Arginase 1 Polypeptide

Abstract

The present invention relates to novel polypeptides derived from Arginase 1. The present invention also relates to polynucleotides encoding the polypeptides. The present invention also relates to compositions comprising the polypeptides and polynucleotides. The present invention also relates to uses of the polypeptides, polynucleotides, and compositions. [Selected figure] Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to novel polypeptides derived from Arginase 1. The present invention also relates to polynucleotides encoding the polypeptides. The present invention also relates to compositions comprising the polypeptides and polynucleotides. The present invention also relates to uses of the polypeptides, polynucleotides, and compositions. [Background technology]

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

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

[0004] The present inventors have previously identified a 50 amino acid region of Arginase 1 that is a "hot spot" for immunogenicity. This region corresponds to positions 161-210 of full-length human Arginase 1 (SEQ ID NO: 10). This region and peptides derived therefrom are described in WO2018065563 (note that the terms "Arginase 1", "Arg1", and "Arginase1" are used interchangeably herein).

[0005] The present inventors have now identified that a specific subset of polypeptides derived from this region is particularly effective in stimulating immune responses. For this reason, the polypeptides of the present invention are expected to be particularly effective in stimulating beneficial immune responses against arginase 1 and arginase 1-expressing cells. The development of novel immunotherapies for cancer requires a thorough understanding of the specific proteins recognized by the immune system, as well as the molecules involved in pathogenesis. In clinical settings, the induction of arginase-specific immune responses may support anti-cancer immune responses by suppressing the immunosuppressive function of arginase-expressing cells in general, and MDSCs and tumor-associated macrophages (TAMs) in particular, in addition to killing cancer cells. Thus, arginase-expressing cells, for example, by vaccination with the polypeptides of the present invention, will antagonize the desired effects of other immunotherapeutic approaches targeting myeloid dendritic cells, and as a result will be highly synergistic with additional anti-cancer immunotherapies.

[0006] The present invention relates to The amino acid sequence: [ka] The present invention provides an isolated polypeptide comprising any one of:

[0007] The present invention further provides a polynucleotide encoding a polypeptide of the present invention, optionally contained within a vector.

[0008] The present invention also provides compositions comprising a polypeptide or polynucleotide of the invention, at least one pharma- ceutically acceptable diluent, carrier or preservative, and optionally an adjuvant.

[0009] The present invention also provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a polypeptide, polynucleotide, or composition of the present invention. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows responses to long Arg1 derived peptides. IFNγ ELISPOT responses to ArgLong, ArgLong2 and ArgLong3 peptides in PBMCS from 4 healthy donors (HD). Bars represent the mean number of spots per well + standard error of the mean. Also shown are corresponding ELISPOT well images with and without peptide addition for each donor. Experiments were performed in triplicate with 5x105 PBMCs / well. TNTC means too large to measure. * means p < 0.05 by distribution-free resampling (DFR) method. [Diagram 2] Figure 2 shows responses to ArgLong2 compared to 20-mer peptides. IFNγ ELISPOT responses to Arg171-190, Arg181-200, Arg191-210 and ArgLong2 in 19 healthy donors (top) and 16 cancer patients (bottom). Each dot represents the average number of peptide-specific spots for a single patient / donor. Responses were calculated by subtracting the average number of spots in control wells from the average number of spots in peptide-stimulated wells. Experiments were performed in triplicates or duplicates with 4.5-5×105 PBMC / well for healthy donors and 2.2-5×105 PBMC / well for cancer patients. Responses to ArgLong2 were not measurable (TNTC) in 6 healthy donors and 2 cancer patients, set at >500 spots. [Figure 3A] Figure 3 shows CD4+ and CD8+ T cell responses to ArgLong2 in vitro and ex vivo. 3A shows the in vitro response to ArgLong2 in CD4+ (left) and CD8+ (right) T cells in intracellular staining of PBMCs from two healthy donors and one breast cancer patient. PBMCs were stimulated with ArgLong2 and low dose IL-2 for one week prior to ELISPOT assay. [Figure 3B]Figure 3 shows CD4+ and CD8+ T cell responses to ArgLong2 in vitro and ex vivo. 3B shows ex vivo responses to ArgLong2 in CD4+ (left) and CD8+ (right) T cells in intracellular staining of PBMCs from two cancer patients (BC-breast cancer, MM-melanoma) without pre-stimulation. [Figure 4] Figure 4 shows that ArgLong2-specific CD4+ and CD8+ cells are memory T cells. 4A shows ex vivo responses to ArgLong2 in four healthy donors (HD) and two cancer patients (BC-breast cancer, MM-melanoma). Experiments were performed in triplicate with 1-5x105 cells / well. 4B shows ex vivo IFNγ ELISPOT of selected CD4+ memory T cells (CD45RO+) from PBMCs of four healthy donors and two cancer patients (breast cancer and melanoma). Experiments were performed in triplicate with 1-3x105 cells / well. 4C shows ex vivo IFNγ ELISPOT of selected CD8+ memory T cells (CD45RO+) from PBMCs of two healthy donors. Experiments were performed in duplicate or once with 1x105 cells / well. Bars represent the mean spot numbers + SEM in peptide stimulated and control wells. [Diagram 5]Figure 5 shows that IL-4 stimulation activates ArgLong2-specific T cells. A shows IFNγ ELISPOT for ArgLong2 responses in PBMCs of melanoma patients stimulated with IL-4 and / or IL-2 for 1 week. Unstimulated PBMCs were used as control. Overlaid bars show mean spot counts + SEM for control and peptide stimulated wells. Experiments were performed in triplicate with 3x105 cells / well. B shows ArgLong2-specific responses in PBMCs from 6 healthy donors (HD) and 2 cancer patients (BC - breast cancer, MM - malignant melanoma) after 1 week of stimulation with IL-2 (120 U / mL) or IL-4 (100 U / mL). Cells without cytokine stimulation were used as control. Responses were calculated as the difference in mean spot counts between peptide stimulated and control wells in ELISPOT. Experiments were performed in triplicate or duplicate with 3x105 cells / well. [Figure 6] Figure 6 shows ArgLong2-specific IFNγ immune responses mounted in vivo after immunization. Four mice were vaccinated with ArgLong2 peptide and the immune responses generated against the peptide were evaluated 7 days later by IFNγ ELISPOT. Cells from spleens and draining lymph nodes from individual mice were subjected to ELISPOT assays. Bars represent the mean number of spots. ELISPOT was performed in triplicate with or without the addition of ArgLong2 peptide. "TNTC" means not measurable majority. ** means p<0.01 by distribution-free resampling method (DFR). [Figure 7] Figure 7 shows that ArgLong2 vaccination induces antitumor effects in MC38 colon adenocarcinoma tumor model. On day 0, 30 mice were subcutaneously inoculated with 0.5x106 MC38 tumor cells in the right flank. Vaccination was started on the same day. 15 mice in the treatment group were treated with 100μg ArgLong2 in emulsion containing Montanide, and 15 mice in the control group were treated with H2O in emulsion containing Montanide. Mixed-effects analysis showed p=0.030. [Figure 8] Figure 8 shows that ArgLong2 vaccination induces antitumor effects in B16F10 melanoma tumor model. On day 0, 0.5x106 B16F10 tumor cells were subcutaneously inoculated into the right flank of 30 mice. Vaccination was started on the same day. 15 mice in the treatment group were treated with 100μg ArgLong2 in emulsion containing Montanide, and 15 mice in the control group were treated with H2O in emulsion containing Montanide. After 3 vaccinations, treatment was stopped. Mice were euthanized when tumor volume exceeded 864mm3. [Figure 9A] Figure 9 shows that ArgLong2-specific T cell clones recognize Arginase 1-expressing THP-1 cells. A and B show that ArgLong2-specific CD4 T cell clones recognize THP-1 cells when Arginase 1 expression is induced by Th2 cytokines as quantified by intracellular cytokine staining. C is qPCR data showing that IL-13 pre-stimulation of THP-1 cells induces Arginase 1 expression. [Figure 9B] Figure 9 shows that ArgLong2-specific T cell clones recognize Arginase 1-expressing THP-1 cells. A and B show that ArgLong2-specific CD4 T cell clones recognize THP-1 cells when Arginase 1 expression is induced by Th2 cytokines as quantified by intracellular cytokine staining. C is qPCR data showing that IL-13 pre-stimulation of THP-1 cells induces Arginase 1 expression. [Figure 9C] Figure 9 shows that ArgLong2-specific T cell clones recognize Arginase 1-expressing THP-1 cells. A and B show that ArgLong2-specific CD4 T cell clones recognize THP-1 cells when Arginase 1 expression is induced by Th2 cytokines as quantified by intracellular cytokine staining. C is qPCR data showing that IL-13 pre-stimulation of THP-1 cells induces Arginase 1 expression. [Figure 10]Figure 10 shows that in vitro cytokine stimulation alone is sufficient to expand T cell responses to ArgLong2 epitopes. PBMCs were treated with either Arginase 1-inducing Th2 cytokines (IL-4 / IL-13) or ArgLong2 peptide for 7 days before quantification of ArgLong2-specific T cells in vitro by IFNγ ELISPOT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] A brief description of the sequence SEQ ID NOs: 1 to 9 are the amino acid sequences of polypeptides derived from regions corresponding to positions 161 to 210 of full-length human or mouse Arginase 1, respectively.

