Arginase 2 vaccine

Abstract

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

Description

Technical Field

[0001] The present invention relates to a novel polypeptide derived from arginase 2 (ARG2), a polynucleotide encoding the polypeptide, and a composition comprising the polypeptide or polynucleotide. The present invention also relates to the use of the polypeptide, polynucleotide and composition. Background Art

[0002] Arginase is an enzyme that catalyzes a reaction that converts the amino acid L-arginine into L-ornithine and urea. This depletes the microenvironment of arginine and leads to the inhibition of tumor-specific cytotoxic T cell responses. Increased arginase activity has been detected in cancer cells from patients with breast, lung, colon or prostate cancer. Both in vitro and in vivo experiments have shown that mouse macrophages transfected with the rat arginase gene promote the proliferation of co-cultured tumor cells. In addition, macrophage induction of arginase expression has been shown to increase tumor vascularization through polyamine synthesis. Results from a mouse lung cancer model showed the presence of a tumor-associated mature myeloid cell subpopulation that expresses high levels of arginase. These tumor-associated myeloid cells consume extracellular L-arginine, thereby inhibiting the antigen-specific proliferation of tumor infiltrating lymphocytes (TILs). Injection of an arginase inhibitor blocked the growth of lung cancer in mice. This suggests that induction of arginase expression in tumor cells and tumor-associated myeloid cells may inhibit antitumor immune responses by negatively affecting TILs, thereby promoting tumor growth.

[0003] Myeloid-derived suppressor cells (MDSCs) inhibit the activation, proliferation, and cytotoxicity of effector T cells and natural killer cells, and induce Treg differentiation and expansion. Both cancer cells and MDSCs can suppress T cells by controlling L-arginine metabolism through nitric oxide synthase (NOS) and arginase. Many tumors show increased expression of arginase and inducible nitric oxide synthase (iNOS), leading to arginine depletion from the tumor microenvironment. Multiple studies have highlighted the importance of this altered tumor arginine metabolism in suppressing tumor-specific T cell responses, and recent studies have shown that acute myeloid leukemia (AML) blasts exhibit arginase-dependent inhibition of T cell proliferation and hematopoietic stem cells. In addition, arginase and iNOS inhibitors reduce the suppressive activity of AML.

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

[0005] The inventors have previously identified a 50 amino acid region of arginase 1 (ARG1) and arginase 2 (ARG2) that is a "hotspot" 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 its derived peptides are described in WO 2018 / 065563.

[0006] The inventors have also found that a specific subset of polypeptides derived from the "hotspot" region of ARG1 is particularly effective in stimulating an immune response. 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). WO 2020 / 064744 describes this subset of polypeptides.

[0007] The inventors further discovered that peptides from a completely different region of human ARG2 are particularly effective in stimulating an immune response. This region spans the C-terminus of the human ARG2 transit peptide (position 22 of SEQ ID NO: 19). WO 2020 / 099582 describes these peptides, including an immunogenic peptide named A2L2, corresponding to positions 2-34 of human ARG2 (SEQ ID NO: 16).

[0008] The present inventors have now identified further immunogenic polypeptides from ARG2. Unexpectedly, the polypeptides of the present invention are not located in the "hotspot" region (i.e. amino acids 180-229 of human ARG2) or the A2L2 region (i.e. amino acids 2-34 of ARG2). The polypeptides of the present invention may advantageously be used to stimulate CD8 + or CD4 + T cells, including cytotoxic CD8 T cells that can lyse cancer cells (e.g., melanoma cells) + T cells.

[0009] Of note, all previously observed immunogenic responses to immunogenic ARG2 peptides were CD4 + T cell responses. However, CD4 + and CD8 +Simultaneous activation of epitopes can result in a powerful synergistic protective effect against tumors. + T cells (e.g. cytotoxic CD8 + The polypeptides of the present invention are particularly advantageous for targeting T cells).

[0010] It has also been demonstrated that 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. Therefore, the polypeptides of the present invention have the utility of regulating the immunosuppressive tumor microenvironment.

[0011] The present invention provides a polypeptide, which is an immunogenic fragment of human arginase 2 (ARG2; SEQ ID NO: 19), which comprises or consists of a sequence of 9 to 19 consecutive amino acids of SEQ ID NO: 19, provided that the fragment does not comprise any of the following sequences:

[0012] - amino acids 2 to 34 of SEQ ID NO: 19;

[0013] - amino acids 180-229 of SEQ ID NO: 19;

[0014] - amino acids 129 to 141 of SEQ ID NO: 19;

[0015] - amino acids 144 to 156 of SEQ ID NO: 19; or

[0016] - amino acids 237-249 of SEQ ID NO: 19.

[0017] 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 be capable of stimulating CD8 + T cells. The CD8 + The positive T cells can be cytotoxic T cells. + Positive 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 following amino acid sequence: NLIVNPRSV (SEQ ID NO: 5).

[0018] The polypeptide may comprise or consist of an HLA class II restricted epitope. The polypeptide may be capable of stimulating CD4 + T cells. The polypeptide may comprise or consist of the following amino acid sequence: GLLSALDLV (SEQ ID NO: 14).

[0019] 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 substituted by the corresponding amide.

[0020] The present invention also provides a polynucleotide encoding the polypeptide of the present invention. The polynucleotide may be contained in a vector.

[0021] The present invention also 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 pharmaceutically acceptable diluent, carrier or preservative. The composition may comprise an adjuvant. The adjuvant may be 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.

[0022] The present invention also provides a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a polypeptide of the present invention, a polynucleotide of the present invention and / or a composition of the present invention.

[0023] The present invention also provides a method for using the polypeptide of the present invention, the polynucleotide of the present invention and / or the composition of the present invention in treating or preventing a disease or disorder in a subject.

[0024] The present invention further provides use of a polypeptide of the present invention, a polynucleotide of the present invention and / or a composition of the present invention in the preparation of a medicament for treating or preventing a disease or disorder in a subject.

[0025] The disease or condition may be characterized, at least in part, by an inappropriate or excessive immunosuppressive function of ARG2. The excessive immunosuppressive function of ARG2 may be mediated, at least in part, by activated Treg cells expressing ARG2. The excessive immunosuppressive function of ARG2 may be mediated, at least in part, by cancer-associated fibroblasts expressing ARG2. The disease or condition may be cancer. The cancer may be melanoma (e.g., malignant metastatic melanoma), chronic myeloid leukemia (CML), or pancreatic cancer.

[0026] In the case where the disease or condition to be treated is cancer, the polypeptide of the invention, the polynucleotide of the invention and / or the composition of the 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.

[0027] The present invention therefore provides a method of treating or preventing cancer in a subject, the method comprising administering to the subject a polypeptide of the present invention, a polynucleotide of the present invention and / or a composition of the present invention, wherein the method further comprises administering to the subject an additional cancer therapy simultaneously or sequentially.

[0028] The present invention also provides a method for using the polypeptide of the present invention, the polynucleotide of the present invention and / or the composition of the present invention to treat or prevent cancer in a subject, the method comprising administering the polypeptide of the present invention, the polynucleotide of the present invention and / or the composition of the present invention to the subject, wherein the method further comprises administering an additional cancer therapy to the subject simultaneously or sequentially.

[0029] The present invention further provides the use of a polypeptide of the present invention, a polynucleotide of the present invention and / or a composition of the present invention in the preparation of a medicament for treating or preventing cancer in a subject, wherein the polypeptide, polynucleotide and / or composition is administered to the subject simultaneously or sequentially with an additional cancer therapy.

[0030] The present invention further provides a method for stimulating ARG2-specific T cells, the method 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. The ARG2-specific T cells may be CD8 + Preferably, ARG2-specific T cells are cytotoxic CD8 + T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shows that CD4 + or CD8 + Identification of T cell responses to ARG2 peptides. (A, left) IFNγ ELISPOT screening of five healthy HLA-A2 + Responses to 15 different HLA-A2 predicted ARG2-derived peptides from donors. 3x10 5 cells. The control and peptide stimulation groups were repeated three times. Peptide-specific IFNγ-secreting cells are represented by the difference in spots between cells in peptide-stimulated wells and control wells. (A, right) Representative examples of ELISPOT responses are shown in (A). (B and C) Representative intracellular cytokine staining of IFNγ and TNFα secretion in samples from three healthy donors stimulated with control or peptide A2S14 (B) or peptide A2S05 (C). The gating strategy is shown in Fig.10 HD = healthy donor. DP = double positive (TNFα + IFNγ + ).

[0032] Figure 2 Peptide A2S05 was shown to be expressed in HLA-A2 + and HLA-A2 - 17 healthy donors to A2L2 and A2S05. 3.5x10 5 cells. Control and peptide stimulation were repeated three times. Peptide-specific IFNγ-secreting cells are represented by the difference in spots between cells in peptide-stimulated wells and control wells. Each dot represents one donor and the bar represents the mean. (B) IFNγ ELISPOT response to A2S05 in 30 healthy donors and 13 cancer patients with known HLA types (two prostate cancer patients, 11 melanoma cancer patients). Each dot represents one donor and the bar represents the mean. (C) IFNγ ELISPOT response to two HLA-A2 + and two HLA-A2 - Donor responses. HD = healthy donor. (D) HLA-type data from three healthy donors and one cancer patient (AA01, melanoma) with a strong response to A2S05 as shown in (C). HLA types shared between donors are highlighted in yellow. (E, left) In vitro ELISPOT responses to A2S05. 9x10 5 Cells, controls and peptide stimulations were performed in 3-6 replicates. *p≤0.05 (according to the no-distribution resampling rule) (E, right) Representative examples of in vitro ELISPOT reactions.

[0033] Figure 3 Display of ARG2-specific CD8 + T cells recognize A2S05 in an HLA-B8 background. (A) ARG2-specific CD8 T cells were expanded from three healthy donors (HD) and one cancer patient (AA01, melanoma). + T cells. The specificity of a particular T cell culture was assessed by intracellular cytokine staining for TNFα and IFNγ. CD107a was included as a marker for cytotoxicity. The bar graph shows CD8 + Percentage of T cells. (BD) To determine the HLA restriction of ARG2-specific T cell cultures, ARG2-specific T cells from two donors (HD78 and HD93) were evaluated in IFNγ ELISPOT with cancer cell lines pre-pulsed with A2S05 peptide. The same cell lines without peptide stimulation served as controls. 3x10 4 ARG2-specific T cells and 1x104 Cancer cells were inoculated together (the ratio of effector cells to target cells was 3 to 1). The cell line was HLA-A1 + (B), HLA-C7 + (C) or HLA-B8 + (D). T cells inoculated alone (-) or together with A2S05 (+pep) were used as negative and positive controls, respectively. (E) 51 Cr-release assay showed cleavage of FM6 as well as cleavage of FM6 stimulated with IFNγ for 24 h prior to the assay.

[0034] Figure 4 ARG2-specific T cells recognize and respond to stimulated regulatory T cells (Treg). (A) IFNγ ELISPOT response of ARG2-specific T cells to ex vivo activated and expanded regulatory T cells (Treg) or effector T cells (Teff). 5x10 5 T cells and 5x10 3 target cells (ratio of effector cells to target cells was 10:1). Bar graphs show mean ± standard deviation (SD). Treg and Teff background has been corrected in the bar graphs. All conditions were repeated six times. *p≤0.05 (according to the distribution-free resampling rule). (BC) RT-qPCR analysis of ARG2 expression (B) or FOXP3 expression (C) in Treg and Teff used for ELISPOT in (A). ARG2 or FOXP3 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Teff, where the bar graph shows the mean ± SD of technical triplicates. (D) RT-qPCR analysis of ARG2 expression in bulk cultures of Treg, Teff, and activated cells isolated from subsets. ARG2 expression was normalized to POL2RA and presented as relative expression (arbitrary units). Bar graphs show the mean ± SD of technical triplicates.

