WO2023280287A1 - Interleukin-15 variants and uses thereof - Google Patents

Interleukin-15 variants and uses thereof Download PDF

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WO2023280287A1
WO2023280287A1 PCT/CN2022/104519 CN2022104519W WO2023280287A1 WO 2023280287 A1 WO2023280287 A1 WO 2023280287A1 CN 2022104519 W CN2022104519 W CN 2022104519W WO 2023280287 A1 WO2023280287 A1 WO 2023280287A1
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amino acid
cell
acid residue
cancer
polypeptide
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PCT/CN2022/104519
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French (fr)
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Shuai Yang
Xiaojie TU
Chengming GE
Wenwen ZENG
Yanmin CHEN
Shu Wu
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Nanjing Legend Biotech Co., Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • This disclosure relates to modified human interleukin-15 (IL-15) polypeptides, and fusion proteins and cells comprising such IL-15 polypeptides.
  • the present disclosure also provides methods of modulating immune responses and treating a disease or disorder using the modified IL-15 polypeptides, fusion proteins, or cells (e.g., immune cells) .
  • Cytokines mediate proliferative and activation signals in the innate and adaptive immune system and play roles in normal physiology, with tightly regulated controls of their production, localization, and activity (see, e.g., Silk AW &Margolin K. 2019. Cytokine Therapy. Hematology/Oncology Clinics of North America 33 (2) : 261-274) .
  • IL-15 is a pleiotropic cytokine that can activate both the innate immune cells such as NK cells and the adaptive immune cells such as CD8+ cytotoxic T lymphocytes. It plays an important role in formation and maintenance of immunological memory. It is also an essential factor for NK development and homeostasis.
  • IL-15 as a therapeutic agent are limited by its short half-life and more importantly toxicity due to the triggering of downstream molecules, including other inflammatory cytokines, such as IL-6 and interferon gamma (IFN ⁇ ) (see, e.g., Conlon KC et al. 2015. J Clin Oncol 33 (1) : 74-82) .
  • IFN ⁇ interferon gamma
  • the disclosure relates to modified human IL-15 polypeptides that include one or more amino acid substitutions.
  • the IL-variants confer improved properties such as increased or decreased binding to IL-15 receptors (e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c) , modulating immune responses, and reducing toxicity of immune therapies (e.g., anti-cancer therapies) .
  • IL-15 receptors e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c
  • immune therapies e.g., anti-cancer therapies
  • the disclosure also relates to fusion proteins and modified immune cells that comprise the modified human IL-15 polypeptides described herein.
  • the disclosure relates to a modified human interleukin 15 (IL-15) polypeptide comprising one or more amino acid substitutions at positions 1, 3, 4, 7, 8, 10, 11, 61, 62, 64, 65, 68, and 69, wherein numbering of amino acid residue positions is according to SEQ ID NO: 3.
  • IL-15 human interleukin 15
  • amino acid residue at position 1 is not N;
  • amino acid residue at position 68 is not I;
  • amino acid substitutions comprise one or more of the following:
  • amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;
  • amino acid residue at position 4 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
  • amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 10 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 11 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 61 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;
  • amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 65 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 68 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 69 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y.
  • the modified human IL-15 has a decreased binding affinity to human IL-2 receptor beta (IL-2R ⁇ ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2R ⁇ .
  • IL-2R ⁇ human IL-2 receptor beta
  • the modified human IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 3 is D, R, S, or T;
  • amino acid residue at position 4 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W;
  • amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
  • amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 10 is A, D, E, F, G, I, L, M, N, P, Q, S, T, V, W, or Y;
  • amino acid residue at position 11 is A, D, F, G, H, I, L, N, P, T, V, W, or Y;
  • amino acid residue at position 61 is F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, or Y;
  • amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 65 is A, E, F, G, H, I, K, L, M, P, Q, R, T, V, W, or Y;
  • amino acid residue at position 68 is A, D, E, F, H, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 69 is D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or W.
  • the modified human IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 1 is Q
  • amino acid residue at position 4 is G, L, P, V, M, or W;
  • amino acid residue at position 7 is D, E, F, G, I, K, N, P, V, W, or Y;
  • amino acid residue at position 61 is H, R, or S;
  • amino acid residue at position 62 is E, F, G, I, M, Q, or V;
  • amino acid residue at position 64 is A, F, H, L, M, N, S, T, V, W, or Y;
  • amino acid residue at position 68 is M, Q, S, T, V, W, or Y;
  • the amino acid residue at position 69 is H, M, N, Q, S, T, or V.
  • the modified human IL-15 polypeptide has an increased binding affinity to human IL-2R ⁇ as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2R ⁇ .
  • the modified IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 3 is A, E, F, H, L, M, N, Q, W, or Y;
  • amino acid residue at position 10 is H or R;
  • amino acid residue at position 61 is A or E.
  • the modified IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 3 is A, E, F, H, L, N, Q, W, or Y;
  • amino acid residue at position 10 is H or R;
  • the modified human IL-15 polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 3.
  • the modified human IL-15 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 4-22.
  • the disclosure relates to a fusion protein comprising any one of the modified human IL-15 polypeptide described herein.
  • the fusion protein further comprises an antigen binding moiety.
  • the antigen binding moiety is selected from the group consisting of an antibody or antigen binding fragment thereof, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain, a variable domain of light chain or a V H H) .
  • variable domains include, e.g., Fab, Fab’, F (ab’) 2, and Fv fragments, scFv, and V H H.
  • the antigen binding moiety is an antibody comprising an Fc region.
  • the antigen binding moiety and the modified human IL-15 polypeptide is fused via a linker.
  • the disclosure relates to a nucleic acid comprising a polynucleotide encoding any one of the modified human IL-15 polypeptides described herein, or any one of the fusion proteins described herein.
  • the disclosure relates to an expression vector comprising any one of the nucleic acids described herein.
  • the disclosure relates to a cell comprising any one of the vectors described herein.
  • the cell is an immune cell.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-cell, an iNK-T cell, an NK-T like cell, an ⁇ T cell and a ⁇ T cell.
  • the immune cell is an NK cell.
  • the immune cell is a cytotoxic T cell.
  • the modified human IL-15 polypeptide is secreted.
  • the modified human IL-15 polypeptide is membrane bound.
  • the cell expresses a chimeric antigen receptor (CAR) , a T-cell antigen coupler (TAC) receptor, or a T-cell receptor (TCR) .
  • CAR chimeric antigen receptor
  • TAC T-cell antigen coupler
  • TCR T-cell receptor
  • the disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising any one of the modified human IL-15 polypeptides described herein, any one of the fusion proteins described herein, or any one of the cells described herein and a pharmaceutically acceptable carrier.
  • the disclosure relates to a method of producing a modified human IL-15 polypeptide, or a fusion protein comprising a modified human IL-15 polypeptide, the method comprising: (a) culturing any one of the cells described herein under conditions sufficient for the cell to produce the modified human IL-15 polypeptide or the fusion protein; and (b) collecting the modified human IL-15 polypeptide or the fusion protein produced by the cell.
  • the disclosure relates to a method of producing a modified cell, comprising: introducing into a cell any one of the expression vector described herein.
  • the cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, an NK cell, an NK-T cell, an iNK-T cell, an NK-T like cell, an ⁇ T cell and a ⁇ T cell.
  • the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the modified human IL-15 polypeptides described herein, any one of the fusion proteins described herein, any one of the pharmaceutical compositions described herein, or any one of the cells described herein, thereby treating cancer in the subject.
  • the cancer is selected from the group consisting of gastric cancer, small intestine cancer, sarcoma, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, biliary cancer, neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, renal cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma, brain tumor, bone cancer, eye cancer, and melanoma.
  • the cancer is a hematological malignancy.
  • the hematological malignancy is selected from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin’s lymphoma, nodular lymphocyte predominant Hodgkin’s lymphoma, Kahler’s disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin’s lymphoma (NHL) , acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL) , acute lymphocytic leukemia (ALL) , chronic myeloid leukemia (CML) , follicular lymphoma, Burkitt’s lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom’s
  • the cancer is relapsed, refractory, or metastatic.
  • the method further comprises administering to the individual an additional therapy.
  • the additional therapy is surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof.
  • the method further comprises administering an effective amount of an additional therapeutic agent to the subject.
  • the additional therapeutic agent is an immune therapy.
  • the additional therapeutic agent is a CAR-T therapy.
  • the additional therapeutic agent is an antibody, a cytokine, or a chemotherapy.
  • FIG. 1 shows a sequence alignment of two isoforms of IL-15 (SEQ ID NO: 1 and SEQ ID NO: 2) produced by alternative splicing.
  • FIG. 2 shows the amino acid sequence of a human IL-15 peptide (SEQ ID NO: 3) .
  • the amino acid positions of the one or more substitutions are underlined.
  • FIG. 3 shows the effect of IL-2R ⁇ binding affinity on the activation of NK cell and CD8+ cytotoxic T cells.
  • FIGs. 4A-4B show the effect of IL-2R ⁇ binding affinity on NK and CD8+ cytotoxic T cell proliferation.
  • FIG. 4A shows the level of IFN- ⁇
  • FIG. 4B shows the level of TNF- ⁇ secreted to the medium.
  • modified human interleukin-15 (IL-15) polypeptides and fusion proteins and cells comprising such IL-15 polypeptides.
  • the modification on the human IL-15 polypeptide improves the therapeutic index.
  • the therapeutic index is a quantitative measurement of the relative safety of a drug. It is a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes toxicity.
  • the modified human IL-15 polypeptides have a greater therapeutic window.
  • the therapeutic window or safety window refer to a range of doses which optimize between efficacy and toxicity, achieving the greatest therapeutic benefit without resulting in unacceptable side-effects or toxicity.
  • the present disclosure also provides methods of modulating immune responses and treating a disease or disorder using the modified IL-15 polypeptides, fusion proteins, or cells (e.g., immune cells) .
  • IL-15 belongs to the four ⁇ -helix bundle family of cytokines. Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (IL-2/IL-15R ⁇ , also known as CD122) and the common gamma chain (CD132) . Unlike IL-2, the predominant mechanism of IL-15 signaling is trans-presentation which is mediated by membrane-bound complex IL-15/IL-15R ⁇ (see, e.g., Dubois S et al., 2002.
  • IL-2/IL-15 receptor beta chain also known as CD122
  • CD132 common gamma chain
  • Immunity 17 (5) : 537-547) i.e., signaling pathway of IL-15 begins with binding to IL-15R ⁇ receptor which is usually expressed by antigen presenting cells like dendritic cells or monocytes, with subsequent presentation to surrounding effector cells like NK cells and T cells bearing IL-2R ⁇ and common gamma chain complex on their cell surface.
  • IL-15/IL-15R ⁇ /IL-2R ⁇ / ⁇ c From the X-ray structure of IL-15 with its three receptors simultaneously (IL-15/IL-15R ⁇ /IL-2R ⁇ / ⁇ c) , the average number of contacts involving the IL-2R ⁇ chain is around 30, whereas the corresponding value is 20 for ⁇ c chains. Also from a binding free energy perspective, ⁇ Gbind free energy calculated for the IL-15/IL-2R ⁇ interface is about -28 kcal/mol, greater than the IL-15/ ⁇ c (-16 kcal/mol) (see, e.g., Sousa RP et al., 2019. Molecules 24 (18) : 3261) . IL-2R ⁇ binds tighter than ⁇ c. Mutating IL-15 residues on IL-15/IL-2R ⁇ interface can modulate the activity of IL-15, thus can improve the therapeutic index of IL-15.
  • IL-15 is produced constitutively by different cell types such as monocytes, macrophages, dendritic cells, stromal cells, epithelial cells. Rather complex biological features characterize IL-15. Regarding IL-15, it exists under several functional forms: (1) a soluble monomeric form (sIL-15) , (2) the soluble complex sIL-15/IL-15R ⁇ , (3) the transpresented form (tp-IL-15) and (4) the transmembrane form (tmb-IL-15) (see, e.g., Fiore PF et al., Journal for Immuno Therapy of Cancer 2020; 8: e001428) .
  • sIL-15 soluble monomeric form
  • tp-IL-15 the transpresented form
  • tmb-IL-15 transmembrane form
  • IL-15 functions on binding to the IL-2R ⁇ / ⁇ c heterodimer, shared with IL-2, formed by IL-2R ⁇ and ⁇ c chains.
  • IL-2R ⁇ / ⁇ c dimeric receptor binds IL-15 and transduce the signal within the cells by activating the Jak/Stat pathway (Jak 1 and 3, Stat 3 and 5) .
  • the IL-15R ⁇ chain confers specificity to IL-15.
  • IL-15R ⁇ forms a high affinity trimeric receptor with IL-2R ⁇ and ⁇ c chains, allowing the cells to respond to low concentrations of IL-15 (see, e.g., Fiore PF et al., Journal for Immuno Therapy of Cancer 2020; 8: e001428) .
  • Cytokines such as IL-2 and IL-15 induce immune responses through immunological checkpoints such as cytotoxic T-lymphocyte-associated 4 (CTLA-4) , programmed death protein-1 (PD-1, CD279) , and PD-1 ligands PD-L1 (CD274, B7-H1) and PD-L2 (CD272, B7-DC) .
  • CTLA-4 cytotoxic T-lymphocyte-associated 4
  • PD-1, CD279 programmed death protein-1
  • PD-1 ligands PD-L1 (CD274, B7-H1) and PD-L2 (CD272, B7-DC) CD274, B7-DC
  • Cytokine stimulation also induces the secretion of inhibitory factors such as IL-10 and transforming growth factor (TGF- ⁇ ) , the expression of inhibitors such as triosephosphate isomerase (TIM) , and the activation of immune-dampening cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) , as well as the activation of intracellular suppressors of cytokine signaling (CIS, SOCS) proteins that terminate the CD4 T cell immune response (see, e.g., Sckisel GD et al., 2015, Immunity 43 (2) : 240-250) .
  • cytokines such as IL-2 or IL-15 dramatically increase the number of activated natural killer (NK) cells
  • NK natural killer
  • MHC major histocompatibility complex
  • Ig recognition and killer-cell immunoglobulin
  • KIRs killer-cell immunoglobulin-like receptors
  • CRS cytokine release syndrome
  • IL-6 interleukin 6
  • IL-2R ⁇ binding affinity of IL-15 By tuning the IL-2R ⁇ binding affinity of IL-15, the therapeutic window can be significantly improved without compromising the efficacy of IL-15.
  • modified IL-15 polypeptides e.g., human IL-15
  • the one or more substitutions are at positions 1, 3, 4, 7, 8, 10, 11, 61, 62, 64, 65, 68, and/or 69, and numbering of the amino acid residue positions is according to SEQ ID NO: 3.
  • modified human IL-15 peptides comprising one or more of the following:
  • amino acid residue at position 1 is not N;
  • amino acid residue at position 3 is not V;
  • amino acid residue at position 4 is not N;
  • amino acid residue at position 7 is not S
  • amino acid residue at position 8 is not D
  • amino acid residue at position 10 is not K
  • amino acid residue at position 11 is not K
  • amino acid residue at position 61 is not D
  • amino acid residue at position 62 is not T;
  • amino acid residue at position 64 is not E;
  • amino acid residue at position 65 is not N;
  • amino acid residue at position 68 is not I;
  • amino acid residue at position 69 is not L.
  • the numbering of the amino acid residue positions is according to SEQ ID NO: 3.
  • the one or more positions can have any suitable amino acid residue other than the excluded amino acid residue.
  • IL-15 modified human interleukin 15 polypeptides comprising one or more amino acid substitutions at positions that correspond to N1, V3, N4, S7, D8, K10, K11, D61, T62, E64, N65, I68, and L69 of SEQ ID NO: 3.
  • polypeptides e.g., modified IL-15 polypeptides, comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 3, wherein the polypeptide comprises at least one amino acid substitution at positions that correspond to N1, V3, N4, S7, D8, K10, K11, D61, T62, E64, N65, I68, and L69 of SEQ ID NO: 3.
  • the polypeptide disclosed herein e.g., the modified human IL-15 polypeptide, contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the polypeptide contains about 1-10, about 1-20, about 1-30, about 1-40, or about 1-50 amino acid substitutions.
  • the modified IL-15 polypeptide disclosed herein has an amino acid sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to the amino acid sequence of SEQ ID NO: 1, 2, or 3.
  • the modified IL-15 polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 3.
  • the modified IL-15 polypeptide described herein can be in any suitable form of IL-15 (e.g., human IL-15) .
  • the modified IL-15 polypeptide is a soluble monomeric IL-15 (sIL-15) .
  • the modified IL-15 polypeptide is a soluble complex (sIL-15/IL-15R ⁇ ) .
  • the modified IL-15 polypeptide is in a transpresented form (tp-IL-15) .
  • the modified IL-15 polypeptide is in a transmembrane form (tmb-IL-15) .
  • amino acid substitutions comprise one or more of the following:
  • amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;
  • amino acid residue at position 4 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
  • amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 10 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 11 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 61 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;
  • amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 65 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 68 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • the amino acid residue at position 69 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y.
  • the numbering of the amino acid residue positions is according to SEQ ID NO: 3.
  • amino acid substitutions comprise one or more of the following:
  • amino acid residue at position 1 is M
  • amino acid residue at position 3 is E, H, L, T, or Y;
  • amino acid residue at position 4 is F, M, P, or V;
  • amino acid residue at position 7 is F, P, W, or Y;
  • amino acid residue at position 8 is E or L
  • amino acid residue at position 61 is E, R, or S;
  • amino acid residue at position 62 is A, E, G, H, I, L, M, Q, S, or V;
  • amino acid residue at position 68 is Q, V, or W;
  • amino acid residue at position 69 is H or N.
  • the modified human IL-15 polypeptide disclosed herein can have an altered binding affinity to an IL-15 receptor (e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c) compared to the binding affinity of a human IL-15 polypeptide without such substitution (s) (e.g., a wild-type IL-15) to the same receptor.
  • the modified human IL-15 polypeptide disclosed herein has a decreased binding affinity to an IL-15 receptor (e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c) compared to the binding affinity of a human IL-15 polypeptide without such substitution (s) (e.g., a wild-type IL-15) to the same receptor.
  • the modified human IL-15 polypeptide disclosed herein has an increased binding affinity to an IL-15 receptor (e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c) compared to the binding affinity of a human IL-15 polypeptide without such substitution (s) (e.g., a wild-type IL-15) to the same receptor.
  • an IL-15 receptor e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c
  • substitution e.g., a wild-type IL-15
  • the modified human IL-15 polypeptide disclosed herein has a decreased binding affinity to human IL-2 receptor beta (IL-2R ⁇ ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2R ⁇ .
  • the modified IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 3 is D, R, S, or T;
  • amino acid residue at position 4 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W;
  • amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
  • amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 10 is A, D, E, F, G, I, L, M, N, P, Q, S, T, V, W, or Y;
  • amino acid residue at position 11 is A, D, F, G, H, I, L, N, P, T, V, W, or Y;
  • amino acid residue at position 61 is F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, or Y;
  • amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 65 is A, E, F, G, H, I, K, L, M, P, Q, R, T, V, W, or Y;
  • amino acid residue at position 68 is A, D, E, F, H, K, L, M, P, Q, R, S, T, V, W, or Y;
  • amino acid residue at position 69 is D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or W.