[0012] SEQ ID NOs:10 and 11 are the amino acid sequences of full-length human and mouse Arginase 1, respectively.

[0013] SEQ ID NO: 12 is the amino acid sequence of a region corresponding to positions 161 to 210 of full-length human Arginase-1.

[0014] SEQ ID NO: 13 is the amino acid sequence of a region corresponding to positions 161 to 210 of full-length mouse Arginase 1.

[0015] Detailed Description of the Invention It is to be understood that different applications of the disclosed products and methods may be tailored to particular needs in the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0016] Further, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides," and the like.

[0017] In this specification, "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics.Thus, the term "polypeptide" encompasses short peptide sequences and longer polypeptides and proteins.As used herein, the term "amino acid" refers to any natural and / or unnatural amino acid or synthetic amino acid, including both D-optical isomers and L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0018] The terms "patient" and "subject" are used interchangeably and typically refer to a human.

[0019] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0020] The present inventors have previously identified a 50 amino acid region of human and mouse Arginase 1 that is a "hot spot" for immunogenicity. This region corresponds to positions 161-210 of full-length human Arginase 1 (SEQ ID NO: 10) or full-length mouse Arginase 1 (SEQ ID NO: 11). This region and peptide fragments derived therefrom are described in WO2018065563. The present inventors have now surprisingly identified that a polypeptide consisting of a specific contiguous amino acid sequence of this region is immunogenic compared to other amino acid sequences from the same region.

[0021] "Immunogenic" in this specification preferably means that the polypeptide can induce an immune response against Arginase 1 protein when the Arginase 1 protein is present in or on a cell expressing the Arginase 1 protein. In other words, the polypeptide can be described as being immunogenic against Arginase 1. Alternatively, the polypeptide can be described as an immunogenic fragment of Arginase 1. The immune response is preferably a T cell response. The immune response can be detected in at least one individual (or sample) after administration of the polypeptide to the individual (or sample taken from the individual).

[0022] A polypeptide can be identified as immunogenic using any suitable method, including in vitro methods. For example, a peptide can be identified as immunogenic if it has at least one of the following characteristics (i) to (iii): (i) capable of inducing IFN-γ producing cells in PBL populations of healthy subjects and / or cancer patients as determined by an ELISPOT assay; and / or (ii) CTLs reactive with Arginase 1 can be detected in situ in a sample of tumor tissue; and / or (iii) It is capable of inducing the in vitro development of specific T cells. Suitable methods for determining whether a polypeptide is immunogenic active are also described in the Examples section below.

[0023] The polypeptide of the invention may consist of: - an amino acid sequence corresponding to positions 169 to 206 of full-length human Arginase 1. This polypeptide is also referred to herein as ArgLong2. The sequence is provided as SEQ ID NO:1. - an amino acid sequence corresponding to positions 169 to 206 of full-length mouse Arginase 1. This polypeptide is also referred to herein as mArgLong2. The sequence is provided as SEQ ID NO:2. - an amino acid sequence corresponding to positions 169-200 of full-length human Arginase 1. This polypeptide is also referred to herein as ArgLong3. The sequence is provided as SEQ ID NO:3. - an amino acid sequence corresponding to positions 169-200 of full-length mouse Arginase 1. This polypeptide is also referred to herein as mArgLong3. The sequence is provided as SEQ ID NO:4. - an amino acid sequence corresponding to positions 169 to 210 of full-length human Arginase 1. This polypeptide is also referred to herein as ArgLong. The sequence is provided as SEQ ID NO:5. - an amino acid sequence corresponding to positions 169-210 of full-length mouse Arginase 1. This polypeptide is also referred to herein as mArgLong. The sequence is provided as SEQ ID NO:6.

[0024] The polypeptide preferably consists of an amino acid sequence corresponding to positions 169 to 206 of the full-length Arginase 1 of human or mouse, i.e., it consists of the amino acid sequence of SEQ ID NO: 1 or 2. That is, the polypeptide is preferably ArgLong2 or mArgLong2.

[0025] The polypeptide most preferably consists of an amino acid sequence corresponding to positions 169 to 206 of full-length human Arginase 1, that is, it consists of the amino acid sequence of SEQ ID NO: 1. That is, the polypeptide is preferably ArgLong2.

