Improved stability IL-2 variant and composition thereof

Abstract

Provided in this disclosure are IL-2 variants, polypeptide conjugates containing IL-2 variants, compositions containing polypeptide conjugates, methods for producing them, and uses thereof. The IL-2 variants and polypeptide conjugates of this disclosure may be used as potent agents for the treatment of cancer, autoimmune diseases, and inflammatory diseases.

Description

[Technical Field]

[0001] cross reference This application claims priority to International Patent Application PCT / CN2023 / 084766, filed on 29 March 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing which is incorporated in its entirety herein by reference.

[0003] field This application generally relates to IL-2 variants, fusion proteins, and polypeptide complexes containing IL-2 variants, methods for preparing them, and their uses. [Background technology]

[0004] Interleukin-2 (IL-2) is a group of four α-helix bundle cytokines identified as growth factors for T cells, and is involved in the growth and proliferation of many immune cells [Morgan DA, Ruscetti FW, and Gallo R. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 1976; 193: 1007 - 1008]. IL-2 is primarily produced by activated T cells. The biological activity of IL-2 is mediated through three transmembrane receptor subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and the common gamma chain (CD132). NK cells and stationary T cells express IL-2Rβ and γ, while only regulatory T cells (Treg) and activated T cells express IL-2Rα in addition to IL-2Rβ and γ. IL-2Rα plays a role in promoting the delivery of IL-2 to the other two subunits, while IL-2Rβ / γ transmits activation signals via the STAT5-associated pathway. The IL-2Rα / β / γ trimers on Tregs and activated T cells are high-affinity receptors for IL-2 (K D about 10 -11It functions as M, and IL-2Rβ / γ (on NK cells and stationary T cells) is an intermediate affinity receptor (K D about 10 -9 M) [Takeshita T, Asao H, Ohtani K, et al. Cloning of the gamma chain of the human IL-2 receptor. Science 1992; 257: 379 - 382]. High-affinity receptors are important for capturing low concentrations of IL-2, making Tregs and activated T cells more sensitive to IL-2 and supporting their respective functions. In particular, in cancer immunotherapy, the binding of IL-2Rα to IL-2 plays a crucial role in programming stem-like T cells [Hashimoto M, Araki K, Cardenas MA, et al. PD-1 combination therapy with IL-2 modifies CD8+ T cell exhaustion program. Nature. 2022; 610:173-181].

[0005] IL-2 and its variants have been tested in various clinical trials. While IL-2 has shown promising efficacy in cancer immunotherapy, its toxicity profile has limited its effectiveness and use. Recombinant IL-2 has a short half-life and has shown serious side effects such as vasoleap syndrome (VLS). While IL-2 variants have been developed to mitigate these problems, they have presented challenges such as reduced efficacy and increased immunogenicity. Fusion proteins containing IL-2 or its variants, such as IL-2-Fc fusion proteins, are structurally fragile, unstable, and have poor pharmacokinetic profiles. These characteristics significantly limit their clinical potential.

[0006] Cancer immunotherapy using immune checkpoint inhibitors such as anti-PD(L)1 antibodies and other immunomodulatory drugs has made groundbreaking progress in recent years. However, a significant proportion of cancer patients remain resistant or refractory to existing cancer immunotherapies. As a cytokine that modulates T cells and NK cells, IL-2 may enhance responses induced by existing therapies such as therapeutic antibodies against checkpoints (PD1, PD-L1, CTLA4, LAG-3, etc.) or tumor-associated antigens (rituximab, trastozumab, daratumumab, etc.).

[0007] Treg cells play a crucial role in maintaining immune homeostasis and self-tolerance, and are critical to controlling the development of allergies and autoimmune diseases. IL-2 can induce Treg cell proliferation through signaling via its highly affinity IL-2 receptor. Therefore, expanding the Treg population with IL-2 therapy may balance and limit pathogenic T cells in inflammatory diseases. Currently, IL-2 therapy is being validated in clinical trials for inflammatory diseases such as graft-versus-host disease, type 1 diabetes, and systemic lupus erythematosus [Ye C, Brand D, and Zheng SG Targeting IL-2: an unexpected effect in treating immunological diseases. Sig Transduct Target Ther 2018]. Nevertheless, existing IL-2 molecules remain burdensome due to their rapid clearance and potential toxicity.

[0008] Therefore, therapeutic IL-2 requires further exploration and optimization to produce an ideal drug molecule with potent efficacy, in vivo stability, good pharmacokinetics, and limited toxicity. IL-2-based therapies have the potential to meet enormous medical needs in the fields of immuno-oncology and autoimmune disease treatment.

[0009] This disclosure provides reduced-potency, stabilized IL-2 variants and their IL-2 fusion proteins having improved pharmacokinetics, which may function as novel immunotherapeutic agents with improved therapeutic efficacy. [Overview of the Initiative]

[0010] These and other objectives are, in a broad sense, provided by this disclosure directed toward compounds, methods, compositions and manufactured articles that provide proteins with improved efficacy. The advantages provided by this disclosure are widely applicable in the fields of therapeutics and diagnostics and can be used in combination with antibodies that react with various targets.

[0011] The present invention is directed toward human IL-2 variants, fusion proteins, and polypeptide complexes containing IL-2 variants. IL-2 is manipulated herein by a unique approach to produce variants with better in vivo stability and pharmacokinetics, and with reduced affinity for IL-2Rβ / γc, IL-2Rα, or both, and the combined IL-2Rα / β / γc complex. Stabilization results in IL-2 variants gaining better thermal stability, better serum stability, extended in vivo half-life, and slower in vivo clearance, while maintaining limited toxicity. Despite being attenuated, IL-2 variants and polypeptide complexes containing IL-2 variants still retain binding ability to IL-2Rα, IL-2Rβ / γc, and the combined IL-2Rα / β / γc complex. IL-2 variants have a higher affinity for the IL-2Rα / β / γc complex than IL-2Rα or IL-2Rβ / γ and remain stable in vivo.

[0012] IL-2 variants and polypeptide conjugates containing IL-2 variants exhibited different potencies and toxicityes. Therefore, IL-2 variants and polypeptide conjugates containing IL-2 variants may function as novel immunotherapies for cancer and autoimmune diseases, either as monotherapy or in combination with other treatment options. Furthermore, IL-2 variants and polypeptide conjugates containing IL-2 variants may function as immunomodulators for autoimmune and other inflammatory diseases.

[0013] In some embodiments, the Disclosure provides IL-2 variants, wherein the IL-2 variant has a modified (more specifically, reduced) binding affinity to at least one of IL-2Rα, IL-2Rβ, the common γ chain, and the IL-2Rα / β / γc complex, or improved in vivo stability and pharmacokinetics, and has an amino acid sequence that, compared to the amino acid sequence described in Sequence ID No. 1, includes one or more mutations selected from C-terminal cleavage, positions 32, 129, 13, 18, 19, 20, 22, 28, 38, 42, 52, 71, 76, 78, 82, 84, 87, 88, 89, 91, 92, 94, 95, 110, 119, 122, 123, 125, and 126, and any combination thereof.

[0014] In some embodiments, the Disclosure provides compositions comprising a polypeptide complex or a nucleic acid molecule(s) encoding the polypeptide complex as an active ingredient, and an excipient, wherein the polypeptide complex comprises an interleukin-2 (IL-2) variant domain, a first dimerization domain and a second dimerization domain, the IL-2 variant domain being cleaved 1 to 20 amino acids from the C-terminus, and, in relation to Sequence ID No. 1, at positions 32, 129, 13, 18, 19, 20, 22, 28, 38, 42, 52, 71, 76, and 78. The polypeptide complex comprises an amino acid sequence having substitutions at one or more positions selected from positions 82, 84, 87, 88, 89, 91, 92, 94, 95, 110, 119, 122, 123, 125, and 126, wherein the first dimerization domain and the second dimerization domain associate to form a dimer, and the polypeptide complex has reduced binding affinity to at least one of the IL-2Rα, IL-2Rβ / γc, and IL-2Rα / β / γc complexes compared to other identical polypeptide complexes containing wild-type IL-2 instead of the IL-2 variant.

[0015] In some embodiments, the polypeptide complex or nucleic acid molecules encoding the polypeptide complex constitute less than 90% by weight, less than 80% by weight, less than 70% by weight, less than 60% by weight, or less than 50% by weight of the composition.

[0016] In some embodiments, the IL-2 variant includes a cleavage of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from the C-terminus, as per SEQ ID NO: 1.

[0017] In some embodiments, the IL-2 variant includes substitutions at one or more positions selected from positions 32, 28, 52, 76, 78, and 82, in combination with substitutions at one or more positions selected from positions 129, 110, and 122, in relation to SEQ ID NO: 1.

[0018] In some embodiments, the IL-2 variant, in combination with substitutions at one or more positions selected from positions 32, 42, and 129, in relation to SEQ ID NO:1, includes substitutions at one or more positions selected from positions 13, 84, 87, 88, 91, 92, 94, 119, and 123.

[0019] In some embodiments, the IL-2 variant, in combination with substitutions at one or more positions selected from positions 32, 42, and 129, in relation to SEQ ID NO:1, includes substitutions at one or more positions selected from positions 3, 18, 19, 20, 22, 71, 125, and 126.

[0020] In some embodiments, the IL-2 variant includes a substitution at position 32 selected from K32D, K32A, K32E, K32G, K32V, K32F, K32M, K32W, K32P, K32Q, K32I, K32S, K32T, K32N, K32R, K32H, K32L, and K32Y, preferably, the K residue is substituted by an amino acid residue having an opposite charge. Further or alternatively, the IL-2 variant includes a substitution at position 129 selected from I129L, I129A, I129E, I129G, I129V, I129F, I129M, I129W, I129P, I129Q, I129K, I129S, I129T, I129N, I129D, I129R, I129H, and I129Y.

[0021] In some embodiments, the IL-2 variant includes a substitution selected from any of the following: (a) A substitution at position 110 selected from E110R, E110A, E110K, E110G, E110V, E110F, E110M, E110W, E110P, E110Q, E110I, E110S, E110T, E110N, E110D, E110H, E110L, and E110Y; (b) Substitutions at position 52 selected from E52G, E52A, E52K, E52V, E52F, E52M, E52W, E52P, E52Q, E52I, E52S, E52T, E52N, E52D, E52R, E52H, E52L and E52Y; (c) Substitution at 76 positions selected from K76R, K76A, K76E, K76G, K76V, K76F, K76M, K76W, K76P, K76Q, K76I, K76S, K76T, K76N, K76D, K76H, K76L and K76Y; (d) Substitutions at 78 positions selected from F78G, F78A, F78K, F78V, F78E, F78M, F78W, F78P, F78Q, F78I, F78S, F78T, F78N, F78D, F78R, F78H, F78L and F78Y; (e) Substitutions at position 82 selected from P82Y, P82E, P82G, P82V, P82F, P82M, P82W, P82K, P82Q, P82I, P82S, P82T, P82N, P82D, P82R, P82H, P82L and P82A; (f) Substitutions at 28 positions selected from I28P, I28A, I28E, I28G, I28V, I28F, I28M, I28W, I28Q, I28K, I28S, I28T, I28N, I28D, I28R, I28H, I28L and I28Y; and (g) Substitutions at position 122 that are not selected from I122Y, I122E, I122G, I122V, I122F, I122M, I122W, I122P, I122Q, I122K, I122S, I122T, I122N, I122D, I122R, I122H, I122L and I122A; (h) Substitutions at position 13 selected from Q13P, Q13A, Q13E, Q13G, Q13V, Q13F, Q13M, Q13W, Q13I, Q13K, Q13S, Q13T, Q13N, Q13D, Q13R, Q13H, Q13L, and Q13Y; (i) Substitutions in 42 positions selected from F42P, F42A, F42E, F42G, F42V, F42Q, F42M, F42W, F42I, F42K, F42S, F42T, F42N, F42D, F42R, F42H, F42L and F42Y; (j) Substitutions at 84 positions selected from D84P, D84A, D84E, D84G, D84V, D84F, D84M, D84W, D84I, D84K, D84S, D84T, D84N, D84Q, D84R, D84H, D84L and D84Y; (k) Substitutions at position 88 selected from N88P, N88A, N88E, N88G, N88V, N88F, N88M, N88W, N88I, N88K, N88S, N88T, N88Q, N88D, N88R, N88H, N88L, and N88Y; (l) Substitution at position 91 selected from V91P, V91A, V91E, V91G, V91Q, V91F, V91M, V91W, V91I, V91K, V91S, V91T, V91N, V91D, V91R, V91H, V91L, and V91Y; (m) Substituents at position 92 selected from I92Y, I92E, I92G, I92V, I92F, I92M, I92W, I92P, I92Q, I92K, I92S, I92T, I92N, I92D, I92R, I92H, I92L and I92A; (n) Substitution at 94 positions selected from L94P, L94A, L94E, L94G, L94V, L94F, L94M, L94W, L94I, L94K, L94S, L94T, L94N, L94D, L94R, L94H, L94Q and L94Y; (o) Substitution at position 95 selected from E95G, E95A, E95K, E95V, E95F, E95M, E95W, E95P, E95Q, E95I, E95S, E95T, E95N, E95D, E95R, E95H, E95L and E95Y; (p) A 119-position substituent selected from N119P, N119A, N119E, N119G, N119V, N119F, N119M, N119W, N119I, N119K, N119S, N119T, N119Q, N119D, N119R, N119H, N119L, and N119Y; (q) A substitution at position 123 selected from T123G, T123A, T123K, T123V, T123F, T123M, T123W, T123P, T123Q, T123I, T123S, T123E, T123N, T123D, T123R, T123H, T123L and T123Y.

[0022] In some embodiments, the IL-2 variant comprises or consists of the amino acid sequences shown in any of SEQ ID NOs: 2-13, 79-103, and 111-114.

[0023] The polypeptide complexes disclosed herein may be IL-2 bivalent fusion proteins. In some embodiments, the polypeptide complex comprises two IL-2 variants in two chains, each chain containing one IL-2 variant from the N-terminus to the C-terminus operably linked to one dimerization domain.

[0024] The polypeptide complexes disclosed herein may be bifunctional fusion proteins. In some embodiments, the polypeptide complex further comprises one or more antigen-binding moieties. The antigen-binding moieties may be in the form of Fab, Fab', VHH, or scFv.

[0025] In some embodiments, the polypeptide complex comprises one IL-2 variant in Fab form and one antigen-binding moiety, and the polypeptide complex comprises two heavy chains and one light chain, where N-terminus to C-terminus: The first heavy chain comprises an IL-2 variant operably linked to the first dimerization domain; The second heavy chain comprises Fab's heavy chain operably linked to the second dimerization domain; The light chain includes the Fab light chain.

[0026] In some embodiments, the polypeptide complex comprises one IL-2 variant of the VHH form and two antigen-binding moieties, and the polypeptide complex comprises two chains, where from the N-terminus to the C-terminus: The first chain contains an IL-2 variant operably linked to the first dimerization domain; The second chain contains two VHHs in tandem, operably linked to the second dimerization domain.

[0027] In some embodiments, the polypeptide complex comprises one IL-2 variant in VHH form and one antigen-binding moiety, and the polypeptide complex comprises two chains, where N-terminus to C-terminus: The first chain contains an IL-2 variant operably linked to the first dimerization domain; The second chain contains a VHH operably linked to the second dimerization domain.

[0028] In some embodiments, the polypeptide complex comprises two IL-2 variants and two antigen-binding moieties of Fab form, and the polypeptide complex comprises two heavy chains and two light chains, where from the N-terminus to the C-terminus: The heavy chain comprises an IL-2 variant operably coupled to a first or second dimerization domain, which is operably coupled to the heavy chain of Fab; The light chain includes the Fab light chain.

[0029] In some embodiments, the polypeptide complex comprises two IL-2 variants of VHH or scFv form and two antigen-binding moieties, and the polypeptide complex comprises two chains, from the N-terminus to the C-terminus: Each chain contains an IL-2 variant operably linked to a first or second dimerization domain operably linked to VHH or scFv.

[0030] In some embodiments, the antigen-binding moiety is a tumor-associated antigen (TAA), I / O checkpoint, tumor microenvironment target, autoimmune-related target, and inflammatory disease-related target, including, but not limited to, PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, TIM-4, 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, BTN1A 1, BTN3A1, CD70, RANKL, CSF1R, CSF2RB, TNFRSF1 / 1a / 1b, BDCA2, BTLA, C5aR, NKG2A, NKG2D, NKp30 , NKp46, CD16a, CD56, CD166, FCGR3, CD2, neurophilin-1, CCR8, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, GCGR, 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, de2-7EGFR, EGFRvIII, PSMA, PSCA, PSA, TAG-72, SEZ6, SEZ6L, SEZ6L2, SEMA4D, DLL3, GD2, GPC3, KLB, KLRB1, KLRG1, GPC1, PCSK9, EpCAM, p-cadherin, Calzin 6, Calzin 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 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, CD4 6, 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, GloboH, IGF-1R, TEM1, TEM8, TAX1BP3, ADAM-9, ENPP3, EphA2, E PhA3, FcRH5, NaPi3b, TWEAK, DLK1, SORT1, SSTR2, STEAP1, CD25, CD39, GARP, LRRC33, LAIR1, LAMP3, LAP, LEPR, LILRB1, LILRB2, LILRB4, RAGE, FGL1, TPBG, PDGFRB, TGFBR2, CEACAM1, CEACAM5, CEACAM6, carcinoembryonic antigen (CEA), ICAM1, A33, CAMPATH-1 (CDw52), 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, CD37, CD38, CD43, CD52, CD55, CD94, CD99, CD7, CD71, CD73, CD74, CD79A, CD79B, CD229, CD132, CD 133, G250, CSF1R (CD115), HLA-DR, 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-4R A, IFNAR, ActRIIb, IL-7R, SLAMF7, Fms-like tyrosine kinase 3 (FLT-3, CD135), GFRA1, BTLA, GloboH, CSF2RB, chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), ITGA4, Clec5a, Clec7a, Clec9a, Clec12a, CLEC14, CD205, CD206, CD200R1, It specifically binds to antigens selected from CD228, CD229, CD40, CD40L, FcRn, TLR8, TLR9, TNFR2, LTBR, ​​CD44, CD93, PDGF, PDGFR-α (CD140a), PDGFR-β (CD140b), CD146, CD147, CRTH2, TNF-α, TGF-β, IL1RAcP, TSLP, DR5, ST2, fibroblast-activating protein (FAP), CDCP1, Derlin1, Tenacin, frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), endoglin, and Tie2.

