Slow-release cytokine conjugates

Abstract

To provide improved agents to enable low-dose, long-term therapies for various diseases including cancers and autoimmune disorders using cytokines.SOLUTION: The present disclosure provides cytokines and variants thereof having an attached releasable linker suitable for conjugation of the proteins to macromolecular carriers. These linkers control the rate of release of the proteins from the carrier, thus determining the concentration and duration of the cytokine or variant in the body.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 839,112, filed April 26, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application has been submitted with a Sequence Listing in electronic format, which is provided as a file entitled 670572002240SeqList.txt, created on April 24, 2020, and having a size of 26,622 bytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Technical Field The present disclosure relates generally to releasable cytokine conjugates and methods of using same. [Background technology]

[0004] Cytokines are small (up to ~20 kDa) proteins involved in cell signaling and include the broad categories of interleukins (ILs), interferons (IFs), tumor necrosis factors (TNFs), chemokines, and lymphokines. They are produced by a wide range of cells and are particularly important in the immune system, regulating the balance between humoral and cellular immune responses. Interleukins constitute one group of cytokines that play a particularly important role in immunity. The majority of interleukins are expressed in helper CD4 T lymphocytes and promote the development and differentiation of T and B lymphocytes and hematopoietic cells.

[0005] Interleukin-2 (IL-2) (SEQ ID NO: 1) is a ~16 kDa cytokine important in the natural response to microbial infection and in discriminating between native and foreign cells. IL-2 plays an essential role in the immune system, tolerance, and key immune functions, primarily through its direct effects on T cells. In the thymus, where T cells mature, specific immature T cells give rise to regulatory T cells (T reg IL-2 prevents autoimmune diseases by promoting the differentiation of regulatory T cells into effector T cells (T cells) that suppress other T cells that are primed to attack normal, healthy cells in the body. IL-2 enhances activation-induced cell death (AICD). IL-2 also promotes the differentiation of T cells from early T cells into effector T cells (T cells) when the early T cells are stimulated by antigen. eff ) and memory T cells (T mem ), thereby helping the body fight infection. IL-2, along with other polarizing cytokines, promotes differentiation of naive CD4 + It stimulates the differentiation of T cells into Th1 and Th2 lymphocytes, while inhibiting the differentiation into Th17 and follicular Th lymphocytes.

[0006] The α subunit (CD25) of the IL-2 receptor (IL-2R) binds to the low affinity (K d ~10 -8 IL-2 binds to IL-2 at the intermediate affinity dimeric CD122 / CD132 IL-2Rβγ receptor (K d ~10 -9 M), or the high affinity trimeric CD25 / CD122 / CD132 IL-2Rαβγ receptor (K d ~10 -11 The dimeric IL-2Rβγ can signal via either CD8 + T mem It is expressed by T cells and NK cells, but not by regand activated T cells express high levels of the trimeric IL-2Rαβγ. The γ subunit (CD132) is shared among the receptors for IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21.

[0007] Regulatory T cells (T reg ) are a subset of T lymphocytes important in maintaining self-tolerance. IL-2 stimulates the differentiation of regulatory T cells and effector (T eff ) cells, but T eff T than cells reg The high expression of high affinity receptors on T cells suggests that low doses of IL-2 may be effective in inhibiting T reg Autoimmune responses in diseases such as type 1 diabetes, multiple sclerosis, Crohn's disease, and systemic lupus erythematosus are driven by T reg Therefore, the high affinity receptor-mediated T reg Selective, long-term stimulation of cells holds promise for the treatment of autoimmune diseases.

[0008] High-dose IL-2 therapy using aldesleukin (recombinant IL-2) has been approved for the treatment of metastatic melanoma and renal cell carcinoma. However, objective response rates are low, and high-dose therapy is associated with a high incidence of end-organ toxicity. Most of the antitumor activity of IL-2 results from stimulation of T cells via the high-affinity IL-2Rαβγ receptor, and most of the toxicity is thought to result from the release of inflammatory proteins by natural killer cells via the low-affinity IL-2Rβγ receptor. An IL-2 mutein with an arginine replacing asparagine at position 88 (SEQ ID NO: 2; IL2-N88R, BAY50-4798) selectively binds to the high-affinity IL-2Rαβγ receptor and inhibits T eff T cells and NK cells reg This increases selectivity for cell activation by 3,000-fold. Consistent with this notion, rodent models have demonstrated comparable efficacy of BAY50-4798 and aldesleukin, but the mutant is less toxic. A phase 1 human trial of BAY50-4798 demonstrated effT cells and NK cells reg Although the expected differential activation of cells was observed, the antitumor response was limited and the development of muteins was halted.

[0009] Attempts to extend the in vivo half-life of IL-2 and analogs, and thereby improve their efficacy, have been reported. Various fusions of IL-2 with antibodies and antibody fragments have been disclosed (WO2014 / 023752A1). Some researchers have combined Fc or IgG with IL-2 {Bell, 2015#2} or T reg Fusions with specific variants, such as Fc-IL-2N88R (Greve, J. US2017 / 0204154A1), or IgG-IL-2N88D {Peterson, 2018#1}, have been disclosed. The half-life of IgG-IL-2N88D was ~8 hours when injected IV and only 14 hours when injected SC in cynomolgus monkeys, much shorter than the expected 14 days for IgG, and the t 1 / 2 The short time to expression was attributed to receptor-mediated endocytosis (RME). Nevertheless, a single SC injection of IgG-IL-2N88ND led to a long-lasting increase in regulatory T cells that was comparable to daily injections of low-dose IL-2. reg The CD4+ and CD8+ CD25+ FOXP3+ T cells expanded for up to 4 days and persisted for 14 days. reg increased IL-2 expression by 10-14 fold, but had no effect on CD4+ or CD8+ memory effector T cells. However, such fusion proteins suffer from several defects, such as loss of potency and increased immunogenicity relative to the native protein. Specific permanent and releasable conjugates of IL-2 with water-soluble polymers have been disclosed (U.S. Patent No. 9,861,705). IL-2 isomers containing unnatural amino acids have been disclosed to alter the selectivity between the receptor and its water-soluble conjugates (WO2019 / 028425; WO2019 / 028419).

[0010] Interleukin-15 is a related cytokine that acts through a unique receptor α chain, but through similar β and γ receptor chains as IL-2. IL-15 is a pleiotropic cytokine important for both adaptive and innate immunity. IL-15 mediates the activation of natural killer (NK) and CD8 + Effector T mem It promotes cell activation and maintenance and is of interest as an immunotherapeutic agent for the treatment of cancer and immune disorders. Exogenous IL-15 inhibits CD8 + T mem Low-dose therapy with IL-15 has been shown to stimulate the proliferation of tumor-specific CD8 + T mem It is hypothesized that this may promote cell maintenance and function, and delay or prevent tumor recurrence in the setting of failed adoptive immunotherapy (Roychowdhury et al., Cancer Research 64: 8062-7 (2004)). Low-dose therapy administered by continuous infusion in monkeys for 10 days reduced peripheral blood CD8 + Effector T memIt resulted in a 100-fold increase in cells, which was more effective than daily bolus dosing regimens (Sneller et al., Blood 118: 6845-8 (2011)). Stabilized mutants of IL-15 have been reported (Nellis et al., Pharm. Res. 29: 722-38 (2012)). Specific permanent and releasable conjugates of IL-15 with water-soluble polymers have been disclosed (PCT Publication WO2015 / 153753A2). Mutants of IL-15 that exhibit improved receptor agonism have been disclosed (Zhu et al., J. Immunology 2009, 183(6): 3598). IL-15[N72D] showed a 4-5-fold increase in bioactivity over native IL-15 in cell proliferation assays. IL-15 and IL-15 receptor agonists, including the sushi domain of IL-15Rα (IL-15RαSu), have also been reported as complexes and fusion proteins (Han et al., Cytokine 2011, 56(3):804-10; Mortier et al., J. Biological Chem. 2006, 281: 1612-9; U.S. Patent No. 10,358,477). A multimeric complex of IL-15RαSuFc and IL-15[N72D] fused to the Fc domain of IgG1 (ALT-803) is currently in clinical trials.

[0011] Mutants of IL-2 with reduced affinity for the trimeric receptor have been disclosed (U.S. Pat. No. 9,206,243). These mutants are capable of stimulating T helper cells, CD8 T cells, and natural killer (NK) cells while retaining the ability to stimulate T reg Such IL-2 mutants show a reduced ability to stimulate T cells. reg It has been proposed that the lack of immune suppression by the cells may result in enhanced antitumor activity.

[0012] IL-7 is a cytokine required for T cell development and for the survival and homeostasis of mature T cells. -CD8 - IL-7 signaling is required for thymocyte precursor migration, but high doses of IL-7 block the progression of DN. Once in the periphery, survival of naive T cells is dependent on IL-7. The IL-7 receptor contains a specific α chain (CD127) that is expressed almost exclusively on lymphocytes, along with a common γ chain (CD132) used for IL-2, IL-15, IL-9, and IL-21. IL-7 is in clinical trials as an immunotherapeutic agent in cancer patients who have undergone T cell depletion therapy to increase the levels of CD4+ and CD8+ T cells. Administration of IL-7 preferentially proliferates naive T cells, expanding the T cell repertoire regardless of the patient's age, suggesting the possibility of treatment with IL-7 to enhance immune responses in patients with low naive T cell populations (ElKassar & Gress, J. Immunotoxicol. (2010) 7: 1-7.).

[0013] IL-9 is responsible for mast cells, NK cells, TH2, TH17, T reg IL-9 is another pleiotropic cytokine structurally related to IL-2 and IL-15, produced by CD4+ T cells, ILC2, and Th9 cells, which are considered the major CD4+ T cell producers. The IL-9 receptor contains a specific alpha chain (CD129) along with a common gamma chain (CD132). Low-dose therapy with IL-9 has been proposed to prevent chemotherapy-induced thrombocytopenia and promote platelet recovery (Xiao et al., Blood 129: 3196-3209 (2017)).

[0014] IL-10 (human cytokine synthesis inhibitor) is an anti-inflammatory, immunosuppressive cytokine produced by Th2 cells, B cells, and macrophages. It inhibits the synthesis of several cytokines produced by Th1 cells, including gamma interferon, IL-2, and tumor necrosis factor-alpha (TNF-α), and inhibits the production of IL-1, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), and TNF-α by monocytes and macrophages. IL-10 has been shown to induce NK cell activation and target cell destruction in a dose-dependent manner (Zheng et al. J. Exp.Med. 184:579-84 (1996)). It is under investigation for the treatment of autoimmune diseases, septic shock, and bacterial sepsis. Pegylated derivatives of IL-10 have been disclosed (PCT Publication WO2010 / 077853). PEGylated IL10 has been shown to induce interferon gamma and CD8+ T cell-dependent antitumor immunity (Emmerich et al., Cancer Res. 72: 3570-81 (2012); Mumm et al., Cancer Cell 20:781-96 (2011); Chan et al., J Interferon Cytokine Res. 35: 948-55 (2015)). Investigations of PEGylated IL10 suggest that in human therapy, IL-10 is immunostimulatory primarily via activation of CD8+ T cells; a phase 3 trial for the treatment of metastatic stage 4 pancreatic cancer failed to meet its primary endpoint, but a phase 2 trial for non-small cell lung cancer is ongoing.

[0015] IL-21 is expressed on activated CD4+ T cells and upregulated in Th2 and Th17 T helper cells, as well as T follicular cells. It is expressed on NK cells and regulates their function. The IL-21 receptor (IL21R) is expressed on the surface of T cells, B cells, and NK cells and functions in combination with the common gamma chain (CD132). The use of IL-21 in the treatment of allergies, viral infections, and cancer has been proposed, and it is in clinical trials for the treatment of metastatic melanoma and renal cell carcinoma. IL-21 has been reported to improve HIV-specific cytotoxic T cell responses and NK cell function in HIV-infected subjects, suggesting its possible use in the treatment of HIV.

[0016] Continuous infusion shows promise for sustained low-dose therapy with cytokines, however, is difficult to implement in practice in human therapy. Thus, improved agents are needed that allow low-dose, long-term treatment of a variety of diseases, including cancer and autoimmune disorders, with cytokines. Summary of the Invention

[0017] In one embodiment, a compound of formula (I): ZLD (I) [wherein Z, L, and D are as described herein.] In one embodiment, a linker-drug is provided, represented by the formula:

[0018] In some embodiments, the linker-drug ZLD has the formula (Ia): [ka] (Ia) [where Z, S, n, R 1 , R 2 , R 4 , Y, and D are as described herein.] It is a compound represented by the formula:

[0019] In another embodiment, the compound of formula (IIa): [ka] (IIa) [where n, Z, S, R 1 , R 2 , R 4 and X is as described herein. A linker represented by the formula:

[0020] In another embodiment, a compound of formula (III): M-[Z * -LD] q (III) [In the formula, M, Z * , L, D, and q are as described herein.] A conjugate represented by the formula:

[0021] In another embodiment, the compound of formula (IV): [ka] (IV) [In the formula, P 1 , P 2 ,r,A * , B, C * , n, R 11 , R 12 , R 14 , x, y, and z are as described herein.] A degradable crosslinked hydrogel is provided,

[0022] In another aspect, methods of making the compounds disclosed herein and methods of using them are provided. In another aspect, pharmaceutical compositions are provided that include the conjugate of formula (III), or the hydrogel of formula (IV). [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows the general structure of the linker-drug conjugate attached to the hydrogel.

[0024] [Diagram 2] FIG. 2 shows binding of IL-2[N88R,C125S] to cells containing αβγ and βγ receptors.

[0025] [Diagram 3] FIG. 3 shows an SDS-PAGE gel with a band corresponding to the linker-protein product from reductive alkylation of IL-2[N88R,C125S].

[0026] [Figure 4] Figure 4 shows C vs t plots of plasma IL2[N88R] in rats after treatment with microsphere-IL2-N88R. Figure 4A shows the release of IL-2[N88R,C125] from the random acylated conjugate administered at 0.25 μmol / kg, and Figure 4B shows the release of AP-IL-2[N88R,C125S] from the reduced alkylated conjugate administered at 0.12 μmol / kg.

[0027] [Diagram 5] Figure 5 shows the pharmacodynamics of IL-2[N88R,C125S] in the spleen. Left: percentage of CD4+ effector / memory T cells; Right: percentage of CD8+ effector / memory T cells.

[0028] [Figure 6] Figure 6 shows the pharmacodynamics of IL-2[N88R,C125S] in pancreatic islets. Top left: percentage of Foxp3+CD4+ T cells; top right: percentage of CD4+; bottom left: percentage of CD8+; bottom right: innate lymphocytes. NOD mice were injected daily with PBS vehicle, Proleukin (25000 units), or IL-2[N88R,C125S] (25000 units) and sacrificed 2 hours after the last injection on the fifth day.

[0029] [Figure 7]FIG. 7 shows the pharmacokinetics of [aminopropyl]-IL-2[N88R,C125S] released from microsphere-IL-2[N88R,C125S] ("MS-IL-2 variant") in mice. FIG. 7A: BALB / c mice (n=6) were administered a single subcutaneous injection containing 28 nmol (19 mg / kg) or 9.9 nmol (6.5 mg / kg) of microsphere-IL-2[N88R,C125S] in the flank. A t1 / 2 of 31 hours was measured. FIG. 7B: NOD mice (n=6) were administered microsphere-IL-2[N88R,C125S] (0.5, 1, 5, 10, or 19 mg / kg) in the flank. A t1 / 2 of 18 hours was measured. Figure 7C: NSG (n=6) or NOD (n=6) mice were administered microsphere-IL-2[N88R,C125S] (5 mg / kg) in the flank. In NSG mice, [aminopropyl]-IL2[N88R,C125S] measured a t1 / 2 of 152 hours. In all cases, plasma was analyzed using Thermofisher ELISA to quantify IL-2[N88R,C125S] concentrations.

[0030] [Figure 8] FIG. 8 shows the effect of IL-2[N88R,C125S] ("IL-2 variant") on the proliferation of Foxp3+CD4+ and CD8+ cell populations. FIG. 8A shows the proliferation of Foxp3+CD4+ T cells in spleen and peripheral blood mononuclear cells (PBMCs). FIG. 8B shows the proliferation of CD8+ T cells in spleen and PBMCs. The percentage of CD8+ cells found in spleen and PBMCs was approximately 11% and 19%, respectively. When treated with microsphere-IL-2[N88R,C125S], these percentages increased to approximately 25% and 60%, respectively. NOD mice were administered IL2-variant (QD×5, 25,000 units), a single injection of empty microspheres, or microsphere-IL-2[N88R,C125S] (18 mg / kg). Mice were sacrificed 2 hours after the last dose on the fifth day.