[0026] In any of the polypeptides described herein, the amino acid sequence may be modified by one, two, three, four, or five (i.e., up to five) additions, deletions, or substitutions, so long as the polypeptide with the modified sequence exhibits equivalent or increased immunogenicity against Arginase 1 compared to the polypeptide with the unmodified sequence. By "equivalent," it should be understood that the polypeptide with the modified sequence does not exhibit significantly reduced immunogenicity against Arginase 1 compared to the polypeptide with the unmodified sequence. Any comparison of immunogenicity between sequences should be performed using the same assay. Unless otherwise stated, the modifications to the polypeptide sequence are preferably conservative amino acid substitutions. In conservative substitutions, amino acids are replaced 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 as the amino acid it replaces. Alternatively, in conservative substitutions, another amino acid that is aromatic or aliphatic may be introduced in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected according to the properties of the 20 major amino acids defined in Table A1 below. If the amino acids have similar polarity, this can be determined by referring to the hydropathic index for the amino acid side chains in Table A2.

[0027] [Table 1]

[0028] [Table 2]

[0029] In any of the polypeptides disclosed herein, any one or more of the following modifications may be made to improve physiochemical properties (e.g., stability), provided that the polypeptide exhibits equivalent or increased immunogenicity to Arginase 1 compared to a polypeptide having an unmodified sequence: a) replacement of the C-terminal amino acid with the corresponding amide (which can increase resistance to carboxypeptidases); b) replacement of the N-terminal amino acid with the corresponding acylated amino acid (which can increase resistance to aminopeptidases); c) replacement of one or more amino acids with corresponding methylated amino acids (which can improve resistance to proteolysis); d) Substitution of one or more amino acids with the corresponding amino acid in the D-configuration, which can improve proteolytic resistance. With regard to modifications of types (c) and (d), a preferred example is modification of the Tyr residue at the position corresponding to position 29 of SEQ ID NO: 1 (e.g., substitution with N-methyl (Tyr) or D-configuration Tyr), because Tyr occurs immediately after the Lys residue at position 28 and is therefore at a potential site for proteolysis by trypsin-like proteases (which normally cleave after Lys).

[0030] Any of the polypeptides disclosed herein may have at least one additional moiety attached to the N- and / or C-terminus to improve solubility, stability, and / or aid in production / isolation, so long as the polypeptide exhibits equivalent or increased immunogenicity to Arginase 1 compared to the polypeptide lacking the additional moiety. Suitable moieties include hydrophilic amino acids. For example, the amino acid sequences KK, KR, or RR may be added to the N- and / or C-terminus. Other suitable moieties include albumin or PEG (polyethylene glycol).

[0031] The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides can be directly synthesized using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, the polypeptides can be produced by transforming a cell, usually a bacterial cell, with a nucleic acid molecule or vector encoding said polypeptide. The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the present invention. The present invention also provides host cells comprising such nucleic acids or vectors.

[0032] 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. The polynucleotides of the present invention can be provided in isolated or substantially isolated form. By substantially isolated, it is meant that there can be substantial, but not complete, isolation of the polypeptide from any surrounding medium. Polynucleotides may be mixed with carriers or diluents that do not interfere with their intended use and still be considered substantially isolated. A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example in an expression vector. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the present invention, such nucleic acid sequences 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. A transcription termination sequence can be located 3' to the coding sequence.

[0033] Polynucleotides can be synthesized according to methods well known in the art, as illustrated by examples in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the invention can be provided in the form of expression cassettes that contain regulatory sequences operably linked to the insert sequence, thereby allowing the expression of the polypeptides of the invention in vivo. These expression cassettes are then usually provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be administered directly to the host subject. Alternatively, vectors containing the polynucleotides of the invention can be administered to the host subject. Preferably, the polynucleotides are prepared and / or administered using genetic vectors. A suitable vector can be any vector that can carry a sufficient amount of genetic information and allow the expression of the polypeptides of the invention.

[0034] Therefore, the present invention includes expression vectors that contain such polynucleotide sequences.Such expression vectors can be constructed conventionally in the field of molecular biology, and can involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as polyadenylation signals, that may be necessary and positioned in the correct direction to allow the expression of the peptide of the present invention.Other suitable vectors will be clear to those skilled in the art.For further examples in this regard, see Sambrook et al.

[0035] The present invention also includes cells that have been modified to express a polypeptide of the present invention. Such cells usually include prokaryotic cells, such as bacterial cells, e.g., E. coli. Such cells may be cultured using conventional methods to produce the polypeptide of the present invention.

[0036] The polypeptide of the present invention can be in substantially isolated form.The polypeptide of the present invention can be mixed with carriers, preservatives or diluents (discussed below) and / or adjuvants (also discussed below) that do not interfere with the intended use, and can still be considered as substantially isolated.The polypeptide of the present invention can also be in substantially purified form, in which case the polypeptide of the present invention generally comprises at least 90%, for example at least 95%, 98% or 99% of the protein in the preparation.

[0037] Compositions Comprising Polypeptides or Polynucleotides In another aspect, the present invention provides a composition comprising the polypeptide of the present invention. The present invention also provides a composition comprising a polynucleotide encoding the polypeptide of the present invention. For example, the present invention provides a composition comprising one or more polypeptides of the present invention and at least one pharma-ceutically acceptable carrier, preservative or excipient. Alternatively, the present invention provides a composition comprising one or more polynucleotides encoding the polypeptide of the present invention and at least one pharma-ceutically acceptable carrier, preservative or excipient. The carrier, preservative and excipient must be "acceptable" in the sense of being compatible with the other components of the composition and not harmful to the subject to which the composition is administered. Usually, all components and the final composition are sterile and pyrogen-free. The composition may be a pharmaceutical composition. The composition may preferably include an adjuvant.

[0038] An adjuvant is any substance whose incorporation into a composition enhances or otherwise modifies the immune response elicited by the composition. Broadly defined, an adjuvant is a substance that promotes an immune response. The adjuvant may also preferably have a depot effect, in that it also provides a slow and sustained release of the active agent from the site of administration. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986) pp. 61-63.

[0039] Adjuvants include AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP11687, also known as nor-MDP), N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1'2'-Dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, also known as MTP-PE), RIBI in 2% squalene / Tween-80.RTM. emulsion (MPL+TDM+CWS), lipopolysaccharide and its various derivatives including lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g., poly IC and poly AU acid), Mycobacterium tuberculosis (Mycobacterium tuberculosis), The adjuvant may be selected from the group consisting of wax D from B. tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the Brucella genus, Titermax, ISCOMS, Quil A, ALUN (see US58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrix or GMDP, interleukin 1, interleukin 2, Montanide ISA-51, and QS-21. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Granulocyte-macrophage colony stimulating factor (GM-CSF) may also be used as an adjuvant.

[0040] Preferred adjuvants for use with the present invention include oil / surfactant-based adjuvants, such as Montanide adjuvant (available from Seppic, Belgium), preferably Montanide ISA-51.Other preferred adjuvants are bacterial DNA-based adjuvants, such as adjuvants that contain CpG oligonucleotide sequences.Yet other preferred adjuvants are viral dsRNA-based adjuvants, such as poly I:C.GM-CSF and imidazoquiniline are also examples of preferred adjuvants.

[0041] The adjuvant is most preferably a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA51 or Montanide ISA720.