[0031] In some embodiments, the antigen-binding moiety is a PD-1-binding Fab or a PD-1-binding VHH. The polypeptide complex thus formed may also be designated as a PD-1 / IL-2 fusion protein.

[0032] In some embodiments, the first dimerization domain is one chain of the immunoglobulin Fc region, the second dimerization domain is the other chain of the immunoglobulin Fc region, and optionally, the Fc region further comprises a partial or whole hinge region.

[0033] In some embodiments, the Fc region is an IgG4, IgG1, IgG2, or IgG3 Fc region and optionally includes one or more substitutions compared to wild-type human Fc to promote heterodimerization or homodimerization, extend half-life, or remove N-glycosylation.

[0034] In some embodiments, the Fc region is (a) Human IgG1 Fc regions that have been optionally modified to include one or more of the following: L234A / L235A mutations, M252Y / S254T / T256E mutations, G236R / L328R mutations, and "knob-into-hole" structures; (b) Human IgG4 Fc regions that have been optionally modified to include one or more of the following: S228P mutation, F234A / L235A mutation, M252Y / S254T / T256E mutation, and a "knob-into-hole" structure. Selected from.

[0035] In some embodiments, the IL-2 variant and / or antigen-binding moiety is operably linked to the Fc region via a linker, which is optionally a GS linker such as a (G4S)n linker, where n is an integer ≥ 0, such as 0-30, 0-20, 0-15, 0-10, and 0-5.

[0036] In some embodiments, the polypeptide complex has improved stability compared to other identical polypeptide complexes containing wild-type IL-2 instead of IL-2, where stability is one or more selected from heat resistance (e.g., measured by DLS), serum stability, extended serum half-life (e.g., by pharmacokinetic analysis), and structural stability.

[0037] In some embodiments, the polypeptide complex comprises any of the amino acid sequences of SEQ ID NOs: 32, 29, 30, and 31.

[0038] In some embodiments, the polypeptide complex is as follows: A first heavy chain containing the amino acid sequence of any of SEQ ID NOs. 52-56; a second heavy chain containing the amino acid sequence of SEQ ID NO. 57; and a light chain containing the amino acid sequence of SEQ ID NO. 58. Includes.

[0039] In some embodiments, the polypeptide complex is The first chain contains the amino acid sequence of one of the sequence numbers 59-74, 76-78, and 104-110; the second chain contains the amino acid sequence of sequence number 75. Includes.

[0040] In some embodiments, the polypeptide complex is The first heavy chain containing any of the amino acid sequences from sequence numbers 17 to 28; A second heavy chain containing the amino acid sequence of SEQ ID NO: 33; and Light chain containing the amino acid sequence of SEQ ID NO: 34 Includes.

[0041] In some embodiments where the polypeptide complex is a homodimer containing two chains, the composition includes a nucleic acid molecule encoding one chain of the polypeptide complex as an active ingredient. Specifically, the nucleic acid molecule may encode any of the amino acid sequences of SEQ ID NOs: 32, 29, 30, and 31.

[0042] In some embodiments, where the polypeptide complex is a heterodimer comprising two heavy chains and one light chain, the composition includes three nucleic acid molecules as active ingredients, each encoding one of the chains of the polypeptide complex. Specifically, the nucleic acid molecules may each encode one of the amino acid sequences of SEQ ID NOs: 19-28, one of SEQ ID NOs: 33, and one of SEQ ID NOs: 34.

[0043] In some embodiments, where the polypeptide complex is a heterodimer comprising two heavy chains and one light chain, the composition includes three nucleic acid molecules as active ingredients, each encoding one of the chains of the polypeptide complex. Specifically, the nucleic acid molecules may each encode one of the amino acid sequences of SEQ ID NOs. 52-56, one of SEQ ID NOs. 57, and one of SEQ ID NOs. 58.

[0044] In some embodiments, where the polypeptide complex is a heterodimer comprising two heavy chains and one light chain, the composition includes three nucleic acid molecules as active ingredients, each encoding one of the chains of the polypeptide complex. Specifically, the nucleic acid molecules may each encode one of the amino acid sequences of SEQ ID NOs. 59-74, 76-78, 104-110, or the amino acid sequence of SEQ ID NO. 75.

[0045] In some embodiments, the Disclosure provides a polypeptide complex comprising an interleukin-2 (IL-2) variant domain, a first dimerization domain, and a second dimerization domain, wherein the IL-2 variant domain comprises any of the amino acid sequences described in SEQ ID NOs. 81-103 and 112-114.

[0046] In some embodiments, the polypeptide complex comprises one IL-2 variant of the VHH form and two antigen-binding moieties, where the polypeptide complex comprises two chains from the N-terminus to the C-terminus: The first chain contains an IL-2 variant operably linked to the first dimerization domain; the second chain contains two tandem VHHs operably linked to the second dimerization domain.

[0047] In some embodiments, the polypeptide complex comprises a first chain containing any of the amino acid sequences of SEQ ID NOs. 59-74, 76-78, and 104-110; and a second chain containing the amino acid sequence of SEQ ID NO. 75.

[0048] In some embodiments, the disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding polypeptide complexes disclosed herein.

[0049] In some embodiments, the present disclosure provides a vector or host cell comprising nucleic acid molecules disclosed herein.

[0050] In some embodiments, the present disclosure provides methods for improving the stability and / or pharmacokinetic properties of IL-2 polypeptides compared to wild-type IL-2 (preferably also attenuating the binding of IL-2 polypeptides to IL-2Rβ / γc, IL-2Rα, or both, and the combined IL-2Rα / β / γc complex), (a) The process of introducing a substitution at one or more positions selected from positions 32, 129, 13, 18, 19, 20, 22, 28, 38, 42, 52, 71, 76, 78, 82, 84, 87, 88, 89, 91, 92, 94, 95, 110, 119, 122, 123, 125, and 126 of the amino acid sequence of SEQ ID NO: 1, and cleaving 1 to 20 amino acids from the C-terminus; (b) optionally, a step of fusing the IL-2 polypeptide to a non-IL-2 moiety that extends its half-life. Includes.

[0051] In some embodiments, the substitution at position 32 is selected from K32D, K32A, K32E, K32G, K32V, K32F, K32M, K32W, K32P, K32Q, K32I, K32S, K32T, K32N, K32R, K32H, K32L, and K32Y, and preferably the K residue is substituted with an amino acid residue having the opposite charge.

[0052] In some embodiments, the method, as related to SEQ ID NO: 1, involves cleaving 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) from the C-terminus.

[0053] In some embodiments, the method further includes introducing substitution at position 129, selected from I129L, I129A, I129E, I129G, I129V, I129F, I129M, I129W, I129P, I129Q, I129K, I129S, I129T, I129N, I129D, I129R, I129H, and I129Y.

[0054] In some embodiments, the non-IL-2 moiety is selected from PEG, lipids, immunoglobulin Fc regions, human serum albumin (HSA), and anti-HSA moieties.

[0055] In some embodiments, stability is one or more selected from heat resistance, serum stability, extended serum half-life, and structural stability, and pharmacokinetic properties include in vivo serum half-life and clearance rate.

[0056] In some embodiments, the non-IL-2 portion further includes an antigen-binding portion.

[0057] In some embodiments, the methods disclosed herein provide IL-2 comprising a polypeptide complex containing any of the amino acid sequences of SEQ ID NOs. 32, 29, 30, and 31.

[0058] In some embodiments, the methods disclosed herein yield IL-2 comprising a polypeptide complex comprising two heavy chains and one light chain, wherein the first heavy chain comprises any of the amino acid sequences of SEQ ID NOs. 19 to 28; the second heavy chain comprises the amino acid sequence of SEQ ID NOs. 33; and the light chain comprises the amino acid sequence of SEQ ID NOs. 34.

[0059] In some embodiments, the methods disclosed herein yield IL-2 comprising a polypeptide complex comprising two heavy chains and one light chain, wherein the first heavy chain comprises any of the amino acid sequences of SEQ ID NOs. 52 to 56; the second heavy chain comprises the amino acid sequence of SEQ ID NO. 57; and the light chain comprises the amino acid sequence of SEQ ID NO. 58.

[0060] In some embodiments, the methods disclosed herein yield IL-2 comprising a polypeptide complex comprising two chains, wherein the first chain comprises any of the amino acid sequences of SEQ ID NOs. 59-74, 76-78, and 104-110; and the second chain comprises the amino acid sequence of SEQ ID NO. 75.

[0061] In some embodiments, the methods disclosed herein yield IL-2 comprising a polypeptide complex comprising two chains, where the first chain comprises any of the amino acid sequences of SEQ ID NOs. 59-74, 76-78, or 104-110; and the second chain comprises a heavy chain of an anti-PD-1 antibody. In some embodiments, IL-2 is an IL-2 variant disclosed herein.

[0062] Preferably, a polypeptide complex as disclosed herein includes one or more of the following properties: (a) Improved stability compared to other identical polypeptide complexes containing wild-type IL-2 instead of IL-2, where stability is one or more selected from heat resistance (e.g., measured by DLS), serum stability, extended serum half-life (e.g., by pharmacokinetic analysis), and structural stability; (b) Reduced binding affinity to at least one of the IL-2Rα, IL-2Rβ / γc, and IL-2Rα / β / γc complexes compared to other identical polypeptide complexes containing wild-type IL-2 instead of the IL-2 variant; and (c) Reduced activity compared to other identical polypeptide complexes containing wild-type IL-2 instead of IL-2.

[0063] In some embodiments, the Disclosure provides methods for modulating an immune response in a subject, comprising administering a composition disclosed herein to the subject, optionally, the immune response being NK cell, CD8+ cell, or CD4+ T cell (particularly Treg) related.

[0064] In some embodiments, the present disclosure provides a method for treating or preventing cancer in a subject, which includes administering an effective amount of a composition such as those disclosed herein to the subject.

[0065] In some embodiments, the method further includes administering additional antitumor therapies, such as cellular immunotherapy, targeted therapy, chemotherapy, and gene therapy (e.g., gene therapy using lentivirus, AAV, poxvirus, herpes zoster virus, oncolytic virus, or other RNA / DNA vectors).

[0066] In some embodiments, the cancer is selected from colon cancer, breast cancer, lung cancer (such as NSCLC), ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma (such as non-Hodgkin lymphoma and diffuse large B-cell lymphoma), leukemia (such as chronic lymphocytic leukemia), and multiple myeloma, with colon cancer being an optional choice. In some embodiments, the cancer is PD-1-related cancer.

[0067] In some embodiments, the Disclosure provides a method for treating or preventing an autoimmune or inflammatory disease in a subject, which includes administering an effective amount of a composition as disclosed herein to the subject.

[0068] In some embodiments, the autoimmune or inflammatory disease is selected from inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, aplastic anemia, celiac disease, type 1 diabetes, Graves' disease, psoriasis, and scleroderma.

[0069] In some embodiments, the Disclosure provides the use of compositions such as those disclosed herein in the manufacture of pharmaceuticals for the treatment or prevention of cancer, autoimmune diseases, or inflammatory diseases.

[0070] In some embodiments, the Disclosure provides the use of compositions such as those disclosed herein for use in treating or preventing cancer, autoimmune diseases, or inflammatory diseases.

[0071] In some embodiments, the present disclosure provides a kit comprising a container containing a composition such as those disclosed herein.

[0072] The above is a summary and therefore inevitably includes simplifications, generalizations, and omissions of details; as a result, those skilled in the art will understand that the summary is illustrative only and not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions and / or devices and / or other subjects described herein will become apparent in the teachings provided herein. [Brief explanation of the drawing]

[0073] [Figure 1] Figure 1 provides a schematic description of various fusion protein and polypeptide complex forms containing wild-type IL-2 or IL-2 variants according to several embodiments of the present disclosure. Conical shapes represent the IL-2 portion, ladder shapes represent the hinge region, and elliptical shapes represent the antigen-binding portion and Fc region. (a,b) Both one IL-2 site and one antigen site (a,Fab; b,VHH) at the N-terminus; (c) Two IL-2 sites at the N-terminus; (d,e,f) Two IL-2 sites at the N-terminus and two antigen sites at the C-terminus (d,Fab; e,VHH; f,scFv); (g) Both one IL-2 site and two VHHs at the N-terminus. The F114 form contains two Fabs at the N-terminus and one IL-2 at the C-terminus. These forms can accommodate different antigen-binding sites such as Fabs, VHH, and scFv, which contain variable regions targeting different epitopes / antigens. [Figure 2] Figure 2 shows that IL-2 variants exhibited different efficacy in primary human CD8+ T cells determined by the STAT5 phosphorylation assay. [Figure 3] Figure 3 shows IL-2 variants that exhibited different efficacy in activated CD8+ T cells (a) and primary human CD8+ T cells (b) as determined by the STAT5 phosphorylation assay. [Figure 4] Figure 4 shows the serum stability of the divalent IL-2 variants Z20-1(a), BMK8(b), Z20-4(c), and Z20-5(d), which exhibited different potencies in human activated CD8+ T cells, as determined by the STAT5 phosphorylation assay. [Figure 5] Figure 5 shows the serum concentration profiles of Z20-1, Z20-5, and BMK7 in C57BL / 6 mice, as detected by Fc-Fc ELISA. [Figure 6] Figure 6 shows a model of the interaction between the receptor and T2U0.E44-49(a) and between the receptor and Z20-5(b). [Figure 7] Figure 7 shows the change in tumor volume in an MC38 colon cancer model after treatment with an IL-2 variant. [Figure 8] Figure 8 shows IHC in the lungs of an MC38 colon cancer model after IL-2 variant administration, with arrows indicating inflammatory cell infiltration. [Figure 9] Figure 9 shows that the PD-1 / IL-2 fusion protein exhibited similar binding affinity to the CHO-PD1 modified cell line. [Figure 10] Figure 10 shows that the PD-1 / IL-2 fusion protein exhibited different efficacy in activated CD8+ T cells (a) and primary human CD8+ T cells (b) as determined by the STAT5 phosphorylation assay. [Figure 11] Figure 11 shows the PD-1 / IL-2 fusion protein that exhibited different efficacy in activated CD8+ T cells (a) and primary human CD8+ T cells (b), as determined by the STAT5 phosphorylation assay. [Figure 12]Figure 12 shows that PD-1 / IL-2 fusion proteins exhibited different potencies in activated CD8+T as determined by the STAT5 phosphorylation assay. [Figure 13] Figure 13 shows the change in tumor volume of a CT-26 colon cancer model after treatment with PD-1 / IL-2 fusion protein. [Modes for carrying out the invention]

[0074] While this disclosure can be embodied in many different forms, what is disclosed herein are specific exemplary embodiments illustrating the principles of this disclosure. It should be emphasized that this disclosure is not limited to the specific embodiments shown. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein.

[0075] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" shall include multiple reference words unless explicitly indicated by context. Thus, for example, a reference to "protein" shall include multiple proteins, and a reference to "cell" shall include a mixture of cells, etc. In this application, the use of "or" shall mean "and / or" unless otherwise stated. Furthermore, the use of the term "contains," as well as other forms such as "contains" and "contained," is not limiting. Furthermore, the scope described herein and in the appended claims shall include both endpoints and all points between the endpoints.

[0076] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids described herein are well known and commonly used in the art. The methods and techniques described herein are generally carried out in accordance with the prior art known herein and, unless otherwise noted, are as described in the various general and more specific literature referenced and discussed throughout this specification. For example, Abbas et al., Cellular and Molecular Immunology, 6 th See also: 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). For example, the nomenclature, experimental procedures, and techniques used in relation to analytical chemistry, organic synthesis chemistry, and medicinal chemistry described herein are well known and commonly used in the art.

[0077] definition To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0078] The terms “IL-2” or “interleukin-2,” as used herein, are intended to encompass, for example, 1) the native, unprocessed IL-2 molecule, the “full-length” IL-2 protein, or naturally occurring variants of IL-2; 2) any form of IL-2 resulting from intracellular processing; or 3) any form of IL-2, such as full-length, fragment (e.g., cleaved form), or modified form. IL-2 is a cytokine primarily produced by activated T cells and contributes to the proliferation and activation of various immune cells. Mature human IL-2 has a molecular weight of approximately 15 kDa (133 amino acids, as shown in SEQ ID NO: 1) and has a 4-α-helix bundle structure. As used herein, “IL-2” may be either wild-type IL-2 or an IL-2 variant, and “IL-2 domain” includes wild-type IL-2 or an IL-2 variant peptide.

[0079] The term “variant” refers to a biologically active polypeptide containing one or more amino acid mutations in the native protein sequence of a polypeptide or protein. Optionally, one or more amino acid mutations include amino acid substitutions, deletions, and / or insertions at specific positions in the amino acid sequence. A variant has at least about 80%, preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% (e.g., at least 96%, 97%, 98%, or 99% or more) amino acid sequence identity with the corresponding native sequence polypeptide. Such variants include, for example, polypeptides in which one or more amino acid residues (naturally occurring and / or unnaturally occurring amino acids) are added to or deleted from the N-terminus and / or C-terminus of the polypeptide. Variants also include typically biologically active polypeptide fragments of the native sequence (e.g., partial sequences, cleavages, etc.).

[0080] As used herein, the term "IL-2 variant" includes all proteins produced by making some modification to wild-type IL-2 and having the same functions as wild-type IL-2, such as specific binding to IL-R2α, IL-2Rβ / γc, and / or the combined IL-R2α / β / γc complex (preferably with binding affinity attenuated as compared to wild-type IL-2), activation of immune cells such as T cells (e.g., CD8+ T cells, NK cells, and Treg cells), phosphorylation of STAT5, etc. Examples of variants include IL-2 variants in which wt IL-2 is modified by amino acid modification (e.g., deletion, substitution or addition), IL-2 variants in which wt IL-2 is modified by sugar modification, and IL-2 variants in which wt IL-2 is modified by chemical modification. Depending on the context, the term "IL-2 variant" may also refer to polypeptide complexes containing an IL-2 variant, such as the E44, Z20 or Z73 forms as shown in FIG. 1.

