IL-10 / FC fusion protein useful as an enhancer in immunotherapy

Abstract

The present disclosure relates to new agents useful in anti-cancer therapies, such as anti-cancer adoptive T cell transfer (ACT) immunotherapy or immune checkpoint inhibitor therapy, and their related compositions, uses and methods.

Description

[Technical Field]

[0001] The present invention relates generally to the field of anti-cancer therapy, and in particular to adjuvants useful in anti-cancer immunotherapy such as adoptive T cell transfer (ACT) immunotherapy and immune checkpoint inhibition. [Background technology]

[0002] Adoptive T cell transfer (ACT) immunotherapy has recently produced remarkable clinical results. Unlike conventional chemotherapy and radiotherapy, immunotherapy activates the host immune system to attack malignant tumors, potentially providing long-term protection from recurrence with less toxicity compared to conventional chemotherapy and radiotherapy. In adoptive CD8+ T cell therapy, large numbers of tumor-specific T cells are provided by the patient, expanded in vitro, and infused back into the patient (Jiang et al., 2019, Cancer Lett., 10;462:23-32; Levine et al., 2017, Cell Therapy. Mol. Ther.-Methods Clin. Dev., 4, 92-101). T cells can be expanded from naturally derived tumor-specific CD8+ T cells isolated from tumor-infiltrating lymphocytes (TILs) or from genetically engineered autologous circulating CD8+ T cells. Genetically engineered T cells express tumor-specific antigen receptors, including chimeric antigen receptors (CARs) and T cell receptors (TCRs), respectively, prepared from cultured T cell clones. The most successful ACT, anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy for B-cell lymphoma, has already been approved for use based on evidence of efficacy (June et al., 2018, Science 359, 1361-1365). Despite significant success in treating hematologic malignancies, solid tumors remain a major challenge for ACT, and cures remain rare (Lim et al., 2017, Cell 168, 724-740; Jiang et al., 2019, supra). Tumor-infiltrating lymphocytes (TILs) have been reported to exhibit a gradual loss of effector function and proliferative capacity in the tumor microenvironment (TME), defined as T cell "exhaustion" (Thommen et al., 2018, Cancer Cell 33, 547-562). From a metabolic perspective, persistent antigen stimulation and other metabolic stresses in the TME profoundly alter T cell signaling and weaken their antitumor immune responses (Schietinger et al., 2016, Immunity 45, 389-401; Vodnala et al., 2019, Science 363).Metabolic reprogramming of adoptively transferred T cells by ex vivo pretreatment has been shown to enhance ACT immunotherapy (Li et al., 2019, Nat. Rev. Clin. Oncol., doi:10.1038 / s41571-019-020). Few preclinical studies have been conducted on in vivo metabolic intervention of tumor-infiltrating CD8+ T cells (Chamoto et al., 2017, Proc. Natl. Acad. Sci. USA, 114, E761 E770; Chowdhury et al., 2018, Cancer Immunol. Res. 6, 1375-1387). However, these methods lacked specificity for target cells. Several preclinical studies have been conducted on ex vivo metabolic intervention using tumor antigen-specific CD8+ T cells or CART cells (Klebanoff et al., 2004, Proc. Natl. Acad. Sci. USA 101, 1969-1974; Vodnala et al., 2019, supra; Klebanoff et al., 2017, JCI Insight 2). However, simple and safe in vivo metabolic interventions that can eradicate solid tumors and induce durable cures in combination with ACT remain lacking.

[0003] Interleukin-10 (IL-10), a member of the IL-10 family of cytokines, is generally considered an immunosuppressant because it reduces tissue damage caused by uncontrolled inflammatory responses (Moore et al., 2001, Annu. Rev. Immunol. 19, 683-765). Clinical application of IL-10's anti-inflammatory properties has been limited by its short serum circulating half-life. Therefore, a fusion protein containing human IL-10 and an IgG Fc fragment (hIL-10 / Fc) was engineered and expressed in Pichia pastoris (Guo et al., 2012, Protein Expr Purif., 83(2):152-6). This extended the circulating half-life of human IL-10 in vivo, suggesting its immunosuppressive effects in the treatment of autoimmune diseases. A heterologous multimeric protein (CmAb-(IL-10)2) containing cetuximab and two IL-10 molecules covalently linked by a polypeptide linker was developed to extend the half-life and enable tumor-targeted delivery of IL-10 (Qiao et al., 2019, Cancer Cell, 35(6), 901-915).

[0004] Therefore, there is an urgent need for safe tools for metabolic reprogramming of adoptively transferred T cells that, in combination with ACT, can induce durable cures of solid tumors. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Jiang et al., 2019, Cancer Lett., 10;462:23-32 [Non-patent document 2] Levine et al., 2017 Cell Therapy.Mol.Ther.-Methods Clin.Dev.4, 92-101 [Non-patent document 3] June et al., 2018, Science 359, 1361-1365 [Non-patent document 4] Lim et al., 2017, Cell 168, 724-740 [Non-patent document 5] Thommen et al., 2018, Cancer Cell, 33, 547-562 [Non-patent document 6] Schietinger et al., 2016, Immunity 45, 389-401 [Non-Patent Document 7] Vodnala et al., 2019, Science 363 [Non-patent document 8] Li et al., 2019, Nat.Rev.Clin.Oncol., doi:10.1038 / s41571-019-020 [Non-Patent Document 9] Chamoto et al., 2017, Proc. Natl. Acad. Sci. USA, 114, E761 E770 [Non-Patent Document 10] Chowdhury et al., 2018, Cancer Immunol. Res. 6, 1375-1387 [Non-Patent Document 11] Klebanoff et al., 2004, Proc. Natl. Acad. Sci. USA 101, 1969-1974 [Non-Patent Document 12] Klebanoff et al., 2017, JCI Insight 2 [Non-Patent Document 13] Moore et al., 2001, Annu.Rev.Immunol.19, 683-765 [Non-Patent Document 14] Guo et al., 2012, Protein Expr Purif., 83(2):152-6 [Non-Patent Document 15] Qiao et al., 2019, Cancer Cell, 35(6), 901-915 Summary of the Invention [Means for solving the problem]

[0006] The present invention is based on the unexpected discovery that metabolic reprogramming of tumor-infiltrating lymphocytes with the fusion protein IL-10 / Fc significantly enhanced the efficacy of ACT against established solid tumors in a syngeneic tumor-bearing mouse model. Surprisingly, it was observed that the fusion protein IL-10 / Fc of the present invention selectively expanded tumor-specific PD-1+TIM-3+CD8+ T cells in the tumor microenvironment, indicating that the fusion protein had little systemic effect on other cell subsets with a favorable safety profile compared to IL-12- or IL-15-based therapies (Wang et al., 2017, Nat. Commun., 8, 1-15; Momin et al., 2019, Sci. Transl. Med. 11, eaaw2614; Berger et al., 2009, Blood, 114, 2417-2426; Huntington et al., 2011, Proc. Natl. Acad. Sci. USA, 108, 6217-6222; Guo et al., 2015, J. Immunol. 195, 2353-2364). This direct effect on ultimately exhausted CD8+ T cells could activate the function and proliferation of tumor-infiltrating CD8+ T cells through metabolic reprogramming, which is expected to have far fewer side effects than other therapeutic strategies that enhance CD8+ T cell-dependent tumor responses by modulating IFNγ production by dendritic cells (DCs) via the IL-10 receptor on DCs, such as the heterologous multimeric protein CmAb-(IL-10)2 proposed by Qiao et al., 2019, supra.

[0007] It should be noted that the observed dramatic efficacy of the combination of the fusion protein IL-10 / Fc of the present invention with ACT (90% of treated mice with highly aggressive tumors were completely cured) was completely unexpected, since IL-10 / Fc is typically considered an immunosuppressive cytokine and is thought to have a negative impact on cancer therapy.

[0008] Since there are currently no in vivo metabolic intervention strategies against tumor-infiltrating CD8+ T cells available for highly effective ACT cancer immunotherapy, there is great potential for the use of the fusion protein IL-10 / Fc or its variants of the present invention as an enhancer of immunotherapies such as ACT therapy or immune checkpoint inhibition.