[0042] It is also stated in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) pp. 61-63 that coupling to an immunogenic carrier is recommended when the antigen of interest is low molecular weight or poorly immunogenic. Thus, the polypeptide of the present invention may be coupled to a carrier. The carrier may exist independently of the adjuvant. The function of the carrier may be to increase the molecular weight of the polypeptide fragment, for example, to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life. Furthermore, the carrier may help present the polypeptide or its fragment to T cells. Thus, in the composition, the polypeptide may be associated with a carrier as described below.

[0043] 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, etc.), or hormones (such as insulin) or palmitic acid. Alternatively, the carrier protein can be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier can be dextran, such as sepharose. The carrier must be physiologically acceptable and safe for humans.

[0044] If the composition contains an excipient, the excipient must be "pharmaceutical acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient thereof. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in the excipient. These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include liquids such as, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts may also be included, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts, such as acetates, propionates, malonates, benzoates, and the like. A complete discussion of pharmaceutical acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0045] The formulation of suitable compositions can be carried out 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 bolus or continuous administration. Injectable compositions can be prepared, packaged, or sold in unit dosage form, for example, in ampoules or in multi-dose containers, optionally containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. In one embodiment of the composition, the active ingredient is provided in a dry (e.g., powder or granules) form, for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to administration of the reconstituted composition. The composition can be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and can include, in addition to the active ingredient, additional components such as adjuvants, excipients, and auxiliary substances described herein. Such sterile injectable preparations can be prepared using non-toxic parenterally acceptable diluents or solvents, such as, for example, water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic saline, and fixed oils, such as synthetic mono- or diglycerides. Other compositions that are useful include compositions that contain the active ingredient in microcrystalline form, in liposomal preparations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation can include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. Alternatively, the active ingredient of the composition can be encapsulated, adsorbed to, or associated with particulate carriers. Suitable particulate carriers include carriers derived from polymethylmethacrylate polymers, and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368.Other microparticle systems and polymers can also be used, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

[0046] How to use The polypeptides, polynucleotides or compositions of the present invention may be used in a method for treating or preventing a disease or condition in a subject. The polypeptides, polynucleotides or compositions of the present invention may be used in the manufacture of a medicament for use in 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. The administration may be of a therapeutically or prophylactically effective amount of the polypeptide, polynucleotide or composition to a subject in need thereof.

[0047] The disease or condition may be characterized at least in part by inappropriate or excessive immunosuppressive function of Arginase 1. The disease or condition may be cancer, preferably a cancer that expresses Arginase 1 and / or is associated with inappropriate or excessive immunosuppressive function of Arginase 1. The cancer may be breast cancer, lung cancer, colon cancer or prostate cancer, or may be leukemia, preferably acute myeloid leukemia (AML), or may be melanoma.

[0048] The method may include simultaneous or sequential administration with a concomitant cancer therapy, which may be selected from cytokine therapy, T cell therapy, NK therapy, immune system checkpoint inhibitors, chemotherapy, radiation therapy, immune stimulants, gene therapy, or antibodies.

[0049] The antibodies are: abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, atinumab, atolizumab (= tocilizumab), atollimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevasimumab, Zumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, con Cizumab, crenezumab, CR6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab situxetan, epratuzumab, erlizumab, ertumaxomab, Taracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fullanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, GS6624,Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Incracumab, Indatuximab avtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Ligelizumab, Lintuzumab, Lirilumab, Roderucizumab, Lorvotuzumab mertansine, Lukatumumab, Rumiriximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimuab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab passudotox, muromonab-CD3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narutumumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, obinutuzumab, ocralizumab, ocrelizumab, ozlimomab , ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, plakmab, polatuzumab vedotin, ponezumab, priliximab, pritoxakis Simab, Pritumumab, PRO140, Kirizumab, Racotumomab, Ladolezumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Lorezumab, Romosozumab, Rontalizumab, Rovelizumab, Rupirizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab,Sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab (= tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab (= atlizumab), toralizumab, tositumomab, It may be tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, beparimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab or zolimomab alitox.

[0050] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, eculizumab, omalizumab, canakinumab, mepolizumab, reslizumab, tocilizumab, ustec, and serovar 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 numab, briakinumab, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, trastuzumab, gemtuzumab, ozogamicin, ibritumomab, tiuxetan, tositumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab and vedotin.

[0051] Particularly preferred antibodies that may be used in the methods of the invention include daratumumab, nivolumab, pembrolizumab, avelumab, rituximab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, panitumumab, tositumomab and ofatumumab. Daratumumab is particularly preferred.

[0052] The concomitant cancer therapy may be selected from the group consisting of actimid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, irinotecan, lenalidomide, leucovorin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, revlimid, temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, vindesine, and vinorelbine.

[0053] The polypeptide of the composition of the present invention can also be used in a method for stimulating Arginase 1-specific T cells, such as CD4 and CD8 T cells, comprising contacting cells with said polypeptide or composition.The method can be performed ex vivo.The cell can be present in a sample, such as a tumor sample, taken from a healthy subject or a cancer patient.

[0054] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples, both separately and in any combination thereof, may be material for realizing the invention in diverse forms thereof.

[0055] Example 1 Materials and Methods patient material PBMCs from healthy donors were isolated using density gradient separation on Lymphoprep™ (STEMCELL Technologies) and stored frozen at −150° C. in FBS supplemented with 10% DMSO. PBMCs from cancer patients were isolated from blood samples a minimum of 4 weeks after the end of all anticancer therapy. The protocol was approved by the Scientific Ethics Committee for The Capital Region of Denmark and was performed in accordance with the provisions of the Declaration of Helsinki. Written informed consent from patients was obtained prior to study enrollment.

[0056] peptide Peptides were synthesized by standard methods and dissolved in DMSO to obtain a stock concentration of 10 mM. The sequences of the peptides used in these experiments are shown in full length alignment with each other and are also described in the section entitled "Sequences". Peptides are indicated by SEQ ID NO:, name, or the start and end position of each peptide sequence in the full length sequence of Arginase 1. Each may be used interchangeably. For example, the peptide of SEQ ID NO:1 may be referred to by the name ArgLong2 on the one hand, or by the name Arg169-206 on the other hand (indicating a start position of 169 and an end position of 206). The intended reference in each example will be clear from the context. Long peptide sequences: [ka]

[0057] ELISPOT assay For in vitro ELISPOT, PBMCs from cancer patients and healthy donors were pulsed with 20 μM arginase 1-derived peptide and 120 U / mL IL-2 in 24-well plates for 7 days before use in the ELISPOT assay. Cells were seeded into 96-well nitrocellulose ELISPOT plates (MultiScreen MAIP N45; Millipore) precoated with IFNγ capture antibody (Mabtech). Arginase peptide was added to a final concentration of 5 μM, and plates were incubated at 37 °C for 14–16 h. After incubation, cells were washed off and secondary biotinylated Ab (Mabtech, catalog no. 3420-6-1000) was added for 2 h at room temperature. Unbound secondary antibody was washed off and streptavidin-alkaline phosphatase (AP) conjugate (Mabtech, catalog no. 3310-10) was added for 1 h at room temperature. The assay was developed by washing away unbound conjugated enzyme and adding BCIP / NBT substrate (Mabtech, Cat. No. 3650-10). Developed ELISPOT plates were analyzed on a CTL ImmunoSpot S6 Ultimate-V analyzer using Immunospot software v5.1. Responses were reported as the difference between the average counts in wells stimulated with Arginase 1 and wells without peptide addition.