[0081] As used herein, the term "antibody" or "Ab" is used in the broadest sense to encompass various antibody structures, including polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and polypeptide complexes as disclosed herein. Native intact antibodies are generally Y-shaped tetrameric proteins composed of two heavy (H) and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. The light chains of antibodies are classified into κ light chains and λ light chains. Heavy chains are classified into μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain further contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1, C H 2, C H 3). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (CL ) consists of V H and V L The region is further divided into hyper-variable regions (called complementary decision regions (CDRs)), with relatively conservative regions (called framework regions (FRs)) in between. Each V H and V L It consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, arranged from the N-terminus to the C-terminus. The variable region (V) of each heavy / light chain pair. H and V L These each form an antigen-binding moiety. The distribution of amino acids in various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883; Contact numbering, AbM numbering, or IMGT numbering. Antibodies may be different antibody isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. In a broader sense, polypeptide complexes containing antigen-binding moieties, such as those disclosed herein, also belong to the category of antibodies.

[0082] As used herein, the term “antigen-binding moiety” refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or another antibody fragment that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, VHH, bivalent domain antibodies, and TCRs. As used herein, the term “Fab” means a polypeptide containing VH, CH1, VL, and CL immunoglobulin domains. An antigen-binding moiety can bind to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may contain one or more CDRs from a particular human antibody grafted onto a framework region from one or more different human antibodies. More detailed information on the antigen-binding moiety can be found in Spiess et al., Molecular Immunology, 67(2), pp.95-106 (2015) and Brinkman et al., mAbs, 9(2), pp.182-212 (2017), which are incorporated herein by reference.

[0083] The term “hinge region,” as used herein, has the same meaning as it does with respect to antibodies and refers to a short sequence of the heavy chain (H) of an immunoglobulin that links the Fab (fragment antigen-binding) region and the Fc (fragment crystallizable) region. The hinge region may be a complete or partial hinge region. The hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids, thereby resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. In some embodiments, the hinge region contained in the polypeptide complex disclosed herein is a C-terminal or N-terminal cleavage hinge region. It will be understood that the hinge region is a specific linker and may preferably be substituted with other linker sequences when constructing the fusion protein or polypeptide complex herein.

[0084] The term "Fc," as used herein, has the same meaning as when used in relation to antibodies and refers to a portion of an antibody that includes the second (CH2) and third (CH3) constant regions of the first heavy chain, which are bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region of an antibody performs various effector functions, such as ADCC and CDC, but generally does not function in antigen binding. In this disclosure, the term "Fc" includes both wild-type Fc, Fc variants, and grafted Fc.

[0085] As used herein, the term “modification” with respect to amino acid residues / positions refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, the change resulting from a sequence alteration, including the amino acid residue / position. Typical modifications include, for example, the substitution of a residue (or position) with another amino acid (e.g., a conservative or non-conservative substitution), the insertion of one or more amino acids adjacent to the residue / position, and the deletion of the residue / position. “Amino acid substitution” or its variations refers to the substitution of an existing amino acid residue in a given (start) amino acid sequence with a different amino acid residue. Generally, this modification results in a change in at least one physicobiochemical activity of the mutant polypeptide compared to the polypeptide containing the starting (or “wild-type”) amino acid sequence. For example, in an IL-2 variant, the altered physicobiochemical activity may be 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 using “+” or “ / ” between each substitution.

[0086] As used herein, the term “fusion protein” refers to a chimeric polypeptide comprising an operably linked IL-2 moiety and a non-IL-2 moiety (and optionally more moieties), where each moiety is a polypeptide having 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, such as binding to a target antigen or catalysis of a reaction. The two moieties may be directly linked by a single peptide bond or linked via a peptide linker comprising one or more amino acid residues. Generally, the two moieties and the linker are in a leading frame relative to each other. Preferably, the non-IL-2 moiety can extend the half-life of IL-2 in vivo. In some embodiments, the non-IL-2 moiety is an immunoglobulin constant region including a hinge region and an Fc region, and thus the resulting fusion protein is referred to as an IL-2 / Fc fusion protein. IL-2 / Fc fusion proteins are generally dimers, containing an IL-2 moiety operably linked to the Fc region (optionally linked via a hinge region).

[0087] As used herein, the term “polypeptide complex” refers to a polypeptide complex (i.e., an IL-2 / Fc polypeptide complex) that contains an IL-2 domain operably linked to a dimerizing domain such as an immunoglobulin Fc region. Such polypeptide complexes are structurally similar to conventional antibodies in that the IL-2 domains replace the Fab or VHH of conventional antibodies, which are located on two antigen-binding arms. As used herein, the term “divalent” means that there are two IL-2 domains in the polypeptide complex or fusion protein. As used herein, the term “monovalent” means that there is one IL-2 domain in the polypeptide complex or fusion protein.

[0088] As used herein, the term “operably linked” means a juxtaposition of two or more biological sequences of interest, with or without spacers or linkers, such that they are related in a way that enables them to function in the manner intended. When used in reference to polypeptides, it is intended to mean that polypeptide sequences are linked in such a way that the linked product has the intended biological function. For example, an antigen-binding moiety may be operably linked to an Fc region to provide a stable product having antigen-binding activity. “Operatably linked” means that the antigen-binding moiety may be directly linked to the Fc region, or more preferably, indirectly linked to the Fc region via a linker sequence such as a hinge region, as long as the two parts function properly. The term can also be used in reference to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, or silencer sequence), it is intended to mean that the polynucleotide sequence is linked in such a way that it enables the regulated expression of the polypeptide from the polynucleotide.

[0089] As used herein, the term "gene therapy" refers to a treatment aimed at treating a disease by replacing, inactivating, or introducing a gene into cells, either in vivo or ex vivo. In some embodiments, the DNA or RNA sequences encoding IL-2 variants disclosed herein are administered to a subject. Gene therapy includes the use of lentiviruses, AAVs, poxviruses, herpes zoster viruses, oncolytic viruses, and other RNA / DNA vectors to deliver the DNA or RNA encoding IL-2 variants.

[0090] As used herein, the term “cell therapy” refers to a therapy aimed at treating a disease by restoring or altering a specific set of cells, or by using cells to deliver therapy throughout the body. In cell therapy, cells are cultured or modified outside the body before being injected into the patient. Cells can be derived from the patient (autologous cells) or from a donor (allogeneic cells). Current cell therapies include the use of CAR-T, TCR-T, TIL, CAR-NK, CAR-γδT, CAR-macrophages, and other artificial immune cells. In some embodiments herein, the therapeutic method further includes cell therapy.

[0091] The term "EC" as used herein 50 The term "half-maximal effective concentration" also refers to the concentration of a drug, antibody, or toxin that induces an intermediate response between baseline and maximum after a specified exposure time.

[0092] As used herein, the term “isolated” refers to a state obtained from its natural state by artificial means. When an “isolated” substance or component exists in nature, it may be in an altered natural environment, isolated from its natural environment, or both. For example, if unisolated polynucleotides or polypeptides exist naturally within the body of an animal, a highly purified version of the same polynucleotide or polypeptide isolated from such a natural state is referred to as an isolated polynucleotide or polypeptide. The term “isolated” does not include mixed artificial or synthetic substances or other impurities that do not affect the activity of the isolated substance.

[0093] As used herein, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it enables the expression of a protein encoded by the polynucleotides inserted therein. A vector can express a supported genetic material element in a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-derived artificial chromosomes (PACs); phages such as lambda phages and M13 phages; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The vector includes, but is not limited to, a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, and a reporter gene, and may also include multiple elements for controlling expression. Furthermore, the vector may include an origin of replication.

[0094] The term “host cell,” as used herein, refers to a cell line that can be manipulated to produce the protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissues or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. This term encompasses not only specific target cells but also their offspring. Such offspring may not be identical to the parent cells because certain modifications may occur in the progeny due to mutations or environmental influences, but they are still included in the scope of the term “host cell.”

[0095] As used herein, the term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. “Identity Percentage” means the percentage of identical residues between amino acids or nucleotides in the molecules being compared, calculated based on the size of the smallest of the molecules being compared. For these calculations, any alignment gaps are preferably addressed by a specific mathematical model or computer program (i.e., “algorithm”). Methods that may 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.

[0096] As used herein, the term “transfection” refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, chemical and physical methods such as lipid transfection and electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, 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; and Chu et al., 1981, Gene 13:197. In certain embodiments of this disclosure, vectors encoding heavy and / or light chains of polypeptide complexes were transfected into 293F cells.

[0097] As used herein, the term “fluorescence-activated cell sorting” or “FACS” refers to a specific type of flow cytometry. This 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 2017-11-09). Apparatus for performing FACS is known to those skilled in the art and is generally commercially available. Examples of such apparatus include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson, Inc. (Foster City, California), the Epics C from Coulter Epics Division, Inc. (Hyalia, Florida), and the MoFlo from Cytomation, Inc. (Colorado Springs, Colorado).

[0098] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, macrophages) enables these cytotoxic effector cells to specifically bind to target cells containing antigens, and subsequently kill the target cells through cytotoxins. Antibodies “arm” the cytotoxic cells and are absolutely necessary for such killing. Primary cells that mediate ADCC, such as NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a target molecule may be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0099] The term "subject" includes any human or non-human animal, preferably a human.

[0100] As used herein, the term "cancer" refers to solid tumors and non-solid tumors such as leukemia, which involve the growth, proliferation, or metastasis of any tumor or malignant cell that can initiate a disease.

[0101] As used herein, the term “autoimmune disease” refers to any condition that may result from an abnormal immune response against functional parts of the body, such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and multiple sclerosis.

[0102] In this specification, the terms “treatment,” “to treat,” or “treated” as used in the context of treating a disease generally relate to treatments and therapies, whether in humans or animals, that achieve some desired therapeutic effect, such as inhibition of disease progression, and include reduction in the rate of disease progression, cessation of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a preventive measure (i.e., prevention, prophylaxis) is also included. In the case of cancer, “to treat” may mean inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or a combination thereof. In the case of tumors, “treatment” includes the removal of all or part of a tumor, inhibition or delay of tumor growth and metastasis, prevention or delay of tumor development, or a combination thereof.

[0103] As used herein, the term "effective dose" refers to an amount of an active compound, or a material, composition, or dosage form containing an active compound, that is effective in producing some desired therapeutic effect in proportion to a reasonable benefit-risk ratio when administered according to a desired therapeutic regimen. For example, when used in connection with the treatment of a disease or condition such as cancer, "effective dose" refers to an amount or concentration of the active agent, drug, or antibody or antigen-binding moiety that is effective in treating the disease or condition.

[0104] In this specification, the terms “prevent,” “prevent,” or “prevention” as used in reference to specific disease conditions in mammals mean preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms.

[0105] As used herein, the term “pharmaceutically acceptable” means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0106] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmaceutically and / or physiologically compatible with the subject and activator, including, but not limited to, pH modifiers, surfactants, adjuvants, and ionic strength enhancers, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween®-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0107] As used herein, the term “adjuvant” refers to a nonspecific immunostimulant that, when delivered to an organism with an antigen or prior to the organism, enhances the immune response to an antigen or alters the type of immune response in the organism. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund’s adjuvants (e.g., complete Freund’s adjuvant, incomplete Freund’s adjuvant), Corynebacter parvam, lipopolysaccharides, and cytokines. Freund’s adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0108] IL-2 variant In some embodiments, the present disclosure provides IL-2 variants comprising one or more modifications, such as insertions, substitutions, and / or deletions, compared to a wild-type IL-2 protein, such as human wild-type IL-2 protein. A mature form of human wild-type IL-2 protein is exemplified in SEQ ID NO: 1.

[0109] In some embodiments, the IL-2 variant has one or more amino acid cleavages from the C-terminus of wild-type IL-2. The one or more amino acid cleavages may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more amino acids, as long as the IL-2 variant can retain (preferably attenuate) its binding ability to IL-R2α, IL-2Rβ / γc, and / or the complex IL-2Rα / β / γc. The IL-2 variant may have an amino acid sequence different from SEQ ID NO: 1 only by the C-terminal cleavage. In some specific embodiments, the amino acid sequence of the IL-2 variant is as described in SEQ ID NO: 2.

[0110] In some embodiments, the IL-2 variant has at least one amino acid substitution compared to SEQ ID NO: 1. The substitution may occur in an amino acid involved in the stability of IL-2. Specifically, the substitution may occur at one or more of the positions corresponding to SEQ ID NO: 3, 13, 18, 19, 20, 22, 28, 32, 42, 52, 72, 71, 76, 78, 82, 84, 87, 88, 91, 92, 94, 110, 119, 122, 123, 125, 126, and 129. In some specific embodiments, the amino acid sequences of the IL-2 variant are as described in SEQ ID NOs: 3-13, 79-103, and 111-114.

[0111] In some embodiments, the IL-2 variant has at least one amino acid substitution compared to SEQ ID NO: 1. The substitution may occur at an amino acid located at the IL-2 / Rα or IL-2Rβ / γc binding interface. Specifically, the substitution may occur at one or more of the positions 110, 122, and 129 corresponding to SEQ ID NO: 1. Preferably, in relation to SEQ ID NO: 1, the IL-2 variant includes a combination of a substitution at one or more positions selected from 32, 28, 52, 76, 78, and 82 that may promote the stabilization of the IL-2 variant, and a substitution at one or more positions selected from 129, 110, and 122 that may weaken the binding of IL-2 to its receptor.

[0112] In some embodiments, the IL-2 variant has at least one amino acid substitution compared to SEQ ID NO: 1. The substitution may occur at an amino acid located at the IL-2 / Rα or IL-2Rβ / γc binding interface. Specifically, the substitution may occur at one or more of the positions 110, 122, and 129 corresponding to SEQ ID NO: 1. Preferably, in relation to SEQ ID NO: 1, the IL-2 variant includes a combination of a substitution at one or more positions selected from positions 3, 13, 18, 19, 20, 22, 42, 71, 84, 87, 88, 91, 92, 94, 119, 123, and 126, and a substitution at one or more positions selected from positions 129, 110, and 122 that can attenuate the binding of IL-2 to its receptor.

[0113] In some embodiments, the IL-2 variant includes one or more substitutions at positions 3, 13, 18, 19, 20, 22, 25, 28, 32, 37, 38, 41, 42, 43, 45, 52, 61, 62, 65, 68, 71, 72, 76, 78, 82, 84, 87, 88, 91, 92, 94, 95, 107, 110, 111, 119, 122, 123, 125, 126, 127, 128, and 129 of the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the IL-2 variant includes a substitution at position 28 of an amino acid sequence, as described in SEQ ID NO: 1, where the original amino acid isoleucine(I) can be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, N, P, Q, R, S, L, V, W, Y, and more specifically P.

[0114] In some embodiments, the IL-2 variant includes a substitution at position 32 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid lysine (K) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, I, M, N, P, Q, R, S, L, V, W, Y, and more specifically D.

[0115] In some embodiments, the IL-2 variant includes a substitution at position 52 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid glutamic acid (E) may be substituted with any amino acid other than cysteine, such as A, D, F, G, H, T, K, M, N, P, Q, R, S, L, V, W, Y, and more specifically G.

[0116] In some embodiments, the IL-2 variant includes a substitution at position 76 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid lysine (K) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, I, M, N, P, Q, R, S, L, V, W, Y, and more specifically R.

[0117] In some embodiments, the IL-2 variant includes a substitution at position 78 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid phenylalanine (F) may be substituted with any amino acid other than cysteine, such as A, D, E, K, G, H, T, I, M, N, P, Q, R, S, L, V, W, Y, and more specifically G.

[0118] In some embodiments, the IL-2 variant includes a substitution at position 82 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid proline (P) may be substituted with any amino acid other than cysteine, such as A, D, E, K, G, H, T, I, M, N, F, Q, R, S, L, V, W, Y, and more specifically Y.

[0119] In some embodiments, the IL-2 variant comprises a substitution at position 110 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid glutamic acid (E) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, N, P, Q, R, S, L, V, W, Y, more specifically R, I, or T.

[0120] In some embodiments, the IL-2 variant includes a substitution at position 122 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid isoleucine(I) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, K, M, N, P, Q, R, S, T, L, V, W, Y, more specifically Y, T, or V.

[0121] In some embodiments, the IL-2 variant comprises a substitution at position 129 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid isoleucine(I) may be substituted with any amino acid other than cysteine ​​and isoleucine, such as A, D, E, F, G, H, T, K, M, N, P, Q, R, S, L, V, W, Y, more specifically L, V, A, S, or T.

[0122] In some embodiments, the IL-2 variant includes a substitution at position 13 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid glutamine (Q) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, N, P, R, S, L, V, W, Y, I, more specifically D or R.

[0123] In some embodiments, the IL-2 variant includes a substitution at position 84 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid aspartic acid (D) may be substituted with any amino acid other than cysteine, such as A, E, F, G, H, T, K, M, N, P, Q, R, S, L, V, W, Y, I, more specifically K or T.

[0124] In some embodiments, the IL-2 variant includes a substitution at position 87 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid serine (S) may be substituted with any amino acid other than cysteine, such as A, E, F, G, H, T, K, M, N, P, Q, R, L, V, W, Y, I, more specifically K or I.

[0125] In some embodiments, the IL-2 variant includes a substitution at position 88 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid asparagine (N) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, P, Q, R, S, L, V, W, Y, I, and more specifically K.

[0126] In some embodiments, the IL-2 variant includes a substitution at position 91 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid valine(V) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, N, P, Q, R, S, L, W, Y, I, more specifically E or S.

[0127] In some embodiments, the IL-2 variant comprises a substitution at position 92 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid isoleucine(I) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, N, P, Q, R, S, L, W, Y, more specifically D and R.

[0128] In some embodiments, the IL-2 variant includes a substitution at position 94 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid leucine (L) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, N, P, Q, R, S, V, W, Y, I, and more specifically D.

[0129] In some embodiments, the IL-2 variant comprises a substitution at position 95 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid glutamic acid (E) may be substituted with any amino acid other than cysteine, such as A, D, F, G, H, T, K, M, N, P, Q, R, S, L, V, W, Y, I, more specifically R or Y.

[0130] In some embodiments, the IL-2 variant comprises a substitution at position 119 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid asparagine (N) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, T, K, M, P, Q, R, S, L, V, W, Y, I, more specifically R or Q.

[0131] In some embodiments, the IL-2 variant includes a substitution at position 123 of the amino acid sequence described in SEQ ID NO: 1, where the original amino acid threonine (T) may be substituted with any amino acid other than cysteine, such as A, D, E, F, G, H, K, M, N, P, Q, R, S, L, V, W, Y, I, more specifically K or V.