[0031] [Figure 9] Figure 9 shows dose-dependent proliferation of Foxp3+CD4+T cells in PBMCs. Figure 9A shows the effect of microsphere-IL-2[N88R,C125S] on proliferation of Foxp3+CD4+T cells, and Figure 9B shows its effect on CD8+ cell activation (right) in NOD mice (n=3 / dose group). Microsphere-IL-2[N88R,C125S] preferentially expands Foxp3+CD4+T cells and avoids CD8+ cell activation in NOD mice (n=3 / dose group). Proliferation of Foxp3+CD4+T cells peaks at day 4 at all doses and returns to baseline levels by day 14.

[0032] [Figure 10] Figure 10 shows an SDS-PAGE gel with bands corresponding to the linker-protein products from reductive alkylation of IL-15. From left to right: molecular weight markers; IL-15; IL-15 + PEG5kDa-DBCO; IL-15 + 1Eq(IIb) + PEG5kDa-DBCO; IL-15 + 3Eq(IIb) + PEG5kDa-DBCO; and IL-15 + 5Eq(IIb) + PEG5kDa-DBCO.

[0033] [Figure 11]Figure 11 shows the pharmacokinetics of [aminopropyl]-IL-15 released from MS-IL-15 in C57BL / 6J mice. Normal male C57BL / 6J mice were administered MS~IL-15 (50 μg) at t = 0 and t = 240 hours. Plasma samples were prepared and analyzed using a human IL-15 Quantikine ELISA (R&D systems). Two distinct t1 / 2 were observed throughout 240 hours. A t1 / 2 > 200 hours was observed until 120 hours, after which a second t1 / 2 of 27 hours was observed from 120 to 240 hours. A second injection of MS~IL-15 (50 μg) was administered immediately after the 240 hour bleed (data in blue). A t1 / 2 of 23 hours was observed from 264 to 360 hours.

[0034] [Figure 12] Figure 12 shows the dose-dependence of the pharmacokinetics of [aminopropyl]-IL-15 released from microsphere-IL-15 in C57BL / 6J mice. Normal male C57BL / 6J mice were administered MS-IL-15 (12.5, 25, or 50 μg). Plasma samples were prepared and analyzed using a human IL-15 Quantikine ELISA (R&D systems). A t1 / 2 of 115-207 hours was observed with data fits out to 120 hours.

[0035] [Figure 13] Figure 13 shows the pharmacokinetics of [aminopropyl]-IL-15 released from microsphere-IL-15 in subcutaneously or intraperitoneally administered C57BL / 6J mice. Normal male C57BL / 6J mice were administered MS-IL-15 (50 μg) by subcutaneous (sc) (black, ●) or intraperitoneal (ip) injection (blue, ■). Plasma samples were prepared and analyzed using a human IL-15 Quantikine ELISA (R&D systems). Similar t1 / 2 was observed for subcutaneous (115 h) and intraperitoneal (129 h) administration up to 120 h.

[0036] [Figure 14] FIG. 14 shows the effect of microsphere-IL15 conjugates on NK cells and CD44hiCD8+T cells. Microsphere-IL15 conjugates expand CD44hiCD8+T cells and NK cells. FIG. 14A: CD44hiCD8+T cell expansion. FIG. 14B: NK cell expansion. Normal male C57BL / 6J mice were administered a single subcutaneous injection of microsphere-IL-15 (2.5, 12.5, 25, or 50 μg of IL-15), empty microspheres (black), or a single subcutaneous injection of rhIL15 (2.5 μg). Flow cytometry was used to monitor the expansion of NK cells and CD44hiCD8+T cells in PBMCs. CD44hiCD8+T cell expansion was followed for 28 days after a single 50ug injection of microsphere-IL15.

[0037] [Figure 15] Figure 15 shows an SDS-PAGE gel with bands corresponding to the linker-protein products from reductive alkylation of receptor-bound interleukin (RLI) with linker (IIb) visualized after gel shift reaction with PEG5kDa-DBCO. From left to right: molecular weight marker; RLI; RLI + PEG5kDa-DBCO; RLI + 1.5Eq(IIb) + PEG5kDa-DBCO; RLI + 2Eq(IIb) + PEG5kDa-DBCO; RLI + 3Eq(IIb) + PEG5kDa-DBCO; and RLI + 5Eq(IIb) + PEG5kDa-DBCO.

[0038] [Figure 16] Figure 16 shows the results of an IL-2Rβγ receptor binding cell-based assay for RLI. Using a U2OS cell-based assay, the binding activity of [aminopropyl]-RLI released from the conjugate at pH 7.4 (EC50=180 pM) was measured and compared to native RLI (EC50=160 pM).

[0039] [Figure 17] Figure 17 shows the pharmacokinetics of [aminopropyl]-RLI released from the microsphere conjugate in C57BL / 6J mice. Normal male C57BL / 6J mice were administered microsphere-RLI conjugate (1.5 nmol). Plasma samples were prepared and analyzed using R&D systems DuoSet hIL15 / IL15Rα complex ELISA (DY6924).

[0040] [Figure 18] Figure 18 shows the pharmacodynamics of [aminopropyl]-RLI released from microsphere conjugates and measuring the proliferation of CD8+ memory T cells in PBMCs. Figure 18A: Percentage of CD8+ memory T cells in PBMCs and Figure 18B: Proliferation of CD8+ T cells. Normal male C57BL / 6J mice were administered empty MS, MS~RLI (34 μg, 1.5 nmol), or native RLI (2.5 μg, 0.11 nmol, QD x 4) subcutaneously in the flank. After blood collection, PBMCs were prepared and stained for flow cytometry analysis.

[0041] [Figure 19] Figure 19 shows the proliferation of NK cells in PBMCs treated with microsphere-RLI. Figure 19A: Cell percentage of NK cells in PBMCs, and Figure 19B: Proliferation of NK cells. Normal male C57BL / 6J mice were subcutaneously injected with empty MS, MS~RLI (34 μg, 1.5 nmol), or native RLI (2.5 μg, 0.11 nmol, QD x 4) into the flank. After blood collection, PBMCs were prepared and stained for analysis by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] The present disclosure provides releasable conjugates of cytokine proteins, including mutants thereof, that provide sustained delivery of low doses of these protein therapeutics over long periods of time, making them useful for treating a variety of diseases.

[0043] In one aspect, the present disclosure provides cytokines and variants thereof having attached releasable linkers suitable for conjugating the protein to a polymeric carrier. These linkers control the release rate of the protein from the carrier, and thus determine the concentration and duration of the cytokine or variant in the body.

[0044] In another aspect, the present disclosure provides conjugates that release cytokines and variants thereof from polymeric carriers, which are soluble or insoluble depots that extend the duration of the protein in the body.

[0045] In another embodiment, the present disclosure provides methods of production and use for the linker-cytokines and conjugates of the present disclosure.

[0046] definition In this specification, unless otherwise stated, the use of the terms "a", "an", etc. means one or more.

[0047] As used herein, unless otherwise indicated, the terms "about" or "approximately" when used in connection with a numerical value contemplate a numerical value within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5%.

[0048] The term "alkyl" includes linear, branched, or cyclic saturated hydrocarbon groups of 1 to 20, 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In some embodiments, the alkyl is linear or branched. Examples of linear or branched alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, the alkyl is cyclic. Examples of cyclic alkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, and the like.

[0049] The term "alkoxy" includes alkyl groups linked to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0050] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon double bond and having 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0051] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond and having 2 to 20, 2 to 12, 2 to 8, 2 to 6, or 2 to 4 carbon atoms.

[0052] The term "aryl" includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" includes aromatic rings containing at least one N, O, or S atom and containing 3 to 15 carbons, preferably containing at least one N, O, or S atom and containing 3 to 7 carbons, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0053] In some instances, the alkenyl, alkynyl, aryl, or heteroaryl moiety may be attached to the remainder of the molecule via an alkyl bond. In these situations, the substituent is referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl to indicate that an alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl moiety and the molecule to which it is attached.

[0054] The term "halogen" or "halo" includes bromo, fluoro, chloro, and iodo.

[0055] The term "heterocycle" or "heterocyclyl" refers to a 3-15 membered aromatic or non-aromatic ring containing at least one N, O, or S atom. Examples include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided above for the term "heteroaryl". In some embodiments, the heterocycle or heterocyclyl is non-aromatic. In some embodiments, the heterocycle or heterocyclyl is aromatic.

[0056] The term "polymer" refers to a molecule or residue of a molecule having a molecular weight of 5,000 to 1,000,000 daltons, preferably 10,000 to 500,000 daltons, more preferably 10,000 to 250,000 daltons. Polymers include, but are not limited to, proteins, including antibodies, antibody fragments, and enzymes; polypeptides, including poly(amino acids), such as poly(lysine) and poly(valine), and mixed sequence polypeptides; synthetic polymers, including poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(ethyleneimine) (PEI), and copolymers thereof; and polysaccharides, such as dextran. In some embodiments, the polymer contains at least one functional group suitable for conjugation, either naturally or after chemical conversion, such as an amine, carboxylic acid, alcohol, thiol, alkyne, azide, or maleimide group, as described above. In certain embodiments of the present disclosure, the polymer is polyethylene glycol. The polyethylene glycol may be linear or branched, terminated at one end with a functional group suitable for conjugation and at the other end (or ends) with a capping group (e.g., methyl), or may comprise multiple arms, each arm terminated with a functional group suitable for conjugation. In a preferred embodiment of the present disclosure, the polyethylene glycol is a linear, branched, or multi-armed polymer having an average molecular weight of 20,000-200,000 daltons, preferably 20,000-100,000 daltons, and most preferably about 40,000 daltons. Examples of such polyethylene glycols are known in the art and are commercially available, for example, from NOF Corporation (Tokyo, Japan).

[0057] Unless otherwise specified, "optionally substituted" means that the group can be unsubstituted or substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituents, which can be the same or different. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, halogen, -CN, -OR, -CH, -CH- ...aa , -SR aa , -NR aa R bb , -NO2, -C=NH(OR aa ), -C(O)R aa , -OC(O)R aa , -C(O)OR aa , -C(O)NR aa R bb , -OC(O)NR aa R bb , -NR aa C(O)R bb , -NR aa C(O)OR bb , -S(O)R aa , -S(O)2R aa , -NR aa S(O)R bb , -C(O)NR aa S(O)R bb , -NR aa S(O)2R bb , -C(O)NR aa S(O)2R bb , -S(O)NR aa R bb , -S(O)NR aa R bb , -P(O)(OR aa )(OR bb ), heterocyclyl, heteroaryl, or aryl, wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently optionally selected from R cc may be substituted by, where R aa and R bb are each independently H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl; R aa and R bb together with the nitrogen atom to which they are attached form a heterocyclyl, which is optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN, where Each R ccis independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0058] Typically, the active form of the drug is released directly from the conjugates of the present disclosure, although in some cases it is possible to release the active drug in its prodrug form.

[0059] Linker-drug In one embodiment, a compound of formula (I): ZLD (I) where Z is a linker-functional group that allows for connection of the drug to a polymeric carrier, L is a cleavable linker, and D is a cytokine or cytokine variant. In one embodiment, a linker-drug is provided, represented by the formula: In some embodiments, the releasable linker is suitable for conjugation of the protein to a polymeric carrier, in some embodiments, the linker controls the release rate of the cytokine or variant from the carrier, thus determining the concentration and duration of active protein in the body. In one embodiment, a compound of formula (I): ZLD (I) where Z is a linker-functional group that allows for connection of the drug to a polymeric carrier, L is a cleavable linker, and D is a cytokine or cytokine variant. In one embodiment, a linker-drug is provided, represented by the formula:

[0060] In some embodiments of the linker-drug of formula (I), the cytokine D is IL-2, IL-4, IL-7, IL-9, IL-10, IL-15, IL-21, or a cytokine variant thereof. D can also be NH(CHCHO). p (CH2) mIn certain embodiments, the present invention includes cytokines having specific chemical modifications to the cytokine, such as: wherein in said formula m is an integer from 2 to 6 and p is an integer from 0 to 1000, which is attached to the amine group resulting from reductive amination to attach a linker L. In certain embodiments, the modification is attached to the N-terminal alpha-amino group of the protein sequence.

[0061] "Cytokine variant" refers to an altered sequence protein ("mutein") having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to a native cytokine. In some embodiments, the cytokine variant has at least 90% sequence identity to the native cytokine. In some embodiments, the cytokine variant contains 1-10 altered amino acids from the native sequence and is selected based on improved protein stability and / or receptor binding affinity or selectivity. Depending on the expression system used to produce the recombinant cytokine, the sequence may or may not include an initial methionine residue. For example, IL-2 variants useful in the present disclosure may be selected that have increased binding affinity to the trimeric αβγ receptor over the dimeric βγ receptor. In some embodiments, the IL-2 variants have a mutation at asparagine-88, e.g., N88R or N88D, which may be combined with other mutations, such as C125S, to confer additional stability or selectivity. Other IL-2 variants suitable for use in the present disclosure include variants altered at aspartic acid-20, such as IL-2 D20T, or variants with reduced affinity for trimeric receptors, as disclosed in U.S. Patent No. 9,206,243. Particular embodiments of IL-2 and variants are those set forth in SEQ ID NOs: 1-11.

[0062] SEQ ID NO: 1 Native human IL-2 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS LT

[0063] SEQ ID NO: 2 IL-2-N88R (BAY50-4798) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0064] SEQ ID NO: 3 IL-2-N88R,C125S APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFSQSIISTLT

[0065] SEQ ID NO: 4 IL-2-D20T APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0066] SEQ ID NO: 5 IL-2-D20T,C125S APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE TTFMCEYADETATIVEFLNRWITFSQSIISTLT

[0067] SEQ ID NO: 6 IL-2-R38K,F42I,Y45N,E62L,E68V APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TIKFNMPKKA TELKHLQCLE ELLKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0068] SEQ ID NO: 7 IL-2-R38K,F42Q,Y45E,E68V APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TQKFEMPKKA TELKHLQCLE EELKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0069] SEQ ID NO: 8 IL-2-R38A,F42I,Y45N,E62L,E68V APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TIKFNMPKKA TELKHLQCLE ELLKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0070] SEQ ID NO: 9 IL-2-R38K,F42K,Y45R,E62L,E68V APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TKKFRMPKKA TELKHLQCLE ELLKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0071] SEQ ID NO: 10 IL-2 R38K,F42I,Y45E,E68V APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TIKFEMPKKA TELKHLQCLE EELKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0072] SEQ ID NO: 11 IL-2 R38A,F42A,Y45A,E62A APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TAKFAMPKKA TELKHLQCLE EALKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT

[0073] Similarly, native IL-15 can be replaced with mutants that improve activity, receptor binding selectivity, or stability.For example, native IL-15 (SEQ ID NO: 12) can be replaced with mutants that improve resistance to degradation by asparagine deamidation, such as IL-15-[N77A] (SEQ ID NO: 13), or IL-15-[N71S,N72A,N77A] (SEQ ID NO: 14), which have been shown to retain biological activity (Nellis et al., Pharm. Res. 29:722-38 (2012)), or IL-15[N72D] (SEQ ID NO: 15), which shows enhanced receptor agonism (Zhu et al., J. Immunology 2009, 183(6): 3598).

[0074] SEQ ID NO: 12 IL-15 NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0075] SEQ ID NO: 13 IL-15[N77A] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSAGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0076] SEQ ID NO: 14 IL-15[N71S,N72A,N77A] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA SASLSSAGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0077] SEQ ID NO: 15 IL-15[N72D] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0078] Complexes and fusion proteins of IL-15 and IL-15RαSu can also be used, such as receptor-binding interleukin RLI (SEQ ID NO: 16) and its variants (Mortier et al., J. Biological Chem. 2006, 281: 1612-9; U.S. Patent No. 10,358,477). These fusion proteins can optionally include the IL-15RαSu signal sequence and sequences known in the art to facilitate protein isolation and purification, such as His and Flag tags, or these components can be absent (SEQ ID NO: 17).

[0079] SEQ ID NO:16 RLI MAPRRARGC RTLGLPALLL LLLLRPPATR GDYKDDDDKI EGRITCRRRM SVEHADIWVK SYSLYSRERY ICNSGFKRKA GTSSLTECVL NKATNVAHWT TPSLKCIRDP ALVHQRPAPP SGGSGGGGSG GGSGGGGSLQ NWVNVISDLK KIQDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0080] Sequence number 17 RLI[N77A] ITCPPPMSVE HADIWVKSY SLYSRERYIC NSGFKRKAGT SSLTECVLNK ATNVAHWTTP SLKCIRDPAL VHQRPAPPSS GGSGGGGSGG GSGGGGSLQN WVNVISDLKK IEDLIQSMHI DATLYTESDV HPSCKVTAMK CFLLELQVIS LESGDASIHD TVENLIILAN NSLSSAGNVT ESGCKECEEL EEKNIKEFLQ SFVHIVQMFI NTS

[0081] Other cytokines include IL-7, IL-9, IL-10, and IL-21 (SEQ ID NOs: 18-21).