[0058] To confirm IL-4-induced Arg1 responses, PBMCs were stimulated with IL-4 (100 or 50 U / mL) and / or IL-2 (120 or 60 U / mL) for 1 week before being set up in the ELISPOT assay as described above.

[0059] Intracellular staining PBMCs were stimulated with arginase-derived peptides in the presence of BD GolgiPlug™ (added after the first hour of peptide stimulation) for 5 hours before intracellular staining of cell cultures. Stimulated cells were stained with fluorescently labeled antibodies against surface markers (CD3, CD4, CD8) and then permeabilized with Fixation / Permeabilization and Permeabilization Buffer (eBioscience, Cat. No. 00-5123-43) according to the manufacturer's instructions. Permeabilized cells were then stained with fluorescently labeled antibodies against IFNγ and TNFα. Flow cytometry analysis was performed on a FACSCanto™ II (BD Biosciences). The following antibodies were used according to the manufacturer's instructions: IFNγ-APC (cat. no. 341117), TNFα-455 BV421 (cat. no. 562783), CD4-FITC (cat. no. 347413), CD8-PerCP (cat. no. 345774), CD3-APC-H7 (cat. no. 560275) (all from BD Biosciences), Dead Cell Stain-FVS510 (564406, BD Biosciences).

[0060] Sorting of memory T cells CD4+ and CD8+ memory T cells were sorted from freshly thawed healthy donor or cancer patient PBMC samples using magnetic bead sorting kits: Memory CD4+ T Cell Isolation Kit, human (cat. no. 130-091-893, Miltenyi Biotec) and CD8+ Memory T Cell Isolation Kit, human (cat. no. 130-094-412, Miltenyi Biotec). Purity of isolated cells was assessed by staining for CD4-FITC, CD8-PerCP, CD45RO-PE (all from BD Biosciences), and dead cells were stained using the LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen™, ThermoFisher Scientific).

[0061] result Arginase 1 peptide length determines the efficiency of T cell stimulation Based on a previously identified 50 amino acid long Arg1 hotspot region from positions 161 to 210 of Arginase 1, three different peptides were selected that covered a major portion of this region, and also excluded the cysteine ​​at position 168 of Arginase 1, which is expected to improve manufacturability, solubility and stability. The three peptides were 42-mer ArgLong (positions 169 to 210), 38-mer ArgLong2 (positions 169 to 206) and 32-mer ArgLong3 (positions 169 to 200).

[0062] To test whether these peptides can be used to identify Arginase 1 responses, PBMCs from six healthy donors were screened for responses in IFNγ ELISPOT. PBMCs were stimulated with ArgLong2 peptide and low dose IL-2 for one week prior to ELISPOT. Despite sequence similarity, ArgLong2 peptide appeared to be superior in stimulating T cell responses in IFNγ ELISPOT. As shown in Figure 1, high responses to ArgLong2 peptide were observed in four of six donors, while low or no responses were observed to ArgLong and ArgLong3 peptides.

[0063] ArgLong2 is only 4 amino acids shorter than ArgLong and 6 amino acids longer than ArgLong3. This suggests that peptide length and sequence may play an important role in ensuring optimal processing and presentation of arginase peptides. As a result, it may have an effect on the ability of such peptides to activate Arg1-specific T cells.

[0064] To determine whether the ArgLong2 peptide is comparable to the individual 20-mer peptides covering the same sequence previously described (see Examples 1, 2 and 3 of WO2018065563), PBMCs from 19 healthy donors and 16 cancer patients (8 melanoma, 6 multiple myeloma, 1 breast cancer and 1 renal cell carcinoma) were screened for responses to the three 20-mer peptides and the 38-mer ArgLong2. In both cancer patients and healthy donors, ArgLong2 produced the strongest response compared to all three 20-mer peptides (see Figure 2). Strong responses to ArgLong2 were observed in 14 of 19 healthy donors and 8 of 16 cancer patients. Responses were also observed to the 20-mer peptides, but these were fewer and less severe. In summary, ArgLong2 produced the strongest response compared to all three 20-mer peptides (see Figure 2). 171-190 showed responses in three healthy donors and three cancer patients, with Arg 181-200 showed responses in four healthy donors and six cancer patients, with Arg 191-210 demonstrated responses in seven healthy donors and three cancer patients.

[0065] CD4+ and CD8+ responses to arginase 1 Previously described T cell responses to 20-mer Arg1 peptides have shown mainly CD4+ T cell responses in cancer patients and healthy donors (see Example 2 of WO2018065563). Since the ArgLong2 peptide appeared to be more efficiently processed and subsequently recognized by T cells, the type of T cell response to ArgLong2 was examined by intracellular staining.

[0066] PBMCs from two healthy donors (HD384, HD400) and one breast cancer patient (BD30) that had previously shown strong responses to ArgLong2 in ELISPOT were examined by intracellular staining one week after in vitro stimulation with ArgLong2 and low-dose IL-2. Notably, both CD4+ and CD8+ T cell responses to the ArgLong2 peptide were identified in the cancer patient and the healthy donor. Both CD4+ (left panel of FIG. 3A) and CD8+ (right panel of FIG. 3A) responses were detected in the PBMCs of one healthy donor and one breast cancer patient. The other healthy donor showed only CD8+ T cell responses.

[0067] Interestingly, detectable CD4+ and CD8+ responses to ArgLong2 were also observed in intracellular staining of PBMCs without peptide pre-stimulation. PBMCs from two cancer patients, one malignant melanoma (MM27) and one breast cancer (BC30), were thawed and directly tested for response to ArgLong2 peptide in ex vivo intracellular staining. We were able to detect ex vivo CD4+ responses in PBMCs from both cancer patients (Figure 3B, left) and ex vivo CD8+ responses in PBMCs from a breast cancer patient (Figure 3B, right).

[0068] Memory CD4+ and CD8+ responses to arginase 1 The strong responses observed against ArgLong2 indicated that the frequency of Arg1-specific T cells was more generally detectable in PBMCs that had not been subjected to any peptide pre-stimulation. PBMCs from four healthy donors and two cancer patients (one breast cancer and one melanoma) that had shown strong responses in in vitro ELISPOT were directly tested for responses to ArgLong2 peptides in ex vivo IFNγ ELISPOT. We found significant spontaneous responses in all six donors (see FIG. 4A).

[0069] The presence of strong spontaneous ex vivo responses against ArgLong2 peptide in PBMCs of healthy donors and cancer patients suggested that Arginase 1-specific cells are a natural part of the immune system. To test the sorting of CD4+ and CD8+ memory T cells from PBMCs of cancer patients and healthy donors that showed strong spontaneous immune responses ex vivo, CD4+ or CD8+ memory T cells were sorted from PBMCs of four healthy donors and two cancer patients using magnetic bead sorting and setting in ex vivo IFNγ ELISPOT. The purity of memory T cell isolation was confirmed by flow cytometry analysis for CD4+CD45RO+ and CD8+CD45RO+ T cells, which was more than 95%. We found CD4+ memory responses against ArgLong2 peptide in two cancer patients and three healthy donors (see Figure 4B). A clear CD8+ memory response could be detected in two healthy donors (see FIG. 4C).