[0132] In some embodiments, the IL-2 variant includes both a C-terminal cleavage and one or more substitutions selected from positions 3, 13, 18, 19, 20, 22, 28, 32, 42, 52, 72, 76, 78, 82, 84, 87, 88, 91, 92, 94, 110, 119, 122, 125, 126, and 129.

[0133] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 28, such as I28P. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of I28P.

[0134] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 32, such as K32D. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of K32D.

[0135] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 52, such as E52G. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and the substitution of E52G.

[0136] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 76, such as K76R. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a K76R substitution.

[0137] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 78, such as F78G. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and the substitution of F78G.

[0138] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 82, such as P82Y. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of P82Y.

[0139] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 110, such as E110R, E110I, or E110T. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of E110R, E110I, or E110T.

[0140] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 122, such as I122Y, I122T, or I122V. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of I122Y, I122T, or I122V.

[0141] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and a substitution at position 129, such as I129L, I129V, I129A, I129S, or I129T. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of I129L, I129V, I129A, I129S, or I129T.

[0142] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and a substitution at position 42, such as F42A or F42I. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids, and a substitution of F42A or F42I.

[0143] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and a substitution at position 38, such as R38W. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and a substitution of R38W.

[0144] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and substitutions at positions 42, 110, and 129, such as F42I, E110R, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of F42I, E110R, and I129L.

[0145] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 32, 42, and 129, such as K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of K32D, F42I, and I129L.

[0146] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 13, 32, 42, and 129, such as Q13D, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of Q13D, K32D, F42I, and I129L.

[0147] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 13, 32, 42, and 129, such as Q13R, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of Q13R, K32D, F42I, and I129L.

[0148] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and substitutions at positions 84, 32, 42, and 129, such as D84K, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of D84K, K32D, F42I, and I129L.

[0149] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 84, 32, 42, and 129, such as D84T, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of D84T, K32D, F42I, and I129L.

[0150] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 87, 32, 42, and 129, such as S87R, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of S87R, K32D, F42I, and I129L.

[0151] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 88, 32, 42, and 129, such as N88K, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids, and substitutions of N88K, K32D, F42I, and I129L.

[0152] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 91, 32, 42, and 129, such as V91E, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of V91E, K32D, F42I, and I129L.

[0153] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 91, 32, 42, and 129, such as V91S, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids, and substitutions of V91S, K32D, F42I, and I129L.

[0154] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 92, 32, 42, and 129, such as I92D, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of I92D, K32D, F42I, and I129L.

[0155] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 92, 32, 42, and 129, such as I92R, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of I92R, K32D, F42I, and I129L.

[0156] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 94, 32, 42, and 129, such as L94D, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of L94D, K32D, F42I, and I129L.

[0157] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 95, 32, 42, and 129, such as E95R, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids and substitutions of E95R, K32D, F42I, and I129L.

[0158] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 95, 32, 42, and 129, such as E95S, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids, and substitutions of E95S, K32D, F42I, and I129L.

[0159] In some embodiments, the IL-2 variant includes C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and substitutions at positions 119, 32, 42, and 129, such as N119R, K32D, F42I, and I129L. Specifically, the IL-2 variant may include C-terminal cleavage of 4 or 5 amino acids, and substitutions of N119R, K32D, F42I, and I129L.

[0160] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and substitutions at positions 119, 32, 42, and 129, such as N119Q, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of N119Q, K32D, F42I, and I129L.

[0161] In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids and substitutions at positions 123, 32, 42, and 129, e.g., T123K, K32D, F42I, and I129L. Specifically, the IL-2 variant may include a C-terminal cleavage of 4 or 5 amino acids and substitutions of T123K, K32D, F42I, and I129L. In some embodiments, the IL-2 variant includes a C-terminal cleavage of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, as well as substitutions at positions 123, 32, 42, and 129, e.g., T123V, K32D, F42I, and I129L. Specifically, IL-2 variants may include C-terminal cleavage of 4 or 5 amino acids and substitutions of T123V, K32D, F42I, and I129L.

[0162] In some specific embodiments, the amino acid sequence of the IL-2 variant is selected from those described in SEQ ID NOs: 2, 17, 29, and their homologous sequences having at least 95% identity, for example, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity or more.

[0163] In some embodiments, the IL-2 variant includes at least two substitutions occurring at one or more of the positions corresponding to SEQ ID NO: 3, 13, 18, 19, 22, 28, 32, 42, 52, 72, 76, 78, 82, 84, 88, 91, 94, 110, 119, 122, 125, 126, and 129. In some specific embodiments, the amino acid sequence of the IL-2 variant is selected from SEQ ID NOs: 3-13, 18-32, 52-56, 59-74, 76-78, 79-103, and homologous sequences thereof having at least 95% identity.

[0164] The percentage of identity between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput. Appl. Biosci., 4:11-17 (1988)), which is incorporated into the ALIGN program (version 2.0), by using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J. Mol. Biol. 48:444-453 (1970)), which is incorporated into the GAP program of the GCG software package (http: / / www.gcg.com), using either the Blossum62 matrix or the PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0165] Additionally or alternatively, the protein sequences of this disclosure may be used, for example, as “query sequences” for performing searches against public databases to identify relevant sequences. Such searches may be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. To obtain gapped alignments for comparative purposes, gapped BLAST can be used as described in Altschul et al, (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. www.ncbi.nlm.nih.gov.

[0166] IL-2-containing fusion proteins and polypeptide complexes In some embodiments, the disclosure provides fusion proteins containing IL-2 fused to a non-IL-2 moiety. Incorporation of non-IL-2 moieties such as PEG, functional analogs of PEG, lipids, and long-lived serum proteins may be useful for the purpose of half-life extension. In some embodiments, the non-IL-2 moiety is an antibody Fc region. In some other embodiments, the non-IL-2 moiety is human serum albumin (HSA). In some other embodiments, the non-IL-2 moiety is an anti-HSA moiety. The resulting fusion protein may be monomeric or dimeric.

[0167] As those skilled in the art will understand, both Fc-albumin fusions achieve an extended half-life not only by increasing the size of the fusion protein but also by utilizing the neonatal Fc receptor FcRn, a natural recycling mechanism of the body. The pH-dependent binding of these proteins to FcRn prevents the degradation of the fusion protein in endosomes. Fusion to antibody Fc can improve the solubility and stability of the fusion protein.

[0168] The main difference between the Fc and HSA / anti-HSA moieties is that the Fc moiety is dimeric, while the Has / anti-HSA moiety is monomeric. This means that the fusion protein is presented as either a dimer or a monomer depending on the choice of fusion partner. The dimeric nature of the Fc fusion protein can lead to an avidity effect if the target receptor is sufficiently close or is itself a dimer.

[0169] In addition, fusion proteins such as those disclosed herein may further reduce their affinity for IL-2Rβ / γc, IL-2Rα, or both, and the combined IL-2Rα / β / γc complex, through steric hindrance, hydrogen bonding, salt bridging, hydrophobic effects, or other intramolecular interactions.

[0170] In some embodiments, the disclosure provides polypeptide complexes comprising an IL-2 moiety, an antigen-binding moiety, and a complete or partial hinge region, in addition to an Fc region. The antigen-binding moiety may be a Fab, scFv, nanobody (VHH), or TCR.

[0171] In some embodiments, the antigen-binding portion is Fab. The IL-2 portion may be constructed on a different chain from Fab. Alternatively, the IL-2 portion may be on the same heavy chain as the VH region of Fab. If the IL-2 portion is on the same heavy chain as the VH region, the IL-2 portion may be operably linked (optionally via a linker) to a hinge region and an Fc region, which in turn is operably linked to the VH region; that is, the IL-2 portion and Fab are separated by the intervening hinge region and Fc region. In some other embodiments, the IL-2 portion and Fab are on the same side (usually the N-terminus) of the Fc region.

[0172] In some embodiments, the antigen-binding portion is VHH. The IL-2 portion may be constructed on a different chain from VHH. Alternatively, the IL-2 portion may be on the same heavy chain as VHH. If the IL-2 portion is on the same chain as the VHH region, the IL-2 portion may be operably linked (optionally via a linker) to a hinge region and an Fc region, which in turn is operably linked to the VHH region; that is, the IL-2 portion and VHH are separated by the intervening hinge region and Fc region. In some other embodiments, the IL-2 portion and VHH are on the same side (usually the N-terminus) of the Fc region.

[0173] In some embodiments, the antigen-binding portion is the TCR. The IL-2 portion may be constructed on a different chain from the TCR. Alternatively, the IL-2 portion may be on the same heavy chain as one of the TCR's chains (α or β). If the IL-2 portion is on the same chain as the α or β chain of the TCR region, the IL-2 portion may be operably linked (optionally via a linker) to a hinge region and an Fc region, which in turn is operably linked to the TCR region; that is, the IL-2 portion and the TCR are separated by the intervening hinge region and Fc region. In some other embodiments, the IL-2 portion and the TCR are on the same side (usually the N-terminus) of the Fc region.

[0174] Depending on the desired biochemical properties (e.g., solubility and stability) and pharmacokinetic properties, different construction forms can be applied. For example, the polypeptide complex may contain one or more IL-2 moieties or one or more antigen-binding moieties.

[0175] As depicted in Figure 1(a), the polypeptide complex may contain two heavy chains and one light chain, the antigen-binding moiety being either Fab or TCR, and the first heavy chain from the N-terminus to the C-terminus containing one chain with: (a) an IL-2 site; (b) optionally a linker; and (c) a hinge region and an Fc region; The second heavy chain includes (d) the Fab or TCR heavy chain from the N-terminus to the C-terminus; and (e) the other chain of the hinge region and Fc region; and The light chain includes the light chain of Fab or TCR.

[0176] As depicted in Figure 1(b), the polypeptide complex can contain two chains, the antigen-binding portion being VHH, and the first chain from the N-terminus to the C-terminus containing: (a) the IL-2 portion; (b) optionally the linker; and (c) the hinge region and the Fc region of one of the chains; The second strand includes the other strand from the N-terminus to the C-terminus: (d) VHH; and (e) the hinge region and Fc region.

[0177] As depicted in Figure 1(c), the polypeptide complex may contain two chains, each chain comprising (a) an IL-2 moiety from the N-terminus to the C-terminus; (b) an optional linker; and (c) a hinge region and an Fc region on one of the chains.

[0178] As depicted in Figure 1(d), the polypeptide complex can consist of two heavy chains and two light chains, the antigen-binding portion being Fab or TCR, and the first chain from the N-terminus to the C-terminus: (a) IL-2 portion; (b) optionally a linker; and (c) one chain containing a hinge region and an Fc region; (d) the heavy chain of Fab or TCR; The light chain includes the light chain of Fab or TCR.

[0179] As shown in Figure 1(e), the polypeptide complex may contain two chains, the antigen-binding portion being VHH, and the first chain, from the N-terminus to the C-terminus, includes: (a) the IL-2 portion; (b) optionally a linker; and (c) one of the chains consisting of a hinge region and an Fc region; and (d) VHH.

[0180] As shown in Figure 1(f), the polypeptide complex may contain two chains, the antigen-binding portion being scFv, and the first chain comprising (a) an IL-2 portion from the N-terminus to the C-terminus; (b) an optional linker; and (c) a hinge region and an Fc region; and (d) scFv.

[0181] As shown in Figure 1(g), the polypeptide complex may contain two chains, the antigen-binding portion containing two VHHs, and the first chain containing one chain from the N-terminus to the C-terminus: (a) IL-2 portion; (b) optionally a linker; and (c) a hinge region and an Fc region. The second chain includes (d) two tandem VHHs from the N-terminus to the C-terminus; (e) an optional linker; and (f) the other chain with a hinge region and an Fc region.

[0182] The target antigen of the antigen-binding portion can be selected from checkpoint molecules such as PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, A2aR, TIGIT, and VISTA, or tumor-associated antigens such as HER2 and BCMA, or angiogenesis-related factors such as VEGF and PDGF. A wide variety of antibodies against such antigens have already been developed and are well known to those skilled in the art.

[0183] The antigen-binding portion may be derived from an antibody that is already known, marketed, or developed de novo, for example, any of the following antibodies: trastuzumab, pertuzumab, sacituzumab, absiximab, adalimumab, alefecept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, bevacizumab, canakinumab, certolizumab pegol, cetuki Simab, daclizumab, denosumab, efalizumab, golimumab, gemtuzumab, infliximab, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, ofatumumab, palivizumab, panitumumab, pembrolizumab, rituximab, ranibizumab, tocilizumab, trastuzumab, secukinumab, ustekinumab. The antigen-binding variable region (or at least the CDR region) may be the same as that of a known antibody or may be de novo-developed. "Derived" means that the variable region is identical to the variable region of the parent antibody, or has at least 80% homology (e.g., at least 85%, 90%, 95% or more), and retains the ability to bind to the target antigen. For example, the variable region of the parent antibody may be humanized, affinity matured, or glycosylated before being constructed into the polypeptide complex disclosed herein. Methods for modifying the variable region, including the CDR and framework region, are well known to those skilled in the art.

[0184] ii)E44 format Polypeptide complexes such as those disclosed herein can be constructed as IL-2 / Fab fusion proteins comprising a Fab moiety operably linked to one chain of the Fc region and an IL-2 moiety operably linked (optionally via a linker) to the N-terminus of the other chain of the Fc region. A series of such polypeptide complexes of the E44 format are provided herein.

[0185] In some embodiments, the IL-2 moiety consists of the wild-type IL-2 protein. Specifically, the polypeptide complex includes a first heavy chain as described in SEQ ID NO: 15, a second heavy chain as described in SEQ ID NO: 33, and a light chain as described in SEQ ID NO: 34 (W3XX115-T2U0.E44-1.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2 by C-terminal cleavage (e.g., cleavage by 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids), as described above. Specifically, the polypeptide complex includes a first heavy chain as described in SEQ ID NO: 17, a second heavy chain as described in SEQ ID NO: 33, and a light chain as described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-6.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position I129, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 18, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-26.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at the E110 position, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 19, a second heavy chain described in SEQ ID NO: 33, and a light chain as described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-40.uIgG4V322).

[0186] In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position I122, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 20, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-41.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position I28, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 21, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-42.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at the K32 position, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 22, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-43.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at the E52 position, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 23, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-44.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at the K76 position, as described above. Specifically, the polypeptide complex includes a first heavy chain as described in SEQ ID NO: 24, a second heavy chain as described in SEQ ID NO: 33, and a light chain as described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-45.uIgG4V322).In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position F78, as described above. Specifically, the polypeptide complex includes a first heavy chain as described in SEQ ID NO: 25, a second heavy chain as described in SEQ ID NO: 33, and a light chain as described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-46.uIgG4V322).

[0187] In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position P82, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 26, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-47.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions E110 and I129, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 27, a second heavy chain described in SEQ ID NO: 33, and a light chain described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-48.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at the K32 and I129 positions, as described above. Specifically, the polypeptide complex includes a first heavy chain as described in SEQ ID NO: 28, a second heavy chain as described in SEQ ID NO: 33, and a light chain as described in SEQ ID NO: 34 (corresponding to W3XX115-T2U0.E44-49.uIgG4V322).

[0188] In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2 by both the C-terminal cleavage described above (e.g., 4-amino acid cleavage). Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 52, a second heavy chain described in SEQ ID NO: 7, and a light chain described in SEQ ID NO: 58 (corresponding to W3XX115-T2U3.E44-6.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2 by both the C-terminal cleavage described above (e.g., 4-amino acid cleavage) and substitution at position I129. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 53, a second heavy chain described in SEQ ID NO: 57, and a light chain described in SEQ ID NO: 58 (corresponding to W3XX115-T2U3.E44-26.uIgG4V322).

[0189] In some embodiments, the IL-2 moiety polypeptide consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position I129, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 54, a second heavy chain described in SEQ ID NO: 57, and a light chain described in SEQ ID NO: 58 (corresponding to W3XX115-T2U3.E44-26.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2, obtained by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position R38W, as described above. Specifically, the polypeptide complex includes a first heavy chain described in SEQ ID NO: 55, a second heavy chain described in SEQ ID NO: 57, and a light chain described in SEQ ID NO: 58 (corresponding to W3XX115-T2U3.E44-20.uIgG4V322). In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I and I129, as described above. Specifically, the polypeptide complex includes a first heavy chain as described in SEQ ID NO: 56, a second heavy chain as described in SEQ ID NO: 57, and a light chain as described in SEQ ID NO: 58 (corresponding to W3XX115-T2U3.E44-33.uIgG4V322).

[0190] iii) Z20 format The polypeptide complexes disclosed herein may be constructed as IL-2 / Fc fusion proteins comprising an IL-2 moiety operably linked (optionally via a linker) to the N-terminus of each chain of the Fc region. In some embodiments, the IL-2 moiety consists of an IL-2 variant different from WT IL-2 by C-terminal cleavage (e.g., cleavage by amino acids 1, 2, 3, 4, 5, 6, 7, 8, or 9) as described above. Specifically, the polypeptide complex comprises the heavy chain described in SEQ ID NO: 29 (corresponding to W3XX115-T2.Z20-1.uIgG4V322).

[0191] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at position I129, as described above. Specifically, the polypeptide complex contains the first heavy chain described in SEQ ID NO: 30 (corresponding to W3XX115-T2.Z20-2.uIgG4V322).

[0192] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage), substitution at position I129, and substitution at position E110, as described above. Specifically, the polypeptide complex contains the first heavy chain described in SEQ ID NO: 31 (corresponding to W3XX115-T2.Z20-4.uIgG4V322).

[0193] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage), substitution at position I129, and substitution at position K32, as described above. Specifically, the polypeptide complex contains the first heavy chain described in SEQ ID NO: 32 (corresponding to W3XX115-T2.Z20-5.uIgG4V322).

[0194] v)Z73 format Polypeptide complexes such as those disclosed herein can be constructed as IL-2 / VHH fusion proteins comprising two tandem VHHs operably linked to one chain of the Fc region and an IL-2 moiety operably linked (optionally via a linker) to the N-terminus of the other chain of the Fc region. A series of such polypeptide complexes of the Z73 form are provided herein.

[0195] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, E110R, and I129, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 59 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-50.uIgG4V322).

[0196] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 60 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-51.uIgG4V322).

[0197] In some embodiments, the IL-2 portion consists of an IL-2 variant that differs from WT IL-2 by substitutions at positions L18R, Q22E, F42I, and Q126K, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 61 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-52.uIgG4V322).

