IL-21 fusion proteins useful as immunotherapy enhancers

Abstract

The present invention relates generally to the field of anti-cancer therapy, and in particular to the use of drugs or adjuvants useful in anti-cancer immunotherapy, such as adoptive T cell transfer (ACT) immunotherapy and immune checkpoint inhibition. The present invention provides a fusion protein for use in the prevention and / or treatment of cancer, the fusion protein comprising (i) an immunoglobulin IgG Fc domain or a human serum albumin (HSA) polypeptide and (ii) a polypeptide comprising the sequence of an interleukin-21 (IL-21) polypeptide, a fragment thereof, or a variant thereof, wherein the IL-21 polypeptide, a fragment thereof, or a variant thereof is covalently fused to the N-terminus or C-terminus of the IgG Fc domain or the HSA polypeptide via a linker.

Description

[Technical Field]

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

[0002] Natural killer (NK) cells are a type of cytotoxic lymphocyte belonging to the innate immune system that have the ability to eliminate infectious and cancerous cells independent of major histocompatibility complex (MHC) restriction (Wolf, NK et al., 2022). NK cell-based therapy is considered an effective and safe treatment, with impressive in vitro data reported on NK cell cytotoxicity against cancer.

[0003] However, clinical trials have not yet shown satisfactory results, as NK cells acquire higher cytotoxicity despite gradually losing stemness and homeostasis during differentiation and maturation (Bald, T. et al. 2020). In particular, in the tumor microenvironment (TME), terminally differentiated NK cells partially lose their killing ability and are unable to eliminate cancer (Li, Z.-Y. et al. 2021). The immunosuppressive microenvironment impedes NK cell function through various physiological factors, such as hypoxia and nutrient deprivation (Bi, J. & Tian, ​​Z. 2017). Maintaining the persistence of NK cells in the TME ensures their sustainable cytotoxicity against cancer cells.

[0004] NK cell metabolism meets the biosynthetic and energy demands for survival, proliferation, and specialized functions (O'Brien, KL & Finlay, DK 2019). Therefore, metabolically maintaining the stem-like characteristics of tumor-infiltrating NK cells is a potential target for enhancing their antitumor activity and therapeutic efficacy against cancer.

[0005] Interleukin-21 (IL-21) is a common cytokine receptor gamma chain family involved in regulating the differentiation of NK cells and their multiple functions (Parrish-Novak, J. et al. 2000). Here, we report that engineered IL-21 (IL-21 / Fc) preserves NK cell stemness, restores their antitumor activity in the TME, and, when combined with IL-15SA treatment, enhances efficacy against established solid tumors in multiple syngeneic tumor-bearing mouse models.

[0006] Therefore, there remains an urgent need to engineer processes and molecules that support immune cell metabolic fitness, proliferation, and survival within the TME and to develop effective immune cells with enhanced anti-tumor activity. Summary of the Invention

[0007] The present invention provides a fusion protein for use in the prevention and / or treatment of cancer, the fusion protein comprising (i) an immunoglobulin IgG Fc domain or a human serum albumin (HSA) polypeptide and (ii) a polypeptide comprising the sequence of an interleukin-21 (IL-21) polypeptide, a fragment or a variant thereof, wherein the IL-21 polypeptide, a fragment or variant thereof is covalently fused to the N-terminus or C-terminus of the IgG Fc domain or the HSA polypeptide via a linker.

[0008] Further provided is a fusion protein comprising (i) an immunoglobulin IgG Fc domain or a human serum albumin (HSA) polypeptide and (ii) a polypeptide comprising the sequence of an interleukin-21 (IL-21) polypeptide, a fragment or a variant thereof, wherein the IL-21 polypeptide, a fragment or variant thereof is covalently fused to the N-terminus or C-terminus of the IgG Fc domain or the HSA polypeptide via a linker.

[0009] Additionally provided are nucleic acids encoding one or more fusion proteins according to the invention.

[0010] Additionally provided is a plasmid or vector comprising a nucleic acid according to the invention.

[0011] Also provided is an isolated host cell or population of cells comprising a plasmid or vector according to the invention.

[0012] Also provided is a pharmaceutical composition comprising i) a fusion protein for use according to the invention, ii) a fusion protein according to the invention, iii) a plasmid or vector according to the invention, or iv) an isolated host cell or cell population according to the invention, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0013] Also provided is a method for treating and / or preventing cancer in a subject, comprising administering a pharmaceutical composition of the present invention.

[0014] Also provided are methods of treatment and / or prevention comprising: (i) removing and isolating immune cells, preferably natural T cells, from a patient or subject; (ii) genetically engineering the T cells to encode a chimeric antigen receptor (CAR), T cell receptor (TCR) or any other synthetic tumor-targeting motif or antigen; (iii) expanding the engineered T cells ex vivo to a larger population; and (iv) reintroducing the engineered T cells into the patient or subject.

