Heterodimeric polypeptide complexes containing IL-15 variants and uses thereof

Abstract

Provided herein are IL-15 variants, IL-15 / IL-15Rα-Fc fusion proteins comprising the IL-15 variants, and IL-15 / IL-15Rα fusion proteins comprising the IL-15 variants, methods for their production, and uses. The IL-15 variants, fusion proteins, and polypeptide complexes of the present disclosure can be used as potent agents for the treatment of cancer and infectious diseases.

Description

[Technical field]

[0001] cross reference This application claims priority to International Patent Application No. PCT / CN2022 / 078326, filed February 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] Field This application relates generally to IL-15 proteins and polypeptide complexes comprising IL-15 proteins. More specifically, the application relates to IL-15 variants, fusion proteins, heterodimeric IL-15 / IL-15Rα-Fc polypeptide complexes, methods for preparing same, and uses thereof. [Background technology]

[0003] background Although recombinant proinflammatory cytokines (such as IL-2, IFNα, and TNFα) have been approved for cancer treatment for decades, their efficacy is often limited, mainly due to their short half-life and toxicity. IL-15 is an IL-2 family proinflammatory cytokine important for anti-viral and anti-tumor immunity, and is involved in the proliferation and survival of many immune cells (especially cytolytic T / NK cells) without the induction of immunosuppressive Tregs and activation-induced cell death in T / NK cells. IL-15-based therapy may enhance the T / NK cells to benefit anti-tumor immunity and enhance the therapeutic efficacy of current immunotherapies.

[0004] IL-15 is produced in monocytes and dendritic cells and exists primarily as a membrane-bound heterodimeric complex with the IL-15 receptor alpha (IL-15Rα) present in the same cells. Its effects are mediated by trans-presentation of the IL-15 / IL-15Rα complex to NK cells and T cells that express the IL-2Rβ and common γ chain.

[0005] IL-15 and its variants have been tested in various clinical trials. However, its potential in cancer immunotherapy is still greatly limited by its immuno-toxicity and short half-life. There is still a need to develop novel IL-15 variants, conjugates, fusion proteins, and polypeptide complexes to improve therapeutic efficacy.

[0006] The present disclosure provides IL-15 mutants and corresponding heterodimeric polypeptide complexes with reduced toxicity and enhanced PK / PD profiles. These peptides and complexes may serve as novel immunotherapeutic agents with improved antitumor efficacy, especially in combination with other treatment options. Summary of the Invention

[0007] overview These and other objects are provided by the present disclosure, which is broadly directed to compounds, methods, compositions, and articles of manufacture that provide proteins with improved efficacy. The benefits provided by the present disclosure are broadly applicable in the therapeutic and diagnostic fields and may be used in conjunction with antibodies reactive with a variety of targets.

[0008] In some aspects, the disclosure provides IL-15 variant peptides, wherein the IL-15 variant peptides have reduced potency in stimulating immune cell proliferation (such as NK and / or CD8+ T cell proliferation) compared to wild-type IL-15 and comprise an amino acid sequence with a truncation of 1 to 30, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids from the N-terminus, C-terminus, or both the N- and C-terminus compared to SEQ ID NO:1.

[0009] In some embodiments, the IL-15 mutant peptide consists of a truncation of SEQ ID NO: 1, which is truncated by 1 to 12 amino acids forming the C-terminus, the N-terminus, or both the N- and C-terminus. In some embodiments, the IL-15 mutant peptide is truncated by 1 to 12 amino acids (more preferably 1 to 5 amino acids) forming the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-15 mutant peptide is truncated by 1 to 12 amino acids forming the N-terminus of SEQ ID NO: 1. In some embodiments, the IL-15 mutant peptide consists of a truncation of SEQ ID NO: 1, which is truncated at both the C- and N-terminus, and the total number of truncated amino acids is between 2 and 12.

[0010] In some embodiments, the IL-15 mutant peptide comprises the amino acid sequence of any one of SEQ ID NOs: 51 to 69. For example, the IL-15 mutant peptide may consist of the amino acid sequence of SEQ ID NO: 51. In some embodiments, the IL-15 mutant peptides have low potency (i.e., reduced levels but retain stimulatory capacity) in stimulating proliferation and activation of immune cells such as NK cells, DC cells and T cells. The immune cells are not limited to human immune cells, but can also be from other species such as mouse, rat and cynomolgus monkey.

[0011] In some embodiments, the potency of IL-15 variant peptides disclosed herein in stimulating NK and / or CD8+ T cell proliferation is reduced by more than 10-fold, more than 50-fold, more than 100-fold, more than 200-fold, more than 300-fold, more than 400-fold, more than 500-fold, more than 600-fold, more than 700-fold, more than 800-fold, more than 900-fold, more than 1000-fold, more than 1500-fold, more than 2000-fold, or even more, compared to wild-type IL-15, as measured in proliferation and activation assays by FACS. The proliferation and activation assays may use Ki-67 and / or pSTAT5, among others, as markers or indicators.

[0012] In some embodiments, the disclosure provides a fusion protein comprising an IL-15 variant disclosed herein operably linked to a non-IL-15 moiety, which can be a moiety that extends the half-life of the fusion protein in vivo, such as a moiety selected from PEGs, lipids, Fc regions, human serum albumin (HSA), and anti-HSA moieties. In some embodiments, the non-IL-15 moiety is an Fc region, such as human wild-type IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof. The non-IL-15 moiety can also be an IL-15Rα domain. In some embodiments, the IL-15Rα domain comprises or consists of a wild-type IL-15Rα protein, an IL-15Rα variant, or any fragment thereof that retains IL-15 binding activity, such as the fragment set forth in SEQ ID NO:2.

[0013] In some embodiments, the IL-15 variant peptide is linked directly to the non-IL-15 moiety or indirectly to the non-IL-15 moiety via a linker, such as a peptide linker, including a GS linker, such as (G4S)n, with n=1-10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0014] In some embodiments, the present disclosure provides a polypeptide complex comprising an IL-15 mutant domain, an IL-15Rα domain, a first dimerization domain, and a second dimerization domain, Where: The IL-15 variant domain comprises an IL-15 variant peptide as described above, i.e., which has a reduced potency in stimulating the proliferation or activation of immune cells (such as NK and / or CD8+ T cells) compared to wild-type IL-15, and which comprises an amino acid sequence truncated by 1 to 30 (e.g., 1 to 12) amino acids from the C-terminus, the N-terminus, or both the N- and C-terminus, compared to SEQ ID NO:1; The first dimerization domain and the second dimerization domain bind together to form a dimer.

[0015] In some embodiments, the polypeptide complexes disclosed herein have reduced potency in stimulating the proliferation or activation of immune cells (such as NK and / or CD8+ T cells) compared to corresponding polypeptide complexes that comprise wild-type IL-15 instead of an IL-15 mutant.

[0016] In some embodiments, the IL-15 mutant peptide included in the polypeptide complex comprises the amino acid sequence of any of SEQ ID NOs: 51-69, in particular SEQ ID NO: 51. In some embodiments, the IL-15Rα domain of the polypeptide complex comprises or consists of a wild-type IL-15Rα protein, an IL-15Rα mutant, or any fragment thereof that retains IL-15 binding activity, such as a fragment set forth in SEQ ID NO:2.

[0017] The IL-15Rα domain may be on the same chain as the IL-15 variant domain or on a separate chain. In some embodiments, the polypeptide complex comprises two chains, where from N-terminus to C-terminus: the first chain comprises an IL-15 variant domain operably linked to a first dimerization domain; and The second chain comprises an IL-15Rα domain operably linked to a second dimerization domain.

[0018] In some embodiments, the polypeptide complex comprises one or more targeting moieties, such as an antigen-binding moiety. The antigen-binding moiety may be located at the N-terminus or C-terminus of the dimerization domain. The antigen-binding moiety may adopt a VHH, Fab, scFv (single chain variable fragment), VH or VL format.

[0019] In some embodiments, the polypeptide complex comprises two antigen-binding moieties, where a first antigen-binding moiety is operably linked to a first dimerization domain and a second antigen-binding moiety is operably linked to a second dimerization domain.

[0020] The antigen-binding moieties disclosed herein may specifically bind to a variety of antigens, including, but not limited to, PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, TIM4, 4-1BB, OX-40, OX-40L, GITR, A2aR, TIGIT, CD96, PVRIG, CD226, 5T4, VISTA, VSIG3, VSIG4, ICOS, CD28, CD3, CD4, CD8, CD45, CD44v6, CD27, CD47, SIRPAα, SLAMF7, CD24, Siglec10, Siglec15, Siglec8, VSIR, VSIG4, PSGL-1, C5AR1, BTN1A1, BTN3A1, CD70, RANKL, C SF1R, CSF2RB, TNFRSF1 / 1a / 1b, BDCA2, BTLA, C5aR, NKG2A, NKG2D, NKp30, NKp46, CD16a, CD56, C D166, FCGR3, CD2, Neuropilin-1, CCR8, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, GCG R, CXCR2, CXCR4, CXCR5, CALCRL, ETAR, GLP1R, CX3CR1, GPR1, GPR17, GPR20, GPR30, GPR34, GPR- 65, GPCR78, GPRC5D, GPR84, LGR4, LGR5, VEGF, VEGFR, HER2, HER3, Trop2, pCAD, ERα, EGFR, de2-7 EGFR, EGFRvIII, PSMA, PSCA, PSA, TAG-72, SEZ6, SEZ6L, SEZ6L2, SEMA4D, DLL3, GD2, GPC3, KLB, KLRB1, KLRG1, GPC1, PCSK9, EpCAM, p-cadherin, Claudin 6, Claudin 18.218.2) FGFR2b, FGFR3, FGFR4, MUC1, MUC13, MUC16, MUC17, MUCL3, FolRa, TfR, TF, TFR, TFPI, c-Met, NY-ESO-1, GUCY2C LIV-1 αvβ6 α10β1 α(Intergrin)α3 α5β4, αvβ3, αvβ8, ROR1, ROR2, PRLR. PTK7, B7-H3, Paris-4, NetG1, Ax1, CD147, LRRC15, Napi2b. STEAP1, LY6G6D, LYPD1, MACRO, MerTK, MICA, MICB, MSLN, Mk ars, G12D, CDH3, CDH6, CDH17, APLA2, CAIX, CD46, CD47, CLD N6, EphA3, Liquid-GM1, ITGA3, Liquid, MISRII, RON ROBO1, PAUF, PLA2, PRLR, PTK7, TM4SF1, TMEFF2, TREAKR, TRE M-1, TREM-2, uPARAP, TYRP1, KAAG1, RU2AS, CD146, CD63, Balloon H, IGF-1R, TEM1, TEM8, TAX1BP3, ADAM-9, ENPP3, EphA2, EphA3, FcRH5, NaPi3b, TW EAK, DLK1, SORT1, SSTR2, STEAP1, CD25, CD39, GARP, LRRC33, LAIR1, LAMP3, LAP EPR, LILRB1, LILRB2, LILRB4, RAGE, FGL1, TPBG, PDGFRB, TGFBR2, CEACAM1, CEACA M5, CEACAM6, Certain Enzymatic Activation Aggregate (CEA), ICAM1, A33, CAMPATH-1 (CDw52), Catalyst IX (MN / CAIX), CD248, PDPN, ITGB1, ITGAV, CD20, CD19, CD21, CD22, CLL, BCMA, DCLK1, DDR1, DLK1, DPEP3, DKK1, CD5, CD13, CD30, CD33, CD34, CD 36, CD37, CD38, CD43, CD52, CD55, CD94, CD99, CD7, CD71, CD73, CD74, CD79a, CD79b, CD229, CD132, CD133, G250, CSF1R(CD115), HLA-D R, HLA-G, HTRA1, TRA-1-60, IGFR, IL-2 receptor, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), ART1, ASGR1, B7H3, B7-H4, B7H6, CD124, c-Kit (CD117), CD7, Clex12A, Clever-1, IL-13RA2, IL-11RA, IL-31RA, IL-4RA, IFNAR, ActRIIb, IL-7R, SLAMF7, Fms-like tyrosine kinase 3 (FLT-3, CD1 35), GFRA1, BTLA, GloboH, CSF2RB, chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), ITGA4, Clec5a, Clec7a, Clec9a, Clec12a, CLEC14, CD205, CD206, CD200R1, CD228, CD229, CD40, CD40L, FcRn, TLR8, TLR9, TNFR2, LTBR, ​​CD44, CD93, PDGH, PDGFR-α, (CD140 a), PDGFR-β (CD140b), CD146, CD147, CRTH2, TNF-α, TGF-β, IL1RAcP, TSLP, DR5, ST2, fibroblast activation protein (FAP), CDCP1, delrin 1, tenascin, frizzled 1-10, vascular antigen VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), endoglin, Tie2 and other TAAs, I / O checkpoints, tumor microenvironment targets, or autoimmune and inflammatory disease related targets.

[0021] In some embodiments, the first and second antigen binding moieties are in the VHH format and specifically bind to PD-1. Preferably, the first and second antigen binding moieties have the same amino acid sequence. In some embodiments, the first and second antigen binding moieties comprise the amino acid sequence set forth in SEQ ID NO:80.

[0022] The term operably linked, as referred to herein, encompasses direct linkage and linkage via a linker, such as a peptide linker. The peptide linker may be a partial or complete IgG hinge region. The peptide linker may also be a conventionally used peptide linker, such as a GS series linker, including (GS)n, (G2S)n, (G3S)n, (G4S)n linkers, and the like. In some embodiments, the dimerization domain is operably linked to the antigen binding moiety via a (G4S)n linker with n=1-10.

[0023] In some embodiments, the first dimerization domain is one chain of an immunoglobulin Fc region and the second dimerization domain is the other chain of an immunoglobulin Fc region. The Fc region comprises a human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof. In some embodiments, the Fc variants contain one or more substitutions compared to a wild-type human Fc to enhance stability of the Fc region, promote heterodimerization, and / or alter binding to an Fc receptor.

[0024] In some embodiments, the Fc variants comprise: (a) a human IgG4 Fc that has been engineered to contain one or more of the following: an S228P mutation, an F234A / L235A mutation, an M252Y / S254T / T256E mutation, and a "knob-into-hole" structure; or (b) a human IgG1 Fc modified to include one or more of the following: M252Y / S254T / T256E mutations, G236R / L328R mutations, and a "knob-into-hole" structure; Includes.

[0025] In some embodiments, the Fc variants comprise: (a) an amino acid sequence having at least 80% identity to SEQ ID NO:3 on one chain and an amino acid sequence having at least 80% identity to SEQ ID NO:4 on the other chain; (b) an amino acid sequence having at least 80% identity to SEQ ID NO:5 on one chain and an amino acid sequence having at least 80% identity to SEQ ID NO:6 on the other chain; or (c) an amino acid sequence having at least 80% identity to SEQ ID NO:7 on one chain and an amino acid sequence having at least 80% identity to SEQ ID NO:8 on the other chain; Includes.