  • the modified IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 1 is Q
  • amino acid residue at position 4 is G, L, P, V, M, or W;
  • amino acid residue at position 7 is D, E, F, G, I, K, N, P, V, W, or Y;
  • amino acid residue at position 61 is H, R, or S;
  • amino acid residue at position 62 is E, F, G, I, M, Q, or V;
  • amino acid residue at position 64 is A, F, H, L, M, N, S, T, V, W, or Y;
  • amino acid residue at position 68 is M, Q, S, T, V, W, or Y;
  • the amino acid residue at position 69 is H, M, N, Q, S, T, or V.
  • the modified human IL-15 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-22.
  • the modified human IL-15 polypeptide disclosed herein has an increased binding affinity to human IL-2 receptor beta (IL-2R ⁇ ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2R ⁇ .
  • the modified IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 3 is A, E, F, H, L, M, N, Q, W, or Y;
  • amino acid residue at position 10 is H or R;
  • amino acid residue at position 61 is A or E.
  • the modified IL-15 polypeptide comprises one or more of the following:
  • amino acid residue at position 3 is A, E, F, H, L, N, Q, W, or Y;
  • amino acid residue at position 10 is H or R;
  • the modified human IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 4.
  • the modified IL-15 specifically binds to IL-2R ⁇ (e.g., human IL-2R ⁇ ) with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • KD is less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, less than 1 ⁇ 10 -10 M, or less than 1 ⁇ 10 -11 M. In some embodiments, the KD is less than 50nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
  • KD is greater than 1 ⁇ 10 -6 M, 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, greater than 1 ⁇ 10 -10 M, greater than 1 ⁇ 10 -11 M, or greater than 1 ⁇ 10 -12 M.
  • the modified IL-15 has a KD with IL-2R ⁇ that is less than the KD of D61R. In some embodiments, the modified IL-15 has a KD with IL-2R ⁇ that is less than 1.5 ⁇ 10 -5 M. In some embodiments, the modified IL-15 has a KD with IL-2R ⁇ that is between 1.5 ⁇ 10 -5 M and 2.3 ⁇ 10 -8 M.
  • the modified IL-15 has a KD with IL-2R ⁇ or IL-2R ⁇ c that is between 1 ⁇ 10 -4 M and 1 ⁇ 10 -5 M, between 1 ⁇ 10 -5 M and 1 ⁇ 10 -6 M, between 1 ⁇ 10 -6 M and 1 ⁇ 10 -7 M, between 1 ⁇ 10 -7 M and 1 ⁇ 10 -8 M, between 1 ⁇ 10 -8 M and 1 ⁇ 10 -9 M, or between 1 ⁇ 10 -9 M and 1 ⁇ 10 -10 M.
  • the modified IL-15 has a KD that is higher than 1.8 ⁇ 10 -8 M, 2.3 ⁇ 10 -8 M, 2 ⁇ 10 -8 M, 3 ⁇ 10 -8 M, 4 ⁇ 10 -8 M, 5 ⁇ 10 -8 M, 6 ⁇ 10 -8 M, 7 ⁇ 10 -8 M, 8 ⁇ 10 -8 M, 9 ⁇ 10 -8 M, 1 ⁇ 10 -7 M, 5 ⁇ 10 -7 M, or 1 ⁇ 10 -6 M.
  • the modification does not change the binding affinity with IL-15R ⁇ .
  • the change of KD can be less than 20%, 10%, or 5%after such modifications.
  • the modified IL-15 has a KD with IL-15Ra that is about 1 ⁇ 10 -11 M.
  • the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from Table 1, e.g., N1A, N1D, N1E, N1F, N1G, N1H, N1I, N1K, N1L, N1M, N1P, N1Q, N1R, N1S, N1T, N1V, N1W, N1Y, V3A, V3D, V3E, V3F, V3G, V3H, V3I, V3K, V3L, V3M, V3N, V3P, V3Q, V3R, V3S, V3T, V3W, V3Y, N4A, N4D, N4E, N4F, N4G, N4H, N4I, N4K, N4L, N4M, N4Q, N4R, N4S, N4T, N4
  • the modified IL-15 has one or more substitutions selected from N1M, V3E, V3H, V3L, V3T, V3Y, N4F, N4M, N4P, N4V, S7F, S7P, S7W, S7Y, D8E, D8L, D61E, D61R, D61S, T62A, T62E, T62G, T62H, T62I, T62L, T62M, T62Q, T62S, T62V, E64W, E64Y, N65V, I68Q, I68V, I68W, L69H and L69N.
  • the modified IL-15 does not have D8A, D8N, D61A, and/or N65A amino acid substitutions. In some embodiments, the modified IL-15 does not have T62D, I68F, I68H, I68D, and/or I68K amino acid substitutions. In some embodiments, the modified IL-15 does not have V3I, V3M, V3R, N4H, K11L, K11M, and/or K11R amino acid substitutions.
  • the modified IL-15 does not have E64D, E64K, E64R, N65D, N65E, N65K, N65R, I68D, I68E, I68K, I68R, L69D, L69E, L69K, and/or L69R amino acid substitutions. In some embodiments, the modified IL-15 does not have N1D, N4D, D8N, D61N, E64Q, and/or N65D amino acid substitutions. In some embodiments, the modified IL-15 does not have D8E, D8Q, D8R, D8S, D8V, D8G, D8I, D8L, and/or D8T amino acid substitutions. In some embodiments, the modified IL-15 does not have S7N, K10Q, D61Q, N65Q, I68A, L69A, and/or L69V amino acid substitutions.
  • the modified IL-15 does not have amino acid substitutions at a position that corresponds to N1 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to V3 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to N4 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to S7 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to D8 of SEQ ID NO: 3.
  • the modified IL-15 does not have amino acid substitutions at a position that corresponds to K10 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to K11 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to D61 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to T62 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to E64 of SEQ ID NO: 3.
  • the modified IL-15 does not have amino acid substitutions at a position that corresponds to N65 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to I68 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to L69 of SEQ ID NO: 3.
  • the modified IL-15 has a weaker binding affinity to IL-2R ⁇ as compared to a wild-type IL-15.
  • the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from e.g., N1A, N1D, N1E, N1F, N1G, N1H, N1I, N1K, N1L, N1M, N1P, N1Q, N1R, N1S, N1T, N1V, N1W, N1Y, V3D, V3R, V3S, V3T, N4A, N4D, N4E, N4F, N4G, N4L, N4M, N4Q, N4S, N4W, S7A, S7E, S7G, S7H, S7I, S7K, S7L, S7M, S7N, S7Q, S7R,
  • the modified IL-15 has a KD that is greater than 1 ⁇ 10 -7 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from N1G, N1K, N1Q, N1R, N1W, N4A, N4G, N4L, N4P, N4V, N4Q, N4S, N4W, S7E, S7G, S7I, S7K, S7N, S7P, S7R, S7T, S7V, S7Y, D8E, K10W, K11D, K11G, K11H, K11W, D61H, D61N, D61R, D61S, T62A, T62F, T62G, T62I, T62N, T62Q, T62V, T62Y, E64A, E64F, E64H, E64K, E64L, E64M,
  • the modified IL-15 has a KD that is greater than 1 ⁇ 10 -6 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions selected from N4P, N4V, S7P, D8E, K11D, D61R, E64K, E64S, I68Q I68W, L69H, and/or L69E.
  • the modified IL-15 has a KD that is greater than 1 ⁇ 10 -5 M. In some embodiments, the modified IL-15 has 1 or 2 amino acid substitutions selected from D8E, and/or D61R.
  • the modified IL-15 has a KD that is between 1 ⁇ 10 -7 M and 3 ⁇ 10 -6 M.
  • the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from N1G, N1K, N1P, N1Q, N1R, N1W, N4A, N4G, N4L, N4P, N4Q, N4S, N4W, S7E, S7F, S7G, S7I, S7K, S7N, S7P, S7R, S7T, S7V, S7Y, K10D, K10V, K10W, K11G, K11H, K11W, D61H, D61N, D61S, T62A, T62F, T62G, T62N, T62Q, T62V, T62Y, E64A, E64F, E64H, E64K, E64L, E64
  • the modified IL-15 has a KD that is between 5 ⁇ 10 -8 M and 1 ⁇ 10 -7 M.
  • the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from N1A, N1E, N1F, N1I, N1L, N1M, N1P, N1V, N1Y, N4D, N4E, N4F, S7H, S7L, S7M, K10D, K10F, K10L, K10V, K11A, K11F, K11I, K11N, K11T, K11Y, D61G, T62S, E64I, and/or L69I.
  • the modified IL-15 has a higher binding affinity to IL-2R ⁇ as compared to a wild-type IL-15.
  • the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from e.g., V3A, V3E, V3F, V3H, V3L, V3M, V3N, V3Q, V3W, V3Y, N4H, N4Y, K10H, K10R, K11E, K11M, K11Q, D61A, and/or D61E.
  • the modified IL-15 has a KD that is less than 1 ⁇ 10 -8 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from V3F, V3H, V3L, V3W, V3Y, N4H, K10H, K11Q, and/or D61E.
  • the modified IL-15 polypeptide can be different from a wild-type IL-15 polypeptide (e.g., SEQ ID NO: 3) .
  • the modified IL-15 polypeptide is an engineered IL-15 polypeptide or an IL-15 polypeptide variant or an IL-15 mutein.
  • a fusion protein e.g., an antibody-IL-15 fusion protein
  • a modified human IL-15 polypeptide described herein comprising a modified human IL-15 polypeptide described herein.
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope in an antigen.
  • CDR complementary determining region
  • Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies, tri-specific antibodies) , single-chain antibodies, single variable domain (V H H) antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., multi-specific antibodies, bi-specific antibodies, single-chain antibodies, diabodies, and linear antibodies formed from these antibodies or antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain, a variable domain of light chain or a V H H) .
  • variable domains include, e.g., Fab, Fab’, F (ab’) 2, and Fv fragments, scFv, and V H H.
  • the fusion protein further comprises an antigen binding moiety.
  • Any suitable antigen binding moiety can be used in the fusion protein described herein.
  • the antigen binding moiety can be an antibody or antigen binding fragment thereof, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule.
  • the antigen binding moiety is an antibody comprising an Fc region.
  • the antibodies or antigen-binding fragments thereof useful in the IL-15 fusion proteins described herein include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, single variable domain (V H H) antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the antigen binding moiety and the modified human IL-15 polypeptide is fused via a linker.
  • Any suitable linker can be used in the fusion protein described herein.
  • the linker is a polypeptide linker.
  • the fusion protein comprises one or more tags.
  • a polypeptide tag include, but not are not limited to a FLAG tag, a 6-His tag, an 8-His tag, or an AVI tag.
  • the IL-15 fusion protein described herein e.g., antibody-IL-15 fusion protein
  • has decreased or no binding to the IL-15 receptor e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c
  • IL-15 receptor e.g., IL-15R ⁇ , IL-2R ⁇ , and/or ⁇ c
  • Such IL-15 fusion protein can deliver cytokines to a desired cell type while minimizing peripheral exposure (e.g., NK cells which are a major site of action) and thus toxicities.
  • a modified IL-15 polypeptide as described herein can be coupled to a PD-1 antibody, which acts as a marker to antigen-specific tumor-resident CD8+ cells, thereby maximizing anti-tumor efficacy and minimizing exposure to peripheral immune cell subsets.
  • the modified IL-15 polypeptide is linked to an Fc region.
  • the present disclosure also provides the introduction of modifications to the modified IL-15 peptides and/or the fusion proteins.
  • the modification improves at least one physical property of the peptide (e.g., solubility, bioavailability, serum half-life, and circulation time) .
  • Other modifications include introducing means for blocking receptor cleavage and increasing affinity for the IL-15 receptor (s) (or modifying the off-rate so that the IL-15 molecule will be docked with the receptor (s) for a longer duration) .
  • modification of the IL-15 peptides does not cause a detrimental effect on immunogenicity of a level that is therapeutically relevant, and in some embodiments the modified IL-15 is less immunogenic than unmodified IL-15.
  • the modification is pegylation and the modified peptide is PEG-IL-15.
  • the pegylated peptides can comprise at least one PEG molecule covalently attached to at least one amino acid residue of IL-15 (e.g., N-terminal or C- terminal pegylation) .
  • the PEG molecule can be conjugated to IL-15 through a linker; linkers are described in detail hereafter.
  • two or more different sites on IL-15 can be modified (e.g., pegylated) by introducing more than one mutation and then modifying each of them.
  • the N-terminus can be modified (e.g., pegylated) in combination with the introduction of one or more mutations, and the modification (e.g., pegylation) thereof, elsewhere within the IL-15 protein.
  • the C-terminus can be modified (e.g., pegylated) in combination with the introduction of one or more mutations, and the modification (e.g., pegylation) thereof, elsewhere within the IL-15 protein.
  • the modified human IL-15 polypeptides, fusion proteins, and immune cells as described herein can modulate (e.g., increase or decrease) immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds, as compared to immune responses without the administration of the modified human IL-15 polypeptides, fusion proteins, and immune cells as described herein.
  • immune cells e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells
  • the binding affinity of the modified human IL-15 polypeptide or fusion protein described herein to the human IL-2 receptor beta (IL-2R ⁇ ) is decreased compared to the binding affinity of a human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the modified human IL-15 polypeptide or fusion protein has a K D for the human IL-2 receptor beta (IL-2R ⁇ ) that is about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 90, about 10 to about 100, about 10 to about 150, about 10 to about 200, about 10 to about 250, about 10 to about 300, about 10 to about 350, about 10 to about 400, about 10 to about 450, about 10 to about 500, about 10 to about 550, about 10 to about 600, about 10 to about 650, about 10 to about 700, about 10 to about 750, about 10 to about 800, about 10 to about 850, about 10 to about 900, about 10 to about 950, about 10 to about 1000, about 100 to about 150, about 100 to about 200, about 100 to about 250, about 100 to about 300, about 100 to about 350, about 100 to about 400, about 1000 to about 450, about
  • the modified human IL-15 polypeptide or fusion protein has a K D for the human IL-2 receptor beta (IL-2R ⁇ ) that is about 10 folds to about 200 folds higher than the K D of a human IL-15 polypeptide, or fusion protein comprising SEQ ID NO: 3.
  • IL-2R ⁇ human IL-2 receptor beta
  • the binding affinity of the modified human IL-15 polypeptide described herein to the human IL-2 receptor beta (IL-2R ⁇ ) is increased compared to the binding affinity of a human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the modified human IL-15 polypeptide or fusion protein has a K D for the human IL-2 receptor beta (IL-2R ⁇ ) that is about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 90, about 10 to about 100, about 10 to about 150, about 10 to about 200, about 10 to about 250, about 10 to about 300, about 10 to about 350, about 10 to about 400, about 10 to about 450, about 10 to about 500, about 10 to about 550, about 10 to about 600, about 10 to about 650, about 10 to about 700, about 10 to about 750, about 10 to about 800, about 10 to about 850, about 10 to about 900, about 10 to about 950, about 10 to about 1000, about 100 to about 150, about 100 to about 200, about 100 to about 250, about 100 to about 300, about 100 to about 350, about 100 to about 400, about 1000 to about 450, about
  • ELISA enzyme-linked immunosorbent assay
  • RIA Radioimmunoassay
  • FACS fluorescence-activated cell sorting
  • SPR surface plasmon resonance
  • the effect of modified human IL-15 polypeptide or fusion protein described herein is evaluated by quantifying a signaling pathway measured by phosphorylation of certain factors, such as STAT5 phosphorylation.
  • STAT5 represents a master regulator of NK and CD8+ cytotoxic T cell function, therefore STAT5 phosphorylation is used to determine the effect of IL-2R ⁇ binding affinity on the activation of NK cell and CD8+ cytotoxic T cells.
  • the activity of modified human IL-15 polypeptide or fusion protein described herein in activating NK cells and CD8+ cytotoxic T cells is comparable to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the activity of modified human IL-15 polypeptide or fusion protein described herein in activating NK cells and CD8+ cytotoxic T cells is decreased compared to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the effect of the modified human IL-15 polypeptide or fusion protein described herein is evaluated by immune cell proliferation assays, using for example Ki67 intracellular staining of immune effector cells.
  • Ki67 is a protein strictly associated with cell proliferation and the percentage of Ki67 on CD8+ T and NK cells may be measured by FACS, which indicates their activities in stimulating CD8+ T and NK cells.
  • the activity of modified human IL-15 polypeptide or fusion protein described herein on the proliferation of NK cells and CD8+ cytotoxic T cells is comparable to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the activity of modified human IL-15 polypeptide or fusion protein described herein on the proliferation of NK cells and CD8+ cytotoxic T cells is decreased compared to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the effect of modified human IL-15 polypeptide or fusion protein described herein is evaluated by assessing NK cells and CD8+ cytotoxic T cells activity measured by inflammatory cytokine production (e.g., TNF- ⁇ and IFN- ⁇ ) .
  • inflammatory cytokine production e.g., TNF- ⁇ and IFN- ⁇
  • the TNF- ⁇ and IFN- ⁇ levels of NK cells and CD8+ cytotoxic T cells treated with the modified human IL-15 polypeptide or fusion protein described herein is comparable to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the TNF- ⁇ and IFN- ⁇ levels of NK cells and CD8+ cytotoxic T cells treated with the modified human IL-15 polypeptide or fusion protein described herein is decreased compared to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
  • the NK cells and CD8+ cytotoxic T cells treated with the modified human IL-15 polypeptide or fusion protein has levels of TNF- ⁇ and IFN- ⁇ that is about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 90, about 10 to about 100, about 10 to about 150, about 10 to about 200, about 10 to about 250, about 10 to about 300, about 10 to about 350, about 10 to about 400, about 10 to about 450, about 10 to about 500, about 10 to about 550, about 10 to about 600, about 10 to about 650, about 10 to about 700, about 10 to about 750, about 10 to about 800, about 10 to about 850, about 10 to about 900, about 10 to about 950, about 10 to about 1000, about 100 to about 150, about 100 to about 200, about 100 to about 250, about 100 to about 300, about 100 to about 350, about 100 to about 400,
  • modified human IL-15 polypeptide or fusion protein described herein decreases, the level of TNF- ⁇ and IFN- ⁇ decrease, especially for modified human IL-15 polypeptide or fusion protein with IL-2R ⁇ binding affinity of around 670 nM ( ⁇ 30-fold affinity decrease compared to wild-type IL-15) to 34 ⁇ M (1500-fold affinity decrease compared to wild-type IL-15) , their functional activities of activation NK cells and CD8+ cytotoxic T cells and promoting cell proliferation maintained, but the level of secreted TNF- ⁇ and IFN- ⁇ dropped significantly.
  • the present disclosure provides cells (e.g., engineered immune cells, T cells, NK cells, tumor-infiltrating lymphocytes) that express modified human IL-15 polypeptides or fusion proteins described herein. These engineered cells can be used to treat various disorders or disease as described herein (e.g., cancer) .
  • the cells comprise a nucleic acid encoding the modified human IL-15 polypeptides or fusion proteins described herein.
  • the cell that is engineered can be obtained from e.g., humans and non-human animals.
  • the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species.
  • the cell is from humans, rats or mice.
  • the cells are mouse lymphocytes and engineered (e.g., transduced) to express a modified human IL-15 polypeptide, a fusion protein, or a CAR that includes a modified human IL-15 polypeptide.
  • the cell is obtained from humans.
  • the cell that is engineered is a blood cell.