[0015] Also provided is a method of enhancing ACT anti-tumor activity in a subject, comprising administering a pharmaceutical composition according to the invention. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram of a homodimerized IL-21 / IgG Fc fusion protein. [Figure 2]Characterization of IL-21 / Fc production and function. a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified IL-21 / Fc. βME, β-mercaptoethanol. b. Activated NK cells were cultured in the presence of IL-21 or IL-21 / Fc at the indicated concentrations for 24 hours. The fold change in IFN-γ expression of NK cells was normalized to NK cells treated with PBS. c. Pharmacokinetic profile and half-life of IL-21 or IL-21 / Fc. Blood from C57 / BL6J mice was collected at the indicated time points after a single intravenous injection (n = 5, data are mean ± SEM), and IL-21 concentrations were measured by ELISA. e. Killing efficacy of activated NK cells against B16F10_β2m (left) and CT26_β2m (right) cells at an E:T ratio of 0.5 in the presence of 100 ng / ml of IL-21 / Fc (n=5, data are mean ± SEM). f. Mean fluorescence intensity of CD107a, granzyme B, and IFN-γ expression of activated NK cells in the presence of 100 ng / ml of IL-21 / Fc. [Figure 3] IL-21 / Fc inhibits tumor growth and promotes IL-15SA antitumor activity. a. Experimental timeline. Mice were inoculated subcutaneously (sc) with B16F10_β2m melanoma cells (5 × 10 / mouse), CT26_β2m colon carcinoma cells (5 × 10 / mouse), or RMA-S lymphoma cells (8 × 10 / mouse). Treatment began on day 7, with eight intratumoral (it) injections of IL-21 / Fc (20 μg / mouse) every other day and / or two weekly it injections of IL-15SA (5 μg / mouse), with PBS as a control. b–d. Mean tumor area and survival curves for mice bearing B16F10_β2m melanoma (left), CT26_β2m colon carcinoma (center), and RMA-S lymphoma (right) (n = 8, data are mean ± SEM). [Figure 4]IL-21 combination therapy suppresses tumor growth. IL-21 / Fc combined with other therapies (other than ACT) for cancer immunotherapy demonstrates that the fusion protein enhances various immunotherapies. Mice were subcutaneously inoculated with B16F10 melanoma cells (5x10), followed by eight doses of IL-21 / Fc (20µg) injected every two days starting on day 7 in addition to the combination treatment. IL-15SA (5µg) was administered itally on days 7 and 14, three doses of anti-PD-1 (100µg) were injected i.p. every three days starting on day 7, and eight doses of R837 (1mg / kg) were administered subcutaneously every two days starting on day 7. Mean tumor area and survival curves for B16F10 melanoma-bearing mice (n=5, data are mean ± SEM). [Figure 5] The antitumor activity of IL-21 / Fc is dependent on NK cells in the tumor. a. Analysis of tumor-infiltrating immune cell subsets. BALB / c mice bearing CT26_β2m tumors received IL-21 / Fc (20 μg / mouse) every two days and / or IL-15SA (5 μg / mouse) weekly, starting on day 7. One week after treatment, tumors were resected, and tumor-infiltrating CD45+ lymphocytes (TILs), NK cells, CD8+ T cells, CD4+ T cells, B cells, dendritic cells (DCs), macrophages, neutrophils, and eosinophils were analyzed by FACS. b. Average tumor area in C57 / BL6J mice bearing B16F10_β2m tumors. Mice received IL-21 / Fc (20 μg / mouse) eight times weekly and IL-15SA (5 μg / mouse) twice weekly, starting on day 7. Three doses of anti-NK1.1 (PK136, BioXcell, 400 μg / mouse) were injected intraperitoneally (ip) every 4 days, one day before treatment (n=5, data are mean±SEM). [Figure 6]IL-21 / Fc enhances NK cell function by promoting glycolytic metabolism. a. Real-time analysis of extracellular acidification rate (ECAR). b. Mean basal glycolysis, glycolytic reserve, and the ratio of OCR (oxygen consumption rate) to ECAR in activated NK cells in the presence of IL-21 / Fc after 24 h of incubation. c. Changes in glycolysis-related gene expression in activated NK cells in the presence of IL-21 / Fc after 24 h of incubation. d. Ratios of changes in the frequency of granzyme B+ and IFN-γ+ in activated NK cells in the presence of IL-21 / Fc and the indicated inhibitors after 24 h of incubation: 2-DG, 2 mM; oligomycin, 1 μM. e-f. CT26_β2m tumor-bearing BALB / c mice received IL-21 / Fc (at 20 μg / mouse) every 2 days and / or IL-15SA (5 μg / mouse) weekly, starting from day 7. One week after treatment, tumors were excised and analyzed by FACS. (e) Expression frequency of glucose transporter 1 (Glut1) and MFI of 2-NBDG-uptake cells. (f) Expression frequency of granzyme B, IFN-γ, and TNF-α in tumor-infiltrating NKp46+ TILs. [Figure 7]IL-21 / Fc enriches NK cells, promotes stemness, and induces sustained protection against tumor reinfection. a. CT26_β2m tumor-bearing BALB / c mice received IL-21 / Fc (at 20 μg / mouse) every 2 days and / or IL-15SA (5 μg / mouse) weekly starting on day 7. Spleens and tumors were excised 1 week after treatment and analyzed by FACS. Representative flow cytometry plots showing the frequency of double-negative (DN), CD27+, and CD11b+ NK cell populations among all CD45.2+NKp46+ cells in the spleen (top) or tumor (bottom). b. Percentages of CD11b+ and CD27+ cells among CD45.2+NKp46+ cells in the spleen or tumor. c. Frequency of granzyme B+, IFN-γ+, and TNF-α+ among NKp46+ TILs. d. Frequency of CD27+ and Sca1+ populations in D45.2+NKp46+ tumor-infiltrating cells; MFI of TCF1 and Ki67 in NKp46 TILs. e. Survival curve and number of long-term surviving mice against reinfection. CT26_β2m tumor-bearing BALB / c mice or B16F10_β2m tumor-bearing C57BL / 6 mice were administered IL-21 / Fc (20 μg / mouse) and IL-15SA (5 μg / mouse) every 2 days. Surviving mice from the combination group were subcutaneously reinfected with CT26_β2m or B16F10_β2m, respectively, 90 days after the primary inoculation. [Figure 8] Human IL-21 / Fc promotes human NK cell function and glycolysis. a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human IL-21 / Fc (hIL-21 / Fc). βME, β-mercaptoethanol. b. Killing potency of activated NK92MI cells against K562 cells at the indicated E:T ratios in the presence of 100 ng / ml hIL-21 / Fc (n=5, data are mean ± SEM). c. Basal glycolysis of NK92MI cells in the presence of 100 ng / ml hIL-21 / Fc after 24 hours of incubation. [Figure 9]IL-21 / Fc enhances the killing efficiency of PBMC-derived human NK cells. Human NK cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting (MACS). Isolated human NK cells were activated and cultured for 5 days in the presence of human IL-21 (50 U / ml). Killing efficacy of activated NK cells against target cells in the presence of hIL-21 / Fc (100 ng / mL) at an E / T ratio of 0.5 after 5 hours of incubation. [Figure 10] IL-21 / Fc promotes the antitumor efficacy of transplanted human NK92MI cells against K562 human lymphoma in a xenograft model. Immunodeficient NSG mice were subcutaneously inoculated with K562 lymphoma cells (8 x 10). NK92MI cells (3 x 10) were transferred to tumor-bearing mice on day 7, followed by 8 injections of IL-21 / Fc (20 μg) every 2 days. Average tumor area and survival curves of K562 lymphoblastoid tumor-bearing mice (n = 7, data are mean ± SEM). DETAILED DESCRIPTION OF THE INVENTION

[0017] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0018] In case of conflict, the present specification, including definitions, will control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this specification belongs. As used herein, the following definitions are provided to facilitate the understanding of the present invention.

[0019] The term "comprise" is generally used in the sense of including, i.e., allowing for the presence of one or more features or components. The term "comprise(s)" encompasses the more specific terms "consist" and "consisting," respectively, as well as "consist essentially of."

[0020] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0021] As used herein, "at least one" means "one or more," "two or more," "three or more," etc.

[0022] As used herein, the terms "subject" / "subject in need thereof" or "patient" / "patient in need thereof" are well-recognized in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some cases, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other aspects, the subject may be a normal subject. The term does not designate a particular age or sex. Thus, adult and neonatal subjects, regardless of gender, are also intended to be encompassed. Preferably, the subject is a human, most preferably a human suffering from or at risk of suffering from cancer and / or cancer metastasis. The cancer may be a solid or liquid cancer.

[0023] In one aspect, the solid cancer is selected from the non-limiting group including 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, and skin cancer, particularly melanoma, or one or more combinations thereof.

[0024] In one particular embodiment, the cancer is an MHC-I expression altered cancer, preferably an MHC-I deficient solid cancer.

[0025] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any type of deoxyribonucleotide (e.g., DNA, cDNA, etc.) or ribonucleotide (e.g., RNA, mRNA, etc.) polymer, or combination of deoxyribonucleotide and ribonucleotide (e.g., DNA / RNA) polymers, in linear or circular conformation and in either single- or double-stranded form. These terms should not be construed as limiting with respect to the length of the polymer and can encompass known analogues of natural nucleotides as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.

[0026] The term "vector," as used herein, refers to a viral vector or nucleic acid (DNA or RNA) molecule (e.g., a plasmid or other vehicle) that contains one or more heterologous nucleic acid sequences of the invention and is preferably designed for transfer between different host cells. The terms "expression vector," "gene delivery vector," and "gene therapy vector" refer to any vector effective to incorporate and express one or more nucleic acids of the invention in a cell, preferably under the control of a promoter. A cloning vector or expression vector may contain additional elements in addition to a promoter, such as regulatory elements and / or post-transcriptional regulatory elements.

[0027] The term "about," particularly with respect to a given amount, number, or percentage, is meant to encompass a variation of plus or minus ten percent (±10). For example, about 5% encompasses any value between 4.5% and 5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5, 4.1, 5.2, 5.3, 5.4, or 5.5.

[0028] As used herein, interleukin-21 (IL-21) refers to a member of the IL-21 family of cytokines. IL-21 is a common cytokine receptor gamma chain family involved in regulating the differentiation of NK cells and their multiple functions (O'Brien, KL & Finlay, DK 2019). "IL-21, a fragment or variant thereof" preferably includes sequences including the sequence of native human IL-21 as well as the sequences of fragments and variants thereof. In one embodiment, the IL-21 sequence is the human IL-21 amino acid sequence set forth in SEQ ID NO: 1.

[0029] The term "variant," when referring to IL-21, refers to one or more biologically active derivatives of IL-21, preferably the human IL-21 sequence of the present invention. Generally, the term "variant" refers to a molecule having a native sequence with one or more additions, substitutions (generally conservative in nature), and / or deletions compared to the native molecule, provided that the modifications do not destroy its biological activity and the molecule is "substantially homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). Generally, the sequence of such a variant has a high degree of sequence homology or identity to the reference sequence, e.g., greater than 25%, generally greater than 50% to 70%, and even more specifically, 80% or 85% or more, e.g., at least 90% or 95% or more, when the two sequences are aligned. Preferably, the reference sequence is the human IL-21 amino acid sequence shown in SEQ ID NO:1.

[0030] As used herein, a "fragment" of IL-21, preferably human IL-21, of the present invention refers to a sequence that is shorter in amino acid or nucleotide length than the respective polypeptide or nucleic acid sequence. Preferably, the polypeptide sequence or fragment comprises less than 90%, preferably less than 60%, in particular less than 30% of the nucleotide length of the respective polypeptide or nucleic acid sequence, e.g., the human IL-21 amino acid sequence shown in SEQ ID NO: 1.