[0198] In some embodiments, the IL-2 portion consists of an IL-2 variant different from WT IL-2 by substitutions at the positions of L19H, F42I, C125I, and Q126E, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 62 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-53.uIgG4V322).

[0199] In some embodiments, the IL-2 portion consists of an IL-2 variant different from WT IL-2 by substitutions at positions T3A, D20N, F42I, N71K, and Q125S, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 63 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-54.uIgG4V322).

[0200] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitutions at positions Q13D, F42I, K32D, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 64 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-55.uIgG4V322).

[0201] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, D84K, and I129, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 65 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-56.uIgG4V322).

[0202] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, S87R, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 66 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-57.uIgG4V322).

[0203] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, N88K, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 67 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-58.uIgG4V322).

[0204] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitutions at positions F42I, K32D, V91E, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 68 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-59.uIgG4V322).

[0205] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitutions at positions F42I, K32D, I92R, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 69 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-60.uIgG4V322).

[0206] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, I92D, and I129, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 70 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-61.uIgG4V322).

[0207] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, L94D, and I129, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 71 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-62.uIgG4V322).

[0208] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, E95R, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 72 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-63.uIgG4V322).

[0209] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitutions at positions F42I, K32D, N119R, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 73 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-64.uIgG4V322).

[0210] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, T123K, and I129, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 74 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-65.uIgG4V322).

[0211] In some embodiments, the IL-2 portion consists of an IL-2 variant that differs from WT IL-2 by substitutions at positions L18R, Q22E, and Q126K, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 76 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-66.uIgG4V322).

[0212] In some embodiments, the IL-2 portion consists of an IL-2 variant that differs from WT IL-2 by substitutions at the positions of L19H, C125I, and Q126E, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 77 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-67.uIgG4V322).

[0213] In some embodiments, the IL-2 portion consists of an IL-2 variant that differs from WT IL-2 by substitutions at positions T3A, D20N, N71K, and Q125S, as described above. Specifically, the polypeptide complex includes a first chain as described in SEQ ID NO: 78 and a second chain as described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-68.uIgG4V322).

[0214] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitutions at the Q13R, F42I, K32D, and I129 positions, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 104 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-69.uIgG4V322).

[0215] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, D84T, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 105 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-70.uIgG4V322).

[0216] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitutions at positions F42I, K32D, S87I, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 106 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-71.uIgG4V322).

[0217] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, V91S, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 107 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-72.uIgG4V322).

[0218] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, E95Y, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 108 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-73.uIgG4V322).

[0219] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, N119Q, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 109 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-74.uIgG4V322).

[0220] In some embodiments, the IL-2 moiety consists of an IL-2 variant that differs from WT IL-2 by both C-terminal cleavage (e.g., 4-amino acid cleavage) and substitution at positions F42I, K32D, T123V, and I129, as described above. Specifically, the polypeptide complex includes a first chain described in SEQ ID NO: 110 and a second chain described in SEQ ID NO: 75 (corresponding to W3XX115-T2U10.Z73-75.uIgG4V322).

[0221] FC Domain This disclosure provides Fc fusion proteins and polypeptide complexes comprising an Fc domain and the above-described human IL-2 variant. The Fc domain may be wild-type Fc or an Fc variant. Wild-type Fc may be human IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, wild-type Fc is human IgG1 Fc. The Fc variant includes one or more amino acid residue modifications (e.g., substitutions, insertions, and / or deletions) compared to wild-type Fc (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc). In some embodiments, the Fc variant includes one or more amino acid residue modifications (e.g., substitutions, insertions, and / or deletions) compared to wild-type human IgG1 Fc. In some embodiments, the Fc variant includes one or more amino acid residue modifications (e.g., substitutions, insertions, and / or deletions) compared to wild-type human IgG4 Fc.

[0222] Numerous mutations are known to increase or decrease ADCC, ADCP, and CDC. One example of a human IgG1 heavy chain mutation is the LALA mutation, which impairs all effector functions, i.e., functions for ADCC, ADCP, and CDC. The hIgG1 LALA sequence has two mutations, L234A and L235A (EU numbering), which suppress FcgR binding. The "EU numbering system" or "EU index" is commonly used to refer to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al. Sequences of Proteins of Immunological Interest (5th edition), US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242). "EU numbering in Kabat" or "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. In this specification, unless otherwise specified, references to residue numbers in the constant domain of the Fc region mean residue numbering according to the EU numbering system.

[0223] 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 fusion protein contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants bind to FcRn at acidic pH, thus extending their pharmacokinetic half-life, thereby escaping degradation in lysosomes and subsequently transferring to the extracellular space for release. Methods for engineering antibodies and their antigen-binding fragments to improve binding affinity to FcRn are well known in this art. For example, see 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).

[0224] The two strands of the Fc domain may associate together via a disulfide bond. In some embodiments, the Fc domain includes one or more amino acid modifications (e.g., substitutions) at the interface of the Fc region to promote and / or facilitate heterodimerization. For example, the two strands of the Fc domain are manipulated to form a “knob-in-hole” structure to promote heterodimerization, which involves introducing a protrusion ("knob") into the first Fc polypeptide and a cavity ("hole") into the second Fc polypeptide, where the protrusion may be positioned in the cavity to promote interaction between the first and second Fc polypeptides to form a heterodimer or complex. Methods for producing antibodies having such modifications are known in the art and are described, for example, in U.S. Patent No. 5,731,166 and No. 5,731,168. Specifically, the Fc domain may contain at least one “knob” (projection) and at least one “hole” (cavity), and the presence of the “knob” and “hole” promotes the formation of a complex or heterodimer (see WO2005 / 063816 for details). In some embodiments, the Fc domain as disclosed herein comprises first and second Fc polypeptide chains, each containing one or more mutations with respect to wild-type human IgG1 Fc. The IL-2 domain may be fused to one chain of the Fc domain containing the “knob” mutation, while the VH region of the antigen-binding moiety is fused to the other chain containing the “hole” mutation, or vice versa. In at least one embodiment, the “hole” mutations are Y349C, T366S, L368A, and / or Y407V, and the “knob” mutations are S354C and / or T366W.

[0225] In certain embodiments, the Fc domain of the fusion protein contains one or more amino acid substitutions that alter antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Specific amino acid residues in the CH2 domain of the Fc region may be substituted to provide reduced ADCC activity.

[0226] In some embodiments, the Fc domain is an IgG4 Fc variant containing a "FALA" mutation (i.e., F234A / L235A) that reduces binding to the Fc receptor or complement receptor. In some other embodiments, the Fc domain is an IgG4 Fc variant having a cleaved hinge region. In some yet other embodiments, the Fc domain is an IgG4 Fc variant containing an S228P mutation that can reduce IgG4 Fab-arm exchange.

[0227] In some specific embodiments, the Fc variant comprises two chains, and the amino acid sequence of the first chain has at least 80%, e.g., 80%, 85%, 90%, 95% or more (e.g., 100%) sequence identity with wild-type human IgG1, IgG2, IgG4, or IgG4 having a hinge cleavage.

[0228] Characteristics of polypeptide complexes This disclosure provides affinity-reduced and potency-reduced IL-2 variants, as well as polypeptide conjugates containing IL-2 variants. The IL-2 variants have regulated potency / toxicity, PK and PD potential, and therefore can function as novel immunotherapeutic agents with improved antitumor efficacy and treatment of autoimmune diseases.

[0229] The functionality of IL-2, including these IL-2 variants and polypeptide complexes, can be evaluated by in vitro or in vivo assays.

[0230] In some embodiments, the affinity of IL-2, including the polypeptide complex, to IL-2Rα or IL-2β is determined by a BIAcore binding assay.

[0231] Alternatively, the binding affinity of IL-2, including polypeptide complexes, is determined by FACS using cell lines expressing IL-2Rβ (intermediate affinity receptor) and / or IL-2Rα (high affinity receptor), such as HH cells, NK92, and MJ20 cells.

[0232] In some embodiments, the effects of IL-2 variants and polypeptide complexes are evaluated by quantifying signaling pathways measured by the phosphorylation of specific factors, such as STAT5 phosphorylation. STAT5 plays a crucial role in maintaining normal immune function and homeostasis, both of which are regulated by specific members of the IL-2 family of cytokines.

[0233] Regulatory T cells (Tregs) play a crucial role in maintaining immune homeostasis and self-tolerance, and are essential in controlling the development of allergies and autoimmune diseases. Activated CD8+ T cells are very important for immune defense against tumors. IL-2 can induce the proliferation of Treg cells and activated CD8+ T cells through signaling via high-affinity IL-2 receptors. In some embodiments, the effects of IL-2 variants and polypeptide complexes are evaluated by assessing T cell activation, such as CD8+ T cell or Treg cell activation.

[0234] IL-2 comprising the polypeptide complex of this disclosure provides at least one of the following properties: (a) The binding affinity to IL-2Rα, IL-2Rβ / γc, and at least one of the combined IL-2Rα / β / γc complex is reduced; (b) Moderate efficacy in simulating immune cell proliferation, such as CD8+ T cell and NK cell proliferation; (c) The ability to activate immune cells such as activated CD8+ T cells or Treg cells is reduced but retained; and (d) Increased thermal stability, serum stability, and in vivo PK; (e) No apparent toxicity in vivo; (f) Significant antitumor effect in mouse models.

[0235] Nucleic acid molecules encoding IL-2 variants In some embodiments, this disclosure is directed to isolated nucleic acid molecules containing nucleic acid sequences encoding IL-2 variants or Fc fusion proteins disclosed herein.

[0236] The nucleic acids of this disclosure can be obtained using standard molecular biological techniques. Isolated nucleic acids encoding IL-2 variants can be operably ligated to another DNA molecule encoding an Fc domain. Similarly, nucleic acids encoding antigen-binding regions can be operably ligated to another DNA molecule encoding an Fc domain. DNA fragments containing these regions can be obtained by standard PCR amplification.

[0237] Once DNA fragments encoding the IL-2 moiety, antigen-binding moiety, and Fc domain (or a specific region) are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques and, for example, incorporated into an expression vector as known in the art. In some embodiments, the nucleic acids encoding these DNA fragments are generally contained within a single expression vector under the control of different or the same promoter. In some other embodiments, the nucleic acids encoding these DNA fragments are operably ligated and contained within a single expression vector under the control of the same promoter. As used in this context, the term “operably ligated” is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0238] host cell The host cells disclosed herein may be any cells suitable for expressing the fusion proteins herein, such as bacterial cells, yeast, or mammalian cells. Mammalian host cells for expressing the fusion proteins herein include Chinese hamster ovary (CHO) cells (DHFR-selectable markers as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, e.g., 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 in NSO myeloma cells is the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the fusion protein is produced by culturing the host cells for a period sufficient to allow expression of the fusion protein in the host cells or secretion of the fusion protein into the culture medium in which the host cells are growing. The fusion protein can be recovered from the culture medium using standard protein purification methods.

[0239] Pharmaceutical composition In some embodiments, this disclosure is directed to pharmaceutical compositions comprising a fusion protein or polypeptide complex disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure is directed to pharmaceutical compositions comprising a nucleic acid molecule encoding a fusion protein or polypeptide complex disclosed herein and a pharmaceutically acceptable carrier.

[0240] Composition components The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as antibodies. The pharmaceutical composition of this disclosure may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine. Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspensions / dispersants, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, and various combinations or more of other components known in the art.

[0241] Suitable components include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propylgalacte. As disclosed herein, a composition may contain one or more antioxidants, such as methionine, a reducing antibody, or an antigen-binding fragment thereof, which can be oxidized. Redox reactions can prevent or reduce a decrease in binding affinity, thereby enhancing the stability and shelf life of the protein. Accordingly, in some embodiments, the disclosure provides a composition comprising a fusion protein and one or more antioxidants such as methionine. The disclosure further provides various methods for mixing fusion proteins with one or more antioxidants, such as methionine, so that the fusion proteins can be protected from oxidation, thereby extending their shelf life and / or increasing their activity.

[0242] Further examples of pharmaceutically acceptable carriers include aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or glucose and lactated Ringer's injection; non-aqueous vehicles such as plant-derived fixing oils, cottonseed oil, corn oil, sesame oil, and peanut oil; antibacterial agents at bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride and glucose; buffers such as phosphate buffer and citrate buffer; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspensions and dispersants such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); chelating agents such as EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers containing phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methyl p-hydroxybenzoate and propyl esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients include, for example, water, physiological saline, glucose, glycerol, or ethanol. Suitable non-toxic auxiliary substances include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or drugs such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0243] Administration, formulation, and dosage The pharmaceutical compositions of this disclosure may be administered in vivo to subjects requiring them by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, intrabuccal, intrarectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The compositions may be formulated in solid, semi-solid, liquid, or gaseous form; examples include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols. Appropriate formulations and routes of administration may be selected according to the intended use and treatment regimen.

[0244] Formulations suitable for enteral administration include hard or soft gelatin capsules, tablets, coated tablets, elixirs, suspensions, syrups, or inhalants, as well as controlled-release formulations thereof.

[0245] Suitable formulations for parenteral administration (e.g., injection) include aqueous or nonaqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquid formulations may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the blood (or other relevant body fluids) of the recipient in which the formulation is intended. Examples of excipients include, for example, water, alcohol, polyols, glycerol, and vegetable oils. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific administration regimens, including dosage, timing, and repetition, depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0246] The frequency of administration can be determined and adjusted throughout the course of treatment, based on reducing the number of proliferative or oncogenic cells, maintaining a reduction in such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dosage may be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, a sustained-release formulation of the therapeutic composition in question may be appropriate.

[0247] Those skilled in the art will understand that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against any risks or adverse side effects. The selected dose level depends on a variety of factors, including, but not limited to, the activity of the particular 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 concomitantly, the severity of the condition, and the patient's species, sex, age, weight, condition, general health, and prior medical history. The amount of compound and the route of administration are ultimately left to the discretion of the physician, veterinarian, or clinician, but generally the dosage is selected to achieve a local concentration at the site of action that produces the desired effect without causing substantially harmful or adverse side effects.

[0248] In general, the polypeptide conjugates of this disclosure can be administered in a range of doses. These include approximately 100 μg / kg body weight to approximately 10 mg / kg body weight per dose; approximately 100 μg / kg body weight to approximately 1 mg / kg body weight per dose; and approximately 1 μg / kg body weight to approximately 10 mg / kg body weight per dose. Other ranges include approximately 100 μg / kg body weight to approximately 200 μg / kg body weight per dose; approximately 200 μg / kg body weight to approximately 300 μg / kg body weight per dose; approximately 300 μg / kg body weight to approximately 400 μg / kg body weight per dose; approximately 400 μg / kg body weight to approximately 0.5 μg / kg body weight per dose; and approximately 0.5 mg / kg body weight to approximately 1 mg / kg body weight per dose. In certain embodiments, the dose 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, or at least about 10 mg / kg body weight.

[0249] In any case, the polypeptide complex of this disclosure is preferably administered as needed to the subject who needs it. The frequency of administration can be determined by a person skilled in the art, such as the attending physician, taking into consideration the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health status of the subject being treated, etc.

[0250] In certain preferred embodiments, the course of treatment comprising the polypeptide complex of the Disclosure comprises multiple administrations of the selected drug over a period of several weeks or months. More specifically, the polypeptide complex of the Disclosure may be administered every four days, once a week, once every ten days, once every two weeks, once every three weeks, once a month, once every six weeks, once every two months, once every ten weeks, or once every three months. In this regard, it will be understood that the dosage may be modified or the interval may be adjusted based on the patient's response and clinical practice.

[0251] Dosages and regimens may also be empirically determined for the disclosed therapeutic compositions in individuals who have received one or more doses. For example, an increasing dose of the therapeutic composition prepared as described herein may be given to an individual. In selected embodiments, doses may be gradually increased, decreased, or attenuated, respectively, based on empirically determined or observed side effects or toxicity. To evaluate the efficacy of the selected composition, markers of specific diseases, disorders, or conditions may be tracked as described above. In the case of cancer, these 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; measurement of indirect tumor markers (e.g., PSA for prostate cancer identified according to the methods described herein) or oncogenic antigens; reduction of pain or paralysis; improvement of speech, vision, respiration, or other tumor-related impairments; increase in appetite; or improvement in quality of life as measured by accepted tests; or extension of survival. It will be apparent to those skilled in the art that the dosage varies depending on the individual, the type of neoplasm, the stage of the neoplasm, whether the neoplasm has begun to metastasize to other parts of the individual, and the treatments previously used and concurrently used.

[0252] Applications of this disclosure The polypeptide complexes, pharmaceutical compositions, and methods of this disclosure have numerous in vitro and in vivo uses, for example, in enhancing immune responses. For example, these molecules can enhance immunity in a variety of situations when administered to cultured cells, in vitro or ex vivo, or to human subjects, for example, in vivo. The immune response can be modulated, for example, by enhancement, stimulation, or upregulation.

[0253] For example, the subject includes human patients who require enhancement of the immune response. This method is particularly suitable for treating human patients having a disorder that can be treated by enhancing an immune response (e.g., an NK / T cell-mediated immune response). In certain embodiments, the method is particularly suitable for the treatment of cancer in vivo. To achieve enhancement of immunity, the polypeptide complex can be administered alone or in combination with another therapy. When the polypeptide complex is administered with another agent, the two can be administered in either order or simultaneously.

[0254] For example, the subject includes human patients who require suppression of the immune response. This method is particularly suitable for treating human patients having a disease that can be treated by suppressing an immune response (e.g., a Treg-mediated immunosuppression). In certain embodiments, the method is particularly suitable for the treatment of autoimmune diseases in vivo. To achieve suppression of immunity, the polypeptide complex can be administered alone or in combination with another therapy. When the polypeptide complex is administered with another agent, the two can be administered in either order or simultaneously.

[0255] Treatment of disorders including cancer In some aspects, the present disclosure provides a method for treating a disorder or disease in a mammal, the method comprising administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of the polypeptide complex disclosed herein. In some aspects, the present disclosure provides a method for treating a disorder or disease in a mammal, which comprises administering to a subject (e.g., a human) in need of treatment a therapeutically effective amount of a nucleic acid molecule encoding the polypeptide complex disclosed herein. The disorder or disease may be cancer.