  • the cell is a leukocyte (e.g., a T cell) , lymphocyte or any other suitable blood cell type.
  • the cell is a peripheral blood cell.
  • the cell is a tumor-infiltrating lymphocyte (TIL) .
  • TIL tumor-infiltrating lymphocyte
  • the cell is a T cell, B cell or NK cell.
  • the cells are human peripheral blood mononuclear cells (PBMCs) .
  • the human PBMCs are CD3+ cells.
  • the human PBMCs are CD8+ cells or CD4+ cells.
  • the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-cell, an iNK-T cell, an NK-T like cell, an ⁇ T cell and a ⁇ T cell.
  • the disclosure also provides modified immune cells that include the modified human IL-15 polypeptide described herein and an engineered receptor.
  • the engineered receptor is a chimeric antigen receptor (CAR) .
  • CAR chimeric antigen receptor
  • a “chimeric antigen receptor” or “CAR” refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, and an intracellular region comprising one or more intracellular signaling domains derived from signal transducing proteins. These intracellular signaling domains are typically different from the polypeptide from which the extracellular domain is derived.
  • the extracellular domain can be any proteinaceous molecule or part thereof that can specifically bind to a predetermined antigen.
  • the extracellular domain comprises an antibody or antigen binding fragment thereof.
  • the intracellular signaling domain can be any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell.
  • Chimeric antigen receptors combine many facets of normal T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell when an antigen is bound.
  • CARs typically have the following regions: an antigen binding domain, an extracellular hinge region, a transmembrane region, and an intracellular region.
  • the intracellular region comprises an intracellular signaling domain or an intracellular signaling region.
  • the antigen binding domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. It interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule.
  • the antigen binding domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scFv) .
  • An scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobulins, connected with a short linker peptide.
  • the antigen binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) heavy chain single variable domains (V H Hs) .
  • the V H Hs are connected with a linker peptide (e.g., a flexible linker) .
  • the linker peptide between the two V H Hs includes hydrophilic residues with stretches of glycine and serine in it for flexibility as well as stretches of glutamate and lysine for added solubility.
  • the linker peptide comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker peptide comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues. In some embodiments, the linker peptide comprises at least or about 1, 2, 3, 4, 5, 6, 7, or 8 serine residues. In some embodiments, the linker peptide comprises or consists of both glycine and serine residues.
  • the linker peptide comprises or consists of a sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100%identical to GGGGS (SEQ ID NO: 23) or GGGGSGGGGSGGGGS (SEQ ID NO: 24) .
  • the linker sequence comprises at least 1, 2, 3, 4, 5, 6, 7, or 8 repeats of GGGGS (SEQ ID NO: 23) .
  • the linker sequence has no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues.
  • the linker peptide comprises 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions.
  • the cell is a T cell.
  • the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell.
  • the cell surface receptor can be a wild-type or recombinant T cell receptor (TCR) , a chimeric antigen receptor (CAR) , or any other surface receptor capable of recognizing an antigenic moiety that is associated with the target cell.
  • T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients) , with a viral vector.
  • the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells.
  • the T cells are T helper type 1 T cells and T helper type 2 T cells.
  • the T cell expressing this receptor is an ⁇ T cell. In alternate embodiments, the T cell expressing this receptor is a ⁇ T cell.
  • the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are T cells.
  • the cell is an NK cell.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the binding molecule, e.g., CAR can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells are stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs) .
  • the cells can be primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the stem cells are cultured with additional differentiation factors to obtain desired cell types (e.g., T cells) .
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity-or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • nucleic acids, compositions, and kits for expressing the IL-15 polypeptides as described herein, and for producing the genetically engineered cells expressing the heterologous IL-15 polypeptides.
  • the genetic engineering generally involves introduction of a nucleic acid encoding the therapeutic molecule, e.g., the modified IL-15 polypeptides, CAR, fusion proteins, into the cell, such as by retroviral transduction, transfection, or transformation.
  • gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical application.
  • a stimulus such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker
  • recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40) , adenoviruses, adeno-associated virus (AAV) .
  • recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors.
  • the retroviral vector has a long terminal repeat sequence (LTR) , e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV) , myeloproliferative sarcoma virus (MPSV) , murine embryonic stem cell virus (MESV) , murine stem cell virus (MSCV) , or spleen focus forming virus (SFFV) .
  • LTR long terminal repeat sequence
  • MoMLV Moloney murine leukemia virus
  • MPSV myeloproliferative sarcoma virus
  • MSV murine embryonic stem cell virus
  • MSCV murine stem cell virus
  • SFFV spleen focus forming virus
  • retroviral vectors are derived from murine retroviruses.
  • the retroviruses include those derived from any avian or mammalian cell source.
  • the retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including
  • the vector is a lentivirus vector.
  • recombinant nucleic acids are transferred into T cells via electroporation.
  • recombinant nucleic acids are transferred into T cells via transposition.
  • Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment and strontium phosphate DNA co-precipitation. Many of these methods are descried e.g., in WO2019195486, which is incorporated herein by reference in its entirety.
  • the T cells are pre-activated, e.g., using anti-CD3/CD28 particles, for about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 60 hours prior to transduction.
  • the transduced T cells are harvested on day 5, day 6, day 7, day 8, day 9, day 10, day 11, or day 12 post transduction.
  • the transfection efficiency of the virus-infected T cells described herein is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.
  • the viability of the transduced T cells is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%on day 0, day 1, day 2, day 3, day 4, or day 5 post transduction.
  • the viability of the transduced T cells is at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%as compared to the viability of untransduced T cells, on day 0, day 1, day 2, day 3, day 4, or day 5 (e.g., on day 5) post transduction.
  • the T cell expansion fold is at least 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 15 folds, 20 folds, 25 folds, 30 folds, 35 folds, 40 folds, 45 folds, or 50 folds, on day 0, day 1, day 2, day 3, day 4, or day 5 post transduction.
  • the T cell expansion fold of the transduced T cells is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%as compared to that of untransduced T cells, on day 0, day 1, day 2, day 3, day 4, or day 5 (e.g., on day 5) post transduction.
  • populations of engineered cells, compositions containing such cells and/or enriched for such cells such as in which cells expressing the modified IL-15 polyeptides make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as T cells, CD8+ or CD4+ cells.
  • the cells are human PBMCs and engineered (e.g., transduced) to express the modified IL-15 polypeptides, CAR, or antigen-binding fragment thereof.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a modified human IL-15 polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a modified human IL-15 polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a
  • a vector is a construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-A tail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • the present disclosure provides a recombinant vector comprising a nucleic acid construct suitable for genetically modifying a cell, which can be used for treatment of pathological disease or condition.
  • Any vector or vector type can be used to deliver genetic material to the cell.
  • vectors include but are not limited to plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs) , yeast artificial chromosomes (YACs) , and human artificial chromosomes (HACs) .
  • Viral vectors can include but are not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems.
  • AAV adeno-associated viral
  • Other vectors that are known in the art can also be used in connection with the methods described herein.
  • the vector is a viral vector.
  • the viral vector can be grown in a culture medium specific for viral vector manufacturing. Any suitable growth media and/or supplements for growing viral vectors can be used in accordance with the embodiments described herein.
  • the viral vector contains constitutive promoters to facilitate expression, exemplary constitutive promoters contemplated herein include, but are not limited to, Cytomegalovirus (CMV) promoters, human elongation factors-1alpha (hEF1 ⁇ ) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , and chicken ⁇ -Actin promoter coupled with CMV early enhancer (CAGG) .
  • the constitutive promoter is a hEF1 ⁇ promoter.
  • the vector used is a recombinant retroviral vector.
  • a retroviral vector is capable of directing the expression of a nucleic acid molecule of interest.
  • a retrovirus is present in the RNA form in its viral capsule and forms a double-stranded DNA intermediate when it replicates in the host cell.
  • retroviral vectors are present in both RNA and double-stranded DNA forms.
  • the retroviral vector also includes the DNA form which contains a recombinant DNA fragment and the RNA form containing a recombinant RNA fragment.
  • the vectors can include at least one transcriptional promoter/enhancer, or other elements which control gene expression.
  • Such vectors can also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used.
  • LTRs long terminal repeats
  • LTRs are identical sequences of DNA that repeat many times (e.g., hundreds or thousands of times) found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes.
  • the vectors can also include a signal which directs polyadenylation, selectable markers such as Ampicillin resistance, Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • retroviral vector used herein can also refers to the recombinant vectors created by removal of the retroviral gag, pol, and env genes and replaced with the gene of interest.
  • the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules.
  • promoters can be multicistronic (bicistronic or tricistronic) .
  • transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site) , which allows coexpression of gene products (e.g., encoding the modified IL-15 polypeptides, CAR and an antibody or antigen binding fragment thereof) by a message from a single promoter.
  • IRES internal ribosome entry site
  • a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF) , two or three genes (e.g., encoding the modified IL-15 polypeptides, CAR and/or an antibody or antigen binding fragment thereof) separated from one another by sequences encoding a self-cleavage peptide (e.g., P2A or T2A) or a protease recognition site (e.g., furin) .
  • the ORF thus encodes a single polyprotein, which, either during (in the case of 2A e.g., T2A) or after translation, is cleaved into the individual proteins.
  • the peptide such as T2A
  • T2A can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.
  • eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; HEK293 cells, including HEK293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; cells; and NSO cells.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the proteins or polypeptides.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in HEK293 cells.
  • the disclosure relates to a cell comprising the vector or the pair of vectors as described herein.
  • vectors encoding modified human IL-15 polypeptides, fusion proteins, CARs and/or fragments thereof.
  • sequence of the vectors are codon-optimized.
  • nucleic acid sequence comprising a nucleotide sequence encoding any of the modified human IL-15 polypeptides, fusion proteins, CARs, and/or CAR-derived binding molecules (including e.g., functional portions and functional variants thereof, polypeptides, or proteins described herein) .
  • Nucleic acid as used herein can include “polynucleotide, ” “oligonucleotide, ” and “nucleic acid molecule, ” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources, which can contain natural, non-natural or altered nucleotides.
  • the nucleic acid comprises complementary DNA (cDNA) . It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it can be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • the present disclosure also provides the nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • the nucleic acid is synthetic.
  • the nucleic acid is cDNA.
  • the polypeptide comprises a signal peptide (e.g., a secretory signal peptide) .
  • the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein.
  • the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
  • the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
  • the present disclosure provides a method or process for preparing, manufacturing and/or using the engineered cells for treatment of pathological diseases or conditions (e.g., cancer) .
  • pathological diseases or conditions e.g., cancer
  • the cells for introduction of the polypeptides described herein can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • a sample such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector) , washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs) , leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.
  • the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • cancer refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • malignancies of the various organ systems such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine.
  • Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections.
  • a carcinogen s
  • cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene and cancer caused by infections, e.g., viral infections.
  • the term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • an “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • the term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin.
  • a hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • a hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided.
  • Veterinary and non-veterinary applications are contemplated in the present disclosure.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • the disclosure provides methods for reducing toxicity of a cancer treatment regimen in a subject undergoing the treatment, including administering to the subject an effective amount of the modified human IL-15 polypeptides, fusion proteins, or modified immune cells.
  • the reducing toxicity of a cancer treatment regimen involves reducing the affinity of the modified human IL-15 polypeptide for its receptor (s) , e.g., IL-2R ⁇ .
  • the toxicity of a cancer treatment regimen is measured by the adverse effects of the cancer treatment regimen on the subject under treatment.
  • the adverse effect is a grade 3 or grade 4 adverse effect.
  • the toxicity of a cancer treatment regimen is a systemic toxicity, which include, but are limited to, hypotension, acute renal insufficiency, respiratory failure, and neuropsychiatric symptoms.
  • the modification can increase therapeutic index and/or therapeutic window e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70%.
  • the disclosure provides methods for optimizing a therapeutic window of a cancer treatment regimen in a subject undergoing the treatment, including administering to the subject an effective amount of the modified human IL-15 polypeptides, fusion proteins, or modified immune cells.
  • the therapeutic window between effective doses of the modified human IL-15 polypeptides or the pharmaceutical composition and those at which they produce adverse toxic effects is broadened.
  • the therapeutic window is changed based on the adjustment of the dosages and administration frequencies of the polypeptide, fusion proteins, and/or immune cells described herein.
  • the disclosure features methods that include administering a therapeutically effective amount of the modified human IL-15 polypeptides, fusion proteins, or modified immune cells to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) .
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • the cancer is gastric cancer, small intestine cancer, sarcoma, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, biliary cancer, neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, renal cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma, brain tumor, bone cancer, eye cancer, or melanoma.
  • the cancer is a hematological malignancy.
  • the hematological malignancy is selected from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin’s lymphoma, nodular lymphocyte predominant Hodgkin’s lymphoma, Kahler’s disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin’s lymphoma (NHL) , acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL) , acute lymphocytic leukemia (ALL) , chronic myeloid leukemia (CML) , follicular lymphoma, Burkitt’s lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B- lymphoblastic
  • the cancer is relapsed, refractory, or metastatic.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent and/or therapeutic compositions is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • the term “delaying development of a disease” refers to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer) .
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • a late stage cancer such as development of metastasis, can be delayed.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of compositions used.
  • Effective amounts and schedules for administrations may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the treatment, the route of administration, the particular type of therapeutic agents and other drugs being administered to the mammal. Guidance in selecting appropriate doses can be found in the literature. In addition, a treatment does not necessarily result in the 100%or complete treatment or prevention of a disease or a condition. There are multiple treatment/prevention methods available with a varying degree of therapeutic effect which one of ordinary skill in the art recognizes as a potentially advantageous therapeutic mean.
  • Also included are methods of stimulating immune responses in a subject comprising administering to the subject an effective amount of the modified IL-15 polypeptide, fusion protein, or engineered immune cell as described herein. Also included are methods of suppressing immune responses in a subject comprising administering to the subject an effective amount of the modified IL-15 polypeptide, fusion protein, or engineered immune cell as described herein.
  • methods of stimulating immune responses in a subject comprising administering to the subject an effective amount of the modified IL-15 polypeptide, fusion protein, or engineered immune cell as described herein.
  • methods of suppressing immune responses in a subject comprising administering to the subject an effective amount of the modified IL-15 polypeptide, fusion protein, or engineered immune cell as described herein.
  • a modified IL-15 polypeptide that lacks the ability to bind the IL-2R ⁇ c complex may be particularly advantageous.
  • the modified IL-15 polypeptides, fusion proteins, or engineered immune cells can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • the modified human IL-15 polypeptides and at least one additional therapeutic agent are administered in two different administrations.
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule.
  • the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • the one or more additional therapeutic agents can be administered to the subject prior to, concurrently with, or after administering the modified IL-15 polypeptides to the subject.
  • the additional therapeutic agent is an immune therapy.
  • the additional therapeutic agent is a CAR-T therapy.
  • the additional therapeutic agent is an antibody, a cytokine, or a chemotherapy (e.g., a small molecule agent) .
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) .
  • an inhibitor of B-Raf an EGFR inhibitor
  • an inhibitor of a MEK an inhibitor of ERK
  • K-Ras an inhibitor of c-Met
  • ALK an inhibitor
  • the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) .
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, prala
  • therapeutic agents
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
  • TNF tumor necrosis factor
  • carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
  • the additional therapeutic agent is selected from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and/or combinations thereof.
  • the disclosure also provides methods of improving efficacy of a therapy (e.g., an immune therapy, an antibody therapy or a cell therapy) .
  • a therapy e.g., an immune therapy, an antibody therapy or a cell therapy.
  • the methods involve administering to the subject an effective amount of the modified IL-15 polypeptide or the fusion protein as described herein in combination with the therapy.
  • compositions including pharmaceutical and therapeutic compositions
  • methods e.g., therapeutic methods for administrating the modified human IL-15 polypeptides, fusion proteins, immune cells, and compositions thereof to subjects, e.g., patients or animal models (e.g., mice) .
  • compositions including the modified human IL-15 polypeptides, fusion proteins, immune cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof are provided.
  • the pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carrier or excipient.
  • the composition includes at least one additional therapeutic agent.
  • a pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient.
  • the pharmaceutically acceptable carrier does not interfere with the active ingredient and is nontoxic to a subject.
  • a pharmaceutically acceptable carrier can include, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the pharmaceutical formulation refers to process in which different substances and/or agents are combined to produce a final medicinal product. The formulation studies involve developing a preparation of drug acceptable for patient. Additionally, a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the pharmaceutical composition can contain preservatives.
  • Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride.
  • a mixture of two or more preservatives is used.
  • the preservative or mixtures thereof are typically present in an amount of about 0.0001%to about 2%by weight of the total composition.
  • Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) .
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001%to about 4%by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005) .
  • the formulations can include aqueous solutions.
  • the formulation or composition can also contain more than one active ingredient useful for a particular indication, disease, or condition, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition can further include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • other pharmaceutically active agents or drugs such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutic agents e.g., asparaginase
  • the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • the desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
  • the cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous.
  • immunoresponsive T cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject after genetically modifying them in accordance with various embodiments described herein.
  • Peripheral blood derived immunoresponsive T cells or their progeny e.g., in vivo, ex vivo or in vitro derived
  • a therapeutic composition e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell
  • it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion) .
  • Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the cell populations are administered parenterally.
  • parenteral, ” as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as a suitable carrier, diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose) , pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts can in some aspects be consulted to prepare suitable preparations.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, and sorbic acid.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.
  • compositions e.g., compositions including the modified human IL-15 polypeptides or fusion proteins
  • uses of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancers e.g., the methods described herein can reduce the risk of the developing diseases, conditions, and disorders as described herein.
  • the cells, populations, and compositions, described herein are administered to a subject or patient having a particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • adoptive cell therapy such as adoptive T cell therapy.
  • cells and compositions prepared by the provided methods such as engineered compositions and end-of-production compositions following incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition.
  • the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in cancer expressing an antigen recognized by the engineered T cells.
  • adoptive T cell therapy methods are described, e.g., in U.S. 2003/0170238; U.S. Pat. No. 4,690,915; Rosenberg, Nature reviews Clinical oncology 8.10 (2011) : 577; Themeli et al., Nature biotechnology 31.10 (2013) : 928; Tsukahara et al., Biochemical and biophysical research communications 438.1 (2013) : 84-89;Davila et al., PloS one 8.4 (2013) ; each of which is incorporated herein by reference in its entirety.
  • the cell therapy e.g., adoptive T cell therapy
  • the cell therapy is carried out by autologous transfer, in which the T cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the subject has been treated with a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells.
  • a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells.
  • the subject is refractory or non-responsive to the other therapeutic agent.
  • the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT.
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
  • the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time.
  • the subject has not relapsed.
  • the subject is determined to be at risk for relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
  • the subject has not received prior treatment with another therapeutic agent.
  • the compositions or cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type (s) and/or a desired ratio of cell types.
  • the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio.
  • the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types.
  • the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
  • compositions comprising the modified human IL-15 polypeptides are administered at one or more doses of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 mcg/kg/d. Any other suitable dosages can also be used in the methods described herein.
  • multiple doses can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) .
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) .
  • the IL-15 polypeptides in the compositions described herein are PEGylated. In some embodiments, the compositions comprising the modified human IL-15 polypeptides are administered at a dose of about 1-100 mcg/kg about every 21 days. In some embodiments, the compositions comprising the modified human IL-15 polypeptides are administered at a dose of about 1-100 mcg/kg about every three weeks.
