Slow-releasing cytokine conjugates

Abstract

The present invention relates generally to releasable cytokine conjugates and methods of use thereof.

Description

[0001] Cross-reference to related applications

[0002] 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.

[0003] Reference inclusion of sequence listing

[0004] The electronic sequence list is submitted with this application. The sequence list is provided as a file titled 670572002240SeqList.txt, created on April 24, 2020, and is 26,622 bytes in size. Information regarding the electronic sequence list is incorporated herein by reference in its full text. Technical Field

[0005] This invention generally relates to releaseable cytokine conjugates and methods of using them. Background Technology

[0006] Cytokines are small (up to approximately 20 kDa) proteins involved in cell signaling, including a wide range of interleukins (ILs), interferons (IFs), tumor necrosis factor (TNF), chemokines, and lymphokines. They are produced by a variety of cells and are particularly important in the immune system, regulating the balance between humoral and cellular immune responses. Interleukins comprise a group of cytokines that play a particularly important role in immunity. Most interleukins are expressed on helper CD4 T lymphocytes, where they promote the development and differentiation of T and B lymphocytes and hematopoietic cells.

[0007] Interleukin-2 (IL-2) (SEQ ID No:1) is a cytokine of approximately 16 kDa that plays a crucial role in the natural response to microbial infection and in differentiating between native and exogenous cells. IL-2 primarily exerts its influence through direct action on T cells, playing a vital role in key functions of the immune system, including tolerance and immunity. In the thymus, where T cells mature, it promotes the differentiation of certain immature T cells into regulatory T cells (T cells). reg To prevent autoimmune diseases, regulatory T cells (T cells) reg IL-2 suppresses other T cells that would otherwise attack normal, healthy cells in the body. IL-2 enhances activation-induced cell death (AICD). When naive T cells are also stimulated by antigens, IL-2 also promotes the differentiation of T cells into effector T cells (T cells). eff ) and memory T cells (T mem This helps the body fight infection. Along with other polarizing cytokines, IL-2 stimulates primary CD4+. + T cells differentiate into Th1 and Th2 lymphocytes, while being prevented from differentiating into Th17 and phyllotype Th lymphocytes.

[0008] The α subunit of the IL-2 receptor (IL-2R) (CD25) has a low affinity (Kd~10). -8 M) binds to IL-2. Due to the short intracellular chain, the interaction between IL-2 and CD25 alone does not lead to signal transduction, but it has the ability (when binding to the β and γ subunits) to increase the affinity of IL-2R by 1000-fold. Heterodimerization of the β and γ subunits of IL-2R is crucial for signal transduction in T cells. IL-2 can be transduced via the intermediate-affinity dimer CD122 / CD132 IL-2Rβγ receptor (Kd~10). -9 M) or high-affinity trimeric CD25 / CD122 / CD132 IL-2Rαβγ receptor (Kd~10 -11 M) transmits signals. The dimer IL-2Rβγ is generated by CD8+T mem Cells and NK cells express this, while T cells express it. reg Activated T cells express high levels of the trimer 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.

[0009] Regulatory T cells (T cells) reg IL-2 is a subset of T lymphocytes that is crucial for maintaining self-tolerance. While IL-2 is involved in regulation and effector T lymphocytes... eff ) cell activation, but with T eff Compared to cells, T reg Higher expression of medium- and high-affinity receptors means that low-dose IL-2 preferentially supports T cells. reg Cell maintenance. Autoimmune responses and T cells in diseases such as type 1 diabetes, multiple sclerosis, Crohn's disease, and systemic lupus erythematosus. reg Defect-related. Therefore, by using high-affinity receptors to target T... reg Selective and sustained stimulation of cells holds promise for treating autoimmune diseases.

[0010] High-dose interleukin-2 therapy with ade-interleukin (recombinant interleukin-2) has been approved for the treatment of metastatic melanoma and renal cell carcinoma. However, high-dose therapy has a low objective response rate and a high incidence of end-organ toxicity. It is believed that most of the antitumor activity of IL-2 is generated by stimulation of T cells through the high-affinity IL-2Rαβγ receptor, and most toxicity is due to the release of inflammatory proteins by natural killer cells through the low-affinity IL-2Rβγ receptor. A mutant IL-2 protein (SEQ ID NO:2; IL2-N88R, BAY 50-4798) with an arginine substitution for asparagine at position 88 selectively binds to the high-affinity IL-2Rαβγ receptor, leading to T cell toxicity. regSelectivity of cell activation relative to T eff It increased NK cell activation by 3000-fold. Consistent with this, rodent models showed that BAY 50-4798 and adrenaline had equivalent efficacy, but the mutant protein was less toxic. Phase 1 human trials of BAY 50-4798 confirmed the T... reg Cells relative to T eff The mutant protein exhibited the expected differential activation compared to NK cells, but its anti-tumor response was limited, leading to the cessation of its development.