[0026] In some embodiments, a first chain of the polypeptide complex comprises an amino acid sequence set forth in SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74 or 76, and / or a second chain of the polypeptide complex comprises an amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, 48, 50, 71, 73, 75 or 77.

[0027] In some embodiments, the polypeptide complex comprises two chains: Where: (a) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 19 and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 20; (b) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 70 and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 71; (c) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 72 and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 73; (d) the first chain comprises the amino acid sequence set forth in SEQ ID NO:74 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:75; (e) the first chain comprises the amino acid sequence set forth in SEQ ID NO:21 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:22; (f) the first chain comprises the amino acid sequence set forth in SEQ ID NO:23 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:24; (g) the first chain comprises the amino acid sequence set forth in SEQ ID NO:25 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:26; or (h) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 76, and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 77.

[0028] In some embodiments, the present disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding the first and / or second chain of a polypeptide complex, a fusion protein, or an IL-15 mutant peptide disclosed herein. In some aspects, the disclosure provides a vector comprising a nucleic acid molecule disclosed herein. In some aspects, the disclosure provides a host cell comprising a vector or a nucleic acid molecule disclosed herein. In some aspects, the present disclosure provides a pharmaceutical composition comprising a polypeptide complex, a fusion protein, or a nucleic acid molecule disclosed herein and a pharma- ceutically acceptable carrier.

[0029] In some aspects, the present disclosure provides an immunoconjugate comprising a polypeptide conjugate or fusion protein disclosed herein conjugated to a moiety selected from a chemical agent (such as a cytotoxic agent, a cytostatic agent, etc.), a polypeptide, a carbohydrate, a lipid, a nucleic acid, or a combination thereof.

[0030] In some aspects, the disclosure provides a method of making a polypeptide complex or a fusion protein as disclosed herein, comprising the steps of: - culturing a host cell containing the vector(s) encoding one or both chains of said polypeptide complex or fusion protein, and - isolating the polypeptide complex or fusion protein from the host cell culture; The method further comprises:

[0031] In some aspects, the disclosure provides a method of modulating an immune response in a subject, comprising administering to the subject a polypeptide complex or pharmaceutical composition disclosed herein, optionally wherein the immune response is associated with NK cells or T cells (such as CD8+ T cells).

[0032] In some aspects, the disclosure provides a method of treating or preventing cancer, an infectious disease, an immunodeficiency, or lymphocytopenia in a subject, comprising administering to the subject an effective amount of a polypeptide complex or pharmaceutical composition disclosed herein.

[0033] In some embodiments, the method further comprises administering an additional anti-tumor therapy, such as tumor infiltrating lymphocyte (TIL) therapy, T cell receptor T cell (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy, NK cell therapy, oncolytic virus therapy, targeted therapy, chemotherapy, and cellular immunotherapy, including gene therapy. The administration of the additional anti-tumor therapy may be performed before, after, or simultaneously with the administration of the polypeptide complex or pharmaceutical composition disclosed herein.

[0034] In some aspects, the disclosure provides methods of treating or preventing cancer, an infectious disease, an immunodeficiency, or lymphocytopenia in a subject comprising administering a cell therapy or gene therapy, wherein the cell therapy and gene therapy comprises delivering a nucleic acid molecule disclosed herein to the subject.

[0035] In some embodiments, the cell therapy is selected from tumor infiltrating lymphocyte (TIL) therapy, T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, and NK cell therapy. In some embodiments, the gene therapy uses lentivirus, AAV, poxvirus, varicella zoster virus, oncolytic virus, or other RNA / DNA vectors for gene delivery.

[0036] In some embodiments, the cancer is selected from breast cancer, lung cancer (such as NSCLC), colon cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma (such as non-Hodgkin's lymphoma and diffuse large B-cell lymphoma), leukemia (such as chronic lymphocytic leukemia), and multiple myeloma. In some embodiments, the infectious disease is caused by a viral, bacterial, fungal, or parasitic infection.

[0037] In some aspects, the disclosure provides for the use of a polypeptide complex, fusion protein, or nucleic acid molecule disclosed herein in the manufacture of a medicament for preventing, treating and / or managing cancer, an infectious disease, an immunodeficiency, or lymphocytopenia.

[0038] In some aspects, the disclosure provides a polypeptide complex, a fusion protein, or a nucleic acid molecule disclosed herein for use in treating or preventing cancer, an infectious disease, an immunodeficiency, or lymphocytopenia.

[0039] In some aspects, the disclosure provides a kit comprising a container comprising a polypeptide complex, fusion protein, or nucleic acid molecule disclosed herein.

[0040] The above is a summary, and therefore includes, by necessity, simplifications, generalizations, and omissions of details; as a result, those skilled in the art will appreciate that this summary is merely illustrative and is not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and / or devices and / or other subject matter described herein will become apparent in the teachings set forth herein. This summary is presented to introduce selected concepts in a simplified form, which are further described in the detailed description below. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to help define the scope of the claimed subject matter. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 illustrates the structure of an IL-15 / Rα fusion protein. (A) A heterodimeric IL-15 / Rα-Fc polypeptide complex according to some embodiments of the present disclosure. (B) An IL-15 / Rα fusion protein according to some embodiments of the present disclosure. (C) A heterodimeric IL-15 / Rα-Fc polypeptide complex further comprising an antigen-binding moiety or moieties according to some embodiments of the present disclosure. [Diagram 2]FIG. 2 illustrates that heterodimeric IL-15 / Rα-Fc polypeptide complexes promote proliferation of CD8+ T cells (A) and NK cells (B) in human PBMCs. Human PBMCs were cultured with the indicated proteins for 4 days. Proliferation of CD8+ T cells (CD3+CD8+) and NK cells (CD3-CD56+) was shown as Ki67+ (%) of CD8+ T cells and Ki67+ (%) of NK cells, respectively. [Diagram 3] Figure 3 illustrates that W327151-TXU1.P1-V0013.uIgG4v322 promotes proliferation of cynomolgus monkey CD8+ cells (A) and CD16+ cells (B). Cynomolgus monkey PBMCs were cultured with the indicated proteins for 4 days. Proliferation of CD8+ and CD16+ cells was shown as Ki67+ (%) of CD8+ cells and Ki67+ (%) of CD16+ NK cells, respectively. [Figure 4] FIG. 4 illustrates that W327151-TXU1.P1-V0013.uIgG4v322 promotes proliferation of mouse CD8+ T cells (A) and NK cells (B). Mouse splenocytes were cultured with the indicated proteins for 4 days. Proliferation of CD8+ T cells (CD3+CD8a+) and NK cells (CD3-CD335+) was shown as Ki67+ (%) of CD8+ T cells and Ki67+ (%) of NK cells, respectively. [Diagram 5] FIG. 5 illustrates the binding curve of W327151-TXU1.P1-V0013.uIgG4v322 to the HH cell line. [Figure 6] FIG. 6 illustrates an SPR curve showing binding of precomplexed human IL-2Rβ with the heterodimeric IL-15 / Rα-Fc polypeptide complex (1:1) to human IL2Rγc immobilized on a sensor chip. [Figure 7] Figure 7 illustrates the efficacy of the fusion proteins in an MLR assay. T cell activation was measured by IFN-γ (A) and IL-2 (B) in the supernatants detected by ELISA. [Figure 8]FIG. 8 illustrates the serum concentration profiles of W327151-TXU1.P1-V0013.uIgG4v322 and W327151-BMK1 in C57BL / 6 mice detected by ELISA for Fc-Fc (A) or Fc-IL-15 (B). [Figure 9] 9 illustrates the body weight change for a maximum tolerated dose (MTD) assay. Mice were treated with various doses of IL-15 / Rα-Fc polypeptide complexes. [Figure 10] FIG. 10 illustrates the pharmacokinetic effects of treatment with various doses of W327151-TXU1.P1-V0013.uIgG4V322 or WT IL-15 on mouse peripheral lymphocyte, NK, T, CD8+T, CD4+T, and Treg cell numbers. [Figure 11] FIG. 11 illustrates the proliferation and activation of mouse peripheral NK and CD8+ T cells upon treatment with various doses of W327151-TXU1.P1-V0013.uIgG4V322 or WT IL-15. [Figure 12] FIG. 12 illustrates the pharmacokinetic effects of treatment with various doses of W327151-TXU1.P1-V0013.uIgG4V322 or WT IL-15 on peripheral Tcm, Tem, and naive CD8+ T cell numbers. [Figure 13] FIG. 13 illustrates albumin concentration in mouse plasma (A) and inflammatory cytokine release (B) following treatment with various doses of W327151-TXU1.P1-V0013.uIgG4V322 or WT IL-15. [Figure 14] FIG. 14 illustrates the change in tumor volume in a CT26 colon carcinoma model following treatment with various doses of W327151-TXU1.P1-V0013.uIgG4V322 or WT IL-15. [Figure 15] Figure 15 illustrates the change in tumor volume in an A375 humanized model following treatment with W327151-TXU1.P1-V0013.uIgG4V322, W327151-BMK1 or W327151-BMK2. [Figure 16]FIG. 16 illustrates the change in tumor volume in the Daudi CDX model following treatment with various doses of W327151-TXU1.P1-V0013.uIgG4V322 or WT IL-15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Detailed Description Although the present disclosure can be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the present disclosure. It should be emphasized that the present disclosure is not limited to the specific embodiments described. Moreover, the headings of sections used herein are for clarity of organization only and should not be construed as limiting the subject matter described.

[0043] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, singular terms include the plural and plural terms include the singular unless the context requires otherwise. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes a mixture of a plurality of cells, and so forth. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "comprising" and its other forms (such as "comprises" and "comprised") is open ended. Additionally, the ranges set forth in this specification and the appended claims include both endpoints and every point between those endpoints.

[0044] Generally, the nomenclature used with respect to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and the nomenclature used in these techniques, are well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. See, e.g., Abbas et al., Cellular and Molecular Immunology, 6th ed., WB Saunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature used for the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry laboratory procedures and techniques described herein are well known and commonly used in the art.

[0045] definition To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0046] The term "IL-15" as used herein refers to interleukin 15 and is intended to encompass all forms of IL-15, e.g., 1) the native unprocessed IL-15 molecule, the "full-length" IL-15 protein or naturally occurring variants of IL-15; 2) any form of IL-15 resulting from processing in cells; or 3) full-length or modified forms. Wild-type IL-15 is a 14-15 kDa member of the 4-α-helix bundle family of cytokines involved in natural killer (NK) cell differentiation, T cell function, and host responses to intracellular pathogens. An example amino acid sequence of human wild-type IL-15 is shown in SEQ ID NO:1. As used in this disclosure, the term "IL-15" or "IL-15 domain" encompasses both wild-type IL-15 and IL-15 variants.

[0047] The term "IL-15Rα" or "IL-15 receptor α" as used herein refers to a high affinity receptor for IL-15, which binds IL-15 and transduces a signal in the presence of IL-15Rβ and γc (common γ chain). Full-length human IL-15Rα is a type-1 transmembrane protein with a signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail. As used in this disclosure, the term "IL-15Rα" or "IL-15Rα domain" encompasses both wild-type IL-15Rα and IL-15Rα variants, and fragments thereof. As described herein and known in the art, the IL-15Rα protein contains the "sushi domain", which is the shortest region of the receptor that retains IL-15 binding activity (set forth in SEQ ID NO:2). In some embodiments, the "sushi domain", i.e., the peptide set forth in SEQ ID NO:2, is used to construct the IL-15 / Rα-Fc polypeptide complexes disclosed herein.

[0048] With respect to a polypeptide or protein, the term "variant" refers to a biologically active polypeptide that includes one or more amino acid mutations in the native protein sequence. Optionally, the one or more amino acid mutations include amino acid substitutions, insertions, and deletions at specific positions in the amino acid sequence. A variant has preferably at least about 80%, more preferably at least about 90%, and more preferably at least about 95% amino acid sequence identity with the corresponding native sequence polypeptide. Such variants include, for example, polypeptides in which one or more amino acid residues are truncated at the N- and / or C-terminus of the polypeptide. The variants for use in the present disclosure may be prepared by various methods well known in the art, such as synthesizing or recombinantly producing DNA encoding the variant and then expressing the DNA in cell culture. In certain embodiments disclosed herein, the IL-15 variants are truncated by one or more amino acid residues at the N- and / or C-terminus compared to the wild-type IL-15 protein.

[0049] The term "Fc" as used herein has the same meaning as it does with respect to an antibody, and refers to that portion of an antibody that includes the second (CH2) and third (CH3) constant regions of a first heavy chain linked to the second and third constant regions of a second heavy chain via disulfide bonds. The Fc region may also include part or the entire hinge region. The Fc region of an antibody is involved in various effector functions, such as ADCC and CDC, but does not function in antigen binding. In this disclosure, the term "Fc" encompasses both wild-type Fc and Fc variants.

[0050] The term "modification" as used herein refers to a change in the primary amino acid sequence with respect to an amino acid residue / position, compared to the starting amino acid sequence, where the change results from a sequence change with respect to said amino acid residue / position. For example, typical modifications include the substitution of a residue (or at said position) by another amino acid (e.g., conservative or non-conservative substitution), and the insertion of one or more amino acids adjacent to said residue / position. An "amino acid substitution" or mutation thereof refers to the replacement of an existing amino acid residue in a predetermined (starting) amino acid sequence by a different amino acid residue. Generally, a modification results in an alteration in at least one physico-biochemical activity of a mutant polypeptide, compared to a polypeptide comprising a starting (or "wild type") amino acid sequence. For example, in an IL-15 mutant, the altered physico-biochemical activity may be the binding affinity, binding ability, and / or binding effect to a target molecule. As used herein, two or more substitutions in an amino acid sequence may be represented by a "+" or " / " between each substitution.

[0051] The term "fusion protein" as used herein refers to a polypeptide having two (or more) parts operatively linked together, each part being a polypeptide with a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, catalysis of a reaction, etc. The two parts may be directly linked via a single peptide bond or indirectly linked via a linker, such as a peptide linker comprising one or more amino acid residues. Generally, the two parts and the peptide linker are in reading frame with each other. In a particular embodiment, the two parts of the fusion protein are an IL-15 protein domain and an Fc domain, or an IL-15Rα protein domain and an Fc domain, or an IL-15 protein domain and an IL-15Rα protein domain. The two fusion proteins may be bound together to form a heterodimer. In a broad sense, when referring to a "fusion protein" herein, depending on the context, it may refer to a single chain IL-15-containing fusion protein or a heterodimeric polypeptide complex, as defined below.