[0035] Figure 5 ELISPOT responses to ARG2-derived peptides are shown. IFNγ ELISPOT responses to A2S15 (A), A2S14 (B) or A2S05 (C) from three healthy donors. 3x10 5 cells. Control and peptide stimulation were performed in triplicate. TNTC = too many to count (>500).

[0036] Figure 6Results of experiments characterizing FM6 cells are shown. (A) Flow cytometric analysis of HLA-ABC and HLA-B8 expression in the metastatic melanoma cell line FM6. The bar graph represents the expression of HLA-ABC in the viable cell population. + or HLA-B8 + (B) HLA-B8 in FM6 cells and FM6 cells stimulated with IFNγ (100 U / ml) for 24 hours. + Mean fluorescence intensity (MFI) of HLA-B8 in the population. (C) RT-qPCR analysis of ARG2 expression in FM6. Data are presented as relative expression (arbitrary units) relative to the housekeeping gene POL2RA, with bar graphs showing mean ± SD of technical triplicates.

[0037] Figure 7 A2S05-specific T cells showed that they were sensitive to HLA-B8 + ELISPOT responses of cell lines. (A) ARG2-specific T cells from two donors (HD81 and AA01) were evaluated by IFNγ ELISPOT with the metastatic melanoma cell line FM6 (FM6 pep) that had been pre-pulsed with A2S05 peptide. FM6 without peptide stimulation (FM6) was used as a control. 3x10 4 ARG2-specific T cells and 1x10 4 Cancer cells were inoculated together (effector to target ratio of 3:1). All conditions were set up in triplicate. Bar graphs represent mean ± SD. TNTC = too many to count (>500) (B) ARG2-specific T cells from HD93 were evaluated in IFNγ ELISPOT as described above. FM6 cells were pre-pulsed with ARG1-derived peptide (FM6 pep(ARG1)) or A2S05 peptide (FM6 pep(ARG2)). *p≤0.05 (according to the no-distribution resampling rule). (C) IFNy ELISPOT was used to validate the expression of ARG2 in three additional HLA-B8 + Cell lines (two metastatic melanoma cell lines (FM28 and FM82) and one AML cell line (OCI-M2) HLA-B8 restriction of A2S05 in four A2S05-specific T cell cultures. The experiments were performed as described in (A).

[0038] Figure 8 Results of sorting and characterization of regulatory T cells (Treg) and effector T cells (Teff) are shown. (A) Flow cytometry plots showing the sorting strategy for Treg and Teff from three donors. Treg were sorted for CD25 高 CD127 - , while Teff is sorted as CD25低 CD127 + . Fig. 9 The complete gating strategy for sorting is shown. (B) Mean fluorescence intensity (MFI) of FOXP3-PE in sorted Tregs and Teffs from three donors. (CD) RT-qPCR analysis of IL-10 expression (C) or CTLA4 expression (D) in Tregs and Teffs. IL-10 or CTLA4 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Teff, where the bar graphs show the mean ± SD of technical triplicates. (E) Representative examples of RT-qPCR analysis of ARG1 and ARG2 expression in Tregs and Teffs isolated from HD93. Data are expressed as relative expression of the housekeeping gene POL2RA (arbitrary units), where the bar graphs show the mean ± SD of technical triplicates.

[0039] Fig. 9 Gating strategies for sorting regulatory T cells (Treg) or effector T cells (Teff) are shown. (A) Gating strategies for sorting Treg or Teff. Cells were initially gated as lymphocytes > single cells > live cells > CD3 + >CD4 + The fluorescence minus one (FMO) control of CD127 was used to set the gating to distinguish CD127 + Cells and CD127 - Based on this, Tregs are classified as CD25 高 CD127 - , while Teff is classified as CD25 低 CD127 + Place Treg and Teff gates to target approximately 5% of CD4 + T cells are sorted. (B) For CD127 + CD127-FITC FMO gating setup. (C) Gated CD127-FITC FMO gating setup for FOXP3 + Gated FOXP3-PE FMO. (DE) Representative examples of purity assessment of sorting quality of Treg (D) and Teff (E).

[0040] Fig.10 Gating strategy for intracellular cytokine staining of IFNγ and TNFα is shown. Gating strategy for intracellular cytokine staining of IFNγ and TNFα. Cells were initially sorted by lymphocytes > monocytes > live cells > CD3 + Gate, then press CD4 + or CD8 + Gating was performed. CD4 +(right) and CD8 + (Left) CD107a expression and IFNγ and TNFα secretion by T cells. Samples without peptide stimulation (control) were used to secrete IFNγ + TNFα + and CD107a + Set up the gating.

[0041] Fig.11 ARG2-specific T cells are shown to recognize and respond to stimulated regulatory T cells (Treg). (AC) ARG2-specific CD8 + T cells activated and expanded ex vivo regulatory T cells (Treg) or resting CD4 + IFNγ ELISPOT reaction of T cells (Trest). 5x10 4 ARG2-specific CD8 + T cells and 5x10 3 target cells (effector to target ratio of 10:1). Bar graphs show mean IFNγ spot counts ± SD. Bar graphs are corrected for background of Tregs and Trest. All conditions were repeated six times. *p ≤ 0.05 (according to the no-distribution resampling rule). (D) Activated PBMCs were mixed with autologous CD8 + After 6 hours of co-culture with T cells (control T cells) or ARG2-specific T cells, the Treg population was evaluated by flow cytometry. The ratio of activated PBMC to T cells in the co-culture was 3:2. Activated PBMCs were used alone to set the gate for Treg, which is similar to the gate used to sort Treg in the ELISPOT experiment in (C). The bar graph shows the average reduction in the Treg population when co-cultured with control T cells or ARG2-specific T cells compared to PBMCs inoculated alone ± the SD of three technical replicates (represented by points). *p≤0.0194 (unpaired t-test). (E) RT-qPCR analysis of ARG2 expression in Treg and Trest used in ELISPOT in (AC). Also included are bulk cultures of activated PBMCs from which Treg and Trest were isolated. ARG2 expression is standardized to the housekeeping gene POL2RA and is expressed as a fold change relative to Trest. The bar graph shows the mean ± SD of technical triplicates. (F) Western Blot analysis evaluating ARG2 expression in Treg and Trest sorted from HD93. Cell lines Set2 and UKE-1 were included as ARG2 positive and negative controls. (GH) ARG2-specific CD4 + IFNγ ELISPOT response of T cells to ex vivo activated and expanded Treg or Trest. 5x104 ARG2-specific CD4 + T cells and 5x10 3 target cells (effector to target ratio 10:1). Bar graphs show mean IFNγ spot counts ± SD for six technical replicates. Bar graphs are corrected for Treg and Trest background. All conditions were repeated six times. *p ≤ 0.05 (by the no-distribution resampling rule).

[0042] Fig.12 Regulatory T cells (Treg) and resting CD4 + T cell (Trest) sorting and characterization results. (A) Flow cytometry images showing the sorting strategy for Treg and Trest in four donors. Treg were sorted for CD25 高 CD127 - , while Trest was sorted as CD25 低 CD127 + The complete gating strategy used for sorting is shown in Fig. 9 As shown. (B) Representative examples of RT-qPCR analysis of ARG1 and ARG2 expression in Treg and Trest isolated from HD93. Data are expressed as relative expression of the housekeeping gene POL2RA (arbitrary units), where the bar graph represents the mean ± SD of technical triplicates. (C) RT-qPCR analysis of FOXP3 expression in sorted and purified Treg and Trest. FOXP3 expression was normalized to the housekeeping gene POL2RA and expressed as fold change relative to Trest. The bar graph shows the mean ± SD of technical triplicates. (D) Treg and Trest isolated from HD93 and Figure 4 Autologous ARG2-specific CD8 + Representative example of RT-qPCR analysis of ARG2 expression in T cells. Data are presented as relative expression (arbitrary units) relative to the housekeeping gene POL2RA, and the bar graph represents the mean ± SD of technical triplicates. (E) CD8 + Purity analysis of enriched fractions. CD8 + Live CD3 before and after MACS enrichment (pre-sorting) + (F, left) Flow cytometry images showing the sorting strategy for Treg and Trest in donor AA01. (F, right) Western Blot analysis of ARG2 expression in sorted Treg and Trest from AA01. Cell lines Set2 and UKE-1 were included as ARG2 positive and negative controls, respectively.

[0043] Fig.13 Characterization of sorted and purified Tregs is shown. (A) Mean fluorescence intensity (MFI) of FOXP3-PE in sorted Tregs and Trest from three donors after intracellular staining. (BF) RT-qPCR analysis of the expression of IL2RA (B), TNFRSF18 (C), IKZF2 (D), CTLA4 (E), or IL10 (F) in Treg and Trest. The expression of each Treg signature gene was normalized to the housekeeping gene POL2RA and expressed as the fold change relative to Trest, where the bar graph shows the mean ± SD of technical triplicates. (G) RT-qPCR analysis of PDCD1 expression in Treg and Trest. Data are expressed as relative expression of the housekeeping gene POL2RA (arbitrary units), and the bar graph shows the mean ± SD of technical triplicates. (H) Ex vivo Treg suppression assay. (Left) Undivided CD8 + Percentage of T cells. The bar graph represents the mean of three technical replicates (represented by dots). **p=0.0069 (unpaired t-test). (Middle) Proliferation index of CD8 T cells after 5 days of co-culture with Treg or Trest. The bar graph shows the mean of three technical replicates (represented by dots). **p=0.0022 (unpaired t-test). (Right) CD8 + CFSE staining of T cells. The dark grey bar (far right) represents the non-dividing population.

[0044] Fig.14 The ARG2-based immunomodulatory vaccine showed an effect on tumor growth, ARG2-specific CD4 + and CD8 + Activation of T cells and the impact of the proinflammatory tumor microenvironment. (A) Overview of the experimental timeline. (B) Average Pan02 tumor growth in mice receiving control (Ctrl) or ARG2 peptide immunomodulatory vaccine. Mice (n=10 per group) were vaccinated and vaccinated according to the experimental overview in A. Data are expressed as mean ± SEM. ****p<0.0001 (two-way ANOVA test). (C) IFNγ ELISPOT was performed on spleen cells isolated from mice receiving control (n=3) or ARG2-based (n=6) vaccines from the study shown in B. Briefly, 8x10 5splenocytes were inoculated into each well. Control and peptide stimulation were performed in triplicate. Peptide-specific IFNγ-secreting cells were quantified as the difference in the number of spot counts between peptide-stimulated and control wells. Each dot represents one mouse and the bar graph represents the mean ± SEM. *p = 0.0238 (Mann Whitney test). (D) ARG1- and ARG2-specific IFNγ-secreting cells in splenocytes of Pan02 tumor-bearing mice that received an ARG2-based peptide vaccine were determined using IFNγ ELISPOT. In this assay, 8x10 5 Each well was inoculated with either ARG1 or ARG2 peptide. Each dot represents one mouse and the bar graph represents the mean ± SEM. *p = 0.0022 (Mann Whitney test). (E) CD4+ Tgp ... + and CD8 + The presence of ARG2-specific IFNγ-secreting cells in sorted T cells was measured. In this assay, 2.8x10 5 T cells and 6x10 5 (Ag-presenting cells (splenocytes from naive mice) with or without ARG2 peptide. Each dot represents one sample (pooled from two mice) and the bar graph represents the mean ± SEM. (F) Randomly selected tumors from Pan02 tumor-bearing mice in the experiment in B were harvested on day 31 (n = 4-6 per group). Tumor RNA was extracted and bulk RNAseq was performed. Differentially expressed genes in Pan02 tumors from ARG2-vaccinated mice compared to control-vaccinated mice (FDR < 0.05 and absolute log2 fold change > 0) were identified and presented in a volcano plot. n = 282 upregulated genes and n = 33 downregulated genes (see also Figure 18). (G) Immune-related biological processes (gene ontology analysis) associated with genes significantly upregulated in the RNAseq data described in F (see also Figure 19). (H, J) Bar graphs show absolute immune infiltration scores (arbitrary units) for all immune populations (H) or immune score ratios for M1 to M2 macrophages (I) and CD8 T cells to Tregs (J). Immune population scores were generated using the ImmuCC algorithm using bulk tumor RNAseq as described in F. Each point represents a single mouse and bars represent mean ± SEM.