  • the cells or individual populations of sub-types of cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values) , such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values) , and in some cases about 100 million cells to about 50 billion cells (e.g., about 120
  • the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about 10 4 and at or about 10 9 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg, or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 6 T cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells are administered at or within a certain range of error of, greater than, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 CD4+ cells, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 CD8+ cells, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 T cells.
  • the cells are administered at or within a certain range of error of between about 10 8 and 10 12 or between about 10 10 and 10 11 T cells, between about 10 8 and 10 12 or between about 10 10 and 10 11 CD4+ cells, and/or between about 10 8 and 10 12 or between about 10 10 and 10 11 CD8+ cells.
  • the cells described herein can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
  • the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the modified human IL-15 polypeptides, fusion proteins, or immune cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the modified human IL-15 polypeptides, fusion proteins, or immune cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered after the one or more additional therapeutic agents. In some embodiments, the methods comprise administration of a chemotherapeutic agent. In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered in combination with cancer-directed monoclonal antibodies to increase the antibody-dependent cellular cytotoxicity (ADCC) of these antibodies, thereby augmenting their antitumor efficacy.
  • ADCC antibody-dependent cellular cytotoxicity
  • the modified human IL-15 polypeptides, fusion proteins, or immune cells induce lower levels of immune responses compared to those induced by the human IL-15 polypeptides, or fusion proteins or immune cells including IL-15 polypeptides without any substitution (e.g., the polypeptide having an amino acid sequence of SEQ ID NO: 3) .
  • the modified human IL-15 polypeptides, fusion proteins, or immune cells induce higher levels of immune responses compared to those induced by the human IL-15 polypeptides, or fusion proteins or immune cells including IL-15 polypeptides without any substitution (e.g., the polypeptide having an amino acid sequence of SEQ ID NO: 3) .
  • Example 1 IL-15 quaternary complex structure analysis and single-point saturation mutation library construction
  • residues that are on the surface of wild-type (WT) IL-15 are: N1, V3, N4, S7, D8, K10, K11, D61, T62, E64, N65, I68, and L69 (FIG. 2) .
  • IL-15 single-point mutant library To create IL-15 single-point mutant library, the afore-mentioned amino acids were mutated to the other amino acids except cysteine.
  • the site-directed mutation was carried out using Q5 Site-Directed Mutagenesis Kit Mix (NEB, Cat. No: E0552S) according to manufacturer’s manual. The site-directed mutation was confirmed by DNA sequencing. The DNA coding Avi-tag was added to the 3’-end of the IL-15 variants.
  • Individual mutant plasmid was Maxipreped and was subsequently used to transfect HEK293 cells. Transfected HEK293 cells were cultured for 96 hours, and IL-15 muteins were secreted in the medium.
  • Example 2 Affinity measurement of crude IL-15 muteins to IL-2R ⁇
  • HEK293 cells were pelleted and the crude IL-15 mutein supernatants were used for affinity measurement by Surface Plasmon Resonance (SPR) .
  • SPR Surface Plasmon Resonance
  • the experiment was performed on a Biacore T200 SPR biosensor (GE Healthcare) at room temperature.
  • the anti-Avi tag sensor chips were prepared at 25°C with a running buffer of 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.005% (v/v) Tween-20, pH 7.4. All surfaces of a Biacore CM5 sensor chip were activated with a 1: 1 (v/v) mixture of 400 mM EDC and 100 mM NHS for 7 minutes, at a flow rate of 10 ⁇ L/min.
  • An anti-Avi reagent (Genscript, Cat. No: A00674-200) was diluted to 30 ⁇ g/mL in 10 mM sodium acetate (pH 5.0) and injected on all flow cells for 7 minutes at 10 ⁇ L/min. All flow cells were blocked with 1 M Ethanolamine-HCl, pH 8.5 for 7 minutes at 10 ⁇ L/min.
  • the affinity determination of all mutein supernatants was performed at 25°C on Biacore T200 using a running buffer of 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% (v/v) Tween-20, pH 7.4.
  • Avi-tagged IL-15 variants were captured on flow cells 2, 3 and 4 at a flow rate of 10 ⁇ L/min for 30 seconds.
  • the flow cell 1 was used as a reference surface.
  • analytes buffer, 250 nM, 500 nM, and 1000 nM concentrations of human IL-2R ⁇ was injected at a flow rate of 30 ⁇ L/min in all flow cells for 100 seconds.
  • the binding affinity range of these muteins are wide, from about 5-fold affinity increase (i.e. D61E mutein) to no binding to IL-2R ⁇ at all.
  • IL-15 muteins (Table 2) were selected to study to what extent IL2R ⁇ -binding affinity affects the efficacy and safety of IL-15.
  • An avi-tag and a 6 ⁇ Histidine tag were added to the C-terminus of IL-15 muteins, and a tobacco etch virus (TEV) protease cleavage sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly was inserted between IL15 mutein and avi-tag.
  • TSV tobacco etch virus
  • the Nineteen IL-15 muteins comprises an amino acid substitution set forth in Table 3 below.
  • STAT5 represents a master regulator of NK and CD8+ cytotoxic T cell function, therefore STAT5 phosphorylation was used to determine the effect of IL-2R ⁇ binding affinity on the activation of NK cell and CD8+cytotoxic T cells.
  • 2 ⁇ 10 5 human PBMCs were treated with 100 nM wild-type IL-15 and IL-15 muteins and incubated at 37°C for 15 minutes. Cells were washed once with FACS buffer (2%(v/v) FBS/PBS) and first stained with surface marker antibodies, including anti-human CD56-Alexa Fluor 647 (Biolegend, Cat.
  • NK and CD8+ cytotoxic T cell proliferation upon IL-15 mutein treatment was also studied. Briefly, 1 ⁇ 10 6 human PBMCs were treated with 100 nM wild-type IL-15 and IL-15 muteins and incubated at 37°C for 3 days. On Day 3, cells were washed once with FACS buffer (2% (v/v) FBS/PBS) and first stained with NK and CD8+ T cell surface marker antibodies as described above. Then cells were fixed and permed using FOXP3 Fix/Perm buffer Set (Biolegend, Cat. No: 421403) , and stained with anti-human Ki67-PE (Biolegend, Cat. No: 350504) .
  • FACS buffer 2% (v/v) FBS/PBS
  • TNF- ⁇ tumor necrosis factor alpha
  • IFN- ⁇ interferon gamma
  • the level of TNF- ⁇ and IFN- ⁇ decrease especially for IL-15 muteins with IL-2R ⁇ binding affinity of around 670 nM ( ⁇ 30-fold affinity decrease compared to wild-type IL-15) to 34 ⁇ M (1500-fold affinity decrease compared to wild-type IL-15) , their functional activities of activation NK cells and CD8+ cytotoxic T cells and promoting cell proliferation maintained, but the level of secreted TNF- ⁇ and IFN- ⁇ dropped significantly.

Abstract

Provided herein are modified human IL-15 polypeptides, fusion proteins, and cells expressing the modified human IL-15 polypeptides. Further provided are methods of treating cancer using the modified human IL-15 polypeptides, fusion proteins, or cells.

Description

INTERLEUKIN-15 VARIANTS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATION
This application claims priority benefits of International Application No. PCT/CN2021/105484 filed July 09, 2021, entitled “INTERLEUKIN-15 VARIANTS AND USES THEREOF” , the contents of which is incorporated herein by reference in its entirety.
SEQUENCE STATEMENT
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: P11195-PCT. 220707. Sequence listing. xml, date recorded: July 07, 2022, size: 24 kilobytes) .
TECHNICAL FIELD
This disclosure relates to modified human interleukin-15 (IL-15) polypeptides, and fusion proteins and cells comprising such IL-15 polypeptides. The present disclosure also provides methods of modulating immune responses and treating a disease or disorder using the modified IL-15 polypeptides, fusion proteins, or cells (e.g., immune cells) .
BACKGROUND
The last decade has seen the clinical success of immunotherapeutic strategies which include adoptive transfer of chimeric antigen receptor (CAR) -T and natural killer (NK) cells, and administration of antibodies or recombinant proteins that either co-stimulate immune cells or block the immune checkpoint pathways. Cytokines mediate proliferative and activation signals in the innate and adaptive immune system and play roles in normal physiology, with tightly regulated controls of their production, localization, and activity (see, e.g., Silk AW &Margolin K. 2019. Cytokine Therapy. Hematology/Oncology Clinics of North America 33 (2) : 261-274) .
IL-15 is a pleiotropic cytokine that can activate both the innate immune cells such as NK cells and the adaptive immune cells such as CD8+ cytotoxic T lymphocytes. It plays an important role in formation and maintenance of immunological memory. It is also an essential factor for NK development and homeostasis. However, current uses of IL-15 as a therapeutic  agent are limited by its short half-life and more importantly toxicity due to the triggering of downstream molecules, including other inflammatory cytokines, such as IL-6 and interferon gamma (IFNγ) (see, e.g., Conlon KC et al. 2015. J Clin Oncol 33 (1) : 74-82) . To address these major therapeutic barriers, there is an urgent need for IL-15 polypeptides with improved therapeutic index.
SUMMARY
The disclosure relates to modified human IL-15 polypeptides that include one or more amino acid substitutions. The IL-variants confer improved properties such as increased or decreased binding to IL-15 receptors (e.g., IL-15Rα, IL-2Rβ, and/or γc) , modulating immune responses, and reducing toxicity of immune therapies (e.g., anti-cancer therapies) . The disclosure also relates to fusion proteins and modified immune cells that comprise the modified human IL-15 polypeptides described herein.
In one aspect, the disclosure relates to a modified human interleukin 15 (IL-15) polypeptide comprising one or more amino acid substitutions at  positions  1, 3, 4, 7, 8, 10, 11, 61, 62, 64, 65, 68, and 69, wherein numbering of amino acid residue positions is according to SEQ ID NO: 3.
In some embodiments,
(1) the amino acid residue at position 1 is not N;
(2) the amino acid residue at position 3 is not V;
(3) the amino acid residue at position 4 is not N;
(4) the amino acid residue at position 7 is not S;
(5) the amino acid residue at position 8 is not D;
(6) the amino acid residue at position 10 is not K;
(7) the amino acid residue at position 11 is not K;
(8) the amino acid residue at position 61 is not D;
(9) the amino acid residue at position 62 is not T;
(10) the amino acid residue at position 64 is not E;
(11) the amino acid residue at position 65 is not N;
(12) the amino acid residue at position 68 is not I; and/or
(13) the amino acid residue at position 69 is not L.
In some embodiments, the amino acid substitutions comprise one or more of the following:
(1) the amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(2) the amino acid residue at position 3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;
(3) the amino acid residue at position 4 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(4) the amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
(5) the amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(6) the amino acid residue at position 10 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(7) the amino acid residue at position 11 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(8) the amino acid residue at position 61 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(9) the amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;
(10) the amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(11) the amino acid residue at position 65 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(12) the amino acid residue at position 68 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; and
(13) the amino acid residue at position 69 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y.
In some embodiments, the modified human IL-15 has a decreased binding affinity to human IL-2 receptor beta (IL-2Rβ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2Rβ.
In some embodiments, the modified human IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(2) the amino acid residue at position 3 is D, R, S, or T;
(3) the amino acid residue at position 4 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W;
(4) the amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
(5) the amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(6) the amino acid residue at position 10 is A, D, E, F, G, I, L, M, N, P, Q, S, T, V, W, or Y;
(7) the amino acid residue at position 11 is A, D, F, G, H, I, L, N, P, T, V, W, or Y;
(8) the amino acid residue at position 61 is F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(9) the amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, or Y;
(10) the amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(11) the amino acid residue at position 65 is A, E, F, G, H, I, K, L, M, P, Q, R, T, V, W, or Y;
(12) the amino acid residue at position 68 is A, D, E, F, H, K, L, M, P, Q, R, S, T, V, W, or Y; and
(13) the amino acid residue at position 69 is D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or W.
In some embodiments, the modified human IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 1 is Q;
(2) the amino acid residue at position 4 is G, L, P, V, M, or W;
(3) the amino acid residue at position 7 is D, E, F, G, I, K, N, P, V, W, or Y;
(4) the amino acid residue at position 8 is E;
(5) the amino acid residue at position 10 is W;
(6) the amino acid residue at position 11 is D, H, or W;
(7) the amino acid residue at position 61 is H, R, or S;
(8) the amino acid residue at position 62 is E, F, G, I, M, Q, or V;
(9) the amino acid residue at position 64 is A, F, H, L, M, N, S, T, V, W, or Y;
(10) the amino acid residue at position 65 is V;
(11) the amino acid residue at position 68 is M, Q, S, T, V, W, or Y; and
(12) the amino acid residue at position 69 is H, M, N, Q, S, T, or V.
In some embodiments, the modified human IL-15 polypeptide has an increased binding affinity to human IL-2Rβ as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2Rβ.
In some embodiments, the modified IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 3 is A, E, F, H, L, M, N, Q, W, or Y;
(2) the amino acid residue at position 4 is H or Y;
(3) the amino acid residue at position 10 is H or R;
(4) the amino acid residue at position 11 is E, M, or Q; and
(5) the amino acid residue at position 61 is A or E.
In some embodiments, the modified IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 3 is A, E, F, H, L, N, Q, W, or Y;
(2) the amino acid residue at position 4 is Y;
(3) the amino acid residue at position 10 is H or R;
(4) the amino acid residue at position 11 is E or Q; and
(5) the amino acid residue at position 61 is E.
In some embodiments, the modified human IL-15 polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 3.
In some embodiments, the modified human IL-15 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 4-22.
In one aspect, the disclosure relates to a fusion protein comprising any one of the modified human IL-15 polypeptide described herein.
In some embodiments, the fusion protein further comprises an antigen binding moiety.
In some embodiments, the antigen binding moiety is selected from the group consisting of an antibody or antigen binding fragment thereof, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule.
In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain, a variable domain of light chain or a V HH) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F (ab’) 2, and Fv fragments, scFv, and V HH.
In some embodiments, the antigen binding moiety is an antibody comprising an Fc region.
In some embodiments, the antigen binding moiety and the modified human IL-15 polypeptide is fused via a linker.
In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding any one of the modified human IL-15 polypeptides described herein, or any one of the fusion proteins described herein.
In one aspect, the disclosure relates to an expression vector comprising any one of the nucleic acids described herein.
In one aspect, the disclosure relates to a cell comprising any one of the vectors described herein.
In some embodiments, the cell is an immune cell.
In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-cell, an iNK-T cell, an NK-T like cell, an αβT cell and a γδT cell.
In some embodiments, the immune cell is an NK cell.
In some embodiments, the immune cell is a cytotoxic T cell.
In some embodiments, the modified human IL-15 polypeptide is secreted.
In some embodiments, the modified human IL-15 polypeptide is membrane bound.
In some embodiments, the cell expresses a chimeric antigen receptor (CAR) , a T-cell antigen coupler (TAC) receptor, or a T-cell receptor (TCR) .
In one aspect, the disclosure relates to a pharmaceutical composition comprising any one of the modified human IL-15 polypeptides described herein, any one of the fusion proteins described herein, or any one of the cells described herein and a pharmaceutically acceptable  carrier.
In one aspect, the disclosure relates to a method of producing a modified human IL-15 polypeptide, or a fusion protein comprising a modified human IL-15 polypeptide, the method comprising: (a) culturing any one of the cells described herein under conditions sufficient for the cell to produce the modified human IL-15 polypeptide or the fusion protein; and (b) collecting the modified human IL-15 polypeptide or the fusion protein produced by the cell.
In one aspect, the disclosure relates to a method of producing a modified cell, comprising: introducing into a cell any one of the expression vector described herein.
In some embodiments, the cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, an NK cell, an NK-T cell, an iNK-T cell, an NK-T like cell, an αβT cell and a γδT cell.
In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of any one of the modified human IL-15 polypeptides described herein, any one of the fusion proteins described herein, any one of the pharmaceutical compositions described herein, or any one of the cells described herein, thereby treating cancer in the subject.
In some embodiments, the cancer is selected from the group consisting of gastric cancer, small intestine cancer, sarcoma, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, biliary cancer, neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, renal cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma, brain tumor, bone cancer, eye cancer, and melanoma.
In some embodiments, the cancer is a hematological malignancy.
In some embodiments, the hematological malignancy is selected from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin’s lymphoma, nodular lymphocyte predominant Hodgkin’s lymphoma, Kahler’s disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin’s lymphoma (NHL) , acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL) , acute lymphocytic leukemia (ALL) , chronic myeloid leukemia (CML) , follicular  lymphoma, Burkitt’s lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom’s macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type) , EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other hematopoietic cells related cancer.
In some embodiments, the cancer is relapsed, refractory, or metastatic.
In some embodiments, the method further comprises administering to the individual an additional therapy.
In some embodiments, the additional therapy is surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof.
In some embodiments, the method further comprises administering an effective amount of an additional therapeutic agent to the subject.
In some embodiments, the additional therapeutic agent is an immune therapy.
In some embodiments, the additional therapeutic agent is a CAR-T therapy.
In some embodiments, the additional therapeutic agent is an antibody, a cytokine, or a chemotherapy.
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 this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and  examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the disclosure will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
FIG. 1 shows a sequence alignment of two isoforms of IL-15 (SEQ ID NO: 1 and SEQ ID NO: 2) produced by alternative splicing.
FIG. 2 shows the amino acid sequence of a human IL-15 peptide (SEQ ID NO: 3) . The amino acid positions of the one or more substitutions are underlined.
FIG. 3 shows the effect of IL-2Rβ binding affinity on the activation of NK cell and CD8+ cytotoxic T cells.
FIGs. 4A-4B show the effect of IL-2Rβ binding affinity on NK and CD8+ cytotoxic T cell proliferation. In addition, FIG. 4A shows the level of IFN-γ and FIG. 4B shows the level of TNF-α secreted to the medium.
DETAILED DESCRIPTION
This disclosure provides modified human interleukin-15 (IL-15) polypeptides, and fusion proteins and cells comprising such IL-15 polypeptides. The modification on the human IL-15 polypeptide improves the therapeutic index. The therapeutic index is a quantitative measurement of the relative safety of a drug. It is a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes toxicity. In one aspect, the modified human IL-15 polypeptides have a greater therapeutic window. The therapeutic window or safety window refer to a range of doses which optimize between efficacy and toxicity, achieving the greatest therapeutic benefit without resulting in unacceptable side-effects or toxicity.
The present disclosure also provides methods of modulating immune responses and treating a disease or disorder using the modified IL-15 polypeptides, fusion proteins, or cells  (e.g., immune cells) .
INTERLEUKIN-15 (IL-15)
IL-15 belongs to the four α-helix bundle family of cytokines. Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (IL-2/IL-15Rβ, also known as CD122) and the common gamma chain (CD132) . Unlike IL-2, the predominant mechanism of IL-15 signaling is trans-presentation which is mediated by membrane-bound complex IL-15/IL-15Rα (see, e.g., Dubois S et al., 2002. Immunity 17 (5) : 537-547) , i.e., signaling pathway of IL-15 begins with binding to IL-15Rα receptor which is usually expressed by antigen presenting cells like dendritic cells or monocytes, with subsequent presentation to surrounding effector cells like NK cells and T cells bearing IL-2Rβ and common gamma chain complex on their cell surface. Upon binding IL-2Rβ subunit activates Janus kinase 1 (Jak1) and γc subunit Janus kinase 3 (Jak3) , which leads to phosphorylation and activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 (see, e.g., Okada S et al., Immunology and Cell Biology 93 (5) : 461-71. ) .