[0009] In a first aspect, the present invention provides an Fc fusion protein for use in the prevention and / or treatment of cancer, which is a homodimer of two polypeptides, each comprising (i) an immunoglobulin IgG Fc domain and (ii) a heterologous polypeptide a comprising the sequence of human IL-10 or a variant thereof, wherein said heterologous polypeptide is covalently linked to the N-terminus or C-terminus of the Fc domain by a polypeptide linker (e.g., a flexible hinge), and the two heterologous polypeptides are non-covalently assembled within the homodimer.

[0010] In another aspect, the present invention provides a pharmaceutical composition comprising at least one Fc-fusion protein according to the invention, a pharmaceutically acceptable carrier, diluent or excipient thereof, and at least one agent useful in anti-cancer immunotherapy.

[0011] In another aspect, the present invention provides the use of an Fc-fusion protein according to the present invention for the preparation of a pharmaceutical composition for use in the prevention and / or treatment of cancer.

[0012] In another aspect, the present invention provides a method for preventing or treating cancer, comprising administering a therapeutically effective amount of at least one Fc-fusion protein of the present invention to a subject in need thereof.

[0013] In another aspect, the invention provides a method of inducing immunity or restoring responsiveness to immunotherapy in a subject, said method comprising administering to a subject in need thereof an Fc-fusion protein of the invention in combination with immune checkpoint inhibition therapy. [Brief explanation of the drawings]

[0014] [Figure 1(1)] Figure 1 depicts IL-10 / Fc, which promotes OXPHOS in CD8+ T cells during the priming phase and promotes T cell proliferation. (a) Left: Schematic diagram of the fusion protein of the present invention as a homodimer (IL-10 / Fc) described herein, comprising two polypeptides, each comprising (i) an immunoglobulin IgG Fc domain and (ii) a heterologous polypeptide a comprising the sequence of human IL-10 or a variant thereof. Each heterologous polypeptide is covalently linked to the N- or C-terminus of the Fc domain by a polypeptide linker L, and the two IL-10 polypeptides are noncovalently assembled into the homodimer (dotted line between the heterologous IL-10 polypeptides); Right: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of recombinant human IL-10 and IgG1 Fc fusion protein. The disulfide bond between each monomer was cleaved in the presence of the reducing agent DTT (dithiothreitol). (b and c) Freshly isolated Pmel-1 mouse splenocytes were labeled with the proliferation-tracking dye CFSE (carboxyfluorescein succinimidyl ester) and stimulated for 3 days with different concentrations of the cognate peptide hgp 100 in the presence or absence of IL-10 / Fc. CD8+ T cell numbers (b) and the percentage of non-dividing cells (c) were analyzed by flow cytometry. Data represent at least three independent experiments. Error bars indicate SEM. [Figure 1(2)] Figure 1 shows that IL-10 / Fc promotes OXPHOS in CD8+ T cells during the priming phase and promotes T cell proliferation. (d) shows a representative oxygen consumption rate (OCR) trace of CD8+ T cells stimulated as in (b and c). (e) shows a statistical analysis of the maximum OCR from (d). (f) shows a representative extracellular acidification rate (ECAR) trace of CD8+ T cells stimulated as in (b and c). (g) shows a statistical analysis of the maximum ECAR from (f). (h) shows the ratio of OCR / ECAR from (e) and (g). Data represent at least three independent experiments. Error bars indicate SEM. [Figure 2(1)]Figure 2 shows that IL-10 / Fc reprograms T cell metabolism in a pyruvate-dependent manner following TCR stimulation. (a) Pmel-1 murine CD8+ T cells were activated by stimulation with hgp 100 peptide for 3 days, rested for 4 days, and then cocultured with B16F10 murine melanoma cells for an additional 2 days in the presence or absence of IL-10 / Fc. Control Pmel-1 CD8 T cells were maintained in the resting phase without B16F10 cells. (a) shows representative OCR and ECAR traces of CD8+ T cells isolated from the coculture. Data are representative of at least three independent experiments. [Figure 2(2)] Figure 2 shows that IL-10 / Fc reprograms T cell metabolism in a pyruvate-dependent manner following TCR stimulation. (b) Statistical analysis of maximum OCR from (a). (c) Statistical analysis of maximum ECAR from (a). Data are representative of at least three independent experiments. Error bars indicate SEM. [Figure 2 (3)] Figure 2 shows that IL-10 / Fc reprograms T cell metabolism in a pyruvate-dependent manner following TCR stimulation. (d) shows the ratio of OCR / ECAR from (c) and (d). (e) shows the number of B16F10 tumor cells from (a). (f) shows the number of CD8+ T cells from (a). Data are representative of at least three independent experiments. Error bars indicate SEM. [Figure 2(4)] Figure 2 shows that IL-10 / Fc reprograms T cell metabolism in a pyruvate-dependent manner following TCR stimulation. (g) Schematic diagram of the metabolic pathway and inhibitors. (h) Pmel-1 mouse CD8+ T cells were activated by stimulation with hgp 100 peptide for 3 days, rested for 4 days, and then restimulated with anti-CD3 tetramer for an additional 2 days in the presence or absence of IL-10 / Fc and in the presence of the different inhibitors listed in (g). CD8+ T cell fold change represents the number of cells treated with and without IL-10 / Fc. (i) shows the basal OCR of CD8+ T cells from (h). Data are representative of at least three independent experiments. Error bars indicate SEM. [Figure 3(1)]Figure 3 shows that IL-10 / Fc enhances adoptive T cell therapy to eradicate established, highly aggressive murine melanoma tumors. (a) shows the experimental scheme of Pmel CD8+ T cell ACT and IL-10 / Fc combination therapy in a murine B16F10 melanoma model. (b) shows individual tumor growth curves for mice treated with PBS control (b), IL-10 / Fc monotherapy (c), ACT monotherapy (d), and combination therapy (e) as in (a). Data represent at least three independent experiments. Error bars indicate SEM. [Figure 3(2)] Figure 3 shows that IL-10 / Fc enhances adoptive T cell therapy to eradicate established, highly aggressive murine melanoma tumors. (f) shows the experimental scheme of Pmel CD8+ T cell ACT and IL-10 / Fc combination therapy in a murine B16F10 melanoma model using reduced doses. (g) Tumor growth was monitored in mice treated as in (f). (h) shows the survival plot of mice treated as in (f). (i and j) show individual tumor growth curves for mice treated with ACT monotherapy (i) and combination therapy (j) as in (f). Data represent at least three independent experiments. Error bars indicate SEM. [Figure 4(1)] Figure 4 shows the combination of IL-10 / Fc with adoptive T cell therapy to eradicate established tumors in multiple syngeneic tumor mouse models. (a) Tumor growth was monitored in mice treated as in Figure 3(a). (b) Survival plots for mice treated as in Figure 3(a). (c) Surviving mice from the combination group as in Figure 3(a) were rechallenged with 1 x 10 B16F10 cells subcutaneously 90 days after primary inoculation. Naive wild-type mice were inoculated with the same number of tumor cells as the control. Survival plots for mice were monitored. Data represent at least three independent experiments for (a) and (b). Data from (c) are pooled from two independent experiments. Error bars indicate SEM. [Figure 4(2)]Figure 4 shows the combined use of IL-10 / Fc with adoptive T cell therapy to eradicate established tumors in multiple syngeneic tumor mouse models. (d) 1 x 106 YUMM1.7-OVA mouse melanoma cells were subcutaneously inoculated into C57BL / 6J mice. In this model, OVA-specific OT-I CD8+ T cells were administered as ACT. The experimental schedule was the same as in Figure 3(a). Tumor growth was monitored. (e) shows the survival plot of mice treated as in (d). Data from (c) are a pool of two independent experiments. Error bars indicate SEM. [Figure 4(3)] Figure 4 shows the combination of IL-10 / Fc with adoptive T cell therapy to eradicate established tumors in multiple syngeneic mouse models. (f) 1 x 10 MC38-HER2 murine colon cancer cells were subcutaneously inoculated into C57BL / 6J mice. In this model, HER2-CAR-T cells were administered as ACT. The experimental schedule was the same as in Figure 3(a). Tumor growth was monitored. (g) shows the survival plot of mice treated as in (f). Data from (c) are a pool of two independent experiments. Error bars indicate SEM. [Figure 5] Figure 5 shows that IL-10 / Fc enhances antitumor immunity. (a) shows the experimental scheme for flow cytometry mechanistic studies in a mouse B16F10 melanoma model. Mice were sacrificed on day 14, and TILs were analyzed by flow cytometry. (b) shows the density of total CD45.2+ leukocytes, NK cells, and CDllc+ dendritic cells (DCs) within the tumor. (c) shows the percentage of Foxp3+ regulatory T cells (Tregs) among total CD4+ T cells, and the ratio of total CD8+ T cells to Treg cells. Data represent at least three independent experiments. Error bars indicate SEM. [Figure 6(1)]Figure 6 shows that IL-10 / Fc specifically expands PD-1+TIM-3+ cytotoxic T cells. B16F10 TILs were analyzed by flow cytometry as in Figure 5(a). (a) shows the density of total CD3+ T cells, total CD8+ T cells, and CD4+ T cells within the tumor. (b) shows immunofluorescence staining of CD3 in the tumor section of Figure 5(a). Scale bar: 50 μm. Data represent at least three independent experiments. Error bars indicate SEM. [Figure 6(2)] Figure 6 shows that IL-10 / Fc specifically expands PD-1+TIM-3+ cytotoxic T cells. B16F10 TILs were analyzed by flow cytometry as in Figure 5(a). (c) Representative flow cytograms of endogenous CD8+ T cells and transferred Pmel CD8+ T cells are shown. Statistical analysis of the percentage of PD-1+TIM-3+ in endogenous CD8+ T cells and transferred Pmel CD8+ T cells. Data represent at least three independent experiments. Error bars indicate SEM. [Figure 6(3)] Figure 6 shows that IL-10 / Fc specifically expands PD-1+TIM-3+ cytotoxic T cells. B16F10 TILs were analyzed by flow cytometry as in Figure 5(a). (d and e) show the density of PD-1+TIM-3+ endogenous CD8+ T cells (d) and PD-1+TIM-3+-transferred Pmel CD8+ T cells (e) within the tumor. (f) shows IL-10R expression on different subpopulations of endogenous CD8+ T cells from the PBS group of B16F10 TILs. (g) shows PD-1 expression on PD-1+TIM-3+ endogenous CD8+ T cells and PD-1+TIM-3+-transferred Pmel CD8+ T cells. (h) Percentage of polyfunctional (IFNγ-TNFα+Granzyme B+) CD8+ T cells among endogenous CD8+ T cells and transferred Pmel CD8+ T cells. Data are representative of at least three independent experiments. Error bars indicate SEM. [Figure 6(4)]Figure 6 shows that IL-10 / Fc specifically expands PD-1+TIM-3+ cytotoxic T cells. B16F10 TILs were analyzed by flow cytometry as in Figure 5(a). (i) shows the percentage of polyfunctional (IFNγ+TNFα+Granzyme B+) CD8+ T cells among endogenous PD-1+TIM-3+CD8+ T cells and transferred PmelPD-1+TIM-3+CD8+ T cells. (j and k) show the different subpopulations of CD8+ T cells classified according to the expression of PD-1 and TIM-3 in Figure 2(h) after coculture with B16F10 tumor cells for 2 days. The number of CD8+ T cells (j) and the killing efficiency of B16F10 cells (k) were assessed by flow cytometry. Data represent at least three independent experiments. Error bars indicate SEM. [Figure 6(5)] Figure 6 shows that IL-10 / Fc specifically expands PD-1+TIM-3+ cytotoxic T cells. B16F10 TILs were analyzed by flow cytometry as in Figure 5(a). (l) Memory phenotype analysis (CD62L expression and CD44 expression) of PD-1+TIM-3+ endogenous CD8+ T cells from TILs and splenocytes. (m) Representative OCR traces and maximum OCR of PD-1+TIM-3+CD8+ T cells as in Figure 2(h). Data represent at least three independent experiments. Error bars indicate SEM. [Figure 7(1)] Figure 7 shows that IL-10 / Fc enhances checkpoint blockade therapy to eradicate established murine colon tumors. (a) shows the experimental scheme for the combination therapy of α-PD-1 and IL-10 / Fc in a murine CT26 colon cancer model. BALB / c mice were subcutaneously inoculated with 3 x 10 CT26 murine colon cancer cells. As a control, α-PD-1 antibody (RMP-14) was administered every 3 days. (b) shows the individual tumor growth curves for each group. Data represent at least two independent experiments. Error bars indicate SEM. [Figure 7(2)]Figure 7 shows that IL-10 / Fc enhances checkpoint blockade therapy to eradicate established murine colon tumors. (c) Tumor growth was monitored in mice treated as in (a). (d) shows a survival plot of mice treated as in (a). Data represent at least two independent experiments. Error bars indicate SEM. DETAILED DESCRIPTION OF THE INVENTION