[0045] Fig.15 The immunomodulatory effect of ARG2-based peptide vaccine in a murine tumor model is shown. (A) Figure 6B shows the individual tumor growth of Pan02 tumor-bearing mice. The average tumor growth of each treatment group is also plotted. One mouse showed complete tumor regression (CR), as indicated by the arrow. (B) Average Pan02 tumor growth of mice that received the ARG2-based peptide immunomodulatory vaccine or were untreated. Mice (n=8-9 per group) were vaccinated and vaccinated, which were Figure 6 The experimental overview is similar to that in A. Data are presented as mean ± SEM. **p = 0.0010 (two-way ANOVA). (C) Representative examples of percent body weight change compared to day 0 in Pan02 tumor-bearing mice that were untreated or received an ARG2-based immunomodulatory peptide vaccine. Figure 6 Experimental overview in A. Mice were treated (n=8-9 per group). (D) Protein sequence alignment of 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. Figure 6 D served as a control for specific recognition. (E) 5x10 5 C57BL / 6 were challenged with MC38 cells and received ARG2-based vaccines or control vaccines on days 0 and 7 after tumor inoculation. On day 18, mice were sacrificed and spleens and tumors were recovered. (E, left) Peptide-specific IFNγ-secreting cells identified by IFNγ ELISPOT on splenocytes from control (n=2) and ARG2-vaccinated (n=6) MC38 tumor-bearing mice (left). 8x10 5 splenocytes. Control and peptide stimulation were performed in triplicate. Peptide-specific IFNγ-secreting cells are represented as the difference in spots between splenocytes in peptide-stimulated and control wells. Each dot represents one mouse, and the bar graph represents the mean ± SEM. (E, right) CD45 in MC38 tumors of mice treated with control (n = 2) or ARG2 (n = 2) vaccines + Peptide-specific T cell responses in enriched cells. 5x10 5 CD45 + Cells. Control and peptide stimulation were performed in triplicate. Each point represents one mouse, and the bar graph represents the mean ± SEM. (F) C57BL / 6 mice were treated with 5x10 5 LL2 cells were challenged and received control or ARG2 vaccine on days 0 and 5 after tumor inoculation. The study was terminated on day 12 and spleens and tumors were recovered. (F, left) Peptide-specific IFNγ-secreting cells identified by IFNγ ELISPOT on spleen cells from control (n=8) and ARG2 (n=8) vaccinated mice bearing LL2 tumors. 8x105 splenocytes. Control and peptide stimulation were performed in triplicate. Each dot represents one mouse and the bars represent mean ± SEM. (F, right) CD45 in LL2 tumors of mice treated with control (n = 4) or ARG2 (n = 3) vaccines + Peptide-specific T cell responses in enriched cells. 5x10 5 CD45 + Cells. Control and peptide stimulation were performed in triplicate. Each point represents one mouse, and the bar graph represents the mean ± SEM. (G) C57BL / 6 mice were treated with 5x10 5 B16-F10 cells were challenged and received control or ARG2 vaccine on days 0 and 5 after tumor inoculation. The study was terminated on day 12 and spleens and tumors were recovered. (G, left) Peptide-specific IFNγ-secreting cells identified by IFNγ ELISPOT on spleen cells from control (n=3) vaccinated and ARG2 (n=3) vaccinated mice bearing B16-F10 tumors. 8x10 5 splenocytes. Control and peptide stimulation were performed in triplicate. Each dot represents one mouse and the bar graph represents the mean ± SEM. (G, right) CD45 in B16-F10 tumors of mice treated with control (n = 3) or ARG2 (n = 3) vaccines + Peptide-specific T cell responses in enriched cells. 5x10 5 CD45 + cells. Control and peptide stimulation were performed in triplicate. Each dot represents one mouse and the bar graphs represent the mean ± SEM. (H) Gene ontology (GO) analysis of biological processes was performed using the Gene Ontology Resource software (http: / / geneontology.org / ) with genes significantly upregulated in ARG2-treated mice as input. Enriched biological processes were initially classified as immune- or non-immune-related biological processes and expressed as a percentage of the total number of enriched biological processes (see also Figure 19). (I) The percentage of immune-related biological processes associated with positive anti-tumor responses, negative anti-tumor responses, or immune responses to enriched bacteria in mice receiving the ARG2-based vaccine compared to mice receiving the control vaccine (see also Figure 19).

[0046] Fig.16 The immune population calculated by the ImmuCC algorithm in Pan02 tumor-bearing mice receiving a control vaccine or an ARG2-based immune modulatory vaccine is shown. Figure 6F, ImmuCC analysis of immune populations by analyzing RNAseq data of bulk tumor RNA from Pan02 tumor-bearing mice that received control vaccine or ARG2-based immune modulatory vaccine. Box plots show CD8 + T cells (A), CD4 + Absolute immune scores for T cells (B), regulatory T cells (C), natural killer cells (D), dendritic cells (E), B cells (F), macrophages (G), monocytes (H) and granulocytes (I). Subgroups within the different immune populations are listed in Tables 5 and 6.

[0047] Fig.17 Results of sorting and characterization of regulatory T cells (Treg) and resting T cells (Trest) are shown. (A) Gating strategy used to sort Treg or Trest. Cells were initially gated as lymphocytes > single cells > live cells > CD3 + >CD4 + The fluorescence minus one (FMO) control of CD127 was used to set the gate to distinguish CD127 + Based on this, Tregs were sorted as CD25 高 CD127 - , while Trest was sorted as CD25 低 CD127 + . Treg and Teff gates were set to target approximately 5% of CD4 + T cells are sorted. (B) For CD127 + CD127-FITC FMO gating setup. (C) Gated CD127-FITC FMO gating setup for FOXP3 + FOXP3-PE FMO with gate settings. (DE) Representative examples of purity assessment of Treg (D) and Teff (E) sorting quality. (FG) FOXP3 in Treg (F) and Teff (E) in HD93 + Representative examples of cells.

[0048] Figure 18 shows Fig.14 Supplementary data regarding gene expression information is shown in F.

[0049] Figure 19 shows the Fig.14 G. Fig.15 H and Fig.15 Supplementary data for the gene ontology analysis shown in I.

[0050] A brief description of the sequence

[0051] SEQ ID NOs: 1-15 are the amino acid sequences of polypeptides derived from human ARG2, respectively.

[0052] SEQ ID NO: 16 is the amino acid sequence of the "hotspot" region of human ARG2.

[0053] SEQ ID NO: 17 is the amino acid sequence of an ARG2 peptide designated A2L2.

[0054] SEQ ID NO: 18 is the amino acid sequence of the ARG1 peptide designated ARG1_65-73.

[0055] SEQ ID NO: 19 is the amino acid sequence of full-length human ARG2.

[0056] SEQ ID NO: 20 is the amino acid sequence of full-length human ARG1.

[0057] SEQ ID NOs: 21 and 22 are the amino acid sequences of immunogenic murine ARG2-derived and murine ARG1-derived epitopes, respectively.

[0058] SEQ ID NO:23 is the amino acid sequence of murine ARG1 comprising SEQ ID NO:22.

[0059] SEQ ID NO:24 is the amino acid sequence of murine ARG2 comprising SEQ ID NO:21. DETAILED DESCRIPTION

[0060] It should be understood that different applications of the disclosed products and methods can be customized according to specific needs in the art. It should also be understood that the terminology used herein is only used to describe specific embodiments of the present invention and is not intended to be limiting.

[0061] Furthermore, 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 "a plurality of polypeptides" and the like.

[0062] As used herein, "polypeptide" refers in its broadest sense to a compound of two or more subunit amino acids, amino acid analogs or other peptide mimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0063] The terms "patient" and "subject" are used interchangeably and generally refer to a human.

[0064] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0065] The present inventors have identified additional polypeptides from human ARG2 that are particularly immunogenic. Unexpectedly, these polypeptides are not located within regions of human ARG2 that have previously been shown to be immunogenic. Specifically, the polypeptides of the present invention are not located within the "hotspot" region or the A2L2 region of human ARG2.

[0066] "Immunogenicity" herein refers to the ability of a polypeptide to elicit an immune response to an ARG2 protein, preferably when the protein is present in or on a cell expressing the ARG2 protein. In other words, the polypeptide may be described as being immunogenic to ARG2. The polypeptide may alternatively be described as an immunogenic fragment of ARG2. The immune response is preferably a T cell response, and thus the polypeptide may be described as an immunogenic fragment of ARG2 comprising a T cell epitope. Upon administration of the polypeptide to the individual (or the sample), an immune response may be detected in at least one individual (or a sample collected from the individual).

[0067] Any suitable method (including in vitro methods) can be used to identify whether a polypeptide is immunogenic. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics:

[0068] (i) it is capable of inducing IFN-γ producing cells in PBL populations of healthy subjects and / or cancer patients as determined by ELISPOT, and / or

[0069] (ii) it is capable of in situ detection in tumor tissue samples of CTLs reactive with ARG2; and / or

[0070] (iii) It is able to induce the growth of specific T cells in vitro.

[0071] The Examples section below also describes methods useful for determining whether a polypeptide is immunogenic.

[0072] The polypeptide of the present invention is an immunogenic fragment of human arginase 2 (ARG2; SEQ ID NO: 19), which comprises or consists of a sequence of 9-19 consecutive amino acids of SEQ ID NO: 19, provided that the fragment does not comprise amino acids 2-34 or 180-229 of SEQ ID NO: 19. The maximum length of the polypeptide may be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids and / or wherein the C-terminal amino acid is substituted by the corresponding amide. The polypeptide may be isolated.

[0073] 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 + The T cell can be a cytotoxic T cell. + T cells may be ARG2 specific. An exemplary polypeptide of this type comprises or consists of the following amino acid sequence: NLIVNPRSV (SEQ ID NO: 5; A2S05). Peptide A2S05 is restricted by HLA-B8 and is capable of stimulating ARG2-specific CD8 + Cytotoxic T cells.

[0074] 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 following amino acid sequence: GLLSALDLV (SEQ ID NO: 14).

[0075] In any polypeptide 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, provided that the polypeptide with the modified sequence exhibits the same or increased immunogenicity to ARG2 compared to the polypeptide with the unmodified sequence. With respect to "same", it is understood that the polypeptide with the modified sequence does not exhibit a significantly reduced immunogenicity to ARG2 compared to the polypeptide with the unmodified sequence. Any comparison of immunogenicity between sequences is performed using the same detection method. Unless otherwise specified, modifications to the polypeptide sequence are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids 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 to the amino acid it replaces. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 main amino acids defined in Table A1 below. When amino acids have similar polarity, the hydrophilicity ranking of the amino acid side chains in Table A2 can be referenced for determination.