From the X-ray structure of IL-15 with its three receptors simultaneously (IL-15/IL-15Rα/IL-2Rβ/γc) , the average number of contacts involving the IL-2Rβ chain is around 30, whereas the corresponding value is 20 for γc chains. Also from a binding free energy perspective, ΔGbind free energy calculated for the IL-15/IL-2Rβ interface is about -28 kcal/mol, greater than the IL-15/γc (-16 kcal/mol) (see, e.g., Sousa RP et al., 2019. Molecules 24 (18) : 3261) . IL-2Rβbinds tighter than γc. Mutating IL-15 residues on IL-15/IL-2Rβ interface can modulate the activity of IL-15, thus can improve the therapeutic index of IL-15.
IL-15 is produced constitutively by different cell types such as monocytes, macrophages, dendritic cells, stromal cells, epithelial cells. Rather complex biological features characterize IL-15. Regarding IL-15, it exists under several functional forms: (1) a soluble monomeric form (sIL-15) , (2) the soluble complex sIL-15/IL-15Rα, (3) the transpresented form (tp-IL-15) and (4) the transmembrane form (tmb-IL-15) (see, e.g., Fiore PF et al., Journal for Immuno Therapy of Cancer 2020; 8: e001428) .
The sequences of two isoforms of IL-15 produced by alternative splicing and the IL-15 peptide are shown below:
IL-15 precursor (SEQ ID NO: 1)
Figure PCTCN2022104519-appb-000001
IL-15 precursor (SEQ ID NO: 2)
Figure PCTCN2022104519-appb-000002
IL-15 Peptide (SEQ ID NO: 3)
Figure PCTCN2022104519-appb-000003
IL-15 functions on binding to the IL-2Rβ/γc heterodimer, shared with IL-2, formed by IL-2Rβ and γc chains. IL-2Rβ/γc dimeric receptor binds IL-15 and transduce the signal within the cells by activating the Jak/Stat pathway (Jak 1 and 3, Stat 3 and 5) . The IL-15Rα chain confers specificity to IL-15. IL-15Rα forms a high affinity trimeric receptor with IL-2Rβ and γc chains, allowing the cells to respond to low concentrations of IL-15 (see, e.g., Fiore PF et al., Journal for Immuno Therapy of Cancer 2020; 8: e001428) .
Cytokines such as IL-2 and IL-15 induce immune responses through immunological checkpoints such as cytotoxic T-lymphocyte-associated 4 (CTLA-4) , programmed death protein-1 (PD-1, CD279) , and PD-1 ligands PD-L1 (CD274, B7-H1) and PD-L2 (CD272, B7-DC) . Cytokine stimulation also induces the secretion of inhibitory factors such as IL-10 and transforming growth factor (TGF-β) , the expression of inhibitors such as triosephosphate isomerase (TIM) , and the activation of immune-dampening cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) , as well as the activation of intracellular  suppressors of cytokine signaling (CIS, SOCS) proteins that terminate the CD4 T cell immune response (see, e.g., Sckisel GD et al., 2015, Immunity 43 (2) : 240-250) . While cytokines such as IL-2 or IL-15 dramatically increase the number of activated natural killer (NK) cells, these effector cells are inhibited by the interaction of self-class I-A, B, major histocompatibility complex (MHC) molecules with recognition and killer-cell immunoglobulin (Ig) -like receptors (KIRs) , and MHC self-class I-E with NKG2A that are upregulated by immune stimulation.
The last decade has witnessed impressive progress in the field of cancer immunotherapy, such as immune checkpoint inhibition, and T-cell engaging therapies like bispecific T-cell engager and chimeric antigen receptor (CAR) T cells. However, along with these potent immunotherapeutic agents comes the increasing awareness of their inherent adverse effects, most notably the cytokine release syndrome (CRS) . One proposed pathomechanism of CRS is activation of manly T cells or lysis of immune cells induces a release of IFN-γ or TNF-α. This leads to the activation of macrophages, dendritic cells, other immune cells and endothelial cells. These cells further release proinflammatory cytokines. Importantly, macrophages and endothelial cells produce large amounts of interleukin 6 (IL-6) which in a positive feedback loop manner activates T cells and other immune cells leading to a cytokine storm (Shimabukuro-Vornhagen et al.Journal for ImmunoTherapy of Cancer (2018) 6: 56) . By tuning the IL-2Rβ binding affinity of IL-15, the therapeutic window can be significantly improved without compromising the efficacy of IL-15.
MODIFIED IL-15 POLYPEPTIDES
Disclosed herein are modified IL-15 polypeptides (e.g., human IL-15) comprising one or more amino acid substitutions. In some embodiments, the one or more substitutions are at  positions  1, 3, 4, 7, 8, 10, 11, 61, 62, 64, 65, 68, and/or 69, and numbering of the amino acid residue positions is according to SEQ ID NO: 3.
Also disclosed herein are modified human IL-15 peptides (or IL-15 muteins) comprising one or more of the following:
the amino acid residue at position 1 is not N;
the amino acid residue at position 3 is not V;
the amino acid residue at position 4 is not N;
the amino acid residue at position 7 is not S;
the amino acid residue at position 8 is not D;
the amino acid residue at position 10 is not K;
the amino acid residue at position 11 is not K;
the amino acid residue at position 61 is not D;
the amino acid residue at position 62 is not T;
the amino acid residue at position 64 is not E;
the amino acid residue at position 65 is not N;
the amino acid residue at position 68 is not I; and
the amino acid residue at position 69 is not L.
In some embodiments, the numbering of the amino acid residue positions is according to SEQ ID NO: 3. In some embodiments, the one or more positions can have any suitable amino acid residue other than the excluded amino acid residue.
Also disclosed herein are modified human interleukin 15 (IL-15) polypeptides comprising one or more amino acid substitutions at positions that correspond to N1, V3, N4, S7, D8, K10, K11, D61, T62, E64, N65, I68, and L69 of SEQ ID NO: 3.
The disclosure also provides are polypeptides, e.g., modified IL-15 polypeptides, comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 3, wherein the polypeptide comprises at least one amino acid substitution at positions that correspond to N1, V3, N4, S7, D8, K10, K11, D61, T62, E64, N65, I68, and L69 of SEQ ID NO: 3.
In some embodiments, the polypeptide disclosed herein, e.g., the modified human IL-15 polypeptide, contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions. In some embodiments, the polypeptide contains about 1-10, about 1-20, about 1-30, about 1-40, or about 1-50 amino acid substitutions.
In some embodiment, the modified IL-15 polypeptide disclosed herein has an amino acid sequence that is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to the amino acid sequence of SEQ ID NO: 1, 2, or 3. In some embodiments, the modified IL-15 polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 3.
The modified IL-15 polypeptide described herein can be in any suitable form of IL-15  (e.g., human IL-15) . In some embodiments, the modified IL-15 polypeptide is a soluble monomeric IL-15 (sIL-15) . In some embodiments, the modified IL-15 polypeptide is a soluble complex (sIL-15/IL-15Rα) . In some embodiments, the modified IL-15 polypeptide is in a transpresented form (tp-IL-15) . In some embodiments, the modified IL-15 polypeptide is in a transmembrane form (tmb-IL-15) .
In some embodiments, the amino acid substitutions comprise one or more of the following:
(1) the amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(2) the amino acid residue at position 3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;
(3) the amino acid residue at position 4 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(4) the amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
(5) the amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(6) the amino acid residue at position 10 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(7) the amino acid residue at position 11 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(8) the amino acid residue at position 61 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(9) the amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;
(10) the amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(11) the amino acid residue at position 65 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
(12) the amino acid residue at position 68 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; and
(13) the amino acid residue at position 69 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, the numbering of the amino acid residue positions is according to SEQ ID NO: 3.
In some embodiments, the amino acid substitutions comprise one or more of the following:
(1) the amino acid residue at position 1 is M;
(2) the amino acid residue at position 3 is E, H, L, T, or Y;
(3) the amino acid residue at position 4 is F, M, P, or V;
(4) the amino acid residue at position 7 is F, P, W, or Y;
(5) the amino acid residue at position 8 is E or L,
(6) the amino acid residue at position 61 is E, R, or S;
(7) the amino acid residue at position 62 is A, E, G, H, I, L, M, Q, S, or V;
(8) the amino acid residue at position 64 is W or Y,
(9) the amino acid residue at position 65 is V;
(10) the amino acid residue at position 68 is Q, V, or W; and
(11) the amino acid residue at position 69 is H or N.
The modified human IL-15 polypeptide disclosed herein can have an altered binding affinity to an IL-15 receptor (e.g., IL-15Rα, IL-2Rβ, and/or γc) compared to the binding affinity of a human IL-15 polypeptide without such substitution (s) (e.g., a wild-type IL-15) to the same receptor. In some embodiments, the modified human IL-15 polypeptide disclosed herein has a decreased binding affinity to an IL-15 receptor (e.g., IL-15Rα, IL-2Rβ, and/or γc) compared to the binding affinity of a human IL-15 polypeptide without such substitution (s) (e.g., a wild-type IL-15) to the same receptor. In some embodiments, the modified human IL-15 polypeptide disclosed herein has an increased binding affinity to an IL-15 receptor (e.g., IL-15Rα, IL-2Rβ, and/or γc) compared to the binding affinity of a human IL-15 polypeptide without such substitution (s) (e.g., a wild-type IL-15) to the same receptor.
In some embodiments, the modified human IL-15 polypeptide disclosed herein has a decreased binding affinity to human IL-2 receptor beta (IL-2Rβ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2Rβ. In some embodiments, the modified IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W,  or Y;
(2) the amino acid residue at position 3 is D, R, S, or T;
(3) the amino acid residue at position 4 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W;
(4) the amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
(5) the amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(6) the amino acid residue at position 10 is A, D, E, F, G, I, L, M, N, P, Q, S, T, V, W, or Y;
(7) the amino acid residue at position 11 is A, D, F, G, H, I, L, N, P, T, V, W, or Y;
(8) the amino acid residue at position 61 is F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
(9) the amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, or Y;
(10) the amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
(11) the amino acid residue at position 65 is A, E, F, G, H, I, K, L, M, P, Q, R, T, V, W, or Y;
(12) the amino acid residue at position 68 is A, D, E, F, H, K, L, M, P, Q, R, S, T, V, W, or Y; and
(13) the amino acid residue at position 69 is D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or W.
In some embodiments, the modified IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 1 is Q;
(2) the amino acid residue at position 4 is G, L, P, V, M, or W;
(3) the amino acid residue at position 7 is D, E, F, G, I, K, N, P, V, W, or Y;
(4) the amino acid residue at position 8 is E;
(5) the amino acid residue at position 10 is W;
(6) the amino acid residue at position 11 is D, H, or W;
(7) the amino acid residue at position 61 is H, R, or S;
(8) the amino acid residue at position 62 is E, F, G, I, M, Q, or V;
(9) the amino acid residue at position 64 is A, F, H, L, M, N, S, T, V, W, or Y;
(10) the amino acid residue at position 65 is V;
(11) the amino acid residue at position 68 is M, Q, S, T, V, W, or Y; and
(12) the amino acid residue at position 69 is H, M, N, Q, S, T, or V.
In some embodiments, the modified human IL-15 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 5-22.
In some embodiments, the modified human IL-15 polypeptide disclosed herein has an increased binding affinity to human IL-2 receptor beta (IL-2Rβ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2Rβ. In some embodiments, the modified IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 3 is A, E, F, H, L, M, N, Q, W, or Y;
(2) the amino acid residue at position 4 is H or Y;
(3) the amino acid residue at position 10 is H or R;
(4) the amino acid residue at position 11 is E, M, or Q; and
(5) the amino acid residue at position 61 is A or E.
In some embodiments, the modified IL-15 polypeptide comprises one or more of the following:
(1) the amino acid residue at position 3 is A, E, F, H, L, N, Q, W, or Y;
(2) the amino acid residue at position 4 is Y;
(3) the amino acid residue at position 10 is H or R;
(4) the amino acid residue at position 11 is E or Q; and
(5) the amino acid residue at position 61 is E.
In some embodiments, the modified human IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 4.
In some embodiments, the modified IL-15 specifically binds to IL-2Rβ (e.g., human IL-2Rβ) with a dissociation rate (koff) of less than 0.1 s -1, less than 0.01 s -1, less than 0.001 s -1, less than 0.0001 s -1, or less than 0.00001 s -1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1, greater than 0.001 s -1, greater than 0.0001 s -1, greater than 0.00001 s -1, or greater than 0.000001 s -1. In some embodiments, kinetic association rates (kon) is greater than 1×10 2/Ms, greater than 1×10 3/Ms, greater than 1×10 4/Ms, greater than 1×10 5/Ms, or greater than 1×10 6/Ms. In some embodiments, kinetic association rates (kon) is less than 1×10 5/Ms, less than  1×10 6/Ms, or less than 1×10 7/Ms. KD can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M, or less than 1×10 -11 M. In some embodiments, the KD is less than 50nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10 -6 M, 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10  -11 M, or greater than 1×10 -12 M.
In some embodiments, the modified IL-15 has a KD with IL-2Rβ that is less than the KD of D61R. In some embodiments, the modified IL-15 has a KD with IL-2Rβ that is less than 1.5×10 -5 M. In some embodiments, the modified IL-15 has a KD with IL-2Rβ that is between 1.5×10 -5 M and 2.3×10 -8 M.
In some embodiments, the modified IL-15 has a KD with IL-2Rβ or IL-2Rβγc that is between 1×10 -4 M and 1×10 -5 M, between 1×10 -5 M and 1×10 -6 M, between 1×10 -6 M and 1×10 -7 M, between 1×10 -7 M and 1×10 -8 M, between 1×10 -8 M and 1×10 -9 M, or between 1×10 -9 M and 1×10 -10 M. In some embodiments, the modified IL-15 has a KD that is higher than 1.8×10 -8 M, 2.3×10 -8 M, 2×10 -8 M, 3×10 -8 M, 4×10 -8 M, 5×10 -8 M, 6×10 -8 M, 7×10 -8 M, 8×10 -8 M, 9×10 -8 M, 1×10 -7 M, 5×10 -7 M, or 1×10 -6 M.
In some embodiments, the modification does not change the binding affinity with IL-15Rα. For example, the change of KD can be less than 20%, 10%, or 5%after such modifications. In some embodiments, the modified IL-15 has a KD with IL-15Ra that is about 1×10 -11 M.
In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from Table 1, e.g., N1A, N1D, N1E, N1F, N1G, N1H, N1I, N1K, N1L, N1M, N1P, N1Q, N1R, N1S, N1T, N1V, N1W, N1Y, V3A, V3D, V3E, V3F, V3G, V3H, V3I, V3K, V3L, V3M, V3N, V3P, V3Q, V3R, V3S, V3T, V3W, V3Y, N4A, N4D, N4E, N4F, N4G, N4H, N4I, N4K, N4L, N4M, N4P, N4Q, N4R, N4S, N4T, N4V, N4W, N4Y, S7A, S7D, S7E, S7F, S7G, S7H, S7I, S7K, S7L, S7M, S7N, S7P, S7Q, S7R, S7T, S7V, S7W, S7Y, D8A, D8E, D8F, D8G, D8H, D8I, D8K, D8L, D8M, D8N, D8P, D8Q, D8R, D8S, D8T, D8V, D8W, D8Y, K10A, K10D, K10E, K10F, K10G, K10H, K10I, K10L, K10M, K10N, K10P, K10Q, K10R, K10S, K10T, K10V, K10W, K10Y, K11A, K11D, K11E, K11F, K11G, K11H, K11I, K11L, K11M, K11N, K11P, K11Q, K11R, K11S, K11T, K11V,  K11W, K11Y, D61A, D61E, D61F, D61G, D61H, D61I, D61K, D61L, D61M, D61N, D61P, D61Q, D61R, D61S, D61T, D61V, D61W, D61Y, T62A, T62D, T62E, T62F, T62G, T62H, T62I, T62K, T62L, T62M, T62N, T62P, T62Q, T62R, T62S, T62V, T62W, T62Y, E64A, E64D, E64F, E64G, E64H, E64I, E64K, E64L, E64M, E64N, E64P, E64Q, E64R, E64S, E64T, E64V, E64W, E64Y, N65A, N65D, N65E, N65F, N65G, N65H, N65I, N65K, N65L, N65M, N65P, N65Q, N65R, N65S, N65T, N65V, N65W, N65Y, I68A, I68D, I68E, I68F, I68G, I68H, I68K, I68L, I68M, I68N, I68P, I68Q, I68R, I68S, I68T, I68V, I68W, I68Y, L69A, L69D, L69E, L69F, L69G, L69H, L69I, L69K, L69M, L69N, L69P, L69Q, L69R, L69S, L69T, L69V, L69W, and/or L69Y. In some embodiments, the modified IL-15 has 1 amino acid substitution selected from Table 1.
In some embodiments, the modified IL-15 has one or more substitutions selected from N1M, V3E, V3H, V3L, V3T, V3Y, N4F, N4M, N4P, N4V, S7F, S7P, S7W, S7Y, D8E, D8L, D61E, D61R, D61S, T62A, T62E, T62G, T62H, T62I, T62L, T62M, T62Q, T62S, T62V, E64W, E64Y, N65V, I68Q, I68V, I68W, L69H and L69N.
In some embodiments, the modified IL-15 does not have D8A, D8N, D61A, and/or N65A amino acid substitutions. In some embodiments, the modified IL-15 does not have T62D, I68F, I68H, I68D, and/or I68K amino acid substitutions. In some embodiments, the modified IL-15 does not have V3I, V3M, V3R, N4H, K11L, K11M, and/or K11R amino acid substitutions. In some embodiments, the modified IL-15 does not have E64D, E64K, E64R, N65D, N65E, N65K, N65R, I68D, I68E, I68K, I68R, L69D, L69E, L69K, and/or L69R amino acid substitutions. In some embodiments, the modified IL-15 does not have N1D, N4D, D8N, D61N, E64Q, and/or N65D amino acid substitutions. In some embodiments, the modified IL-15 does not have D8E, D8Q, D8R, D8S, D8V, D8G, D8I, D8L, and/or D8T amino acid substitutions. In some embodiments, the modified IL-15 does not have S7N, K10Q, D61Q, N65Q, I68A, L69A, and/or L69V amino acid substitutions.
In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to N1 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to V3 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to N4 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to S7 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that  corresponds to D8 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to K10 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to K11 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to D61 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to T62 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to E64 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to N65 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to I68 of SEQ ID NO: 3. In some embodiments, the modified IL-15 does not have amino acid substitutions at a position that corresponds to L69 of SEQ ID NO: 3.