[0031] Focusing on the development of novel and efficient approaches to treat tumors, the present inventors surprisingly showed that an engineered IL-21 / IgG Fc fusion protein (IL-21 / Fc) exhibits increased half-life and promotes NK cell cytotoxicity and stemness through metabolic modulation, thus enhancing the efficacy of NK cells against established tumors, particularly MHC-I-deficient tumors. These promising results overcome major barriers to current NK cell-based immunotherapy in the clinic and provide a novel and efficient approach to improve response rates.

[0032] In one aspect, the present invention provides a fusion protein comprising: (i) a polypeptide comprising the sequence of an interleukin-21 (IL-21) polypeptide, a fragment or a variant thereof; and (ii) a molecule that extends the half-life of the IL-21 polypeptide, a fragment or a variant thereof.

[0033] As used herein, a molecule that increases the half-life of an IL-21 polypeptide, a fragment or a variant thereof is preferably selected from the group comprising the Fc domain of IgG and human serum albumin (HSA), such as a sequence comprising or consisting of SEQ ID NO: 17, any one of its fragments or variants.

[0034] As used herein, the Fc domain of IgG is preferably the silent Fc domain of immunoglobulin (Ig) G, preferably mouse or human IgG, most preferably human IgG1, IgG2, IgG3, or IgG4, a fragment or variant thereof. In one aspect, the Fc domain of human IgG is selected from the group comprising: IgG1 Fc (SEQ ID NO: 2), IgG2 Fc (SEQ ID NO: 8), IgG3 Fc (SEQ ID NO: 11), and IgG4 Fc (SEQ ID NO: 14), a fragment, variant, or a combination of one or more of these sequences.

[0035] In one embodiment, IL-21, a fragment, or variant thereof, is covalently fused to the N- or C-terminus of an Fc domain or HAS polypeptide by or through a linker, e.g., a polypeptide linker. In one embodiment, the polypeptide linker is a stretch of predominantly Gly and Ser residues (a "GS" linker) or Gly-Gly and Ser residues (a "GGS" linker), followed or not by one or more Arg residues (an "R" residue). Typically, the linker comprises 10-30 amino acids, preferably 10-25 amino acids, and more preferably 15-25 amino acids. Non-limiting examples of GGS and GGGGS linkers are disclosed herein.

[0036] According to one embodiment, the IgG Fc domain may be an Fc domain obtained from a murine IgG1, IgG2a, IgG2b and / or IgG3 isoform, or a variant of said fragment.

[0037] According to one embodiment, the IgG Fc domain may be an Fc domain obtained from a human IgG1, IgG2, IgG3 and / or IgG4 isoform, or a variant of said fragment.

[0038] In one embodiment, the IgG Fc domain of the fusion protein dimerizes with a second IgG Fc domain, thereby forming a homodimer, and the second IgG Fc domain is covalently fused via its N- or C-terminus to an IL-21 polypeptide, fragment, or variant thereof. This homodimerization occurs through a non-covalent bond between the first and second IgG Fc domains, thereby extending the half-life of the fusion protein. Typically, the first and second IgG Fc domains are similar (e.g., two human IgG1, two human IgG2, ...).

[0039] The term "variant," when referring to an IgG Fc fragment, refers to one or more biologically active derivatives of an IgG Fc fragment, preferably a human IgG Fc fragment sequence of the present invention. Generally, the term "variant" refers to a molecule having a native sequence with one or more additions, substitutions (generally conservative in nature), and / or deletions compared to the native molecule, provided that the modifications do not destroy its biological activity and the molecule is "substantially homologous" to the reference molecule. Generally, the sequence of such a variant has a high degree of sequence homology or identity to the reference sequence, e.g., greater than 25%, generally greater than 50% to 70%, and even more specifically, 80% or 85% or more, e.g., at least 90% or 95% or more, when the two sequences are aligned. Preferably, the reference sequence is a human IgG Fc fragment amino acid sequence set forth in any one of the sequences IgG1 Fc (SEQ ID NO: 2), IgG2 Fc (SEQ ID NO: 8), IgG3 Fc (SEQ ID NO: 11), and IgG4 Fc (SEQ ID NO: 14), or a fragment or combination of one or more of these sequences.

[0040] Mutants of IgG Fc fragments can be mutated to reduce antibody-dependent cell-mediated cytotoxicity (ADCC), as described, for example, in Czajkowsky et al., 2012, EMBO Mol. Med, 1015-1028, or to improve the half-life or in vivo levels of IgG (e.g., IL-10 / Fc), as described, for example, in Zalevsky et al., 2010, Nat. Biotechnol. 28, 157-159 and Vaccaro et al., 2005, Nat. Biotechnol. 23, 1283-1288.

[0041] Point mutations can also be introduced into an IgG Fc fragment, e.g., into an IgG1 Fc domain as described in Armour et al., 1999, Eur. J. Immunol. 29, 2613-2624 or Zheng XX et al., 1995, J. Immunol. 154, 5590-5600, to generate a non-cytolytic IgG1 Fc domain.

[0042] According to an embodiment of the invention, the variant of SEQ ID NO: 2 comprises at least one mutation selected from C220A, L234A, L235A and P329G. In a further embodiment of the invention, the variant of SEQ ID NO: 2 comprises at least two, at least three or at least four mutations selected from C220A, L234A, L235A and P329G. The positions of these mutations are indicated with reference to the complete human IgG1 sequence.

[0043] According to an embodiment of the invention, the variant of SEQ ID NO: 8 comprises at least one mutation selected from A330S and P331S. In a further embodiment of the invention, the variant of SEQ ID NO: 8 comprises at least two mutations selected from A330S and P331S. The positions of these mutations are indicated with reference to the complete human IgG2 sequence.

[0044] According to an embodiment of the invention, the variant of SEQ ID NO: 14 comprises at least one mutation selected from S228P and L235E. In a further embodiment of the invention, the variant of SEQ ID NO: 14 comprises at least two mutations selected from S228P and L235E. The positions of these mutations are indicated with reference to the complete human IgG4 sequence.

[0045] According to certain embodiments, the Fc-fusion proteins and HSA-fusion proteins of the invention can alternatively be modified to further extend their half-life in vivo by standard strategies, including pegylation (e.g., pegylation of the human IL-21 sequence as described in Mumm et al., 2011). The Fc domain of the Fc-fusion protein IL-21 / Fc of the invention can also be replaced with an antibody or human serum albumin or a variant thereof, as described or reviewed in Qiao, et al., 2019, Cancer Cell 35, 901-915.e4; Kontermann, 2011, Curr. Opin. Biotechnol., 22, 868-876.

[0046] In one aspect the IL-21 is a murine IL-21 sequence comprising or consisting of SEQ ID NO: 20, a fragment or variant thereof.

[0047] In one aspect the IL-21 is a human IL-21 sequence comprising or consisting of SEQ ID NO: 1, a fragment or variant thereof.

[0048] Non-limiting examples of fusion proteins of the invention comprise or consist of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22, a fragment or variant of any one of the sequences.

[0049] The term "variant," when referring to a fusion protein, refers to one or more biologically active derivatives of the fusion protein, preferably of the sequences described herein. Generally, the term "variant" refers to a molecule having a native sequence with one or more additions, substitutions (generally conservative in nature), and / or deletions compared to the native molecule, provided that the modifications do not destroy its biological activity and the molecule is "substantially homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). Generally, the sequence of such a variant has a high degree of sequence homology or identity to the reference sequence, e.g., greater than 25%, generally greater than 50% to 70%, and even more specifically, 80% or 85% or more, e.g., at least 90% or 95% or more, when the two sequences are aligned.

[0050] As used herein, a "fragment" of a fusion protein of the invention refers to a sequence of amino acids shorter in length than the respective polypeptide sequence. Preferably, the polypeptide sequence or fragment comprises less than 90%, preferably less than 60%, and in particular less than 30% of the nucleotides in length of the respective polypeptide sequence as described herein.

[0051] In one aspect, the fusion proteins described herein are for use in the prevention and / or treatment of cancer. Preferably, the cancer is a solid or liquid cancer. More preferably, the cancer is a solid cancer selected from the group including 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, and skin cancer, particularly melanoma, or a combination of one or more thereof.