[0076] Table Al-Chemical properties of amino acids

[0077] Ala(A) Aliphatic, hydrophobic, neutral Met(M) Hydrophobic, neutral Cys(C) Polar, hydrophobic, neutral Asn(N) Polar, hydrophilic, neutral Asp(D) Polar, hydrophilic, charged (-) Pro(P) Hydrophobic, neutral Glu(E) Polar, hydrophilic, charged (-) Gln(Q) Polar, hydrophilic, neutral Phe(F) Aromatic, hydrophobic, neutral Arg(R) Polar, hydrophilic, charged (+) Gly(G) Aliphatic, neutral Ser(S) Polar, hydrophilic, neutral His(H) Aromatic, polar, hydrophilic, charged (+) Thr(T) Polar, hydrophilic, neutral Ile(I) Aliphatic, hydrophobic, neutral Val(V) Aliphatic, hydrophobic, neutral Lys(K) Polar, hydrophilic, charged (+) Trp(W) Aromatic, hydrophobic, neutral Leu(L) Aliphatic, hydrophobic, neutral Tyr(Y) Aromatic, polar, hydrophobic

[0078] Table A2 - Hydrophilicity rating

[0079]

[0080]

[0081] In any polypeptide disclosed herein, any one or more of the following modifications may be made to improve physicochemical properties (e.g. stability), provided that the polypeptide exhibits the same or increased immunogenicity to ARG2 compared to a polypeptide having an unmodified sequence:

[0082] a) Replacement of the C-terminal amino acid with the corresponding amide (may increase resistance to carboxypeptidases);

[0083] b) replacing the N-terminal amino acid with the corresponding acylated amino acid (which may increase resistance to aminopeptidases);

[0084] c) replacing one or more amino acids with the corresponding methylated amino acids (which may improve resistance to proteolysis);

[0085] d) Substitution of one or more amino acids with the corresponding amino acids in the D-configuration (may improve resistance to proteolysis).

[0086] Any polypeptide disclosed herein may be linked to at least one additional moiety at the N- and / or C-terminus to improve solubility, stability and / or aid in manufacturing / isolation, provided that the polypeptide exhibits the same or enhanced immunogenicity to ARG2 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequence KK, KR or RR may be added to the N- and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0087] The polypeptides disclosed herein can be produced by any suitable method. 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 by solution phase peptide synthesis. Alternatively, the polypeptides can be produced by transforming cells (usually bacterial cells) with nucleic acid molecules or vectors encoding the polypeptides.

[0088] The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the present invention. The present invention also provides a host cell comprising such nucleic acid or vector.

[0089] 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.

[0090] When the polynucleotide of the present invention is mRNA, it can be provided as an mRNA vaccine. The mRNA vaccine can be formulated as a lipid nanoparticle composition, such as a lipid nanoparticle composition comprising an mRNA encoding a polypeptide of the present invention and an ionizable lipid.

[0091] The polynucleotides of the present invention can be provided in an isolated or substantially isolated form. With regard to substantially isolated, it means that the polypeptide can be substantially (but not completely) isolated from any surrounding culture medium. The polynucleotide can be mixed with a carrier or diluent that does not interfere with its intended use, and is still considered to be substantially isolated. The nucleic acid sequence of a "coding" selected polypeptide is a nucleic acid molecule that is transcribed (with respect to DNA) and translated (with respect to mRNA) into a polypeptide in vivo when placed under the control of an appropriate regulatory sequence, such as in an expression vector. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) terminal and the translation termination codon at the 3' (carboxyl) terminal. For purposes of the present invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence can be located at the 3' end of the coding sequence.

[0092] Polynucleotides can be synthesized according to methods known in the art, such as described in Sambrook et al. (1989, Molecular Cloning-a Laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the present invention can be provided in the form of an expression cassette, which includes a control sequence operably linked to an insertion sequence, thereby allowing the polypeptide of the present invention to be expressed in vivo. In turn, these expression cassettes are usually provided in a vector (e.g., a plasmid or a recombinant viral vector). These expression cassettes can be directly administered to a host subject. Alternatively, a vector comprising a polynucleotide of the present invention can be administered to a host subject. Preferably, a genetic vector is used to prepare and / or administer the polynucleotide. A suitable vector can be any vector that can carry sufficient genetic information and allow expression of the polypeptide of the present invention.

[0093] Therefore, the present invention includes expression vectors containing such polynucleotide sequences. Such expression vectors are routinely constructed in the field of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as polyadenylation signals that may be required and that are positioned in the correct orientation to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, we refer to Sambrook et al. (1989, Molecular Cloning-a Laboratory manual; Cold Spring Harbor Press).

[0094] The present invention also includes cells that have been modified to express the polypeptides of the present invention. Such cells generally include prokaryotic cells, such as bacterial cells, such as Escherichia coli. Such cells can be cultured using conventional methods to produce the polypeptides of the present invention.

[0095] The polypeptide of the present invention may be in substantially isolated form. It may be mixed with carriers, preservatives or diluents (as described below) and / or adjuvants (also described below) that do not interfere with the intended use and still be considered substantially isolated. It may also be in substantially purified form, in which case it typically accounts for at least 90%, such as at least 95%, 98% or 99% of the protein in the preparation.

[0096] Compositions comprising polypeptides or polynucleotides

[0097] In another aspect, the present invention provides a composition comprising a polypeptide of the present invention and / or a polynucleotide of the present invention. For example, the present invention provides a composition comprising one or more polypeptides of the present invention and / or one or more polynucleotides of the present invention, and optionally at least one adjuvant, a pharmaceutically acceptable carrier, a preservative and / or an excipient.

[0098] 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 present invention, and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0099] 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 present invention and optionally at least one adjuvant, pharmaceutically acceptable carrier, preservative and / or excipient.

[0100] The carrier, preservative and excipient must be "acceptable", i.e., compatible with the other ingredients of the composition and not harmful to the subject to which the composition is administered. Typically, all ingredients and the final composition are sterile and non-pyrogenic. The composition may be a pharmaceutical composition. The composition may preferably contain an adjuvant.

[0101] An adjuvant is any substance that, when mixed into a composition, enhances or alters the immune response elicited by the composition. An adjuvant, in a broad sense, is a substance that promotes an immune response. Adjuvants may also preferably have a reservoir effect, as they also result in a slow and sustained release of the active agent from the site of administration. Goding discusses adjuvants generally in Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63.

[0102] The adjuvant may be selected from the group consisting of AlK(SO4)2, AlNa(SO4)2, AINH4(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 (CGP 11687, also known as nor-MDP (nor-MDP)), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), RIBI (MPL+TDM+CWS) in 2% squalene / Tween-80.RTM. emulsion, lipopolysaccharide and its various derivatives (including lipid A), Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (e.g., polyIC and polyAU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, immunostimulating complexes (ISCOMS), Quil A, ALUN (see US 58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, heat shock protein (HSP) derivatives, lipopolysaccharide (LPS) derivatives, synthetic peptide matrix or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21. Various saponin extracts have also been proposed to be used as adjuvants in immunogenic compositions. Granulocyte-macrophage colony stimulating factor (GM-CSF) can also be used as an adjuvant.

[0103] Preferred adjuvants for use in the present invention include oil / surfactant based adjuvants, such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA based adjuvants, such as adjuvants comprising CpG oligonucleotide sequences. Yet other preferred adjuvants are viral dsRNA based adjuvants, such as poly I:C. GM-CSF and imidazoquinoline are also examples of preferred adjuvants.

[0104] The adjuvant is most preferably Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

[0105] Goding also pointed out in "Monoclonal Antibodies: Principles and Practice" (2nd edition, 1986) pages 61-63 that when the molecular weight of the antigen of interest is low or the immunogenicity is poor, it is recommended to couple it to an immunogenic carrier. Therefore, the polypeptide of the present invention can be coupled to a carrier. The carrier can exist independently of the adjuvant. The role of the carrier can be, for example, to increase the molecular weight of the polypeptide fragment to increase activity or immunogenicity, impart stability, increase biological activity or increase serum half-life. In addition, the carrier can help present the polypeptide or its fragment to T cells. Therefore, in the composition, the polypeptide can be associated with a carrier as described below.

[0106] The carrier can be any suitable carrier known to those skilled in the art, such as 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 or hormones (such as insulin) or palmitic acid. Alternatively, the carrier protein can be a tetanus toxoid or a diphtheria toxoid. Alternatively, the carrier can be a dextran, such as sepharose. The carrier must be physiologically acceptable to humans and safe.

[0107] If the composition contains an excipient, the excipient must be "pharmaceutically acceptable", that is, compatible with the other ingredients of the composition and harmless to the recipient. Auxiliary substances may be present in the excipient, such as wetting agents or emulsifiers, pH buffer substances, etc. These excipients and auxiliary substances are generally agents that do not induce an immune response in the individual receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts may also be included, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991) discusses pharmaceutically acceptable excipients, carriers and auxiliary substances in depth.

[0108] Suitable compositions can be formulated using standard pharmaceutical formulation chemistry and methods, 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 push injection or continuous administration. Injectable compositions can be prepared, packaged or sold in unit dose forms, such as ampoules or multi-dose containers optionally containing preservatives. Compositions include, but are not limited to suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable preparations. In one embodiment of the composition, the active ingredient is provided in a dry form (e.g., powder or granules) so that it can be reconstituted with a suitable solvent (e.g., sterile pyrogen-free water) before administering the reconstructed 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 prepared according to known techniques and, in addition to the active ingredient, may also contain additional ingredients, such as adjuvants, excipients, and auxiliary substances as described herein. Such sterile injectable preparations can be prepared using non-toxic parenteral acceptable diluents or solvents (e.g., water or 1,3-butanediol). Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, fixed oils (e.g., synthetic monoglycerides or diglycerides, etc.). Other useful compositions include compositions containing active ingredients in microcrystalline form, liposome formulations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation may include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts. Alternatively, the active ingredient of the composition may be encapsulated, adsorbed on, or combined with a particle carrier. Suitable particle carriers include carriers derived from polymethyl methacrylate polymers, and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolides). See, for example, Jeffery et al. (1993) Pharm. Res. 10: 362-368. Other particle systems and polymers may also be used, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and the like, as well as conjugates of these molecules.

[0109] How to use

[0110] The polypeptide, polynucleotide or composition of the present invention can be used in a method for treating or preventing a disease or condition in a subject. The polypeptide, polynucleotide or composition of the present invention can be used to manufacture a medicament for a method for treating or preventing a disease or condition in a subject. The method comprises administering the polypeptide, polynucleotide or composition to the subject. A therapeutically or prophylactically effective amount of the polypeptide, polynucleotide or composition can be administered to a subject in need.

[0111] The disease or condition may be characterized, at least in part, by an inappropriate or excessive immunosuppressive function of ARG2. The disease or condition may be a cancer, preferably a cancer expressing ARG2 and / or associated with an inappropriate or excessive immunosuppressive function of ARG2. The cancer may be renal 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 a melanoma, or may be a leukemia, such as acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML). Preferably, the cancer is a melanoma (eg, a malignant melanoma), CML, or pancreatic cancer. The cancer may be CML, characterized by an inappropriate or excessive immunosuppressive function of ARG2. The cancer may be a melanoma, characterized by an inappropriate or excessive immunosuppressive function of ARG2. The cancer may be a malignant melanoma, characterized by an 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 ARG2 expression. The excessive immunosuppressive function of ARG2 may be mediated, at least in part, by cancer-associated fibroblasts (CAFs) characterized by ARG2 expression. Cancers may be resistant to other cancer therapies, and in particular may be resistant to immune system checkpoint inhibitors such as anti-PD1 therapy.