In some embodiments, the modified IL-15 has a weaker binding affinity to IL-2Rβ as compared to a wild-type IL-15. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from e.g., N1A, N1D, N1E, N1F, N1G, N1H, N1I, N1K, N1L, N1M, N1P, N1Q, N1R, N1S, N1T, N1V, N1W, N1Y, V3D, V3R, V3S, V3T, N4A, N4D, N4E, N4F, N4G, N4L, N4M, N4Q, N4S, N4W, S7A, S7E, S7G, S7H, S7I, S7K, S7L, S7M, S7N, S7Q, S7R, S7T, S7V, S7Y, D8E, K10A, K10D, K10E, K10F, K10I, K10L, K10M, K10N, K10Q, K10S, K10T, K10V, K10W, K10Y, K11A, K11D, K11F, K11G, K11H, K11I, K11L, K11N, K11T, K11V, K11W, K11Y, D61G, D61H, D61N, D61Q, D61R, D61S, T62A, T62D, T62F, T62G, T62H, T62I, T62N, T62Q, T62S, T62V, T62Y, E64A, E64D, E64F, E64H, E64I, E64K, E64L, E64M, E64N, E64Q, E64R, E64S, E64T, E64V, E64Y, N65H, I68F, I68L, I68M, I68T, I68V, I68W, I68Y, L69E, L69H, L69I, and/or L69M.
In some embodiments, the modified IL-15 has a KD that is greater than 1×10 -7 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from N1G, N1K, N1Q, N1R, N1W, N4A, N4G, N4L, N4P, N4V, N4Q, N4S, N4W, S7E, S7G, S7I, S7K, S7N, S7P, S7R, S7T, S7V, S7Y, D8E, K10W, K11D, K11G, K11H, K11W, D61H, D61N, D61R, D61S, T62A, T62F, T62G, T62I, T62N, T62Q, T62V, T62Y, E64A, E64F, E64H, E64K, E64L, E64M, E64N, E64Q, E64R,  E64S, E64T, E64V, E64Y, I68F, I68M, I68Q, I68T, I68V, I68W, I68Y, L69E, L69H, and/or L69M.
In some embodiments, the modified IL-15 has a KD that is greater than 1×10 -6 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions selected from N4P, N4V, S7P, D8E, K11D, D61R, E64K, E64S, I68Q I68W, L69H, and/or L69E.
In some embodiments, the modified IL-15 has a KD that is greater than 1×10 -5 M. In some embodiments, the modified IL-15 has 1 or 2 amino acid substitutions selected from D8E, and/or D61R.
In some embodiments, the modified IL-15 has a KD that is between 1×10 -7 M and 3×10 -6 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from N1G, N1K, N1P, N1Q, N1R, N1W, N4A, N4G, N4L, N4P, N4Q, N4S, N4W, S7E, S7F, S7G, S7I, S7K, S7N, S7P, S7R, S7T, S7V, S7Y, K10D, K10V, K10W, K11G, K11H, K11W, D61H, D61N, D61S, T62A, T62F, T62G, T62N, T62Q, T62V, T62Y, E64A, E64F, E64H, E64K, E64L, E64M, E64N, E64Q, E64R, E64S, E64T, E64V, E64W, E64Y, I68F, I68K, I68M, I68Q, I68T, I68S, I68V, I68W, I68Y, L69D, L69E, L69S, L69T, and/or L69M.
In some embodiments, the modified IL-15 has a KD that is between 5×10 -8 M and 1×10 -7 M.In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from N1A, N1E, N1F, N1I, N1L, N1M, N1P, N1V, N1Y, N4D, N4E, N4F, S7H, S7L, S7M, K10D, K10F, K10L, K10V, K11A, K11F, K11I, K11N, K11T, K11Y, D61G, T62S, E64I, and/or L69I.
In some embodiments, the modified IL-15 has a higher binding affinity to IL-2Rβ as compared to a wild-type IL-15. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acid substitutions selected from e.g., V3A, V3E, V3F, V3H, V3L, V3M, V3N, V3Q, V3W, V3Y, N4H, N4Y, K10H, K10R, K11E, K11M, K11Q, D61A, and/or D61E.
In some embodiments, the modified IL-15 has a KD that is less than 1×10 -8 M. In some embodiments, the modified IL-15 has 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from V3F, V3H, V3L, V3W, V3Y, N4H, K10H, K11Q, and/or D61E.
The modified IL-15 polypeptide can be different from a wild-type IL-15 polypeptide  (e.g., SEQ ID NO: 3) . In some embodiments, the modified IL-15 polypeptide is an engineered IL-15 polypeptide or an IL-15 polypeptide variant or an IL-15 mutein.
IL-15 FUSION PROTEINS
Also disclosed herein is a fusion protein (e.g., an antibody-IL-15 fusion protein) comprising a modified human IL-15 polypeptide described herein.
As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope in an antigen. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies, tri-specific antibodies) , single-chain antibodies, single variable domain (V HH) antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., multi-specific antibodies, bi-specific antibodies, single-chain antibodies, diabodies, and linear antibodies formed from these antibodies or antibody fragments.
As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain, a variable domain of light chain or a V HH) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F (ab’) 2, and Fv fragments, scFv, and V HH.
In some embodiments, the fusion protein further comprises an antigen binding moiety. Any suitable antigen binding moiety can be used in the fusion protein described herein. For example, the antigen binding moiety can be an antibody or antigen binding fragment thereof, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule. In some embodiments, the antigen binding moiety is an antibody comprising an Fc region.
The antibodies or antigen-binding fragments thereof useful in the IL-15 fusion proteins described herein include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies  (e.g., bi-specific antibodies) , single-chain antibodies, single variable domain (V HH) antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody.
In some embodiments, the antigen binding moiety and the modified human IL-15 polypeptide is fused via a linker. Any suitable linker can be used in the fusion protein described herein. In some embodiments, the linker is a polypeptide linker.
In some embodiments, the fusion protein comprises one or more tags. Examples of a polypeptide tag include, but not are not limited to a FLAG tag, a 6-His tag, an 8-His tag, or an AVI tag.
In some embodiments, the IL-15 fusion protein described herein (e.g., antibody-IL-15 fusion protein) has decreased or no binding to the IL-15 receptor (e.g., IL-15Rα, IL-2Rβ, and/or γc) compared to the binding affinity of a fusion protein comprising a human IL-15 polypeptide without such substitution (s) . Such IL-15 fusion protein can deliver cytokines to a desired cell type while minimizing peripheral exposure (e.g., NK cells which are a major site of action) and thus toxicities.
In some embodiments, a modified IL-15 polypeptide as described herein can be coupled to a PD-1 antibody, which acts as a marker to antigen-specific tumor-resident CD8+ cells, thereby maximizing anti-tumor efficacy and minimizing exposure to peripheral immune cell subsets.
In some embodiments, the modified IL-15 polypeptide is linked to an Fc region.
The present disclosure also provides the introduction of modifications to the modified IL-15 peptides and/or the fusion proteins. In some embodiments, the modification improves at least one physical property of the peptide (e.g., solubility, bioavailability, serum half-life, and circulation time) . Other modifications include introducing means for blocking receptor cleavage and increasing affinity for the IL-15 receptor (s) (or modifying the off-rate so that the IL-15 molecule will be docked with the receptor (s) for a longer duration) . In some embodiments, modification of the IL-15 peptides does not cause a detrimental effect on immunogenicity of a level that is therapeutically relevant, and in some embodiments the modified IL-15 is less immunogenic than unmodified IL-15. In some embodiments, the modification is pegylation and the modified peptide is PEG-IL-15. The pegylated peptides can comprise at least one PEG molecule covalently attached to at least one amino acid residue of IL-15 (e.g., N-terminal or C- terminal pegylation) . The PEG molecule can be conjugated to IL-15 through a linker; linkers are described in detail hereafter. In some embodiments, two or more different sites on IL-15 can be modified (e.g., pegylated) by introducing more than one mutation and then modifying each of them. In some embodiments, the N-terminus can be modified (e.g., pegylated) in combination with the introduction of one or more mutations, and the modification (e.g., pegylation) thereof, elsewhere within the IL-15 protein. In some embodiments, the C-terminus can be modified (e.g., pegylated) in combination with the introduction of one or more mutations, and the modification (e.g., pegylation) thereof, elsewhere within the IL-15 protein.
IL-15 POLYPEPTIDES CHARACTERISTICS
In some embodiments, the modified human IL-15 polypeptides, fusion proteins, and immune cells as described herein can modulate (e.g., increase or decrease) immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds, as compared to immune responses without the administration of the modified human IL-15 polypeptides, fusion proteins, and immune cells as described herein.
In some embodiments, the binding affinity of the modified human IL-15 polypeptide or fusion protein described herein to the human IL-2 receptor beta (IL-2Rβ) is decreased compared to the binding affinity of a human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the modified human IL-15 polypeptide or fusion protein has a K D for the human IL-2 receptor beta (IL-2Rβ) that is about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 90, about 10 to about 100, about 10 to about 150, about 10 to about 200, about 10 to about 250, about 10 to about 300, about 10 to about 350, about 10 to about 400, about 10 to about 450, about 10 to about 500, about 10 to about 550, about 10 to about 600, about 10 to about 650, about 10 to about 700, about 10 to about 750, about 10 to about 800, about 10 to about 850, about 10 to about 900, about 10 to about 950, about 10 to about 1000, about 100 to about 150, about 100 to about 200, about 100 to about 250, about 100 to about 300, about 100 to about 350, about 100 to about 400, about 1000 to about 450, about  100 to about 500, about 100 to about 550, about 100 to about 600, about 100 to about 650, about 100 to about 700, about 100 to about 750, about 100 to about 800, about 100 to about 850, about 100 to about 900, about 100 to about 950, about 100 to about 1000, about 1000 to about 2000, about 1000 to about 3000, about 1000 to about 4000, about 1000 to about 5000, about 1000 to about 6000, about 1000 to about 7000, about 1000 to about 8000, about 1000 to about 9000, about 1000 to about 10 4, about 10 4 to about 10 5, about 10 5 to about 10 6 folds higher than the K D of a human IL-15 polypeptide or fusion protein comprising SEQ ID NO: 3.
In some embodiments, the modified human IL-15 polypeptide or fusion protein has a K D for the human IL-2 receptor beta (IL-2Rβ) that is about 10 folds to about 200 folds higher than the K D of a human IL-15 polypeptide, or fusion protein comprising SEQ ID NO: 3.
In some embodiments, the binding affinity of the modified human IL-15 polypeptide described herein to the human IL-2 receptor beta (IL-2Rβ) is increased compared to the binding affinity of a human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the modified human IL-15 polypeptide or fusion protein has a K D for the human IL-2 receptor beta (IL-2Rβ) that is about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 90, about 10 to about 100, about 10 to about 150, about 10 to about 200, about 10 to about 250, about 10 to about 300, about 10 to about 350, about 10 to about 400, about 10 to about 450, about 10 to about 500, about 10 to about 550, about 10 to about 600, about 10 to about 650, about 10 to about 700, about 10 to about 750, about 10 to about 800, about 10 to about 850, about 10 to about 900, about 10 to about 950, about 10 to about 1000, about 100 to about 150, about 100 to about 200, about 100 to about 250, about 100 to about 300, about 100 to about 350, about 100 to about 400, about 1000 to about 450, about 100 to about 500, about 100 to about 550, about 100 to about 600, about 100 to about 650, about 100 to about 700, about 100 to about 750, about 100 to about 800, about 100 to about 850, about 100 to about 900, about 100 to about 950, about 100 to about 1000, about 1000 to about 2000, about 1000 to about 3000, about 1000 to about 4000, about 1000 to about 5000, about 1000 to about 6000, about 1000 to about 7000, about 1000 to about 8000, about 1000 to about 9000, about 1000 to about 10 4, about 10 4 to about 10 5, about 10 5 to about 10 6 folds lower than the K D of a human IL-15 polypeptide or fusion protein comprising SEQ ID NO: 3.
General techniques for measuring the affinity of an antibody for an antigen include, e.g., enzyme-linked immunosorbent assay (ELISA) , Radioimmunoassay (RIA) , fluorescence-activated cell sorting (FACS) , and surface plasmon resonance (SPR) .
In some embodiments, the effect of modified human IL-15 polypeptide or fusion protein described herein is evaluated by quantifying a signaling pathway measured by phosphorylation of certain factors, such as STAT5 phosphorylation. STAT5 represents a master regulator of NK and CD8+ cytotoxic T cell function, therefore STAT5 phosphorylation is used to determine the effect of IL-2Rβ binding affinity on the activation of NK cell and CD8+ cytotoxic T cells.
In some embodiments, the activity of modified human IL-15 polypeptide or fusion protein described herein in activating NK cells and CD8+ cytotoxic T cells is comparable to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In In some embodiments, the activity of modified human IL-15 polypeptide or fusion protein described herein in activating NK cells and CD8+ cytotoxic T cells is decreased compared to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the effect of the modified human IL-15 polypeptide or fusion protein described herein is evaluated by immune cell proliferation assays, using for example Ki67 intracellular staining of immune effector cells. Ki67 is a protein strictly associated with cell proliferation and the percentage of Ki67 on CD8+ T and NK cells may be measured by FACS, which indicates their activities in stimulating CD8+ T and NK cells.
In some embodiments, the activity of modified human IL-15 polypeptide or fusion protein described herein on the proliferation of NK cells and CD8+ cytotoxic T cells is comparable to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the activity of modified human IL-15 polypeptide or fusion protein described herein on the proliferation of NK cells and CD8+ cytotoxic T cells is decreased compared to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the effect of modified human IL-15 polypeptide or fusion protein described herein is evaluated by assessing NK cells and CD8+ cytotoxic T cells activity  measured by inflammatory cytokine production (e.g., TNF-α and IFN-γ) .
In some embodiments, the TNF-α and IFN-γ levels of NK cells and CD8+ cytotoxic T cells treated with the modified human IL-15 polypeptide or fusion protein described herein is comparable to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the TNF-α and IFN-γ levels of NK cells and CD8+ cytotoxic T cells treated with the modified human IL-15 polypeptide or fusion protein described herein is decreased compared to that of the human IL-15 polypeptide without amino acid substitutions (e.g., a human IL-15 polypeptide represented by SEQ ID NO: 3) .
In some embodiments, the NK cells and CD8+ cytotoxic T cells treated with the modified human IL-15 polypeptide or fusion protein has levels of TNF-α and IFN-γ that is about 1 to about 10, about 1 to about 20, about 1 to about 30, about 1 to about 40, about 1 to about 50, about 1 to about 60, about 1 to about 70, about 1 to about 80, about 1 to about 90, about 10 to about 100, about 10 to about 150, about 10 to about 200, about 10 to about 250, about 10 to about 300, about 10 to about 350, about 10 to about 400, about 10 to about 450, about 10 to about 500, about 10 to about 550, about 10 to about 600, about 10 to about 650, about 10 to about 700, about 10 to about 750, about 10 to about 800, about 10 to about 850, about 10 to about 900, about 10 to about 950, about 10 to about 1000, about 100 to about 150, about 100 to about 200, about 100 to about 250, about 100 to about 300, about 100 to about 350, about 100 to about 400, about 1000 to about 450, about 100 to about 500, about 100 to about 550, about 100 to about 600, about 100 to about 650, about 100 to about 700, about 100 to about 750, about 100 to about 800, about 100 to about 850, about 100 to about 900, about 100 to about 950, about 100 to about 1000, about 1000 to about 2000, about 1000 to about 3000, about 1000 to about 4000, about 1000 to about 5000, about 1000 to about 6000, about 1000 to about 7000, about 1000 to about 8000, about 1000 to about 9000, about 1000 to about 10 4, about 10 4 to about 10 5, about 10 5 to about 10 6 folds lower than the TNF-α and IFN-γ levels of NK cells and CD8+ cytotoxic T cells treated with a human IL-15 polypeptide or fusion protein comprising SEQ ID NO: 3.
In some embodiments, as the binding affinity of modified human IL-15 polypeptide or fusion protein described herein decreases, the level of TNF-α and IFN-γ decrease, especially for modified human IL-15 polypeptide or fusion protein with IL-2Rβ binding affinity of around 670 nM (～30-fold affinity decrease compared to wild-type IL-15) to 34 μM (1500-fold affinity  decrease compared to wild-type IL-15) , their functional activities of activation NK cells and CD8+ cytotoxic T cells and promoting cell proliferation maintained, but the level of secreted TNF-α and IFN-γ dropped significantly.
ENGINEERED CELLS
The present disclosure provides cells (e.g., engineered immune cells, T cells, NK cells, tumor-infiltrating lymphocytes) that express modified human IL-15 polypeptides or fusion proteins described herein. These engineered cells can be used to treat various disorders or disease as described herein (e.g., cancer) . In some embodiments, the cells comprise a nucleic acid encoding the modified human IL-15 polypeptides or fusion proteins described herein.
In various embodiments, the cell that is engineered can be obtained from e.g., humans and non-human animals. In various embodiments, the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species. Preferably, the cell is from humans, rats or mice. In some embodiments, the cells are mouse lymphocytes and engineered (e.g., transduced) to express a modified human IL-15 polypeptide, a fusion protein, or a CAR that includes a modified human IL-15 polypeptide. In some embodiments, the cell is obtained from humans. In various embodiments, the cell that is engineered is a blood cell. Preferably, the cell is a leukocyte (e.g., a T cell) , lymphocyte or any other suitable blood cell type. In some embodiments, the cell is a peripheral blood cell. In some embodiments, the cell is a tumor-infiltrating lymphocyte (TIL) . In some embodiments, the cell is a T cell, B cell or NK cell. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs) . In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells or CD4+ cells. In some embodiments, the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-cell, an iNK-T cell, an NK-T like cell, an αβT cell and a γδT cell.
The disclosure also provides modified immune cells that include the modified human IL-15 polypeptide described herein and an engineered receptor. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) . A “chimeric antigen receptor” or “CAR” refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, and an intracellular region comprising one or more intracellular signaling domains derived from signal transducing proteins. These intracellular signaling domains are typically  different from the polypeptide from which the extracellular domain is derived. The extracellular domain can be any proteinaceous molecule or part thereof that can specifically bind to a predetermined antigen. In some embodiments, the extracellular domain comprises an antibody or antigen binding fragment thereof. In some embodiments, the intracellular signaling domain can be any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell.
Chimeric antigen receptors (CARs) combine many facets of normal T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell when an antigen is bound. CARs typically have the following regions: an antigen binding domain, an extracellular hinge region, a transmembrane region, and an intracellular region. In some embodiments, the intracellular region comprises an intracellular signaling domain or an intracellular signaling region.
The antigen binding domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. It interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule. The antigen binding domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scFv) . An scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobulins, connected with a short linker peptide. In some embodiments, the antigen binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) heavy chain single variable domains (V HHs) . In some embodiments, the V HHs are connected with a linker peptide (e.g., a flexible linker) . The linker peptide between the two V HHs includes hydrophilic residues with stretches of glycine and serine in it for flexibility as well as stretches of glutamate and lysine for added solubility.
In some embodiments, the linker peptide comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker peptide comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues. In some embodiments, the linker peptide comprises at least or about 1, 2, 3, 4, 5, 6, 7, or 8 serine residues. In some embodiments, the linker peptide comprises or consists of both glycine and serine residues. In some embodiments, the linker peptide comprises or consists of a sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least  or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100%identical to GGGGS (SEQ ID NO: 23) or GGGGSGGGGSGGGGS (SEQ ID NO: 24) . In some embodiments, the linker sequence comprises at least 1, 2, 3, 4, 5, 6, 7, or 8 repeats of GGGGS (SEQ ID NO: 23) . In some embodiments, the linker sequence has no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker peptide comprises 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions.