[0015] As used herein, "Fc fusion protein" refers to an Fc fusion protein (homodimer) comprising the sequences of human IL-10 or a variant thereof and an IgG Fc fragment covalently linked via a flexible hinge. In one embodiment, the IgG Fc fragment may be an Fc fragment from an IgG1, IgG2, IgG3, or IgG4 isoform. The IgG Fc fragment may be mutated to reduce antibody-dependent cellular cytotoxicity (ADCC), as described in Czajkowsky et al., 2012, EMBO Mol. Med., 1015-1028, or to increase the half-life or in vivo levels of IgG (e.g., IL-10 / Fc), as described in Zalevsky et al., 2010, Nat. Biotechnol. 28, 157-159; Vaccaro et al., 2005, Nat. Biotechnol. 23, 1283-1288.

[0016] In certain embodiments, point mutations may be introduced into the IgG1 Fc domain to generate a non-cytolytic IgG1 Fc domain, as described in Armour et al., 1999, Eur. J. Immunol. 29, 2613-2624 or Steele et al., 1995, J. Immunol. 154, 5590-5600.

[0017] In a further particular embodiment, at least three mutations selected from L234V, L235A and P331S in the IgG1 Fc domain of SEQ ID NO:2 are made.

[0018] In another more particular embodiment, at least two mutations selected from A330S and P331S in the IgG2 Fc domain of SEQ ID NO:5 are made.

[0019] In another further particular embodiment, at least one mutation P329G in the IgG4 Fc domain of SEQ ID NO: 11 is made.

[0020] As used herein, "human IL-10 or variants thereof" includes sequences including the sequence of native human IL-10 and variants thereof as described in Mumm et al., 2011, Cancer Cell, 20, 781-796; Guo et al., 2012, Protein Expr. Purif., 83, 152-156 (2012); Zheng et al., 1997, J. Immunol., 158, 4507-13; Qiao et al., 2019, Cancer Cell 35, 901-915.e4.

[0021] In certain embodiments, the Fc-fusion proteins of the invention can be modified to increase their half-life in vivo by standard strategies, including pegylation (e.g., pegylation of the sequence of human IL-10 or variants thereof as described in Mumm et al., 2011, supra), or the Fc domain of the Fc-fusion protein IL-10 / Fc of the invention can also be replaced by an antibody, human serum albumin or variants thereof as described or reviewed in Qiao et al., 2019, Cancer Cell 35, 901-915.e4; Kontermann, 2011, Curr. Opin. Biotechnol., 22, 868-876.

[0022] As used herein, a "flexible hinge" useful in the context of the present invention may be peptidic or non-peptidic. If the flexible hinge is peptidic, it generally contains about 3 to 20 amino acids. For example, a suitable hinge of the present invention may be selected from those described in Klein et al., 2014, Protein Eng. Des. Sel. 27, 325-330. A non-peptidic flexible hinge may be selected from the linkers described in Capon et al., 2011, Proc. Japan Acad. Ser. B Phys. Biol. Sci., 87, 603-616.