[0052] In one embodiment, the cancer is an MHC-I expression-altered cancer (e.g., downregulation or complete loss of MHC-I expression), preferably an MHC-I-deficient solid cancer. Alterations in MHC class I (MHC-I) expression are a frequent event during cancer progression, allowing tumor cells to evade the immune system. MHC-I expression-altered cancers are discussed, for example, in Cornell, AM, Mimpen, IL, & Nierkens, S. (2020). MHC Class I Downregulation in Cancer: Underlying Mechanisms and Potential Targets for Cancer Immunotherapy. Cancers, 12(7), 1760.

[0053] In one aspect, the fusion proteins described herein are used in combination with another cancer therapy. Preferably, the other cancer therapy is an anti-cancer immunotherapy selected from the group including ACT therapy, immune checkpoint blockade therapy, cytokine therapy, cancer vaccine therapy, bispecific antibody therapy, and other cancer immunotherapies (such as chemotherapy, radiation therapy, and hormone therapy), or one or more combinations thereof.

[0054] Chemotherapy according to the present invention may involve agents that damage DNA and / or prevent cells from proliferating, eg, genotoxic agents.

[0055] The genotoxic agent may be selected from the group consisting of alkylating agents, antimetabolites, DNA cutters, DNA binders, topoisomerase poisons and spindle poisons. Examples of alkylating agents are lomustine, carmustine, streptozocin, mechlorethamine, melphalan, uracil, nitrogen mustard, chlorambucil, cyclosphamide, ifosfamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazine, procarbazine, hexamethylmelamine, triethylenemelamine, busulfan, pipobroman, mitotane and other platin derivatives.

[0056] An example of a DNA cutter is bleomycin.

[0057] The topoisomerase poison may be selected from the group including topotecan, irinotecan, camptothecin sodium salt, daurubicin, doxorubicin, idarubicin, mitoxantrone teniposide, adriamycin and etoposide.

[0058] Examples of DNA binders are dactinomycin and mithramycin, while spindle poisons can be selected from the group including vinblastine, vincristine, navelbine, paclitaxel and docetaxel.

[0059] The chemotherapy of the present invention may involve an antimetabolite selected from the following compounds: methotrexate, trimetrexate, pentostatin, cytarabine, ara-CMP, fludarabine phosphate, hydroxyurea, fluorouracil, phyoxuridine, chlorodeoxyadenosine, gemcitabine, thioguanine, and 6-mercaptopurine.

[0060] Radiation therapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, but are not limited to, external radiation therapy and internal radiation therapy (also called brachytherapy).

[0061] External radiation therapy is the most common and typically involves directing a beam of direct or indirect ionizing radiation at the tumor or cancer site. The radiation beam, photons, cobalt, or microparticle therapy, is focused on the tumor or cancer site, but it is almost impossible to avoid exposing normal, healthy tissue. The energy source for external radiation therapy is selected from the group including direct or indirect ionizing radiation (e.g., x-rays, gamma rays, and particle beams, or a combination thereof).

[0062] Internal radiation therapy involves implanting radiation-emitting sources, such as beads, wires, pellets, or capsules, inside the body or at or near the tumor site. The energy source for internal radiation therapy is selected from the group of radioisotopes, including iodine (Iodine-125 or Iodine-131), strontium-89, phosphorus, palladium, cesium, indium, phosphate, or cobalt radioisotopes, and combinations thereof. Such implants can be removed after treatment or left inactive in the body. Types of internal radiation therapy include, but are not limited to, interstitial and intracavitary brachytherapy (high-dose rate, low-dose rate, pulsed-dose rate).

[0063] Currently less common forms of internal radiation therapy involve biological carriers of radioisotopes, such as by radioimmunotherapy, in which tumor-specific antibodies conjugated to radioactive material are administered to the patient or subject. The antibodies bind to tumor antigens, thereby effectively administering a dose of radiation to the relevant tissue.

[0064] Methods for administering radiation therapy are well known to those skilled in the art.

[0065] A variety of other additional therapeutic agents can be used in conjunction with the compositions described herein.

[0066] Additional therapeutic agents suitable for use in combination with the present invention include ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), immature leukemia (IMMUNO), leukemia (LEKT® ... Tetuximab (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sucralose ... Nitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaquinanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pazopanib, regorafenib, semaquinanib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pazopanib, sunitin ... These include, but are not limited to, mTOR inhibitors such as critinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as the CDK inhibitor (palbociclib).

[0067] In a further embodiment, the additional therapeutic agent may be an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, and methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or proporxifene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), TNF antagonists, cytokine inhibitors such as etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0068] ACT therapy, as used herein, is selected from the non-limiting group including T cells, chimeric antigen receptor (CAR)-T cells, T cell receptor (TCR)-transgenic T cells, tumor infiltrating lymphocytes (TIL), NK cells, NK-T cells, CAR-NK cells, CAR-NKT cells, TCR-transgenic NK cells, TCR-transgenic NK-T cells, dendritic cells, macrophages, CAR-macrophages, or any synthetic tumor-specific immune cells. In a preferred embodiment, the ACT therapy is selected from the group including TCR-T, CAR-T, TIL, and NK cell therapy, or a combination of one or more thereof.

[0069] Non-limiting examples of ACT immunotherapies are listed in Fan et al., 2018, Theranostics, 8(20):5784-5800; Rosenberg et al., 2008, Nat. Rev. Cancer 8, 299-308.

[0070] Cytokine therapy, as used herein, is selected from the non-limiting group including GM-CSF, IFNγ, IL-7, IL-10, IL-12, IL-15, and fusion proteins thereof comprising a cytokine and an immunoglobulin IgG Fc domain, human serum albumin (HAS), or one or more combinations thereof.

[0071] In one aspect, the cytokine therapy is selected from the non-limiting group including IL-15, a fusion protein comprising IL-15, IL-15Rα and an immunoglobulin IgG Fc domain, or one or more combinations thereof.

[0072] In a preferred embodiment, a fusion protein comprising IL-15, IL-15Rα and an immunoglobulin IgG Fc domain is an IL-15 superagonist (see, e.g., Karin M. Knudson et al., Expert Opin Biol Ther. 2020).

[0073] NK cell therapy, as used herein, is selected from the non-limiting group including induced or activated NK cells, iPSC-NK cells, hESC-NK cells, CAR-NK cells, CB-NK cells and PBNK cells, or a combination of one or more thereof.

[0074] The NK cells may be autologous or allogeneic NK cells.

[0075] Induced or activated NK cells refer to cells that are cultured and expanded in vitro, usually in the presence of IL-2, and maintained until (re)injected into a patient in need thereof.

[0076] iPSC-NK cells refer to NK cells derived from induced pluripotent stem cells.

[0077] hESC-NK cells refer to NK cells derived from human embryonic stem cells.

[0078] CAR-NK cells refer to NK cells that have been engineered to express a chimeric antigen receptor (CAR) (see, e.g., Daher M, et al. Clin Transl Immunology. 2021 Apr 28;10(4)).

[0079] PBNK cells or PB-NK cells both refer to peripheral blood NK cells that are collected from donors by apheresis and expanded before use (Fujisaki H, et al., Cancer Res. 2009).

[0080] CB-NK cells are typically obtained and expanded from umbilical cord blood units (Shah N, et al., PloS One 2013).

[0081] Any source of NK cells is contemplated in the present invention. Currently, clinical-grade NK cells can be produced on a large scale from multiple sources, including the NK92 cell line, peripheral blood mononuclear cells (PBMCs), umbilical cord blood cells (UBCs), CD34+ hematopoietic progenitor cells (HPCs), and induced pluripotent stem cells (iPSCs).

[0082] Immune checkpoint blockade therapy, as used herein, includes an inhibitor selected from the group including a CTLA-4 inhibitor, a TIM3 inhibitor, a PD-1 inhibitor, a TIGIT inhibitor, a LAG inhibitor, and a PD-L1 inhibitor, or a combination of one or more thereof.

[0083] Non-limiting examples of PD-1 inhibitors include inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0084] Non-limiting examples of PD-L1 inhibitors include inhibitors such as atezolizumab, avelumab, AMP-224, MEDI-0680, RG-7446, GX-P2, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, CX-072, and BMS-936559.

[0085] Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly ticilimumab, CP-675, 206).

[0086] Non-limiting examples of TIGIT inhibitors include tiragolumab (MTIG7192A; RG6058), AB154 (Arcus Biosciences), MK-7684 (Merck), BMS-986207 (Bristol-Myers Squibb), ASP8374 (Astellas Pharma), and ASP8374 (Astellas Pharma).