[0011] There have been reports attempting to prolong the in vivo half-life of IL-2 and its analogues, thereby enhancing their potency. Various fusions of IL-2 with antibodies and antibody fragments have been disclosed (WO2014 / 023752A1). Some researchers have also disclosed Fc or IgG with IL-2 {Bell, 2015#2} or T... reg Fusions of specific mutant proteins, such as Fc-IL-2N88R (Greve, J.US2017 / 0204154 A1) or IgG-IL-2N88D {Peterson, 2018#1}. In cynomolgus monkeys, the half-life of intravenously administered IgG-IL-2N88D was only about 8 hours, or the half-life of subcutaneously administered IgG was 14 hours, far shorter than the expected 14 days for IgG. This short-lived t 1 / 2 This is due to receptor-mediated endocytosis (RME). In any case, compared to daily injections of low-dose IL-2, a single subcutaneous (SC) injection of IgG-IL-2N88ND prolonged the time of regulatory T cell proliferation. That is, after a single injection, T... reg It reaches its maximum at approximately 4 days and lasts for about 14 days. CD4+ and CD8+CD25+FOXP3+T reg Increased by 10-14 times, but had no effect on CD4+ or CD8+ memory effector T cells. However, this fusion protein has some drawbacks, such as loss of potency and enhanced immunogenicity compared to the native protein. Certain permanent and releasable conjugates of IL-2 with water-soluble polymers have been disclosed (US Patent 9,861,705). Mutant IL-2 proteins containing non-natural amino acids have been disclosed to alter the selectivity between the receptor and its water-soluble conjugates (WO2019 / 028425; WO2019 / 028419).

[0012] Interleukin-15 is a related cytokine that functions via a unique α-receptor chain, but shares the same β and γ receptor chains as interleukin-2. IL-15 is a pleiotropic cytokine important for both adaptive and innate immunity. IL-15 promotes natural killer (NK) and CD8+ receptor activation. + Effect T memCell activation and maintenance are areas of interest for immunotherapeutic agents used to treat cancer and immunodeficiency. Exogenous IL-15 can stimulate CD8+ cells both in vivo and in vitro. + T mem Cell proliferation. It is hypothesized that low-dose treatment with IL-15 can promote tumor-specific CD8 proliferation. + T mem The maintenance and function of cells, thereby delaying or preventing tumor recurrence in the event of failed adaptive immunotherapy (Roychowdhury et al., Cancer Research 64:8062-7 (2004)). Continuous low-dose treatment in monkeys for more than 10 days resulted in increased CD8+ levels in peripheral blood. + Effect T mem Cells expanded 100-fold, which was more effective than a once-daily pill dosing regimen (Sneller et al., blood118:6845-8 (2011)). Stable mutant proteins of IL-15 have been reported (Nellis et al., Pharm.Res.29:722-38 (2012)). Certain permanent and releasable conjugates of IL-15 with water-soluble polymers have been disclosed (PCT Publication No. WO2015 / 153753A2). Mutant proteins of IL-15 have been found to exhibit improved receptor agonism (Zhu et al., J. Immunology 2009, 183(6):3598). In cell proliferation assays, the bioactivity of IL-15[N72D] was 4-5 times higher than that of native IL-15. IL-15 receptor agonists containing the sushi domain of IL-15 and IL-15Rα (IL-15RαSu) have also been reported, both as complexes and fusion proteins (Han et al., Cytokine 2011, 56(3): 804-10; Mortier et al., J. Biological Chem. 2006, 281: 1612-9; US Patent 10,358,477). A multimeric complex of IL-15[N72D] and IL-15RαSuFc fused to the IgG1 Fc domain (ALT-803) is currently in clinical trials.

[0013] IL-2 mutant proteins with reduced affinity for the trimer receptor have been disclosed (US Patent 9,206,243). These mutant proteins, while maintaining their ability to stimulate CD4+ T helper cells, CD8+ T cells, and natural killer (NK) cells, exhibit stimulation of T cells. reg The cells' ability is reduced. It has been suggested that this is due to T... reg In the absence of cellular immunosuppression, this mutant IL-2 protein may exhibit enhanced antitumor activity.

[0014] IL-7 is a cytokine essential for T cell development, survival, and T cell homeostasis. Double-negative (DN) CD4+ is found in the thymus. - CD8 - Transformation of thymic progenitor cells requires IL-7 signaling, although high-dose IL-7 blocks DN progression. Once in the periphery, the survival of primary T cells depends on IL-7. The IL-7 receptor contains a specific α chain (CD127), which is expressed almost exclusively on lymphocytes, and a γ chain (CD132), which is shared with IL-2, IL-15, IL-9, and IL-21. IL-7 has been used as an immunotherapeutic agent in clinical trials in cancer patients who received T-cell depletion therapy in an attempt to increase the levels of CD4+ and CD8+ T cells. Administration of IL-7 resulted in preferential expansion of primary T cells, regardless of patient age, and generated a broader T-cell pool, suggesting that IL-7 has the potential to enhance immune responses in patients with low primary T-cell populations (ElKassar and Gress, J. Immunotoxicol. (2010) 7:1-7).

[0015] IL-9 is another cell that is structurally associated with mast cells, NK cells, TH2, TH17, and T cells. reg IL-2 and IL-15 are pleiotropic cytokines produced by ILC2 and Th9 cells, with Th9 cells considered the primary producers of CD4+ T cells. The IL-9 receptor consists of a specific α chain (CD129) and a shared γ chain (CD132). Low-dose IL-9 therapy has been proposed to prevent chemotherapy-induced thrombocytopenia and accelerate platelet recovery (Xiao et al., Blood 129:3196-3209 (2017)).

[0016] 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 interferon-gamma, IL-2, and tumor necrosis factor-α (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 appears 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 currently being investigated for the treatment of autoimmune diseases, septic shock, and bacterial sepsis. A polyethylene glycol derivative of IL-10 has been disclosed (PCT Publication No. WO2010 / 077853). PEGylated IL-10 has been shown to induce interferon-γ 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)). Studies of PEGylated IL-10 have shown that in human treatment, IL-10 mainly exerts its immunostimulatory effect by activating CD8+ T cells. Although a phase 3 trial for treating metastatic stage 4 pancreatic cancer failed to meet the primary endpoint, a phase 2 trial for non-small cell lung cancer is underway.