[0052] As used herein, the terms "heterodimer", "heterodimeric fusion protein", "heterodimeric IL-15 polypeptide complex", and "IL-15 / Rα-Fc polypeptide complex" may be used interchangeably and refer to a heterodimeric protein comprising two chains, each chain containing a dimerization domain capable of binding together to form a dimer, and the IL-15 domain may be on the same or different chains with the IL-15Rα domain. In some embodiments, the first chain is a fusion protein of an IL-15 protein domain and an Fc domain, and the second chain is a fusion protein of an IL-15Rα protein domain and an Fc domain. Such heterodimeric fusion proteins are structurally similar to antibodies in that the Fab portion in the antibody is replaced by the IL-15 / IL-15Rα complex formed by the interaction of IL-15 with the IL-15Rα domain. Additionally, the polypeptide complex may be fused to one or more antigen-binding portions or antigen-binding fragments derived from an antibody. Such polypeptide complexes combine the immune cell proliferation and activation function of the IL-15 / IL-15Rα portion with the site-specific targeting of the antigen-binding portion.

[0053] The term "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without spacers or linkers, in a manner that allows them to function in a planned manner. When used in reference to a polypeptide, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have an intended biological function, and in which the respective functions of each polypeptide sequence are retained or the function of one of the polypeptide sequences is reduced due to steric hindrance imposed by the other polypeptide(s). For example, an antibody variable region may be operably linked to a constant region to provide a stable product with antigen-binding activity; an IL-15 domain may be operably linked (directly or indirectly via a linker) to an Fc domain. The term may also be used in reference to polynucleotides. As an example, it is intended to mean that the polynucleotide sequences are linked in a manner that allows regulated expression of the polypeptide from the polynucleotide when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.).

[0054] The term "gene therapy" as used herein refers to therapy that aims to treat disease by replacing, inactivating or introducing genes into cells - either in vivo or ex vivo. In some embodiments, DNA or RNA sequences encoding the IL-15 variant(s) and fusion proteins disclosed herein are administered to a subject. Gene therapy includes the use of lentivirus, AAV, poxvirus, varicella zoster virus, oncolytic virus, and other RNA / DNA vectors to deliver DNA or RNA encoding the heterodimeric polypeptide complexes or pharmaceutical compositions disclosed herein.

[0055] The term "cell therapy" as used herein refers to a therapy that aims to treat a disease by restoring or altering a specific set of cells or by using cells to carry a therapy throughout the body. With cell therapy, cells are cultured or modified outside the body before being infused into the patient. The cells may be of patient origin (autologous cells) or donor origin (allogeneic cells). Current cell therapies include using CAR-T, TCR-T, TIL, CAR-NK, CAR-γδT, CAR-macrophage, and other engineered immune cells. In some embodiments herein, the method of treatment further includes cell therapy.

[0056] The term "EC50", also known as "half maximal effective concentration", means herein the concentration of a drug, antibody, or toxin that induces a response halfway between the baseline and maximum after a specified exposure time. In the context of this application, EC50 is expressed in units of "nM".

[0057] The term "isolated" as used herein refers to a state obtained by artificial means from a natural state. If a substance or component is "isolated" in nature, this can be because its natural environment has been altered, or because the substance has been isolated from its natural environment, or both. If a non-isolated polynucleotide or polypeptide naturally occurs, for example in a living animal, the same polynucleotide or polypeptide isolated in high purity from such a natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude mixed artificial or synthetic substances, or other impurities, that do not affect the activity of the isolated substance.

[0058] The term "vector" as used herein refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector allows for the expression of a protein encoded by a polynucleotide inserted therein, the vector is called an expression vector. A vector can express an element of genetic material introduced into a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art, and non-limiting examples include plasmids, phages, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)); phages (such as lambda or M13 phages), and animal viruses. Non-limiting examples of animal viruses that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). A vector can contain a number of elements for controlling expression, non-limiting examples of which include promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain an origin of replication.

[0059] The term "host cell" as used herein refers to a cell line engineered to produce a protein, protein fragment, or peptide of interest. Non-limiting examples of host cells include cultured cells (e.g., mammalian cultured cells CHO, BHK, NSO, SP2 / 0, YB2 / 0, etc., derived from rodents (rat, mouse, guinea pig, or hamster); or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular subject cell, but also the progeny of such cells. Because certain modifications may occur in successive generations due to mutations or environmental influences, such progeny may not be identical to the parent cell, but are still within the scope of the term "host cell."

[0060] The term "identical" as used herein refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as revealed by aligning and comparing the sequences. "Percent identity" refers to the percentage of matching residues among the amino acids or nucleotides in the compared molecules, and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignment, if any, are preferably addressed by a special mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48:1073.

[0061] The term "transfection" as used herein refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Non-limiting examples of protocols and techniques for transfection include lipid transfection and chemical and physical methods, such as electroporation. Numerous transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197. In one particular embodiment of the present disclosure, the human IL-15 gene is transfected into 293F cells.

[0062] The term "fluorescence activated cell sorting" or "FACS" as used herein refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". Retrieved November 09, 2017). Equipment for performing FACS is known to those of skill in the art and is commercially available. Examples of such equipment include the FACS Star Plus, FACScan, and FACSort instruments manufactured by Becton Dickinson (Foster City, Calif.), Epics C manufactured by Coulter Epics Division (Hialeah, Fla.), and MoFlo manufactured by Cytomation (Colorado Springs, Colo.).

[0063] The term "subject" includes any human or non-human animal, preferably a mammal, and more preferably a human. The term "cancer" as used herein means any or a certain neoplasm or solid tumors and non-solid tumors (such as leukemia) resulting from the growth, proliferation, or metastasis of malignant cells and initiating a certain medical condition.

[0064] The terms "treatment," "treat," or "treated" in the context of treating a condition, as used herein, generally relate to human or animal treatments and therapies in which some desired therapeutic effect (e.g., inhibition of progression of a condition) is achieved, including slowing the rate of progression, stopping the rate of progression, regressing the condition, improving the condition, and curing the condition. Treatment as a preventative measure (i.e., prevention, prevention) is also included. With respect to cancer, "treat" can mean slowing or retarding the growth, proliferation, or metastasis of tumors or malignant cells, or any combination thereof. With respect to tumors, "treatment" includes removal of all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or retarding the progression of tumors, or any combination thereof.

[0065] The term "effective amount" as used herein relates to an amount of an active compound or material, or an amount of a composition or dosage form containing an active compound, that when administered in accordance with a desired treatment regimen, produces some desired therapeutic effect at a reasonable benefit / risk ratio. For example, when used in reference to the treatment of a disease or condition such as cancer, an "effective amount" refers to an amount or concentration of an active agent, drug, or antibody or antigen-binding portion thereof effective to treat said disease or condition.

[0066] The terms "prevent," "prevention," or "preventing," in reference to a disease state in a mammal, means arresting or delaying the onset of the disease or preventing the appearance of clinical or subclinical symptoms thereof.

[0067] The term "pharmaceutical acceptable" as used herein means that the vehicle, diluent, excipient, and / or salts thereof are chemically and / or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient.

[0068] As used herein, the term "pharmaceutical acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active agent and are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including non-limiting examples of pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, non-limiting examples of pH adjusters include phosphate buffers; non-limiting examples of surfactants include cationic, anionic, or nonionic surfactants (e.g., Tween®-80); and non-limiting examples of ionic strength enhancers include sodium chloride.

[0069] As used herein, the term "adjuvant" refers to a non-specific immune enhancing agent that, when delivered to an organism along with an antigen or when delivered to the organism beforehand, can enhance or change the type of immune response in the organism to the antigen. A wide variety of adjuvants exist, non-limiting examples of which include aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete and Freund's incomplete), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0070] IL-15, IL-15Rα protein domain, and IL-15 / IL-15Rα complex In some embodiments, the present disclosure provides IL-15 mutant peptides that include one or more modifications, e.g., truncations from the N-terminus or C-terminus, compared to a wild-type IL-15 protein, such as a human wild-type IL-15 protein. The present disclosure further provides IL-15-Fc fusion proteins that include an IL-15 protein domain fused to an Fc domain. The IL-15 protein domain may include a wild-type IL-15 protein as set forth in SEQ ID NO:1, or may include an IL-15 mutant that includes one or more deletions compared to the wild-type IL-15 protein.

[0071] Residues are designated herein by the one-letter amino acid code followed by the IL-15 amino acid position, e.g., W2 is a Trp residue at position 2 of SEQ ID NO: 1, which is the second residue from the N-terminus. F110 is a Phe residue at position 110 of SEQ ID NO: 1, which is the fifth residue from the C-terminus. Substitutions are designated herein by the one-letter amino acid code followed by the IL-15 amino acid position that they are replacing, e.g., D30N is the substitution of an Asp residue at position 30 of SEQ ID NO: 1 with an Asn residue.

[0072] In some embodiments, the IL-15 variant has a truncation of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids or more from the C-terminus compared to SEQ ID NO:1.

[0073] In some embodiments, the IL-15 variant has a truncation of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, or more amino acids from the N-terminus compared to SEQ ID NO:1.

[0074] In some embodiments, the IL-15 variant is truncated by 1, 2, 3, 4, 5, 7, 8, 9, 10 or more amino acids from the C-terminus and by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids from the N-terminus compared to SEQ ID NO: 1. Specifically, the IL-15 variant may comprise or consist of any of SEQ ID NOs: 51-69.

[0075] Truncating amino acid residues at the N- and / or C-termini may reduce the interface interaction between IL-15 and IL-2Rβ / γ, for example, by disrupting hydrogen bond formation, salt bridges, and / or van der Waals interactions at the interface. By deleting the terminal amino acid residues, the IL-15 variants disclosed herein provide reduced binding affinity to the IL-15Rβ / γ chain and reduced (but still present) potency in stimulating immune cells, thereby resulting in reduced toxicity in vivo.

[0076] In one aspect, provided herein is an IL-15 / IL-15Rα complex comprising an IL-15 domain associated with an IL-15Rα domain. The IL-15 domain may be on a different chain than the IL-15Rα domain or may be operably linked to the IL-15Rα domain on the same chain. The IL-15Rα protein domain may comprise a wild-type IL-15Rα protein, an IL-15Rα variant, or any fragment thereof that retains IL-15 binding activity. In some embodiments, the IL-15Rα protein domain comprises an IL-15 binding fragment thereof as set forth in SEQ ID NO:2. In some embodiments, the IL-15Rα variant has one or more modifications, e.g., insertions, substitutions, and / or deletions, compared to SEQ ID NO:2.

[0077] The IL-15 protein domain may bind to the IL-15Rα protein domain in different ways to form the IL-15R / IL-15Rα complex. In some embodiments, the IL-15 protein domain and the IL-15Rα protein domain self-assemble through ligand-ligand interactions, i.e., the binding affinity between the two domains. In certain other embodiments, the two domains are covalently linked via a linker, preferably a peptide linker. In some further embodiments, both domains are modified to include cysteine ​​amino acid(s) such that disulfide bond(s) are formed between the two domains.

[0078] IL-15-Fc fusion proteins and heterodimeric IL-15 / IL-15Rα-Fc polypeptide complexes In some embodiments, the present disclosure provides a single chain IL-15-Fc fusion protein comprising an IL-15 protein domain operably linked to an Fc domain, and an IL-15Rα-Fc fusion protein comprising an IL-15Rα protein domain operably linked to an Fc domain. Furthermore, the two fusion proteins may heterodimerize to form an IL-15 / IL-15Rα-Fc polypeptide complex comprising two chains. Each of the IL-15 domain, the IL-15Rα domain, and the Fc domain that constitute the heterodimeric IL-15 / IL-15Rα-Fc polypeptide complex may be in wild-type form or a corresponding mutant, as described above. The IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises an Fc domain linked to an IL-15 / IL-15Rα domain, although it is contemplated that other dimerization domains may also be used. For example, an IL-15 domain can be operably linked to a first dimerization domain and an IL-15Rα domain can be operably linked to a second dimerization domain, and the first dimerization domain and the second dimerization domain combine to form a dimer.

[0079] As used herein, the term "long-acting" means that the IL-15 protein is fused or operably linked to a moiety that can extend the half-life of IL-15 in vivo. Preferably, the moiety may be an immunoglobulin Fc region that provides an extended pharmacokinetic (PK) profile, as manifested, for example, in a longer serum half-life, enhanced Cmax, lower serum clearance and improved drug exposure (AUC), or extended pharmacokinetic (PD) efficacy on lymphocyte expansion, particularly NK and CD8+ T cell proliferation.

[0080] In some embodiments, the heterodimeric IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, a first chain comprising a fusion of one chain of an Fc domain with an IL-15 domain and a second chain comprising a fusion of the other chain of an Fc domain with an IL-15Rα domain. In some embodiments, the IL-15 / IL-15Rα domain is operably linked to the Fc domain. Preferably, the IL-15 or IL-15Rα domain is operably linked to the N-terminus of the Fc domain.

[0081] In certain other embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, where the first chain comprises a fusion of one chain of an Fc domain with an IL-15 domain and an IL-15Rα domain, and the second chain comprises the other chain of an Fc domain. For example, the IL-15 domain may be operably linked to the IL-15Rα domain (optionally via a linker) and further operably linked, from N-terminus to C-terminus, to one chain of an Fc domain, such as IL-15-IL-15Rα-Fc or IL-15Rα-IL-15-Fc, where "-" represents an operable linkage.

[0082] In some embodiments, the IL-15 / IL-15Rα domain is directly linked to the Fc domain, i.e., without a linker. This type of more rigid structure may reduce the desired potency of IL-15 (wild type or mutant), possibly due to the structural disorder caused by the Fc domain. Alternatively, the IL-15 / IL-15Rα domain is linked to the Fc domain via a linker, such as a peptide linker with one or more amino acid residues. Many suitable linkers are available in the art and are used herein. In some embodiments, the linker may be (G4S)n, (GS)n, (GSGGS)n, or (G3S)n, where n can be 0-5, such as 0, 1, 2, 3, 4, or 5.

[0083] In some embodiments, the IL-15 / IL-15Rα-Fc fusion protein disclosed herein comprises two chains, a first chain comprising an IL-15 domain operably linked to one chain of an IgG Fc domain, a second chain comprising an IL-15Rα protein domain operably linked to the other chain of an IgG Fc domain, and the IL-15 domain comprises an IL-15 variant as described above.

[0084] In some exemplary embodiments, the IL-15 variant has a truncation of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids or more from the C-terminus compared to SEQ ID NO:1.

[0085] In some exemplary embodiments, the IL-15 variant has one amino acid, two amino acids, three amino acids, four amino acids, five amino acids, six amino acids, seven amino acids, eight amino acids, nine amino acids, ten amino acids truncated, eleven amino acids truncated, twelve amino acids truncated, or more amino acids truncated from the N-terminus compared to SEQ ID NO:1.

[0086] In some exemplary embodiments, the IL-15 variant has a truncation of 1, 2, 3, 4, 5, 7, 8, 9, 10 or more amino acids from the C-terminus and a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids from the N-terminus compared to SEQ ID NO: 1. Specifically, the IL-15 variant may comprise or consist of any of SEQ ID NOs: 51-69.