[0087] In one aspect, a fusion protein for use described herein, e.g., an IL-21-Fc fusion protein, increases the efficacy of anti-cancer immunotherapy by about 2% or more, about 5% or more, about 20% or more, about 40% or more, about 60% or more, about 500% or more when compared to the efficacy of the anti-cancer treatment in the absence of the fusion protein (e.g., in the absence of the Fc-IL-21 fusion protein).

[0088] Additionally provided are nucleic acid sequences encoding one or more recombinant constructs of the invention, such as fusion proteins of the invention. Non-limiting examples of fusion proteins of the invention comprise or consist of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22, a fragment or variant of any one of the sequences.

[0089] Generally, the term "variant" refers to a molecule having a native sequence with one or more additions, substitutions (generally conservative in nature), and / or deletions compared to the native molecule, provided that the modifications do not destroy its biological activity and the molecule is "substantially homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). Generally, the sequence of such a variant has a high degree of sequence homology or identity to the reference sequence, e.g., greater than 25%, generally greater than 50% to 70%, and even more specifically, 80% or 85% or more, e.g., at least 90% or 95% or more, when the two sequences are aligned. Preferably, the reference sequence is selected from SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:22.

[0090] As used herein, a "fragment" refers to a sequence that is shorter in amino acid or nucleotide length than the respective polypeptide or nucleic acid sequence. Preferably, the sequence or fragment contains less than 90%, preferably less than 60%, and in particular less than 30% of the amino acid or nucleotide length of the respective polypeptide or nucleic acid sequence, such as SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22.

[0091] Additionally, a plasmid or vector comprising a nucleic acid sequence of the present invention is also contemplated.

[0092] For cloning of the polynucleotides of the present invention, the vector can be introduced into a host cell (autologous, allogeneic, or heterologous) to allow the vector to replicate itself, thereby amplifying copies of the polynucleotides contained therein. The cloning vectors of the present invention may generally contain sequence components, including, but not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements may be selected appropriately by those skilled in the art. For example, an origin of replication may be selected to promote autonomous replication of the vector in the host cell.

[0093] The term "autologous" refers to any material derived from the same individual that is later reintroduced.

[0094] The term "allogeneic" refers to any material derived from one individual and then introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0095] In certain aspects, the present disclosure provides isolated host cells or populations of cells containing the vectors or plasmids provided herein. Host cells or populations of cells containing the vectors or plasmids may be useful for expressing or cloning polynucleotides contained in the vectors. Suitable host cells can include, but are not limited to, oncolytic viruses, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Prokaryotic cells suitable for this purpose include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive bacteria, for example, Enterobacteriaceae, e.g., Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, as well as Bacillus, e.g., B. subtilis and B. licheniformis, Pseudomonas, e.g., P. aeruginosa, and Streptomyces.

[0096] Vectors or plasmids can be introduced into a host cell or cell population using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, biolistics, receptor-mediated gene delivery, polylysine, histones, chitosan, and peptide-mediated delivery. Standard methods for transfection and transformation of cells for expression of a vector or plasmid of interest are well known in the art.

[0097] The present invention also contemplates compositions and pharmaceutical compositions.

[0098] In an aspect of the invention, a pharmaceutical composition of the invention comprises a therapeutically effective amount of a fusion protein described herein, a pharmaceutically acceptable carrier, diluent and / or excipient.

[0099] In an aspect of the invention, a pharmaceutical composition of the invention comprises a therapeutically effective amount of a plasmid or vector described herein, a pharmaceutically acceptable carrier, diluent and / or excipient.

[0100] In an aspect of the invention, a pharmaceutical composition of the invention comprises a therapeutically effective amount of an isolated host cell or cell population described herein, a pharmaceutically acceptable carrier, diluent and / or excipient.

[0101] The pharmaceutical composition as described above may further comprise an anti-cancer immunotherapy as described herein, preferably a cytokine therapy, more preferably a cytokine therapy selected from the group comprising IL-15, a fusion protein comprising IL-15, IL-15, IL-15Rα and an immunoglobulin IgG Fc domain, or a combination of one or more thereof.

[0102] The pharmaceutical composition may further comprise, in addition to the fusion protein and / or anti-cancer therapy described herein, an ACT therapy selected from the group including TCR-T, CAR-T, TIL and NK cell therapy, or a combination of one or more thereof.

[0103] Methods of treating and / or preventing cancer in a subject in need thereof are also contemplated.

[0104] In an embodiment of the present invention, a method for treating and / or preventing cancer in a subject in need thereof comprises administering a pharmaceutical composition of the present invention alone or in combination with an anti-cancer immunotherapy as described herein, preferably a cytokine therapy, more preferably a cytokine therapy selected from the group comprising IL-15, a fusion protein comprising IL-15, IL-15, IL-15Rα and an immunoglobulin IgG Fc domain, or a combination of one or more thereof.

[0105] The pharmaceutical composition may further comprise, in addition to the fusion protein and / or anti-cancer therapy described herein, an ACT therapy selected from the group including TCR-T, CAR-T, TIL and NK cell therapy, or a combination of one or more thereof.

[0106] In this particular aspect, a method of treating and / or preventing cancer in a patient or subject comprises the steps of (i) removing and isolating immune cells, preferably natural T cells, from said patient or subject, (ii) genetically engineering said T cells to encode a chimeric antigen receptor (CAR), T cell receptor (TCR), or any other synthetic tumor-targeting motif or antigen, (iii) expanding the engineered T cells ex vivo to a larger population, and (iv) reintroducing said engineered T cells into the patient or subject. After the engineered T cells are reintroduced into the patient or subject, they mediate an immune response against cells expressing the tumor-targeting motif or antigen described herein.

[0107] In one aspect, a method of treating and / or preventing cancer comprises (i) removing and isolating immune cells, preferably natural T cells, from a patient or subject, or providing immune cells, preferably natural T cells; (ii) genetically engineering the T cells to encode at least a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif or antigen; (iii) expanding the engineered T cells ex vivo to a larger population; and (iv) reintroducing them into the patient or subject.

[0108] Also contemplated is a method of enhancing ACT anti-tumor activity in a subject, comprising administering a pharmaceutical composition of the present invention.

[0109] Kits for carrying out one or more methods according to the invention are also contemplated.

[0110] In one embodiment, the kit comprises the composition or pharmaceutical composition of the present invention in one or more containers. The composition may be in liquid form or frozen. Suitable containers for the composition include, for example, bottles, vials, syringes, and test tubes. The containers can be made of various materials, including glass or plastic. The kit may further comprise instructions, which may include information or instructions on prescription, drug amounts, composition, etc.

[0111] The invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from its spirit or essential characteristics. The invention also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any and all combinations or any two or more of said steps or features. Accordingly, the present disclosure is to be considered as non-limiting in all exemplified aspects, and the scope of the invention is indicated by the appended claims, with all changes that come within the meaning and range of equivalents intended to be embraced therein. Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety. The above description will be more fully understood with reference to the following examples. [Example]

[0112] IL-21 enhances NK cell effector function We first sought to investigate whether IL-21 could enhance NK cell effector function. We designed and produced a recombinant mouse IL-21 and mutant IgG1 Fc fusion protein (IL-21 / Fc) (Figure 1), which had a longer half-life and retained comparable biological activity compared to native IL-21 (Figures 2a and 2b). The fused IL-21 / Fc protein exhibited prolonged circulation as measured by IL-21 concentration in peripheral blood. Compared to native mouse IL-21 due to its smaller molecular weight and size, the half-life of IL-21 / Fc was approximately 11.1 hours, corresponding to a 37-fold increase compared to native mouse IL-21 at 0.3 hours (Figures 2c and 2d). NK cells isolated from mouse spleens were cultured in the presence of IL-2 to activate NK cells in vitro and maintain their survival. After 6 days of activation and expansion, the activated NK cells were cocultured with B16F10_β2m cells (a β2 microglobulin knockout B16F10 cell line) and CT26_β2m cells (a β2 microglobulin knockout CT26 cell line described in Nicolai Christopher, J. et al. 2020) at various E:T ratios up to 0.5:1 in the presence of 100 ng / ml IL-21 / Fc. β2 microglobulin is a component of the MHC I molecule. These β2 microglobulin knockout cell lines all have dysfunctional MHC I molecules and therefore cannot be recognized by T cells, but are susceptible to NK cell-mediated killing. Killing efficacy was determined by LDH assay after 5 hours of incubation. IL-21 / Fc clearly demonstrated that NK cell killing efficacy against target cells was significantly enhanced (Figure 2e). More importantly, we found that IL-21 / Fc rapidly exerted NK cell killing function at a relatively low E:T ratio of <1, which resembled the actual situation in the tumor microenvironment with little immune cell infiltration. Furthermore, cytokine and degranulation activity were measured via CD107a expression and IFN-γ and granzyme B secretion. IL-21 / Fc supported the antitumor activity of NK cells through promoting degranulation and cytokine secretion (Figure 2f).