[0017] IL-21 is expressed in activated CD4+ T cells and upregulated in Th2 and Th17 helper T cells as well as T follicular cells. It is also expressed in NK cells and regulates their function. The IL-21 receptor (IL21R) is expressed on the surface of T, B, and NK cells and functions by binding to a common γ chain (CD132). The role of IL-21 in the treatment of allergies, viral infections, and cancer has been reported, and its efficacy has been confirmed 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 individuals, suggesting its potential for HIV treatment.

[0018] Continuous infusion shows promise for low-dose, sustained use of cytokines, but it is difficult to implement in practice in humans. Therefore, there is a need to improve drugs to enable low-dose, extended-duration treatment of various diseases, including cancer and autoimmune diseases, using cytokines.

[0019] Overview

[0020] On the one hand, it provides a connector-drug of type (I):

[0021] ZLD(I),

[0022] Z, L, and D are detailed in this article.

[0023] In some embodiments, the connector-drug ZLD is a compound of formula (Ia):

[0024]

[0025] Where Z,S,n,R 1 ,R 2 ,R 4 Y and D are detailed in this article.

[0026] On the other hand, a connector of type (IIa) is provided:

[0027]

[0028] Where n, Z, S, R 1 ,R 2 ,R 4 And X is detailed in this article.

[0029] On the other hand, a coupling of formula (III) is provided:

[0030] M-[Z*-LD] q (III),

[0031] M, Z*, L, D and q are described in detail in this paper.

[0032] On the other hand, a degradable crosslinked hydrogel of formula (IV) is provided:

[0033]

[0034] Where P 1 ,P 2 ,r,A*,B,C*,n,R 11 ,R 12 ,R 14 x, y, and z are described in detail in this article.

[0035] In another aspect, methods for preparing the compounds disclosed herein and methods for using them are provided. In yet another aspect, pharmaceutical compositions comprising a conjugate of formula (III) or a hydrogel of formula (IV) are provided.

[0036] Brief description of the attached figures

[0037] Figure 1 The general structure of the coupling is shown, in which the connector-drug is attached to the hydrogel.

[0038] Figure 2The binding of IL-2 [N88R, C125S] to cells containing αβγ and βγ receptors was shown.

[0039] Figure 3 The SDS-PAGE gel shows bands corresponding to the reductively alkylated linker-protein product of IL-2 [N88R, C125S].

[0040] Figure 4 The C-p-t plot of plasma IL2[N88R] in rats after treatment with microspheres-IL2-N88R is shown. Figure 4 A shows the release of IL-2 [N88R, C125] from the randomized acylated conjugate administered at a dose of 0.25 μmol / kg. Figure 4 B shows the release of AP-IL-2 [N88R, C125S] from the reduced alkylated conjugate administered at a dose of 0.12 μmol / kg.

[0041] Figure 5 The pharmacokinetics of IL-2 [N88R, C125S] in the spleen are shown. Left: Percentage of CD4+ effector / memory T cells; Right: CD8+ effector / memory T cells. + Percentage of effector / memory T cells.

[0042] Figure 6 The pharmacokinetics of IL-2 [N88R, C125S] in the pancreas are shown. Top left: Foxp3 + CD4 + T cell percentage; Top right: CD4 + Percentage; Bottom Left: CD8 + Percentage; bottom right: innate lymphocytes. NOD mice were injected daily with PBS carrier, adeleukin (25,000 units), or IL-2 [N88R, C125S] (25,000 units) and sacrificed 2 hours after the last injection on day 5.

[0043] Figure 7 The pharmacokinetics of [aminopropyl]-IL-2[N88R,C125S] released from microsphere-IL-2[N88R,C125S] (“MS-IL-2 mutant protein”) in mice are shown. Figure 7 A: BALB / c mice (n=6) received a single subcutaneous injection in the flank containing microspheres of IL-2 [N88R, C125S] at 28 nmol (19 mg / kg) or 9.9 nmol (6.5 mg / kg). The t-test results were measured at 31 hours. 1 / 2 . Figure 7B: NOD mice (n=6) were injected laterally with microspheres of IL-2 [N88R, C125S] (0.5, 1, 5, 10, or 19 mg / kg). The t-test results were measured at 18 hours. 1 / 2 . Figure 7 C: NSG mice (n=6) or NOD mice (n=6) were administered microspheres of IL-2 [N88R, C125S] (5 mg / kg) to the flank. The t-value of [aminopropyl]-IL-2 [N88R, C125S] at 152 h was measured in NSG mice. 1 / 2 In all cases, plasma was analyzed using the Thermofisher ELISA to quantify IL-2 [N88R, C125S] concentrations.

[0044] Figure 8 This demonstrates the effect of IL-2 [N88R, C125S] (“IL-2 mutant protein”) on Foxp3. + CD4 + and CD8 + The impact of cell population expansion. Figure 8 A shows Foxp3 in spleen and peripheral blood mononuclear cells (PBMCs). + CD4 + T cell expansion. Figure 8 B shows CD8 in the spleen and PBMCs. + T cell expansion. CD8 cells were found in the spleen and PBMCs. + The cell percentages were approximately 11% and 19%, respectively. When treated with microspheres-IL-2 [N88R, C125S], these percentages increased to approximately 25% and 60%, respectively. NOD mice were given a single injection of IL-2 mutant protein (QDx5, 25,000 units) or microspheres-IL-2 [N88R, C125S] (18 mg / kg). Mice were sacrificed 2 hours after the last administration on day 5.