[0082] SEQ ID NO:18 IL-7 DCDIEGKDGK QYESVLMVSI DQLLDSMKEI GSNCLNNEFNFFKRHICDAN KEGMFLFRAA RKLRQFLKMN STGDFDLHLL KVSEGTTILL NCTGQVKGRK PAALGEAQPT KSLEENKSLK EQKKLNDLCF LKRLLQEIKT CWNKILMGTK EH

[0083] SEQ ID NO: 19 IL-9 QGCPTLAGIL DINFLINKMQ EDPASKCHCS ANVTSCLCLG IPSDNCTRPC FSERLSQMTN TTMQTRYPLI FSRVKKSVEV LKNNKCPYFS CEQPCNQTTA GNALTFLKSL LEIFQKEKMR GMRGKI

[0084] SEQ ID NO: 20 IL-21 QGQDRHMIRM RQLIDIVDQL KNYVNDLVPE FLPAPEDVET NCEWSAFSCF QKAQLKSANT GNNERIINVS IKKLKRKPPS TNAGRRQKHR LTCPSCDSYE KKPPKEFLER FKSLLQKMIH QHLSSRTHGS EDS

[0085] SEQ ID NO: 21 IL-10 MSPGQGTQSE NSCTHFPGNL PNMLRDLRDA FSRVKTFFQM KDQLDNLLLK ESLLEDFKGY LGCQALSEMI QFYLEEVMPQ AENQDPDIKA HVNSLGENLK TLRLRLRRCH RFLPCENKSK AVEQVKNAFN KLQEKGIYKA MSEFDIFINY IEAYMTMKIR N

[0086] In certain embodiments, cytokines can be chemically modified, for example, by attaching water soluble polymers such as polyethylene glycol to one or more positions to extend the duration of the protein in the body upon release from the conjugate and / or to alter receptor selectivity.

[0087] These proteins can be produced using methods known in the art. If produced recombinantly, they can be expressed in either prokaryotic or eukaryotic systems.

[0088] Various cleavable linkers L can be used, including those disclosed in U.S. Patent No. 8,680,315; PCT Publication No. WO2013 / 036857; PCT Publication No. WO2006 / 138572; PCT Publication No. WO2005 / 099768; PCT Publication No. WO2006 / 136586; PCT Publication No. WO2011 / 012722; PCT Publication No. WO2011 / 089214; PCT Publication No. WO2011 / 089215; PCT Publication No. WO2011 / 089216; and PCT Publication No. WO2016 / 020373. Linker L comprises a covalent bond that cleaves at a specific rate under appropriate conditions. Such cleavage can be by catalytic or non-catalytic hydrolysis, proteolysis, or elimination reaction. Suitable conditions for cleavage are those typically found in a physiological environment, typically a pH of about 6.5-7.5 and a temperature of 30-45°C, preferably a pH of about 7.4 and a temperature of about 37°C.

[0089] In some embodiments, the linker-drug of formula (I) has formula (Ia): [ka] (Ia) [In the formula, n is an integer from 0 to 6; R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of is an electron withdrawing group; Each R 4 are independently C1-C3 alkyl, or two R 4 form a 3- to 6-membered ring together with the carbon atom to which they are attached; Z is a group for connecting the linker to the polymeric carrier; S is absent or (CH2CH2O) h (CH2) g CONH, where g is an integer from 1 to 6 and h is an integer from 0 to 1000; Y is absent or NH(CH2CH2O) p (CH2) m where m is an integer from 2 to 6, and p is an integer from 0 to 1000; and D is an amine residue of a cytokine or cytokine variant disclosed herein. It is a compound represented by the formula:

[0090] In some embodiments of the linker-drug of formula (Ia), n=1-6, R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of R is an electron-withdrawing group; 4 are independently H, or C1-C3 alkyl, or may be joined together to form a 3-6 membered ring; Z is a group for connecting the linker to the polymeric carrier; S is absent or (CH2CH2O) h (CH2) g CONH, where g=1 to 6, and h=0 to 1000; Y is absent or NH(CH2CH2O) p (CH2) m where m=2-6 and p=0-1000; and D is an amine residue of an IL-2, IL-2 variant, IL-15, or IL-15 variant cytokine.

[0091] R 1 and R 2A description of electron-withdrawing groups can be found in U.S. Patent No. 8,680,315, which is incorporated herein by reference. Electron-withdrawing groups are defined as groups with a Hammett sigma value greater than 0 (see, for example, Hansch et al. 1991 Chemical Reviews 91: 165-195). Representative examples of electron-withdrawing groups include, but are not limited to, nitrile, nitro, sulfone, sulfoxide, carbonyl, optionally substituted aryl, and optionally substituted heteroaryl.

[0092] In some embodiments of the linker-drug of formula (Ia), R 1 and R 2 The electron-withdrawing group is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR 5 , -SOR 5 , or -SO2R 5 , [Where, R 5 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 6 , or -NR 6 2, where each R 6 are independently H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R 6 the groups taken together with the nitrogen to which they are attached form a heterocycle; or S.R. 7 [Where, R 7is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. It is.

[0093] In some embodiments of the linker-drug of formula (Ia), R 1 and R 2 The electron withdrawing group in R is -CN. 1 and R 2 The electron withdrawing group in is -NO. In some embodiments, R 1 and R 2 The electron withdrawing group is an optionally substituted aryl containing 6 to 10 carbons. For example, in some embodiments, R 1 and R 2 The electron withdrawing group in is optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, R 1 and R 2 The electron withdrawing group is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. For example, in some embodiments, R 1 and R 2 The electron withdrawing group of is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which may be optionally substituted. 1 and R 2 The electron withdrawing group is an optionally substituted alkenyl containing 2 to 20 carbon atoms. 1 and R 2 The electron withdrawing group is an optionally substituted alkynyl containing 2 to 20 carbon atoms. 1 and R 2 The electron-withdrawing group is -COR 5, -SOR 5 , or -SO2R 5 where R 5 is H, optionally substituted alkyl containing 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 6 , or -NR 6 2, where each R 6 are independently H or optionally substituted alkyl containing 1 to 20 carbon atoms, or two R 6 The groups taken together with the nitrogen to which they are attached form a heterocycle. In some embodiments, R 1 and R 2 The electron-withdrawing group is -SR 7 where R 7 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl containing 1 to 20 carbon atoms.

[0094] In some embodiments of the linker-drug of formula (Ia), R 1 and R 2 At least one of the following is -CN, -SOR 5 , or -SO2R 5 In some embodiments, R 1 and R 2 At least one of the groups is -CN or -SO2R. 5 In some embodiments, R 1 and R 2 At least one of the groups is -CN or -SO2R. 5 where R 5 is optionally substituted alkyl, optionally substituted aryl. In some embodiments, R 1 and R 2At least one of is -CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0095] In some embodiments of the linker-drug of formula (Ia), each R 4 is independently C1-C3 alkyl. In some embodiments, R 4 At least one of R is methyl. 4 are both methyl.

[0096] In some embodiments of the linker-drug of Formula (Ia), n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is an integer from 0 to 3. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0097] In some embodiments of the linker-drug of formula (Ia), R 1 is CN, or -SO2R 5 where R 5 is C1-C6 alkyl, aryl, heteroaryl, or -NR 6 2, where R 6 is independently C1-C6 alkyl, aryl, or heteroaryl, and R 2 =H, where R 5 and R 6 Each of may independently be optionally substituted.

[0098] In some embodiments of the linker-drug of formula (Ia), Z can include any functional group known in the art for conjugation. Such functional groups include, but are not limited to, for example, amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected variants thereof. In some embodiments, Z includes protected amine, protected aminooxy, ketone or protected ketone, aldehyde or protected aldehyde, maleimidyl, protected thiol, protected alcohol, azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, or cyclooctynyl. In some embodiments, Z includes azide, ketone, or protected ketone. In some embodiments, Z selectively reacts with an associated functional group Z′ on the polymeric support to form a connecting functional group Z * In some embodiments, the connecting functional group Z * is a carboxamide when Z / Z' is an amine / carboxylate or active ester; an oxime when Z / Z' is an NH2O / ketone or aldehyde; a thioether when Z / Z' is a thiol / maleimide or halocarbonyl; or a triazole when Z / Z' is an azide / cyclooctyne.

[0099] In some embodiments of the linker-drug of formula (Ia), S is absent. In some embodiments, S is (CH2CH2O) h (CH2) g CONH.

[0100] In some embodiments of the linker-drug of formula (Ia), Y is absent. In some embodiments, Y is NH(CH2CH2O) p (CH2) m It is.

[0101] In the description herein, it is understood that every description, variation, embodiment, or aspect of a moiety can be combined with every description, variation, embodiment, or aspect of any other moiety, just as if each and every combination of descriptions was specifically and individually set forth. For example, R 1 All descriptions, variations, embodiments, or aspects provided herein with respect to Z, S, n, R, 2 , R 4 , Y, and / or D. It is also understood that all descriptions, variations, embodiments, or aspects of formulas such as formula (I), (Ia), (IIa), (IIIa), (IV), (V), or (VI), if applicable, apply equally to other formulas described herein, and all descriptions, variations, embodiments, or aspects are described equally as if they were described separately and individually for all formulas. For example, all descriptions, variations, embodiments, or aspects of formula (I) apply equally to any of the formulas described herein, such as formula (Ia), (IIa), (IIIa), (IV), (V), or (VI), if applicable, and all descriptions, variations, embodiments, or aspects are described equally as if they were described separately and individually for all formulas.

[0102] Linker In another embodiment, the compound of formula (IIa): [ka] (IIa) [where n, Z, S, R 1 , R 2 , and R 4is as disclosed herein for formula (Ia); and X is a halogen, an active ester (e.g., N-succinimidyloxy, nitrophenoxy, or pentahalophenoxy), or NH(CHCHO) p (CH2) (m-1) CHO, where m is an integer from 2 to 6, and p is an integer from 0 to 1000. A linker is provided, shown as: In some embodiments, X is a halogen. In some embodiments, X is an active ester, such as succinimidyloxy. In some embodiments, X is a halide, succinimidyloxy, or nitrophenoxy. In some embodiments, X is NH(CH2CH2O) p (CH2) (m-1) CHO. X is NH(CH2CH2O) p (CH2) (m-1) A linker that is CHO can be attached to a cytokine by reductive alkylation, in which an aldehyde group of the linker forms an imine with an amine group of the cytokine, and the imine is reduced to an amine in the presence of a reducing agent such as sodium cyanoborohydride. This method is usually selective for attaching the linker to the N-terminal alpha-amine group of the cytokine. In this embodiment, the cytokine released from the conjugate upon cleavage of the linker is NH2(CH2CHO) p (CH2) m These linkers are prepared as described in Schneider et al. (2016) Bioconjugate Chem 27: 2534-9, incorporated herein by reference. In some embodiments, p is 0 and the cytokine released from the conjugate upon cleavage of the linker is NH2(CH2) m is modified at the N-terminal alpha-amine by the addition of

[0103] In some embodiments of the linker of Formula (IIa), n=1 to 6, R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of R is an electron-withdrawing group; 4 are independently H, or C1-C3 alkyl, or may be taken together to form a 3-6 membered ring; Z is a group for connecting the linker to the polymeric carrier; S is absent or (CH2CH2O) h (CH2) g CONH, where g=1-6, and h=0-1000; and X is a halide, succinimidyloxy, or nitrophenoxy.

[0104] The preparation of these linker reagents is disclosed in U.S. Patent No. 8,680,315, and PCT patent application PCT / US2020 / 026726 (filed April 3, 2020), both of which are incorporated herein by reference.

[0105] These linkers can be attached to the cytokine or cytokine variant by methods known in the art, for example by reacting with a protein buffer at pH 6-9, preferably pH 7-8, to acylate the amine groups on the protein to form a linker-protein of formula (I). If multiple amine groups on the protein are available for reaction, multiple linkers can be attached to each protein. The selectivity of the number of linkers attached to the protein can be controlled using the stoichiometry of the linker reagent to the protein. If only one linker is attached, the protein released from the conjugate upon linker cleavage will not have additional modifications.

[0106] Conjugates In another embodiment, a compound of formula (III): M-[Z * -LD] q (III) [In the formula, M is a polymeric carrier, and Z * is a connecting functional group, L is a cleavable linker, D is a cytokine or cytokine mutant protein, and q is an integer from 1 to 10 when M is a soluble polymeric carrier, or q is a large number when M is an insoluble polymeric carrier. A conjugate is provided, represented by the formula: It is understood that when M is an insoluble polymeric carrier, such as an insoluble matrix or support, multiple linker-drugs can be attached to M. For example, in some embodiments, M is a hydrogel of formula (IV), P 1 and P 2 If both P are 4-arm polymers, one, two, three, or four linker-drugs can be added to each P 1 -P 2 The linker-drug can be linked to the M unit. Thus, by reacting the linker-drug with M in an appropriate ratio, a desired number of multiplicities can be obtained. In this way, an appropriate drug concentration in the volume of the matrix can be obtained.

[0107] In some embodiments, the conjugate of formula (III) has formula (IIIa): [ka] (IIIa) [In the formula, M, Z * , S, n, R 1 , R 2 and R 4 , Y and D are defined as described herein for formula (I), (Ia), or (IIa). This is shown by:

[0108] In some embodiments, M is a soluble polymeric carrier, such as polyethylene glycol, dextran, a protein, or an antibody; * is a connecting group; q=1 to 10. In each case, M reacts with group Z on the compound of formula (I) to form the connecting group Z *The connecting group Z * is a carboxamide when Z / Z' is an amine / carboxylate or active ester; an oxime when Z / Z' is an aminooxy / ketone or aldehyde; a thioether when Z / Z' is a thiol / maleimide or halocarbonyl; or a triazole when Z / Z' is an azide / cyclooctyne. In some embodiments, Z * includes an amide, carboxamide, oxime, triazole, thioether, thiosuccinimide, or ether.

[0109] In some embodiments, M is a polyethylene glycol having an average molecular weight of 1,000-100,000 daltons, preferably 10,000-60,000 daltons, and most preferably 20,000-40,000 daltons. M can be single-chain, branched, or multi-armed. M contains one or more functional groups Z' for connecting to a linker-drug. Z' can be attached to commercially available polymers M using methods known in the art; for example, if M contains an amine group, it can be further derivatized to introduce Z'=aminooxy by acylation, by reaction with (Boc-aminooxy)acetic acid followed by deprotection; Z'=cyclooctyne by acylation, by reaction with an active ester or carbonate of cyclooctyne (e.g., 4-cyclooctynyl succinimidyl carbonate or (1R,8S,9s)-bicyclo[6.1.0]nona-4-yn-9-yl methoxy succinimidyl carbonate (BCN-OSu) or its (1R,8S,9r) diastereomer); or a maleimide group can be introduced by acylation, by reaction with 3-maleimidopropionic acid.

[0110] In some embodiments, M is an insoluble polymeric carrier, such as a hydrogel or a surgical device. In such embodiments, q is a number determined by the number of reactive groups Z' attached to the insoluble support. In some embodiments, M is represented by formula (IV): [ka] (IV) [In the formula, P 1 and P 2 is independently an r-arm polymer, where r is an integer from 2 to 8; n is an integer from 0 to 6; x, y, and z are each independently an integer from 0 to 6; B is a group containing Z'; A * and C * are each independently a connecting group, such as a carboxamide, oxime, ether, thioether, or triazole; R 11 and R 12 are each independently H, C1-C4 alkyl, or an electron-withdrawing group, where R 11 or R 12 at least one of is an electron withdrawing group; and Each R 14 are independently C1-C3 alkyl, or two R 14 form a 3- to 6-membered ring together with the carbon atom to which they are attached. It is a degradable crosslinked hydrogel represented by the formula:

[0111] electron withdrawing group R 11 and R 12 A description of this can be found in U.S. Pat. No. 8,680,315, which is incorporated herein by reference. In some embodiments of the hydrogel of formula (IV), R 11 and R 12 The electron-withdrawing group is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR 15 , -SOR 15 , or -SO2R 15 , [Where, R 15 is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 16 , or -NR 16 2, where each R 16 are independently H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R 16 the groups together with the nitrogen to which they are attached form a heterocycle; or S.R. 17 [Where, R 17 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. It is.