[0256] Various types of cancer, whether malignant or benign, and whether primary or secondary, can be treated or prevented by the methods provided herein. The cancers may be solid tumors or hematological malignancies.Examples of such cancers include lung cancers such as bronchogenic lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyoma; bone cancers such as osteochondroma, chondromycoma, chondroblastoma, chondromyxofibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticular sarcoma; and brain tumors such as gliomas (e.g., polymorphic gliocytoma). Brain tumors such as blastoma, anaplastic astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pineal glandoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ blastoma, teratoma, dermid cyst, and hemangioma; digestive system cancers such as colon cancer, leiomyoma, epidermal carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colon polyps, and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibroporous carcinoma, bile duct cancer, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, renal epithelioma, and transitional cell carcinoma of the renal pelvis. Cancer; bladder cancer; acute lymphoblastic leukemia, acute myeloid (myelocytic, myelogenic, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sézary syndrome, pilocytic cell leukemia), chronic myeloid (myeloid, myelogenic, granulocytic) leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, mycosis fungoides, myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myeloid leukemia); cancers of the skin, basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, Paget's disease, head and neck cancer, retinoblastoma Examples include eye-related cancers such as cystic tumors and intraocular melanoma; cancers of the male reproductive system such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., breast cancer; cancers of the female reproductive system such as endometrial cancer, cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, and medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid gland; pancreatic cancer; and blood cancers such as leukemia, myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma. In a specific embodiment, cancer is colon cancer.

[0257] In some embodiments, examples of cancer include, but are not limited to, B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-necrotic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and transplantation Post-lymphoblastic leukemia (PTLD), as well as facomatosis, edema (such as that associated with brain tumors), B-cell proliferative disorders, and abnormal angiogenesis associated with Meigs syndrome. More specific examples include, but are not limited to, relapsed or refractory non-Hodgkin lymphoma, frontline low-grade non-Hodgkin lymphoma, stage III / IV non-Hodgkin lymphoma, chemotherapy-resistant non-Hodgkin lymphoma, precursor B-cell lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or pre-lymphoblastic leukemia and / or small lymphocytic lymphoma. B-cell lymphoma, B-cell prelymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, nodular marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasmacytotic myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular non-Hodgkin lymphoma, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate-grade diffuse non-Hodgkin lymphoma, diffuse large B-cell lymphoma, invasive non-Hodgkin lymphoma (invasive frontline non-Hodgkin lymphoma and These include invasive relapsed non-Hodgkin lymphoma (including invasive relapsed non-Hodgkin lymphoma), non-Hodgkin lymphoma that has relapsed after autologous stem cell transplantation or is resistant to autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary pleural effusion lymphoma, high-grade immunoblastic non-Hodgkin lymphoma, high-grade lymphoblastic non-Hodgkin lymphoma, high-grade small non-necrotic cell non-Hodgkin lymphoma, bulky disease non-Hodgkin lymphoma, Burkitt lymphoma, progenitor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sézary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, and vascular central lymphoma.

[0258] In some embodiments, examples of cancers further include B cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, the following. a) Follicular lymphoma, b) Small non-necrotic 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), nodular 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, angiocentric lymphoma - pulmonary B cell lymphoma), f) Hairy cell leukemia, g) Lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, h) Acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell lymphocytic leukemia, i) Plasma cell neoplasms, plasmacytoma, multiple myeloma, plasmacytosis, and / or j) Hodgkin's disease.

[0259] In other embodiments, the disorder or disease may be an autoimmune and inflammatory disease including, but not limited to, type 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis, systemic lupus erythematosus, autoimmune hepatitis, antiphospholipid syndrome, Wegener's granulomatosis, bullous pemphigoid, Churg-Strauss syndrome, thyroid diseases including Graves' disease, inflammatory bowel disease, Guillain-Barré syndrome, psoriasis, myasthenia gravis, vasculitis.

[0260] Stimulation / suppression of immune responses without cytotoxicity In some embodiments, the Disclosure also provides methods for enhancing (e.g., stimulating) or suppressing an immune response in a subject, comprising administering the polypeptide complex of the Disclosure to the subject such that the immune response in the subject is enhanced without producing undesirable side effects. For example, the subject is a mammal. In specific embodiments, the subject is a human.

[0261] The term "enhance immune response," or its grammatical variations, means stimulating, eliciting, increasing, improving, or enhancing any response of the mammalian immune system. The immune response may be a cellular response (i.e., a cell-mediated response, such as one mediated by cytotoxic T lymphocytes) or a humoral response (i.e., an antibody-mediated response), and may be a primary or secondary immune response. An example of enhancing the immune response is CD4 + Examples include increased activity of T cells, particularly helper T cells, and the generation of cytolytic T cells. Enhancement of the immune response can be evaluated using many 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-15 production or IFN-γ production), tumor regression, survival of animals with tumors, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, the methods of this disclosure enhance the immune response in a mammal compared to the immune response in an untreated mammal or a mammal not treated with a method such as those disclosed herein. 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. Conversely, “suppressing the immune response” means reducing, mitigating, lowering or reducing the response of the mammalian immune system, which is often desired in autoimmune diseases where the immune system is overactivated. The polypeptide conjugates disclosed herein may be used as monotherapies, but more frequently, they are used in combination with cellular immunotherapy, targeted therapy, chemotherapy, or radiotherapy.

[0262] Applications in cell immunotherapy and gene therapy In some embodiments, the IL-2 fusion proteins and variants disclosed herein are used in cellular immunotherapy involving IL-2 variant secretion. The IL-2 variant genes are constructed in therapeutic artificial immune cells, including but not limited to tumor-infiltrating lymphocytes (TILs), artificial T cell receptors (TCR-Ts), chimeric antigen receptor (CAR) T cells, CAR NK cells, CAR macrophages, and natural killer (NK) cells.

[0263] In some embodiments, the IL-2 fusion proteins and variants disclosed herein are used in gene therapy. The genes encoding the IL-2 variants are incorporated into therapeutic vectors such as lentiviruses, AAVs, poxviruses, herpes zoster viruses, Oncoltoviruses, and other RNA / DNA vectors.

[0264] Combination therapy with cellular immunotherapy In some embodiments, the IL-2 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 from the human body's immune system to eliminate cancer. These approaches range from directly isolating our own immune cells and simply increasing their numbers (e.g., by activating and increasing the patient's immune cells outside the body and then injecting them into the patient) to genetically engineering immune cells (through gene therapy) to enhance their ability to fight cancer. Cellular immunotherapy includes, but is not limited to, tumor-infiltrating lymphocyte (TIL) therapy, genetically modified 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.

[0265] Combination with gene therapy In some embodiments, the IL-2 fusion proteins disclosed herein are used in combination with gene therapy. Genes encoding IL-2 variants can be delivered to the target by therapeutic vectors, including viruses such as lentiviruses, AAVs, poxviruses, herpes zoster viruses, and Oncolyte viruses. Gene introduction can be carried out by transformation, in which the gene is directly taken up by bacterial cells under specific conditions; transduction, in which genetic material is introduced using bacteriophages; and finally, transfection, in which the gene is forcibly introduced using viral or nonviral vectors. Nonviral transfection methods are classified into physical, chemical, and biological methods. Physical methods include electroporation, bioristic methods, microinjection, lasers, high temperatures, ultrasound, and hydrodynamic gene transfer. Chemical methods include calcium phosphate, DAE-dextran, liposomes, and nanoparticles. In biological methods, viruses are increasingly used for gene introduction; these viruses are integrated into the genome of the host cell, resulting in stable gene expression, whereas other non-integrating viruses are mostly episomal and their expression is diluted in proportion to cell division.

[0266] Combination therapy with targeted therapies and chemotherapy The heterodimer and homodimer fusion proteins disclosed herein may be administered, for example, as single active ingredients, as adjuvants to other agents, such as anticancer agents, immunomodulators, or other anti-inflammatory agents, or in combination thereof, for the treatment or prevention of the aforementioned diseases.

[0267] "Anticancer" or "antiproliferative agent" means any drug that can be used to treat cell proliferation disorders such as cancer, and includes, but is not limited to, therapeutic antibodies, cytotoxic agents, cell activators, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy agents and radiotherapeutic agents, targeted anticancer agents, BRMs, cancer vaccines, cytokines, hormone therapy agents, radiotherapy agents, anti-metastatic agents and immunotherapy agents.

[0268] For example, the fusion proteins described herein can be used in combination with antibodies against tumor-associated antigens, stromal-associated antigens or pathways, such as PD-1 / PD-L1, TIM-3, LAG-3, VEGF, HER2, CTLA-4; antibodies against leukocyte receptors, such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86 or their ligands; CD3 engager antibodies, NK engager antibodies; ADCCs that enable antitumor-associated antigens; monoclonal antibodies against TNF, etc. Antibodies include, but are not limited to, absiximab, adalimumab, alefecept, alemtuzumab, basiliximab, belimumab, bezlotoxumab, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, efalizumab, golimumab, infractra, ipilimumab, ixekizumab, natalizumab, nivolumab, olaratumab, omalizumab, palivizumab, panitumumab, pembrolizumab, rituximab, tocilizumab, trastuzumab, secukinumab, and ustekinumab.

[0269] Antibodies may be monospecific or multispecific. For example, an antibody may be a trispecific antibody (TrAbs or TrioMabs) having two variable segments for antigen binding and an Fc component for recruiting immune cells. An example of a TrAb is cetuximab, used to treat EpCam-positive gastric and ovarian tumors. Antibodies may also be bispecific T-cell engager antibodies (BiTEs), such as blinatumomab, MEHD7945A, ABT-122, and XmAb5871.

[0270] In some embodiments, the heterodimer and homodimer fusion proteins described herein can be used in combination with immunomodulatory compounds, such as recombinant binding molecules having at least a portion of the extracellular domain of CTLA4 or a variant thereof; adhesion molecule inhibitors, such as LFA-1 antagonists, ICAM-1 or 3 antagonists, VCAM-4 antagonists or VLA-4 antagonists; inflammatory cytokine blockers, IL-1 blockers; chemokine blockers; or chemotherapeutic agents.

[0271] For the purposes of this disclosure, “chemotherapeutic agents” include chemical compounds (e.g., cytotoxic agents or cytostatic agents) that nonspecifically reduce or inhibit the proliferation, growth, and / or survival of cancer cells. Such chemical agents are often directed at intracellular processes necessary for cell proliferation or division, and are therefore generally particularly effective against rapidly growing and dividing cancer cells. For example, vincristine depolymerizes microtubules and inhibits cells from entering mitosis. Generally, chemotherapeutic agents include chemical agents that inhibit or are designed to inhibit cancer cells or cells that may become cancerous or cells that may produce oncogenic progeny (e.g., TICs). Such agents are often administered in combination, for example, in regimens such as CHOP and FOLFIRI, and are often most effective in combination. Examples of anticancer agents that may be used in combination with the site-specific constructs of this disclosure (as components of a site-specific conjugate or in an unconjugated state) include, but are not limited to, paclitaxel, gemcitabine, cisplatin, doxorubicin, 5-fiurouracil, capecitabine, combretastatin, and leucovorin.

[0272] For example, the heterodimer and homodimer fusion proteins described herein include DMARDs, e.g., gold salts, sulfasalazine, antimalarial drugs, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, refeunomide, glocorticoids; calcineurin inhibitors, e.g., cyclosporine A or FK506; lymphocyte recirculation modulators, e.g., FTY720 and FTY720 analogs; mTOR inhibitors, e.g., rapamycin. , 40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573 or TAFA-93; ascomycin with immunosuppressive properties, e.g., ABT-281, ASM981; corticosteroids; cyclophosphamide; azathioprene; methotrexate; refeunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxysperguarin or their immunosuppressive homologs, analogs or derivatives; can be used in combination with immunosuppressive agents.

[0273] As described above, in selected embodiments, such anticancer agents may include conjugates and may associate with heterodimer and homodimer polypeptide complexes disclosed prior to administration. More specifically, in certain embodiments, selected anticancer agents are linked to unpaired cysteine ​​in an artificial polypeptide complex to provide an artificial conjugate as defined herein. Thus, such artificial conjugates are expressly intended to be within the scope of this disclosure. In other embodiments, the disclosed anticancer agents are administered in combination with site-specific conjugates containing different therapeutic agents as described above.

[0274] An anti-cancer agent or immunomodulatory agent used in combination with a heterodimeric and homodimeric polypeptide complex as disclosed herein should be compatible with the polypeptide complex, i.e., it will not reduce, interfere with, or eliminate the effects of the polypeptide complex as disclosed herein, and preferably will even provide a modulating or synergistic effect, which will be readily understood.

[0275] As an immune-enhancing component / protein in a multispecific antibody A polypeptide complex as disclosed herein can associate with a second antigen-specific binding moiety to form a multispecific antibody complex. For example, an antigen-binding moiety (e.g., comprising a heavy-chain variable region and a light-chain variable region) can be fused to the N-terminus or C-terminus of the IL-2 moiety. Such a multispecific antibody complex not only has a high affinity for the target antigen but also has the potency of IL-2 to promote the activation of immune cells.

[0276] Combined use with radiotherapy The present disclosure also provides for the combined use of its heterodimeric and homodimeric polypeptide complexes with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells such as gamma-ray irradiation, X-ray irradiation, ultraviolet irradiation, microwave, electron emission, etc.). Combined therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in association with a targeted anti-cancer agent or other targeting means. Typically, radiotherapy is administered in pulses over a period of about one week to about two weeks. Radiotherapy can be administered to a subject having head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple consecutive doses.

[0277] As a non-cytotoxic immunosuppressant In some embodiments, the Disclosure also provides methods for suppressing an immune response in a subject, comprising administering the polypeptide complex of the Disclosure to the subject such that the immune response in the subject is reduced without producing undesirable side effects. For example, the subject is a mammal. In a specific embodiment, the subject is a human.

[0278] The term “suppression of the immune response,” or its grammatical variation, means reducing any response of the mammalian immune system. The immune response may be a cellular response (i.e., a cell-mediated response such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., an antibody-mediated response), and may be a primary or secondary immune response. Examples of suppression of the immune response include increased Treg cell activity and proliferation. Suppression of the immune response can be evaluated using many in vitro or in vivo measurements known to those skilled in the art, including but not limited to Treg lymphocyte assays, cytokine release (e.g., IL-15 production or IFN-γ production), regression of autoimmune disease, survival of autoimmune animals, autoantibody production, proliferation of immune cells, and cytotoxicity. Typically, the methods of this disclosure enhance the immune response in a mammal compared to the immune response in an untreated mammal or a mammal not treated using methods such as those disclosed herein. In one embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-17 production. In another embodiment, the immune response is suppression of B cell activity.

[0279] Polypeptide conjugates such as those disclosed herein may be used alone as monotherapy, but more often they are used in combination with cellular immunotherapy, gene therapy, targeted therapy, or chemotherapy.

[0280] Pharmaceutical packs and kits Pharmaceutical packs and kits are also provided, each containing one or more containers, each containing one or more doses of a polypeptide conjugate. In certain embodiments, unit doses are provided, where the unit dose contains, for example, a predetermined amount of a composition containing a polypeptide conjugate, with or without one or more additional agents. In other embodiments, such unit doses are supplied in single-use pre-filled syringes for injection. In yet another embodiment, the composition contained in the unit dose may be formulated within a stable and effective pH range, such as saline, sucrose, etc.; a buffer such as a phosphate; and / or within a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition may be provided as a lyophilized powder that can be reconstituted by adding a suitable liquid, such as sterile water or saline. In certain preferred embodiments, the composition contains one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container(s) indicates that the enclosed conjugate composition is used to treat a selected neoplastic disease condition.

[0281] This disclosure also provides kits for producing site-specific conjugates and, optionally, single-dose or multi-dose units of one or more anticancer agents. The kit comprises a container and labels or packaging inserts on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic and contain a pharmaceutically effective amount of the disclosed conjugate in conjugate or unconjugate form. In other preferred embodiments, the container includes a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). Such kits generally include a pharmaceutically acceptable formulation of the manipulated conjugate and, optionally, one or more anticancer agents in the same or different containers, in a suitable container. The kit may also include other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the polypeptide conjugates of this disclosure, such kits may include one or more anticancer agents, such as chemotherapeutic agents or radiotherapeutic agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0282] More specifically, the kit may have a single container containing the disclosed polypeptide conjugate, with or without additional components, or separate containers for each desired drug. If the combined therapeutic agent is provided for conjugation, the single solution may be pre-mixed in molar equivalent combinations or with one component in excess of the other. Alternatively, the kit's conjugate and any anticancer drugs may be managed separately in separate containers before administration to the patient. The kit may also include second / third container means for containing sterile, pharmaceutically acceptable buffers or other diluents, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0283] If the kit components are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, with sterile aqueous solutions or physiological saline being particularly preferred. However, the kit components may also be provided as dry powders. If the reagents or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent. The solvent may also be provided in a separate container.

[0284] As briefly outlined above, the kit may also include means for administering the polypeptide complex and any optional components to a patient, such as one or more needles, IV bags or syringes, or droppers, pipettes, or other such instruments, from which the formulation can be injected or introduced into an animal or applied to a diseased area of ​​the body. The kit of the present disclosure also typically includes means for housing vials, or such, and other components in a tightly sealed state for commercial sale, such as injection-molded or blow-molded plastic containers in which the desired vials and other instruments are placed and held.

[0285] Overview of the array list Table A below provides a description of the polypeptide complex constructs, and Tables B-C provide the sequences of the IL-2 variants and each chain of the polypeptide complex as used herein. The designation W3XX115-T2U0.E44-[N].UIGG4V322 (which may be abbreviated as "T2U0.E44-[N]") indicates the structure of the polypeptide complex: [n] is numbering, and "E44" indicates the form shown in Figure 1, which includes two heavy chains and one light chain. The designation W3XX115-T2.Z20-[n].uIgG4V322 (which may be abbreviated as "T2.Z20-[n]" or "Z20-[n]") indicates that the polypeptide complex is in the "Z20" form as shown in Figure 1, each containing two chains with IL-2. W3XX115-T2U10.Z73-[N].UIGG4V322 indicates that the polypeptide complex is in the "Z73" format as shown in Figure 1, and contains one strand containing the IL-2 variant and the other strand containing VHH. "uIgG4V322" indicates an IgG4 FC with the F234A / L235A mutation (EU numbering). "uIgG1V320" refers to an IgG1 FC with the L234A / L235A mutation. For simplicity, the prefix "W3xx115-" may be omitted in this specification.