[0113] In vivo antitumor effect of IL-21 / Fc Next, we investigated the in vivo antitumor effects of IL-21 / Fc to verify whether it enhances NK cell function in cancer immunotherapy (Figure 3a). Because tumors lacking MHC I molecules generally escape immune surveillance and are poorly recognized by CD8+ T cells, resulting in limited killing, we established an MHC I-deficient tumor model to identify the antitumor activity of NK cells. First, we evaluated the therapeutic effect in a highly aggressive and poorly immunogenic B16F10_β2m knockout mouse melanoma model. Treatment with IL-21 / Fc significantly contributed to controlling tumor growth in the early stage compared with the PBS group, but the mice ultimately died due to increased tumor burden. Considering the "cold" tumor immune microenvironment, to achieve better therapeutic efficacy, we combined IL-15SA (an IL-15 superagonist) to increase NK cell infiltration and promote the antitumor potential of IL-21 / Fc. IL-15 is a promising cytokine for NK cell translational research because it exhibits robust effects on stimulating NK cell proliferation. Surprisingly, combined treatment with IL-21 / Fc and IL-15SA induced significant tumor regression and durable cure in 75% of B16F10_β2m tumor-bearing mice compared with IL-21 / Fc treatment alone (Figure 3b). Compared to the B16F10_β2m tumor model, IL-21 / Fc itself was able to eliminate CT26_β2m and RMA-S tumors, but sustained efficacy remained limited. IL-15SA did not further regress tumor growth compared with initial IL-21 / Fc treatment, but it did substantially control and eradicate tumor growth, ultimately leading to prolonged mouse survival (Figures 3c and 3d). To broaden IL-21 / Fc-based immunotherapy, multiple combination therapies were explored and tested in the B16 melanoma tumor model. Combining IL-21 with currently used therapies, such as the immune checkpoint blockade antibody anti-PD-1 and the innate immune stimulator R837, significantly suppressed tumor growth in an MHC I-competent model, comparable to the combination with IL-15SA (Figure 4). Taken together, these findings suggest that IL-21 / Fc could enhance NK cell antitumor activity and control tumor growth.Furthermore, the combination of IL-15SA further enhanced the therapeutic effect of NK cells in established solid tumors.

[0114] NK cells are the dominant immune cell subset that contributes to controlling tumor growth To verify whether NK cells were the dominant immune cell subset contributing to tumor growth control, we analyzed tumor-infiltrating CD45.2+ immune cells, including NK cells, CD4+ T cells, CD8+ T cells, B cells, macrophages, dendritic cells, neutrophils, and eosinophils, from CT26_β2m tumors. The changes in immune cell subsets following different treatments revealed that IL-15 promoted the infiltration of all CD45.2+ immune cells. Notably, NK cells accounted for the majority of infiltrating CD45.2+ immune cells in the tumor microenvironment in all groups, consistent with the typical characteristics of tumor models lacking MHC I molecules. As expected, IL-15SA increased NK cell infiltration compared with the PBS group, likely due to IL-15SA-mediated stimulation and NK cell proliferation. Treatment with IL-21 / Fc in addition to IL-15SA further significantly increased NK cell infiltration. Although CD4+ and CD8+ T cells were slightly increased under IL-15SA treatment, their absolute cell numbers were lower than those of NK cells. Furthermore, there were no significant differences in T cells among the four groups, regardless of the type of treatment (Fig. 5a).

[0115] Based on NK cells as the major immune cell subset in the tumor microenvironment, we attempted to further confirm the dominant role of NK in tumor eradication. We maintained the same IL-21 / Fc and IL-15SA injection schedule as in the efficacy study, but depleted NK cells in tumor-bearing mice. The antitumor effects of IL-21 / Fc and IL-15SA were completely abolished when we depleted NK cells. Tumor growth in these mice was similar to that in the PBS group. In contrast, IL-21 / Fc and IL-15SA demonstrated greater antitumor efficacy and mean survival rates than mice that additionally received an isotype control antibody, in which NK cells remained (Figure 5b). Analysis of tumor-infiltrating immune cells and efficacy testing under specific immune cell depletion clearly revealed the dominant role of NK cells in the context of IL-21 / Fc and IL-15SA combination therapy. Furthermore, IL-21 / Fc further favored increased numbers and enhanced cytolytic function of NK cells in the presence of sufficient IL-15SA-mediated NK cell infiltration.

[0116] The antitumor activity of IL-21 / Fc depends on NK cells in the tumor Considering that metabolism satisfies the biosynthetic and energy demands for cell fate and function, we evaluated metabolic changes in NK cells in the presence of IL-21 / Fc using a seahorse assay. IL-21 / Fc significantly enhanced NK cell glycolysis, including basal glycolysis levels and preserved glycolytic capacity, but did not significantly alter NK cell oxidative phosphorylation (OXPHOS) metabolism, and the metabolic profile of NK cells shifted toward glycolysis in the presence of IL-21 / Fc (Figures 6a and 6b). Furthermore, glycolysis-related genes were identified, and most of these genes were increased upon IL-21 / Fc treatment (Figure 6c). IL-21 / Fc-mediated NK cell function was maintained in the presence of oligomycin, an OXPHOS metabolism inhibitor, but the functional enhancement was abolished by application of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) (Figure 6d). These results suggest that IL-21 / Fc affects NK cell metabolism by enhancing glycolysis and that changes in metabolic activity are essential for NK cell function. To verify the attribution of IL-21 / Fc-mediated NK cell effector function enhancement to glycolytic metabolism, we measured tumor-infiltrating NK cell effector function and glycolytic activity upon IL-21 / Fc treatment. Tumor-infiltrating NK cells showed increased expression of glucose transporter 1 (Glut1) and increased uptake of 2-NBDG, a fluorescent glucose analog widely used to monitor glucose uptake in live cells (Figure 6e). Correspondingly, tumor-infiltrating NK cells receiving IL-21 / Fc showed enhanced effector function in the IL-15SA combination scenario (Figure 6f).

[0117] IL-21 / Fc enriches NK cells, promotes stemness, and induces sustained protection against tumor reinfection In addition to metabolic changes, we also investigated phenotypic changes. By comparing the differences in NK cell phenotype and function between tumors and spleens, we observed a dramatic decrease in the CD27+ NK population among tumor-infiltrating NK cells, and the effector function of tumor-infiltrating NK cells was similarly reduced. In contrast, the CD11b+ NK cell population remained largely unchanged (Figures 7a and 7b). CD27+ NK cells have been reported to have memory and stem-like characteristics during infection (Kujur, W. et al. 2020). The decrease in CD27+ NK cells in the tumor microenvironment may explain the gradual loss of function of NK cells in controlling tumor growth (Figure 7c). Interestingly, IL-21 / Fc significantly increased the CD27+ NK cell population in the TME. Furthermore, the expression of Sca1, TCF1, and Ki67 was increased in NK cells treated with IL-21 / Fc (Figure 7d). Due to the increase in memory-like CD27+ NK cells, cured mice showed rejection of secondary tumor transplants (Figure 7e) (Venkatasubramanian, S. et al. 2017). Taken together, these results suggest that IL-21 / Fc enhances the memory and stem-like properties of tumor-infiltrating NK cells, which may be related to the sustained function of NK cells and tumor reinfection.