[0112] The method may include administering an additional cancer therapy simultaneously or sequentially. The additional cancer therapy may be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiotherapy, immunostimulatory substances, gene therapy or antibodies.

[0113] Immune system checkpoint inhibitors are preferred as additional cancer therapy. Vaccination against ARG2 combined with immune system checkpoint inhibitors may have a synergistic effect. Examples of immune system checkpoints include:

[0114] a) Interactions between indoleamine 2,3-dioxygenase (IDO1) and its substrates;

[0115] b) Interactions between PD1 and PDL1 and / or PD1 and PDL2;

[0116] c) Interaction between CTLA4 and CD86 and / or CTLA4 and CD80;

[0117] d) interactions between B7-H3 and / or B7-H4 and their respective ligands;

[0118] e) Interaction between HVEM and BTLA;

[0119] f) Interaction between GAL9 and TIM3;

[0120] g) interaction between MHC class I or class II and LAG3; and

[0121] h) Interaction between MHC class I or class II and KIR.

[0122] Inhibition of checkpoints (a), (b) and (c) is particularly preferred as an additional cancer therapy. A checkpoint inhibitor can be any immunomodulator (e.g., an antibody) that blocks or inhibits an immune system checkpoint, or it can be an immunotherapeutic composition comprising a component of an immune system checkpoint or an immunogenic fragment of said component that stimulates the immune system to target the checkpoint.

[0123] Additional cancer therapies may be antibodies.

[0124] The antibody can be Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab, pegol), ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (= tocilizumab), Atollizumab Atorolimucil, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine), Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumabmertansine, Cantuzumab ravtansine, Caplacizumab, Capromabpendetide), Carlumab, Catumaxomab, CC49, Cedelizumab, Certolizumab Pegol, Cetuximab, Ch.14.18, Citatuzumabbogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan), Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox), Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecoromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegolpegol), Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FB TA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab Ozomicin ozogamicin), Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximabravtansine), Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab ), Lirilumab, Lodelcizumab, Lorvotuzumabmertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox), Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenoxinestafenatox、Narnatumab、Natalizumab、Nebacumab、Necitumumab、Nerelimomab、Nesvacumab、Nimotuzumab、Nivolumab、Nofetumomab merpentan), Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumabmonatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibacon Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin vedotin), Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140. Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab mab), Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab Pandide pendetide), Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomabaritox), Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (= tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab ilizumab) (= atlizumab), toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuviruzumab Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesinkumab esencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, or zolimomab aritox.

[0125] Preferably, the antibody comprises Natalizumab, Vedolizumab, Belimumab, Atacicept, Alefacept, Otelixizumab, Teplizumab, Rituximab, Ofatumumab, Ocrelizumab, Epratuzumab, Alemtuzumab, Abasi Abatacept, Eculizumab, Omalizumab, Canakinumab, Meplizumab, Reslizumab, Tocilizumab, Ustekinumab, Briakinumab, Etanercept, Inlfliximab, Adalimumab, Certolizumab pegol), Golimumab, Trastuzumab, Gemtuzumab, Ozogamicin, Ibritumomab, Tiuxetan, Tostitumomab, Cetuximab, Bevacizumab, Panitumumab, Denosumab, Ipilimumab, Brentuximab, and Vedotin.

[0126] Particularly preferably, antibodies that can be used in the method of the present invention include: daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab and ofatumumab. Anti-PD1 antibodies such as nivolumab and pembrolizumab are also particularly preferred. Pembrolizumab is most preferred.

[0127] The additional cancer therapy can be selected from Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Dauno-rubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Etoposide ... Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide, Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine.

[0128] The polypeptide or composition of the present invention can also be used to stimulate ARG2-specific T cells (e.g., CD4 + and CD8 + T cells) comprising contacting the cells with the polypeptide or composition. The method may be performed ex vivo. The method may be performed in vitro. The cells may be present in a sample collected from a healthy subject or a cancer patient, such as a tumor sample. The method may be to stimulate CD8 + T cell approach. CD8 + T cells can be cytotoxic CD8 +In this type of stimulation (simulating) CD8 + The polypeptide 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 comprising or consisting of the amino acid sequence of NLIVNPRSV (SEQ ID NO: 5) may be used to stimulate CD8 + T cells, especially cytotoxic CD8 + T cell approach.

[0129] The present invention is further illustrated by the following examples, but these examples should not be construed as limiting the scope of protection. The features disclosed in the above description and the following examples can be used alone or in any combination to form various forms of material for implementing the present invention.

[0130] Example

[0131] Example 1 - Materials and Methods

[0132] Donor material

[0133] Using Lymphoprep TM Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by density gradient separation on ELISA (Alere) and cryopreserved at -150°C in fetal bovine serum (FBS, Life Technologies) supplemented with 10% dimethyl sulfide (DMSO). PBMCs from patients with solid cancer tumors were derived from blood samples collected at least four weeks after receiving any therapy. PBMCs were stored in X-Vivo (BioNordika) supplemented with 5% human serum (Sigma Aldrich). All patient protocols were approved by the Scientific Ethics Committee for the Capital Region of Denmark and were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from the patients before entering the study.

[0134] Peptides

[0135] Use www.syfpeithi.de and cbs.dtu.dk (www.syfpeithi.de The peptide sequences were predicted by the algorithm obtained from SYFPEITHI and cbs.dtu.dk. The selected short peptide list only includes peptides with a score higher than 24 (SYFPEITHI) or a grade lower than 1.5 (NetMHC). All peptides were Synthesized with a purity higher than 90%. 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 sterile filtered before use. All peptides used in this study are listed in Table 1.

[0136] Ex vivo stimulation and IFNγ ELISPOT

[0137] PBMCs from healthy donors or cancer patients were stimulated ex vivo with ARG2-derived peptides by inoculating 10 μM peptides into the cells. The next day, cells were treated with low-dose IL-2 (120 U / ml). 10-14 days after peptide stimulation, cells were used for IFNγ ELISPOT. ELISPOT plates were coated overnight with 7.5 μg / μl IFNγ capture antibody (MabTech). The next day, plates were washed and blocked in X-vivo medium, followed by inoculation of 2.5-3.5x10 5 PBMCs were used for ELISPOT with or without restimulation with 5 μM peptide. A minimum of three replicate wells were set up depending on the condition. Cells were incubated for 14-16 hours, then washed and biotin-conjugated secondary antibodies (Mabtech) were added and then incubated for 2 hours.

[0138] Next, the cells were washed again and then incubated for 1 hour with alkaline phosphatase (Mabtech) conjugated with streptavidin. Finally, the spots were developed by adding BCIP / NBT substrate (Mabtech). The reaction was stopped with tap water. The spots corresponding to IFNγ secretion were quantified by visualization using ImmunoSpot software version 5.1 on a CTL ImmunoSpot S6 Ultimate-V analyzer.

[0139] Peptide-specific IFNγ-secreting cells were calculated by subtracting the mean spot counts in control wells from the mean spot counts in peptide-stimulated wells. 4 -5x10 4 Effector cells were added to the ELISPOT wells, followed by 5x10 3 -10 4 Target cells were used to establish ELISPOT assays with ARG2-specific T cells (effector cells) and various immune cells or cancer cells as target cells. Peptide pulsing was performed by incubating target cells with 20 μM peptide for 1 hour, followed by two washes to remove unbound peptide. Effector cells with added peptide served as positive controls, and target cells without added target cells served as negative controls. In ELISPOT assays with immune cells as target cells, wells containing only effector cells were also included as controls. The average spot counts of duplicate wells were subtracted from the spot counts of effector cells seeded with the corresponding target cells.

[0140] In vitro ELISPOT

[0141] Thaw PBMC and place overnight. The next day, 9x10 5 Cells were seeded into each well. Control and peptide stimulation were performed at least three times. The rest of the protocol was performed as described above.

[0142] Cell lines

[0143] 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 MDM (Gibco) with 20% FBS. Cells were passaged every 2-3 days. Adherent cells (FM6, FM28 and FM82) were passaged after being separated from the culture flask using 0.25% trypsin (Gibco). All cell lines were confirmed to be mycoplasma negative.

[0144] Intracellular cytokine staining

[0145] PBMCs were stimulated ex vivo with peptides as described above. Cells were stained for intracellular cytokines 10-14 days after simulation. Cells were incubated with peptides or no peptide controls and CD107a-PE (BD) for 1 hour before the addition of GolgiPlug (BD). After a 4-hour incubation period, cells were washed and stained with extracellular markers CD3-APC / H7, CD4-FITC, and CD8-PerCP, and dead cells were stained with FVS-510. Next, cells were permeabilized using fixation / permeabilization buffer (Invitrogen) and then stained with IFNγ-APC and TNFα-BV421. Data were acquired using a BD Canto II flow cytometer and analyzed using FlowJo. The gating strategy was as follows. Fig.10 The antibodies used in this study are shown in Table 2 below.

[0146] Table 2 - List of antibodies used in this study

[0147]

[0148]

[0149] *Dead cell marker is fixable viability stain 510 and therefore not an antibody. N / A = Not Applicable

[0150] HLA expression analysis based on flow cytometry

[0151] Pan-HLA (HLA-ABC) and HLA-B8 expression levels in cell lines were analyzed using HLA-ABC-FITC (BD) and HLA-B8-PE (Miltenyi Biotec), respectively. + and HLA-B8 + Cells were gated. Data were acquired using a BD Canto II flow cytometer and analyzed using FlowJo. The antibodies used in this study are listed in Table 2 above.

[0152] Generation of ARG2-specific T cell cultures

[0153] ARG2-specific T cell cultures were obtained by stimulating PBMCs with ARG2-S05 peptide and then with low-dose IL-2 (120 U / mL) the next day. Twelve days after peptide stimulation, cells were stimulated again with peptide and IFNγ-secreting cells were isolated using magnetic beads. Next, these cells were expanded using a rapid expansion protocol, including feeder cells, CD3 antibodies, and high-dose IL-2 (3000-6000 U / mL). On days 16-17, culture specificity was determined by stimulation with ARG2-S05 peptide and measuring cytokine release by ICS.

[0154] Activation, expansion and isolation of regulatory T cells (Treg) and effector T cells (Teff) or resting T cells (Trest)

[0155] PBMCs were thawed and stimulated with CD3 / CD28 Dynabeads (Gibco) in X-vivo supplemented with 10% FBS, 1% sodium pyruvate (Gibco), and 1% non-essential amino acids (Gibco) according to the manufacturer's instructions. 300 U / mL of IL-2 was added on days 2, 5, and 7 after bead activation. On day 8, the beads were removed and the cells were plated in medium without IL-2. On day 9, the cells were stained with CD3-APC / H7, CD4-PerCP, CD25-Pe-Cy / 7, and the dead cell stain FVS-510. An aliquot of cells was used as a fluorescence minus one (FMO) control for CD127, and the remaining cells were stained with CD127-FITC. From the CD127-FITC-stained cells, an aliquot of cells was fixed, permeabilized, and stained for FOXP3. From the live population, Tregs were sorted as CD3 + CD4 + CD25 高 CD127 - , while Teffs or Trest were sorted as CD3 + CD4 + CD25低 CD127 + BD FACSMelody TM Sorting was performed and data were visualized using BD FACSChorus software. Figure 8 , 9 and 17 show the gating strategies and specific sorting gates, and the antibodies used are listed in Table 2 .