[0112] In some embodiments of the hydrogel of formula (IV), R 11 and R 12 The electron withdrawing group in R is -CN. 11 and R 12 The electron withdrawing group in is -NO. In some embodiments, R 11 and R 12 The electron withdrawing group is an optionally substituted aryl containing 6 to 10 carbons. For example, in some embodiments, R11 and R 12 The electron withdrawing group in is optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, R 11 and R 12 The electron withdrawing group is an optionally substituted heteroaryl containing 3 to 7 carbons and at least one N, O, or S atom. For example, in some embodiments, R 11 and R 12 The electron withdrawing group of is pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl, each of which may be optionally substituted. 11 and R 12 The electron withdrawing group is an optionally substituted alkenyl containing 2 to 20 carbon atoms. 11 and R 12 The electron withdrawing group is an optionally substituted alkynyl containing 2 to 20 carbon atoms. 11 and R 12 The electron-withdrawing group is -COR 15 , -SOR 15 , or -SO2R 15 where R 15 is H, optionally substituted alkyl containing 1 to 20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR 16 or -NR 16 2, where each R 16 are independently H or optionally substituted alkyl containing 1 to 20 carbon atoms, or two R 16 The groups, together with the nitrogen to which they are attached, form a heterocycle. In some embodiments, R 11 and R 12 The electron-withdrawing group is -SR 17where R 17 is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl containing 1 to 20 carbon atoms.

[0113] In some embodiments of the hydrogel of formula (IV), R 11 and R 12 At least one of the following is -CN, -SOR 15 , or -SO2R 15 In some embodiments, R 11 and R 12 At least one of the groups is -CN or -SO2R. 15 In some embodiments, R 11 and R 12 At least one of the groups is -CN or -SO2R. 15 where R 15 is optionally substituted alkyl, optionally substituted aryl. In some embodiments, R 11 and R 12 At least one of is -CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0114] In some embodiments of the hydrogel of formula (IV), each R 14 is independently C1-C3 alkyl. In some embodiments, R 14 At least one of R is methyl. 14 are both methyl.

[0115] In some embodiments of the hydrogel of formula (IV), R 11 CN, or -SO2R 15where R 15 is C1-C6 alkyl, aryl, heteroaryl, or -NR 16 2, where each R 16 is independently C1-C6 alkyl, aryl, or heteroaryl, and R 12 =H, where R 15 and R 16 Each of may independently be optionally substituted.

[0116] A general formula for such a linker-protein bound hydrogel is shown in FIG.

[0117] In certain embodiments, M is of formula (V) or formula (VI): [ka] (V) [ka] (VI) [In the formula, P 1 , P 2 , r, R 11 , R 12 , and R 14 is as described herein for formula (IV); and Z' comprises a cyclooctyne group. It is a hydrogel represented by the formula: In certain embodiments, Z' is 4-cyclooctynyloxycarbonyl, or (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl.

[0118] The manufacture of hydrogel supports of these formulas is disclosed in U.S. Pat. No. 9,649,385, and PCT / US2020 / 026726 (filed April 3, 2020), each of which is incorporated herein by reference.

[0119] The conjugates described above can be used to provide continuous low doses of cytokines in subjects with a disease or condition that can be treated with the regimen. Particular diseases and conditions treatable with continuous low dose cytokine therapy include tolerogenic CD4 + CD25 + FOXP3 + These include chronic graft-versus-host disease (cGVHD), which is associated with inappropriate reconstitution of regulatory T cells (Koreth et al., Blood 128: 130-7 (2016)); systemic lupus erythematosus; sarcoidosis; hepatitis C-induced vasculitis; alopecia areata; rheumatoid arthritis; inflammatory bowel disease; multiple sclerosis; and type 1 diabetes (Koreth et al., Oncology & Hematology Review 10: 157-63 (2014)). Immune enhancement by exogenous cytokines may be useful in the treatment of cancer and immune deficiencies.

[0120] The conjugates of the present disclosure can be formulated using standard buffers and excipients known in the art. The buffers used are preferably pH 3 to pH 7, more preferably pH 4 to pH 6. Administration can be intravenous, subcutaneous, intravitreal, or intramuscular for soluble conjugates, and subcutaneous, intravitreal, or intramuscular for insoluble conjugates. Intratumoral injection can also be used.

[0121] Pharmaceutical Compositions In another embodiment, a pharmaceutical composition is provided herein, comprising a polymeric carrier-drug conjugate or a pharma- ceutically acceptable salt thereof together with a pharma- ceutically acceptable buffer and / or excipient. The buffer is selected so that the stability of the linker is maintained during storage and upon reconstitution as necessary, typically having a pH of 2-7, preferably 2-6, more preferably 2-5. Acceptable buffers include acetic acid, citric acid, phosphoric acid, histidine, gluconic acid, aspartic acid, glutamic acid, lactic acid, tartaric acid, succinic acid, malic acid, fumaric acid, α-ketoglutaric acid, and the like. Excipients may include tonicity and osmolarity agents, such as sodium chloride; preservatives, such as citric acid or citrate salts, and parabens; antimicrobial agents, such as phenol and cresol; antioxidants, such as butylated hydroxytoluene, vitamin A, C, or E, cysteine, methionine, and the like; density adjusters, such as sucrose, polyols, hyaluronic acid, and carboxymethylcellulose. These formulations are described, for example, in "Remington's Pharmaceutical Science" AR Gennaro, ed., 17 th The pharmaceutical compositions may be prepared by conventional methods known to those skilled in the art, as described in the American Journal of Clinical Chemistry, 1985, Mack Publishing Company, Easton, PA, USA. The pharmaceutical compositions may be provided in liquid solution or suspension, or may be provided as solids, for example, by lyophilization of liquid compositions. Such lyophilized products may further comprise bulking agents for rapid and efficient reconstitution before use.

[0122] How to use In another aspect, the polymeric carrier-drug conjugates described herein and pharmaceutical compositions comprising the same can be used to treat or prevent disease or illness in individuals. In some embodiments, a method for treating disease or illness is provided, comprising administering the polymeric carrier-drug conjugates described herein or pharmaceutical compositions comprising the polymeric carrier-drug conjugates described herein to an individual in need thereof. The "individual" may be a human or an animal, such as a cat, dog, cow, rat, mouse, horse, rabbit, or other domestic animal.

[0123] Also provided is a composition comprising a polymeric carrier-drug conjugate as described herein for use in treating a disease or condition. Also provided herein is the use of a polymeric carrier-drug conjugate as described herein in the manufacture of a medicament for treating a disease or condition.

[0124] The applicable disease or condition requiring treatment will be recognized by one skilled in the art from the nature of the conjugated drug.

[0125] Certain representative embodiments are provided below. EMBODIMENT 1 The formula below M-[Z * -LD] q [In the formula, M is a polymeric carrier; Z * is a connecting functional group; L is a cleavable linker; and D is an amine residue of a cytokine or a variant thereof; and Here, when M is a soluble carrier, q=1 to 10, and when M is an insoluble carrier, q is a large number. The conjugate is shown by: EMBODIMENT 2 Z * is a carboxamide, oxime, thioether, or triazole; L is the following formula: [ka] [In the formula, n=0-6, or n=1-6; R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of is an electron withdrawing group; Each R 4 are independently H or C1-C3 alkyl, or two R 4 together form a 3- to 6-membered ring; S is absent or (CH2CH2O) h (CH2) g CONH, where g=1-6 and h=0-1000; Y is absent or NH(CH2CH2O) p (CH2) m where m=2 to 6 and p=0 to 1000. 2. The conjugate of embodiment 1, wherein EMBODIMENT 3 R 1 , CN, or R 5 SO2, where R 5 is C1-C6 alkyl, aryl, heteroaryl, or (R 6 )2N, where R 6 is C1-C6 alkyl, aryl, or heteroaryl, and R 2 =H, where R 4 ~R 6 wherein each of the groups is optionally substituted. EMBODIMENT 4 The conjugate according to any one of embodiments 1 to 3, wherein M is a soluble polyethylene glycol having an average molecular weight of 1,000 to 100,000 daltons, and q=1 to 10. EMBODIMENT 5 The conjugate of any one of embodiments 1 to 3, wherein M is an insoluble hydrogel or a surgical device and q is a number. EMBODIMENT 6 4. The conjugate according to any of the preceding embodiments, wherein D is IL-2, IL-7, IL-9, IL-10, IL-15, IL-21, or a variant thereof. EMBODIMENT 7 7. The conjugate of embodiment 6, wherein D is an IL-2 mutant having selective binding to trimeric αβγ receptors over dimeric βγ receptors or an IL-2 mutant having selective binding to dimeric βγ receptors over trimeric αβγ receptors. EMBODIMENT 8 4. The conjugate according to any of embodiments 1 to 3, wherein D is an IL-15 mutant stabilized against deamidation. EMBODIMENT 9 The following formula [ka] [Wherein, n=0 to 6, or n=1 to 6, R 1 and R 2 are independently an electron withdrawing group, alkyl, or H, where R 1 and R 2 at least one of R is an electron-withdrawing group; 4 are independently H, or C1-C3 alkyl, or may be taken together to form a 3-6 membered ring; Z is a functional group for connecting the linker to the polymeric carrier; S is absent or (CH2CH2O) h (CH2) g CONH, where g=1 to 6 and h=0 to 1000; Y is absent or NH(CH2CH2O) p (CH2) m where m=2-6 and p=0-1000; and D is an amine residue of a cytokine or a variant thereof. The linker-protein is indicated by EMBODIMENT 10 R 1 , CN, or R 5 SO2, where R 5 is C1-C6 alkyl, aryl, heteroaryl, or (R 6 )2N, where R 6 is C1-C6 alkyl, aryl, or heteroaryl, and R 2 =H, where R 4 ~R 6 wherein each of the linker-proteins is optionally substituted. EMBODIMENT 11 The linker protein of embodiment 9 or 10, wherein D is IL-2, IL-7, IL-9, IL-10, IL-15, IL-21, or a variant thereof. EMBODIMENT 12 The linker protein of embodiment 11, wherein D is an IL-2 mutant having selective binding to trimeric αβγ receptors over dimeric βγ receptors or an IL-2 mutant having selective binding to dimeric βγ receptors over trimeric αβγ receptors. EMBODIMENT 13 13. The linker protein of embodiment 12, wherein D is selected from the group consisting of IL-2, IL-2 N88R, IL-2 N88D, IL-2 N88R,C125S, and IL-2 N88D,C125S. EMBODIMENT 14 The linker-protein of embodiment 9 or 10, wherein D is selected from the group consisting of IL-15, IL-15 N77A, and IL-15-[N71S,N72A,N77A]. EMBODIMENT 15 D is selected from the group consisting of IL-2, IL-7, IL-9, IL-10, IL-15, IL-21, or variants thereof, wherein the N-alpha amine group is NH2(CH2CH2O) p (CH2) m wherein m=2-6 and p=0-1000. EMBODIMENT 16 4. A method for treating a subject with a conjugate according to any one of embodiments 1 to 3, wherein D is IL-2 or an IL-2 mutant. reg A method for selectively growing cells. EMBODIMENT 17 A method of selectively expanding CD8+ effector T cells in a subject, comprising treating the subject with a conjugate of any of embodiments 1 to 3, wherein D is IL-15 or an IL-15 mutant. EMBODIMENT 18 A method of treating a disease or condition in a subject in need of such treatment comprising administering a conjugate according to any one of embodiments 1 to 8. EMBODIMENT 19 The disease or condition is an autoimmune disease, tolerogenic CD4 + CD25 + FOXP3 + The method of embodiment 18, wherein the disease is chronic graft-versus-host disease (cGVHD) associated with inappropriate reconstitution of regulatory T cells; systemic lupus erythematosus; sarcoidosis; hepatitis C-induced vasculitis; alopecia; rheumatoid arthritis; inflammatory bowel disease; multiple sclerosis; or type 1 diabetes. EMBODIMENT 20 A method for enhancing immunotherapy in a subject undergoing such treatment, comprising administering a conjugate according to any one of embodiments 1 to 8. EXAMPLES

[0126] The following examples are intended to be illustrative and not limiting of the scope of the disclosure, and all references cited therein are incorporated herein by reference, including those cited for specific aspects of their disclosure, particularly those aspects and generally.

[0127] Manufacturing A A linker of formula (IIa) in which S is absent [ka] Linkers of formula (IIa) in which S is absent were prepared according to the following general procedure. In one method, the groups Z and R 4 In the presence of a base, typically potassium tert-butoxide or potassium tert-pentoxide, an ester containing R 1 R 2 Condensation with CH2 formed an intermediate ketone, which was reduced to an alcohol using sodium borohydride. This was then activated by reaction with triphosgene and pyridine to give the linker of formula (IIa) where X = Cl. This could be further converted to X = succinimidyloxy by reaction with chloroformate and N-hydroxysuccinimide. In an alternative method, the initial condensation can be carried out by first condensing R 1 R 2 CH2 is reacted with a strong base such as butyllithium, lithium diisopropylamide, or metalated hexamethyldisilazane, and the resulting R 1 R 2 CH - This was accomplished by treating the carbanion with an ester to generate the corresponding ketone intermediate. Some specific examples are shown below.

[0128] (1) 4-azido-1-cyano-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =CN,R 2 =H, R 4 = CH3, Z = N3, and X = succinimidyloxy) A 1M solution of potassium tert-butoxide in THF (3.5 mL, 3.5 mmol) was added to a solution of methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 300 mg, 1.9 mmol) and acetonitrile (0.365 mL, 7.0 mmol) in 7 mL of THF at -30°C. The mixture was stirred at -30°C for 30 min, then warmed to ambient temperature over 1 h and stirred for an additional 30 min. The mixture was cooled in ice and quenched by the addition of 6N HCl (0.62 mL, 3.7 mmol), then partitioned between EtOAc and water. The aqueous phase was extracted twice with EtOAc and the combined organics were washed with brine, dried over MgSO4, filtered and concentrated to give the crude ketone.

[0129] Sodium borohydride (33 mg, 0.88 mmol) was added to a solution of the crude ketone (300 mg, ca. 1.75 mmol) in 7 mL of methanol. The mixture was then stirred for 15 min, quenched by the addition of 6N HCl (0.7 mL), and partitioned between EtOAc and water. The aqueous phase was extracted twice with EtOAc, and the combined organics were washed with brine, dried over MgSO4, filtered, and concentrated to give the crude alcohol. Purification on SiO2 (20-40% EtOAc / hexanes) gave 4-azido-1-cyano-3,3-dimethyl-2-butanol (142 mg, 0.85 mmol). 1 H-NMR (CDCl3, 300 MHz) d 3.83-3.92 (m,1H), 3.43 (d, J=12.1 Hz,1H), 3.21 (d, J=12.1 Hz, 1H), 2.41-2.62 (m,3H), 0.97 (s,3H), 0.96 (s,3H).

[0130] Pyridine (136 μL, 1.7 mmol) was added dropwise to an ice-cold solution of 4-azido-1-cyano-3,3-dimethyl-2-butanol (142 mg, 0.85 mmol) and triphosgene (425 mg, 1.44 mmol) in 8 mL of THF. The resulting suspension was allowed to warm to ambient temperature and stirred for 15 min, then filtered and concentrated to give the crude chloroformate. This was dissolved in 8 mL of THF, cooled on ice, and treated with N-hydroxysuccinimide (291 mg, 2.5 mmol) and pyridine (204 μL, 2.53 mmol). The resulting suspension was allowed to warm to ambient temperature and stirred for 15 min, then partitioned between EtOAc and 5% KHSO4. The aqueous phase was extracted twice with EtOAc and the combined organics were washed with brine, dried over MgSO4, filtered and concentrated to give the crude succinimidyl carbonate. Purification on SiO2 (20-40% EtOAc / Hexanes) gave 4-azido-1-cyano-3,3-dimethyl-2-butyl succinimidyl carbonate (174 mg, 0.56 mmol). 1 H-NMR (CDCl3, 300 MHz) d 5.03 (dd,J=7.0,5.1,1H), 3.27-3.41 (m,6H), 3.43 (d, J=12.1 Hz,1H), 3.21 (d, J=12.1 Hz, 1H), 2.41-2.62 (m,3H), 0.97 (s,3H), 0.96 (s,3H).

[0131] (2) 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH3)2, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). A 1.43 M solution of n-butyllithium in hexanes (70 mL, 100 mmol) was added to a stirred solution of N,N-dimethylmethanesulfonamide (12.33 g, 100 mmol) in 200 mL of anhydrous THF, kept at -50 °C under an inert atmosphere. The mixture was warmed to -20 °C for 1 h and then recooled to -50 °C before adding methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 7.70 g, 50 mmol). The mixture was warmed to +10 °C for 2 h and then quenched with 20 mL of 6N HCl. The mixture was diluted with methyl t-butyl ether (MTBE, 200 mL), washed twice with 100 mL of water and once with 100 mL of brine, dried over MgSO4, filtered, and concentrated to give 14.05 g of crude ketone product. Purification by chromatography on SiO2 (220 g) using a step gradient of 0, 20, 30, 40, and 50% EtOAc / hexanes afforded 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanone (10.65 g, 86%) as a crystalline solid.