[0118] Human IL-21 / Fc promotes human NK cell function and glycolysis The effect of IL-21 on human NK cells was also evaluated. A fusion human IL-21 (hIL-21 / Fc) was generated and its effect on NK92MI cells was tested (Figure 8a). hIL-21 / Fc significantly enhanced the killing potency of NK92MI against K562 cells (Figure 8b). Furthermore, NK92MI cell glycolytic metabolic activity increased in the presence of hIL-21 / Fc (Figure 8c). In addition to verifying the biological activity of hIL-21 / Fc on NK92MI cells, we also tested it on primary human NK cells isolated from human peripheral blood mononuclear cells (PBMCs). PBMC-derived NK (PBMC-NK) cells were pre-activated with hIL-2 for 5 days. Activated PBMC-NK cells were co-cultured with the triple-negative breast cancer cell line MDA-MB-231 or the glioblastoma cell line U87 at an E / T ratio of 0.5:1. Killing efficacy was determined by LDH assay after 5 hours of incubation. Similar to its effect on NK92MI, hIL-21 / Fc significantly increased the killing efficacy of PBMC-NK cells against the two target cells (Figure 9). To further investigate hIL-21 / Fc-mediated antitumor activity in vivo, a subcutaneous lymphoblastoid K562 tumor model was established in NSG mice. Mice received adoptive transfer of NK92MI cells and intratumoral injection of hIL-21 / Fc. Further injection of hIL-21 / Fc significantly suppressed tumor growth, indicating that IL-21 / Fc enhances both NK cell antitumor effector function and antitumor efficacy, which is thought to be dependent on NK cells (Figure 10). The consistent results from human NK cells suggested the promising translational value of IL-21 in the clinic.

[0119] In summary, we have demonstrated that IL-21 / Fc, in combination with IL-15SA, induces significant tumor regression and durable antitumor effects in multiple solid tumor models.

[0120] (References) Wolf, N.K., Kissiov, D.U. & Raulet, D.H. Roles of natural killer cells in immunity to cancer, and applications to immunotherapy. Nat. Rev. Immunol. (2022). Bald, T., Krummel, M.F., Smyth, M.J. & Barry, K.C. The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies. Nat. Immunol. 21, 835-847 (2020). Li, Z.-Y. et al. The transcriptional repressor ID2 supports natural killer cell maturation by controlling TCF1 amplitude. J. Exp. Med. 218 (2021). Bi, J. & Tian, Z. NK Cell Exhaustion. 8 (2017). O’Brien, K.L. & Finlay, D.K. Immunometabolism and natural killer cell responses. Nat. Rev. Immunol. 19, 282-290 (2019). Parrish-Novak, J. et al. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408, 57-63 (2000). Nicolai Christopher, J. et al. NK cells mediate clearance of CD8+ T cell-resistant tumors in response to STING agonists. Science Immunology 5, eaaz2738 (2020). Kujur, W. et al. Memory like NK cells display stem cell like properties after Zika virus infection. PLoS Pathog. 16, e1009132-e1009132 (2020). Venkatasubramanian, S. et al. IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis. Mucosal Immunol. 10, 1031-1042 (2017). Gorby C, Sotolongo Bellon J, Wilmes S, Warda W, Pohler E, Fyfe PK, Cozzani A, Ferrand C, Walter MR, Mitra S, Piehler J, Moraga I. Engineered IL-10 variants elicit potent immunomodulatory effects at low ligand doses. Sci Signal. 2020 Sep 15 Saxton RA, Tsutsumi N, Su LL, Abhiraman GC, Mohan K, Henneberg LT, Aduri NG, Gati C, Garcia KC. Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10. Science. 2021 Mar 19 Armour KL, Clark MR, Hadley AG, Williamson LM. Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities. Eur J Immunol. 1999 Aug;29(8):2613-24. Zheng XX, Steele AW, Nickerson PW, Steurer W, Steiger J, Strom TB. Administration of noncytolytic IL-10 / Fc in murine models of lipopolysaccharide-induced septic shock and allogeneic islet transplantation. J Immunol. 1995 May 15;154(10):5590-600 Mumm JB, Emmerich J, Zhang X, Chan I, Wu L, Mauze S, Blaisdell S, Basham B, Dai J, Grein J, Sheppard C, Hong K, Cutler C, Turner S, LaFace D, Kleinschek M, Judo M, Ayanoglu G, Langowski J, Gu D, Paporello B, Murphy E, Sriram V, Naravula S, Desai B, Medicherla S, Seghezzi W, McClanahan T, Cannon-Carlson S, Beebe AM, Oft M. IL-10 elicits IFNγ-dependent tumor immune surveillance. Cancer Cell. 2011 Dec 13;20(6):781-96 Karin M. Knudson, James W. Hodge, Jeffrey Schlom & Sofia R. Gameiro (2020) Rationale for IL-15 superagonists in cancer immunotherapy, Expert Opinion on Biological Therapy, 20:7, 705-709 Czajkowsky DM, Hu J, Shao Z, Pleass RJ. Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med. 2012 Oct;4(10):1015-28. Zalevsky J, Chamberlain AK, Horton HM, Karki S, Leung IW, Sproule TJ, Lazar GA, Roopenian DC, Desjarlais JR. Enhanced antibody half-life improves in vivo activity. Nat Biotechnol. 2010 Feb;28(2):157-9. Vaccaro C, Zhou J, Ober RJ, Ward ES. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 2005 Oct;23(10):1283-8. Qiao J, Liu Z, Dong C, Luan Y, Zhang A, Moore C, Fu K, Peng J, Wang Y, Ren Z, Han C, Xu T, Fu YX. Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8+ T Cell Apoptosis. Cancer Cell. 2019 Jun 10;35(6):901-915. Kontermann RE. Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol. 2011 Dec;22(6):868-76. Fan J, Shang D, Han B, Song J, Chen H, Yang JM. Adoptive Cell Transfer: Is it a Promising Immunotherapy for Colorectal Cancer? Theranostics. 2018 Nov 10;8(20):5784-5800. Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008 Apr;8(4):299-308. Knudson KM, Hodge JW, Schlom J, Gameiro SR. Rationale for IL-15 superagonists in cancer immunotherapy. Expert Opin Biol Ther. 2020 Jul;20(7):705-709. Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T, Eldridge P, Leung WH, Campana D. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res. 2009 May 1;69(9):4010-7 Daher M, Melo Garcia L, Li Y, Rezvani K. CAR-NK cells: the next wave of cellular therapy for cancer. Clin Transl Immunology. 2021 Apr 28;10(4)

Claims

1. A fusion protein for use in the prevention and / or treatment of cancer, said fusion protein comprising: (i) an immunoglobulin IgG Fc domain or a human serum albumin (HSA) polypeptide; (ii) a polypeptide comprising the sequence of an interleukin-21 (IL-21) polypeptide, a fragment or a variant thereof; Including, The fusion protein for use, wherein said IL-21 polypeptide, fragment or variant thereof is covalently fused via a linker to the N-terminus or C-terminus of said IgG Fc domain or said HAS polypeptide.

2. 2. The fusion protein for use according to claim 1, wherein the IgG Fc domain dimerizes with a second IgG Fc domain, thereby forming a homodimer, and the second IgG Fc domain is covalently fused via its N-terminus or C-terminus to an IL-21 polypeptide, fragment, or variant thereof.

3. The fusion protein for use according to claim 1 or 2, wherein the IL-21 sequence comprises or consists of SEQ ID NO: 1, a fragment or variant thereof.

4. The fusion protein for use according to any one of claims 1 to 3, wherein said IL-21 variant is a biologically active derivative of IL-21.

5. 5. The fusion protein for use according to any one of claims 1 to 4, wherein the fusion protein is used in combination with an anti-cancer immunotherapy selected from the group comprising ACT therapy, immune checkpoint blockade therapy, cytokine therapy, cancer vaccine therapy, bispecific antibody therapy and other cancer immunotherapy, or one or more combinations thereof.

6. The fusion protein for use according to claim 5, wherein the ACT therapy is selected from the group comprising TCR-T, CAR-T, TIL and NK cell therapy, or a combination of one or more thereof.

7. 6. The fusion protein for use according to claim 5, wherein the cytokine therapy is selected from the group comprising GM-CSF, IFN gamma, IL-7, IL-10, IL-12, IL-15, and fusion proteins thereof comprising a cytokine and an immunoglobulin IgG Fc domain, human serum albumin (HAS), or one or more combinations thereof.

8. The fusion protein for use according to any one of claims 5 to 7, wherein the cytokine therapy is selected from the group comprising IL-15, a fusion protein comprising IL-15, IL-15Rα and an immunoglobulin IgG Fc domain, or one or more combinations thereof.