[0156] Total RNA extraction

[0157] Cells were harvested, washed in PBS, and pelleted for RNA extraction. Cell pellets were stored at -80°C until RNA isolation. Total RNA was isolated using the RNEasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions and eluted with 30 μL of RNAse-free water. RNA concentration was determined using a NanoDrop2000 spectrophotometer (Thermo Scientific). RNA was stored at -80°C.

[0158] cDNA synthesis and RT-qPCR

[0159] cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems) with random primers and 400-1000 ng RNA as input. Before RT-qPCR analysis, cDNA was diluted 1:2 to 1:4. RT-qPCR analysis was performed using the TaqMan gene expression assay on the Roche Lightcycler 480 instrument. The assay was repeated three times and the results were analyzed as described previously (Bookout et al., Curr. Protoc. Mol. Biol. 73, 15.8.1-15.8.28 (2006)). During the detection process, the Ct value was set to 40 for low-expression samples that did not undergo amplification. A control lacking reverse transcriptase was included in the primer validation analysis. The primers used in this study are listed in Table 3 below.

[0160] Table 3 - RT-qPCR primers

[0161] Gene Primer ID source ARG1 Hs00163660_m1 Thermo Scientific ARG2 Hs00982833_m1 Thermo Scientific CTLA4 Hs00175480_m1 Thermo Scientific FOXP3 Hs01085834_m1 Thermo Scientific IL-10 Hs00961622_m1 Thermo Scientific POL2RA Hs00172187_m1 Thermo Scientific IKZ2 Hs00212361_m1 Thermo Scientific IL2RA Hs00907777_m1 Thermo Scientific TNFRSF18 Hs00188346_m1 Thermo Scientific

[0162] Preparation of cell lysates for western blotting

[0163] Set2, UKE-1, and sorted Treg and Trest cells were washed twice with sterile PBS, pelleted, and stored at -80°C. The cell pellets were resuspended in ice-cold RIPA lysis buffer (Thermo Scientific) and Halt's was added at a dilution of 1:100.TM Protease inhibitor cocktail (Thermo Scientific). The cell lysate was stirred continuously for 15 minutes at 4°C and then centrifuged at 16800×g, 4°C for 15 minutes. The supernatant was then transferred to a new Eppendorf tube and the protein concentration was measured using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's protocol.

[0164] Western blot analysis of ARG2 expression in sorted Treg and Trest

[0165] A large amount of cell lysate equivalent to 20 μg of total protein was mixed with distilled water, Bolt TM Sample reducing agent (1:10 dilution) and Bolt TM LDS sample buffer (1:4 dilution, Invitrogen) was mixed to a total sample volume of 50 μL. The samples were incubated at 99°C for 10 minutes to aid denaturation and then purified using PowerPac HV (BioRad) and Bolt TM MESSDS electrophoresis buffer (Invitrogen) was used at 200 V in a Bolt TM 4-12% Bis-Tris Plus gel (Invitrogen) for 20 minutes. To achieve quantification of protein size, a BioRad Precision Plus protein dual-color gradient was used. According to the manufacturer's guidelines, the gel was transferred to an iBlot 2PVDF Ministack (Invitrogen) and electroblotted with an iBlot Gel 2 transfer device (Invitrogen). The membrane was cut into two pieces to stain ARG2 and focal adhesion protein separately. Both parts of the membrane were blocked for 1 hour with TBST buffer (TBS buffer (Thermo Scientific) supplemented with 0.1% Tween-20 (Sigma Aldrich)) with 5% nonfat dry milk. Next, one part of the membrane was incubated overnight at 4°C with an ARG2-specific antibody diluted 1:1000 in blocking buffer, while the other half of the membrane was stored in TBST overnight. The next day, the unstained part of the membrane was incubated with a focal adhesion protein-specific antibody diluted 1:100000 in blocking buffer for 1 hour. Subsequently, the membranes were washed three times with TBST for 5 min. The ARG2-stained and vinculin-stained membranes were then incubated with rabbit or mouse secondary antibodies in blocking buffer at a dilution of 1:2000 for 1 h. After three washes, the membranes were visualized with SuperSignal TMThe membrane was developed with West Femto Maximum Sensitivity Substrate (Thermo Scientific) for 5 min and then imaged in Gel Doc using ImageLab software (V.5.2.1). TM Visualization was performed on a XR system (BioRad). The antibodies used are listed in Table 2 above.

[0166] Cytotoxicity assay

[0167] Conventional PCR was performed as previously described by Andersen et al., J. Immunol. 163, 3812-3818 (1999). 51 Cr-release assay to assess ARG2-specific CD8 + T cell cytotoxicity. Briefly, target cells were treated with 100 μCi of radioactive 51 Cr was labeled for 1 hour and then washed twice to remove extracellular 51 Cr. Effector cells and target cells were seeded at different effector cell to target cell (E:T) ratios and incubated for 4 h. Next, 100 μL of the supernatant was recovered and measured using a 2470 automated γ-counter (Perkin Elmer). 51 Cr release. The maximum release was determined in separate wells by adding 100 μL of 10% Triton-X to the target cells. 51 Cr release. Spontaneous target cell lysis in other wells was determined by incubating target cells with medium only. The assay was set up using technical replicates of all E:T ratios; maximum and minimum release wells were set up using six technical replicates.

[0168] Ex vivo Treg suppression assay

[0169] On day 8 after the start of stimulation, the CD8-positive fraction of activated PBMCs for Treg / Trest sorting 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 next day, CD8 T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) dye (Sigma Aldrich) at a concentration of 5 μM. Next, 1.5x10 5 CFSE-labeled CD8 T cells and 0.3x10 5Sorted and purified Treg or Trest cells were co-cultured together in a round-bottom 96-well plate. Cells were stimulated with human T activator anti-CD3 / CD28 Dynabeads (Gibco) at a ratio of 1:25 and supplemented with 300U / mL of IL-2 one day after the start of co-culture. The proliferation status of CD8 T cells was analyzed by flow cytometry on days 0 and 5 after the start of co-culture. Staining cells with CD4-PerCP can separate Treg / Trest cells from CFSE-labeled CD8 T cells. Three technical replicates were set up for each time point analyzed. Data were acquired using a BD Canto II flow cytometer and analyzed using FCSExpress V.7 and FlowJo V.10. The antibodies used are listed in Table 2 above.

[0170] Analysis of Treg / Trest populations in co-cultures of activated PBMCs and effector T cells

[0171] On the 9th day after the start of stimulation, 7.5x10 5 Activated PBMCs were incubated alone or with 5x10 5 ARG2-specific CD8 T cells or 5x10 5 Control CD8 T cells were isolated from activated PBMCs as described above. Cells were cultured in 48-well plates for 6 h and then stained using the same procedure as described above for Tregs and Trest isolation. In addition, a similar gating strategy was used based on samples containing only activated PBMCs ( Fig. 9 ). The Treg and Trest population sizes of samples containing ARG2-specific CD8 T cells or control CD8 T cells were compared to samples containing activated PBMCs alone to determine the percentage reduction of both populations. Three technical replicates were included for both conditions with added T cells, while six technical replicates were included for the activated PBMCs alone condition.

[0172] Animal experiments

[0173] Animal experiments were performed in the animal facility of the Department of Oncology, Copenhagen University Hospital, Herlev. Experiments were approved by the Danish Ethics Committee on Experimental Animal Welfare. C57BL / 6 female mice were bred in-house on a C57BL / 6JBomTac background. Daily care was performed by an animal caretaker within the animal facility.

[0174] Peptide vaccines

[0175] Mouse ARG1 peptide (mARG1_169-177, ISAKDIVYI (SEQ ID NO: 22)) was obtained from Synthesized and dissolved in DMSO to a stock concentration of 10 mM. Mouse ARG2 peptide (mARG2_188-196, LSPPNIVYI (SEQ ID NO: 21)) was obtained from PepScan or Synthesized 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 50 μL sterile water and emulsified with Montanide ISA 51 (Seppic) at a 1:1 ratio. The control vaccine consisted of 50 μL sterile water emulsified with Montanide at a 1:1 ratio. Mice were injected subcutaneously with 100 μL of control or peptide emulsion at the base of the tail. For tumor studies, mice were vaccinated on days 11 and 17 (Pan02), days 0 and 7 (MC38), or days 0 and 5 (B16-F10 and LL2) after tumor inoculation. For the remaining studies, mice were vaccinated 1-3 times with at least 1 week between each vaccination.

[0176] Cancer Research

[0177] Pan02, MC38, B16-F10 and LL2 cell lines were thawed one week before seeding and cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin and streptavidin (P / S) (Life Technologies).

[0178] Female C57BL / 6 mice were inoculated subcutaneously on the right side with 5x10 5 Pan02, MC38, B16-F10, or LL2 cells were placed in the ovary. Tumor volume was measured with a digital caliper and calculated as (length x width 2 ) / 2. The experimental endpoint was defined as a tumor volume exceeding 1000mm 3 or the presence of tumor ulceration. Researchers who performed tumor measurements were blinded to treatment groups.

[0179] Mouse IFNγ ELISPOT

[0180] Mice were sacrificed at the end point of the tumor study and one week after the last vaccination for the remainder of the study. Spleens were harvested, broken through a 70 μm filter, and erythrocytes were lysed with erythrocyte lysis buffer (Qiagen). 8×10 cells / well were seeded in IFNγ ELISPOT plates. 5 splenocytes. 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 I (Worthington), 75 μg / ml DNase I (Worthington), 5 mM CaC12, and 1% P / S. Cells were filtered through a 70 μm cell strainer and erythrocytes were lysed as described above. CD45 was 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). + Tumor infiltrating lymphocytes (TIL). + The cells were left to rest overnight. 5x10 cells were inoculated into each well of the IFNγ ELISPOT plate. 5 CD45 + To phenotype the ARG2 response, CD8 cells were isolated from the spleens of vaccinated mice using CD8a (Ly-2) and CD4 (L3T4) microbeads (Miltenyi Biotec), respectively, following the manufacturer’s instructions. + and CD4 + T cells. The separated T cells were left to stand overnight. 2.8x10 5 CD8 + and CD4 + T cells and 6x10 5 Splenocytes from naive mice (used as antigen presenting cells) were inoculated together in IFNγ ELISPOT plates. Peptide-specific responses were reported as the difference in the mean number of spots between peptide-stimulated and unstimulated wells.

[0181] Tumor RNA extraction and RNA sequencing

[0182] Tumor fragments (≤30 mg) were stored in RNAlater (Invitrogen) at −80°C. Tumors were homogenized on a TissueLyser (Qiagen) and RNA was extracted using the RNEasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. RNA concentration was measured on a Nanodrop 2000 spectrophotometer. Isolated RNA was stored at −80°C. RNAseq was performed on tumors from four untreated and six vaccinated mice as described in et al., Oncogene 41, 1364–1375 (2022). Briefly, 500 ng of purified RNA (RIN score >7) was enriched using oligo dT magnetic beads (Illumina) to obtain polyadenylated mRNA, followed by fragmentation and cDNA synthesis using random primers (NEBNext). cDNA was prepared for Illumina sequencing by adapter ligation followed by indexing using PCR and size selection. The concentration of the cDNA library was determined using the KAPA Library Quantification Kit (Roche) and sequenced using the Novasek 6000 Illumina sequencing platform. RNAseq data are available in the GEO repository (GSE212500). As previously described. Reads were aligned and quantified as described by et al. Soon, sequenced DNA was aligned to the GRCm39 reference genome assembly using STAR-v2.7.8, and reads were quantified using featureCounts and ENSEMBL gene and transcript version 104. Differential gene expression was analyzed by DESeq2. Volcano plots were generated using the EnhancedVolcano package (vl.8.0) based on differential gene expression analysis. The Gene Ontology Resource (http: / / geneontology.org / ) was used to assess the biological processes associated with the differentially upregulated genes, and then the most specific subprocesses were classified.