[0132] The above ketone was dissolved in 200 mL of methanol, cooled on ice, and treated with sodium borohydride (0.96 g, 25 mmol) for 15 min, then quenched with 4 mL of 6 N HCl and concentrated. The resulting slurry was diluted with methyl t-butyl ether (MTBE, 200 mL), washed once with 100 mL of water and once with 100 mL of brine, dried over MgSO4, filtered, and concentrated to give 10.0 g of crystalline 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol.

[0133] Pyridine (10.6 mL, 132 mmol) was added to a stirred mixture of N-hydroxysuccinimide (6.90 g, 60 mmol) and triphosgene (5.93 g, 20 mmol) in 250 mL of ice-cold dichloromethane in 10 min. The mixture was stirred for 15 min on ice and then warmed to ambient temperature in 30 min. A solution of 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol (10.0 g, 40 mmol) in 20 mL of dichloromethane was added and the mixture was further stirred at ambient temperature for 1 h. After cooling on ice, the mixture was treated with 100 mL of water and the phases were separated. The organic phase was washed twice with water, once with 5% KHSO4, and once with brine, dried over MgSO4, filtered, and concentrated. The crude product was crystallized from 100 mL of 30% EtOAc / hexanes to give 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (11.1 g, 71%) as a white crystalline solid. (3) Additional compounds of formula (I) prepared according to these procedures include: 4-Azido-1-(methylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2CH3, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-((4-methylpiperidinyl)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH2CH2)2CHCH3, R 2 =H, R 4 = CH3, Z = N3, and X = succinimidyloxy). LC / MS was [M+H] + =446.15. 4-Azido-1-(phenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2Ph, R 2 =H, R 4=CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(4-chlorophenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2PhCl, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(4-morpholinosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH2CH2)2O, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(isopropylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2CH(CH3)2, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-((N-ethyl-N-methylamino)sulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH3)(CH2CH3), R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-((N,N-bis(2-methoxyethyl)aminosulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2N(CH2CH2OCH3)2, R 2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). 4-Azido-1-(4-methylphenylsulfonyl)-3,3-dimethyl-2-butyl succinimidyl carbonate (wherein n=1, R 1 =SO2PhCH3,R2 =H, R 4 =CH3, Z=N3, and X=succinimidyloxy). If S does not exist, then each R 4 The compound of formula (I), wherein is H, was prepared according to Santi et al., Proc. Natl. Acad. Sci. USA 2012, 109(16): 6211-6.

[0134] Manufacturing B S = (CH2CH2O) h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) A linker of formula (IIa) which is CHO [ka] S = (CH2CH2O) h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) Linkers of formula (IIa) where Z is azide, S is absent, and X=succinimidyloxy were prepared as follows: In one method, a linker of formula (IIa) where Z is azide, S is absent, and X=succinimidyloxy was prepared by the addition of an amine-acetal HN—(CH) where R is alkyl. m-1 Reaction with CH(OR)2 gave the azidocarbamate acetal. The azide group was reduced to the amine by either catalytic hydrogenation with a palladium catalyst or Staudinger reduction with trimethylphosphine in the presence of water, followed by the addition of the spacer-succinimidyl ester Z-(CH2CH2O). h (CH2) g Addition of C(O)OSu gave the linker in the form of an acetal protection. Hydrolysis of the acetal under acidic conditions then gave S=(CH2CHO). h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) A linker of formula (IIa) was obtained, which is CHO. Specific examples are as follows:

[0135] 7-(15-azido-4,7,10,13-tetraoxapentadecanamido)-1-(4-phenylsulfonyl)-2-heptyl N-(3-oxypropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=5, R 1 = (4-methylphenyl)SO2, R 2 =H, each R 4 =H, and X=NH(CH2)2CHO) [ka] (1) 7-Azido-1-(4-methylphenylsulfonyl)-2-heptyl N-(3,3-diethoxypropyl)carbamate 7-Azido-1-(4-methylphenylsulfonyl)-2-heptylsuccinimidyl carbonate (125 mg, 277 μmol, final concentration 50 mM) (Santi et al., Proc. Natl. Acad. Sci. USA 2012, 109(16): 6211-6) was dissolved in 5.5 mL of MeCN and 1-amino-3,3-diethoxypropane (54 μL, 0.33 mmol, final concentration 60 mM) was added. The reaction mixture was stirred at ambient temperature. Within 15 min, the starting carbonate was completely consumed as judged by TLC. The reaction mixture was partitioned with 100 mL of 1:1 EtOAc:NaHCO3 (sat. aq.). The aqueous layer was extracted with 40 mL of EtOAc. The combined organic layers were washed successively with water, KHSO4 (5% aq.), water and brine (1×30 mL each). The organic phase was separated, dried over MgSO4, filtered and concentrated to give 109 mg (81% crude) of the title compound as a colorless oil which was used entirely in the next step without further purification. 1H NMR (300 MHz, CDCl3) δ 7.76 (d, J=8.1 Hz, 2H), 7.33 (d, J=8.1 Hz, 2H), 5.04 (quin, J=6.8 Hz, 1H), 4.91 (t, J=5.4 Hz, 1H), 4.49 (t, J=5.2 Hz, 1H), 3.62 (m, 2H), 3.37-3.53 (m, 3H), 3.10-3.25 (m, 5H), 2.42 (s, 3H), 1.74 (q, J=5.8 Hz, 2H), 1.63 (br q, J=5.7 Hz, 2H), 1.52 (m, 2H), 1.30 (br m, 4H), 1.17 (td, J=7.0, 2.1 Hz, 6H). LC-MS (m / z): calc, 529.2; obsd, 529.6 [M+HCO2] - .

[0136] [ka] (2) 7-(15-azido-4,7,10,13-tetraoxapentadecanamido)-1-(4-methylphenylsulfonyl)-2-heptyl N-(3,3-diethoxypropyl)carbamate 7-Azido-1-(4-methylphenylsulfonyl)-2-heptyl N-(3,3-diethoxypropyl)carbamate (109 mg, 225 μmol, final concentration 0.1 M) was dissolved in 2.3 mL of anhydrous EtOH. Palladium on carbon (10%, activated, 109 mg) was added. The reaction flask was sealed with a rubber septum, then evacuated and filled with hydrogen gas (3×). The reaction mixture was vigorously stirred at ambient temperature under an atmosphere of H2 (balloon). After 90 min, the starting material was completely consumed as judged by TLC. The reaction mixture was filtered through a short pipette plug of Celite and the pad was washed with 10 mL of EtOH. The filtrate was concentrated to dryness to give 90 mg of the intermediate amine as a colorless oil, which was used entirely in the next step without further purification. Crude 7-amino-1-(4-methylphenylsulfonyl)-2-heptyl N-(3,3-diethoxypropyl)carbamate (90 mg, max. 0.20 mmol, final concentration 0.1 M) was dissolved in 2.0 mL of MeCN. Succinimidyl 15-azido-4,7,10,13-tetraoxapentadecanoate (93 mg, 0.24 mmol, final concentration 0.12 M) and DIPEA (42 μL, 0.22 mmol) were added and the reaction was stirred at ambient temperature and monitored by TLC. After 1 h, the reaction mixture was partitioned with 60 mL of 1:1 EtOAc:NaHCO3 (sat. aq.). The organic layer was washed successively with water, citric acid (10% aq.), water and brine (1×30 mL each). The organic phase was separated, dried over MgSO4, filtered and concentrated to dryness. The crude product was purified on a 4 g SiliaSep column eluted with a stepwise gradient of acetone in CH2Cl2: 0%, 10%, 20%, 30%, 40% and 50% (30 mL each). Fractions containing the purified product were combined and concentrated to give the title compound (68 mg, 93 μmol, 41% over two steps) as a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 7.76 (d, J=8.3 Hz, 2H), 7.32 (d, J=8.1 Hz, 2H), 6.60 (br t, J=5.8 Hz, 1H), 4.95-5.08 (m, 2H), 4.50 (br t, J=4.9 Hz, 1H), 3.56-3.74 (m, 18H), 3.40-3.52 (m, 3H), 3.36 (t, J=5.1 Hz, 2H), 3.10-3.26 (m, 5H), 2.44 (t, J=5.8 Hz, 2H, obscured), 2.42 (s, 3H), 1.74 (q, J=6.0 Hz, 2H), 1.62 (br s, 2H), 1.43 (br m, 2H), 1.26 (br s, 4H), 1.17 (td, J=7.0, 2.3 Hz, 6H). LC-MS (m / z): calc, 776.4; obsd, 776.7 [M+HCO2] - .

[0137] [ka] (3) 7-(15-azido-4,7,10,13-tetraoxapentadecanamido)-1-(4-methylphenylsulfonyl)-2-heptyl N-(3-oxypropyl)carbamate 7-(15-Azido-4,7,10,13-tetraoxapentadecanamido)-1-(4-methylphenylsulfonyl)-2-heptyl N-(3,3-diethoxypropyl)carbamate (68 mg, 93 μmol, final concentration 0.1 M) was dissolved in 0.62 mL of CHCl3. Water and TFA (0.16 mL each) were added sequentially. The reaction mixture was vigorously stirred at ambient temperature. After 2 h, the starting acetal was completely consumed as judged by TLC. The reaction mixture was concentrated to dryness and then purified on a 4 g SiliaSep column eluting with a stepwise gradient of acetone in CH2Cl2: 0%, 15%, 30%, 45%, 60% and 75% (30 mL each). Fractions containing the purified product were combined and concentrated to give the title compound (26 mg, 40 μmol, 43%) as a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 9.78 (s, 1H), 7.78 (d, J=8.3 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 6.60 (br s, 1H), 5.09 (m, 1H), 4.98 (t, J=6.0 Hz, 1H), 3.62-3.75 (m, 16H), 3.36-3.44 (m, 5H), 3.13-3.26 (m, 3H), 2.70 (t, J=5.7 Hz, 2H), 2.47 (t, J=5.7 Hz, 2H, obscured), 2.45 (s, 3H), 1.63 (br s, 2H), 1.46 (br t, J=6.6 Hz, 2H), 1.29 (m, 4H). LC-MS (m / z): calc, 656.3; obsd, 656.6 [MH] - ; calc, 702.3; obsd, 702.6 [M+HCO2] -; calc, 734.3; obsd, 734.7 [M+CH3OH+HCO2] - .

[0138] In the second method, a linker of formula (IIa), where Z=Boc-amino, S=absent, and X=OH, was carried out through a similar sequence of steps, except that the Boc group was first removed under acidic treatment to afford the spacer-succinimidyl ester Z-(CH2CH2O) h (CH2) g C(O)OSu was then added. The alcohol was then activated by reaction with triphosgene and pyridine, and the resulting chloroformate was converted to the amine-acetal H2N-(CH2) where R is alkyl. m-1 Reaction with CH(OR)2 gave the acetal protected linker. Subsequent hydrolysis of the acetal under acidic conditions gave S=(CH2CH2O). h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) A linker of formula (IIa) was obtained, which is CHO. Specific examples are as follows:

[0139] [ka] 1-Azido-18,18-dimethyl-20-phenylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3-oxopropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=1, R 1 = PhenylSO2, R 2 =H, each R 4 = methyl, X = NH(CH2)2CHO) Steps 1 and 2 1-Azido-18,18-dimethyl-20-phenylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-aza-19-icosanol Trifluoroacetic acid (1 mL) was added to a solution of 4-[(tert-butoxycarbonyl)amino]-1-phenylsulfonyl-3,3-dimethyl-2-butanol (124 mg of a 58% w / w mixture; 72 mg, 0.20 mmol, final concentration 0.1 M) in 1 mL of CHCl. ​​The reaction was stirred at ambient temperature and monitored by TLC (40% EtOAc in hexanes, cerium molybdate stain). After 10 min, the starting material was converted to a single more polar spot by TLC. The reaction was concentrated to dryness and residual volatiles were removed under high vacuum to give the intermediate amine as a white film. The intermediate was dissolved in 1.8 mL of MeCN and DIPEA (0.17 mL, 1.0 mmol) was added. Neat azido-PEG4-OSu (78 mg, 0.2 mmol) was added. The reaction was stirred at ambient temperature and monitored by C18-HPLC (ELSD). Azide-PEG4-OSu was completely converted to a single faster migrating HPLC peak within 5 min. The reaction was then concentrated to dryness and loaded onto a 4 g SiliaSep silica gel column. The product was eluted with a stepwise gradient of acetone in CH2Cl2 (0%, 10%, 20%, 30%, acetone; each step 30 mL). Fractions containing purified product, as judged by C18-HPLC, were combined and concentrated to dryness. Residual volatiles were removed under high vacuum to give the title compound (85 mg, 0.16 mmol, 80% yield for two steps) as a colorless oil. C18-HPLC, purity determined by ELSD: 98.2% (RV=9.12 mL).

[0140] Process 3 1-Azido-18,18-dimethyl-20-phenylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3,3-diethoxypropyl)carbamate N-Hydroxysuccinimide (92 mg, 0.80 mmol) was added to a solution of triphosgene (0.24 g, 0.80 mmol) in 8.0 mL of anhydrous THF under N2. Pyridine (77 μL, 0.96 mmol) was added dropwise, with immediate formation of a white precipitate. The suspension was stirred at ambient temperature for 15 min and then filtered through a cotton plug. The filtrate was concentrated to dryness and redissolved in 1.6 mL of anhydrous THF. A solution of 1-azido-18,18-dimethyl-20-phenylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-aza-19-icosanol (86 mg, 0.16 mmol, 0.1 M) in 1 mL of anhydrous THF was added. The reaction was stirred at ambient temperature and monitored by C18-HPLC (ELSD). After 1 h, the starting alcohol was consumed. The reaction mixture was partitioned with 50 mL of 1:1 EtOAc:KHSO4 (5% aq). The layers were separated and the organic phase was washed successively with KHSO4 (5% aq), water, NaHCO3 (sat. aq) and brine (25 mL each). The washed organic phase was dried over MgSO4, filtered and concentrated by rotary evaporation. The crude succinimidyl carbonate was dissolved in 1.6 mL of anhydrous THF and 1-amino-3,3-diethoxypropane (86 μL, 0.53 mmol) was added. The reaction was stirred at ambient temperature and monitored by C18-HPLC (ELSD). After 25 min, the succinimidyl carbonate was converted to two slower eluting product peaks. The reaction mixture was partitioned with 30 mL of 1:1 EtOAc:sodium acetate (0.2 M, pH 5.0). The layers were separated and the organic phase was washed successively with water and brine (15 mL each). The washed organic phase was dried over MgSO4, filtered, and concentrated by rotary evaporation. Residual volatiles were removed under high vacuum to give the crude title compound (105 mg, 0.15 mmol, 94% crude yield for two steps) as a yellow oil.

[0141] Process 4 1-Azido-18,18-dimethyl-20-phenylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl (3-oxopropyl)carbamate Water (0.21 mL) and TFA (0.21 mL) were added sequentially to a solution of 1-azido-18,18-dimethyl-20-phenylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3,3-diethoxypropyl)carbamate (105 mg, 0.15 mmol, final concentration 0.1 M) in 1.1 mL of CHCl. ​​The reaction was stirred at ambient temperature and monitored by C-HPLC (ELSD). After 10 min, the reaction was judged to be complete. The mixture was concentrated to dryness. The concentrate was loaded onto a SiliaSep 4g silica gel column and the product was eluted with a stepwise gradient of acetone in CHCl (0%, 20%, 40%, 60% acetone, 30 mL each step). Fractions were analyzed by TLC (cerium molybdate staining). Fractions containing the purified product were combined and concentrated to dryness. Residual volatiles were removed under high vacuum to give the title compound (34 mg, 54 μmol, 36% yield) as a colorless oil. The product was dissolved in 5.0 mL of Gibco HO (0.01 M mass). C18-HPLC, purity determined by ELSD: 99.0% (RV=8.76 mL).