[0087] In some further embodiments, the Fc domain is an IgG1 Fc domain and may comprise SEQ ID NO:3 in one chain and SEQ ID NO:4 in the other chain (also referred to as "V805" configuration). In some further embodiments, the Fc domain is an IgG4 Fc domain and may comprise SEQ ID NO:5 in one chain and SEQ ID NO:6 in the other chain (also referred to as "V1" configuration). In some further embodiments, the Fc domain is an IgG4 Fc domain and may comprise SEQ ID NO:7 in one chain and SEQ ID NO:8 in the other chain (also referred to as "V322" configuration).

[0088] For example, in certain embodiments, an IL-15 variant is operably linked to one chain of an Fc domain defined in SEQ ID NO:3, whereas an IL-15Rα protein domain is operably linked to the other chain of an Fc domain defined in SEQ ID NO:4; in certain other embodiments, an IL-15 variant is operably linked to one chain of an Fc domain defined in SEQ ID NO:4, whereas an IL-15Rα protein domain is operably linked to the other chain of an Fc domain defined in SEQ ID NO:3; in certain embodiments, an IL-15 variant is operably linked to one chain of an Fc domain defined in SEQ ID NO:5, whereas an IL-15Rα protein domain is operably linked to the other chain of an Fc domain defined in SEQ ID NO:6. in certain other embodiments, the IL-15 variant is operably linked to one chain of an Fc domain defined in SEQ ID NO:6, whereas the IL-15Rα protein domain is operably linked to the other chain of an Fc domain defined in SEQ ID NO:5; in a particular embodiment, the IL-15 variant is operably linked to one chain of an Fc domain defined in SEQ ID NO:7, whereas the IL-15Rα protein domain is operably linked to the other chain of an Fc domain defined in SEQ ID NO:8; in certain other embodiments, the IL-15 variant is operably linked to one chain of an Fc domain defined in SEQ ID NO:8, whereas the IL-15Rα protein domain is operably linked to the other chain of an Fc domain defined in SEQ ID NO:7.

[0089] In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, a first chain comprising an IL-15 variant comprising a truncation of four amino acids from the C-terminus operably linked to an IgG4 Fc domain (V1 form) and a second chain comprising an IL-15Rα protein operably linked to the other chain of an IgG4 Fc domain (V1 form). Alternatively, the Fc domain may be of the V322, V805 or V803 form.

[0090] In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, a first chain comprising an IL-15 variant comprising a C-terminal 5 amino acid truncation operably linked to an IgG4 Fc domain (V1 form) and a second chain comprising an IL-15Rα protein operably linked to the other chain of an IgG4 Fc domain (V1 form). Alternatively, the Fc domain may be of the V322, V805 or V803 form.

[0091] In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, a first chain comprising an IL-15 variant comprising a truncation of six amino acids from the C-terminus operably linked to an IgG4 Fc domain (V1 form) and a second chain comprising an IL-15Rα protein operably linked to the other chain of an IgG4 Fc domain (V1 form). Alternatively, the Fc domain may be of the V322, V805 or V803 form.

[0092] In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, a first chain comprising an IL-15 variant comprising a truncation of 7 amino acids from the C-terminus operably linked to an IgG4 Fc domain (V1 form) and a second chain comprising an IL-15Rα protein operably linked to the other chain of an IgG4 Fc domain (V1 form). Alternatively, the Fc domain may be of the V322, V805 or V803 form.

[0093] In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, wherein a first chain comprises the amino acid sequence set forth in SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, or 74, and a second chain comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, 48, 50, 71, 73, or 75. In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains selected from the amino acid sequences set forth in the following sets: SEQ ID NOs: 13 / 14, 15 / 16, 17 / 18, 19 / 20, 21 / 22, 23 / 24, 25 / 26, 27 / 28, 29 / 30, 31 / 32, 33 / 34, 35 / 36, 37 / 38, 39 / 40, 41 / 42, 43 / 44, 45 / 46, 47 / 48, 49 / 50, 70 / 71, 72 / 73, or 74 / 75.

[0094] In some exemplary embodiments, the IL-15 / IL-15Rα-Fc polypeptide complex disclosed herein comprises two chains, where a first chain comprises the amino acid sequence set forth in SEQ ID NO: 19, 21, 71, 73, or 75, and a second chain comprises the amino acid sequence set forth in SEQ ID NO: 20, 71, 73, or 75. Specifically, the IL-15 / IL-15Rα-Fc polypeptide complex comprises a pair of two chains selected from 19 / 20, 70 / 71, 72 / 73, or 74 / 75.

[0095] Fc domain As described above, the single-chain IL-15-Fc fusion protein and the IL-15Rα-Fc protein each contain one chain of an Fc domain, which combine together to form an Fc dimer domain similar to the Fc region of an antibody.

[0096] The Fc domain may be a wild-type Fc or an Fc variant. The wild-type Fc may be a human IgG1, IgG2, IgG3 or IgG4 Fc. In some embodiments, the wild-type Fc is a human IgG1 Fc or IgG4 Fc. The Fc variant comprises one or more amino acid residue modifications (e.g., substitutions, insertions, and / or deletions) compared to a wild-type Fc (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc). In some embodiments, the Fc variant comprises one or more amino acid residue modifications (e.g., substitutions, insertions, and / or deletions) compared to a wild-type human IgG1 Fc or IgG4 Fc.

[0097] The one or more amino acid modifications contained in the Fc variant may alter binding to one or more FcγR receptors, alter binding to the FcRn receptor, etc. In certain embodiments, the Fc domain of the heterodimeric polypeptide complex comprises one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants have an extended pharmacokinetic half-life when bound to FcRn at acidic pH, which allows them to escape degradation in lysosomes and subsequently be translocated and released outside the cell. Methods for modifying antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al, Structure, 6(1): 63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al, J. Immunology, 176:346-356 (2006).

[0098] The two chains of the Fc domain may be bound together via disulfide bonds. In some embodiments, the Fc domain comprises one or more amino acid modifications (e.g., substitutions) at the interface of the Fc region to facilitate and / or promote heterodimerization. For example, the two chains of the Fc domain are modified to comprise a "knob-into-hole" structure to promote heterodimerization, which involves the introduction of a protuberance ("knob") in a first Fc polypeptide and a cavity ("hole") in a second Fc polypeptide, where the protuberance is positioned within the cavity to promote interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168. Specifically, the Fc domain may comprise at least one "knob" (protuberance) and at least one "hole" (cavity), where the presence of the "knob" and "hole" promotes the formation of a complex or heterodimer (see WO2005 / 063816 for further details). In some embodiments, the Fc domain disclosed herein comprises a first and a second Fc polypeptide chain, where the first and second polypeptides each comprise one or more mutations with respect to wild-type human IgG1 Fc. The IL-15 domain may be fused to one chain of the Fc domain that comprises the "knob" mutation, whereas the IL-15Rα domain is fused to the other chain that comprises the "hole" mutation, or vice versa. In at least one embodiment, the "hole" mutation is Y349C, T366S, L368A, and / or Y407V, and the "knob" mutation is S354C, and / or T366W.

[0099] In certain embodiments, the Fc domain of the fusion protein comprises one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Certain amino acid residues in the CH2 domain of the Fc region may be substituted to provide reduced ADCC activity.

[0100] In certain embodiments, the Fc domain is an IgG4 Fc and contains an S228P mutation. As known in the art, the S228P mutation of an antibody can serve to eliminate Fab arm exchange and improve stability. In certain embodiments, the Fc domain contains a triple mutation M252Y / S254T / T256E ("YTE"). This triple mutation has been reported to cause an approximately 10-fold increase in binding to the human neonatal Fc receptor (FcRn) and an approximately 4-fold increase in serum half-life of YTE-containing human IgG in cynomolgus monkeys (Oganesyan V. et al, Mol Immunol. 2009 May;46(8-9):1750-5). In some further embodiments, the Fc domain contains additional mutations to alter the interaction between Fc and human FcRn. In some specific embodiments, the Fc domain comprises a triple "YTE" mutation plus C220S, G236R, and / or L328R substitutions. Double "G236R / L328R" or "RR" substitutions have been reported to reduce binding affinity to FcγR and Fc effector functions (ADCC, CDC, ADCP). In some embodiments, the Fc domain comprises FALA mutations (F234A / L235A) to abolish binding to Fc receptors or complement receptors.

[0101] The "EU numbering system" or "EU index" is commonly used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index as reported in Kabat et al. Sequences of Proteins of Immunological Interest (5th Ed.), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242). "Kabat-like EU numbering" or "Kabat-like "EU index" refers to the numbering of residues in the human IgG1 EU antibody. Unless otherwise stated in this specification, references to residue numbers in the constant domain of an Fc region refer to the numbering of residues according to the EU numbering system.

[0102] In some specific embodiments, the Fc variant comprises two chains, wherein the amino acid sequence of a first chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity to SEQ ID NO:3 and / or the amino acid sequence of a second chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity to SEQ ID NO:4. In some specific embodiments, the first chain of the Fc variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 and the second chain of the Fc variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:4.

[0103] In some specific embodiments, the Fc variant comprises two chains, wherein the amino acid sequence of a first chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity to SEQ ID NO:5 and / or the amino acid sequence of a second chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity to SEQ ID NO:6. In some specific embodiments, the first chain of the Fc variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 and the second chain of the Fc variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:6.

[0104] In some specific embodiments, the Fc variant comprises two chains, wherein the amino acid sequence of a first chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity to SEQ ID NO:7 and / or the amino acid sequence of a second chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity to SEQ ID NO:8. In some specific embodiments, the first chain of the Fc variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:7 and the second chain of the Fc variant comprises or consists of the amino acid sequence set forth in SEQ ID NO:8.

[0105] The phrase "at least 80%" with respect to sequence identity can include, for example, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% (i.e., identical). The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J. Mol. Biol. 48:444-453 (1970)), as incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0106] Additionally or alternatively, a search can be performed against public databases using the protein sequences of the present disclosure as a "query sequence," for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the fusion protein molecules of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0107] Properties of the IL-15 variants and IL-15-Fc fusion proteins disclosed herein The present disclosure provides reduced potency, enhanced PK / PD IL-15 variants and IL-15-Fc polypeptide complexes comprising these IL-15 variants, which exhibit good thermal stability, reduced efficacy / toxicity, prolonged PK, enhanced PD, and may therefore serve as novel immunotherapeutic agents with improved anti-tumor efficacy. The functionality of these IL-15 variants and IL-15-Fc fusion proteins can be assessed in a number of ways, including in vitro or in vivo assays.

[0108] In some embodiments, the effect of IL-15 variants and IL-15-Fc polypeptide complexes is assessed by immune cell proliferation assays, for example using Ki-67 intracellular staining of immune effector cells. Ki-67 is a protein strictly related to cell proliferation, and the percentage of Ki-67 in CD8+ T and NK cells can be measured by FACS, which indicates their activity in stimulated CD8+ T and NK cells. Other immune cells can also be used to assess the effect on cell proliferation, especially CD45+ lymphocytes.

[0109] In some embodiments, the effect of IL-15 variants and IL-15-Fc polypeptide complexes may be evaluated by quantifying signal transduction pathways as measured by phosphorylation of specific factors, such as STAT5 phosphorylation. In some embodiments, the effect of IL-15 variants and IL-15-Fc polypeptide complexes may be evaluated by assessing T cell activity as measured by cytokine production. In some embodiments, the effect of IL-15 variants and IL-15-Fc fusion proteins may be evaluated by assessing lymphocyte expansion (white blood cell, lymphocyte, NK cell, T cell and T cell subsets, monocyte, and basophil counts).

[0110] The IL-15 containing polypeptide complexes and fusion proteins of the present disclosure have the following properties: (a) more moderate potency in simulating immune cell proliferation, such as CD8+ T cell and NK cell proliferation, in humans, cynomolgus monkeys and mice; (b) good thermal stability; (c) an extended pharmacokinetic profile compared to wild-type IL-15 or IL-15 containing polypeptide complexes or fusion proteins; (d) showing prolonged and higher lymphocyte proliferation in pharmacokinetic studies; (e) no apparent toxicity in vivo; Provide at least one of the following:

[0111] Nucleic acid molecules encoding IL-15 variants and fusion proteins In some embodiments, the present disclosure is directed to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an IL-15 variant or a heterodimeric Fc fusion protein disclosed herein.

[0112] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. An isolated nucleic acid encoding an IL-15 variant can be operably linked to another DNA molecule encoding an Fc domain. Similarly, a nucleic acid encoding an IL-15Rα domain (wild-type, variant, or any fragment thereof that retains IL-15 binding activity) can be operably linked to another DNA molecule encoding an Fc domain. DNA fragments encompassing these regions can be obtained by standard PCR amplification.

[0113] Once the DNA fragments encoding IL-15, IL-15Rα and Fc domain are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, incorporated into expression vectors known in the art. In some embodiments, the nucleic acids encoding these DNA fragments are each contained within a single expression vector, typically under the control of different or the same promoters. In certain other embodiments, the nucleic acids encoding these DNA fragments are operably linked and contained within a single expression vector, typically under the control of a promoter. The term "operably linked" as used in this context is intended to mean that two DNA fragments are joined together such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0114] Vectors and host cells DNA encoding IL-15, including IL-15 variants or fusion proteins, can be placed into one or more expression vectors, which are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain synthesis of the desired protein in the genetically engineered host cells. See, e.g., PCT Publication No. WO 87 / 04462.

[0115] In general, the nucleic acid sequence encoding one or all chains of the fusion protein can be cloned into a suitable expression vector operably linked to a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and then joined together by ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell to be used to produce the antibody.

[0116] A variety of promoters can be used to express the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter. Regulatable promoters including repressors with associated operons can be used.

[0117] The host cell disclosed in the present disclosure can be any cell suitable for expressing the fusion protein of the present disclosure, such as bacterial cells, yeast cells, and mammalian cells. Mammalian host cells for expressing the fusion protein of the present disclosure include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, for example, used with DHFR selection marker as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, another expression system for use with NSO myeloma cells is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody is introduced into a mammalian host cell, the fusion protein is produced by culturing the host cell for a period of time sufficient to allow the fusion protein to be expressed in the host cell or secreted into the medium that the host cell is grown in. The fusion protein can be recovered from the medium using standard protein purification methods.

[0118] Pharmaceutical Compositions In some aspects, the present disclosure is directed to a pharmaceutical composition comprising a heterodimeric polypeptide complex as disclosed herein and a pharma- ceutical acceptable carrier.In some aspects, the present disclosure is directed to a pharmaceutical composition comprising a nucleic acid molecule encoding a heterodimeric polypeptide complex or a fusion protein as disclosed herein and a pharma- ceutical acceptable carrier.