[0183] ImmuCC

[0184] The computational framework of the CIBERSORT analysis tool for deconvolution of mouse bulk RNA-Seq data and the developed ImmuCC signature matrix (non-tissue specific) were used to characterize and quantify 25 immune cell subtypes. The ImmuCC signature matrix used consisted of 511 genes, of which 510 genes from our bulk RNA-Seq data were mapped (1 was missing). In order to deconvolute bulk RNA-Seq samples using CIBERSORT, the median of the ratio-normalized data from DEseq2 was used to generate the input mixing matrix. In addition, the analysis also included CIBERSORT-Relative Mode and CIBERSORT-Absolute Mode. While CIBERSORT-Relative Mode represents the proportion of immune cells relative to the total immune content and is therefore suitable for intra-sample comparisons, CIBERSORT-Absolute Mode produces a score that quantifies the abundance of each cell type, making it suitable for intra-sample comparisons between cell types and inter-sample comparisons of the same cell type. CIBERSORT output was generated by performing 1000 permutations and disabling the quantile normalization parameter. In this study, two population schemes (compact and extended, Tables 5 and 6) were defined, resulting in the aggregation of some of the 25 immune subgroups. The total absolute score of the combined subgroups was calculated as the sum of each subgroup. Relative scores were recalculated based on the new total immune content of each scheme.

[0185] Statistical analysis

[0186] ELISPOT responses were statistically analyzed in R studio using the distribution-free resampling rule (DFR) described by Moodie et al. (Methods Mol. Biol. 792, 185–196 (2012)). Descriptive statistics from proliferation assays and changes in Treg population size in Treg / Trest population analysis were statistically analyzed using unpaired t-tests. Two-way ANOVA and comparisons of multiple time points were performed to determine the statistical significance of tumor growth curves. The Mann-Whitney U test and unpaired two-tailed t-test were used to assess the statistical significance of differences in peptide-specific IFNγ-secreting cells and ImmuCC correlation analysis between treatment groups, respectively. Statistical analysis was performed in GraphPad Prism (version 9).

[0187] Example 2 - Spontaneous CD8 to ARG2 + T cell response

[0188] The present inventors studied the ARG2-specific CD8 +T cells. An In silico HLA-prediction algorithm (available at syfpeithi.de and cbs.dtu.dk) was used to generate a library of 15 short ARG2-derived peptides (9-mers and 10-mers) that were predicted to bind strongly to HLA-A2, as shown in Table 4 below.

[0189] Table 4 - Peptide library HLA-A2 predicted AR2-derived peptides

[0190]

[0191]

[0192] These 15 peptides (A2S01 to A2S15) were screened in peripheral blood mononuclear cells (PBMCs) from five healthy donors (HD) who were confirmed to be HLA-A2 positive (HLA-A2 + ). PBMCs were stimulated ex vivo with each peptide and restimulated in IFNγ ELISPOT after 12-14 days in culture.

[0193] 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 1 A). However, the responses observed with A2S15 could not be validated, as repeat IFNγ ELISPOT in HD did not reveal a significant response ( Figure 5 A2S14, on the other hand, elicited a strong IFNγ ELISPOT response in the validation setting ( Figure 5 B) Further characterization of the response to TNFα and IFNγ by intracellular cytokine staining (ICS) showed that the response came from CD4 + T cells ( Figure 1 B). A2S05 was also shown to induce a strong IFNγ ELISPOT response ( Figure 5 C), and ICS demonstrated that the response was from CD8 + T cells ( Figure 1 C). Therefore, A2S05 was identified as a priming agent for CD8 + T cell responses to the ARG2 peptide.

[0194] Next, the 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 to A2S05 were observed in this particular assay ( Figure 2 A). The HD used in this test was of undetermined HLA type. Therefore, it was proposed that the selected donors might have an HLA type that was incompatible with the peptide A2S05. Therefore, 38 donors (13 cancer patients with solid tumors and 25 healthy donors) with known HLA-A2 status (positive or negative) were further screened for reactivity to A2S05. Most of the donors were selected based on their HLA-A2 + (n=29) were selected, but some HLA-A2 - Donors (n=9) were included as controls. Similarly, responses to A2S05 were uncommon, with only 10 of 38 donors experiencing a statistically significant response ( Figure 2 B) Interestingly, in HLA-A2 + and HLA-A2 - Responses to A2S05 were observed in all donors ( Figure 2 C), indicating that A2S05 is not HLA-A2 restricted, as suggested by in silico HLA prediction algorithms.

[0195] Complete HLA typing was performed on the four donors with strong A2S05 reactions. The study found that the four donors shared HLA-A1, HLA-B8, and HLA-C7 ( Figure 2 D). Importantly, in some donors with strong ex vivo responses to A2S05, strong and significant responses were also detected in PBMCs in vitro ( Figure 2 E).

[0196] Example 3 - ARG2-specific CD8 + Characterization of T cells

[0197] To further characterize the ARG2-specific CD8 + T cells, A2S05-specific T cell cultures were established from four donors. Briefly, donor PBMCs were stimulated ex vivo with A2S05. Twelve days later, peptide-specific T cells were restimulated and isolated using an IFNγ capture kit. IFNγ-producing cells were expanded using a rapid expansion protocol and examined for specificity for IFNγ and TNFα by ICS 16-17 days after expansion. Highly specific CD8 + Culture ( Figure 3 A).

[0198] The established cultures were used to determine the HLA restriction of A2S05. IFNγ ELISPOTs were performed using A2S05-specific T cells from HD78 and HD93 co-cultured with different target cell lines pulsed with A2S05 peptide. These results showed that the restriction of A2S05 to HLA-A1 + and HLA-C7 + Cell lines were not reactive ( Figure 3 B and 3C), but for HLA-B8 + Metastatic malignant melanoma cell line FM6 has recognition and reactivity ( Figure 3 D) Flow cytometry confirmed that FM6 is HLA-B8 + RT-qPCR confirmed the expression of ARG2 ( Figure 6 ). Confirmation of the remaining two ARG2-specific CD8 + T cell cultures showed responsiveness to FM6 ( Figure 7 A) and confirmed that all four ARG2-specific CD8 + T cell cultures showed resistance to three additional HLA-B8 + Reactivity of cell lines ( Figure 7 C). The specificity of the ARG2-specific CD8 T cell cultures was confirmed by the lack of reactivity in IFNγ ELISPOT to a peptide with the corresponding ARG1 sequence (ARG1_65-73) ( Figure 7 B).

[0199] Next, the standard 51 Cr cytotoxicity assay to identify ARG2-specific CD8 + Can T cells lyse FM6 cells? In fact, specific T cells lyse FM6 cells in a concentration-dependent manner ( Figure 3 E), thus demonstrating that ARG2-specific CD8 + Cytotoxicity of T cells. IFNγ stimulation increased the mean fluorescence intensity (MFI) of HLA-B8 expression ( Figure 6 ) and resulted in a slight increase in FM6 cell lysis ( Figure 3 E).

[0200] Therefore, the HLA-B8 restriction of the A2S05 peptide was confirmed by the expression of the metastatic melanoma cell line FM6 and three other HLA-B +Specific recognition and cytolytic activity against cancer cell lines were confirmed. This result was surprising given that the peptide had been predicted to bind HLA-A2. Interestingly, the HLA-B8 haplotype has been proposed to be protective against melanoma based on the observation that HLA-B8 frequency is significantly reduced in patients with advanced melanoma compared with healthy donors (Fensterle et al., BMC Med. 4, 1–6 (2006)). Furthermore, in chronic myeloid leukemia (CML), expression of HLA-B8 is associated with a reduced incidence of CML (Posthuma et al., Blood 93, 3863–3865 (1999)). ARG2 upregulation 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 ARG2 has an immunosuppressive effect in acute myeloid leukemia (Mussai et al., Blood 122, 749–758 (2013)). Without being bound by theory, HLA-B8 restriction of class I ARG2 peptides implies that ARG2-specific T cells play a role in immune surveillance of melanoma and CML.

[0201] Example 4 - ARG2-specific T cells specifically recognize activated regulatory T cells

[0202] In a recent study, Lowe and colleagues found that activated effector CD4 + Activated Tregs from peripheral blood exhibit high ARG2 expression compared to activated T cells (Teff), and ARG2-dependent inhibition of T cell proliferation by Tregs has been demonstrated (Lowe et al., JCI Insight 4, (2019)). It was hypothesized that if activated Tregs express higher levels of ARG2 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 according to the experiments described by Lowe et al. On day 9 after activation, purified Tregs and Teffs were purified using FACS ( Figure 8 A and 9). Purified Tregs or Teffs were then used as target cells to inoculate autologous ARG2-specific T cells in the IFNγ ELISPOT assay. Significantly higher responses to Tregs than to Teffs were observed in all three donors ( Figure 4 A), RT-qPCR analysis showed that the expression of ARG2 in Treg was 3-7 times that in Teff ( Figure 4 B), while the expression level of ARG1 was the lowest ( Figure 8 E), thus indicating ARG2-dependent recognition. The Treg phenotype was confirmed by preferential expression of Treg signature genes, including FOXP3 ( Figure 4 C), IL-10 and CTLA-4 ( Figure 8 CD). Finally, the expression levels of ARG2 in Treg and Teff were compared with those in bulk cultures of activated cells from which these two subsets were isolated and found to be comparable to those in Teff ( Figure 4 D).

[0203] Thus, the ability of ARG2-specific T cells to recognize and respond to Tregs highlights their well-established immunomodulatory function. The ability of ARG2-specific T cells to target Tregs suggests that ARG2-specific T cells are anti-Tregs, characterized by their ability to “tune up Tregs.” The preferential expression of ARG2 in activated Tregs compared to activated bulk cultures or activated Teffs suggests that ARG2 induction is an active mechanism that enhances the immunosuppressive capacity of Tregs. Thus, without being bound by theory, it is reasonable to predict that targeting activated Tregs with ARG2-specific T cells has important immunomodulatory potential by eliminating the immunosuppression exerted by activated Tregs with high ARG2 expression. Many tumors are characterized by the presence of large numbers of such Tregs, and thus ARG2-specific anti-Treg targeting of activated immunosuppressive Tregs could alleviate Treg-mediated immunosuppression by tumor-infiltrating T cells (TILs).

[0204] Example 5 - ARG2-specific T cells specifically recognize activated regulatory T cells

[0205] In a recent study, Lowe and colleagues found that activated effector CD4 + Activated Tregs from peripheral blood exhibit high ARG2 expression compared to T cells (Teff), and ARG2-dependent inhibition of T cell proliferation by Tregs has been demonstrated (Lowe et al., JCI Insight 4, (2019)). It is hypothesized that if activated Tregs express higher ARG2 levels than other T cells, they can 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 according to the experiments described by Lowe et al. On day 9 after activation, purified Tregs and resting T cells (Trest) were purified using FACS ( Fig.12A and 13). Purified Treg or Trest were then used as target cells to inoculate autologous ARG2-specific T cells in the IFNγ ELISPOT assay. Significantly higher responses to Treg than to Trest were observed in all three donors ( Fig.11 AC). In addition, with autologous CD8 + Co-culture of activated PBMCs with ARG2-specific T cells resulted in a significant reduction in the Treg population compared to activated PBMCs co-cultured with T cells ( Fig.11 D), indicating that ARG2-specific T cells have cytotoxic activity against Tregs. Importantly, RT-qPCR analysis showed that the expression of ARG2 in Tregs was 3-7 times that in Trests ( Fig.11 E), while the lowest level of ARG1 expression ( Fig.12 B), indicating ARG2-dependent recognition of Tregs. Western blot analysis confirmed the preferential expression of ARG2 in Tregs at the protein level ( Fig.11 F and 12F). ARG2 expression levels in Treg and Trest were also compared with those in bulk cultures of activated PBMCs from which both T cell subsets were isolated and found to express ARG2 levels comparable to those of Trest ( Fig.11 E). In addition, ARG2 expression in ARG2-specific T cells was comparable to that in Trest ( Fig.12 D) Finally, Treg or Trest were used as autologous ARG2-specific CD4 + Interestingly, ARG2-specific CD4 + The response of T cells to Treg was also significantly higher than that to Trest ( Fig.11 FG).

