were prepared. From these samples, CD4 + and CD8 + T cells were isolated. In ELISPOT assays, CD4 + Fractions and CD8 + Responses were observed in both fractions (Figure 14E).

[0122] Due to the small tumor size, the amount of tumor tissue available was limited. However, homing of ARG2-specific T cells to tumors in mice vaccinated with the ARG2-based vaccine was shown in three other mouse tumor models (Figure 15E-G), suggesting that the vaccine induces tumor infiltration of ARG2-specific T cells upon vaccination. To evaluate the consequences of induction of ARG2-specific T cells on the transcriptional landscape in tumors, total tumor RNA was isolated from four mice treated with the control vaccine and six mice treated with the ARG2-based vaccine and RNAseq was performed. Differential gene expression analysis identified a total of 282 upregulated genes and 33 downregulated genes after vaccination with ARG2-peptide (Figure 14F). Biological process-based gene ontology analysis showed that among the upregulated genes in mice treated with the ARG2-based vaccine, 44% of the enriched processes were associated with tumor immunity (Figure 15H). Of these, 76% were associated with positive antitumor immune responses, 10% were associated with negative antitumor immune responses, and the remaining 14% were associated with immune responses to bacteria (Figure 15I). Among the biological processes significantly enriched in mice treated with ARG2-based vaccines, processes related to both innate and adaptive immunity were found. Enrichment was found for processes related to T cells and lymphocytes, type I interferon, cytokine regulation, angiogenesis and immune system processes, as well as calcium ion concentration, overall indicating the development of an immune response in ARG2-vaccinated mice (Figure 14G).

[0123] Following these observations, the ImmuCC algorithm was used to assess the relative composition of infiltrating immune cell types in bulk tumor samples (Tables 5 and 6). Overall, there was a clear trend toward higher mean infiltration of immune cells into the tumor microenvironment following vaccination with ARG2-based immunomodulatory vaccines (Figure 14H). Specifically, mice receiving ARG2 immunomodulatory vaccines showed increased mean absolute proportions of several immune cell types, including NK cells, and a trend toward higher numbers of macrophages and monocytes (Figure 16). Mice receiving ARG2-derived peptide vaccines had higher mean M1 / M2 macrophage and CD8 / Treg ratios (Figures 14I and J), indicating a more inflammatory tumor microenvironment and suggesting the immunomodulatory potential of ARG2-specific T cells induced upon vaccination.

[0124] [Table 5]

[0125] [Table 6]

[0126] Thus, the immune-modulatory function of ARG2-specific T cells was demonstrated in the murine Pan02 tumor model after ARG2 inoculation. Gene expression changes in Pan02 tumors of ARG2-inoculated mice indicate the induction of an antitumor immune response in the form of increased immune cell infiltration, as well as the establishment of a more inflammatory microenvironment with a higher M1 / M2 macrophage and CD8 / Treg ratio. The induction of a more immune-tolerant tumor microenvironment by ARG2 inoculation may explain the observed significant inhibition of Pan02 tumor growth. Furthermore, three other murine tumor models, namely MC38, B16-F10 and LL2, were used to validate the ability of ARG2-specific T cells to infiltrate the tumor bed. Overall, these results highlight the ability of ARG2-specific T cells to change the immune landscape in favor of antitumor immune responses.

[0127] array Start and end positions indicate positions within full length human Arginase 2 (SEQ ID NO: 19) unless otherwise noted.

[0128] TIFF2025508840000008.tif114167TIFF2025508840000009.tif114167

Claims

1. An immunogenic fragment of human arginase 2 (ARG2; SEQ ID NO: 19) comprising or consisting of a sequence of 9 to 19 consecutive amino acids of SEQ ID NO: 19, with the proviso that said fragment does not include amino acids 2 to 34 or 180 to 229 of SEQ ID NO:

19.

2. 2. The polypeptide of claim 1, comprising or consisting of a human leukocyte antigen (HLA) class I restricted epitope.

3. The polypeptide of claim 1, comprising or consisting of an HLA-B8 restricted epitope.

4. CD8 ＋ and optionally, said CD8 ＋ The polypeptide of claim 1, wherein the positive T cells are cytotoxic T cells and / or are ARG2-specific.

5. 2. The polypeptide of claim 1, comprising or consisting of the amino acid sequence: NLIVNPRSV (SEQ ID NO: 5).

6. 2. The polypeptide of claim 1, comprising or consisting of an HLA class II restricted epitope.

7. CD4 ＋ The polypeptide of claim 1, which is capable of stimulating T cells.

8. 2. The polypeptide of claim 1, comprising or consisting of the amino acid sequence: GLLSALDLV (SEQ ID NO: 14).

9. 2. The polypeptide of claim 1, having a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids and / or having the C-terminal amino acid replaced by the corresponding amide.

10. A polynucleotide encoding a polypeptide as defined in claim 1, optionally contained within a vector.

11. A composition comprising the polypeptide of claim 1 and optionally an adjuvant.

12. A composition comprising the polynucleotide of claim 10, and optionally an adjuvant.

13. a. at least one different polypeptide according to claim 1, at least one different polynucleotide according to claim 10; and / or at least one pharmaceutically acceptable diluent, carrier or preservative; and / or b. An adjuvant selected from the group consisting of a bacterial DNA-based adjuvant, an oil / surfactant-based adjuvant, a viral dsRNA-based adjuvant, an imidazoquinoline, and a Montanide ISA adjuvant.

13. The composition of claim 11 or 12, comprising:

14. A polypeptide described in claim 1, a polynucleotide described in claim 10 and / or a composition described in claim 11 or 12 for use in a method for treating or preventing a disease or condition in a subject, the method comprising administering the composition to a subject.

15. a. the disease or condition is characterized, at least in part, by inappropriate or excessive immunosuppressive function of ARG2, and optionally, the excessive immunosuppressive function of ARG2 is mediated, at least in part, by activated Treg cells that express ARG2 and / or cancer-associated fibroblasts (CAFs) that express ARG2; and / or b. the disease or condition is cancer, and optionally the cancer is melanoma (such as malignant metastatic melanoma), chronic myeloid leukemia (CML), or pancreatic cancer; A polypeptide, polynucleotide or composition according to claim 14.

16. 15. The polypeptide, polynucleotide or composition of claim 14, wherein the disease or condition is cancer, and optionally the method further comprises the simultaneous or sequential administration of an additional cancer therapy to the subject, optionally wherein the additional cancer therapy is an immune system checkpoint inhibitor, preferably an antibody, more preferably an anti-PD1 antibody.

17. 13. A method for stimulating ARG2-specific T cells, comprising contacting said cells with a polypeptide of claim 1 or a composition of claim 11 or 12, and optionally a. the cell is present in a sample taken from a healthy subject or a cancer patient, optionally in a tumor sample; and / or b. The ARG-2-specific T cells are CD8+ positive T cells, preferably cytotoxic CD8+ T cells; method.

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