[0070] Upregulated Arginase 1 Expression by IL-4 Augments T Cell Responses to ArgLong2 Because Arg1-specific CD4+ and CD8+ memory T cells exist in both cancer patients and healthy donors, we tested whether these T cells might be involved in immune regulation in response to upregulated Arg1 expression. Arg1 has previously been described to be upregulated in myeloid cells in response to IL-4.

[0071] First, we tested PBMCs from one melanoma patient who had previously shown a strong spontaneous response to ArgLong2 peptide ex vivo. PBMCs were thawed and stimulated with IL-4 (50 U / mL or 100 U / mL), IL-2 (120 U / mL) or a combination of IL-4 and IL-2a (50 U / mL and 60 U / mL, respectively) for one week before testing in IFNγ ELISPOT for response to ArgLong2. Unstimulated cells were used as controls. Strong significant responses were observed in all IL-4-stimulated groups, with the strongest responses observed for the IL-4-stimulated group compared to no stimulation or stimulation with IL-2 alone, suggesting that Arg1-specific T cells are activated in response to upregulated Arg1 expression. See Figure 5A.

[0072] Next, PBMCs from two cancer patients (BC30 (breast cancer), MM27 (malignant melanoma)) and six healthy donors who had previously shown strong spontaneous responses to ArgLong2 ex vivo were stimulated with either low doses of IL-2 (120 U / mL) or IL-4 (100 U / mL) for 7 days. Unstimulated cells were used as controls. After 7 days in culture, PBMCs were tested for reactivity to ArgLong2 in IFNγ ELISPOT. Six of eight cultures showed an increased response to ArgLong2 in IL-4 stimulated cultures compared to unstimulated and IL-2 stimulated controls, further confirming a potential common mechanism of Arg1-specific T cell activation in vivo. See Figure 5B.

[0073] Consideration The existence of Arg1-specific T cells has been described previously. Such T cells can be called "anti-Treg" due to their role in targeting immune-regulatory proteins. Anti-Treg responses against other immune-regulatory proteins, such as PD-L1 and IDO, have also been described. The above experiments indicate that Arginase 1-specific anti-Tregs are not only naturally present in cancer patients and healthy donors, but also as part of the memory T cell repertoire, since responses against ArgLong2 peptides were observed in isolated CD4+ and CD8+ memory T cell populations.

[0074] To maintain immune balance, regulatory immune cells, such as Tregs, different dendritic cell subtypes, bone marrow-derived immunosuppressive cells, and M2 macrophages, suppress or terminate immune responses. This regulatory group ensures unresponsiveness or tolerance to self-antigens. Regulatory immune cells suppress immunity through a number of different cellular and extracellular factors. In contrast, specific anti-Tregs that recognize HLA-restricted derived epitopes arising from intracellularly resolved antigens can directly eliminate regulatory immune cells. In addition, anti-Tregs can increase local immune activation by secreting effector cytokines.

[0075] T H 2-driven pulmonary inflammation increases arginase 1 expression in myeloid cells. In particular, IL-4, a prototypic inducer of the macrophage M2 phenotype, induced the upregulation of arginase 1. Here, arginase 1-specific T cells were expressed as T H It has been shown that arginase 1-specific T cells are activated in response to the cytokine IL-4. H 1 cells infiltrate the IL-4-enriched environment and mount an immune response. HThis suggests that the arginase 1-specific anti-Tregs drive the immune system back to the 1 pathway, which may be a key role in controlling immune suppression and promoting inflammation. However, the arginase 1-specific anti-Tregs themselves are hindered by the inhibitory effect of arginase 1-expressing regulatory immune cells. Thus, in the immune regulatory network, arginase 1-specific anti-Tregs suppress the function of arginase 1+ regulatory immune cells, and vice versa. Therefore, under normal physiological conditions, a balance between immune activation and inhibition may be necessary to maintain immune homeostasis.

[0076] The finding of memory T cells specific for arginase 1 in healthy subjects that proliferate in response to IL-4 is consistent with previous findings of IDO- and PD-L1-specific anti-Tregs. Circulating IDO- or PD-L1-specific anti-Tregs have been identified in healthy donors, although detection was not as frequent as in patients with cancer. Furthermore, the inflammatory cytokines IL-2 and IFN-γ, known to induce IDO and PD-L1, have been found to expand populations of IDO- or PD-L1-specific T cells in human PBMCs without further stimulation.

[0077] Expression of arginase 1 in the tumor microenvironment by immunosuppressive myeloid cells inhibits antitumor T cell responses via L-arginine depletion. In an arginase 1-positive environment, T cells cannot proliferate, and thus a promising therapeutic strategy is to reconstitute adaptive immune responses by activation of proinflammatory arginase 1-specific T cells. Activation of arginase 1-specific anti-Tregs by peptide vaccination provides a novel approach to target specific immunosuppressive mechanisms in cancer.

[0078] The development of novel immunotherapeutic approaches in cancer treatment is likely to be most effective and versatile if they target common suppressive mechanisms. As shown in these experiments, Arginase 1-specific anti-Tregs exist as a natural part of the immune system and can be easily utilized to tip the balance away from immune suppression in cancer.

[0079] The peptide ArgLong2, which covers most of the previously described Arginase 1 hotspot regions, has been shown to be particularly effective in stimulating Arginase-specific CD4+ and CD8+ T cell responses compared to similar peptides of different lengths. Thus, ArgLong2 is particularly likely to be successful in the context of vaccination, given its potential to stimulate Arginase 1-specific anti-Tregs, which can exert both effector and helper functions in the tumor microenvironment.

[0080] Example 2 – Further peptide design To design peptides suitable for use in vaccination experiments in mice and that may also be functional in humans, the sequence of mouse Arginase 1 (SEQ ID NO: 11) was compared to the human Arginase 1 sequence of SEQ ID NO: 10. The level of similarity is particularly high in the hotspot region of human Arginase 1 corresponding to positions 161-210 of SEQ ID NO: 1. An alignment of this region and the corresponding region in mouse Arginase 1 is shown below: [ka]

[0081] Only two of the 50 residues are different, as shown in bold and underlined. The leucine in the human sequence is replaced by isoleucine, a very similar aliphatic amino acid in mouse, and the arginine in the human sequence is replaced by lysine, a similarly basic amino acid in mouse. Both changes are conservative. Thus, the hotspot region is highly conserved between humans and mice. Thus, the polypeptides of the present invention include polypeptides consisting of a particular human sequence from the hotspot region, with substitutions to the corresponding mouse sequence. For example, SEQ ID NO:2 corresponds to SEQ ID NO:1, but with both the leucine-isoleucine and arginine-lysine substitutions described above. A polypeptide consisting of SEQ ID NO:2 may be referred to herein as mArgLong2. Similarly, SEQ ID NO:4 corresponds to SEQ ID NO:3, but with the leucine-isoleucine substitutions described above. A polypeptide consisting of SEQ ID NO:2 may be referred to herein as mArgLong3. Similarly, SEQ ID NO:6 corresponds to SEQ ID NO:5, but with both the leucine-isoleucine and arginine-lysine substitutions described above. A polypeptide consisting of SEQ ID NO:6 may be referred to herein as mArgLong. The mouse peptides are expected to have similar potential as vaccines to their human counterparts.