[0045] Figure 9 Foxp3 in PBMC is displayed + CD4 + Dose-dependent expansion of T cells. Figure 9 A shows the effect of microspheres-IL-2 [N88R, C125S] on Foxp3. + CD4 + The effect of T cell expansion Figure 9 B showed their effect on CD8 in NOD mice (n=3 / dose group) + Effects on cell activation (right). Microsphere-IL-2 [N88R, C125S] preferentially amplifies Foxp3. + CD4 + T cells, and prevented CD8 in NOD mice (n=3 / dose group)+ Cell activation. All doses of Foxp3 + CD4 + T cell expansion peaked on day 4 and returned to baseline levels on day 14.

[0046] Figure 10 The SDS-PAGE gel shows bands corresponding to the reductively alkylated linker-protein products of IL-15. From left to right: molecular weight markers; IL-15; IL-15+PEG. 5kDa -DBCO;IL-15+1Eq(IIb)+PEG 5kDa -DBCO;IL-15+3Eq(IIb)+PEG 5kDa -DBCO; and IL-15+5Eq(IIb)+PEG 5kDa -DBCO.

[0047] Figure 11 The pharmacokinetics of [aminopropyl]-IL-15 released from MS-IL-15 in C57BL / 6J mice are shown. 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 human IL-15 Quantikine ELISA (R&D systems). Two distinct t=240-hour intervals were observed. 1 / 2 t was observed within 120 hours. 1 / 2 >200 hours, then a second t was observed at 27 hours within 120 to 240 hours. 1 / 2 Immediately after a blood draw 240 hours later, a second injection of MS-IL15 (50 μg) was administered (blue data). From 264 to 360 hours, a t-reduction of 23 hours was observed. 1 / 2 .

[0048] Figure 12 The pharmacokinetics of [aminopropyl]-IL-15 released from microspheres-IL-15 in C57BL / 6J mice are shown. 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). The t-values ​​(tc) at 115–207 hours were observed by fitting data over 120 hours. 1 / 2 .

[0049] Figure 13The pharmacokinetics of [aminopropyl]-IL-15 released from microspheres-IL-15 in C57BL / 6J mice (subcutaneous vs. intraperitoneal) are shown. MS-IL-15 (50 μg) was administered subcutaneously (black, ●) or intraperitoneally (blue, ■) to normal male C57BL / 6J mice. Plasma samples were prepared and analyzed using the human IL-15 Quantikine ELISA (R&D systems). Similar t-response rates were observed between subcutaneous and intraperitoneal administrations over 120 hours. 1 / 2 (115 hours (subcutaneous) and 129 hours (intraperitoneal)).

[0050] Figure 14 The microsphere-IL15 conjugate showed its effect on NK cells and CD44hiCD8. + The role of T cells. Microsphere-IL15 conjugates can amplify CD44. hi CD8 + T cells and NK cells. Figure 14 A: Expansion of CD44hiCD8+ T cells. Figure 14 B: NK cell expansion. Normal male C57BL / 6J mice were administered a single subcutaneous injection of microspheres-IL-15 (2.5, 12.5, 25, or 50 μg IL-15), empty microspheres (black), or a single subcutaneous injection of rhIL15 (2.5 μg). Flow cytometry was used to monitor NK cells and CD44 in peripheral blood mononuclear cells. hi CD8 + T cell expansion. Following a single injection of 50 μg microspheres-IL15, CD44-hiCD8... + The expansion of T cells lasted for 28 days.

[0051] Figure 15 The SDS-PAGE gel shows bands corresponding to receptor-linked interleukin (RLI) and adaptor (IIb) reductively alkylated adaptor-protein products, in contrast to PEG. 5kDa - This is visible after DBCO undergoes a gel translocation reaction. From left to right: molecular weight marker; RLI; RLI+PEG 5kDa -DBCO;RLI+1.5Eq(IIb)+PEG 5kDa -DBCO;RLI+2Eq(IIb)+PEG 5kDa -DBCO;RLI+3Eq(IIb)+PEG 5kDa -DBCO; and RLI+5Eq(IIb)+PEG 5kDa -DBCO.

[0052] Figure 16The results of RLI analysis based on IL-2Rβγ receptor binding cells are shown. The assay was performed using U2OS cells at pH 7.4 (EC5). 50 =180pM) The [aminopropyl]-RLI released from the coupling compound and the natural RLI (EC) 50 The binding activity was compared to that of 160 pm.

[0053] Figure 17 The pharmacokinetics of [aminopropyl]-PLI released from the microsphere conjugate in C57BL / 6J mice are shown. Normal male C57BL / 6J mice were administered the microsphere-RLI conjugate (1.5 nmol). Plasma samples were prepared and analyzed using the R&D systems DuoSethIL15 / IL15Rα composite ELISA (DY6924).

[0054] Figure 18 The pharmacokinetics of [aminopropyl]-RLI released from the microsphere conjugate were demonstrated, and CD8 in PBMCs were measured. + Expansion of memory T cells. Figure 18 A: CD8 in PBMC + Percentage of memory T cells Figure 18 B: CD8 + T cell proliferation was assessed in normal male C57BL / 6J mice via subcutaneous injection of empty MS, MS-RLI (34 μg, 1.5 nmol), or native RLI (2.5 μg, 0.11 nmol QDx4). PBMCs were prepared from blood samples and stained for flow cytometry analysis.

[0055] Figure 19 This shows the expansion of NK cells in PBMCs after microsphere-RLI treatment. Figure 19 A: Percentage of NK cells in PBMCs Figure 19 B: NK cell proliferation. 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 QDx4) via subcutaneous injection in the flank. PBMCs were prepared from blood samples and stained for flow cytometry analysis. Detailed Implementation

[0056] This invention provides releasable conjugates of cytokine proteins, including variants thereof. These conjugates deliver these protein therapeutic agents at low, sustained doses over extended periods, and are therefore suitable for treating a variety of diseases.