In some embodiments, the cell is a T cell. In some embodiments, the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell. The cell surface receptor can be a wild-type or recombinant T cell receptor (TCR) , a chimeric antigen receptor (CAR) , or any other surface receptor capable of recognizing an antigenic moiety that is associated with the target cell. T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients) , with a viral vector. In some embodiments, the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells. In some embodiments, the T cells are T helper type 1 T cells and T helper type 2 T cells. In some embodiments, the T cell expressing this receptor is an αβT cell. In alternate embodiments, the T cell expressing this receptor is a γδT cell. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are 
Figure PCTCN2022104519-appb-000004
T cells.
In some embodiments, the cell is an NK cell. In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the binding molecule, e.g., CAR, can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
In some embodiments, the cells are stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs) . The cells can be primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the stem cells are cultured with additional differentiation factors to obtain desired  cell types (e.g., T cells) .
Different cell types can be obtained from appropriate isolation methods. The isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity-or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
Also provided are methods, nucleic acids, compositions, and kits, for expressing the IL-15 polypeptides as described herein, and for producing the genetically engineered cells expressing the heterologous IL-15 polypeptides. The genetic engineering generally involves introduction of a nucleic acid encoding the therapeutic molecule, e.g., the modified IL-15 polypeptides, CAR, fusion proteins, into the cell, such as by retroviral transduction, transfection, or transformation. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical application.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40  (SV40) , adenoviruses, adeno-associated virus (AAV) . In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors. In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR) , e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV) , myeloproliferative sarcoma virus (MPSV) , murine embryonic stem cell virus (MESV) , murine stem cell virus (MSCV) , or spleen focus forming virus (SFFV) . Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In some embodiments, the vector is a lentivirus vector. In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation. In some embodiments, recombinant nucleic acids are transferred into T cells via transposition. Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment and strontium phosphate DNA co-precipitation. Many of these methods are descried e.g., in WO2019195486, which is incorporated herein by reference in its entirety. In some embodiments, the T cells are pre-activated, e.g., using anti-CD3/CD28 particles, for about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 60 hours prior to transduction. In some embodiments, the transduced T cells are harvested on day 5, day 6, day 7, day 8, day 9, day 10, day 11, or day 12 post transduction.
In some embodiments, the transfection efficiency of the virus-infected T cells described herein is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%. In some embodiments, the viability of the transduced T cells is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%on day 0, day 1, day 2, day 3, day 4, or day 5 post transduction. In some embodiments, the viability of the transduced T cells is at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%as compared to the viability of untransduced T cells, on day 0, day 1, day 2, day 3, day 4, or day 5 (e.g., on day 5) post transduction.
In some embodiments, the T cell expansion fold is at least 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 15 folds, 20 folds, 25 folds, 30 folds, 35 folds, 40 folds, 45 folds, or 50 folds, on day 0, day 1, day 2, day 3, day 4, or day 5 post transduction. In some embodiments, the T cell expansion fold of the transduced T cells is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%as compared to that of untransduced T cells, on day 0, day 1, day 2, day 3, day 4, or day 5 (e.g., on day 5) post transduction.
Also provided are populations of engineered cells, compositions containing such cells and/or enriched for such cells, such as in which cells expressing the modified IL-15 polyeptides make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as T cells, CD8+ or CD4+ cells.
In some embodiments, the cells are human PBMCs and engineered (e.g., transduced) to express the modified IL-15 polypeptides, CAR, or antigen-binding fragment thereof.
RECOMBINANT VECTORS
The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a modified human IL-15 polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.
A vector is a construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
A vector can be introduced into the host cell by methods known in the art, e.g.,  electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
The present disclosure provides a recombinant vector comprising a nucleic acid construct suitable for genetically modifying a cell, which can be used for treatment of pathological disease or condition.
Any vector or vector type can be used to deliver genetic material to the cell. These vectors include but are not limited to plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs) , yeast artificial chromosomes (YACs) , and human artificial chromosomes (HACs) . Viral vectors can include but are not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems. Other vectors that are known in the art can also be used in connection with the methods described herein.
In some embodiments, the vector is a viral vector. The viral vector can be grown in a culture medium specific for viral vector manufacturing. Any suitable growth media and/or supplements for growing viral vectors can be used in accordance with the embodiments described herein. In some embodiments, the viral vector contains constitutive promoters to facilitate expression, exemplary constitutive promoters contemplated herein include, but are not limited to, Cytomegalovirus (CMV) promoters, human elongation factors-1alpha (hEF1α) , ubiquitin C promoter (UbiC) , phosphoglycerokinase promoter (PGK) , simian virus 40 early promoter (SV40) , and chicken β-Actin promoter coupled with CMV early enhancer (CAGG) . In some embodiments, the constitutive promoter is a hEF1α promoter.
In some embodiments, the vector used is a recombinant retroviral vector. A retroviral vector is capable of directing the expression of a nucleic acid molecule of interest. A retrovirus is present in the RNA form in its viral capsule and forms a double-stranded DNA intermediate when it replicates in the host cell. Similarly, retroviral vectors are present in both RNA and double-stranded DNA forms. The retroviral vector also includes the DNA form which contains a  recombinant DNA fragment and the RNA form containing a recombinant RNA fragment. The vectors can include at least one transcriptional promoter/enhancer, or other elements which control gene expression. Such vectors can also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used. Long terminal repeats (LTRs) are identical sequences of DNA that repeat many times (e.g., hundreds or thousands of times) found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes. Optionally, the vectors can also include a signal which directs polyadenylation, selectable markers such as Ampicillin resistance, Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence. For example, such vectors can include a 5' LTR, a leading sequence, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3' LTR or a portion thereof. Additionally, retroviral vector used herein can also refers to the recombinant vectors created by removal of the retroviral gag, pol, and env genes and replaced with the gene of interest.
In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such promoters can be multicistronic (bicistronic or tricistronic) . For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site) , which allows coexpression of gene products (e.g., encoding the modified IL-15 polypeptides, CAR and an antibody or antigen binding fragment thereof) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF) , two or three genes (e.g., encoding the modified IL-15 polypeptides, CAR and/or an antibody or antigen binding fragment thereof) separated from one another by sequences encoding a self-cleavage peptide (e.g., P2A or T2A) or a protease recognition site (e.g., furin) . The ORF thus encodes a single polyprotein, which, either during (in the case of 2A e.g., T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.
Various cell lines can be used in connection with the vectors as described herein.  Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; HEK293 cells, including HEK293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; 
Figure PCTCN2022104519-appb-000005
cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the proteins or polypeptides. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in HEK293 cells. In one aspect, the disclosure relates to a cell comprising the vector or the pair of vectors as described herein.
In some embodiments, provided herein are vectors encoding modified human IL-15 polypeptides, fusion proteins, CARs and/or fragments thereof. In some embodiments, sequence of the vectors are codon-optimized.
The present disclosure also provides a nucleic acid sequence comprising a nucleotide sequence encoding any of the modified human IL-15 polypeptides, fusion proteins, CARs, and/or CAR-derived binding molecules (including e.g., functional portions and functional variants thereof, polypeptides, or proteins described herein) . “Nucleic acid” as used herein can include “polynucleotide, ” “oligonucleotide, ” and “nucleic acid molecule, ” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources, which can contain natural, non-natural or altered nucleotides. Furthermore, the nucleic acid comprises complementary DNA (cDNA) . It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it can be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
The present disclosure also provides the nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein. In some embodiments, the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.
In certain embodiments, the polypeptide comprises a signal peptide (e.g., a secretory signal peptide) .
In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any  nucleotide sequences as described herein.
In some embodiments, the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
The present disclosure provides a method or process for preparing, manufacturing and/or using the engineered cells for treatment of pathological diseases or conditions (e.g., cancer) .
The cells for introduction of the polypeptides described herein (e.g., modified human IL-15 polypeptides, fusion proteins, or CARs) , can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector) , washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs) , leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated  lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.
METHODS OF TREATMENT
The modified human IL-15 polypeptides, fusion proteins, and cells disclosed herein can be used for various therapeutic purposes. In one aspect, the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a cancer. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen (s) , cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” is art recognized and  refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. A hematologic cancer is a cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system. Examples of hematologic cancer include e.g., leukemia, lymphoma, and multiple myeloma etc.
As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
In one aspect, the disclosure provides methods for reducing toxicity of a cancer treatment regimen in a subject undergoing the treatment, including administering to the subject an effective amount of the modified human IL-15 polypeptides, fusion proteins, or modified immune cells.
In some embodiments, the reducing toxicity of a cancer treatment regimen involves reducing the affinity of the modified human IL-15 polypeptide for its receptor (s) , e.g., IL-2Rβ. In some embodiments, the toxicity of a cancer treatment regimen is measured by the adverse effects of the cancer treatment regimen on the subject under treatment. In some embodiments, the adverse effect is a grade 3 or grade 4 adverse effect. In some embodiments, the toxicity of a cancer treatment regimen is a systemic toxicity, which include, but are limited to, hypotension, acute renal insufficiency, respiratory failure, and neuropsychiatric symptoms.
In some embodiments, the modification can increase therapeutic index and/or therapeutic window e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, or 70%.
In one aspect, the disclosure provides methods for optimizing a therapeutic window of a cancer treatment regimen in a subject undergoing the treatment, including administering to the subject an effective amount of the modified human IL-15 polypeptides, fusion proteins, or modified immune cells.
In some embodiments, the therapeutic window between effective doses of the modified human IL-15 polypeptides or the pharmaceutical composition and those at which they produce adverse toxic effects is broadened. In some embodiments, the therapeutic window is changed based on the adjustment of the dosages and administration frequencies of the polypeptide, fusion proteins, and/or immune cells described herein.
In one aspect, the disclosure features methods that include administering a therapeutically effective amount of the modified human IL-15 polypeptides, fusion proteins, or modified immune cells to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) .
In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer. Patients with cancer can be identified with various methods known in the art.
In some embodiments, the cancer is gastric cancer, small intestine cancer, sarcoma, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, biliary cancer, neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, renal cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma, brain tumor, bone cancer, eye cancer, or melanoma.
In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is selected from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin’s lymphoma, nodular lymphocyte predominant Hodgkin’s lymphoma, Kahler’s disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin’s lymphoma (NHL) , acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL) , acute lymphocytic leukemia (ALL) , chronic myeloid leukemia (CML) , follicular lymphoma, Burkitt’s lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B- lymphoblastic lymphoma, myeloid leukemia, Waldenstrom’s macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type) , EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, or other hematopoietic cells related cancer.
In some embodiments, the cancer is relapsed, refractory, or metastatic.
As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent and/or therapeutic compositions is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
As used herein, the term “delaying development of a disease” refers to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer) . This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, can be delayed.
An effective amount can be administered in one or more administrations. By way of example, an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount  sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro. As is understood in the art, an effective may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of compositions used.
Effective amounts and schedules for administrations may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the treatment, the route of administration, the particular type of therapeutic agents and other drugs being administered to the mammal. Guidance in selecting appropriate doses can be found in the literature. In addition, a treatment does not necessarily result in the 100%or complete treatment or prevention of a disease or a condition. There are multiple treatment/prevention methods available with a varying degree of therapeutic effect which one of ordinary skill in the art recognizes as a potentially advantageous therapeutic mean.
Also included are methods of stimulating immune responses in a subject comprising administering to the subject an effective amount of the modified IL-15 polypeptide, fusion protein, or engineered immune cell as described herein. Also included are methods of suppressing immune responses in a subject comprising administering to the subject an effective amount of the modified IL-15 polypeptide, fusion protein, or engineered immune cell as described herein. In the case of immune suppression, a modified IL-15 polypeptide that lacks the ability to bind the IL-2Rβγc complex may be particularly advantageous.
In any of the methods described herein, the modified IL-15 polypeptides, fusion proteins, or engineered immune cells, optionally with at least one additional therapeutic agent, can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) . In some embodiments, the modified human IL-15 polypeptides and at least one additional therapeutic agent are administered in two different administrations. In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation. In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, concurrently with, or after administering the modified IL-15 polypeptides to the subject. In some embodiments, the additional therapeutic agent is an immune therapy. In some  embodiments, the additional therapeutic agent is a CAR-T therapy. In some embodiments, the additional therapeutic agent is an antibody, a cytokine, or a chemotherapy (e.g., a small molecule agent) .
In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) . In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) . In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.
In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject. In some embodiments, the additional therapeutic agent is selected from asparaginase, busulfan, carboplatin, cisplatin,  daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and/or combinations thereof.
In one aspect, the disclosure also provides methods of improving efficacy of a therapy (e.g., an immune therapy, an antibody therapy or a cell therapy) . The methods involve administering to the subject an effective amount of the modified IL-15 polypeptide or the fusion protein as described herein in combination with the therapy.
COMPOSITIONS AND FORMULATIONS
The present disclosure provides compositions (including pharmaceutical and therapeutic compositions) containing the modified human IL-15 polypeptides, fusion proteins, or immune cells and populations thereof, produced by the methods disclosed herein. Also provided are methods, e.g., therapeutic methods for administrating the modified human IL-15 polypeptides, fusion proteins, immune cells, and compositions thereof to subjects, e.g., patients or animal models (e.g., mice) .
Compositions including the modified human IL-15 polypeptides, fusion proteins, immune cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof are provided. The pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient. The pharmaceutically acceptable carrier does not interfere with the active ingredient and is nontoxic to a subject. A pharmaceutically acceptable carrier can include, but is not limited to, a buffer, excipient, stabilizer, or preservative. The pharmaceutical formulation refers to process in which different substances and/or agents are combined to produce a final medicinal product. The formulation studies involve developing a preparation of drug acceptable for patient. Additionally, a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A variety of suitable formulations are available. For example, the pharmaceutical  composition can contain preservatives. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001%to about 2%by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) . Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as polyethylene glycol (PEG) .
Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001%to about 4%by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005) .
The formulations can include aqueous solutions. The formulation or composition can also contain more than one active ingredient useful for a particular indication, disease, or condition, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition can further include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents,  e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
The cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous. For example, immunoresponsive T cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject after genetically modifying them in accordance with various embodiments described herein. Peripheral blood derived immunoresponsive T cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. Usually, when administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell) , it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion) .
Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral, ” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose) , pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the  route of administration and the preparation desired. Standard texts can in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
The compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.
Provided are also methods of administering the cells, populations, and compositions (e.g., compositions including the modified human IL-15 polypeptides or fusion proteins) and uses of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancers. In some embodiments, the methods described herein can reduce the risk of the developing diseases, conditions, and disorders as described herein.
In some embodiments, the cells, populations, and compositions, described herein are administered to a subject or patient having a particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and compositions prepared by the provided methods, such as engineered compositions and end-of-production compositions following incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in cancer expressing an antigen recognized by the engineered T cells.
Methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in U.S. 2003/0170238; U.S. Pat. No. 4,690,915; Rosenberg, Nature reviews Clinical oncology 8.10 (2011) : 577; Themeli et al., Nature biotechnology 31.10 (2013) : 928; Tsukahara et al., Biochemical and biophysical research communications 438.1 (2013) : 84-89;Davila et al., PloS one 8.4 (2013) ; each of which is incorporated herein by reference in its  entirety.
In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the T cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.
In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some embodiments, the subject has not received prior treatment with another therapeutic agent.
In some embodiments, the compositions or cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type (s) and/or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
In some embodiments, the compositions comprising the modified human IL-15  polypeptides are administered at one or more doses of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 mcg/kg/d. Any other suitable dosages can also be used in the methods described herein.
In some embodiments, multiple doses can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) .
In some embodiments, the IL-15 polypeptides in the compositions described herein are PEGylated. In some embodiments, the compositions comprising the modified human IL-15 polypeptides are administered at a dose of about 1-100 mcg/kg about every 21 days. In some embodiments, the compositions comprising the modified human IL-15 polypeptides are administered at a dose of about 1-100 mcg/kg about every three weeks.
In certain embodiments, the cells or individual populations of sub-types of cells (e.g., cells expressing the modified IL-15 polypeptides and/or CAR) , are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values) , such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values) , and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value  in between these ranges.
In some embodiments, the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about 10 4 and at or about 10 9 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1×10 5 cells/kg, 1.5×10 5 cells/kg, 2×10 5 cells/kg, or 1×10 6 cells/kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 6 T cells/kg body weight, for example, at least or at least about or at or about 1×10 5 T cells/kg, 1.5×10 5 T cells/kg, 2×10 5 T cells/kg, or 1×10 6 T cells/kg body weight.
In some embodiments, the cells are administered at or within a certain range of error of, greater than, and/or at least about 1×10 6, about 2.5×10 6, about 5×10 6, about 7.5×10 6, or about 9×10 6 CD4+ cells, and/or at least about 1×10 6, about 2.5×10 6, about 5×10 6, about 7.5×10 6, or about 9×10 6 CD8+ cells, and/or at least about 1×10 6, about 2.5×10 6, about 5×10 6, about 7.5×10 6, or about 9×10 6 T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 10 8 and 10 12 or between about 10 10 and 10 11 T cells, between about 10 8 and 10 12 or between about 10 10 and 10 11 CD4+ cells, and/or between about 10 8 and 10 12 or between about 10 10 and 10 11 CD8+ cells.
The cells described herein can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered as part of a combination treatment, such as simultaneously with  or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The modified human IL-15 polypeptides, fusion proteins, or immune cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the modified human IL-15 polypeptides, fusion proteins, or immune cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered after the one or more additional therapeutic agents. In some embodiments, the methods comprise administration of a chemotherapeutic agent. In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells are administered in combination with cancer-directed monoclonal antibodies to increase the antibody-dependent cellular cytotoxicity (ADCC) of these antibodies, thereby augmenting their antitumor efficacy.
In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells induce lower levels of immune responses compared to those induced by the human IL-15 polypeptides, or fusion proteins or immune cells including IL-15 polypeptides without any substitution (e.g., the polypeptide having an amino acid sequence of SEQ ID NO: 3) .
In some embodiments, the modified human IL-15 polypeptides, fusion proteins, or immune cells induce higher levels of immune responses compared to those induced by the human IL-15 polypeptides, or fusion proteins or immune cells including IL-15 polypeptides without any substitution (e.g., the polypeptide having an amino acid sequence of SEQ ID NO: 3) .
EXAMPLES
The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.
Example 1: IL-15 quaternary complex structure analysis and single-point saturation mutation library construction
According to the structure of the IL-15 quaternary complex, residues that are on the surface of wild-type (WT) IL-15 (SEQ ID NO: 3) and are also in close proximity, i.e., with 
Figure PCTCN2022104519-appb-000006
distance, to IL-2Rβ are: N1, V3, N4, S7, D8, K10, K11, D61, T62, E64, N65, I68, and L69 (FIG. 2) .
To create IL-15 single-point mutant library, the afore-mentioned amino acids were mutated to the other amino acids except cysteine. The site-directed mutation was carried out using Q5 Site-Directed Mutagenesis Kit Mix (NEB, Cat. No: E0552S) according to manufacturer’s manual. The site-directed mutation was confirmed by DNA sequencing. The DNA coding Avi-tag was added to the 3’-end of the IL-15 variants. Individual mutant plasmid was Maxipreped and was subsequently used to transfect HEK293 cells. Transfected HEK293 cells were cultured for 96 hours, and IL-15 muteins were secreted in the medium.