[0082] Example 3 – In vivo experiments with human ArgLong2 peptide in mouse tumor models A mouse model was developed to demonstrate the therapeutic potential of vaccination with ArgLong2 peptides. The human ArgLong2 sequence was used for subsequent experiments because the corresponding ArgLong2 sequence in mice differs by only two amino acids. A comparison of the human and mouse ArgLong2 sequences is shown below: [ka]

[0083] Materials and Methods Peptide vaccination of C57BL / 6 mice Animals were subcutaneously vaccinated with 100 μg of peptide in DMSO / H2O in a 1:1 emulsion containing incomplete Montanide ISA51VG. Montanide ISA51VG+DMSO / H2O was inoculated as a control vaccine. For subsequent analysis of the immune response to the ArgLong2 peptide, mice were sacrificed on day 7 and spleens and draining lymph nodes (dLN) were harvested for the setup of ELISPOT.

[0084] ELISPOT analysis of peptide-specific responses Mouse immune cells were subjected to ELISPOT analysis. Single cell suspensions were prepared from the spleen or dLN by passing through a cell strainer. After lysis of red blood cells, 0.6–0.8 × 10 6 Cells / well were seeded on ELISPOT plates coated with anti-IFNγ antibody. Peptides of interest were added to designated wells and cells were incubated with peptides overnight. The next day, cells were removed, plates were washed and incubated with biotinylated detection antibody. Finally, after adding streptavidin-ALP and substrate, visible spots appear. Each spot corresponds to an individual IFNγ producing cell. Plates were analyzed with an immunospot analyzer and plotted.

[0085] Female C57BL / 6 mice, 15-16 / 17-20 weeks old, 15 mice / group, tumor-vaccinated For each animal, 0.5 × 10 6 Syngeneic tumor cells were inoculated subcutaneously in the right flank. Both syngeneic B16 F10 melanoma and MC38 colon adenocarcinoma cell lines were used. Vaccinations began on day 0 and were repeated weekly (days 7 and 14). Peptide and control vaccinations were performed as described above.

[0086] Tumor growth was monitored and tumors were measured approximately every 2 days. Tumor volume was calculated as V [mm 3 ]=L×W 2 / 2 (where L is the longest diameter and W is perpendicular to L). The maximum tumor size before the mice were euthanized was 864 mm 3 (L=12mm, W=12mm).

[0087] result ArgLong2 was found to be strongly immunogenic in C57BL / 6 mice, as evidenced by the high frequency of vaccine-specific T cell responses detected after a single vaccination (Figure 6).

[0088] In tumor studies, ArgLong2 vaccination significantly delayed tumor growth compared to control vaccination. Tumor growth delay was observed in both the MC38 model for colon adenocarcinoma (Figure 7) and the B16 model for melanoma (Figure 8).

[0089] Example 4 – ArgLong2-specific T cell clones recognize Arginase 1-expressing THP-1 cells Materials and Methods Generation of ArgLong2-specific T cell clones ArgLong2 clonal cultures were generated from T cell cultures enriched for ArgLong2-specific cells following a standard rapid proliferation protocol (REP) as follows: ArgLong2-specific cells were isolated by magnetic bead sorting of TNFα-producing T cells and used for limiting dilution cloning in 96-well plates at approximately 1 cell / well. Each well contained 200 μL of REP mixture (irradiated feeder cells from three different buffy coats, αCD3 antibody and 6000 U / mL of IL-2) to promote the growth of ArgLong2-specific T cells.

[0090] Quantification of Arginase 1 mRNA in THP-1 cells by qPCR THP-1 cells were stimulated with 20ng / mL IL-13 (TriChem) for 48 hours. Cells were harvested, washed in PBS, and pelleted by centrifugation at 300g for 5 minutes. The pellet was kept on ice and resuspended in 350μL RLT Plus-Buffer (Qiagen). RNA was purified using the RNAeasy Kit (Qiagen) according to the manufacturer's instructions, with a final elution in 30μL RNA-free water. RNA concentration was measured with a NanoDrop 2000 Spectrophotometer (Thermo Scientific). RNA was stored at -80℃.

[0091] Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). 1000 ng of RNA was reverse transcribed for each reaction. For RT-qPCR, cDNA was diluted 1:5 and subjected to RT-qPCR analysis using TaqMan Gene Expression Assay on a Roche Lightcycler 480 Instrument. RT-qPCR was performed in quadruplicates and data were analyzed using the ddCT method normalized to the expression levels of the housekeeping gene RPLPO and control samples. For low concentration samples that did not amplify, the Ct was set at 40. A no reverse transcriptase control (cDNA reaction started without reverse transcriptase) was used as a control for specific amplification. Arg1 human primers were purchased from Thermofisher (sequence not disclosed, product number Hs00163660_m1).

[0092] Measurement of THP-1 cell recognition by ArgLong2-specific T cells Untreated and IL-4 / IL-13 treated THP-1 cells were washed and divided into two groups: HLA-DR / DQ / DP blocking antibody (clone Tue39) was added to the first group at a concentration of 10 μg / mL for 20 minutes at 37°C. The second group of THP-1 cells was left untreated. After 20 minutes, HLA-II blocked THP-1 cells were washed. Then, THP-1 cells treated with IL-4 / IL-13 (with or without HLA-II blocking) were mixed with ArgLong2 peptide-specific T cells at an effector:target ratio of 2:1 and then processed according to a standard intracellular cytokine staining protocol.

[0093] result To further evaluate the functionality of ArgLong2-specific T cells, T cell clones were generated from healthy donors previously identified as exhibiting strong ArgLong2 responses, and their ability to specifically recognize Arginase 1-expressing targets was examined.

[0094] Th2 cytokines such as IL-4 or IL-13 are known to upregulate ARG1 expression in myeloid cells. To test whether ArgLong2-specific T cells specifically recognize Th2 cytokine-treated myeloid cells, we tested the ability of ArgLong2 T cell clones to recognize HLA-matched monocytic THP-1 cell lines prestimulated with either IL-4 (100 U / mL) or IL-13 (20 U / mL) for 48 hours.

[0095] Th2-cytokine treated THP-1 showed enhanced expression of Arginase 1. In this regard, Figure 9C shows that expression of Arginase 1 is induced in THP-1 cells when pretreated with IL-13, thereby increasing the presentation of Arginase 1-derived peptide epitopes on the surface of these cells.