9. The fusion protein for use according to claim 8, wherein the fusion protein comprising IL-15, IL-15Rα and an immunoglobulin IgG Fc domain is an IL-15 superagonist.

10. 10. The fusion protein for use according to any one of claims 6 to 9, wherein the NK cell therapy is selected from the group comprising induced or activated NK cells, iPSC-NK cells, CAR-NK cells, and PBNK cells, or a combination of one or more thereof.

11. The fusion protein for use according to claim 10, wherein the NK cells are autologous or allogeneic NK cells.

12. The fusion protein for use according to any one of claims 1 to 11, wherein the cancer is a solid cancer or a liquid cancer.

13. 13. The fusion protein for use according to claim 12, wherein the solid cancer is selected from the group comprising 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, and skin cancer, in particular melanoma, or one or more combinations thereof.

14. The fusion protein for use according to any one of claims 1 to 13, wherein said cancer is an MHC-I expression altered cancer, preferably an MHC-I deficient solid cancer.

15. 15. The fusion protein for use according to any one of claims 5 to 14, wherein the immune checkpoint blockade therapy comprises an inhibitor selected from the group comprising a CTLA-4 inhibitor, a TIM3 inhibitor, a PD-1 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, and a PD-L1 inhibitor, or one or more combinations thereof.

16. 16. The fusion protein for use according to any one of claims 1 to 15, wherein the IL-21-Fc fusion protein increases the efficacy of the anti-cancer immunotherapy by about 2% or more, about 5% or more, about 20% or more, about 40% or more, about 60% or more, or about 500% or more when compared to the efficacy of the anti-cancer treatment in the absence of the IL-21-Fc fusion protein.

17. 17. The fusion protein for use according to any one of claims 1 to 16, wherein the IL-21-Fc fusion protein increases the half-life and / or number of NK cells by about 2%, about 5%, about 20%, about 40%, about 60%, about 100%, about 200%, about 300% or more when compared to the half-life and / or number of NK cells in the absence of the Fc-IL-21 fusion protein.

18. The fusion protein for use according to any one of claims 1 to 17, wherein the IgG Fc domain is a silent Fc domain of immunoglobulin (IgG) G.

19. 19. The fusion protein for use according to any one of claims 1 to 18, wherein the IgG Fc domain is selected from the group comprising human IgG1, human IgG2, human IgG3 and human IgG4, fragments or variants thereof.

20. 20. The fusion protein for use according to claim 19, wherein the Fc domain of human IgG comprises or is selected from the group consisting of IgG1 Fc (SEQ ID NO: 2), IgG2 Fc (SEQ ID NO: 8), IgG3 Fc (SEQ ID NO: 11), and IgG4 Fc (SEQ ID NO: 14), fragments, variants, or combinations of one or more of these sequences.

21. 21. The fusion protein for use according to claim 20, wherein said variant of SEQ ID NO: 2 comprises at least one mutation selected from C220A, L234A, L235A and P329G.

22. 21. The fusion protein for use according to claim 20, wherein said variant of SEQ ID NO: 8 comprises at least one mutation selected from A330S and P331S.

23. 21. The fusion protein for use according to claim 20, wherein said variant of SEQ ID NO: 14 comprises at least one mutation selected from S228P and L235E.

24. The fusion protein for use according to any one of claims 1 to 23, wherein the linker is a polypeptide linker.

25. 25. The fusion protein for use according to claim 24, wherein the polypeptide linker consists of a stretch of Gly and Ser residues (a "GS" linker) or Gly-Gly and Ser residues (a "GGS" linker), followed or not by one or more Arg residues (an "R" residue).

26. 26. The fusion protein for use according to any one of claims 1 to 25, wherein the fusion protein comprises or is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:22, a fragment or variant of any one of the sequences thereof.

27. (i) an immunoglobulin IgG Fc domain or a human serum albumin (HSA) polypeptide; (ii) a polypeptide comprising the sequence of interleukin-21 (IL-21), a fragment or a variant polypeptide thereof; A fusion protein comprising: A fusion protein, wherein the IL-21 polypeptide, fragment, or variant thereof is covalently fused to the N-terminus or C-terminus of the IgG Fc domain or the HAS polypeptide via a linker.

28. 28. The fusion protein of claim 27, wherein the IgG Fc domain dimerizes with a second IgG Fc domain, thereby forming a homodimer, and the second IgG Fc domain is covalently fused via its N-terminus or C-terminus to an IL-21 polypeptide, fragment, or variant thereof.

29. 30. The fusion protein of claim 28 or 29, wherein the IL-21 sequence comprises or consists of SEQ ID NO: 1, a fragment or variant thereof.

30. The fusion protein of any one of claims 27 to 29, wherein the IL-21 variant is a biologically active derivative of IL-21.

31. 31. The fusion protein of claim 30, wherein the IgG Fc domain comprises or is selected from the group consisting of IgG1 Fc (SEQ ID NO: 2), IgG2 Fc (SEQ ID NO: 8), IgG3 Fc (SEQ ID NO: 11), and IgG4 Fc (SEQ ID NO: 14), fragments, variants, or combinations of one or more of these sequences.

32. 33. The fusion protein of claim 32, wherein the variant of SEQ ID NO: 2 comprises at least one mutation selected from C220A, L234A, L235A and P329G.

33. 33. The fusion protein of claim 32, wherein the variant of SEQ ID NO: 8 comprises at least one mutation selected from A330S and P331S.

34. 33. The fusion protein of claim 32, wherein the variant of SEQ ID NO: 14 comprises at least one mutation selected from S228P and L235E.

35. The fusion protein of any one of claims 27 to 34, wherein the linker is a polypeptide linker.

36. 36. The fusion protein of claim 35, wherein the polypeptide linker consists of a stretch of Gly and Ser residues (a "GS" linker) or Gly-Gly and Ser residues (a "GGS" linker), followed or not by one or more Arg residues (an "R" residue).

37. 37. The fusion protein of any one of claims 27 to 36, wherein the fusion protein comprises or is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:22, a fragment or variant of any one of the sequences thereof.

38. A nucleic acid sequence encoding one or more fusion proteins according to any one of claims 27 to 37.

39. 39. A plasmid or vector comprising the nucleic acid sequence of claim 38.

40. 29. An isolated host cell or cell population comprising the vector or plasmid of claim 28.

41. 41. A pharmaceutical composition comprising: i) a fusion protein for use according to any one of claims 1 to 26; ii) a fusion protein according to any one of claims 27 to 38; iii) a plasmid or vector according to claim 39; or iv) an isolated host cell or cell population according to claim 40; and a pharmaceutically acceptable carrier, diluent and / or excipient.

42. 42. The pharmaceutical composition according to claim 41, further comprising an anti-cancer immunotherapy, preferably a cytokine therapy, more preferably selected from the group comprising IL-15, a fusion protein comprising IL-15, IL-15Rα and an immunoglobulin IgG Fc domain, or one or more combinations thereof.

43. 43. The pharmaceutical composition of claim 41 or 42, further comprising an ACT therapy selected from the group comprising TCR-T, CAR-T, TIL and NK cell therapy, or a combination of one or more thereof.

44. A method for treating and / or preventing cancer in a subject, comprising administering the pharmaceutical composition according to any one of claims 41 to 43.

45. 45. The method of treatment and / or prevention according to claim 44, wherein the anti-cancer immunotherapy is a cytokine therapy, more preferably a cytokine therapy selected from the group comprising IL-15, a fusion protein comprising IL-15, IL-15Rα and an immunoglobulin IgG Fc domain, or one or more combinations thereof.

46. The method of treatment and / or prevention according to claim 44 or 45, wherein the pharmaceutical composition further comprises an ACT therapy selected from the group comprising TCR-T, CAR-T, TIL and NK cell therapy, or a combination of one or more thereof.

47. 47. A method of treatment and / or prevention according to any one of claims 44 to 46, comprising the steps of: (i) removing and isolating immune cells, preferably natural T cells, from the patient or subject; (ii) genetically engineering the T cells to encode a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor-targeting motif or antigen; (iii) expanding the engineered T cells ex vivo to a larger population; and (iv) reintroducing the engineered T cells into the patient or subject.

48. A method for enhancing ACT antitumor activity in a subject, comprising administering the pharmaceutical composition of any one of claims 41 to 43.

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