Example 2: Affinity measurement of crude IL-15 muteins to IL-2Rβ
HEK293 cells were pelleted and the crude IL-15 mutein supernatants were used for affinity measurement by Surface Plasmon Resonance (SPR) . The experiment was performed on a Biacore T200 SPR biosensor (GE Healthcare) at room temperature. The anti-Avi tag sensor chips were prepared at 25℃ with a running buffer of 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.005% (v/v) Tween-20, pH 7.4. All surfaces of a Biacore CM5 sensor chip were activated with a 1: 1 (v/v) mixture of 400 mM EDC and 100 mM NHS for 7 minutes, at a flow rate of 10 μL/min. An anti-Avi reagent (Genscript, Cat. No: A00674-200) was diluted to 30 μg/mL in 10 mM sodium acetate (pH 5.0) and injected on all flow cells for 7 minutes at 10 μL/min. All flow cells were blocked with 1 M Ethanolamine-HCl, pH 8.5 for 7 minutes at 10 μL/min.
The affinity determination of all mutein supernatants was performed at 25℃ on Biacore T200 using a running buffer of 10 mM HEPES, 150 mM NaCl, 3mM EDTA, 0.05% (v/v) Tween-20, pH 7.4. Avi-tagged IL-15 variants were captured on flow cells 2, 3 and 4 at a flow rate of 10 μL/min for 30 seconds. The flow cell 1 was used as a reference surface. Following capture of IL-15 variants, analytes (buffer, 250 nM, 500 nM, and 1000 nM concentrations of human IL-2Rβ) was injected at a flow rate of 30 μL/min in all flow cells for 100 seconds. After each analyte injection, dissociation was monitored for 300 seconds, followed by regeneration of all flow cells with three 15-second injections of 10mM Glycine-HCl, pH2.0. Sensorgrams of  buffer cycles were collected for double-referencing purposes (see, e.g., Myszka DG. 1999. Journal of molecular recognition 12: 279-284) . For kinetic analysis, the double-referenced sensorgrams were fit globally to a simple 1: 1 Langmuir binding model using Biacore T200 Evaluation Software version 3.0. For steady-state affinity analysis, the double-referenced equilibrium binding responses were fit with a 1: 1 Langmuir steady-state model using Biacore T200 Evaluation Software version 3.0.
As shown in Table 1, the binding affinity range of these muteins are wide, from about 5-fold affinity increase (i.e. D61E mutein) to no binding to IL-2Rβ at all.
Table 1. Binding affinity of IL-15 muteins to IL-2Rβ.
Figure PCTCN2022104519-appb-000007
Figure PCTCN2022104519-appb-000008
Figure PCTCN2022104519-appb-000009
Example 3: Production of IL-15 Muteins and Affinity Measurement Using Purified Proteins
Nineteen IL-15 muteins (Table 2) were selected to study to what extent IL2Rβ-binding affinity affects the efficacy and safety of IL-15. An avi-tag and a 6×Histidine tag were added to  the C-terminus of IL-15 muteins, and a tobacco etch virus (TEV) protease cleavage sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly was inserted between IL15 mutein and avi-tag. DNA sequences of these IL-15 mutein fusion proteins were synthesized and cloned into mammalian expression vector. The fusion proteins were expressed in FreeStyle 293-F cells transiently transfected with aforementioned expression vectors. After six days of cell culture, the supernatant containing secreting protein was harvested for protein purification using Ni 
Figure PCTCN2022104519-appb-000010
Excel (GE Healthcare, Cat. No: 17-3712-01) affinity chromatography. The binding affinity to IL-2Rβ was determined using the same method as described in Example 2, except that the ligand was purified muteins, not crude proteins. The binding affinity of purified muteins was in general comparable to that of crude proteins (Tables 1 and 2) .
Table 2. IL-2Rβ binding affinity and functional activity of IL-15 muteins
Figure PCTCN2022104519-appb-000011
s.d., standard deviation
The Nineteen IL-15 muteins comprises an amino acid substitution set forth in Table 3 below.
Table 3. Amino Acid Sequences
Figure PCTCN2022104519-appb-000012
Figure PCTCN2022104519-appb-000013
Example 4: Functional Characterization of IL-15 Muteins
For functional assays, C-terminal tags were removed by TEV protease. STAT5 represents a master regulator of NK and CD8+ cytotoxic T cell function, therefore STAT5 phosphorylation was used to determine the effect of IL-2Rβ binding affinity on the activation of NK cell and CD8+cytotoxic T cells. Briefly, 2×10 5 human PBMCs were treated with 100 nM wild-type IL-15 and IL-15 muteins and incubated at 37℃ for 15 minutes. Cells were washed once with FACS buffer (2%(v/v) FBS/PBS) and first stained with surface marker antibodies, including anti-human CD56-Alexa Fluor 647 (Biolegend, Cat. No: 362514) and anti-human CD8a-Brilliant Violet 421 (Biolegend, Cat. No: 301036) . After 20-minute incubation and wash, cell pellets were fixed using Fixation Buffer (BD, Cat. No: 554655) and permed using Perm Buffer III (BD, Cat. No: 558050) .  Then cells were washed and stained with anti-human pSTAT5-PE (BioLegend, Cat. No: 936904) at 4℃ for 30 minutes. Finally, collected cells were washed, re-suspended in FACS buffer and acquired on BD FACSCelesta flow cytometer (BD Biosciences) . All selected muteins showed comparable activity to that of wild-type IL-15 on activating NK cells and CD8+ cytotoxic T cells, except N65V which has an inferior activity on CD8+ cytotoxic T cells (FIG. 3) .
NK and CD8+ cytotoxic T cell proliferation upon IL-15 mutein treatment was also studied. Briefly, 1×10 6 human PBMCs were treated with 100 nM wild-type IL-15 and IL-15 muteins and incubated at 37℃ for 3 days. On Day 3, cells were washed once with FACS buffer (2% (v/v) FBS/PBS) and first stained with NK and CD8+ T cell surface marker antibodies as described above. Then cells were fixed and permed using FOXP3 Fix/Perm buffer Set (Biolegend, Cat. No: 421403) , and stained with anti-human Ki67-PE (Biolegend, Cat. No: 350504) . All selected muteins showed comparable activity to that of wild-type IL-15 on NK cells and CD8+ cytotoxic T cells, except N65V which has an inferior activity on CD8+ cytotoxic T cells (FIGs. 4A-4B) . The result is similar to that of the activation of NK and CD8+ cytotoxic T cells. Most of the muteins showed comparable activity to that of wild-type IL-15 on the proliferation of NK cells and CD8+ cytotoxic T cells, except N65V which has a significantly decreased activity on NK and CD8+ cytotoxic T cell proliferation (FIGs. 4A-4B) .
In the same experiment on day 3, levels of cytokines such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) released into the media were also measured using human TNF alpha kit (Cisbio, Cat. No: 62HTNFAPEH) and human IFN gamma kit (Cisbio, Cat. No: 62HIFNGPEH) , respectively. As shown in FIGs. 4A-4B, as the binding affinity of IL-15 mutein decreases, the level of TNF-α and IFN-γ decrease, especially for IL-15 muteins with IL-2Rβbinding affinity of around 670 nM (～30-fold affinity decrease compared to wild-type IL-15) to 34 μM (1500-fold affinity decrease compared to wild-type IL-15) , their functional activities of activation NK cells and CD8+ cytotoxic T cells and promoting cell proliferation maintained, but the level of secreted TNF-α and IFN-γ dropped significantly.
OTHER EMBODIMENTS
It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (41)

  1. A modified human interleukin 15 (IL-15) polypeptide comprising one or more amino acid substitutions at positions 1, 3, 4, 7, 8, 10, 11, 61, 62, 64, 65, 68, and 69, wherein numbering of amino acid residue positions is according to SEQ ID NO: 3.
  2. The modified human IL-15 polypeptide of claim 1, wherein
    (1) the amino acid residue at position 1 is not N;
    (2) the amino acid residue at position 3 is not V;
    (3) the amino acid residue at position 4 is not N;
    (4) the amino acid residue at position 7 is not S;
    (5) the amino acid residue at position 8 is not D;
    (6) the amino acid residue at position 10 is not K;
    (7) the amino acid residue at position 11 is not K;
    (8) the amino acid residue at position 61 is not D;
    (9) the amino acid residue at position 62 is not T;
    (10) the amino acid residue at position 64 is not E;
    (11) the amino acid residue at position 65 is not N;
    (12) the amino acid residue at position 68 is not I;
    (13) the amino acid residue at position 69 is not L.
  3. The modified human IL-15 polypeptide of claim 1 or 2, wherein the amino acid substitutions comprise one or more of the following:
    (1) the amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
    (2) the amino acid residue at position 3 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y;
    (3) the amino acid residue at position 4 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
    (4) the amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
    (5) the amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
    (6) the amino acid residue at position 10 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
    (7) the amino acid residue at position 11 is A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
    (8) the amino acid residue at position 61 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
    (9) the amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;
    (10) the amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
    (11) the amino acid residue at position 65 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
    (12) the amino acid residue at position 68 is A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y; and
    (13) the amino acid residue at position 69 is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y.
  4. The modified human IL-15 polypeptide of any one of claims 1-3, wherein the modified human IL-15 has a decreased binding affinity to human IL-2 receptor beta (IL-2Rβ) as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 to human IL-2Rβ.
  5. The modified human IL-15 polypeptide of claim 4, wherein the modified human IL-15 polypeptide comprises one or more of the following:
    (1) the amino acid residue at position 1 is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;
    (2) the amino acid residue at position 3 is D, R, S, or T;
    (3) the amino acid residue at position 4 is A, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, or W;
    (4) the amino acid residue at position 7 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y;
    (5) the amino acid residue at position 8 is A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
    (6) the amino acid residue at position 10 is A, D, E, F, G, I, L, M, N, P, Q, S, T, V, W, or Y;
    (7) the amino acid residue at position 11 is A, D, F, G, H, I, L, N, P, T, V, W, or Y;
    (8) the amino acid residue at position 61 is F, G, H, I, L, M, N, P, Q, R, S, T, V, W, or Y;
    (9) the amino acid residue at position 62 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, or Y;
    (10) the amino acid residue at position 64 is A, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;
    (11) the amino acid residue at position 65 is A, E, F, G, H, I, K, L, M, P, Q, R, T, V, W, or Y;
    (12) the amino acid residue at position 68 is A, D, E, F, H, K, L, M, P, Q, R, S, T, V, W, or Y; and
    (13) the amino acid residue at position 69 is D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, or W.
  6. The modified human IL-15 polypeptide of claim 4 or 5, wherein the modified human IL-15 polypeptide comprises one or more of the following:
    (1) the amino acid residue at position 1 is Q;
    (2) the amino acid residue at position 4 is G, L, P, V, M, or W;
    (3) the amino acid residue at position 7 is D, E, F, G, I, K, N, P, V, W, or Y;
    (4) the amino acid residue at position 8 is E;
    (5) the amino acid residue at position 10 is W;
    (6) the amino acid residue at position 11 is D, H, or W;
    (7) the amino acid residue at position 61 is H, R, or S;
    (8) the amino acid residue at position 62 is E, F, G, I, M, Q, or V;
    (9) the amino acid residue at position 64 is A, F, H, L, M, N, S, T, V, W, or Y;
    (10) the amino acid residue at position 65 is V;
    (11) the amino acid residue at position 68 is M, Q, S, T, V, W, or Y; and
    (12) the amino acid residue at position 69 is H, M, N, Q, S, T, or V.
  7. The modified human IL-15 polypeptide of any one of claims 1-3, wherein the modified human IL-15 polypeptide has an increased binding affinity to human IL-2Rβ as compared to the binding affinity of a human IL-15 polypeptide comprising SEQ ID NO: 3 (wild-type IL-15) to human IL-2Rβ.
  8. The modified human IL-15 polypeptide of claim 7, wherein the modified IL-15 polypeptide comprises one or more of the following:
    (1) the amino acid residue at position 3 is A, E, F, H, L, M, N, Q, W, or Y;
    (2) the amino acid residue at position 4 is H or Y;
    (3) the amino acid residue at position 10 is H or R;
    (4) the amino acid residue at position 11 is E, M, or Q; and
    (5) the amino acid residue at position 61 is A or E.
  9. The modified human IL-15 polypeptide of claim 7 or 8, wherein the modified human IL-15 polypeptide comprises one or more of the following:
    (1) the amino acid residue at position 3 is A, E, F, H, L, N, Q, W, or Y;
    (2) the amino acid residue at position 4 is Y;
    (3) the amino acid residue at position 10 is H or R;
    (4) the amino acid residue at position 11 is E or Q; and
    (5) the amino acid residue at position 61 is E.
  10. The modified human IL-15 polypeptide of any one of claims 1-9, wherein the modified human IL-15 polypeptide comprises an amino acid sequence that is at least 90%identical to the amino acid sequence of SEQ ID NO: 3.
  11. The modified human IL-15 polypeptide of any one of claims 1-10, wherein the modified human IL-15 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 4-22.
  12. A fusion protein comprising the modified human IL-15 polypeptide of any one of claims 1-11.
  13. The fusion protein of claim 12, wherein the fusion protein further comprises an antigen binding moiety.
  14. The fusion protein of claim 13, wherein the antigen binding moiety is selected from the group consisting of an antibody or antigen binding fragment thereof, a divalent antibody fragment, a monovalent antibody fragment, or a proteinaceous binding molecule.
  15. The fusion protein of claim 13, wherein the antigen binding moiety is an antibody comprising an Fc region.
  16. The fusion protein of any one of claims 12-15, wherein the antigen binding moiety and the modified human IL-15 polypeptide is fused via a linker.
  17. A nucleic acid comprising a polynucleotide encoding the modified human IL-15 polypeptide of any one of claims 1-11, or the fusion protein of any one of claims 12-16.
  18. An expression vector comprising the nucleic acid of claim 17.
  19. A cell comprising the vector of claim 18.
  20. The cell of claim 19, wherein the cell is an immune cell.
  21. The cell of claim 20, wherein the immune cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a natural killer (NK) cell, an NK-cell, an iNK-T cell, an NK-T like cell, an αβT cell and a γδT cell.
  22. The cell of claim 20, wherein the immune cell is an NK cell.
  23. The cell of claim 20, wherein the immune cell is a cytotoxic T cell.
  24. The cell of any one of claims 19-23, wherein the modified human IL-15 polypeptide is secreted.
  25. The cell of any one of claims 19-23, wherein the modified human IL-15 polypeptide is membrane bound.
  26. The cell of any one of claims 19-25, wherein the cell expresses a chimeric antigen receptor (CAR) , a T-cell antigen coupler (TAC) receptor, or a T-cell receptor (TCR) .
  27. A pharmaceutical composition comprising the modified human IL-15 polypeptide of any one of claims 1-11, the fusion protein of any one of claims 12-16, or the cell of any one of claims 19-26 and a pharmaceutically acceptable carrier.
  28. A method of producing a modified human IL-15 polypeptide, or a fusion protein comprising a modified human IL-15 polypeptide, the method comprising:
    (a) culturing the cell of any one of claims 19-23 under conditions sufficient for the cell to  produce the modified human IL-15 polypeptide or the fusion protein; and
    (b) collecting the modified human IL-15 polypeptide or the fusion protein produced by the cell.
  29. A method of producing a modified cell, comprising: introducing into a cell the expression vector of claim 18.
  30. The method of claim 29, wherein the cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, an NK cell, an NK-T cell, an iNK-T cell, an NK-T like cell, an αβT cell and a γδT cell.
  31. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the modified human IL-15 polypeptide of any one of claims 1-11, the fusion protein of any one of claims 12-16, the cell of any one of claims 19-26, or the pharmaceutical composition of claim 27, thereby treating cancer in the subject.
  32. The method of claim 31, wherein the cancer is selected from the group consisting of gastric cancer, small intestine cancer, sarcoma, head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, biliary cancer, neuroendocrine tumors, stomach cancer, thyroid cancer, lung cancer, mesothelioma, ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, renal cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, penile cancer, testicular cancer, anal cancer, choriocarcinoma, colorectal cancer, oral cancer, skin cancer, Merkel cell carcinoma, glioblastoma, brain tumor, bone cancer, eye cancer, and melanoma.
  33. The method of claim 31, wherein the cancer is a hematological malignancy.
  34. The method of claim 33, wherein the hematological malignancy is selected from the group consisting of multiple myeloma, malignant plasma cell neoplasm, Hodgkin’s lymphoma, nodular lymphocyte predominant Hodgkin’s lymphoma, Kahler’s disease and Myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin’s lymphoma (NHL) , acute myeloid leukemia (AML) , chronic lymphocytic leukemia (CLL) , acute lymphocytic leukemia (ALL) , chronic myeloid leukemia (CML) , follicular lymphoma, Burkitt’s lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom’s macroglobulienemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma,  mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large Bcell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type) , EBV positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, B-cell lymphoma unclassified with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other hematopoietic cells related cancer.
  35. The method of any one of claims 31-34, wherein the cancer is relapsed, refractory, or metastatic.
  36. The method of any one of claims 31-35, wherein the method further comprises administering to the individual an additional therapy.
  37. The method of claim 36, wherein the additional therapy is surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof.
  38. The method of any one of claims 31-35, wherein the method further comprises administering an effective amount of an additional therapeutic agent to the subject.
  39. The method of claim 38, wherein the additional therapeutic agent is an immune therapy.
  40. The method of claim 38, wherein the additional therapeutic agent is a CAR-T therapy.
  41. The method of claim 38, wherein the additional therapeutic agent is an antibody, a cytokine, or a chemotherapy.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180044391A1 (en) * 2015-03-06 2018-02-15 Deutsches Krebsforschungszentrum FUSION PROTEINS COMPRISING A BINDING PROTEIN AND AN INTERLEUKIN-15 POLYPEPTIDE HAVING A REDUCED AFFINITY FOR IL15Ra AND THERAPEUTIC USES THEREOF
CN112004547A (en) * 2018-02-26 2020-11-27 新索思股份有限公司 IL-15 conjugates and uses thereof
WO2020252264A1 (en) * 2019-06-12 2020-12-17 AskGene Pharma, Inc. Novel il-15 prodrugs and methods of use thereof
CN112513070A (en) * 2018-02-28 2021-03-16 辉瑞公司 IL-15 variants and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180044391A1 (en) * 2015-03-06 2018-02-15 Deutsches Krebsforschungszentrum FUSION PROTEINS COMPRISING A BINDING PROTEIN AND AN INTERLEUKIN-15 POLYPEPTIDE HAVING A REDUCED AFFINITY FOR IL15Ra AND THERAPEUTIC USES THEREOF
CN112004547A (en) * 2018-02-26 2020-11-27 新索思股份有限公司 IL-15 conjugates and uses thereof
CN112513070A (en) * 2018-02-28 2021-03-16 辉瑞公司 IL-15 variants and uses thereof
WO2020252264A1 (en) * 2019-06-12 2020-12-17 AskGene Pharma, Inc. Novel il-15 prodrugs and methods of use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PATIDAR，M.ET AL.: "Interleukin 15: A key cytokine for immunotherapy", CYTOKINE & GROWTH FACTOR REVIEWS, vol. 31, 7 June 2016 (2016-06-07), pages 49 - 59, XP055453494, DOI: 10.1016/j.cytogfr.2016.06.001 *
SU QIN, FENG JIAN-NAN, WANG JIAXI: "MOLECULAR DESIGN OF IL－15 AND ITS MUTANTS", ACTA BIOPHYSICA SINICA, vol. 16, no. 2, 30 June 2000 (2000-06-30), pages 310 - 314, XP009542424 *

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