[0057] In one aspect, the present invention provides cytokines and variants thereof having releasable linkers adapted for attaching proteins to macromolecular carriers. These linkers control the rate of protein release from the carrier, thereby determining the concentration and duration of the cytokine or variant in vivo.

[0058] In another aspect, the present invention provides conjugates for the release of cytokines and their variants from macromolecular carriers. The carriers can be soluble or insoluble, and can prolong the time proteins remain in vivo.

[0059] On the other hand, this specification provides methods for the preparation and use of the adapter-cytokines and conjugates of the present invention.

[0060] definition

[0061] For the purposes of this document, unless otherwise expressly indicated, the terms “a”, “an”, etc., refer to one or more.

[0062] Unless otherwise specified, the terms “about” or “approximately” as used herein, when relating to a value, mean a value within 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of a given value.

[0063] The term "alkyl" includes linear, branched, or cyclic saturated hydrocarbon groups with 1-20, 1-12, 1-8, 1-6, or 14 carbon atoms. In some embodiments, the alkyl group is straight-chain or branched. Examples of straight-chain or branched alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc. In some embodiments, the alkyl group is cyclic. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, etc.

[0064] The term "alkoxy" includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, etc.

[0065] The term "alkenyl" includes non-aromatic unsaturated hydrocarbons having carbon-carbon double bonds and 2-20, 2-12, 2-8, 2-6, or 2-4 carbon atoms.

[0066] The term "alkynyl" includes non-aromatic unsaturated hydrocarbons having a carbon-carbon triple bond and 2-20, 2-12, 2-8, 2-6, or 2-4 carbon atoms.

[0067] The term "aryl" includes aromatic hydrocarbon groups with 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracene. The term "heteroaryl" includes an aromatic ring consisting of 3 to 15 carbons (preferably 3 to 7 carbons) containing at least one N, O, or S atom, including groups such as pyrrole, pyridinyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indole, indole, and indene.

[0068] In some cases, the alkenyl, ynyl, aryl, or heteroaryl moieties can be coupled to the rest of the molecule via alkyl bonds. In these cases, the substituent is referred to as alkenylalkyl, ynylalkyl, arylalkyl, or heteroarylalkyl, indicating that the alkenyl moieties are located between the alkenyl, ynyl, aryl, or heteroaryl moieties and the molecule coupled to the alkenyl, ynyl, aryl, or heteroaryl group.

[0069] The term "halogen" or "halogenated" includes brominated, fluorinated, chlorinated, and iodinated.

[0070] The term "heterocyclic" or "heterocyclic group" 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, piperazineyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as exemplary groups provided for the term "heteroaryl" above. In some embodiments, the heterocyclic or heterocyclic group is non-aromatic. In some embodiments, the heterocyclic or heterocyclic group is aromatic.

[0071] The term "macromolecule" refers to a molecule or molecular residue with a molecular weight between 5,000 and 1,000,000 Daltons, preferably between 10,000 and 500,000 Daltons, and more preferably between 10,000 and 250,000 Daltons. Examples of macromolecules include, but are not limited to, proteins comprising antibodies, antibody fragments, and enzymes; polypeptides comprising poly(amino acids), such as poly(lysine) and poly(valine), and polypeptides with mixed sequences; synthetic polymers, including polyethylene glycol (PEG), polyethylene oxide (PEO), polyethylene imine (PEI), and copolymers thereof; and polysaccharides such as dextran. In some embodiments, the macromolecule contains at least one functional group suitable for coupling naturally or chemically, such as amine, carboxylic acid, alcohol, thiol, alkyne, azide, or maleimide groups as described above. In some embodiments of this specification, the macromolecule is polyethylene glycol. Polyethylene glycol can be linear or branched, with one end connected to a functional group suitable for coupling and the other end connected to a capping group (e.g., methyl), or it can include multiple arms, each arm ending in a functional group suitable for coupling. In a preferred embodiment of this specification, the polyethylene glycol is a linear, branched, or multi-arm polymer with an average molecular weight between 20,000 and 200,000 Daltons, preferably between 20,000 and 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).

[0072] Unless otherwise specified, “optional substitution” means that a group may be unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4, or 5) identical or different substituents. Examples of substituents include, but are not limited to, alkyl, alkenyl, alkynyl, halogen, -CN, and -OR. 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)2NR aa R bb -P(O)(OR) aa (OR) bb ), heterocyclic, heteroaryl, or aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, heteroaryl, and aryl groups are each independently and optionally represented by R. cc Replace, among which

[0073] R aa and R bb Each is independently H, alkyl, alkenyl, alkynyl, heterocyclic, heteroaryl, or aryl, or

[0074] R aa and R bb Together with the nitrogen atoms attached to them, they form heterocyclic groups, which are optionally substituted with alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or CN, wherein:

[0075] Each R cc It can be independently alkyl, alkenyl, alkynyl, halogen, heterocyclic, heteroaryl, aryl, -CN or -NO2.

[0076] Normally, the active form of the drug is released directly from the conjugate described in this specification, but in some cases, the active drug may be released in its prodrug form.