[0142] 1-Azido-18,18-dimethyl-20-methylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3-oxopropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=1, R 1 =MeSO2, R 2 =H, each R 4 = methyl, and X = NH(CH2)2CHO) Process 3 1-Azido-18,18-dimethyl-20-methylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3,3-diethoxypropyl)carbamate N-Hydroxysuccinimide (98 mg, 0.85 mmol) was added to a solution of triphosgene (0.25 g, 0.85 mmol) in 8.5 mL of anhydrous THF under N2. Pyridine (82 μL, 1.0 mmol) was added dropwise, with immediate formation of a white precipitate. The suspension was stirred at ambient temperature for 15 min and then filtered through a cotton plug. The filtrate was concentrated to dryness and redissolved in 2 mL of anhydrous THF. A solution of 1-azido-18,18-dimethyl-20-methylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-aza-19-icosanol (80 mg, 0.17 mmol, 0.06 M) in 1 mL of anhydrous THF was added. The reaction was stirred at ambient temperature and monitored by C18-HPLC (ELSD). After 2 h, the starting alcohol was consumed. The reaction mixture was partitioned with 50 mL of 1:1 EtOAc:KHSO4 (5% aq). The layers were separated and the washed organic phase was washed successively with KHSO4 (5% aq), water, NaHCO3 (sat. aq) and brine (25 mL each). The organic phase was dried over MgSO4, filtered and concentrated by rotary evaporation. Crude succinimidyl carbonate (91 mg) was dissolved in 2 mL of anhydrous THF and 1-amino-3,3-diethoxypropane (61 μL, 0.37 mmol) was added. The reaction was stirred at ambient temperature and monitored by C18-HPLC (ELSD). After 5 min, succinimidyl carbonate was converted to a single slower eluting product peak. The reaction mixture was partitioned with 30 mL of 1:1 EtOAc:sodium acetate (0.2 M, pH 5.0). The layers were separated and the organic phase was washed successively with water and brine (15 mL each). The washed organic phase was dried over MgSO4, filtered, and concentrated by rotary evaporation. Residual volatiles were removed under high vacuum to give the crude title compound (61 mg, 95 μmol, 56% crude yield for two steps) as a yellow oil.

[0143] Process 4 1-Azido-18,18-dimethyl-20-methylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3-oxopropyl)carbamate Water (135 μL) and TFA (135 μL) were added sequentially to a solution of 1-azido-18,18-dimethyl-20-methylsulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaicosan-19-yl(3,3-diethoxypropyl)carbamate (61 mg, 95 μmol, final concentration 0.1 M) in 0.68 mL of CHCl. ​​The reaction was stirred at ambient temperature and monitored by C-HPLC (ELSD). After 25 min, the reaction was judged to be complete. The mixture was concentrated to dryness. The concentrate was loaded onto a SiliaSep 4g silica gel column and the product was eluted with a stepwise gradient of acetone in CHCl (0%, 20%, 40%, 60%, 80%, 100% acetone, 30 mL each step). Fractions were analyzed by TLC (cerium molybdate staining) and C18-HPLC. Fractions containing the purified product were combined and concentrated to dryness. Residual volatiles were removed under high vacuum to give the title compound (12 mg, 21 μmol, 22% yield) as a colorless oil. After characterization, the product was dissolved in 2.0 mL of Gibco HO (0.01 M mass). C18-HPLC, purity determined by ELSD: 91.3% (RV=5.60 mL). (4) Additional compounds of formula (I) which were prepared according to these methods are listed below. 7-(15-azido-4,7,10,13-tetraoxapentadecanamido)-1-(4-phenylsulfonyl)-2-heptyl N-(3-oxypropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=5, R 1 = PhSO2, R 2 =H, each R 4 =H, and X=NH(CH2)2CHO). 7-(15-azido-4,7,10,13-tetraoxapentadecanamido)-1-(methylsulfonyl)-2-heptyl N-(3-oxypropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=5, R 1 =MeSO2, R 2 =H, each R 4 =H, and X=NH(CH2)2CHO). 7-(15-azido-4,7,10,13-tetraoxapentadecanamido)-1-(morpholinosulfonyl)-2-heptyl N-(3-oxypropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=5, R 1 =O(CH2CH2)2N-SO2, R 2 =H, each R 4 =H, and X=NH(CH2)2CHO). 5-(15-azido-4,7,10,13-tetraoxapentadecanamido)-3,3-dimethyl-1-(thiomorpholinosulfonyl)-2-pentyl N-(3-oxypropyl)carbamate (in formula IIa, Z=N3, S=(CH2CH2O)4(CH2)2C(O)NH, n=1, R 1 =S(CH2CH2)2NSO2, R 2 =H, each R 4 = methyl, and X = NH(CH2)2CHO).

[0144] Example 1 Production and activity of IL-2[N88R,C125S] IL-2[N88R,C125S] was produced by expression in HEK cells. Cell-based receptor binding assays were performed to identify the high affinity αβγ trimer of the IL-2 receptor (T reg ) and intermediate affinity βγ dimers (T eff The activity of the mutein against forms of IL-2Rαβγ was evaluated (Table 1). The mutant binds only 6-fold weaker to IL-2Rαβγ than to IL-2, but approximately 900-fold weaker to IL-2Rβγ. Importantly, the mutant is more than 3,000-fold selective for IL-2Rαβγ versus IL-2Rβγ.

[0145] The U2OS cell-based assay kit for IL-2Rαβγ binding was performed according to the manufacturer's instructions (DiscerX, Part#93-1003E3CP0). Cells were seeded in 100 μL (~10,000 cells / well) in 96-well assay plates and grown for 24 hours at 37°C, 5% CO2. Cells were then treated with a dilution series of either WT IL-2, IL-2 N88R,C125S, or IL-2 N88R,C125S released from microspheres at pH 9.4 for 6 hours at 37°C, 5% CO2. Eleven WT IL-2 concentrations were assayed ranging from 2 pg / mL to 100 ng / mL (0.1 pM to 6 nM). Eleven IL-2 N88R,C125S concentrations and released IL-2 N88R,C125S concentrations were assayed from 200 pg / mL to 10 μg / mL (10 pM to 600 nM). Treated cells were incubated with chemiluminescent substrate in the dark at ambient temperature for 1 hour, then luminescence was read on a Spectramax i3 plate reader with an integration time of 250 ms.

[0146] The U2OS cell-based assay kit for IL-2Rβγ binding was performed according to the manufacturer's instructions (DiscerX, Part#93-0998E3CP5). Cells were seeded in 50μL (~5,000 cells / well) in 96-well assay plates and grown for 48 hours at 37°C, 5% CO2. Cells were then treated with a dilution series of either WT IL-2, IL-2 N88R,C125S, or IL-2 N88R,C125S released from microspheres at pH 9.4 for 6 hours at 37°C, 5% CO2. Eleven WT IL-2 concentrations were assayed from 17pg / mL to 1μg / mL (1pM to 61nM). Eleven IL-2 N88R,C125S concentrations were assayed from 1.7ng / mL to 100μg / mL (100pM to 6μM). Eleven released residual IL-2 N88R,C125S concentrations were assayed from 170 pg / mL to 10 μg / mL (10 pM to 600 nM). Treated cells were incubated with chemiluminescent substrate for 1 hour at ambient temperature in the dark, then luminescence was read on a Spectramax i3 plate reader with an integration time of 250 ms.

[0147] The results of these measurements are shown in FIG. Table 1. Binding of IL-2 and IL-2 N88R,C125S to cells containing αβγ and βγ receptors. [Table 1]

[0148] Example 2 Optimization of cytokine reductive alkylation [ka] The attachment of the linker was by reductive alkylation of the N-terminal amino group of IL-2. 200 μM of IL-2 N88R,C125S was reacted with a linker reagent of formula (IIb) (i.e., in formula (IIa), R 1 =MeSO2, R 2 and R 4=H, S=(CH2CH2O) h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) The N3 group was treated with a concentration series of PEG-cyclooctyne DBCO-PEG (linker, where h=4, g=2, p=0, and m=3). 5kDa After reacting with PEG to induce a gel shift due to PEG attachment, the reactions were analyzed by SDS-PAGE. A linker:protein ratio of 1.5:1 was found to be optimal, resulting in a mixture of 58:34:5:3 unmodified protein:single linker-protein:double linker-protein:triple linker-protein (Table 2). Figure 3 shows the resulting gel bands quantified with ImageJ. C125S (200 μM) was treated with 1, 1.5, 2, or 3 equivalents (Eq.) of linker-CHO (Mod=MeSO2) and 10 mM NaCNBH3 in 50 mM MES, 150 mM NaCl, pH 6.0, at ambient temperature for 20 hours. Table 2. Distribution of linker-protein products from reductive alkylation of IL-2 N88R,C125S [Table 2]

[0149] Example 3 Linker-cytokine production IL-2[N88R,C125S] was attached to a releasable linker by one of two methods.

[0150] (1) Random acylation A mixture of cytokines (3.4 mL, 4.81 mg / mL, 1.00 umol) and 1.44 mL of 100 mM HEPES (pH 7.0) was dissolved in 4-azido-3,3-dimethyl-1-(isopropylsulfonyl)-2-butylsuccinimidyl carbonate [R 1 = i PrSO2, R 2 =H, R 4= Me, Z = N3, n = 1; S = absent; and X = succinimidyloxy] (156 uL, 10 mg / mL in acetonitrile, 4 umol) and kept at 4°C for 20 hours. Hydroxylamine (0.55 mL, 1 M, pH 7.0) was added and kept at 4°C for an additional 23 hours. The mixture was applied to a PD-10 column using 50 mM MES, pH 6.0, 0.05% Tween-20 and OD was measured. 280 Analysis by SDS-PAGE showed the formation of a 57:31:6:6 mixture of unmodified:1 linker:2 linker:3+ linker.

[0151] (2) Reductive alkylation Reductive alkylation was carried out using the method described by Schneider et al., Bioconjugate Chem (2016) 27: 2534-9, which is incorporated herein by reference. IL-2 N88R,C125S (final concentration 250 μM, 1.25 μmol, 20.5 mg) was added to a solution of 4.25 mL of 50 mM MES, 150 mM NaCl, pH 6.0 (reaction buffer) at 0° C. with O-7-[(15-azido-13,10,7,4-tetraoxapentadecanoyl)amino]-1-(methylsulfonyl)-2-heptyl N-3-oxapropylcarbamate [in formula (II), R 1 =MeSO2, R 2 =H, R 4A solution of 20 mM NaOAC (pH 5.0), 20 mM NaCNBH3 (final concentration 10 mM, 1 μmol, 0.5 μL) in reaction buffer was added. The reaction was carried out in the dark at ambient temperature for 22 hours. Excess reagent was removed using a PD-10 column equilibrated with 20 mM MES, 150 mM NaCl, 0.05% Tween-20, pH 6.0. After concentration with an Amicon Ultra 10,000 MW cutoff concentrator, 1.85 mL, 600 μM (by A280)-1.1 μmol of linker-N-terminal aminopropyl-IL-2[N88R,C125S] was recovered, 89%-total peptide.

[0152] Example 4 Fabrication of IL2- and [aminopropyl]-IL2-releasing hydrogel microspheres Microsphere Activation PEG hydrogel microspheres of formula (IV) (prepared according to Henise et al., Engineering Reports (2020) https: / / doi.org / 10.1002 / eng2.12091) (wherein P 1 and P 2 is a 20 kDa 4-arm PEG; * where Z=N3 and Z'=triazole from 5-hydroxycyclooctyne; n=4; R 11 =CN;R 12 =H; each R 14=H; B=NH2; x=4; y=0; z=0; and r=4) were used. These were activated to formula (IV), B=NH-CO-O-(4-cyclooctynyl), as follows: To a 1.3 g MeCN suspension of a slurry of microspheres, B=NH2 (4.2 μmol, NH2), in a 15 mL conical tube, was added a 1 mL MeCN solution of 4-cyclooctynyl succinimidyl carbonate (5 μmol, 1.2 equiv.) and a 1 mL MeCN solution of N,N-diisopropylethylamine (17 μmol, 4 equiv.). The reaction was stirred end-over-end at ambient temperature for 6 hours. The slurry was washed with 4×12 mL MeCN, followed by 4×12 mL 20 mM MES, 150 mM NaCl, 0.05% Tween-20, pH 6.0.

[0153] Using the same method, microspheres of formula (IV) where B=NH2 were activated to those of formula (IV) where B=(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy-CO—NH by reaction with BCN-OSu ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethylsuccinimidyl carbonate) instead of 4-cyclooctynylsuccinimidyl carbonate.

[0154] To attach the linker-cytokine, a suspension of 2 g of activated microsphere slurry in 20 mM MES, 150 mM NaCl, 0.05% tween-20, pH 6.0 was mixed with a solution of 18.3 mg (1.1 nmol) of linker-AP-IL-2 N88R,C125S (37% linker-IL-2 by gel shift assay, Example 3) in 1.9 mL of the same buffer in a 15 mL conical tube. The mixture was incubated for 23 hours at 37° C. on an orbital shaker at 250 rpm. The slurry was washed with 8×12 mL of the above buffer, followed by 4×6 mL of 20 mM MES, 250 mM NaCl, 0.05% tween-20, pH 6.0. The total loading of the microspheres was determined as A280 (ε) of AP-IL-2 released from 29-32 mg aliquots of slurry dissolved in 9 volumes of 50 mM NaOH. 280 =10,095M -1 cm -1 ) and in the slurry, 102 nmol IL-2 N88R,C125S gm -1 It was.

[0155] PEG hydrogel microspheres were loaded with linker-IL-2 prepared by random acylation (Example 3) in the same manner to give an insoluble conjugate loaded at 0.11 mM with the protein having SEQ ID NO:3.

[0156] Example 5 In vitro release kinetics The rate of β-elimination was measured under accelerated release conditions using 257 mg of the microsphere-IL-2 variant slurry of Example 4 in 257 μL of 250 mM NaBorate, 0.05% (v / v) Tween-20, pH 9.4 in Eppendorf tubes at 37° C. At time intervals, samples were removed from the 37° C. water bath and centrifuged at 21,000×g for 1 min, and 100 μL of the supernatant was diluted with 100 μL of ethanol. 280was measured in a cuvette-based UV / Vis spectrophotometer. After measurement, the assayed supernatant was transferred back to the tube containing the microspheres and incubation at 37° C. was continued. The release rate was calculated as the amount of A released. 280 The rates were calculated by fitting the reaction rates vs time to a first-order rate equation in Graphpad Prism. Recognizing that the β-elimination is first order with hydroxide ions, the rates were calculated as k pH7.4 =k pH ×10 (pH-7.4) The release profile of IL-2[N88R,C125S] from the random acylation conjugate of Example 2 was biphasic with half-lives of 0.4 and 41 hours at pH 9.4, which correspond to 40 and 4100 hours at pH 7.4. The release profile of AP-IL-2[N88R,C125S] from the reductive alkylation conjugate of Example 2 was monophasic with a half-life of 11 hours at pH 9.0, which corresponds to 440 hours at pH 7.4.

[0157] Example 6 Pharmacokinetics of IL-2[N88R,C125S] released from hydrogel microspheres in rats Syringes (0.5 mL, 29 gauge, fixed needle, BD) were loaded under sterile conditions with an average of 50 mg or 300 mg (5 nmol or 30 nmol IL-2 [N88R,C125S]) of the microsphere-IL-2 slurry of Example 4 in a dosing buffer consisting of 20 mM MES, 250 mM NaCl, 0.05% (w / v) Tween-20, pH 6.0. The contents of each syringe were administered subcutaneously into the flank of four cannulated male Sprague Dawley rats weighing an average of 250 g. Blood samples (200 μL) were taken at 0, 4, 8, 24, 48, 96, 168, 240, 336, 408, 504, 576, and 672 hours. Plasma was collected, protease inhibitors were added, and samples were frozen at −80° C. until analysis. As shown in FIG. 4, IL-2 (or NH2(CH2)3-IL-2, "AP-IL2") was observed in plasma 96 hours after administration using the microsphere conjugate of Example 2.

[0158] Example 7 Pharmacodynamics of IL2 and IL2[N88R,C125S] in mice The pharmacodynamics of free IL-2[N88R,C125S] was compared to that of native IL-2 in NOD (non-obese diabetic) mice. Three groups of three NOD mice were injected daily for five consecutive days with either PBS vehicle, Proleukin (25,000 units, 63 μg), or IL-2[N88R,C125S] (25,000 units, 63 μg). Mice were sacrificed 2 hours after the last injection, and spleens and pancreases were harvested for flow cytometry analysis to measure changes in the total number of T cells and their differentiation in the spleen and pancreatic islets.

[0159] IL-2[N88R,C125S] inhibits splenic CD4 + and CD8 + Expansion of both T cell populations was increased in native IL-2, while it had little or no effect on effector / memory T cells (Figure 5).

[0160] NOD mice were given daily injections of PBS vehicle, Proleukin (25,000 units), or IL-2[N88R,C125S] (25,000 units) and sacrificed 2 hours after the last injection on day 5. The pharmacodynamics of IL-2[N88R,C125S] in the spleen are shown in FIG.

[0161] Both native IL-2 and IL-2[N88R,C125S] had little to no effect on T cells in the islets compared to PBS vehicle (Figure 6). It should be noted that treating mice with IL-2[N88R,C125S] reduced the total number of islets.

[0162] NOD mice were given daily injections of PBS vehicle, Proleukin (25,000 units), or IL-2[N88R,C125S] (25,000 units) and sacrificed 2 hours after the last injection on day 5. The pharmacodynamics of IL-2[N88R,C125S] in pancreatic islets are shown in FIG.