[0286] Sequence IDs 1-14, 79-103, and 111-114 show the amino acid sequences of the IL-2 domain; Sequence IDs 15-34, 38-78, and 104-110 show the amino acid sequences of the polypeptide complex containing the benchmark antibody used in the example; Sequence IDs 35-37 show the ECD sequences of IL-2RA, β, and γ. "Fused to the N-terminus without a linker" indicates that the IL-2 domain is directly linked to the hinge region.

[0287] [Table 1]

[0288] [Table 2-1]

[0289] Table 2-2

[0290] Table 2-3

[0291] Table 2-4

[0292] Table 3-1

[0293] Table 3-2

[0294] Table 3-3

[0295] Table 3-4

[0296] Table 3-5

[0297] Table 3-6

[0298] Table 3-7

[0299] Table 3-8

[0300] Table 3-9

[0301] Table 3-10

[0302] Table 3-11

[0303] Table 3-12

[0304] Table 3-13

[0305] Table 3-14

[0306] Table 3-15

[0307] Table 3-16

[0308] Table 3-17

[0309] Table 3-18

[0310] [Table 4] [Examples]

[0311] While this disclosure is generally described as such, it will be more readily understood by referring to the following examples. The examples are not intended to represent that the following experiments are all or only experiments.

[0312] Example 1 Preparation of materials, IL-2 variants, and fusion proteins 1.1 Preparation of materials Table 1 shows information regarding the commercially available materials used in the examples.

[0313] [Table 5-1]

[0314] [Table 5-2]

[0315] 1.2 Construction of IL-2 / Fc polypeptide complexes containing wild-type IL-2 or IL-2 variants Plasmid construction for IL-2 / Fc polypeptide complex expression The structure of the IL-2 / FC polypeptide complex is shown in Figure 1. The E44 form indicates that one IL-2 domain is operably ligated to the N-terminus of one strand of the FC region, and the FAB is operably ligated to the N-terminus of the other strand of the FC region. The Z20 form indicates that two IL-2 domains are operably ligated to the N-terminus of one strand of the FC region, respectively. The Z73 form indicates that one IL-2 domain is operably ligated to the N-terminus of one strand of the FC region, and two VHHs are operably ligated to the N-terminus of the other strand of the FC region.

[0316] Polynucleotides encoding the antigen-binding regions VL, VH, Ck, CH1, and VHH from the antibody were amplified by PCR from a DNA template. Polynucleotides encoding wild-type IL-2 and IL-2 variants were synthesized by Sangon Biotech Inc. Polynucleotides encoding the native light chain sequence of the antibody were inserted into a linearized vector containing a CMV promoter and a κ or λ signal peptide. DNA fragments of the anti-target VH-CH1 and wild-type IL-2 or IL-2 variant were inserted, according to format, into a linearized vector containing the constant region CH2-CH3 of human IgG1 or IgG4 with or without a (G4S)n linker, or hinged IgG4. The vectors contained a CMV promoter and a human antibody heavy chain signal peptide.

[0317] The benchmark antibodies W3xx115-BMK7, W3xx115-BMK8, and W3xx115-BMK14 are E44-format benchmark polypeptide complexes containing specific IL-2 variants. W3xx115-BMK7 consists of an IL-2 variant with 101, 141, and 169 amino acid deletions and an L53R+Q162H substitution (SyntheKine). W3xx115-BMK8 contains an IL-2 variant with L18R, Q22E, and Q126K substitutions (SyntheKine). W3xx115-BMK14 contains an IL-2 variant with an N88D substitution. W327199-BMK1 and W327199-BMK2 are F114-format benchmark polypeptide complexes containing IL-15 instead of the IL-2 domain.

[0318] Table 2 shows the generated IL-2 / FC polypeptide complexes and their corresponding IL-2 forms (WT or variants). The ID or designation of the IL-2 / FC polypeptide complexes indicates their structure.

[0319] [Table 6-1]

[0320] [Table 6-2]

[0321] [Table 6-3]

[0322] [Table 6-4]

[0323] Generation of IL-2 / FC polypeptide complex in EXPI293 cells Expi293 cells (Thermofisher, A14635) or ExpiCHO cells (Thermofisher, A29133) were prepared for protein expression and diluted in pre-warmed Expi293 Expression Medium. The transfection reagents included A and B. Reagent A was prepared by adding a plasmid to pre-warmed Opti-MEM, and reagent B was prepared by adding the transfection reagent to Opti-MEM. Reagents A and B were then gently mixed and incubated at room temperature for 20 minutes. For transfection, the above mixture was added to the cells and incubated on a shaker at 37°C, 8% CO2, and 120 rpm for 18-20 hours. After transfection, Enhancer 1 and Enhancer 2 were added to the culture medium, and the cells were cultured for a further 5 days, after which the supernatant was collected.

[0324] Purification of the IL-2 / FC polypeptide complex The supernatant from the above-mentioned Expi293 or ExpiCHO cells was collected and filtered for purification using a Protein A column (GE Healthcare, Cat. 175438) or a Protein G column (GE Healthcare, Cat. 170618). The concentration of the purified Fc-polypeptide complex was measured by absorbance at 280 nm. Molecular weight and purity were tested by SDS-PAGE and SEC-HPLC, respectively.

[0325] The Fc polypeptide complexes of IL-2 and its variants were generated with a purity of over 90%, demonstrating that they are intact and well-associated molecules under physiological conditions.

[0326] 1.3 Production of IL-2Rα, IL-2Rβ, and IL-2Rγ Polynucleotides encoding the extracellular domains of IL2Rα (CD25, SEQ ID NO: 135), IL2Rβ (CD122, SEQ ID NO: 136), and IL2Rγ (CD132, SEQ ID NO: 137), all possessing a 6×His tag at the C-terminus, were synthesized by Sangon Biotech Inc. Vectors containing the CMV promoter were transfected into Expi293 cells. The supernatant from the transfected Expi293 cells was collected as described above and filtered for purification using a Ni-column (GE Healthcare, Cat. 173712). The concentration of the purified His-tagged protein was measured by absorbance at 280 nm, and molecular weight and purity were tested by SDS-PAGE and SEC-HPLC, respectively.

[0327] Example 2 In vitro characteristics of the IL-2 variant 2.1 Human CD8+ T cell pSTAT5 activation assay STAT5 is a downstream signaling maker strictly related to T cell activation. Human quiescent CD8+ T cells in PBMCs were incubated with WT IL-2 or IL-2 variant-containing IL-2 polypeptide complexes for 30 minutes, and then analyzed for STAT5 phosphorylation. Immediately after processing, PBMCs were fixed with BD Phosflow Fix Buffer I and then incubated with pre-chilled BD Phosflow Perm Buffer III. Following incubation and fixation, cells were stained with anti-CD3, anti-CD4, and anti-CD8 antibodies at room temperature for 30 minutes. Subsequently, cells were permeabilized by treatment with Perm Buffer III and stained with anti-pSTAT5 antibody for 30 minutes. Lymphocytes were first gated based on SSCs and FSCs, then based on CD3, and then based on CD4 and CD8 expression to identify CD8+ T cells. Finally, the phosphorylation levels of STAT5 in the CD8+ T population were measured.

[0328] Primary human CD8+ T (intermediate affinity receptor, expressing IL-2Rβ / γc) was activated by an IL-2 polypeptide complex containing an IL-2 variant, which was reflected in varying degrees of STAT5 phosphorylation (Figure 2, Table 3). T2U0.E44-46 showed a 7.6-fold decrease in potency compared to T2U0.E44-1 (WT IL-2), followed by T2U0.E44-44 (11-fold), T2U0.E44-43 (17-fold), T2U0.E44-6 (18-fold), T2U0.E44-47 (27-fold), T2U0.E44-45 (31-fold), and T2U0.E44-40 (37-fold). The activation effect (maximum MFI) induced by T2U0.E44-40 was weaker than the others. Truncation and substitution reduced the affinity of IL-2 to the IL-2Rβ / γc complex, while the stabilization design did not impair the efficacy of the IL-2 variant in vitro.

[0329] E110R or K32D were cleaved into IL-2 mutaine and combined with I129L to further attenuate their potency against the IL-2Rβ / γc complex. As shown in Table 4 and Figure 3b, the IL-2 variants T2U0.E44-48 and T2U0.E44-49 showed equivalent attenuations of 2.7-fold and 3.8-fold, respectively, compared to W3XX115-T2U0.E44-1.uIgG4V322.

[0330] [Table 7]

[0331] 2.2 pSTAT5 activation assay of human activated CD8+ T cells Human CD8+ T cells were isolated from fresh PBMCs using the EasySep® Human CD8+ T Cell Isolation Kit (Stemcell-17953). Human CD8+ T cells were then expanded using the Human T Cell Activation / Expansion Kit (Miltenyi 130-091-441). Human activated CD8+ T cells (expressing high-affinity receptors, IL-2Rα / β / γc) were also analyzed for STAT5 phosphorylation after 30 minutes of incubation with the indicated IL-2 variant. Expanded human activated CD8+ T cells were manipulated in the same manner as described above for standard CD8+ T cells.

[0332] In Figure 3a and Table 4, monovalent IL-2 variants showed decreased potency in activated CD8+T, with T2U0.E44-48 and 49 showing 15-fold and 22-fold decreases in potency compared to T2U0.E44-1, respectively, and being slightly more efficient than BMK8.

[0333] Divalent IL-2 variants showed extreme potency recovery in activated CD8+T, with potency equal to or greater than that of WT IL-2, with a decrease factor of 0.3–0.9 (Table 4). However, the potency of divalent IL-2 in primary CD8+T was similar to or much weaker than that of monovalent variants. Therefore, the potency ratios of IL-2 variants in activated primary CD8+T differed significantly between monovalent and divalent IL-2. Divalent IL-2 variants had potency ratios of activated CD8+T / primary CD8+T exceeding 10,000 and shared similar characteristics to BMK8(∞). As a result, divalent Z20-1, Z20-2, Z20-4, and Z20-5 were biased towards IL-2Rα (i.e., CD25) binding and were stronger than WT IL-2. These results suggest that the dimeric IL-2 variant is more promising in terms of antitumor effects, given that IL-2Rα binding is thought to be necessary for IL-2 to exert its biological function.

[0334] [Table 8]

[0335] Example 3 Stability of IL-2 variants in vitro and in vivo 3.1 Differential Scanning Fluorescence (DSF) A DSF assay was 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 value of the negative derivative of the obtained fluorescence data with respect to temperature. Ton can be roughly determined as the temperature at which the negative derivative plot begins to decrease from the baseline before the transition.

[0336] Table 5 shows the thermal stability of IL-2 variants. Monovalent IL-2 variants showed acceptable Tm1. W3XX115-T2U0.E44-49.uIgG4V322 showed a higher Tm than W3XX115-T2U0.E44-26.uIgG4V322, indicating that the K32D mutation can stabilize the IL-2 molecule and improve its thermal stability. The bivalent IL-2 variants Z20-1 and Z20-2 showed lower Tm1 compared to the monovalent protein. In particular, Z20-5 showed significantly improved thermal stability with a Tm1 of 62.6°C. This is consistent with the difference between T2U0.E44-49 and T2U0.E44-26.

[0337] [Table 9]

[0338] 3.2 Serum Stability Assay The stability of the divalent IL-2 variant in mouse serum was further investigated. In the serum stability assay, Z20-1, Z20-4, Z20-5, and BMK8 were diluted 1:9 with mouse serum. After storage at 37°C and 4°C for 4, 7, 10, and 14 days, samples were collected, immediately frozen in liquid nitrogen, and stored at -80°C. Once all samples were available, efficacy measurements were performed on activated CD8+T.

[0339] As shown in Figure 4, Z20-1 and Z20-4 gradually decreased in potency from 4 to 14 days after incubation in mouse serum at 37°C. The monovalent BMK8 variant was more stable in mouse serum, and the decrease in efficiency (maximum MFI) at 37°C was mild. In contrast, Z20-5 maintained its potency and efficiency even after 14 days of culture at 37°C, and its cell-activating ability did not change over time, as shown in Figure 4d. These data indicate that the K32D mutation in the Z20-5 variant stabilizes IL-2 and significantly improves serum stability. All variants in mouse serum were able to maintain their potency at 4°C.

[0340] 3.3 Pharmacokinetics (PK) A mouse pharmacokinetic study was conducted to investigate the in vivo pharmacokinetics of a divalent IL-2 variant. Female C57BL / 6 mice (8-9 weeks old) from Charles River were randomly assigned to two treatment groups and intravenously injected with different doses of the IL-2 variant. Samples were also collected for immunological and pharmacokinetic analysis. Blood samples were collected in additive-free tubes and kept on ice until processing. All samples were processed within 2 hours of collection.

[0341] Drug concentrations in serum were measured by ELISA. Briefly, ELISA plates were coated with 1 μg / mL goat anti-human IgG Fc (Southern Biotech, 2049-01), and the IL-2 variant concentration in plasma was detected using goat anti-human IgG Fc (Southern Biotech, SB-2049-08), followed by HRP-Streptavidin (Thermo-21127) and TMB substrate (Life Technologies, 002023). The absorbance of the wells was measured at (450-540) nm using a multi-wall plate reader (SpectraMax® M5e). Standard curves were prepared according to standard samples, and serum samples were analyzed using SoftMax.

[0342] As shown in Figure 5, the K32D mutation in Z20-5 significantly prolonged the serum half-life compared to Z20-1. Z20-1 showed rapid clearance during the distribution and excretion phases. Z20-5 (7.2 mg / kg) and BMK7 (10 mg / kg) showed similar C (0)を Furthermore, clearance was significantly slower. Additionally, Z20-5 exhibited linear and dose-response pharmacokinetics, was non-toxic, and produced no ADA. PK results were consistent with DLS and serum stability data. K32D substitution of the divalent IL-2 variant contributed to extended drug exposure and improved pharmacodynamics.

[0343] 3.4 Modeling of the Z20-5 To demonstrate how K32D stabilized Z20-5, the full-length sequence of Z20-5, along with the reported crystal structure of IL-2, was used for modeling analysis using Discovery Studio and Gromacs.

[0344] The diagram is shown in Figure 6a, where the substitution of K to D at position 32 (K32D) forms a salt bridge with K76, thus stabilizing the IL-2 monomer and improving stability and pharmacokinetics. The interaction between K32D and K76 also induces a minor conformational change in helix A of IL-2, thus affecting the binding of IL-2 to the common γ chain, which contributes to further decay of affinity and activity.

[0345] In the divalent IL-2 variant (Figure 6b), a K32D mutation to the opposite charge can form a salt bridge with K76 of the same IL-2 moiety. This, along with the resulting conformational change, can reduce the agglutination tendency of the IL-2 variant in the Z20 form, thus stabilizing the divalent form and resulting in improved serum stability (Figure 4), extended half-life (Figure 5), and better thermal stability (Table 5).

[0346] Example 4 In vivo pharmacology of IL-2 variants All procedures related to the handling, care, and treatment of animals in this study were carried out in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi Biologics' LARC.

[0347] 4.1 Antitumor effect To investigate the antitumor effect of Z20-5, a stable divalent IL-2 variant, the MC38 syngeneic model, was used. MC38 cells were transplanted into the right flank of C57BL / 6N mice. The tumor volume was approximately 60-80 mm².3 Once the tumor was reached, mice with tumors were randomly divided into different groups and administered an IL-2 variant on days 0 and 3. Tumor size was measured two-dimensionally using calipers, and volume was measured in mm². 3 The results were expressed as the mean and standard error (mean ± SEM). Statistical analysis was performed using two-way ANOVA, with P<0.05 considered statistically significant.

[0348] First, as shown in Figure 7, all IL-2 variants that retain CD25 binding (Z20-1, Z20-5, BMK7) showed significant antitumor effects in the MC38 syngenic model. This indicates that IL-2Rα binding is essential for IL-2 variants to exert antitumor effects by stimulating activated CD8+T.

[0349] In the case of the divalent IL-2 variant, high-dose Z20-1 and Z20-5 (7.2 mg / kg, equivalent to 10 mg / kg of BMK7) showed superior antitumor efficacy compared to BMK7, with TGI rates exceeding 77% (101% and 98%). Furthermore, the medium-dose Z20-5 (4.3 mg / kg) also showed superior tumor suppression efficacy compared to 10 mg / kg of BMK7. In addition, while the mean TGI of Z20-5 was similar to that of Z20-1 at the same dose, Z20-5 induced a 100% complete response (CR) rate after treatment, compared to a 60% CR rate at day 17 for Z20-1. These results indicate that improved stability and extended pharmacokinetic (PK) lead to greater antitumor efficacy.

[0350] 4.2 Toxicity of IL-2 Variants For toxicity testing, lungs were collected from three mice in each group at the end of the MC38 trial (4.1). The lungs were fixed in formalin, embedded in paraffin, and examined for inflammatory cell infiltration by intravascular coagulation (IHC).

[0351] As shown in Figure 8, inflammatory cell infiltration was minimal in the lungs of all treatment groups. These data indicate that the toxicity of Z20-5 is limited, and that improved stability and long-term PK do not lead to cumulative toxicity.

[0352] Based on the results of PK, efficacy, and toxicity, Z20-5 is a stable divalent IL-2 variant that exhibits prolonged PK and good antitumor efficacy.

[0353] Example 5 In vitro and in vivo characterization of PD-1 / IL-2 fusion proteins 5.1 PD-1 Binding Assay To measure the PD-1 binding profile, a CHO-PD1 modified cell line was used. Cells were incubated with PD-1 / IL-2 fusion protein at 4°C for 30 minutes. Binding was detected with a PE-anti-human Fc secondary antibody for a further 30 minutes at 4°C. The fluorescence intensity (MFI) of CHO-PD1 cells was measured using FACS.

[0354] As shown in Figure 9, the Z73-formatted PD-1 / IL-2 fusion protein exhibited a PD-1 binding profile comparable to that of W3XX115-BMK14. W3XX115-U3T3.F114-1.uIgG4V322 and W3XX115-T2U3.E44-15.uIgG4V322 also showed similar affinity to the Z73-formatted PD-1 / IL-2 fusion protein, although their maximum binding MFI was slightly lower, which may be dominated by differences in PD-1 sequences.