[0077] Connector-Drug

[0078] On the one hand, it provides a connector-drug of type (I):

[0079] ZLD (I),

[0080] Where Z is a functional group that allows the adapter-drug to bind to a macromolecular carrier, L is a cleavable adapter, and D is a cytokine or a cytokine variant. In some embodiments, the release adapter is adapted to couple a protein to a macromolecular carrier. In some embodiments, the adapter controls the rate of release of the cytokine or variant from the carrier, thereby determining the concentration and duration of the active protein in vivo. In one aspect, an adapter-drug of formula (I) is provided:

[0081] ZLD (I),

[0082] Where Z is a functional group that allows the adapter-drug to attach to the macromolecular carrier, L is a cleavable adapter, and D is a cytokine or cytokine variant.

[0083] In some embodiments of the linker-drug of formula (I), cytokine D is IL-2, IL-4, IL-7, IL-9, IL-10, IL-15, IL-21, or cytokine variants thereof. D may also contain cytokines that have undergone certain chemical modifications, such as NH(CH2CH2O). p (CH2) m , where m is an integer from 2 to 6 and p is an integer from 0 to 1000, is attached to an amino group generated by reductive amination to connect the linker L. In some embodiments, this modification is attached to the N-terminal α-amino group of the protein sequence.

[0084] "Cytokine variant" refers to a protein with altered sequence ("mutant protein") that has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity with the native cytokine. In some embodiments, the cytokine variant has at least 90% sequence identity with the native cytokine. In some embodiments, the cytokine variant comprises 1 to 10 altered amino acids compared to the native sequence, selected based on improvements in 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 a starting methionine residue. For example, IL-2 variants useful in this specification may be selected from those that have a higher binding affinity for the trimeric αβγ receptor than for the dimeric βγ receptor. In some embodiments, the IL-2 variant has a mutation at asparagine-88, such as N88R or N88D, which may be combined with other mutations such as C125S to confer additional stability or selectivity. This invention also discloses other IL-2 mutant proteins applicable to this invention, such as mutant proteins with alterations at aspartate-20, like IL-2D20T, or mutant proteins with reduced affinity for the trimer receptor as disclosed in U.S. Patent No. 9,206,243. Specific embodiments of IL-2 and its variants are given in SEQ ID NO:1-11.

[0085] SEQ ID NO: 1 Natural human IL-2

[0086] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISLT

[0087] SEQ ID NO:2 IL-2-N88R(BAY 50-4798)

[0088] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0089] SEQ ID NO: 3 IL-2-N88R,C125S

[0090] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

[0091] SEQ ID NO: 4 IL-2-D20T

[0092] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0093] SEQ ID NO: 5 IL-2-D20T,C125S

[0094] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

[0095] SEQ ID NO:6 IL-2-R38K,F42I,Y45N,E62L,E68V

[0096] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TIKFNMPKKA TELKHLQCLEELLKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT

[0097] SEQ ID NO:7 IL-2-R38K,F42Q,Y45E,E68V

[0098] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TQKFEMPKKA TELKHLQCLEEELKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT

[0099] SEQ ID NO:8 IL-2-R38A,F42I,Y45N,E62L,E68V

[0100] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TIKFNMPKKA TELKHLQCLEELLKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT

[0101] SEQ ID NO:9 IL-2-R38K,F42K,Y45R,E62L,E68V

[0102] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTKML TKKFRMPKKA TELKHLQCLEELLKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT

[0103] SEQ ID NO:10 IL-2R38K,F42I,Y45E,E68V

[0104] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTKML TIKFEMPKKA TELKHLQCLEEELKPLEVVL NLAQSKNFHL RPRDLISNIN VIVLELKGSETTFMCEYADE TATIVEFLNR WITFCQSIISTLT

[0105] SEQ ID No:11 IL-2R38A,F42A,Y45A,E62A

[0106] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTAML TAKFAMPKKA TELKHLQCLEEALKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT

[0107] Similarly, native IL-15 can be replaced by mutant proteins with improved activity, receptor binding selectivity, or stability. For example, native IL-15 (SEQ ID No:12) can be replaced by mutant proteins with improved resistance to 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 maintain their biological activity (Nellis et al., Pharm.Res.29:722-38(2012)) or by IL-15[N72D] (SEQ ID No:15), which shows enhanced receptor agonism (Zhu et al., J.Immunology 2009,183(6):3598).

[0108] SEQ ID No:12 IL-15

[0109] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0110] SEQ ID No:13 IL-15[N77A]

[0111] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA NNSLSSAGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0112] SEQ ID No:14 IL-15[N71S,N72A,N77A]

[0113] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA SASLSSAGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0114] SEQ ID No:15 IL-15[N72D]

[0115] NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA NDSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0116] Complexes of IL-15 and IL-15RαSu, as well as fusion proteins, such as receptor-linked interleukin RLI (SEQ ID No: 16) and its variants (Mortier et al., J. Biological Chem. 2006, 281:1612-9; U.S. Patent 10,358,477). These fusion proteins may optionally contain the IL-15RαSu signaling sequence and sequences known in the art that facilitate the isolation and purification of proteins, such as His tags and Flag tags, or these elements may be absent (SEQ ID No: 17).

[0117] SEQ ID No:16 RLI

[0118] MAPRRARGC RTLGLPALLL LLLLRPPATR GDYKDDDDKI EGRITCRRRM SVEHADIWVKSYSLYSRERY ICNSGFKRKA GTSSLTECVL NKATNVAHWT TPSLKCIRDP ALVHQRPAPP SGGSGGGGSGG GSGGGGSLQ NWVNVISDLK KIQDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIHDTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

[0119] SEQ ID No:17 RLI[N77A]

[0120] ITCPPPMSVE HADIWVKSY SLYSRERYIC NSGFKRKAGT SSLTECVLNK ATNVAHWTTPSLKCIRDPAL VHQRPAPPSS GGSGGGGSGG GSGGGGSLQN WVNVISDLKK IEDLIQSMHI DATLYTESDVHPSCKVTAMK CFLLELQVIS LESGDASIHD TVENLIILAN NSLSSAGNVT ESGCKECEEL EEKNIKEFLQSFVHIVQMFI NTS

[0121] Other cytokines include IL-7, IL-9, IL-10 and IL-21 (SEQ ID NO:18 - 21).