[0023] A "GS" linker refers to any peptide linker or longer linker comprising amino acids selected from G and S, or combinations thereof, in combination with a CPPCP domain (SEQ ID NO: 15), such as GGSCPPCP (SEQ ID NO: 16), GGGGSCPPCP (SEQ ID NO: 17), GGGGSGGGGSCPPCP (SEQ ID NO: 14). Typically, a GS linker has a length of about 3 to about 50, such as about 3 to 20, e.g., 5 to about 15, amino acids.

[0024] The term "variant" as applied to a peptide or polypeptide referred to herein refers to a peptide or polypeptide that is substantially homologous to a reference peptide sequence but has at least one amino acid that differs from that of the reference sequence due to the deletion, insertion, and / or substitution of one or more amino acids. Substantially homologous refers to a variant amino acid sequence that is identical to the reference peptide sequence except for the deletion, insertion, and / or substitution of one, two, three, four, five, or six amino acid residues. In more specific embodiments, the variant amino acid sequence is identical to the reference peptide sequence except for the deletion and / or conservative substitution of one, two, three, four, five, or six amino acid residues. The identity of two amino acid sequences can be determined by visual inspection and / or mathematical calculation, or more simply by comparing sequence information using known computer programs used for sequence comparison, such as the Clustal package version 1.83. Variants may also include sequences with at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue with similar physicochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for one another), or the substitution of one polar residue for another (e.g., Lys for Arg, Glu for Asp, or Gin for Asn). The hydrophobicity of amino acids can be determined based on known scales, such as those described by Kyte et al., 1982, J. Mol. Biol., 157:105-131; Eisenberg, 1984, Ann. Rev. Biochem., 53:595-623. Other such conservative substitutions, e.g., the substitution of entire regions with similar hydrophobicity properties or secondary structure propensities, are well known (Kyte et al., 1982, supra). For example, a "conservative amino acid substitution" may involve the substitution of a natural amino acid residue with a non-natural residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the time such substitutions are desired. Exemplary amino acid substitutions are shown in Table 1 below.The term "variant" also includes peptides or polypeptides that are substantially homologous to a reference peptide sequence, but have an amino acid sequence that differs from that of the reference sequence because one or more amino acids have been chemically modified or replaced with an amino acid analog. For example, non-naturally occurring residues can be introduced to enhance the pharmacological properties of peptide-based therapeutics (Geurink et al., 2013, J. Med. Chem., 56, 1262; Rand et al., 2012, Med. Chem. Commun., 3, 1282).

[0025] [Table 1]

[0026] In another particular embodiment, the sequences of the invention may optionally be acetylated at the N-terminus and / or amidated at the C-terminus.

[0027] In another particular embodiment, the sequences of the invention can optionally be pegylated.

[0028] As used herein, "anti-cancer immunotherapy" refers to anti-cancer treatment strategies that effector activate immune cells, including adoptive cellular therapy (ACT), and strategies that neutralize immune suppressive mechanisms, such as drugs, particularly antibodies, against immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), as described in Weiden et al., 2018, Nat. Rev. Immunol., 18, 212-219.

[0029] As used herein, "ACT immunotherapy" refers to a treatment technique in which T cells are collected from a patient's blood or tumor and grown in a laboratory. Once there are enough T cells, they are infused back into the patient to help the immune system fight cancer cells. Examples of ACT immunotherapy are found in Fan et al., 2018, Theranostics, 8(20):5784-5800; Rosenberg et al., 2008, Nat. Rev. Cancer 8, 299-308; Wang et al., 2014, Immunotherapy 6, 1265-1278.

[0030] As used herein, "immune checkpoint inhibitor" refers to an agent that antagonizes or inhibits an immune checkpoint molecule, and these agents may be selected from the examples of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, or LAG-3 inhibitors as described in Pardoll et al., 2012, Nat. Rev. Cancer 12, 252-264; Lee et al., 2019, Molecules 4, 1-16.

[0031] As used herein, "PD-1 inhibitors" includes anti-PD-1 monoclonal antibodies, such as Keytruda and Opdivo, as described in Lee et al., 2019, supra.

[0032] As used herein, "PD-L1 inhibitors" include PD-L1 antibodies and PD-1-Ig fusion proteins, such as those described in Pardoll et al., 2012, supra.

[0033] As used herein, "CTLA-4 inhibitors" include anti-CTLA-4 monoclonal antibodies such as ipilimumab and tremelimumab as described in Pardoll et al., 2012, supra.

[0034] As used herein, "treatment" and "treating" generally refer to achieving a desired pharmacological and physiological effect. The effect may be prophylactic, in that a disease, symptom, or condition thereof is prevented or partially prevented, and / or therapeutic, in that a disease, condition, symptom, or adverse effects resulting from a disease are partially or completely cured. The term "treatment" as used herein covers any treatment of a mammalian, particularly a human, disease, and includes (a) preventing a subject who may be susceptible to a disease but has not yet been diagnosed with the disease from acquiring the disease, e.g., prophylactic intervention in an early, asymptomatic state, and (b) inhibiting a disease, i.e., preventing its onset, or alleviating a disease, i.e., causing regression of the disease and / or its symptoms or condition, e.g., ameliorating or repairing damage. In particular, the methods, uses, formulations, and compositions of the present invention are useful for the prevention and / or treatment of cancer.

[0035] The expression "solid tumor cancer" includes lung cancer (small cell and non-small cell), breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer (colon cancer), rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer and brain cancer or skin cancer, in particular melanoma.

[0036] As used herein, the term "subject" refers to a mammal. For example, mammals contemplated by the present invention include humans, primates, farm animals such as cattle, sheep, pigs, and horses, laboratory rodents, other pets, and the like.

[0037] As used herein, the term "effective amount" refers to an amount of at least one compound of the present invention or a pharmaceutical formulation thereof according to the present invention that elicits a desired biological or medical response in a tissue, system, animal, or human. In one embodiment, an effective amount is a "therapeutically effective amount" that alleviates the symptoms of the disease or condition being treated. In another embodiment, an effective amount is a "prophylactically effective amount" that prevents the symptoms of the disease or condition being prevented. The term also includes herein an amount of a compound of the present invention sufficient to reduce disease progression, particularly an amount of a compound of the present invention that elicits a desired response by reducing or inhibiting the progression of a cancerous disorder (i.e., an "effective amount"). Typically, an effective amount can be used to inhibit cancer cell proliferation, i.e., slow down the rate of cancer cell proliferation and / or migration, stop cancer cell proliferation and / or migration, or kill cancer cells, such that the rate of cancer cell proliferation is reduced compared to the observed or predicted rate of growth of untreated control cancer cells. The term "inhibiting growth" can also refer to a reduction in the size or elimination of cancer cells or tumors, as well as a reduction in their metastatic potential. Preferably, such inhibition at the cellular level may reduce the size, slow the growth, make the cancer less aggressive, or prevent or inhibit the metastasis of the cancer in the patient. Those skilled in the art can readily determine whether cancer cell growth is inhibited by any of a variety of suitable indicators.

[0038] The term "efficacy" of a treatment according to the present invention can be measured based on changes in the course of the disease in response to the use or method according to the present invention. According to certain embodiments, efficacy can be measured by measuring the immune response elicited against cancer cells, such as by analyzing tumor-specific T cells or by assessing cancer cell death and / or inhibition of tumor growth, progression, and dissemination, reduction in tumor volume, and / or increased progression-free survival and / or increased health and well-being of the subject (e.g., suppression of cancer). The efficacy of a cancer treatment according to the present invention can be measured by inhibition of cancer cell proliferation, evidenced, for example, by arrest of cancer cells at a specific stage of the cell cycle, e.g., arrest in the G2 / M phase of the cell cycle. Inhibition of cancer cell proliferation can also be evidenced using well-known imaging methods, such as magnetic resonance imaging, computed tomography, PET, SPECT, photoacoustic imaging, X-ray, and fluorescence imaging / detection. Cancer cell proliferation can also be determined indirectly, for example, by determining levels of circulating carcinoembryonic antigen, prostate-specific antigen, or other cancer-specific antigens that correlate with cancer cell proliferation.

[0039] In particular, the efficacy of the combination treatment of the present invention may be assessed by the expansion of tumor-infiltrating CD8+ T cells, reduction in tumor size, disappearance of tumors, survival of tumor-bearing mice, or any biomarker associated with the type of cancer.