[0355] 5.2 pSTAT5 activation assay of PD-1 / IL-2 fusion protein STAT5 analysis was performed in the same manner as described above, and the phosphorylation levels of STAT5 in primary and activated CD8+ T cell populations were measured. Activated CD8+ T cells were isolated from fresh PBMCs and activated for 5-7 days using a human T cell activation / expansion kit (Miltenyi 130-091-441). Activated CD8+ T cells were CD25-positive and PD-1-positive.

[0356] As a result, E44-formula PD-1 / IL-2 fusion proteins showed varying degrees of decreased potency against primary CD8+ T cells (PD-1-) induced by attenuation of IL-2Rβγ. In activated CD8+ T cells (PD-1+), all PD-1 / IL-2 fusion proteins were more potent than in primary CD8+ T cells (PD-1-). T2U3.E44-6 and T2U3.E44-26 (maintaining binding to IL-2Rα) showed stronger activity in activated CD8+ T cells compared to primary cells, with PD-1+ / PD-1 ratios of 3473 and 4205, respectively. Additionally, T2U3.E44-20, which has weak IL-2Rα binding, also showed a bias towards PD-1+ activated CD8+ T cells, with a ratio of 669. In IL-2 variants without IL-2Rα binding, T2U3.E44-15 and 33 had PD-1+ / PD-1- ratios of 496 and 283, respectively, indicating a bias towards PD-1 alone.

[0357] Furthermore, the IL-2 variant of the Z73-formula PD-1 / IL-2 was designed to eliminate IL-2Rα binding, and the activated CD8+ T cell bias was dominated by the PD-1 anchor. T2U10.Z73-50 / 51 / 52 / 53 / 54 showed attenuated efficacy against primary T cells and activated CD8+ T cells to varying degrees (Figure 11 and Table 7). PD-1 fusion showed superior efficacy against activated CD8+ T cells.

[0358] Further IL-2 variants were designed and fused with PD-1 antibodies. T2U10.Z73-55 to T2U10.Z73-64 showed further attenuation against IL-2Rβγ, as shown in Figure 12 and Table 8.

[0359] [Table 10]

[0360] [Table 11]

[0361] [Table 12]

[0362] 5.3 Antitumor effect To investigate the antitumor effect of the PD-1 / IL-2 fusion protein, a CT-26 synchronicity model was used. CT-26 cells were transplanted into the right flank of Balb.c mice. The tumor was approximately 60-80 mm in size. 3 Once the target was reached, mice with tumors were randomly divided into different groups and administered PD-1 / IL-2 fusion protein on days 0 and 3. Tumor size was measured two-dimensionally using calipers, and volume was measured in mm². 3 The results were expressed as the mean and standard error (Mean ± SEM). Statistical analysis was performed using two-way ANOVA, with P<0.05 considered statistically significant.

[0363] As shown in Figure 13, CT-26 is a PD-1 resistance model and showed a partial response to IL-2 (W3XX115-T2U0.E44-1.uIgG4V322). PD-1 fused IL-2 or IL-15 showed a greater tumor suppressor effect than IL-2 alone, and T2U3.E44-6 / 20 / 33 showed a similar effect to T2U3.E44-26, and was superior to T2U3.E44-26. T2U3.E44-15 showed the most significant antitumor effect, slightly better than W327199-BMK1. As a result, PD-1 binding sites fused with non-αIL-2 showed efficient tumor suppressor effects.

[0364] Those skilled in the art will further understand that the present invention can be embodied in other specific forms without departing from its spirit or central attributes. It should be understood that the foregoing description of this disclosure provides only exemplary embodiments, and other modifications are intended to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, the appended claims should be used to illustrate the scope and content of the invention.

Claims

1. A composition comprising a polypeptide complex as an active ingredient or a nucleic acid molecule(s) encoding the polypeptide complex and an excipient, The polypeptide complex comprises an interleukin-2 (IL-2) variant domain, a first dimerization domain, and a second dimerization domain. The IL-2 variant domain comprises one or more mutants selected from the following: (1) cleavage of 1 to 20 amino acids from the C-terminus of SEQ ID NO: 1, and / or (2) substitution of one or more amino acid residues at positions selected from 32, 129, 13, 18, 19, 20, 22, 28, 38, 42, 52, 71, 76, 78, 82, 84, 87, 88, 89, 91, 92, 94, 95, 110, 119, 122, 123, 125 and 126 of SEQ ID NO: 1, The composition wherein the first dimerization domain and the second dimerization domain associate together to form a dimer.

2. The composition according to claim 1, wherein the polypeptide complex or nucleic acid molecules encoding the polypeptide complex constitute less than 90% by weight, less than 80% by weight, less than 70% by weight, less than 60% by weight, or less than 50% by weight of the composition.

3. The composition according to claim 1 or 2, wherein the IL-2 variant includes cleavage of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid from the C-terminus of SEQ ID NO: 1, for example, cleavage of 4 amino acids.

4. The composition according to any one of claims 1 to 3, wherein the IL-2 variant includes a K32D substitution.

5. The composition according to any one of claims 1 to 3, wherein the IL-2 variant comprises or consists of the amino acid sequences shown in any of SEQ ID NOs: 13, 12, 2-3, 79-103, 4-11, and 111-114.

6. The composition according to any one of the preceding claims, wherein the polypeptide complex comprises two IL-2 variants in two chains, each chain comprising one IL-2 variant from the N-terminus to the C-terminus, operably linked to one dimerization domain.

7. The composition according to any one of claims 1 to 5, wherein the polypeptide complex further comprises one or more antigen-binding moieties, for example, the antigen-binding moieties being in the form of Fab, VHH, or scFv.

8. The polypeptide complex contains one IL-2 variant and one antigen-binding moiety in Fab form, and the polypeptide complex contains two heavy chains and one light chain, from the N-terminus to the C-terminus, The first heavy chain comprises an IL-2 variant operably linked to the first dimerization domain; The second heavy chain comprises a Fab heavy chain operably linked to the second dimerization domain; The composition according to claim 7, wherein the light chain comprises a light chain of Fab.

9. The polypeptide complex contains one IL-2 variant and two antigen-binding moieties in VHH form, and the polypeptide complex contains two chains, from the N-terminus to the C-terminus, The first chain comprises an IL-2 variant operably linked to the first dimerization domain; The composition according to claim 7, wherein the second chain comprises two VHHs in tandem operably linked to the second dimerization domain.

10. The polypeptide complex contains one IL-2 variant and one antigen-binding moiety in VHH form, and the polypeptide complex contains two chains, from the N-terminus to the C-terminus, The first chain comprises an IL-2 variant operably linked to the first dimerization domain; The composition according to claim 7, wherein the second chain comprises a VHH operably linked to the second dimerization domain.

11. The polypeptide complex contains two IL-2 variants and two antigen-binding moieties in Fab form, and the polypeptide complex contains two heavy chains and two light chains, from the N-terminus to the C-terminus, The heavy chain comprises an IL-2 variant operably coupled to a first or second dimerization domain operably coupled to the heavy chain of Fab; The composition according to claim 7, wherein the light chain comprises a light chain of Fab.

12. The polypeptide complex comprises two IL-2 variants and two antigen-binding moieties in the form of VHH or scFv, and the polypeptide complex comprises two chains, from the N-terminus to the C-terminus, The composition according to claim 7, wherein each chain comprises an IL-2 variant operably linked to a first or second dimerization domain operably linked to a VHH or scFv.

13. The antigen-binding moiety includes tumor-associated antigens (TAAs), I / O checkpoints, tumor microenvironment targets, autoimmune-related targets, and inflammatory disease-related targets, such as PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM-3, TIM4, 4-1BB, OX-40, OX-40L, GITR, A2aR, TIGIT, CD96, PVRIG, and CD2. 26, 5T4, VISTA, VSIG3, VSIG4, ICOS, CD28, CD3, CD4, CD8, CD45, CD44v6, CD27, CD47, SIRPAα, S LAMF7, CD24, Siglec10, Siglec15, Siglec8, VSIR, VSIG4, PSGL-1, C5AR1, BTN1A1, BTN3A1, CD 70, RANKL, CSF1R, CSF2RB, TNFRSF1 / 1a / 1b, BDCA2, BTLA, C5aR, NKG2A, NKG2D, NKp30, NKp46, CD16a, CD56, CD166, FCGR3, CD2, Neurophilin-1, CCR8, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, GCGR, CXCR2, CXCR4, CXCR5, CALCRL, ETAR, GLP1R, CX3CR1, GPR1, GPR17, GPR20, GPR30, GPR34, GPR-6 5, 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, Calzin6, Calzin18.2, 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 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, CD4 6, 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, GloboH, IGF-1R, TEM1, TEM8, TAX1BP3, ADAM-9, ENPP3, EphA2, E phA3, FcRH5, NaPi3b, TWEAK, DLK1, SORT1, SSTR2, STEAP1, CD25, CD39, GARP, LRRC33, LAIR1, LAMP3, LAP, LEPR, LILRB1, LILRB2, LILRB4, RAGE, FGL1, TPBG, PDGFRB, TGFBR2, CEACAM1, CEACAM5, CEACAM6, Carbofetal Antigen (CEA), ICAM1, A33, CAMPATH-1 (CDw52), Carboanhydrase IX (MN / CAIX), CD248, PDPN, ITGB1, ITGAV, CD20, CD19, CD21, CD22, CLL, BCMA, DCLK1, DDR1, DLK1, DPEP3, DKK1, CD5, CD 13, CD30, CD33, CD34, CD36, CD37, CD38, CD43, CD52, CD55, CD94, CD99, CD7, CD71, CD73, CD74, CD79A, CD79B, C D229, CD132, CD133, G250, CSF1R (CD115), HLA-DR, HLA-G, HTRA1, TRA-1-60, IGFR, IL-2 receptor, MCSP, ART1, ASGR 1, 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, CSF2RB, chondroitin sulfate proteoglycan 4 (CSPG4), ITGA4, Clec5a, Clec7a, Clec9a, Clec12a, CLEC14, CD205, CD206, CD200R1, CD228, CD229, C A composition according to any one of claims 7 to 12, which specifically binds to an antigen selected from D40, CD40L, FcRn, TLR8, TLR9, TNFR2, LTBR, ​​CD44, CD93, PDGF, PDGFR-α (CD140a), PDGFR-β (CD140b), CD146, CD147, CRTH2, TNF-α, TGF-β, IL1RAcP, TSLP, DR5, ST2, fibroblast-activating protein (FAP), CDCP1, Derlin1, Tenacin, frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), endoglin, and Tie2.

14. The composition according to any one of claims 1 to 13, wherein the first dimerization domain is one chain of the immunoglobulin Fc region, and the second dimerization domain is the other chain of the immunoglobulin Fc region, and optionally the Fc region further comprises part or all of the hinge region.

15. The composition according to claim 14, wherein the Fc region is an IgG4, IgG1, IgG2, or IgG3 Fc region, and optionally comprises one or more substitutions compared to wild-type human Fc to promote heterodimerization or homodimerization, extend half-life, alter effector function, or remove N-glycosylation.

16. The Fc region is: (a) Human IgG4 Fc regions that have been modified to optionally include one or more of the following: S228P mutations, F234A / L235A mutations, M252Y / S254T / T256E mutations, and "knob-into-hole" structures; and (b) Human IgG1 Fc region that has been modified to optionally include one or more of the following: L234A / L235A mutation, M252Y / S254T / T256E mutation, G236R / L328R mutation, and a "knob-into-hole" structure. A composition according to claim 15, selected from the following.

17. The composition according to any one of claims 1 to 16, wherein an IL-2 variant and / or antigen-binding moiety is operably linked to an Fc region via a linker, and optionally the linker is a GS linker, for example, a (G4S)n linker (where n is an integer greater than or equal to 0).

18. The polypeptide complex has the following characteristics: (a) Improved stability compared to otherwise identical polypeptide complex containing wild-type IL-2 instead of IL-2, where stability is one or more selected from heat resistance (e.g., measured by DLS), serum stability, extended serum half-life (e.g., measured by pharmacokinetic analysis), and structural stability; (b) Reduced binding affinity to at least one of the IL-2Rα, IL-2Rβ / γc, and IL-2Rα / β / γc complexes compared to other identical polypeptide complexes containing wild-type IL-2 instead of the IL-2 variant; and (c) Reduced activity compared to the same polypeptide complex, which otherwise contains wild-type IL-2 instead of IL-2. A composition according to any one of claims 1 to 17, having one or more of the following.

19. The composition according to claim 6 or 18, wherein the polypeptide complex comprises any of the amino acid sequences of SEQ ID NOs: 32, 29, 30, and 31.

20. The composition according to claim 13, wherein the antigen-binding portion specifically binds to PD-1.

21. Polypeptide complex, A first heavy chain containing any of the amino acid sequences of sequence numbers 52-56; A second heavy chain containing the amino acid sequence of SEQ ID NO: 57; and Light chain containing the amino acid sequence of SEQ ID NO: 58 The composition according to claim 8, comprising:

22. polypeptide complex A first chain containing any of the amino acid sequences of SEQ ID NOs. 59-74, 76-78, and 104-110; The second chain containing the amino acid sequence of SEQ ID NO: 75 The composition according to claim 9, comprising:

23. The first heavy chain contains any of the amino acids from SEQ ID NOs: 17-28; The second heavy chain contains the amino acid sequence of SEQ ID NO: 33; The composition according to claim 8, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

34.

24. An interleukin-2 (IL-2) variant, wherein the IL-2 variant comprises one or more mutations selected from: (1) cleavage of 20 amino acids from the C-terminus of SEQ ID NO: 1, and / or (2) substitution of one or more amino acid residues at positions selected from 13, 18, 19, 20, 22, 28, 32, 38, 42, 52, 71, 76, 78, 82, 84, 87, 88, 89, 91, 92, 94, 95, 110, 119, 122, 123, 125, 126 and 129 of SEQ ID NO:

1.

25. A polypeptide complex comprising an interleukin-2 (IL-2) variant domain, a first dimerization domain, and a second dimerization domain, The IL-2 variant domain comprises one or more mutations selected from (1) cleavage of 20 amino acids from the C-terminus of SEQ ID NO: 1, and / or (2) substitution of one or more amino acid residues at positions selected from 13, 18, 19, 20, 22, 28, 32, 38, 42, 52, 71, 76, 78, 82, 84, 87, 88, 89, 91, 92, 94, 95, 110, 119, 122, 123, 125, 126 and 129 of SEQ ID NO: 1, The polypeptide complex wherein the first dimerizing domain and the second dimerizing domain associate together to form a dimer.

26. The polypeptide complex according to claim 25, wherein the IL-2 variant domain comprises any of the amino acid sequences described in SEQ ID NOs: 81-103 and 112-114.

27. The polypeptide complex comprises one IL-2 variant and two antigen-binding moieties in VHH form, and includes two chains from the N-terminus to the C-terminus. The first chain comprises an IL-2 variant operably linked to the first dimerization domain; The polypeptide complex according to claim 25, wherein the second chain comprises two VHHs in tandem operably linked to the second dimerization domain.

28. The first chain comprises any of the amino acid sequences of SEQ ID NOs. 59-74, 76-78, and 104-110; The polypeptide complex according to claim 27, wherein the second chain comprises the amino acid sequence of SEQ ID NO:

75.

29. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the IL-2 variant described in claim 24 or the polypeptide complex described in any one of claims 25 to 28.

30. A vector or host cell comprising the nucleic acid molecule described in claim 29.

31. A pharmaceutical composition comprising a polypeptide complex according to any one of claims 25 to 28 or a nucleic acid molecule according to claim 29, and a pharmaceutically acceptable carrier.

32. A method for improving the stability and / or pharmacokinetic properties of IL-2 polypeptides compared to wild-type IL-2, (a) A step of introducing a substitution at one or more positions selected from positions 32, 28, 52, 76, 78, 82, 129, 110, and 122 of the amino acid sequence of SEQ ID NO: 1, and cleaving 1 to 20 amino acids from the C-terminus; (b) optionally, a step of fusing the IL-2 polypeptide to a non-IL-2 portion that extends its half-life. The above method, including.

33. The method according to claim 32, wherein the substitution at position 32 is K32D.

34. The method according to claim 32 or 33, wherein one or more of the stability features are selected from heat resistance, serum stability, serum half-life extension, and structural stability.

35. The method according to any one of claims 32 to 34, for obtaining an IL-2 polypeptide comprising a polypeptide complex containing any of the amino acid sequences of SEQ ID NOs. 32, 29, 30, and 31.

36. A method for modulating an immune response in a subject, comprising administering to the subject the composition according to any one of claims 1 to 23 or the polypeptide complex according to any one of claims 25 to 28, wherein the immune response is optionally NK cell, CD8+ cell, or CD4+ T cell (particularly Treg) related.

37. A method for treating or preventing cancer in a subject, comprising administering an effective amount of the composition according to any one of claims 1 to 23 or the polypeptide complex according to any one of claims 25 to 28 to the subject.

38. The method according to claim 32, further comprising administering additional antitumor therapies such as cellular immunotherapy, targeted therapy, chemotherapy, and gene therapy (e.g., gene therapy using lentivirus, AAV, poxvirus, herpes zoster virus, oncolytic virus, or other RNA / DNA vectors).

39. The method according to claim 32 or 33, wherein the cancer is selected from colon cancer, breast cancer, lung cancer (NSCLC, etc.), ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma (non-Hodgkin lymphoma and diffuse large B-cell lymphoma, etc.), leukemia (chronic lymphocytic leukemia, etc.), and multiple myeloma, and the cancer may be PD-1 related cancer.

40. A method for treating or preventing an autoimmune disease or inflammatory disease in a subject, comprising administering an effective amount of the composition according to any one of claims 1 to 23 or the polypeptide complex according to any one of claims 25 to 28 to the subject.

41. The method according to claim 35, wherein the autoimmune disease or inflammatory disease is selected from inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, aplastic anemia, celiac disease, type 1 diabetes mellitus, Graves' disease, psoriasis, and scleroderma.

42. Use of the composition according to any one of claims 1 to 23 or the polypeptide complex according to any one of claims 25 to 28 in the manufacture of a pharmaceutical product for treating or preventing cancer, autoimmune disease, or inflammatory disease.

43. A composition according to any one of claims 1 to 23 or a polypeptide complex according to any one of claims 25 to 28, for use in the treatment or prevention of cancer, autoimmune diseases or inflammatory diseases.

44. A kit comprising a container containing the composition according to any one of claims 1 to 23 or the polypeptide complex according to any one of claims 25 to 28.

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