[0119] Ingredients of the composition The pharmaceutical composition may optionally contain one or more additional pharma- ceutical active ingredients, such as antibodies. The pharmaceutical composition of the present disclosure may also be administered in combination therapy, for example with another immunostimulant, anti-cancer agent, anti-viral agent, or vaccine. Pharmaceutically acceptable carriers may include, for example, pharma- ceutical acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, and various other combinations of ingredients known in the art.

[0120] Suitable ingredients can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers (such as sugars and cyclodextrins). Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in the present disclosure, the composition can include one or more antioxidants (such as methionine) and reduce the antibody or antigen-binding fragment thereof that can be oxidized. Reduction of oxidation can prevent or reduce loss of binding affinity, thereby enhancing protein stability and extending shelf life. Thus, in some embodiments, a composition is provided that includes a fusion protein and one or more antioxidants (such as methionine). The present disclosure further provides a versatile method for preventing oxidation of the fusion protein by mixing the fusion protein with one or more antioxidants (e.g., methionine), thereby extending its shelf life and / or increasing its activity.

[0121] To further illustrate, pharma- ceutically acceptable carriers can include, for example, aqueous vehicles (such as sodium chloride solution, Ringer's solution, isotonic dextrose solution, sterile water, or dextrose and lactated Ringer's solution), non-aqueous vehicles (such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents in bacteriostatic or fungistatic concentrations, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), and the like. , local anesthetics (such as procaine hydrochloride), suspending and dispersing agents (such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone), emulsifying agents (such as polysorbate 80 (TWEEN®-80)), sequestering or chelating agents (such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid). Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl ester and p-hydroxybenzoic acid propyl ester, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can be included, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancing agents, or such agents as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0122] Administration, Formulation, and Dosage The pharmaceutical compositions of the present disclosure can be administered in vivo to a subject in need thereof by various routes, non-limiting examples of which include oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or else by implantation or inhalation. The subject compositions can be formulated into preparations in solid, semi-solid, liquid, or gas form; non-limiting examples of which include tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. The appropriate formulation and route of administration can be selected according to the intended use and treatment plan. Formulations suitable for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalations and controlled release forms thereof.

[0123] Formulations suitable for parenteral administration (e.g., administration by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is provided in dissolved, suspended, or other form (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharma- ceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride solution, Ringer's solution, or lactated Ringer's solution. Similarly, the specific dosing regimen (including dose, timing, and repetition rate) will depend on the specific individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, and the like).

[0124] The frequency of administration can be determined and adjusted over the course of treatment and is based on the reduction in the number of proliferative or tumorigenic cells, the maintenance of a reduction in such neoplastic cells, the reduction in the proliferation of neoplastic cells, or the delay in the development of metastases. In some embodiments, the dose administered can be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, sustained continuous release formulations of the therapeutic composition of interest may be appropriate.

[0125] Those skilled in the art will appreciate that appropriate dosages may vary from patient to patient. Determining optimal dosages will generally involve balancing the level of therapeutic benefit against any risk or adverse side effects. The selected dosage level will depend on a variety of factors, non-limiting examples of which include the activity of the specific compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, general health, and previous medical history. Although the amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, generally the dosage will be selected to achieve a local concentration that achieves the desired effect without causing substantial harmful or toxic side effects at the site of action.

[0126] In general, the IL-15 / IL-15Rα-Fc fusion protein of the present disclosure may be administered in a variety of ranges. The various ranges include from about 100 μg / kg body weight to about 10 mg / kg body weight per dose; from about 100 μg / kg body weight to about 1 mg / kg body weight per dose; from about 1 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include from about 100 μg / kg body weight to about 200 μg / kg body weight per dose; from about 200 μg / kg body weight to about 300 μg / kg body weight per dose; from about 300 μg / kg body weight to about 400 μg / kg body weight per dose; from about 400 μg / kg body weight to about 0.5 μg / kg body weight per dose; and from about 0.5 mg / kg body weight to about 1 mg / kg body weight per dose. In certain embodiments, the dosage is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0127] In any event, the IL-15 / IL-15Rα-Fc fusion protein of the present disclosure is preferably administered when needed to a subject in need thereof. The frequency of administration can be determined by one of skill in the art (such as the attending physician) taking into account the condition being treated, the age of the subject being treated, the severity of the condition being treated, the overall state of the health of the subject being treated, etc.

[0128] In a preferred embodiment, a course of treatment involving the IL-15 / IL-15Rα-Fc fusion protein of the present disclosure will include multiple administrations of the selected pharmaceutical product over a period of several weeks or months. More specifically, the IL-15 / IL-15Rα-Fc fusion protein of the present disclosure can be administered every 4 days, every week, every 10 days, every 2 weeks, every 3 weeks, every month, every 6 weeks, every 2 months, every 10 weeks, or every 3 months. In this regard, it will be appreciated that it is possible to vary the dose or adjust the interval based on the patient's response and clinical practice.

[0129] Dosages and schedules can also be empirically determined in individuals who have received one or more doses of the disclosed therapeutic compositions. For example, the therapeutic compositions prepared as described herein can be given to the individual in incremental doses. In selected embodiments, the dose can be titrated up or down or tapered based on empirically determined or observed side effects or toxicity. To assess the effectiveness of the selected compositions, markers of the particular disease, disorder, or condition can be tracked as previously described. For cancer, markers include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by x-ray or other imaging techniques; improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples; indirect tumor markers (e.g., PSA for prostate cancer) or oncogenic antigens identified according to the methods described herein, measurement of reduced pain or numbness; improvement in speech, vision, breathing, or other tumor-related defects; increased appetite; or improved quality of life or increased survival as measured by accepted tests. It will be apparent to one of skill in the art that dosage will vary depending on the individual, the type of neoplasm, the stage of the neoplasm, whether the neoplasm has begun to metastasize to other locations within the individual, and previous and current treatments.

[0130] Formulations suitable for parenteral administration (e.g., intravenous injection) contain the IL-15 / IL-15Rα-Fc fusion protein disclosed herein at a concentration that is deemed suitable for administration and that can be empirically determined by one of skill in the art, e.g., from about 10 μg / ml to about 10 mg / ml.

[0131] Application of this disclosure The IL-15 / IL-15Rα-Fc fusion proteins, pharmaceutical compositions and methods of the present disclosure have numerous in vitro and in vivo utilities, including, for example, enhancing immune responses. For example, these molecules can be administered to cultured cells, in vitro or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of contexts. The immune response can be, for example, enhanced, stimulated, or upregulated.

[0132] For example, subjects include human patients in need of an enhanced immune response. The method is particularly suitable for treating human patients with disorders that can be treated by enhancing an immune response (e.g., a T cell-mediated immune response). In certain embodiments, the method is particularly suitable for treating cancer in vivo. To achieve immune enhancement, the IL-15 / IL-15Rα-Fc fusion protein can be administered alone or in combination with another therapy. When the IL-15 / IL-15Rα-Fc fusion protein is administered together with another agent, the two can be administered sequentially or simultaneously.

[0133] Treatment of disorders including cancer and infectious diseases In some embodiments, the present disclosure provides a method of treating a disorder or disease in a mammal, comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of an IL-15 / IL-15Rα-Fc fusion protein disclosed herein. The disease or disease can be cancer.

[0134] A variety of cancers, whether malignant or benign, primary or secondary, may be treated or prevented by the methods provided by the disclosure. The cancer may be a solid tumor or a hematological malignancy.Examples of such cancers include lung cancer, such as bronchogenic carcinoma (e.g., non-small cell lung carcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); heart cancer, such as myxoma, fibroma, and rhabdomyoma; bone cancer, such as osteochondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancer, such as glioma (e.g., glioblastoma multiforme). , anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pinealoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germinoma, teratoma, dermoid cyst, hemangioma; cancers of the digestive system, such as colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colon polyp, and colorectal carcinoma; liver cancer, such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and hemangiosarcoma; kidney cancer, such as renal adenocarcinoma, renal cell carcinoma, hypernephroma, and transitional cell carcinoma of the renal pelvis; bladder cancer; Liquid cancers; such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myeloid, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sézary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloid, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, chronic myelogenous leukemia); skin cancers, such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; Eye-related cancers include retinoblastoma and intraocular melanoma; male reproductive system cancers, such as prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers, such as uterine cancer (endometrial carcinoma), cervical cancer (cervical carcinoma), ovarian cancer (ovarian carcinoma), vulvar carcinoma, vaginal carcinoma, fallopian tube carcinoma, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid glands; pancreatic cancer; blood cancers, such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma.In a specific embodiment, the cancer is colon cancer.

[0135] In some embodiments, exemplary cancers include, but are not limited to, B-cell lymphomas, such as low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade non-cleaved cell NHL; giant cell NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomas, edema (such as edema associated with brain tumors), B-cell proliferative disorders, Meigs' syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, frontline low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low-grade / follicular lymphoma, intermediate grade lymphoma, These include: intermediate grade / follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive relapsed NHL), NHL that has relapsed after or is refractory to autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous (skin) lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.

[0136] In some embodiments, exemplary cancers further include, but are not limited to, B-cell proliferative disorders, including lymphomas (e.g., B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example: a) follicular lymphoma, b) small non-cleaved cell lymphoma / Burkitt lymphoma (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, non-Burkitt lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B-cell lymphoma, and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, hemangiocentric lymphoma, pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and / or j) Hodgkin's disease.

[0137] In some embodiments, the IL-15 containing fusion proteins disclosed herein can be used to treat or prevent infectious diseases, which can be caused by viruses, bacteria, fungi, or parasites. In certain other embodiments, the IL-15 containing fusion proteins disclosed herein can be used to treat or prevent immunodeficiency or lymphopenia.

[0138] Stimulating the immune response without cytotoxicity In some aspects, the present disclosure also provides a method of enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject an IL-15 / IL-15Rα-Fc fusion protein of the present disclosure, such that the immune response in the subject is enhanced while at the same time there are no undesirable side effects. For example, the subject is a mammal. In a specific embodiment, the subject is a human.

[0139] The term "enhancing an immune response" or grammatical variations thereof means to stimulate, induce, enhance, improve, or potentiate any response of the immune system of a mammal. The immune response may be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated response), and may be a primary or secondary immune response. Examples of enhanced immune responses include CD4 + These include increased helper T cell activity and generation of cytolytic T cells. Enhanced immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, the disclosed method enhances the immune response by the mammal when compared to a mammal or immune response by a mammal not treated with the disclosed method. In one embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-12 production. In another embodiment, the immune response is enhanced B cell proliferation.

[0140] The IL-15 fusion proteins disclosed herein may be used alone as a monotherapy, or more frequently, in combination with cellular immunotherapy, targeted therapy, chemotherapy, or radiation therapy.

[0141] Applications in gene therapy In some embodiments, the IL-15 variants and IL-15 variant fusion proteins disclosed herein or the nucleic acid molecules encoding them are used in gene therapy. Genes encoding the IL-15 variants and IL-15 variant fusion proteins are incorporated into therapeutic vectors such as lentiviruses, AAV, poxviruses, varicella zoster viruses, oncolytic viruses, and other RNA / DNA vectors.

[0142] Applications in Cellular Immunotherapy In some embodiments, the IL-15 variants and IL-15 variant fusion proteins disclosed herein are used in combination with cellular immunotherapy, also known as adoptive cell therapy. As is commonly known, cellular immunotherapy is a form of treatment that uses cells of the body's immune system to eliminate cancer. Some of these approaches involve directly isolating our own immune cells and simply increasing their numbers (e.g., performed by activating and expanding immune cells outside the patient's body and injecting them back into the patient), while others involve genetically manipulating immune cells (via gene therapy) to enhance their cancer-fighting capabilities. Cellular immunotherapy can be deployed in a variety of forms, including, but not limited to, tumor infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy, chimeric antigen receptor (CAR)-γδ T cell therapy, CAR NK cell therapy, CAR macrophage cell therapy, and natural killer (NK) cell therapy.

[0143] In some embodiments, the IL-15 variants and IL-15 variant fusion proteins disclosed herein are incorporated into cell therapy, while the genes encoding the IL-15 variants and IL-15 variant fusion proteins are integrated into the genome of cells by genetic engineering of immune cells.

[0144] Combination with gene therapy In some embodiments, the IL-15 variants and IL-15 variant fusion proteins disclosed herein are used in combination with gene therapy. Genes encoding IL-15 variants and IL-15 variant fusion proteins may be delivered to a subject by therapeutic vectors such as viruses, including lentiviruses, AAV, poxviruses, varicella zoster viruses, and oncolytic viruses. Gene transfer may be performed under certain conditions by transformation, where the gene is directly taken up by bacterial cells, transduction, where bacteriophages are used to transfer the genetic material, and finally transfection, with forced delivery of the gene using either viral or non-viral vectors. Non-viral transfection methods are subdivided into physical, chemical, and biological. Physical methods include electroporation, biolistic, microinjection, laser, elevated temperature, ultrasound, and hydrodynamic gene transfer. Chemical methods utilize calcium phosphate, DAE-dextran, liposome, and nanoparticle methods for transfection. Biological methods increasingly use viruses for gene transfer; these viruses can either integrate into the genome of the host cell conferring stable gene expression, whereas the few other non-integrating viruses are episomal and therefore their expression fades proportionately with cell division.

[0145] Combination with cellular immunotherapy In some embodiments, the IL-15 variants and IL-15 variant fusion proteins disclosed herein are used in combination with cellular immunotherapy, also known as adoptive cell therapy. As is commonly known, cellular immunotherapy is a form of treatment that uses cells of the body's immune system to eliminate cancer. Some of these approaches involve directly isolating our own immune cells and simply increasing their numbers (e.g., performed by activating and expanding immune cells outside the patient's body and injecting them back into the patient), while others involve genetically manipulating immune cells (via gene therapy) to enhance their cancer-fighting capabilities. Cellular immunotherapy can be deployed in a variety of forms, including, but not limited to, tumor infiltrating lymphocyte (TIL) therapy, engineered T cell receptor (TCR-T) therapy, chimeric antigen receptor (CAR) T cell therapy, CAR NK cell therapy, CAR macrophage cell therapy, and natural killer (NK) cell therapy.

[0146] Combination with targeted therapy and chemotherapy The heterodimeric polypeptide complexes disclosed herein may be administered as the sole active ingredient or in combination as an adjuvant to other agents, such as anti-cancer agents, immunomodulatory agents or other anti-inflammatory agents, or in combination with other agents, for example, for the treatment or prevention of the diseases mentioned above.

[0147] For purposes of this disclosure, a "chemotherapeutic agent" includes chemical compounds that non-specifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells (e.g., cytotoxic or cytostatic agents). Such chemical agents are often directed at intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancerous cells, which generally grow and divide rapidly. For example, vincristine depolymerizes microtubules and inhibits cells from entering mitosis. In general, a chemotherapeutic agent can include any chemical agent that inhibits or is designed to inhibit cancerous cells, or cells likely to become cancerous, or their tumorigenic progeny (e.g., TICs). Such drugs are often administered and are often most effective in combination, for example in regimens such as CHOP or FOLFIRI.