[0040] "Pharmaceutically active derivative" refers to any compound that can directly or indirectly provide the activity disclosed herein upon administration to a recipient. The term "indirectly" also encompasses prodrugs that can be converted into the active form of a drug through endogenous enzymes or metabolism. Prodrugs are derivatives of the compounds of the present invention that exhibit antitumor activity and have a chemically or metabolically degradable group, and are compounds that can be converted into pharmaceutically active compounds in vivo under physiological conditions.

[0041] The term "pharmaceutical formulation" refers to a formulation that does not contain additional ingredients that are toxic to the subject to which the formulation is administered, in a form that allows the biological activity of the active ingredient to be demonstrably effective.

[0042] Fusion protein IL-10 / Fc according to the present invention and method for preparing same Fc fusion proteins (homodimers) suitable for use in the context of the present invention are described herein.

[0043] In a specific embodiment, the Fc fusion protein (IL-10 / Fc) of the present invention is a homodimer of two polypeptides, each comprising (i) an immunoglobulin IgG Fc domain and (ii) a heterologous polypeptide a comprising the sequence of human IL-10 or a variant thereof, wherein the heterologous polypeptide is covalently linked to the N-terminus or C-terminus of the Fc domain by a polypeptide linker (e.g., a flexible hinge), and the two polypeptides are non-covalently assembled into the homodimer.

[0044] In a particular embodiment, the sequence of an Fc-fusion protein according to the invention comprises the sequence of human IL-10, for example, that of SEQ ID NO: 1 or a variant thereof.

[0045] In a particular embodiment, the sequence of the Fc-fusion protein of the invention comprises the sequence of an IgG Fc fragment, wherein the IgG Fc fragment is an IgG1 Fc fragment or a variant thereof, in particular a non-cytolytic IgG1 Fc. For example, the fusion protein IL-10 / Fc comprises the IgG Fc fragment of SEQ ID NO: 2 or a variant thereof (Zheng et al., 1997, supra; Sazinsky et al., 2008, Proc. Natl. Acad. Sci. USA 105, 20167-20172).

[0046] In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises a flexible hinge selected from SEQ ID NO: 3 and a GS linker sequence or a variant thereof (Klein et al., 2014, supra).

[0047] In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises a flexible hinge selected from SEQ ID NO: 3 and a GGS linker sequence or a variant thereof (Klein et al., 2014, supra).

[0048] In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises a flexible hinge or GS linker sequence selected from SEQ ID NO: 6 or a variant thereof.

[0049] In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises a flexible hinge or GS linker sequence selected from SEQ ID NO: 9 or a variant thereof.

[0050] In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises a flexible hinge or GS linker sequence selected from SEQ ID NO: 12 or a variant thereof.

[0051] In a particular embodiment, the sequence of an Fc-fusion protein according to the invention comprises the sequence of SEQ ID NO: 4 or a variant thereof.

[0052] In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises the sequence of an IgG Fc fragment, wherein said IgG Fc fragment is an IgG2 Fc fragment or a variant thereof, in particular a non-cytolytic IgG2 Fc. For example, the fusion protein IL-10 / Fc comprises the IgG Fc fragment of SEQ ID NO: 5 or a variant thereof. In a particular embodiment, the sequence of an Fc-fusion protein of the invention comprises the sequence of SEQ ID NO: 7 or a variant thereof.

[0053] In a specific embodiment, the sequence of an Fc-fusion protein of the invention comprises the sequence of an IgG Fc fragment, which is an IgG3 Fc fragment or a variant thereof. For example, the fusion protein IL-10 / Fc comprises the IgG Fc fragment of SEQ ID NO: 8 or a variant thereof.

[0054] In a particular embodiment, the sequence of an Fc-fusion protein according to the invention comprises the sequence of SEQ ID NO: 10 or a variant thereof.

[0055] In a specific embodiment, the sequence of an Fc-fusion protein of the invention comprises the sequence of an IgG Fc fragment, which is an IgG4 Fc fragment or a variant thereof. For example, the fusion protein IL-10 / Fc comprises the IgG Fc fragment of SEQ ID NO: 11 or a variant thereof.

[0056] In a particular embodiment, the sequence of an Fc-fusion protein according to the invention comprises the sequence of SEQ ID NO: 13 or a variant thereof.

[0057] In a particular embodiment, the Fc fusion protein IL-10 / Fc comprises the sequence of the fusion protein IL-10 / Fc described in Guo et al., 2012, supra or Zheng et al., 1997, J. Immunol., 158, 4507-4513 or Steele et al., 1995, J. Immunol., 154, 5590-5600, or a variant or fragment thereof.

[0058] The Fc fusion protein IL-10 / Fc can be prepared as described herein, or as described in Guo et al., 2012, supra, or Zheng et al., 1997, supra, or Steele et al., 1995, supra.

[0059] Composition according to the present invention The present invention provides pharmaceutical or therapeutic agents as compositions and methods for treating a subject, preferably a mammalian subject, most preferably a human patient, suffering from a medical disorder, particularly cancer, particularly solid tumor cancer.

[0060] In one embodiment, the present invention provides a pharmaceutical composition comprising at least one compound of the present invention and a pharmaceutically acceptable carrier, diluent, or excipient thereof.

[0061] The agents of the present invention or formulations thereof may be administered as pharmaceutical preparations, which may contain one or more agents of the present invention in any of the forms described herein. The compositions of the present invention may be placed in the form of pharmaceutical compositions and unit dosages thereof, together with conventional adjuvants, carriers, diluents, or excipients, and may be used as solids, such as tablets or filled capsules, all for oral use, or as liquids, such as solutions, suspensions, emulsions, elixirs, or filled capsules thereof, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use by injection or continuous infusion. Injectable compositions are typically based on injectable sterile saline or phosphate-buffered saline, or other injectable carriers known in the art. Such pharmaceutical compositions and unit dosage forms thereof may contain conventional proportions of ingredients, with or without additional active compounds or main ingredients, and such unit dosage forms may contain any appropriate effective amount of the active ingredient consistent with the intended daily dosage range used.

[0062] The compositions of the present invention may be liquid preparations, including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The compositions may also be formulated as a dry product to be reconstituted with water or another suitable vehicle before use. Such liquid preparations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include, but are not limited to, poly(ethylene glycol), glycerol, bovine serum albumin, Tween®, and Span®.

[0063] Compositions of the present invention may also be formulated as a depot preparation which may be administered by implantation or by intramuscular injection.

[0064] The solid composition of the present invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, including, but not limited to, binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.

[0065] In a more particular embodiment, a composition is provided wherein the sequence of said fusion protein comprises the sequence of SEQ ID NO: 4 or a variant thereof.

[0066] In a more particular embodiment, a composition is provided wherein the sequence of said fusion protein comprises the sequence of SEQ ID NO: 7 or a variant thereof.

[0067] In a more particular embodiment, a composition is provided wherein the sequence of said fusion protein comprises the sequence of SEQ ID NO: 10 or a variant thereof.

[0068] In a more particular embodiment, a composition is provided wherein the sequence of said fusion protein comprises the sequence of SEQ ID NO: 13 or a variant thereof.

[0069] The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.

[0070] In certain embodiments, the compositions of the present invention are for intravenous use.

[0071] In a particular embodiment, the compositions of the present invention are for intraperitoneal use.

[0072] In a particular embodiment, the compositions of the present invention are for intratumoral use.

[0073] In another particular embodiment, the compositions of the present invention are adapted for delivery by multiple doses.

[0074] In certain embodiments, the compositions of the present invention are veterinary compositions.

[0075] Further materials, pharmaceutical processing techniques, and the like are described in Part 5 of Remington's "The Science and Practice of Pharmacy," 22nd Edition, 2012, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, which is incorporated herein by reference.

[0076] Administration method The compounds of the present invention and their formulations may be administered by any method, including parenteral, intravenous, intratumoral, intrathecal, transmucosal, intranasal, rectal, or a combination thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, and intramuscular. The compositions of the present invention may also be administered in the form of an implant, allowing for sustained release of the composition and slow, controlled intravenous infusion.