[0163] Example 8 Pharmacokinetics / pharmacodynamics of [aminopropyl]-IL2[N88R,C125S] released from microsphere-IL-2[N88R,C125S] in mice Three groups of six NOD mice were used to measure the PK / PD of [aminopropyl]-IL-2[N88R,C125S] released from the microsphere-IL-2[N88R,C125S] conjugate. The first group was injected five times daily with free IL-2[N88R,C125S] (25,000 units, 63 μg). The second group was administered empty microspheres capped with cyclooctyne N3(CH2CHO)7H via subcutaneous injection. The third group was administered a single subcutaneous injection of the microsphere-IL-2[N88R,C125S] of Example 4 (0.5, 1, 5, 10 or 19 mg protein / kg). Plasma, peripheral blood mononuclear cells (PBMCs), and organ tissues were prepared and analyzed as described in the figure legends. To monitor changes in T cell populations, flow cytometric analysis of lymphocytes was performed. Spleens and lymph nodes and pancreatic islets were isolated and single cell suspensions were prepared. Surface staining was performed by standard cell surface immunofluorescence staining for flow cytometry. Fixation and intracellular staining followed the protocol of eBioscience Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific). Antibodies used were against CD3, CD4, CD8, CD25, CD44, CD45, and FoxP3; all were from commercial vendors. Stained single cell suspensions were analyzed using an LSRII flow cytometer (BD Biosciences).

[0164] FIG. 7 shows the pharmacokinetics of [aminopropyl]-IL-2[N88R,C125S] released from microsphere-IL-2[N88R,C125S] ("MS-IL-2 variant") in mice. Panel A: BALB / c mice (n=6) were administered a single subcutaneous injection in the flank containing 28 nmol (19 mg / kg) or 9.9 nmol (6.5 mg / kg) of microsphere-IL-2[N88R,C125S]. The t 1 / 2Panel B: NOD mice (n=6) were administered microsphere-IL-2[N88R,C125S] in the flank. In both cases, plasma was analyzed using Thermofisher ELISA to quantify the concentration of IL-2[N88R,C125S].

[0165] Treatment with microsphere-IL-2[N88R,C125S] significantly reduced Foxp3 expression in both spleen and PBMCs. + CD4 + In spleen and PBMCs, approximately 70% and 55% of T cells were Foxp3 + CD4 + T cells (Figure 8). CD8 + The proportion of CD8 T cells was also increased compared to controls. + The cells increased from 11% to 25% in spleen and from 15% to 60% in PBMC.

[0166] FIG. 8A shows the expression of Foxp3 in spleen and PBMCs. + CD4 + Figure 8B shows the proliferation of CD8 T cells in spleen and PBMCs. + This indicates proliferation of T cells. CD8 + The percentages of cells were approximately 11% and 19%, respectively. When treated with microsphere-IL-2[N88R,C125S], these percentages increased to approximately 25% and approximately 60%, respectively. NOD mice were administered IL2-mutant (QDx5, 25,000 units), a single injection of empty microspheres, or microsphere-IL-2[N88R,C125S] (18 mg / kg). Mice were sacrificed 2 hours after the last administration on the fifth day.

[0167] CD8 + Foxp3 without activating cells + CD4 +A dose titration study of microsphere-IL-2[N88R,C125S] was performed to determine the effective dose to expand the T cell population. Four concentrations of microsphere-IL-2[N88R,C125S] (0.5 mg / kg, 1 mg / kg, 5 mg / kg, and 10 mg / kg) were tested and pharmacodynamics were monitored over a 2-week period by PBMC. Foxp3 + CD4 + A dose-dependent proliferation of T cells was observed in PBMCs after a single injection of microsphere-IL-2[N88R,C125S]. Foxp3 + CD4 + T cell proliferation peaked at day 4 and returned to baseline levels by day 14 at all doses (Figure 9A). Importantly, CD8 + The percentage of cells did not increase at any dose (Fig. 9B).

[0168] FIG. 9A shows that microsphere-IL-2[N88R,C125S] inhibited Foxp3 in NOD mice (n=3 / dose group). + CD4 + Figure 9B shows that they preferentially proliferate CD8 T cells. + As shown, Foxp3 + CD4 + T cell proliferation peaked on day 4 at all doses and returned to baseline levels by day 14.

[0169] Example 9 Production of linker-IL-15 The linker of Example 2 was conjugated to the N-terminus of IL-15 via reductive alkylation with NaCNBH3 as described for IL-2 above. The reaction mixture contained IL-15 (30 μM), N3-PEG4-linker (MeSO2)-CHO (90 μM), and NaCNBH3 (10 mM) in 25 mM sodium phosphate, 250 mM NaCl, pH 7.4. The reaction was carried out for 24 hours at ambient temperature in the dark. Excess reagent was removed using a PD-10 column equilibrated with 20 mM sodium citrate, 500 mM NaCl, 0.05% tween-20, pH 5.86. The desalted reaction mixture was concentrated using an Amicon Ultra 3,500 MW cutoff concentrator.

[0170] Small-scale (2.25 nmol, 75 μL) reductive alkylation reactions with varying linker equivalents were performed with IL-15 to determine optimal reaction conditions. Initial reactions were performed with 1, 1.5, and 2 equivalents of N3-PEG4-L(MeSO2)-CHO linker and demonstrated a 1:1 ratio of one linker attached to the protein (data not shown). Subsequent reactions were performed with 1.5, 3, and 5 equivalents of linker to increase the conversion of the unmodified protein (Figure 10A). When 3 equivalents of linker were used, the reaction resulted in approximately 52% of the IL-15 having only one linker attached to the protein, and approximately 5% of the IL-15 having two linkers attached (Figure 10B). Increasing the linker concentration to 5 equivalents resulted in a small increase in single linker-protein, but approximately 27% of the total protein was attached with two or more linkers.

[0171] The progress of the reaction was monitored by SDS-PAGE as shown in Figure 10. 5kThe percentage of modified IL-15 was measured by gel shift assay. Table 3 shows the percentage of modified IL-15. The bands were quantified using ImageJ software. IL-15 (30 μM) was treated with 1.5, 3, or 5 equivalents of linker-CHO (Mod=MeSO2) and NaCNBH3 (10 mM) in 25 mM sodium phosphate, 500 mM NaCl for 20 hours at ambient temperature in the dark. [Table 3]

[0172] Optimized reaction conditions using 3 equivalents of linker were used in large-scale reactions (0.93 μmol to 1.08 μmol). Large-scale reactions were run in duplicate.

[0173] Example 10 Preparation of microsphere-IL-15 A slurry of BCN-activated microspheres (2.6 μmol BCN, Example 4) was washed five times (~35 mL) with 20 mM sodium citrate, 500 mM NaCl, 0.05% tween-20, pH 5.86 in a sterile syringe. Linker-IL-15 (Example 9) (1 μmol total protein, containing approximately 50% alkylated IL-15) was added to the syringe (0.22 μM) through a sterile filter. The mixture was agitated end-over-end for 18 hours at ambient temperature. The slurry mixture was then washed five times with 20 mM sodium citrate, 500 mM NaCl, 0.05% tween-20, pH 5.86. Unreacted BCN-activated microspheres were capped with N3(CH2CH2O)7H and then washed six more times. The concentration of IL-15 loaded onto the microspheres (216–336 μM) was determined by the A280 (ε) from IL-15 released from a 5 mg aliquot of the slurry dissolved in 4 volumes of 50 mM NaOH. 280 =7240M -1 cm -1 The MS-IL-15 concentrations from three separate loadings were determined to be 336 nmol / mL, 216 nmol / mL, and 232 nmol / mL.

[0174] Example 11 Pharmacokinetics of IL-15 released from microsphere-IL-15 The microsphere-IL-15 slurry of Example 10 (275 nmol protein / mL) was diluted in 25 mM sodium citrate buffer pH 5.9, containing 500 mM NaCl, 0.05% tween-20, and 1.25% (w / v) hyaluronic acid. For studies requiring different doses of microsphere-IL15, serial dilutions were used to obtain the desired microsphere-IL-15 concentrations. In all cases, the microsphere conjugates were handled and prepared using sterile conditions. Syringes with fixed needles (27G) were back-filled with the conjugate (100 μL). The contents of the syringes were administered subcutaneously or intraperitoneally to normal male C57BL / 6J mice. Blood samples were taken from each group of 3 mice at -24, 4, 8, 24, 48, 96, 168, and 240 hours. HALT protease inhibitor cocktail (ThermoFisher Scientific) was added to all plasma samples, which were then frozen at −80°C until analysis.

[0175] ELISA for hIL-15 was performed according to the manufacturer's instructions (R&D Systems, hIL-15 Quantikine, Cat#D1500) to measure rhIL-15 plasma. Plasma samples were thawed on ice and then diluted in standard diluent provided by the manufacturer. 4 and 8 hour samples were diluted 50-fold, 24 hour samples were diluted 25-fold, and prebleed, 48, 96, 168, and 240 hour samples were diluted 10-fold. hIL-15 concentrations were plotted as a function of time and goodness of fit using GraphPad Prism software.

[0176] For flow cytometry analysis, PMBCs were prepared and surface staining was performed to quantify NK1.1, CD3, CD8, and CD44 expressing cells. Commercially available FITC-, PE-, or allophycocyanin-conjugated antibodies were used. Sample data were collected on a FACScan flow cytometer (BD Biosciences) and analyzed using FlowJo cytometry analysis software (TreeStar, Ashland, OR).

[0177] The pharmacokinetics of [aminopropyl]-IL-15 released from the microsphere conjugate was measured in normal C57BL / 6J mice. Mice were administered 2.4 nmol of conjugated protein (200 μL injection). There was no significant change in the mean mouse weight at the beginning (25.1 ± 1.3 g) or at the end (25.1 ± 1.4 g). After approximately 120 hours, the observed concentrations decline rapidly (Figure 11). Fit of a one-phase decay model to the data up to 120 hours yields a half-life of at least 200 hours. Data points from 120 to 240 hours were fitted to a one-phase decay model, with a t of 27 hours. 1 / 2 A second injection of MS-IL-15 (50 μg) increased plasma IL-15 levels measured at 248 hours to levels similar to those measured at the initial dose. 1 / 2 However, it was observed from 264 hours to 360 hours.

[0178] Figure 11 shows the pharmacokinetics of [aminopropyl]-IL-15 released from MS-IL-15 in C57BL / 6J mice. Normal male C57BL / 6J mice were administered MS-IL-15 (50 μg) at t = 0 and t = 240 hours. Plasma samples were prepared and analyzed using a human IL-15 Quantikine ELISA (R&D systems). 1 / 2 has been observed, with a t of at least 115 hours 1 / 2 was observed up to 120 hours, followed by a second t of 43 hours from 120 to 240 hours. 1 / 2A second injection of MS-IL15 (50 μg) was administered immediately after the 240 hour bleed (data in blue).

[0179] Figure 12 shows the dose-dependence of the pharmacokinetics of [amino-propyl]-IL-15 released from microsphere-IL-15 in C57BL / 6J mice. Normal male C57BL / 6J mice were administered MS-IL-15 (12.5, 25, or 50 μg). Plasma samples were prepared and analyzed using a human IL-15 Quantikine ELISA (R&D systems).

[0180] The route of administration (i.e., subcutaneous [sc] and intraperitoneal [ip]) significantly increased the t value of released [aminopropyl]-IL15. 1 / 2 (Figure 13). However, AUC sc (14.9nM * h) compared with AUC ip (25.2nM * h) was almost 2-fold higher, which may indicate an increased bioavailability or increased rate of absorption of IL-15 from the intraperitoneal space.

[0181] Figure 13 shows the pharmacokinetics of [aminopropyl]-IL-15 released from microsphere-IL-15 in C57BL / 6J mice administered subcutaneously or intraperitoneally. Normal male C57BL / 6J mice were administered MS-IL-15 (50 μg) subcutaneously (black, ●) or intraperitoneally (blue, ■). Plasma samples were prepared and analyzed using a human IL-15 Quantikine ELISA (R&D systems). By 120 hours, similar t 1 / 2 was observed.

[0182] Example 12 Pharmacodynamics of [aminopropyl]-IL-15 released from microsphere-IL-15 The pharmacodynamics of [aminopropyl]-IL-15 released from the microsphere-IL-15 of Example 10 was measured in normal male C57BL / 6J mice (n=3 / group) (Figure 14). Mice were administered the microsphere-IL-15 (2.5, 12.5, 25, or 50 μg of conjugated protein) produced in Example 10. PMBCs were prepared and surface stained for flow cytometric analysis of NK1.1, CD3, CD8, and CD44 expressing cells. Commercially available FITC-, PE-, or allophycocyanin-conjugated antibodies were used. Sample data were collected on a FACScan flow cytometer (BD Biosciences) and analyzed using FlowJo cytometry analysis software (TreeStar, Ashland, OR). Clinical observations showed no reactions at the injection site and no significant changes in mean body weight at the start (25.1±1.3 g) and end (25.1±1.4 g).

[0183] A single subcutaneous injection of MS~IL-15 conjugate (12.5 μg, 25 μg, or 50 μg) significantly increased the expression of CD44 in PBMCs. hi CD8 + This resulted in a dose-dependent increase in CD44 T cells. hi CD8 + A 2-4 fold expansion of T cells peaked 5 days after treatment. These cells remained elevated above the control group for 21 days (Figure 14A). Mice receiving the highest dose of MS-IL-15 (50 μg) still had CD44 hi CD8 + During the experimental period, a single dose of native rhIL-15 (2.5 μg) or an equivalent dose of MS-IL-15 (2.5 μg) increased the levels of CD44 T cells. hi CD8 + No increase in T cells was observed.

[0184] Dose-dependent proliferation of NK cells was also observed in PBMCs after a single subcutaneous injection of MS~IL-15 conjugates (Figure 14B). When MS~IL-15 (12.5 μg, 25 μg, or 50 μg) was administered, approximately 2- to 3-fold proliferation of NK cells peaked 5 to 7 days after treatment. NK cells remained elevated for 14 to 21 days. No proliferation of NK cells was observed with a single dose of native rhIL-15 (2.5 μg) or with an equivalent dose of MS~IL-15 (2.5 μg).

[0185] Example 13 Preparation of linker-RLI and microsphere-RLI conjugates RLI (receptor-binding interleukin) is a fusion protein containing IL-15 and the sushi domain of the receptor alpha subunit that acts as a superagonist of the IL-15 receptor beta / gamma complex (Mortier et al., J. Biological Chem. 2006, 281: 1612-9; U.S. Patent No. 10,358,488).

[0186] Small-scale reductive alkylation reactions of RLI (10 nmol, 50 μL) were performed with varying linker concentrations to determine optimal reaction conditions for stoichiometric linker addition. Initial reactions were performed using 1.5, 2, 3, and 5 equivalents of the linker from Example 2 (IIb). Under the conditions tested, when 1.5 equivalents of linker were used, 44% of the RLI was modified with one linker and 46% remained unmodified. At 2 equivalents of linker, approximately 53% of the RLI was observed to result in stoichiometric addition of the linker, 33% of the RLI was unmodified, and 14% was covalently attached with multiple linkers. Increasing the amount of linker to 3 equivalents increased the percentage of attachment of two linkers (27%) and also increased the formation of RLI containing three linkers (6%). These percentages were further increased in the presence of 5 equivalents of linker (Figure 15). [Table 4]

[0187] Reductive alkylation of RLI was confirmed by SDS-PAGE using DBCO-PEG 5K The percentage of modified RLI was measured by gel shift assay. Figure 15 shows the percentage of modified RLI measured from the gel shift assay. The bands were quantified using ImageJ software. RLI (10 nmol) was treated with 1.5, 2, 3, or 5 equivalents of linker-CHO (Mod=MeSO2) and NaCNBH3 (10 mM) in 25 mM MES, 500 mM NaCl, and 0.05% Tween-20 for 20 hours at room temperature in the dark.

[0188] A large-scale reductive alkylation reaction (800 nmol, 4 mL) was carried out using two equivalents of linker. The reductively alkylated RLI was then conjugated to BCN-activated microspheres (Example 4). EDTA (1 mM) and methionine (30 mM) were added to the reaction to minimize oxidation processes. After the conjugation reaction, the microspheres were washed extensively with buffer (25 mM sodium citrate, 500 mM NaCl, 0.05% tween-20, 30 mM methionine, pH 5.9) to remove non-covalently attached RLI. A small aliquot (~25 mg) of the washed microspheres was treated with NaOH (50 mM) to measure the concentration of RLI covalently attached to the microspheres. The RLI concentration on the microspheres was determined to be 175 nmol / mL.