[0206] To confirm the immunosuppressive phenotype of isolated ARG2-expressing Tregs, an aliquot of activated PBMCs was subjected to intracellular staining for FOXP3. Evaluation of FOXP3 expression between cells sorted as Tregs and Trests showed that FOXP3 expression was higher in Tregs than in Trests ( Fig.13 A). This was also confirmed by RT-qPCR analysis of FOXP3 expression in Tregs and Trest ( Fig.12 C). To further validate the sorting and purification strategy, the expression of several other Treg signature genes was evaluated by RT-qPCT, and it was found that IL2RA (CD25), TNFRSF18 (GITR), IKZF2 (HELIOS), CTLA4, and IL10 were preferentially expressed in Tregs ( Fig.13 BF). Interestingly, Tregs also preferentially express PDCD1 (PD-1) relative to Trest ( Fig.13 G). Finally, the functional phenotype of the purified Tregs was confirmed by proliferation assay. + T cell populations ( Fig.12 E) CD8 T cells stimulated with anti-CD3 / CD28 and IL-2 were evaluated over 5 days in co-culture with Trest or Tregs. + T cell proliferation, and found that Treg significantly inhibited CD8 + T cell proliferation ( Fig.13 H).

[0207] Example 6 - ARG2-specific T cells induced by peptide vaccination have an immunomodulatory effect in a murine pancreatic cancer model Inhibit tumor growth

[0208] The inventors previously identified a highly immunogenic murine ARG2-derived epitope (mARG2_188-196, LSPPNIVYI) that elicited strong and frequent T cell responses after a single vaccination. Studies have shown that vaccination with immunogenic ARG2-derived peptides can induce tumor growth delay in a murine lung cancer model. In order to confirm the anti-tumor effect of ARG2-derived peptide vaccination in another tumor model and to study the immunomodulatory function of ARG2-specific T cells, a syngeneic mouse model Pan02 was used. Pan02 is a model of pancreatic ductal adenocarcinoma (PDAC), which is relevant to studying ARG2-based vaccines given that ARG2 expression has previously been shown to be associated with poor prognosis in PDAC patients, and Pan02 tumors have been shown to express high levels of Arg2 at tumor endpoints.

[0209] C57BL / 6 mice were challenged with Pan02 cells and mice with similar mean tumor volumes were assigned to different treatment groups on day 11, when tumors became palpable. Mice received the previously described ARG2-based vaccine or control vaccine ( Fig.14 A). Interestingly, a significant inhibition of tumor growth was observed in ARG2-vaccinated mice; in fact, tumor growth ceased after day 20 ( Fig.14 B). In addition, one mouse in the ARG2-vaccinated group showed complete tumor regression ( Fig.15 A). These results were confirmed in an independent study ( Fig.15 B). The ARG2 vaccine group did not lose weight, which also proved the safety of the vaccine ( Fig.15 C).

[0210] Six ARG2-vaccinated mice and three control-vaccinated mice were randomly selected for ELISPOT analysis on day 31 after tumor inoculation. Here, a strong response was observed in all ARG2-vaccinated mice, while no response was observed in the control-vaccinated mice ( Fig.14 C). Despite the partial sequence similarity, the observed immune response was specific for ARG2, as no cross-reaction was observed with peptides from the corresponding region of ARG1 (mARG1_169-177, ISAKDIVYI) ( Fig.14 D and 15D). Next, pairs of ARG2-vaccinated mice were randomly selected and pooled splenocyte samples were prepared. CD4 + and CD8 + In the ELISPOT assay, CD4 T cells were observed in all three groups of ARG2-vaccinated mice. + and CD8 + The reaction in part ( Fig.14 E).

[0211] The amount of tumor tissue available was limited due to the small size of the tumors. However, in three other murine tumor models, we showed that ARG2-specific T cells homed to tumors in mice vaccinated with an ARG2-based vaccine ( Fig.15 EG), indicating that the vaccine induced tumor infiltration of ARG2-specific T cells after vaccination. To evaluate the impact of inducing ARG2-specific T cells on the tumor transcriptional landscape, whole tumor RNA was isolated from four control vaccine-treated mice and six ARG2-based vaccine-treated mice and subjected to RNAseq. Differential gene expression analysis showed that a total of 282 upregulated genes and 33 downregulated genes were expressed after vaccination with the ARG2 peptide ( Fig.14 F). Gene ontology analysis based on biological processes showed that among the genes upregulated in mice treated with ARG2-based vaccines, 44% of the enriched processes were related to tumor immunology ( Fig.15 H). Of these, 76% were associated with positive anti-tumor immune responses, 10% were associated with negative anti-tumor immune responses, and the remaining 14% were associated with immune responses to bacteria ( Fig.15 I). Among the biological processes significantly enriched in mice treated with the ARG2-based vaccine, processes related to innate and adaptive immunity were found. Enrichment of processes related to T cells and lymphocytes, type I interferons, cytokine regulation, angiogenesis and immune system processes, as well as calcium ion concentrations were observed, together suggesting the development of an immune response in ARG2-vaccinated mice ( Fig.14 G).

[0212] Based on these observations, the ImmuCC algorithm was used to assess the relative composition of infiltrating immune cell types in a large number of tumor samples (Tables 5 and 6). Overall, there was a clear trend toward increased mean infiltration of immune cells into the tumor microenvironment after vaccination with the ARG2-based immunomodulatory vaccine ( Fig.14 H). Specifically, the mice that received the ARG2 immunomodulatory vaccine were found to have an increase in the mean absolute proportions of several immune cell types, including NK cells, and a trend toward increased numbers of macrophages and monocytes ( Fig.16 Mice that received the ARG2-derived peptide vaccine had higher mean M1 / M2 macrophage and CD8 / Treg ratios ( Fig.14 I and J), indicating a more proinflammatory tumor microenvironment and demonstrating that ARG2-specific T cells induced after vaccination have immunomodulatory capacity.

[0213] Table 5 - Overview of populations and subpopulations used in the compact population scheme for the ImmuCC algorithm.

[0214]

[0215]

[0216] Table 6 - Overview of populations and subpopulations used in the augmented population scheme for the ImmuCC algorithm.

[0217]

[0218]

[0219] Thus, the immunomodulatory function of ARG2-specific T cells has been demonstrated in the murine Pan02 tumor model after ARG2 vaccination. Gene expression changes in Pan02 tumors of ARG2-vaccinated mice suggest that an antitumor immune response in the form of increased immune cell infiltration was induced and a more pro-inflammatory microenvironment with a higher M1 / M2 macrophage and CD8 / Treg ratio was established. The more immuno-permissive tumor microenvironment induced after ARG2 vaccination could explain the observed significant inhibition of Pan02 tumor growth. In addition, three additional 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 alter the immune landscape to favor antitumor immune responses.

[0220] sequence

[0221] Unless otherwise indicated, the start and end positions refer to positions in full-length human arginase 2 (SEQ ID NO: 19).

[0222] Table 1

[0223]

[0224]

[0225] *Position relative to full-length human arginase 1

[0226] Full length human arginase 2 (NP_001163.1) (SEQ ID NO: 19)

[0227]

[0228] The region identified as an immunogenic hotspot based on arginase 1 homology is shown in bold and underlined (SEQ ID NO: 16). The transit peptide boundary identified as the center of the A2L2 hotspot (SEQ ID NO: 17) is shown in bold and italics as "KSV".

[0229] Full length human arginase 1 (NP_000036.2) (SEQ ID NO: 20)

[0230]

[0231] Regions identified as immunogenic hot spots are shown in bold and underlined.

Claims

1. A polypeptide which is an immunogenic fragment of human arginase 2 (ARG2; SEQ ID NO: 19), said immunogenic fragment comprising or consisting of a sequence of 9 to 19 consecutive amino acids of SEQ ID NO: 19, provided that said fragment does not comprise amino acids 2 to 34 or 180 to 229 of SEQ ID NO:

19.

2. The polypeptide according to claim 1, which comprises or consists of a human leukocyte antigen (HLA) class I restricted epitope.

3. The polypeptide according to any one of claims 1 or 2, which comprises or consists of an HLA-B8 restricted epitope.

4. A polypeptide according to any one of the preceding claims, which is capable of stimulating CD8 + T cells, optionally wherein the CD8 + Positive T cells are cytotoxic T cells and / or ARG2 specific.

5. A polypeptide according to any one of the preceding claims, comprising or consisting of the following amino acid sequence: NLIVNPRSV (SEQ ID NO: 5).

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

7. The polypeptide according to claim 1 or 6, which is capable of stimulating CD4 + T cells.

8. A polypeptide according to any one of claims 1, 6 or 7, comprising or consisting of the following amino acid sequence: GLLSALDLV (SEQ ID NO: 14).

9. A polypeptide according to any one of the preceding claims, which has a maximum length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids and / or wherein the C-terminal amino acid is substituted by the corresponding amide.

10. A polynucleotide encoding a polypeptide as defined in any one of the preceding claims, optionally comprised in a vector.

11. A composition comprising the polypeptide according to any one of claims 1 to 9 and / or the polynucleotide according to claim 10, and optionally an adjuvant.

12. The composition according to claim 11, comprising: a. at least one different polypeptide according to any one of claims 1 to 9, 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 bacterial DNA based adjuvants, oil / surfactant based adjuvants, viral dsRNA based adjuvants, imidazoquinoline and Montanide ISA adjuvants.

13. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject the polypeptide of any one of claims 1 to 9, the polynucleotide of claim 10, and / or the composition of claim 11 or 12.

14. The method according to claim 13, wherein: a. the disease or disorder is characterized at least in part by an inappropriate or excessive immunosuppressive function of ARG2, optionally wherein the excessive immunosuppressive function of ARG2 is at least in part mediated by activated Treg cells expressing ARG2 and / or cancer associated fibroblasts (CAFs) expressing ARG2; and / or b. The disease or disorder is cancer, optionally wherein the cancer is melanoma (eg, malignant metastatic melanoma), chronic myeloid leukemia (CML) or pancreatic cancer.

15. The method according to claim 13 or 14, wherein the disease or disorder is cancer, and optionally wherein the method further comprises administering to the subject an additional cancer therapy simultaneously or sequentially, and optionally wherein the additional cancer therapy is an immune system checkpoint inhibitor, preferably an antibody, more preferably an anti-PD1 antibody.

16. A method for stimulating ARG2-specific T cells, the method comprising contacting the cells with a polypeptide according to any one of claims 1 to 9 or a composition according to claim 11 or 12, optionally wherein: a. the cell is present in a sample taken from a healthy subject or a cancer patient, optionally a tumor sample; and / or b. The ARG-2-specific T cells are CD8 + Positive T cells, preferably cytotoxic CD8 + T cells.

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