[0077] The dosage administered to an individual as a single or multiple doses will vary depending on a variety of factors, including pharmacokinetic properties, the patient's condition and characteristics (sex, age, weight, health, size), the severity of symptoms, concurrent treatments, frequency of treatment, and the desired effect.

[0078] combination In one embodiment, the compounds of the present invention are to be administered in further combination with at least one therapeutic strategy useful in the prevention and / or treatment of cancer, in particular anti-cancer immunotherapy such as ACT therapy or immune checkpoint inhibitor therapy.

[0079] In another particular embodiment, the compounds of the invention are to be administered in combination with ACT therapy.

[0080] In another particular embodiment, the compounds of the invention are to be administered in combination with at least one immune checkpoint inhibitor.

[0081] In one aspect, a pharmaceutical composition is provided comprising at least one Fc-fusion protein IL-10 / Fc, a pharmaceutically acceptable carrier, diluent or excipient thereof, and at least one agent useful in ACT therapy.

[0082] In one aspect, a pharmaceutical composition is provided comprising at least one Fc-fusion protein IL-10 / Fc, a pharmaceutically acceptable carrier, diluent or excipient thereof, and at least one immune checkpoint inhibitor.

[0083] The present invention encompasses the administration of a compound of the present invention or a formulation thereof administered to a subject prior to, concurrently with, or sequentially with other therapeutic regimens or adjuncts useful in preventing, treating, and / or stabilizing cancer, such as anti-cancer therapies.

[0084] The compounds of the present invention or formulations thereof according to the present invention administered simultaneously with the above-mentioned co-agents may be administered in the same or different compositions and by the same or different administration routes.

[0085] In one embodiment, a pharmaceutical formulation is provided comprising a compound of the present invention in combination with at least one pharmaceutically acceptable carrier and at least one adjunct agent useful in treating and / or stabilizing a neurodegenerative disorder.

[0086] Uses of the Fc fusion protein IL-10 / Fc according to the present invention The present invention provides an Fc fusion protein (IL-10 / Fc) for use in the prevention and / or treatment of cancer.

[0087] In a particular embodiment, an Fc fusion protein (IL-10 / Fc) for anti-cancer immunotherapy is provided.

[0088] In a more particular aspect, there is provided an Fc fusion protein (IL-10 / Fc) for use in combination with ACT immunotherapy, in particular based on TCR-T cell or CAR-T cell or TIL therapy.

[0089] The method according to the present invention In another aspect, the present invention provides a method for preventing or treating cancer, particularly solid tumor cancer.

[0090] In another aspect, the present invention provides methods for preventing and / or treating lung cancer (small cell and non-small cell), breast cancer, prostate cancer, cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer, and skin cancer.

[0091] In another aspect, the present invention provides a method of inducing immunity in a subject, said method comprising administering to a subject in need thereof an immune checkpoint inhibitor, in particular an anti-cancer immune checkpoint inhibitor, in combination with the Fc-fusion protein IL-10 / Fc of the present invention.

[0092] All references cited herein are incorporated by reference in their entirety. The present invention is not limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

[0093] The invention having been described, the following examples are offered by way of illustration and not by way of limitation.

[0094] Synthesis of Compounds of the Invention The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred experimental conditions (i.e., incubation or reaction temperature, time, moles of reagents, solvent, etc.) are given, it is understood that other experimental conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art using routine optimization procedures.

[0095] patient In one embodiment, a patient according to the present invention is suffering from any type of cancer.

[0096] In one embodiment, a patient according to the present invention is suffering from any type of cancer at any stage, including non-metastatic and metastatic.

[0097] In certain embodiments, the patient according to the present invention is suffering from lung cancer (small cell and non-small cell), breast cancer, prostate cancer, carcinoma, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer or skin cancer.

[0098] In a further specific embodiment, the patient according to the present invention is suffering from skin cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, colorectal cancer and head and neck cancer, as well as other solid tumors or any pre-malignant or malignant neoplasm.

[0099] In a further particular embodiment, the patient according to the present invention is suffering from melanoma.

[0100] In a further embodiment, a subject according to the present invention has or is at risk of having cancer.

[0101] The compounds, compositions and methods of the present invention are particularly useful in enhancing pyruvate-dependent OXPHOS of CD8+ T cells, and therefore in combination with immunotherapy as described herein.

[0102] All references cited herein are incorporated by reference in their entirety. The present invention is not limited in scope by the specific embodiments and drawings described herein, which are intended as single illustrations of individual aspects of the invention; functionally equivalent methods and components are within the scope of the invention. The examples illustrating the invention are not intended to limit the scope of the invention in any way. [Example]

[0103] The following studies were performed to support the efficacy of the compounds of the present invention.

[0104] Example 1: Preparation of the Fc fusion protein IL-10 / Fc of the present invention We engineered recombinant human IL-10 and IgG1 Fc fusion protein (IL-10 / Fc of SEQ ID NO: 4) (Fig. 1a), which can cross-react with mouse or human IL-10 receptor (IL-10R) via the IL-10 domain of the IL-10 / Fc fusion protein (Qiao et al., 2019, Cancer Cell 35, 901-915.e4). Thus, the IL-10 / Fc fusion protein may exert its function on both mouse and human CD8+ T cells.

[0105] The IL-10 / Fc fusion protein was expressed in HEK293 free-style cells using commercially available mammalian expression vectors such as pcDNA3.1 or pSectag2A harboring the IL-10 / Fc fusion gene as previously described in Guo et al., 2012, supra, or Zheng et al., 1997, J. Immunol., 158, 4507-4513, or Steele et al., 1995, J. Immunol., 154, 5590-5600. After 7 days of culture, the culture supernatant containing IL-10 / Fc was collected by centrifugation.

[0106] The IL-10 / Fc fusion protein was first captured on a Protein A column and then further purified on a Superdex200 increase column. The purified IL-10 / Fc fusion protein was aliquoted and stored at -80°C for stock. SDS-PAGE and HPLC analysis demonstrated that the purity of IL-10 / Fc reached 95%. Using a HiTrap Protein A affinity chromatography column, recombinant IL-10 / Fc was captured from the clarified expression supernatant, which was filtered through a 0.22 μm membrane, washed with five column volumes of binding buffer, and eluted with elution buffer (0.05 M sodium citrate, 0.3 M NaCl, pH 3.0). The eluted protein was immediately collected in neutralization buffer (1.0 M Tris-HCl, pH 10.0). The eluted protein was concentrated by 10 kDa membrane ultrafiltration (Vivaspin) and further purified by Superdex200 increase size exclusion chromatography using PBS at a flow rate of 1.0 mL / min, and the purified protein was aliquoted and stored at -80°C.

[0107] Example 2: Role of the fusion protein of the invention, IL-10 / Fc, in IL-10-mediated metabolic reprogramming In a co-culture system of B16F10 mouse melanoma cells and activated Pmel CD8+ T cells that recognize the gp100 cognate antigen, the basal and maximal oxygen consumption rates (OCR) of CD8+ T cells were increased upon treatment with the fusion protein IL-10 / Fc of the present invention, whereas the extracellular acidification rate (ECAR) remained unchanged (Figures 2a-c).

[0108] The ratio of OCR to EACR in CD8+ T cells treated with IL-10 / Fc significantly increased (Figure 2d), suggesting that IL-10 signaling actively promotes T cell oxidative phosphorylation (OXPHOS). As a result, T cell numbers and cytotoxicity were significantly improved (Figure 2e and f). Importantly, this metabolic reprogramming effect was not observed in CD8+ T cells without antigen stimulation (Figure 2d), indicating that IL-10-mediated metabolic reprogramming is dependent on TCR signaling. Similarly, the fusion protein IL-10 / Fc was also found to promote OXPHOS in CD8+ T cells during the priming phase and promote T cell proliferation (Figure 1).