[0096] ArgLong2-specific CD4 T cell clones were shown to recognize IL-4 and IL-13 treated THP-1 cells, as indicated by increased production of TNFα and IFNγ against these cells compared to untreated THP-1 (Figure 9A). Blockade of HLA class II molecules inhibited the increased recognition of IL-4 or IL-13 treated THP-1 cells, indicating that the increased recognition of IL-4 and IL-13 treated THP-1 cells is dependent on HLA class II presentation of the ARG1 peptide. Figure 9B illustrates representative data from Figure 9A in dot plot format, showing the production of IFNγ and TNFα in response to IL-4 and IL-13 treated THP-1 cells with or without HLA class II blockade.

[0097] In another experiment, PBMCs from a healthy donor (HD22, known to have a pre-existing ex vivo response to ArgLong2) were treated with IL-4 (100 U / mL) or IL-13 (20 U / mL) for 7 days to increase the presentation of Arginase 1-derived peptides in PBMC cultures and thereby stimulate endogenous ArgLong2-specific T cells present in PBMCs. After 7 days of stimulation with IL-4 or IL-13, PBMCs were analyzed in an IFNγ ELISPOT assay to confirm changes in the frequency of ArgLong2-specific T cells.

[0098] Increased ArgLong2 responses were observed in IL-4 and IL-13 treated PBMC cultures compared to untreated controls. The magnitude of the IL-13 stimulated ArgLong2 response was comparable to that observed after ArgLong2 peptide stimulation in vitro, suggesting robust activation of Arginase 1-specific cells under Th2 cytokine conditions.

[0099] [Table 3] TIFF0007624388000008.tif41165

[0100] * Denotes sequences from mouse Arginase 1 that contain at least one difference compared to the corresponding region of human Arginase 1. Residues that are not identical to the corresponding human sequence are shown in bold and underlined. Mouse and human Arginase 1 are the same length, so the start and end positions are the same.

[0101] [ka] Regions identified as hotspots for immunogenicity are shown in bold and underlined.

[0102] [ka] Regions identified as hotspots for immunogenicity are shown in bold and underlined.

[0103] [ka]

[0104] [ka] Residues that are not identical to the corresponding human sequence are shown in bold and underlined. The present invention includes the following embodiments. [1] A polypeptide having the following amino acid sequence: [ka] g. a sequence according to any one of a. to f., in which up to three amino acids are replaced by conservative substitutions, provided that the polypeptide does not exhibit a significantly reduced immunogenicity against Arginase 1 compared to a polypeptide consisting of the corresponding sequence without the substitutions; or h. A polynucleotide encoding a polypeptide according to any one of a. to g., optionally contained in a vector. A polypeptide consisting of any one of the following: [2] The polypeptide according to [1], which consists of the amino acid sequence of SEQ ID NO:1. [3] The polypeptide according to [1] or [2], wherein the C-terminal amino acid is replaced with the corresponding amide. [4] A composition comprising the polypeptide or polynucleotide according to any one of [1] to [3] and an adjuvant. [5] The composition described in [4], comprising at least one pharma- ceutically acceptable diluent, carrier or preservative. [6] The composition of [4] or [5], wherein the adjuvant is selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, viral dsRNA-based adjuvants, imidazoquinolines, and montanide ISA adjuvants. [7] A method for treating or preventing a disease or pathological condition in a subject, the method comprising administering to the subject a polypeptide or polynucleotide described in any one of [1] to [3] or a composition described in any one of [4] to [6]. [8] The method of [7], wherein the disease or condition is characterized, at least in part, by inappropriate or excessive immunosuppressive function of arginase and / or the disease or condition is cancer. [9] The method of [7] or [8], wherein the disease or condition is cancer, and optionally the method further comprises simultaneous or sequential administration of a combination cancer therapy, preferably an antibody.

[10] The method according to any one of claims [7] to [9], wherein the cancer is breast cancer, lung cancer, colon cancer or prostate cancer, or melanoma, or leukemia, preferably acute myeloid leukemia (AML).

[11] A method for stimulating arginase 1-specific T cells, comprising contacting the cells with a polypeptide described in any one of claims [1] to [3] or a composition described in any one of [4] to [6].

[12] The method of

[11] , wherein the cells are present in a sample taken from a healthy subject or a cancer patient, optionally in a tumor sample.

Claims

1. A polypeptide having the following amino acid sequence: a. a polypeptide consisting of the amino acid sequence of ISAKDIVYIGLRDVDPGEHYILKTLGIKYFSMTEVDRL (SEQ ID NO: 1); and b. The sequence according to a., in which up to three amino acids have been replaced by conservative substitutions, so long as the polypeptide does not exhibit significantly reduced immunogenicity against Arginase 1 compared to a polypeptide consisting of the corresponding sequence without such substitutions; A polypeptide consisting of any one of the following:

2. The polypeptide of claim 1 , wherein the C-terminal amino acid is replaced with the corresponding amide.

3. A polynucleotide encoding the polypeptide described in claim 1 or 2.

4. A vector comprising the polynucleotide described in claim 3.

5. A composition comprising a polypeptide according to claim 1 or 2, a polynucleotide according to claim 3, or a vector according to claim 4, and an adjuvant.

6. The composition of claim 5 further comprising at least one pharma- ceutically acceptable diluent, carrier or preservative.

7. 7. The composition of claim 5 or 6, wherein the adjuvant is selected from the group consisting of bacterial DNA-based adjuvants, oil / surfactant-based adjuvants, viral dsRNA-based adjuvants, imidazoquinolines, and Montanide ISA adjuvants.

8. A polypeptide according to claim 1 or 2, a polynucleotide according to claim 3, a vector according to claim 4, or a composition according to any one of claims 5 to 7, for use in a method of treating or preventing a disease or condition in a subject, the method comprising administering the polypeptide, polynucleotide, vector or composition to the subject.

9. The polypeptide, polynucleotide, vector, or composition of claim 8, wherein the disease or condition is characterized, at least in part, by inappropriate or excessive immunosuppressive function of arginase and / or the disease or condition is cancer.

10. 10. The polypeptide, polynucleotide, vector or composition of claim 8 or 9, wherein the disease or condition is cancer.

11. The polypeptide, polynucleotide, vector, or composition of claim 10, wherein the method comprises simultaneous or sequential administration of a combination cancer therapy.

12. The polypeptide, polynucleotide, vector, or composition of claim 11, wherein the combination cancer therapy is an antibody.

13. The polypeptide, polynucleotide, vector or composition according to any one of claims 10 to 12, wherein the cancer is breast, lung, colon or prostate cancer, or melanoma, or leukemia.

14. The polypeptide, polynucleotide, vector, or composition of claim 13, wherein the leukemia is acute myeloid leukemia (AML).

15. A method for stimulating Arginase 1-specific T cells in vitro, comprising contacting the cells with a polypeptide according to claim 1 or 2, a polynucleotide according to claim 3, a vector according to claim 4, or a composition according to any one of claims 5 to 7.

16. The method of claim 15, wherein the cells are present in a sample taken from a healthy subject or a cancer patient.

17. The method of claim 16, wherein the cells are present in a tumor sample.

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