[0148] Examples of anti-cancer drugs that may be used in combination with the proteins of the present disclosure (either as a component of a complex or uncomplexed) include, but are not limited to, paclitaxel, gemcitabine, cisplatin, doxorubicin, 5-fluorouracil, capecitabine, combretastatin, leucovorin, and the like.

[0149] For example, the heterodimeric polypeptide complexes described herein are useful in treating DMARDs, such as gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporin A, tacrolimus, sirolimus, minocycline, lefiunomide, glococorticoids; calcineurin inhibitors, such as cyclosporin A or FK506; regulators of lymphocyte recirculation, such as FTY720 and FTY720 analogs; mTOR inhibitors. agents such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; ascomycins with immunosuppressive properties such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprene; methotrexate; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualin or an immunosuppressive homologue, analogue or derivative thereof; immunosuppressants.

[0150] It is understood that in selected embodiments described above, such anti-cancer agents may comprise a conjugate and may be conjugated to the disclosed IL-15 / IL-15Rα-Fc fusion protein prior to administration. More specifically, in certain embodiments, the selected anti-cancer agent is linked to an unpaired cysteine ​​of a modified IL-15 / IL-15Rα-Fc fusion protein to provide a modified conjugate as described herein. Thus, such modified conjugates are expressly contemplated as being within the scope of the present disclosure. In other embodiments, the disclosed anti-cancer agents are given in combination with site-specific conjugates comprising various therapeutic agents as described above.

[0151] It is readily understood that anti-cancer or immunomodulatory agents used in combination with the IL-15 fusion proteins disclosed herein should be compatible with the IL-15 fusion proteins, i.e., will not reduce, perturb, or eliminate the effect of the IL-15 fusion proteins disclosed herein, but will preferably provide a modulating or even synergistic effect.

[0152] Combined with radiation therapy The present disclosure also provides for the combination of IL-15 / IL-15Rα-Fc fusion proteins with radiation therapy (i.e., any mechanism that locally induces DNA damage in tumor cells, such as gamma irradiation, X-rays, ultraviolet radiation, microwaves, electronic radiation, etc.). Combination therapy using directed delivery of radioisotopes to tumor cells is also contemplated, and the disclosed fusion protein and polypeptide complexes can be used in conjunction with targeted anticancer drugs or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 to about 2 weeks. Radiation therapy can be administered for about 6 to 7 weeks to subjects with head and neck cancer. Optionally, radiation therapy can be administered as a single dose or as multiple consecutive doses.

[0153] Pharmaceutical Packs and Kits Pharmaceutical packs and kits are also provided that include one or more containers containing one or more doses of IL-15 / IL-15Rα-Fc fusion protein. In certain embodiments, a unit dosage is provided that includes a predetermined amount of a composition that includes an IL-15 / IL-15Rα-Fc fusion protein, with or without one or more additional agents. In other embodiments, such unit dosages are provided in a single-use pre-filled syringe for injection. The composition included in the unit dosage can be formulated in saline, sucrose, and the like; a buffer, such as phosphate; and / or within a stable and effective pH range. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that can be reconstituted by adding an appropriate liquid, e.g., sterile water or saline solution. In certain preferred embodiments, the composition includes one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on or associated with the container indicates that the enclosed composition is used to treat the disease condition of choice.

[0154] The disclosure also provides kits for producing single or multiple dose units of a protein and, optionally, one or more other anti-cancer agents. The kits include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic, and contain a medicamentously effective amount of the disclosed protein. The container(s) may include a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle). Such kits will generally contain, in a suitable container, a medicamentically acceptable formulation of the engineered protein, and, optionally, one or more anti-cancer agents in the same or different containers. The kits may also include other medicamentically acceptable formulations, either for diagnostic or combination therapy. For example, in addition to the disclosed IL-15 / IL-15Rα-Fc fusion protein, such kits can contain one or more anti-cancer agents, such as chemotherapeutic or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents; and / or other anti-cancer agents.

[0155] More specifically, the kits can have a single container containing the disclosed IL-15 / IL-15Rα-Fc fusion protein, with or without additional components, or they can have different containers for each desired agent. If multiple therapeutics are provided for conjugation, a single solution can be premixed, either in molar equivalent combination, or with one component over the other. Alternatively, the IL-15 / IL-15Rα-Fc fusion protein and any anti-cancer agents of the kit can be maintained separately in separate containers prior to administration to a patient. The kits may also comprise second / third container means for containing a sterile, pharma- ceutically acceptable buffer or other diluent, such as bacteriostatic water for injection, phosphate buffered saline, Ringer's solution and dextrose solution.

[0156] When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, with a sterile aqueous solution or a physiological saline solution being particularly preferred. However, the components of the kit can be provided as a dry powder. When the reagent or component is provided as a dry powder, the powder can be reconstituted by adding a suitable solvent. It is envisioned that the solvent can also be provided in other containers.

[0157] As briefly described above, the kits will also include a means for administering the IL-15 / IL-15Rα-Fc fusion protein, and any components, to a patient, such as one or more needles, IV bags or syringes, or eye drops, pipettes, or other similar devices from which the formulations can be injected or introduced into an animal, or applied to an affected area of ​​the body. The kits of the present disclosure will also typically include a means for containing vials and the like, and other components sealed for commercial sale, such as injection- or blow-molded plastic containers into which the desired vials and other devices may be placed and held.

[0158] Sequence Listing Overview Table A below provides sequences of proteins, variants, and exemplary polypeptide complexes disclosed herein. "W327151-TXU1.P1-V0013.uIgG4V1" and "W327151-TXU1.P1-V0013.uIgG4V322" can be abbreviated as "V0013.uIgG4V1" and "0013.uIgG4V322", respectively, and apply to the others as well.

[0159] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] EXAMPLES

[0160] Thus, having generally described the present disclosure, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the present disclosure, and are not intended to represent that the following experiments are all or the only experiments performed.

[0161] Example 1 Materials, preparation of IL-15 mutants and fusion proteins 1.1 Preparation of materials Information regarding commercially available materials used in the examples is provided in Table 1.

[0162] [Table 2-1] [Table 2-2]

[0163] 1.2 Construction of IL-15 mutants, fusion proteins, and BMK Construction of expression vectors for BMK, IL-15 / IL-15Rα-Fc mutant, and PD-1 / IL-15 mutant fusion proteins Two heterodimeric IL-15 / IL-15Rα-Fc fusion proteins, XENP24306 and XENP20818, disclosed in US20180118805A1, were used as benchmarks in the following experiments (referred to herein as "W327151-BMK1" and "W327151-BMK2", respectively). XENP24306 utilizes an IL-15 mutant containing D30N / E64Q / N65D mutations, whereas XENP20818 utilizes the wild-type IL-15 protein. The DNA segments of XENP24306 and XENP20818 were synthesized according to US20180118805A1 (Seq.382-387 and Seq.52-57) and subcloned into pcDNA3.4 expression vectors, followed by transfection for protein production.

[0164] DNA fragments encoding the IL-15 variants, IL-15Rα, IgG4 V1 form, IgG4 V322 form, IgG4 V803 form, and IgG1V805 form listed in Table 2 were also synthesized, subcloned into separate expression vectors, and transfected for protein production. Compared to human wild-type IgG Fc, the IgG4 V1 form contains S228P, the IgG4 V322 form contains S228P and "FALA" (F234A / L235A), the IgG4 V803 form contains S228P, "FALA" and "YTE", whereas the IgG1 V805 form contains "YTE" (M252Y / S254T / T256E) and "RR" (G236R / L328R). The structure of the constructed polypeptide complex is as shown in FIG. 1A. For simplicity, the names of these heterodimeric IL-15 / IL-15Rα polypeptide complexes have also been abbreviated throughout the specification to the form "V00xx.uIgG4v1", "V00xx.uIgG4v322", "V00xx.uIgG4v803" or "V00xx.uIgG1v805", where "V00xx" indicates the number of the IL-15 variant.

[0165] Additionally, an antibody was constructed comprising the above described heterodimeric IL-15 / IL-15Rα-Fc fusion protein fused to a previously developed anti-PD-1 VHH (sequence shown in SEQ ID NO: 80) and designated "W327199-T1U1.P1-13.uIgG4V322". DNA fragments encoding the IgG4V322 forms of the PD-1 / IL-15 variants listed in Table A (SEQ ID NOs: 76-77) were synthesized. The structure of the constructed polypeptide complex is shown in Figure 1C, where IL-15 and IL-15Rα are present at the N-terminus and the PD-1 binding moiety is present at the C-terminus, interrupted by the Fc region.

[0166] [Table 3]

[0167] Small scale protein expression in HEK293 cells Expi293 cells (Thermofisher, A14635) were transfected with the plasmids constructed as described above and cultured for 5 days according to the manufacturer's suggested protocol. The supernatants were collected and analyzed by SDS-PAGE or filtered and used for target protein purification using a Protein A column (GE Healthcare, Cat. 175438). The concentration of purified Fc-tagged proteins was measured by absorbance at 280 nm. Size and purity were tested by SDS-PAGE and SEC-HPLC, respectively, and then stored at -80°C.

[0168] Purity by SEC-HPLC Protein purity was tested by SEC-HPLC using an Agilent 1260 Infinity HPLC. 50 μL of antibody solution was injected onto a TSKgel SuperSW3000 column using 50 mM sodium phosphate, 0.15 M NaCl, pH 7.0 as buffer. Run time was 20 min. Peak retention time on the column was monitored at 280 nm. Data was analyzed using ChemStation software (V2.99.2.0).

[0169] Example 2 In vitro characterization 2.1 Differential Scanning Fluorometry (DSF) DSF assays were performed using a 7500 Fast Real-Time PCR system (Applied Biosystems). Briefly, 19 μL of protein solution was mixed with 1 μL of 62.5×SYPRO Orange solution (TheromFisher-S6650) and added to a 96-well plate. The plate was heated from 26° C. to 95° C. at a rate of 2° C. / min, and the resulting fluorescence data was collected. The data was automatically analyzed by the operating software, and Tm was calculated by taking the maximum of the negative derivative of the resulting fluorescence data with respect to temperature. Ton can be roughly determined as the temperature of the negative derivative plot when it starts to decrease from the baseline before the transition.

[0170] The thermostability of the IL-15 mutants is shown in Table 3, which indicates that mutagenesis by amino acid deletion does not affect the thermostability of IL-15.

[0171] [Table 4]

[0172] 2.2 Thermal stability test by DLS-Tagg Measurement of Tagg expression was investigated using a DynaPro Plate Reader III (Wyatt DynaproTM). 7.5 μL of sample was mixed with 5 μL of silicone oil in a microplate. The plate was heated from 40° C. to 80° C. at a rate of 0.125° C. / min. Three collections were collected for each protein sample, with each collection time of 5 seconds. For each measurement, the scattering coefficient was determined and plotted against temperature. Tagg expression values ​​were automatically calculated by the operating software (DYNAMICS 7.8.1.3). As shown in Table 4, V0013 showed good thermal stability.

[0173] [Table 5]

[0174] 2.3 Measurement of diffusion interaction parameter (kD) by DLS kD measurements were performed using a DynaPro Plate Reader III (Wyatt Dynapro™). First, samples were diluted in the microplate to final concentrations of 2.5, 5, 10, 15, and 20 mg / mL. Data collection was performed by the DYNAMICS operating software (v7.8.1.3). Five collections were collected for each protein sample, with each collection time being 5 seconds. For each measurement, the scattering coefficient was determined and plotted against the protein concentration. The kD values ​​were automatically calculated by the software. The kD values ​​shown in Table 5 were within the normal range, implying a low aggregation risk for V0013.

[0175] [Table 6]

[0176] 2.4 Measurement of self-interaction by AC-SINS Antibody self-interactions were investigated using the AC-SINS method. Goat anti-human IgG Fc antibody (capture) and ChromPure goat IgG antibody (non-capture) were premixed, and then the IgG mixture was mixed with gold nanoparticle (AuNP) solution at a volume ratio of 1:9. All antibodies at 0.1 mg / mL were mixed with the AuNP solution, followed by incubation. The antibody-AuNP mixture was detected by absorbance values ​​from 510 to 570 nm. The Δλmax was calculated by subtracting the maximum absorbance value of the buffer from the maximum absorbance value of the sample. The Δλmax of V0013 was -3 nm (Table 6), indicating no risk of self-interactions.

[0177] [Table 7]

[0178] 2.5 Freeze / Thaw Stress Test Each sample with the same concentration was frozen at -80°C and thawed at 25°C for 1 hour. After five repeated freeze / thaw cycles, the samples before and after treatment were tested by concentration and SEC-HPLC. After five freeze / thaw cycles (5xF / T), V0013 was stable in concentration and purity by SEC-HPLC (Table 7), with little further aggregation. The appearance of V0013 was better than BMK1.

[0179] [Table 8]

[0180] 2.6 Nonspecific binding test by BVP ELISA BVP was incubated on the ELISA plate by adding 1% BVP stock. After blocking, the primary antibody (i.e., test antibody) was added and incubated, and anti-human-IgG-HRP was used for detection. The BVP score was measured by normalizing the absorbance with the control well without test antibody. As shown in Table 8, the BVP scores of V0013 and BMK1 are both acceptable, implying a low risk of non-specific binding.

[0181] [Table 9]

[0182] 2.7 Ki-67 proliferation assay in human / cynomolgus / mouse T / NK cells Human PBMCs from healthy donors were purchased from OriBiotech and cultured in RPMI 1640 medium (Gibco, Cat. 22400105) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Cynomolgus monkey PBMCs were isolated from whole blood using Lymphoprep (STEMCELL, Cat. 07851) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Mouse splenocytes were isolated from wild-type C57BL / 6 mice and cultured in RPMI 1640 medium (Gibco, Cat. A1049101) supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, and 0.05 mM 2-mercaptoethanol.

[0183] Human PBMCs were treated with IL-15 variants at the indicated concentrations. After 4 days of treatment, PBMCs were stained with anti-CD3, anti-CD8, and anti-CD56 and gated for CD8+ T cells (CD3+ / CD8+) and NK cells (CD3- / CD56+). As Ki67 is a protein strictly related to cell proliferation, staining of intracellular Ki67 was performed using anti-Ki67 and Foxp3 / transcription factor staining buffer set. Lymphocytes were first gated based on SSC and FSC. Lymphocytes were then gated based on CD3, CD8, and CD56 expression to identify CD8+ T and NK cells. The percentage of Ki67 in CD8+ T and NK cells was measured using FACS.

[0184] Cynomolgus monkey PBMCs were treated with IL-15 variants at the indicated concentrations. After 4 days of treatment, PBMCs were stained with Abs against cynomolgus monkey CD3, CD8, and CD16, gated for CD8+ T cells (CD3+ / CD8+) and NK cells (CD3- / CD16+), and then stained for intracellular Ki67 using anti-Ki67 and Foxp3 / transcription factor staining buffer set. The percentage of Ki67 in CD8+ T and NK cells was measured using FACS.