[0109] Next, we probed the pathway using several pathway-specific inhibitors, such as 2-deoxy-D-glucose, etomoxir, or UK5099 (Hildyard et al., 2005, Biochim. Biophys. Acta-Bioenerg., 1707, 221-230). Resting Pmel T cells at 1 million / ml were restimulated with soluble αCD3 tetramer (0.1 μg / ml) with or without the indicated inhibitors for 2 days, and then T cell counts were analyzed by FACS assay (Figure 2g). We next found that inhibition of glucose uptake by 2-deoxy-D-glucose (2DG) significantly impaired the effect of IL-10 / Fc on CD8+ T cell proliferation (Figure 2h). Glucose restriction also impaired the effect of IL-10 / Fc on CD8+ T cell proliferation (Figure 1e). Interestingly, inhibition of fatty acid-dependent oxidation by ETO further enhanced IL-10 / Fc-induced OXPHOS (Fig. 2i). These results may indicate that the Fc-fusion proteins of the present invention increased OXPHOS dependent on pyruvate oxidation rather than fatty acid β-oxidation, and were further confirmed by applying the mitochondrial pyruvate carrier (MPC) inhibitor UK5099, which nearly abolished the effects of IL-10 / Fc on T cell proliferation and metabolic reprogramming (Fig. 2h and i).

[0110] These data support that the fusion proteins of the invention can reprogram T cell metabolism and promote T cell proliferation by increasing OXPHOS upon TCR stimulation in a pyruvate-dependent manner, which may indicate that the fusion proteins of the invention may also promote the proliferation of tumor-reactive CD8+ T cells in the TME by reprogramming T cell metabolism.

[0111] Example 3: In vivo antitumor effect of the fusion protein of the present invention Enhancing OXPHOS or inhibiting glycolytic metabolism in CD8+ T cells using various reagents promoted the proliferation, memory development, and antitumor function of CD8+ T cells in the TME (Zhang et al., 2017, Cancer Cell 32, 377-391.e9; Chowdhury et al., 2018, Cancer Immunol. Res. 6, 1375-1387; Sukumar et al., 2013, 123, 4479-4488). Based on the observed metabolic regulatory function of the Fc fusion proteins of the present invention on CD8+ T cells, we investigated whether in vivo metabolic intervention of CD8+ T cells could be achieved with IL-10 / Fc, considering the potential for enhancing the efficacy of adoptive T cell immunotherapy against solid tumors.

[0112] To overcome T cell exhaustion in the TME, we promoted the in vivo antitumor effects of IL-10 / Fc in combination with ACT of TCR-transgenic CD8+ T cells (Pmel CD8+ T cells or OTI CD8+ T cells) or HER2 CAR-T cells in several tumor models, such as B16F10 (a murine melanoma model with low immunogenicity and high aggressiveness), YUMM1.7-OVA (a murine melanoma model), or MC-38-HER2 (a murine colon carcinoma), respectively.

[0113] The treatment scheme shown in Figure 3a was implemented in a B16F10 mouse melanoma model. Treatment with IL-10 / Fc or ACT cells alone was observed to slightly suppress tumor growth compared with the PBS group, but most mice eventually died from increasing tumor burden (Figure 4a, Figures 3b-d). Surprisingly, combined treatment with IL-10 / Fc and ACT therapy (ACT administered intravenously, followed by IL-10 / Fc administered intratumorally) induced significant tumor regression, resulting in permanent cure in 90% of B16F10 mouse melanoma-bearing mice (Figure 4b, Figure 3e). Even with reduced frequency of IL-10 / Fc administration (Figure 3f), the combined therapy with IL-10 / Fc and ACT demonstrated comparable efficacy (Figures 3j-g). Notably, approximately 80% (11 / 14) of treated long-term survivors rejected secondary B16F10 tumor cell inoculation (100,000 tumor cells per mouse) 3 months after the final injection (Figure 4c).

[0114] To test the robustness of the combination therapy, IL-10 / Fc treatment was combined with OT-ICD8+ T cells in the treatment of an OVA-expressing murine melanoma model (YUMM1.7-OVA) (Meeth et al., 2016, Pigment Cell Melanoma Res. 29, 590-597; Lane et al., 2018, J. Exp. Med. 215, 3057-3074). Tumor cells (1 million tumor cells per mouse) were injected subcutaneously, and high tumor burden (size >60 mm) was allowed to develop before treatment began. 2 or 150mm 3 ) (Pai et al., 2019, Immunity, 50, 477-492.e8). Administration of IL-10 / Fc and OT-ICD8+ T cell ACT induced significant tumor regression, whereas OT I CD8+ T cell ACT alone only showed transient tumor regression (Fig. 4d). Furthermore, 60% (5 / 8) of mice in the IL-10 / Fc and OT I CD8+ T cell ACT treatment group were cured, whereas no mice were cured in the ACT alone group (Fig. 4e).

[0115] The combination therapy was also tested with chimeric antigen receptor T (CAR-T) cells, and in vivo antitumor experiments were performed using MC-38, a murine colon adenocarcinoma expressing human epidermal growth factor receptor 2 (HER2), transduced with a retroviral construct containing the human HER2 coding sequence.

[0116] We observed that IL-10 / Fc and HER2 CAR-T cells strongly suppressed the growth of MC-38-HER2 tumors (Fig. 4f), and in the combination therapy group, 80% (4 / 5) of tumor-bearing mice were ultimately cured, whereas HER2 CAR-T cells themselves had a very weak therapeutic effect, with no mice surviving at the end of the experiment (Fig. 4g).

[0117] These data support that the fusion proteins of the invention can enhance adoptive T cell therapy to eradicate established tumors by promoting T cell-mediated tumor regression in both syngeneic and xenograft mouse models with pre-established solid tumors.

[0118] Example 4: In vivo role of the fusion protein of the present invention on immune infiltration of tumors To understand the ability of IL-10 / Fc to dramatically improve the efficacy of ACT, tumor immune infiltration was analyzed in the B16F10 tumor model (Fig. 5a).

[0119] B16F10 tumor cells (1 × 10 6 ) was subcutaneously inoculated into C57B1 / 6 mice and allowed to establish tumors for 6 days. Mice were then treated with PBS, IL-10 / Fc, 5 × 10 6 Mice received single adoptive transfer (ACT) of activated pmel-1 CD8+ T cells or a combination of IL-10 / Fc and ACT (combo) on day 6. Mice were injected four times with IL-10 / Fc or PBS, as shown in the experimental scheme. Mice were then sacrificed on day 14, and TILs were analyzed by flow cytometry.

[0120] IL-10 / Fc significantly increased tumor-infiltrating T cells, particularly CD8+ T cells, in the TME in the group treated with the combination therapy compared with ACT alone (Figure 6a), but did not increase natural killer (NK) cells or dendritic cells (DCs) (Figure 5b). This result was confirmed by immunofluorescence staining of tumor sections (Figure 6b).

[0121] Furthermore, IL-10 / Fc significantly decreased the proportion of Tregs and increased the ratio of CD8+ / Treg cells in the TME (Fig. 5c).

[0122] These data indicated that IL-10 / Fc and ACT synergistically remodel the immune cell composition of the TME, particularly CD8+ T cells, to enhance antitumor immunity. Furthermore, IL-10 / Fc treatment significantly enhanced the polyfunctionality of both endogenous and adoptively transferred Pmel CD8+ T cells within the tumor (Figure 6h).

[0123] To determine which subsets of CD8+ T cells respond to IL-10 / Fc treatment and contribute to its significantly enhanced efficacy, we stained tumor-infiltrating CD8+ T cells for activated / exhausted markers. TILs isolated from tumor tissue were stained with anti-mouse CD45.2, CD4, CD8, PD-1, and TIM-3 fluorescent antibodies and analyzed by FACS assay. It has recently been reported that PD-1+TIM-3+CD8+ T cells, previously defined as exhausted, are in fact a highly proliferative, clonal, and dynamically differentiating population in the highly cytotoxic human tumor microenvironment (Miller et al., 2019, Nat. Immunol. 20, 326-336; Li et al., 2019, Cell. 176, 775-789.el8). These results motivated us to investigate the potential metabolic reprogramming effects of IL-10 / Fc treatment on this specific subset of PD-1+TIM-3+CD8+ T cells.

[0124] This specific population observed in vivo was obtained by using anti-CD3 TCR triggering in vitro, which mimics sustained antigen stimulation in vivo. One million / ml of resting Pmel T cells were restimulated with soluble αCD3 tetramer (0.1 μg / ml) for 2 days.