[0189] Example 14 Biological activity of RLI released from microsphere conjugates After RLI is released from the microsphere conjugate, aminopropyl residues remain at the site of conjugation. To test the biological activity of the released [aminopropyl]-RLI, a cell-based assay was used to measure the ability of [aminopropyl]-RLI to induce receptor dimerization compared to native RLI. The EC 50The curves are superimposable, indicating that the aminopropyl residues do not affect IL-15 activity as assessed by this bioactivity assay (FIG. 16).

[0190] Figure 16 shows the results of an IL-2Rβγ receptor binding cell-based assay for RLI. Using a U2OS cell-based assay, the binding activity (EC 50 = 180 pM) and measure the natural RLI (EC 50 =160pM).

[0191] Example 15 Pharmacokinetics of [aminopropyl]-RLI released from microsphere conjugates The pharmacokinetics of [aminopropyl]-RLI released from the microsphere conjugate was measured in normal C57BL / 6J mice. Mice were injected subcutaneously with the conjugate (1.5 nmol protein, 100 μL injection). Blood was collected at designated time points over 10 days and plasma was prepared. The concentration of [aminopropyl]-RLI in plasma was measured using an RLI-specific ELISA (FIG. 17). Manual inspection of the data suggested a Tmax of 48 hours, and fitting the data to a monophasic decay model gave a half-life of 135 hours. There was no change in mouse body weight (start weight: 21.5±1.1 g; end weight: 21.5±1.1 g).

[0192] Figure 17 shows the pharmacokinetics of [aminopropyl]-RLI released from the microsphere conjugate in C57BL / 6J mice. Normal male C57BL / 6J mice were administered microsphere-RLI conjugate (1.5 nmol). Plasma samples were prepared and analyzed using R&D systems DuoSet hIL15 / IL15Rα complex ELISA (DY6924). Data were fitted to a monophasic decay model resulting in a half-life of 135 hours.

[0193] Example 16 Pharmacodynamics of [aminopropyl]-RLI released from microsphere conjugates The pharmacodynamics of the MS~RLI conjugate (34 μg, 1.5 nmol) was compared to that of empty MS and free RLI (2.5 μg, QD × 4) in C57Black mice (n = 5 / group). Blood was collected over a 13-day period, and PBMCs were surface stained using standard laboratory techniques. Fixation and intracellular staining followed the protocol of the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific). Commercially available antibodies used were directed against NK1.1, CD3, CD8, CD19, CD44, and Ki-67 expressing cells. Stained single cell suspensions were analyzed using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo cytometry analysis software (TreeStar, Ashland, OR). Cell populations of particular interest were CD8 + Memory T cells (CD44 hi CD8 + ), natural killer cells (CD3 - NK1.1 + ), proliferating CD8 + Memory T cells (CD44 hi CD8 + Ki-67 + ), and proliferating natural killer cells (CD3 - NK1.1 + Ki-67 + ), was.

[0194] CD44 hi CD8 + The increase in T cells was significant after 5 days of treatment in the MS-RLI and native RLI groups (Figure 18A). This cell mass was not maintained by native RLI, and the mass returned to baseline levels by day 7. This is due to the short half-life (t 1 / 2 This was expected due to the rapid clearance of free RLI. The MS-RLI conjugates were able to inhibit CD44 for 13 days after treatment.hi CD8 + T cell levels were maintained. All five mice that received the MS~RLI conjugate developed lesions at the injection site and required euthanasia.

[0195] CD8 + T cell proliferation was measured by the proliferation marker Ki-67. Three days after injection, CD8 + An increase in T cells was observed (Figure 18B). + T cell percentages peaked at day 5 in all groups and then rapidly returned to baseline.

[0196] An increase in the percentage of NK cells was also observed in mice administered the MS~RLI conjugate compared to control and native RLI (Figure 19A). Free RLI injection and the MS~RLI conjugate increased the percentage of NK cells in PBMCs by ~4-fold and ~15-fold, respectively. NK cell levels returned to baseline by 10 days after treatment in all groups. NK cell proliferation was significantly increased 3 days after treatment and was maintained for 5 days in each group (Figure 19B). NK cell proliferation returned to baseline by 7 days after treatment.

[0197] Example 17 Fabrication of degradable PEG hydrogels [ka] The hydrogel of the present invention reacts to form a connecting functional group C * The hydrogel is prepared by polymerization of two prepolymers comprising groups C and C' to form: The prepolymer connection to one of C or C' further comprises a cleavable linker, which is introduced by reaction with a cleavable linker, such as the linker of formula (IIa) described herein, to introduce a cleavable linker to each crosslink of the hydrogel.

[0198] In one embodiment, the first prepolymer comprises a 4-arm PEG, each arm of which is terminated with an adaptor unit bearing two mutually unreactive ("heterologous") functional groups B and C. B and C can initially exist in protected form, allowing selective chemical reactions in a subsequent step. In certain embodiments, the adaptor unit is a derivative of an amino acid, particularly lysine, cysteine, aspartic acid, or glutamic acid, including derivatives in which the alpha amine group has been converted to an azide, e.g., the monoester of 2-azidoglutaric acid. The adaptor unit comprises a connecting functional group A formed by condensation of a functional group A on each prepolymer arm with the associated functional group A' on the adaptor unit. * The second prepolymer comprises a 4-arm PEG, each arm terminated with a functional group C' having complementary reactivity to group C of the first prepolymer, and C and C' react to form C * When the polymer is formed, crosslinking between the two prepolymers occurs.

[0199] As an illustrative example, a first prepolymer was prepared as follows: H-Lys(Boc)-OH was acylated with a linker of formula (IIa) where Z=azide to give an adaptor unit where A=COOH, B=Boc-protected NH2, and C=azide. This was coupled to a 20 kDa 4-arm PEG-tetraamine and the Boc group was removed to give A *A first prepolymer was obtained, in which A = amide, B = NH2, and C = azide, and a cleavable linker of formula (IIa) was incorporated into the bond between each arm and group C of the first prepolymer. The corresponding second prepolymer was prepared by acylation of a 20 kDa 4-arm PEG-tetraamine with 5-cyclooctynyl succinimidyl carbonate to obtain a second prepolymer with C' = cyclooctyne. When the first and second prepolymers were mixed, the reaction of the C = azide group with the C' = cyclooctyne group formed the corresponding triazole group, thereby crosslinking the two prepolymers into a three-dimensional network, in which each crosslink contains a cleavable linker obtained by incorporation of a compound of formula (IIa), and in which each node obtained by incorporation of the first prepolymer contains a residual functional group B = NH2 that can be derivatized for the addition of further linkers, drugs, fluorophores, metal chelators, etc.

[0200] All publications, including patents, patent applications, and scientific articles, mentioned in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including patents, patent applications, or scientific articles, was specifically and individually indicated to be incorporated by reference.

[0201] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain minor changes and modifications may be implemented in light of the above teachings. Accordingly, the specification and examples should not be construed as limiting the scope of the invention.

Claims

1. Formula: 【Chemical 1】 [In the formula, n is an integer from 0 to 6; R 1 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 2 is H or alkyl; each R 4 is independently H or C 1 -C 3 alkyl, or two R 4 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; S is absent or (CH 2 CH 2 O) h (CH 2 ) g CONH, where g=1-6 and h=0-1000; Y is NH(CH 2 CH 2 O) p (CH 2 ) m where m=2-6 and p=0-1000; D is IL-2, IL-7, IL-9, IL-10, or IL-21; and 【Chemistry 2】 indicates the attachment point between the linker-drug unit and the polymeric carrier. A hydrogel comprising a linker-drug unit represented by:

2. The hydrogel of claim 1, wherein the linker-drug unit is attached to the polymeric carrier through a carboxamide, amide, oxime, triazole, thioether, thiosuccinimide, or ether moiety.

3. The hydrogel described in claim 2, wherein the linker-drug unit is attached to the polymeric carrier through a triazole moiety.

4. The hydrogel of claim 1, wherein R 1 is -CN or -SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl, and R 2 is H, where each of R 5 and R 6 may independently be optionally substituted.

5. The hydrogel described in claim 4, wherein R 1 is -CN, SO 2 N(CH 3 ) 2 , -SO 2 CH 3 , -SO 2 Ph, -SO 2 PhCl, -SO 2 N(CH 2 CH 2 ) 2 O, -SO 2 CH(CH 3 ) 2 , -SO 2 N(CH 3 )(CH 2 CH 3 ), or -SO 2 N(CH 2 CH 2 OCH 3 ) 2 .

6. The hydrogel of claim 1, wherein R 2 is H.

7. The hydrogel described in claim 1, wherein n is 4, each R 4 is H, R 1 is -SO 2 R 5 , where R 5 is methyl, R 2 is H, S is (CH 2 CH 2 O) h (CH 2 ) g CONH, where g = 2 and h = 4, and Y is NH(CH 2 CH 2 O) p (CH 2 ) m , where m = 3 and p = 0.

8. The hydrogel of claim 1, wherein the polymeric carrier comprises a polymer having multi-arm chains.

9. The hydrogel of claim 8, wherein the polymeric carrier comprises an r-arm polymer, where r is an integer from 2 to 8.

10. The hydrogel of claim 9, wherein r is 4.

11. The hydrogel of claim 9, wherein the r-arm polymer is a pegylated polymer.

12. The hydrogel of claim 11, wherein r is 4.

13. A hydrogel according to any one of claims 8 to 12, wherein the r-arm polymer is attached through a cleavable linker.

14. The cleavable linker of claim 1, wherein the cleavable linker has the formula: 【Chemistry 3】 [In the formula, A* and C* are independently connecting groups connecting the cleavable linker and the r-arm polymer; n is an integer from 0 to 6; x, y, and z are independently integers from 0 to 6; R 11 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 12 is H or alkyl; each R 14 is independently C 1 -C 3 alkyl, or two R 14 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; and 【Chemistry 4】 indicates the point of attachment to the linker-drug unit of claim 1.

14. The hydrogel of claim 13, wherein:

15. The hydrogel of claim 14, wherein the linker-drug unit is attached to a cleavable linker through a carboxamide, oxime, thioether, or triazole moiety.

16. The cleavable linker of claim 1, wherein the cleavable linker has the formula: 【Chemistry 5】 15. The hydrogel of claim 14, wherein:

17. The hydrogel of claim 16, wherein the linker-drug unit is attached to the cleavable linker through a reaction between a cyclooctyne group on the cleavable linker and an azide group attached to the linker-drug unit of claim 1.

18. The hydrogel of claim 16, wherein the linker-drug unit is attached to a linker that is cleavable through a reaction between 4-cyclooctynyloxycarbonyl or (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl and an azide group attached to the linker-drug unit of claim 1.

19. The cleavable linker of claim 19, wherein the cleavable linker has the formula: 【Chemistry 6】 16. The hydrogel of claim 15, wherein:

20. The hydrogel of claim 19, wherein the linker-drug unit is attached to the cleavable linker through a reaction between a cyclooctyne group on the cleavable linker and an azide group attached to the linker-drug unit of claim 1.

21. The hydrogel of claim 19, wherein the linker-drug unit is attached to a linker that is cleavable through a reaction between 4-cyclooctynyloxycarbonyl or (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl and an azide group attached to the linker-drug unit of claim 1.

22. A hydrogel described in any one of claims 1 to 12, wherein D is IL-2 of sequence number 1.

23. The hydrogel of any one of claims 1 to 12, wherein D is IL-2 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

24. The hydrogel of any one of claims 1 to 12, wherein each R 4 is H.

25. Formula: 【Chemistry 7】 [In the formula, P 1 and P 2 are independently r-arm polymers, where r is an integer from 2 to 8; A* is a connecting group; C* is a connecting group; each n is independently an integer from 0 to 6; x, y, and z are independently integers from 0 to 6; R 11 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 12 is H or alkyl; each R 14 is independently C 1 -C 3 alkyl, or two R 14 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Z* is a connecting group; S is absent or (CH 2 CH 2 O) h (CH 2 ) g CONH, where g=1-6 and h=0-1000; Y is NH(CH 2 CH 2 O) p (CH 2 ) m where m=2-6 and p=0-100; R 1 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 2 is H or alkyl; each R 4 is independently H or C 1 -C 3 alkyl, or two R 4 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; and D is IL-2, IL-7, IL-9, IL-10, or IL-21. A hydrogel comprising a repeating unit represented by:

26. The hydrogel of claim 25, wherein P 1 and P 2 are PEGylated polymers.

27. The hydrogel of claim 25 or 26, wherein r is 4.

28. The hydrogel of claim 25, wherein A* and C* are independently a carboxamide, an oxime, an ether, a thioether, or a triazole.

29. The hydrogel of claim 25, wherein each R 14 group is methyl.

30. Formula: 【Chemistry 8】 [In the formula, P 1 and P 2 are independently r-arm polymers, where r is an integer from 2 to 8; A* is a connecting group; each n is independently an integer from 0 to 6; R 11 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 12 is H or alkyl; each R 14 is independently C 1 -C 3 alkyl, or two R 14 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Z* is a connecting group; S is absent or (CH 2 CH 2 O) h (CH 2 ) g CONH, where g=1-6 and h=0-1000; Y is NH(CH 2 CH 2 O) p (CH 2 ) m where m=2-6 and p=0-100; R 1 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 2 is H or alkyl; each R 4 is independently H or C 1 -C 3 alkyl, or two R 4 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; and D is IL-2, IL-7, IL-9, IL-10, or IL-21. A hydrogel comprising a repeating unit represented by:

31. The hydrogel of claim 30, wherein P 1 and P 2 are PEGylated polymers.

32. The hydrogel of claim 30 or 31, wherein r is 4.

33. The hydrogel of claim 30, wherein A* is a carboxamide, oxime, ether, thioether, or triazole.

34. The hydrogel of claim 30, wherein each R 14 group is methyl.

35. Formula: 【Chemistry 9】 [In the formula, P 1 and P 2 are independently r-arm polymers, where r is an integer from 2 to 8; A* is a connecting group; each n is independently an integer from 0 to 6; R 11 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 12 is H or alkyl; each R 14 is independently C 1 -C 3 alkyl, or two R 14 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Z* is a connecting group; S is absent or (CH 2 CH 2 O) h (CH 2 ) g CONH, where g=1-6 and h=0-1000; Y is NH(CH 2 CH 2 O) p (CH 2 ) m where m=2-6 and p=0-100; R 1 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 2 is H or alkyl; each R 4 is independently H or C 1 -C 3 alkyl, or two R 4 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; and D is IL-2, IL-7, IL-9, IL-10, or IL-21. A hydrogel comprising a repeating unit represented by:

36. The hydrogel of claim 35, wherein P 1 and P 2 are PEGylated polymers.

37. The hydrogel of claim 35 or 36, wherein r is 4.

38. The hydrogel of claim 35, wherein A* is a carboxamide, oxime, ether, thioether, or triazole.

39. The hydrogel of claim 35, wherein each R 14 group is methyl.

40. Formula (Ia) 【Chemistry 10】 (Ia) [In the formula, n is an integer from 0 to 6; R 1 is —CN, —NO 2 , —COR 5 , —SOR 5 , or —SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl; R 2 is H or alkyl; each R 4 is independently H or C 1 -C 3 alkyl, or two R 4 s together with the carbon atoms to which they are attached form a 3- to 6-membered ring; Z is a group connecting the linker and the polymeric carrier; S is absent or (CH 2 CH 2 O) h (CH 2 ) g CONH, where g is an integer from 1 to 6 and h is an integer from 0 to 1000; Y is NH(CH 2 CH 2 O) p (CH 2 ) m , where m is an integer from 2 to 6 and p is an integer from 0 to 1000; and D is IL-2, IL-7, IL-9, IL-10, or IL-21. The linker-drug is represented by

41. The linker-drug of claim 40, wherein R 1 is -CN or -SO 2 R 5 , where R 5 is C 1 -C 6 alkyl, aryl, heteroaryl, or NR 6 2 , where each R 6 is independently C 1 -C 6 alkyl, aryl, or heteroaryl, and R 2 is H, where each of R 5 and R 6 may independently be optionally substituted.

42. The linker-drug of claim 40, wherein Y is NH(CH 2 CH 2 O) p (CH 2 ) m , where m=3 and p=0.

43. The linker-drug of claim 40, wherein n is 4, each R 4 is H, R 1 is -SO 2 R 5 , where R 5 is methyl, R 2 is H, S is (CH 2 CH 2 O) h (CH 2 ) g CONH, where g = 2 and h = 4, and Y is NH(CH 2 CH 2 O) p (CH 2 ) m , where m = 3 and p = 0.

44. A linker-drug described in any one of claims 40 to 43, wherein D is IL-2 of sequence number 1.

45. A linker-drug described in any one of claims 40 to 43, wherein D is IL-2 of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

46. A hydrogel according to any one of claims 1 to 39 for selectively expanding T reg cells in a subject.

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