[0122] SEQ ID No:18 IL-7

[0123] DCDIEGKDGK QYESVLMVSI DQLLDSMKEI GSNCLNNEFNFFKRHICDAN KEGMFLFRAARKLRQFLKMN STGDFDLHLL KVSEGTTILL NCTGQVKGRK PAALGEAQPT KSLEENKSLK EQKKLNDLCFLKRLLQEIKT CWNKILMGTK EH

[0124] SEQ ID No:19 IL-9

[0125] QGCPTLAGIL DINFLINKMQ EDPASKCHCS ANVTSCLCLG IPSDNCTRPC FSERLSQMTNTTMQTRYPLI FSRVKKSVEV LKNNKCPYFS CEQPCNQTTA GNALTFLKSL LEIFQKEKMR GMRGKI

[0126] SEQ ID No:20 IL-21

[0127] QGQDRHMIRM RQLIDIVDQL KNYVNDLVPE FLPAPEDVET NCEWSAFSCF QKAQLKSANTGNNERIINVS IKKLKRKPPS TNAGRRQKHR LTCPSCDSYE KKPPKEFLER FKSLLQKMIH QHLSSRTHGSEDS

[0128] SEQ ID No:21 IL-10

[0129] N

[0130] In some embodiments, cytokines may be chemically modified, for example, by attaching a water-soluble polymer (e.g., polyethylene glycol) to one or more sites, to prolong the duration of protein release from the conjugate in vivo and / or modify receptor selectivity.

[0131] These proteins can be prepared using methods known in the art. When prepared recombinantly, they can be expressed in prokaryotic or eukaryotic systems.

[0132] A variety of cleavable linkers L can be used, including those disclosed in U.S. Patent No. 8,680,315; 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. The linker L contains covalent bonds that cleave at a specific rate under suitable conditions. This cleavage can be carried out by catalytic or non-catalytic hydrolysis, protein hydrolysis, or elimination reactions. Suitable cleavage conditions are typically those found in the physiological environment, generally with a pH of approximately 6.5-7.5 and a temperature of 30-45°C, preferably with a pH of approximately 7.4 and a temperature of approximately 37°C.

[0133] In some embodiments, the linker-drug of formula (I) is a compound of formula (Ia):

[0134]

[0135] in:

[0136] n is an integer between 0 and 6;

[0137] R 1 and R 2 Independently an electron-withdrawing group, alkyl group, or H, wherein R 1 and R 2 At least one of them is an electron-withdrawing group;

[0138] Each R 4 Independently C1-C3 alkyl or two R 4 Together with the carbon atoms they are attached to, they form a 3-6 membered ring;

[0139] Z is a group used to connect the connector to the macromolecular carrier;

[0140] S either does not exist or is (CH2CH2O). h (CH2) g CONH, where g is an integer from 1 to 6 and h is an integer from 0 to 1000;

[0141] Y either does not exist or is NH(CH2CH2O). p (CH2) m , where m is an integer from 2 to 6, and p is an integer from 0 to 1000; and

[0142] D is an amine residue of a cytokine or a cytokine variant as disclosed herein.

[0143] In some embodiments of the connector-drug of formula (Ia), n = 1-6, R 1 and R 2 Independently an electron-withdrawing group, alkyl group, or H, wherein R 1 and R 2 At least one of them is an electron-withdrawing group; each R 4 Independently, it is H or a C1-C3 alkyl group, or together they can form a 3-6 membered ring; Z is a group used to connect the linker to the macromolecular support; S is absent or is (CH2CH2O). h (CH2) g CONH, where g = 1-6, h = 0-1000; Y is absent or is NH(CH2CH2O). p (CH2) m , where m = 2-6, p = 0-1000; D is an amine residue of the IL-2, IL-2 variant, IL-15, or IL-15 variant cytokine.

[0144] R 1 and R 2 A description of electron-withdrawing groups can be found in U.S. Patent No. 8,680,315, which is incorporated herein by reference. An electron-withdrawing group is defined as a group with a Hammett sigma value greater than 0 (see, for example, Hansch et al. 1991, Chemical Reviews 91:165-195). Typical examples of electron-withdrawing groups include, but are not limited to, nitriles, sulfones, sulfoxides, carbonyls, optionally substituted aryl groups, and optionally substituted heteroaryl groups.

[0145] In some embodiments of the formula (Ia) connector-drug, R 1 and R 2 The electron-withdrawing group is

[0146] -CN;

[0147] -NO2;

[0148] Optionally substituted aryl groups;

[0149] Optionally substituted heteroaryl groups;

[0150] Optional substituted alkenyl groups;

[0151] Optionally substituted alkynyl groups;

[0152] -COR 5 ,-SOR 5 , or -SO2R 5 ,

[0153] Where R 5H, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaryl, -OR 6 or -NR 6 2, where each R 6 Independently H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted aralkyl, or two R 6 The groups together with the nitrogen atoms they are attached to form heterocycles; or

[0154] SR 7 , where R 7 It can be an alkyl group, an aryl group, an aralkyl group, a heteroaryl group, or a heteroaryl group that has been optionally substituted.