[0125] It was observed that IL-10 / Fc treatment significantly increased the proportion of the PD-1+Tim-3+ subset among CD8+T cells, which have a similar phenotype of “exhausted T cells” (Fig. 6c).

[0126] Further analysis showed that IL-10 / Fc selectively expanded PD-1+Tim-3+, but not other subsets, among both endogenous (5.9-fold) and adoptively transferred Pmel CD8+ T cells (12.9-fold) within the tumor (Fig. 6d and e). Consistent with this observation, IL-10 receptor expression was highly upregulated in the PD-1+Tim-3+CD8+ T subset in the TME (Fig. 6f).

[0127] Although the PD-1+Tim-3+ subset has traditionally been considered "exhausted T cells," we noted that this subset in the combination treatment group showed reduced PD-1 expression levels on both endogenous and PMEL CD8+ T cells (Figure 6g), which may indicate reactivation of effector function. Consistent with our speculation, polyfunctional effector cells within the PD-1+Tim-3+CD8+ T cell subset were significantly increased by IL-10 / Fc treatment (Figure 6i). A high proportion of intratumoral PD-1+Tim-3+CD8+ T cells exhibited high cytotoxicity and proliferative capacity (Figures 6j and k). Further analysis indicated that PD-1+Tim-3+CD8+ T cells acquired a memory phenotype in central lymphoid organs, which may contribute to the development of memory CD8+ T cells (Figure 6l).

[0128] Consistently, both the number and killing efficiency of PD-1+Tim-3+CD8+ T cells were increased by IL-10 / Fc treatment in the T cell and B16F10 coculture system (Figures 6j and k).

[0129] As expected, the OCR of the PD-1+Tim-3+CD8+ T subset was significantly increased by IL-10 / Fc treatment (Fig. 6m), indicating that the PD-1+Tim-3+CD8+ T subset preferentially used OXPHOS over glycolysis after metabolic reprogramming by IL-10 / Fc.

[0130] Taken together, these results indicate that metabolic intervention using the Fc-fusion proteins of the present invention effectively reactivates CD8+ TILs, particularly tumor-infiltrating PD-1+Tim-3+CD8+ T cells (cytotoxic T cells), by increasing OXPHOS, and they regain / maintain their cytokine secretion, proliferation, and long-term memory formation. The PD-1+TIM-3+CD8+ T cell subset expanded to 12.9- and 5.9-fold greater numbers in adoptively transferred and endogenous CD8+ T cells, respectively, compared to mice not receiving IL-10 / Fc treatment.

[0131] Similarly, IL-10 / Fc also enhanced the efficacy of checkpoint blockade therapy against established solid tumors in mice (Figure 7). Combination therapy of IL-10 / Fc and α-PD-1 antibody significantly suppressed tumor growth and promoted survival in CT-26 tumor-bearing mice (Figures 7b-d).

[0132] Therefore, for personalized cancer immunotherapy, the Fc-fusion proteins of the present invention provide a novel metabolic intervention strategy for highly effective ACT cancer immunotherapy against solid tumors, such as TCR-T, CAR-T, and TILs isolated from tumor tissue. Furthermore, based on these data, the use of the Fc-fusion proteins of the present invention is considered a safe and potent potential enhancer for immune checkpoint inhibitor therapy.

[0133] [Table 2] [Table 3]

Claims

1. 1. An Fc fusion protein for use in the prevention and / or treatment of cancer, which is a homodimer of two polypeptides, each comprising (i) a non-cytolytic immunoglobulin IgG Fc domain and (ii) a heterologous polypeptide a comprising the sequence of human IL-10 as set forth in SEQ ID NO: 1 or a variant thereof, the variant has deletions and / or conservative substitutions of 1, 2, 3, 4, 5 or 6 amino acid residues; the heterologous polypeptide is covalently linked to the N-terminus or C-terminus of the Fc domain by a polypeptide linker, and the two heterologous polypeptides are non-covalently assembled into a homodimer; the polypeptide linker is a flexible hinge selected from the sequence of SEQ ID NO: 3, SEQ ID NO: 6, and a GS linker; The Fc fusion protein, wherein the non-cytolytic immunoglobulin IgG Fc domain is selected from the group consisting of or including the following i) to iv): i) a non-cytolytic IgG1 Fc fragment comprising the sequence of SEQ ID NO: 2 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues. ii) a non-cytolytic IgG2 Fc fragment comprising the sequence of SEQ ID NO: 5 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues. iii) a non-cytolytic IgG3 Fc fragment comprising the sequence of SEQ ID NO: 8 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues; and iv) a non-cytolytic IgG4 Fc fragment comprising the sequence of SEQ ID NO: 11 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

2. 2. The fusion protein for use according to claim 1, wherein the sequence of the fusion protein comprises the sequence of SEQ ID NO: 4 or a variant thereof, the variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

3. 3. A fusion protein for use according to claim 1 or 2, wherein the sequence of the fusion protein comprises the sequence of SEQ ID NO: 7 or a variant thereof, the variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

4. 2. The fusion protein for use according to claim 1, wherein the sequence of the fusion protein comprises the sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

5. 2. The fusion protein for use according to claim 1, wherein the sequence of the fusion protein comprises the sequence of SEQ ID NO: 13 or a variant thereof, wherein the variant comprises deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

6. The fusion protein for use in the treatment of cancer according to any one of claims 1 to 5, wherein said use in the prevention and / or treatment of cancer is anti-cancer immunotherapy.

7. 7. The fusion protein for use according to claim 6, wherein the anti-cancer immunotherapy is selected from ACT therapy or immune checkpoint inhibitor therapy.

8. The fusion protein for use according to claim 7, wherein the ACT therapy is selected from TCR-T, CAR-T or TIL therapy.

9. The fusion protein for use according to any one of claims 1 to 8, wherein the cancer is a solid tumor cancer.

10. 10. The fusion protein for use according to claim 9, wherein the solid tumor cancer is lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, gastric cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer and brain cancer or skin cancer.

11. The fusion protein for use according to claim 10, wherein the skin cancer is melanoma.

12. A pharmaceutical composition comprising at least one Fc fusion protein which is a homodimer of two polypeptides each comprising (i) a non-cytolytic immunoglobulin IgG Fc domain and (ii) a heterologous polypeptide a comprising the sequence of human IL-10 as set forth in SEQ ID NO: 1 or a variant thereof, wherein the heterologous polypeptide is covalently linked to the N-terminus or C-terminus of the Fc domain by a polypeptide linker, and the two heterologous polypeptides are non-covalently organized within the homodimer, a pharmaceutically acceptable carrier, diluent or excipient therefor, and at least one agent useful for anti-cancer immunotherapy, the variant has deletions and / or conservative substitutions of 1, 2, 3, 4, 5 or 6 amino acid residues; the polypeptide linker is a flexible hinge selected from the sequence of SEQ ID NO: 3, SEQ ID NO: 6, and a GS linker; The pharmaceutical composition as described above, wherein the non-cytolytic immunoglobulin IgG Fc domain is selected from the group consisting of or including the following i) to iv): i) a non-cytolytic IgG1 Fc fragment comprising the sequence of SEQ ID NO: 2 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues. ii) a non-cytolytic IgG2 Fc fragment comprising the sequence of SEQ ID NO: 5 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues. iii) a non-cytolytic IgG3 Fc fragment comprising the sequence of SEQ ID NO: 8 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues; and iv) a non-cytolytic IgG4 Fc fragment comprising the sequence of SEQ ID NO: 11 or a variant thereof, said variant comprising deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

13. 13. The pharmaceutical composition of claim 12, wherein the sequence of the Fc fusion protein comprises the sequence of SEQ ID NO: 4, 7, 10, 13 or a variant thereof, wherein the variant comprises deletion and / or conservative substitution of 1, 2, 3, 4, 5 or 6 amino acid residues.

14. 14. A pharmaceutical composition according to claim 12 or 13 for use in the treatment of cancer, in particular adoptive cell transfer (ACT) therapy.

15. The pharmaceutical composition for use according to claim 14, wherein the ACT therapy is selected from TCR-T, CAR-T or TIL therapy.

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