[0185] Mouse splenocytes were harvested and treated with IL-15 variants at the indicated concentrations. After 4 days of treatment, cells were stained with Abs against mouse CD3, CD8, and CD335, gated for CD8+ T cells (CD3+ / CD8+) and NK cells (CD3- / CD335+), and then stained for intracellular Ki67 using anti-Ki67 and Foxp3 / transcription factor staining buffer set. The percentage of Ki67 in CD8+ T and NK cells was measured using FACS.

[0186] Human PBMC CD8 + To measure the effect of V0013 on T and NK cells, human PBMCs from healthy donors were cultured with V0013, V0016, V0017, V0018, W327151-BMK1, W327151-BMK2, and human IgG isotype control. Ki67 levels were measured in CD8 + Used to measure proliferation of T and NK cells.

[0187] The results showed that W327151-BMK2 inhibited human CD8 + The results demonstrated that V0013 was most potent against T (Figure 2A) and NK cell proliferation (Figure 2B), whereas V0013 inhibited human CD8 + Moderately promoted T and NK cell proliferation. V0016, V0017, and V0018 caused a greater than 1000-fold reduction in potency compared to BMK2.

[0188] [Table 10]

[0189] Cynomolgus CD8 +To investigate the potency of IL-15 variants on T and NK cells, cynomolgus PBMCs were cultured with W327151-TXU1.P1-V0013.uIgG4V322 and W327151-BMK2. As shown in Figure 3, compared to wt IL-15 (W327151-BMK2), W327151-TXU1.P1-V0013.uIgG4V322 inhibited cynomolgus CD8 T cells with a potency that was substantially reduced by hundreds of fold. + T and CD16 + It mildly promoted the proliferation of NK cells, showing a pattern similar to that of human T / NK cells.

[0190] Mouse splenocytes also express mouse CD8 + The effect of the IL-15 variant W327151-TXU1.P1-V0013.uIgG4V322 on T and NK cells was measured. After 4 days of incubation with V0013, W327151-BMK1, W327151-BMK2 and human IgG isotype control, CD3 + CD8a + or CD3 - CD335 + Cellular Ki67 + The percentages of CD8 + The proliferation of T cells or NK cells was measured.

[0191] As shown in Figure 4, only W327151-BMK2 and V0013 expressed mouse CD8 + V0013 was able to proliferate T (Figure 4A) and NK cells (Figure 4B), whereas BMK1 (XENP24306) was completely inactive. + It showed better potency against mouse NK than T. Furthermore, V0013 showed a reduced potency in stimulating mouse T / NK proliferation to a similar extent as in humans, suggesting the feasibility of using mouse tumor models to measure the in vivo efficacy of this IL-15 variant.

[0192] [Table 11]

[0193] 2.8 Binding assay in HH cell line The relative binding of W327151-TXU1.P1-V0013.uIgG4V322 to human lymphoma (HH) cell lines was measured. As can be seen in Figure 5, W327151-TXU1.P1-V0013.uIgG4V322 (EC50 = 91.89nM), W327151-BMK1 (EC50 = 171.70nM), and W327151-BMK2 (EC50 = 0.89nM) bound to the HH cell line in a dose-dependent manner.

[0194] 2.9 Receptor binding test by SPR The binding affinity of IL-15 mutants to their receptors was detected by SPR using Biacore8K. Each mutant was captured on an anti-human Fc immobilized CM5 sensor chip (cytiva). Various concentrations of W327151-hPro2.ECD.His were injected onto the sensor chip at a flow rate of 30 μL / min for a 120 s binding phase, followed by a 120 s dissociation. The chip was then regenerated with 10 mM glycine-HCl pH 1.5 after each binding cycle.

[0195] As summarized in Table 11, V0013 retained receptor β binding affinity and did not or only weakly bound to common γc compared to BMK2 (wt IL-15). W327151-BMK1 showed attenuated affinity for receptor β and common γc. For common γc binding, V0013 was much weaker than BMK1 in FIG. 6. As a result, V0013 was a non-consensus γc binding IL-15.

[0196] [Table 12]

[0197] 2.10 Nonspecific binding assay A full panel non-specific binding assay was performed on a series of tumor or modified cell lines and detected by flow cytometry. There was no detectable binding of V0013 to the cell lines (Table 12), indicating that there was no risk of non-specific binding.

[0198] [Table 13]

[0199] 2.10 Allogeneic Mixed Lymphocyte Reaction Assay For human allogeneic mixed lymphocyte reaction, purified CD4 + T cells were co-cultured with allogeneic dendritic cells and various concentrations of antibodies were added to the cultures. After 3 days of incubation, IFN-γ was detected by ELISA.

[0200] As shown in Figure 7A, W327199-T1U2.P13-1.uIgG4V322 promoted IFN-γ secretion in a dose-dependent manner, which was much more potent than that of PD-1 antibody (W3056-AP17R1-2H2-Z1-R1-14A1-FC(IgG4.SP), US20200002420A1) or combination therapy of W327151-TXU1.P1-V0013.uIgG4V322 and PD-1 antibody. In the IL-2 secretion assay, the efficacy of W327199-T1U2.P13-1.uIgG4V322 was comparable to that of the PD-1 antibody or combination group (Figure 7B).

[0201] Example 3 In vivo characterization 3.1 Animals All procedures related to the handling, care, and treatment of animals in the study were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi Biologics, which follows the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0202] 3.2 Pharmacokinetics (PK) Female C57BL / 6 mice (8-9 weeks) obtained from Charles River were randomly assigned to two dosing groups and injected intravenously with IL-15 variants at a dose of 0.2 mg / kg on day 0. Sample collection for immunological and pharmacokinetic analysis was also performed. The quarantine period was 21 days (prior to the pre-dosing period). The pre-dosing period was 1 day before dosing began. Intravenous administration is the planned clinical route of administration. Systemic exposure to the test article was assessed in all mice. Blood samples were collected pre-dosing (day 0) and 15 seconds, 4 hours, days 2 (48 hours), 3 (72 hours), 5 (120 hours), 7 (168 hours), 10 (240 hours), 12 (288 hours), 14 (336 hours), and 21 (504 hours) after dosing. At each time point, approximately 0.03 mL of blood was drawn from the saphenous vein or other site suitable for collecting serum. Utmost care was taken during blood collection. Blood samples were collected in tubes without additives and kept on ice until they were processed. All samples were processed within 2 hours of collection.

[0203] Measurement of serum drug concentrations by ELISA Serum concentrations of drugs were measured by ELISA. Briefly, ELISA plates were coated with 1 μg / mL goat anti-human IgG Fc (Southern Biotech, 2049-01). After washing and blocking, serially diluted serum samples were added, and then biotin-labeled goat anti-human IgG Fc (Southern Biotech, SB2049-08) or anti-human IL-15 (R&D, BAM247) was used as detection antibody. HRP-streptavidin (Thermo-21127) and TMB substrate (Life Technologies, 002023) were used for color development. The absorbance of the wells was measured at (450-540) nm using a multi-plate reader (SpectraMax® M5e). A calibration curve was made according to the standard samples, and serum samples were analyzed using SoftMax.

[0204] Immunogenicity (ADA) Mouse serum was collected on days 5, 7, 14, and 21 after dosing. Anti-drug antibody (ADA) production was measured by ELISA. ELISA plates were coated with 1 μg / mL of W327151-TXU1.P1-V0013.uIgG4v322 and W327151-BMK1. After washing and blocking, serially diluted serum samples were added, and then goat anti-mouse IgG Fc fragment cross-adsorbed HRP (BETHYL, A90-231P) was used as the detection antibody. TMB substrate (Life Technologies, 002023) was then used for color development. Absorbance was measured as described above. Pre-dosing serum was used as the baseline.

[0205] Data analysis Data were evaluated for treatment-related effects by making comparisons between individual values ​​and, where appropriate, intervals. The significance of any findings was determined based on the prior experience of the scientists involved. Serum concentrations were subjected to non-compartmental pharmacokinetic analysis by using Phenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). The linear / log trapezoidal rule was applied in obtaining PK parameters, and data were expressed as mean ± SD.

[0206] As shown in FIG. 8 and Table 13, the reduction in potency in V0013 and BMK1 (XENP24306) was consistent with the increase in wild-type IL-15 (t 1 / 2 This clearly led to an extended serum half-life compared to V0013 (~0.5 days, US2018118805A1). V0013 and BMK1 had similar C 0 and t 1 / 2 Furthermore, V0013 showed lower clearance and better drug exposure (AUC) compared to BMK1 without causing toxicity and producing little ADA (Table 14). The extended PK profile is expected to contribute to extended drug exposure and improved pharmacokinetics.

[0207] As shown in Figures 8A and B, the plasma drug concentrations detected by the different detection methods of either Fc-Fc or IL-15-Fc were consistent, suggesting good in vivo stability of the tested IL-15 variants.

[0208] [Table 14]

[0209] [Table 15]

[0210] 3.3 Mouse maximum tolerated dose (MTD) Female C57BL / 6 mice (8-9 weeks) obtained from Charles River were randomly assigned to various groups and were intravenously injected with IL-15 variants at various doses every 3 days (Q3D). Mice were observed after dosing and body weights were collected every 3 days.

[0211] As shown in Figure 9, mice treated with 0.8-3.2 mg / kg V0013 were well tolerated as they did not lose weight. Dosing with 6.4 and 10 mg / kg V0013 (Q3D) resulted in a distinction between mice that died at day 7 and the remaining mice that endured more than 10% weight loss. WT IL-15 (BMK2) was more toxic compared to V0013, with only mice in the 0.2 mg / kg treatment group (Q2D) surviving. As a result, V0013 achieved a 16-fold improvement in the MTD (3.2 mg / kg, Q3D) compared to WT IL-15 (0.2 mg / kg, Q3D).

[0212] 3.4 Pharmacokinetics (PD) The pharmacokinetics of IL-15 variants due to mouse cross-reactivity of V0013 was measured in female C57BL / 6 mice. Mice were treated with various doses of V0013 or WT IL-15, and blood was collected at various time points after dosing for analysis. Peripheral lymphocytes were measured by flow cytometry. The pre-dosing period was 1 day before initiation.

[0213] In Figure 10, V0013 expanded peripheral lymphocytes, especially CD8+T and NK cells. Here, NK cells were more sensitive to V0013 with a long-acting response (day 3-57) compared to WT IL-15 (day 7-10). The maximum expansion of CD8+T and NK cells caused by V0013 was at day 17, suggesting a prolonged PD effect compared to WT IL-15 (day 7). Moreover, both CD8+T and NK cell proliferation were dose-responsive. Moreover, Tregs were slightly expanded by V0013 at high doses, whereas CD4+T cells were unresponsive.

[0214] Regarding the detailed activation of NK and CD8+ T cells, V0013 or WT IL-15 induced a longer period of proliferation (Ki-67) in NK compared to CD8+ T (Figure 11). NK cytotoxicity, as determined by granzyme B expression, was stronger and more sustained compared to CD8+ T. In conclusion, V0013 and WT IL-15 induced a long-lasting PD in NK compared to CD8+ T.

[0215] Meanwhile, CD44 and CD62L were used to distinguish memory and naive subtypes of CD8+ T cells. As summarized in Figure 12, V0013 mainly induced proliferation of central memory CD8+ T (Tcm), slightly increased effector memory CD8+ T (Tem), and did not increase naive CD8+ T. There was no peripheral albumin loss or robust cytokine release during PD (Figure 13).

[0216] As a result, V0013 (weak IL-15 with limited adverse events) caused better CD8+ T and NK cell activation and proliferation, and induced memory subtypes compared to WT IL-15. Prolonged and enhanced PD may be paramount for better antitumor efficacy.

[0217] 3.4 Antitumor Efficacy CT26 syngeneic, Daudi CDX and A375 humanized models were used for efficacy studies. CT26 cells were implanted (sc) into the right flank of Balb / c mice. Daudi cells were implanted (sc) into the right flank of SCID mice. A375 cells were implanted (sc) into the right flank of NPG mice followed by human PBMCs implanted via ip. When the tumors reached the corresponding volume, the tumor-bearing mice were randomized into different groups and dosed with IL-15 variants. The size of the tumors was measured bidimensionally using calipers and the volume was expressed in mm 3Results were expressed as mean and standard error (mean±SEM). Statistical analysis was performed by two-way ANOVA, and P<0.05 was considered statistically significant.

[0218] In the CT26 syngeneic model (Figure 14), treatment with V0013 at 2.5 / 5 / 10 mg / kg showed significant antitumor effects compared to PBS treatment, with TGIs of 15.6%, 55.6% and 64.9%, among others. WT IL-15 showed a similar dose response. Compared to WT IL-15, V0013 showed better antitumor effects at the MTD dose. The efficiency of V0013 is influenced by CD8+ T and NK cells.

[0219] A375 humanized model was used to measure the effect of V0013 on T cells, in which human T cells were reconstituted. As shown in Figure 15, V0013 significantly inhibited tumor growth as a single agent, and it was superior to BMK2 and BMK1. The activation and proliferation of human T cells by V0013 is of paramount importance for better antitumor effect.

[0220] The Daudi CDX model was used to measure NK efficiency. In Figure 16, BMK2 showed limited antitumor effect even at high dose (0.4mg / kg). In comparison, V0013 showed significant antitumor effect and dose response, which means that V0013 induced proliferation and activation of mouse NK cells, which are most important for antitumor effect. As a result, V0013 showed more significant effects than any of the CD8+ T or NK cell-mediated effects compared to WT IL-15. The prolonged PK and enhanced PD of V0013 induced better efficacy.

[0221] Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central attributes. It is to be understood that the foregoing description of the present disclosure provides only exemplary embodiments thereof, and other variations are contemplated within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims as indicating the scope and content of the present invention.

Claims

1. 1. A polypeptide complex comprising an IL-15 mutant domain, an IL-15Rα domain, a first dimerization domain and a second dimerization domain, wherein: the IL-15 variant domain has reduced potency in stimulating immune cell proliferation or activation compared to wild-type IL-15, and the IL-15 variant peptide comprises an amino acid sequence that is truncated by 1 to 12 amino acids from the C-terminus, the N-terminus, or both the N- and C-terminus compared to SEQ ID NO:1; the first dimerization domain and the second dimerization domain bind together to form a dimer; The polypeptide complex.

2. The polypeptide complex of claim 1 , wherein the immune cells are NK and / or CD8+ T cells.

3. 3. The polypeptide complex of claim 1 or 2, wherein the immune cell is derived from a human, a mouse, or a cynomolgus monkey.

4. The polypeptide complex of any one of claims 1 to 3, wherein the IL-15 mutant peptide comprises an amino acid sequence of any of SEQ ID NOs: 51 to 69, for example SEQ ID NO:

51.

5. 5. The polypeptide complex of any one of claims 1 to 4, wherein the potency in stimulating NK and / or CD8+ T cell proliferation or activation is reduced by more than 10 fold, more than 50 fold, more than 100 fold, more than 200 fold, more than 300 fold, more than 500 fold, more than 700 fold, more than 900 fold, more than 1000 fold, more than 1500 fold, more than 2000 fold, or even more, compared to wild type IL-15, as measured in proliferation and activation assays by FACS.

6. 6. The polypeptide complex of any one of claims 1 to 5, wherein the IL-15Rα domain comprises or consists of a wild-type IL-15Rα protein, an IL-15Rα mutant, or any fragment thereof that retains IL-15 binding activity, such as the fragment set forth in SEQ ID NO:

2.

7. comprising two chains, where from N-terminus to C-terminus: the first chain comprises an IL-15 variant domain operably linked to the first dimerization domain; and The polypeptide complex of any one of claims 1 to 6, wherein the second chain comprises an IL-15Rα domain operably linked to the second dimerization domain.

8. The polypeptide complex of any one of claims 1 to 7, further comprising one or more antigen-binding moieties.

9. 9. The polypeptide complex of claim 8, wherein a first antigen-binding portion is operably linked to the first dimerization domain and a second antigen-binding portion is operably linked to the second dimerization domain.

10. The antigen binding parts mentioned above are as follows: PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, TIM4, 4-1BB, OX-40, OX-40L, GITR, A2aR, TIGIT, CD96, PVRIG, CD226, 5T4, VISTA, VSIG3, VSIG4, ICOS, CD28, CD3, CD4, CD8, CD45, CD44v6, CD27, CD47, SIRPAα, SLAMF7, CD24, Siglec10, Siglec15, Siglec8, VSIR, VSIG4, PSGL-1, C5AR1, BTN1A1, BTN3A1, CD70, RANKL, CSF1R, CSF2RB, TNFR SF1 / 1a / 1b, BDCA2, BTLA, C5aR, NKG2A, NKG2D, NKp30, NKp46, CD16a, CD56, CD166, FCG R3, CD2, ニューロピリン-1, CCR8, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, GCGR, CXCR2, CXCR4, CX CR5, CALCRL, ETAR, GLP1R, CX3CR1, GPR1, GPR17, GPR20, GPR30, GPR34, GPR-65, GPCR78, GPRC5D, GPR84, LGR4, LGR5, VEGF, VEGFR, HER2, HER3, Trop2, pCAD, ERα, EGFR, de2-7EGFR, EGFRvIII, PSMA, PSCA, PSA, TAG-72, SEZ6, SEZ6L, SEZ6L2, SEMA4D, DLL3, GD2, GPC3, KLB, KLRB1, KLRG1, GPC1, PCSK9, EpCAM, p-cadherin, claudin 6, claudin 18.2, FGFR2b, FGFR3, FGFR4, MUC1, MUC13, MUC16, MUC17, MUCL3, FolRa, TfR, TF, TFR, TFPI, c-Met, NY-ESO-1, GUCY2C, LIV-1, integrin αvβ6, integrin α10β1, integrin Integrin α3, Integrin α5β4, Integrin αvβ3, Integrin αvβ8, ROR1, ROR2, PRLR, PTK7, B7-H3, Nectin-4, NetG1, Ax1, CD147, LRRC15, Napi2b, STEAP1, LY6G6D, LYPD1, MACRO, MerTK, MICA, MICB, MSLN, Mkars, G12D, CDH3, CDH6, CDH17, APLA2, CAIX, CD46, CD47, CLDN6, EphA3, Fucosyl-GM1, ITGA3, Kallikrein, MISRII, Podocalyxin, RON ROBO1, PAUF, PLA2, podocalyxin, PRLR, PTK7, TM4SF1, TMEFF2, TREAKR, TREM-1, TREM-2, uPARAP, TYRP1, KAAG1, RU2AS, CD146, CD63, endoglin, Globo H, IGF-1R, TEM1, TEM8, TAX1BP3, ADAM-9, ENPP3, EphA2, EphA3, FcRH5, NaPi3b, TWEAK, DLK1, SORT1, SSTR2, STEAP1, CD25, CD39, GARP, LRRC33, LAIR1, LAMP3, LAP, LEPR, L ILRB1, LILRB2, LILRB4, RAGE, FGL1, TPBG, PDGFRB, TGFBR2, CEACAM1, CEACAM5, CEACAM6, carcinoembryonic antigen (CEA), ICAM1, A33, CAMPATH-1 (CDw52), Carboanhydrase IX (Carboanhydrase IX)(MN / CAIX), CD248, PDPN, ITGB1, ITGAV, CD20, CD19, CD21, CD22, CLL, BCMA, DCLK1, DDR1, DLK1, DPEP3, DKK1, CD5, CD13, CD30, CD33, CD34, CD36, CD3 7, CD38, CD43, CD52, CD55, CD94, CD99, CD7, CD71, CD73, CD74, CD79a, CD79b, CD229, CD132, CD133, G250, CSF1R(CD115), HLA-DR, HLA-G, HTRA 1, TRA-1-60, IGFR, IL-2 receptor, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), ART1, ASGR1, B7H3, B7-H4, B7H6, CD124, c-Kit (CD117), CD7, Clex12A, Clever-1, IL-13RA2, IL-11RA, IL-31RA, IL-4RA, IFNAR, ActRIIb, IL-7R, SLAMF7, Fms-like tyrosine kinase 3 (FLT-3, CD135), GFRA1, BTLA, GloboH, CS F2RB, chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), ITGA4, Clec5a, Clec7a, Clec9a, Clec12a, CLEC14, CD205, CD206, CD200R1, CD228, CD229, CD40, CD40L, FcRn, TLR8, TLR9, TNFR2, LTBR, ​​CD44, CD93, PDGH, PDGFR-α, (CD140a), PDGFR-β (CD140b), CD146, CD147, CRTH2 10. The polypeptide complex of any one of claims 8 to 9, which specifically binds to a target antigen selected from TNF-α, TGF-β, IL1RAcP, TSLP, DR5, ST2, fibroblast activation protein (FAP), CDCP1, Delrin 1, tenascin, frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), endoglin, Tie2 and other TAAs, I / O checkpoints, tumor microenvironment targets, or autoimmune and inflammatory disease associated targets.

11. The polypeptide complex according to any one of claims 8 to 10, wherein the antigen-binding portion is present in a VHH, Fab, scFv, VH or VL format.

12. 12. The polypeptide complex of any one of claims 8-11, wherein the first and second antigen-binding moieties are in a VHH format and specifically bind to PD-1, and optionally the first and second antigen-binding moieties have the same amino acid sequence as set forth in SEQ ID NO:

80.

13. The polypeptide complex according to any one of claims 1 to 12, wherein operably linked is a direct link or a link via a linker, such as a peptide linker.

14. 14. The polypeptide complex of claim 13, wherein the peptide linker is an IgG hinge region or a GS series linker, such as a (G4S)n linker with n=1-10.

15. 15. The polypeptide complex of any one of claims 1 to 14, wherein the first dimerization domain is one chain of an immunoglobulin Fc region and the second dimerization domain is the other chain of the immunoglobulin Fc region.

16. 16. The polypeptide complex of claim 15, wherein the Fc region comprises human IgG Fc, such as human IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof.

17. The polypeptide complex of claim 16, wherein the Fc variant contains one or more substitutions compared to a wild-type human Fc to enhance the stability of the Fc region, promote heterodimerization, and / or alter binding to an Fc receptor.

18. The Fc variant comprises: (a) a human IgG4 Fc that has been modified to include one or more of the following: an S228P mutation, an F234A / L235A mutation, an M252Y / S254T / T256E mutation, and a "knob-into-hole" structure; or (b) a human IgG1 Fc modified to include one or more of the following: M252Y / S254T / T256E mutations, G236R / L328R mutations, and a "knob-into-hole" structure; 18. The polypeptide complex of claim 17, comprising:

19. The Fc variant comprises: (a) an amino acid sequence having at least 80% identity to SEQ ID NO:3 on one chain and an amino acid sequence having at least 80% identity to SEQ ID NO:4 on the other chain; (b) an amino acid sequence having at least 80% identity to SEQ ID NO:5 on one chain and an amino acid sequence having at least 80% identity to SEQ ID NO:6 on the other chain; or (c) an amino acid sequence having at least 80% identity to SEQ ID NO:7 on one chain and an amino acid sequence having at least 80% identity to SEQ ID NO:8 on the other chain; 19. The polypeptide complex of claim 17 or 18, comprising:

20. 20. The polypeptide complex of any one of claims 1 to 19, wherein the first chain comprises the amino acid sequence set forth in SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 70, 72, 74 or 76 and / or the second chain comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 46, 48, 50, 71, 73, 75 or 77.

21. (a) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 19 and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 20; (b) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 70 and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 71; (c) the first chain comprises the amino acid sequence set forth in SEQ ID NO:72 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:73; (d) the first chain comprises the amino acid sequence set forth in SEQ ID NO:74 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:75; (e) the first chain comprises the amino acid sequence set forth in SEQ ID NO:21 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:22; (f) the first chain comprises the amino acid sequence set forth in SEQ ID NO:23 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:24; (g) the first chain comprises the amino acid sequence set forth in SEQ ID NO:25 and the second chain comprises the amino acid sequence set forth in SEQ ID NO:26; or (h) the first chain comprises the amino acid sequence set forth in SEQ ID NO: 76 and the second chain comprises the amino acid sequence set forth in SEQ ID NO: 77; 21. The polypeptide complex of claim 20.

22. A fusion protein comprising an IL-15 mutant peptide according to any one of claims 1 to 4 operably linked to a non-IL-15 moiety.

23. 23. The fusion protein of claim 22, wherein the non-IL-15 moiety is a moiety that can enhance the half-life of the fusion protein in vivo, such as a moiety selected from PEGs, lipids, Fc regions, human serum albumin (HSA), and anti-HSA moieties, and optionally the Fc region is human wild-type IgG1 Fc, IgG2 Fc, IgG3 Fc, IgG4 Fc, or a variant thereof.

24. 23. The fusion protein of claim 22, wherein the non-IL-15 moiety is an IL-15Rα domain, optionally comprising or consisting of a wild-type IL-15Rα protein, an IL-15Rα mutant, or any fragment thereof that retains IL-15 binding activity, such as a fragment as set forth in SEQ ID NO:

2.

25. The fusion protein of any one of claims 22 to 24, wherein the IL-15 mutant peptide is directly linked to the non-IL-15 moiety or indirectly linked to the non-IL-15 moiety via a linker, such as a peptide linker, comprising a GS linker, such as (G4S)n, with n=1 to 10.

26. An IL-15 mutant peptide having reduced potency in stimulating immune cell proliferation or activation compared to wild-type IL-15 and comprising an amino acid sequence that is truncated by 1 to 12 amino acids from the C-terminus, the N-terminus, or both the N- and C-terminus compared to SEQ ID NO:

1.

27. 27. The IL-15 mutant peptide of claim 26, comprising an amino acid sequence of any of SEQ ID NOs: 51 to 69, for example SEQ ID NO:

51.

28. A nucleic acid sequence encoding the first and / or second strand of a polypeptide complex according to any one of claims 1 to 21, a fusion protein according to any one of claims 22 to 25, or A nucleic acid molecule comprising an IL-15 mutant peptide according to any one of claims 26 to 27.

29. A vector comprising the nucleic acid molecule of claim 28.

30. 30. A host cell comprising the vector of claim 29 or the nucleic acid molecule of claim 28.

31. A pharmaceutical composition comprising a polypeptide complex according to any one of claims 1 to 21, a fusion protein according to any one of claims 22 to 25 or a nucleic acid molecule according to claim 28, and a pharma- ceutical acceptable carrier.

32. 26. An immunoconjugate comprising a polypeptide conjugate according to any one of claims 1 to 21 or a fusion protein according to any one of claims 22 to 25, conjugated to a moiety selected from a chemical (such as a cytotoxic agent, a cytostatic agent), a polypeptide, a carbohydrate, a lipid, a nucleic acid or a combination thereof.

33. A method for producing a polypeptide complex according to any one of claims 1 to 21 or a fusion protein according to any one of claims 22 to 25, comprising the steps of: - culturing a host cell containing a vector encoding one or both chains of said polypeptide complex or said fusion protein, and - isolating said polypeptide complex or said fusion protein from the host cell culture; The method comprising:

34. 32. A method for modulating an immune response in a subject, comprising administering to said subject a polypeptide complex according to any one of claims 1 to 21 or a pharmaceutical composition according to claim 31, optionally wherein said immune response is associated with NK cells or CD8+ T cells.

35. A method for treating or preventing cancer, an infectious disease, an immunodeficiency or lymphocytopenia in a subject, the method comprising administering to the subject an effective amount of a polypeptide complex of any one of claims 1 to 21 or a pharmaceutical composition of claim 31.

36. 35. The method of claim 34, further comprising administering an additional anti-tumor therapy, such as cell therapy, targeted therapy, chemotherapy, and gene therapy.

37. 29. A method for treating or preventing cancer, an infectious disease, an immunodeficiency or lymphocytopenia in a subject, comprising administering a cell therapy or gene therapy, wherein the cell therapy or gene therapy comprises delivering to the subject a nucleic acid molecule described in claim 28.

38. 38. The method of claim 36 or 37, wherein the cell therapy is selected from tumor infiltrating lymphocyte (TIL) therapy, T cell receptor (TCR) therapy, chimeric antigen receptor (CAR) T cell therapy, and NK cell therapy.

39. 38. The method of claim 36 or 37, wherein the gene therapy uses lentivirus, AAV, poxvirus, varicella zoster virus, oncolytic virus, or other RNA / DNA vectors for gene delivery.

40. 40. The method of any one of claims 35 to 39, wherein the cancer is selected from breast cancer, lung cancer (such as NSCLC), colon cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma (such as non-Hodgkin's lymphoma and diffuse large B-cell lymphoma), leukemia (such as chronic lymphocytic leukemia), and multiple myeloma.

41. The method according to any one of claims 35 to 39, wherein the infectious disease is caused by a viral, bacterial, fungal or parasitic infection.

42. Use of a polypeptide complex according to any one of claims 1 to 21, a fusion protein according to any one of claims 22 to 25 or a nucleic acid molecule according to claim 28 in the manufacture of a medicament for the prevention, treatment and / or management of cancer, an infectious disease, an immunodeficiency or lymphocytopenia.

43. A polypeptide complex according to any one of claims 1 to 21, a fusion protein according to any one of claims 22 to 25 or a nucleic acid molecule according to claim 28 for use in treating or preventing cancer, an infectious disease, an immunodeficiency or lymphocytopenia.

44. A kit comprising a container comprising a polypeptide complex according to any one of claims 1 to 21, a fusion protein according to any one of claims 22 to 25 or a nucleic acid molecule according to claim 28.

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