[0155] In some embodiments of the formula (Ia) connector-drug, R 1 and R 2 The electron-withdrawing group is -CN. In some embodiments, R 1 and R 2 The electron-withdrawing group is -NO2. In some embodiments, R 1 and R 2 The electron-withdrawing group is an optionally substituted aryl group containing 6-10 carbons. For example, in some embodiments, R 1 and R 2 The electron-withdrawing group is optionally substituted with phenyl, naphthyl, or anthracene. In some embodiments, R 1 and R 2 The electron-withdrawing group is an optionally substituted heteroaryl group containing 3-7 carbons and at least one N, O, or S atom. For example, in some embodiments, R 1 and R 2 The electron-withdrawing group is pyrrole, pyridinyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indole, or indenyl, each of which may be optionally substituted. In some embodiments, R 1 and R 2 The electron-withdrawing group is an optionally substituted alkenyl group containing 2-20 carbons. In some embodiments, R 1 and R 2 The electron-withdrawing group is an optionally substituted alkynyl group containing 2-20 carbons. In some embodiments, R 1 and R 2 The electron-withdrawing group is -COR 5 -SOR 5 or -SO2R 5 , where R 5H, optionally substituted alkyl groups containing 1-20 carbon atoms, optionally substituted aryl groups, optionally substituted aralkyl groups, optionally substituted heteroaryl groups, optionally substituted heteroaralkyl groups, -OR 6 or -NR 6 2, where each R 6 Independently H or an alkyl group with 1-20 carbon atoms, or two R 6 The groups, together with the nitrogen atoms they are attached to, form heterocycles. In some embodiments, R 1 and R 2 The electron-withdrawing group is -SR 7 , where R 7 It is an optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl or optionally substituted heteroaryl containing 1-20 carbon atoms.

[0156] In some embodiments of the formula (I) connector-drug, R 1 and R 2 At least one of them is -CN or -SOR 5 or -SO2R 5 In some implementations, R 1 and R 2 At least one of them is -CN or -SO2R 5 In some implementations, R 1 and R 2 At least one of them is –CN or -SO2R 5 , where R 5 It can be an optionally substituted alkyl group, an optionally substituted aryl group, or... In some embodiments, R... 1 and R 2 At least one of them is -CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2HCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3) or -SO2N(CH2CH2OCH3)2.

[0157] In some embodiments of the formula (Ia) connector-drug, each R 4 Independently, it is a C1-C3 alkyl group. In some embodiments, at least one R 4 It is a methyl group. In some embodiments, the two R groups are... 4 They are all methyl groups.

[0158] In some embodiments of the connector-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.

[0159] In some embodiments of the connector-drug of formula (Ia), R 1 For CN or -SO2R 5 , where R 5 It is a C1-C6 alkyl, aryl, heteroaryl or -NR 6 2, where R 6 Independently C1-C6 alkyl, aryl, or heteroaryl, and R 2 =H, where R 5 and R 6 Each of them can be arbitrarily replaced independently.

[0160] For some embodiments of the (Ia) linker-pharmaceutical, Z may comprise any functional group known in the art for coupling. Examples of such functional groups include, but are not limited to, amines, aminooxy groups, ketones, aldehydes, maleimides, thiols, alcohols, azides, 1,2,4,5-tetraazine, trans-cyclooctenyl, bicyclononyl, cyclooctyneyl, and their protected variants. In some embodiments, Z comprises a protected amine, a protected aminooxy group, a ketone or a protected ketone, an aldehyde or a protected aldehyde, a maleimide, a protected thiol, a protected alcohol, an azide, 1,2,4,5-tetraazine, trans-cyclooctenyl, bicyclononyl, or cyclooctyneyl. In some embodiments, Z comprises an azide, a ketone, or a protected ketone. In some embodiments, Z comprises a functional group capable of selectively reacting with the associated functional group Z' on a macromolecular support to form a linking functional group Z*. In some embodiments, when Z / Z' is an amine / carboxylate or an active ester, the linking functional group Z* is a carboxamide; when Z / Z' is NH2O / ketone or aldehyde, it is an oxime; when Z / Z' is a thiol / maleimide or a halocarbonyl, it is a thioether; or when Z / Z' is an azide / cyclooctyne, it is a triazole.

[0161] In some embodiments of the (Ia) connector-drug, S is absent. In some embodiments, S is (CH2CH2O). h (CH2) g CONH.

[0162] In some embodiments of the formula (Ia) connector-pharmaceutical, Y is absent. In some embodiments, Y is NH(CH2CH2O).p (CH2) m .

[0163] It should be understood in this specification that each description, variation, implementation, or aspect of one part may be combined with each description, variation, implementation, or aspect of other parts, as if each and every combination described were specifically and individually listed. For example, the R provided herein regarding formula (I) 1 Each description, variation, implementation, or aspect may be related to Z,S,n,R 2 ,R 4 Each description, variation, implementation, or aspect of Y and / or D is combined in the same way as if each and each combination were specifically and individually listed. It should also be understood that all descriptions, variations, implementations, or aspects (if applicable) of any general formula (such as formula (I), (Ia), (IIa), (IIIa), (IV), (V), or (VI)) are equally applicable to other general formulas detailed herein and are described in the same way as if each description, variation, implementation, or aspect were individually listed for each general formula. For example, all descriptions, variations, implementations, or aspects (if applicable) of formula (I) are equally applicable to formulas (Ia), (IIa), (IIIa), (IV), (V), or (VI) detailed herein and are described in the same way as if each description, variation, implementation, or aspect were individually listed for each general formula.

[0164] Connector:

[0165] On the other hand, a connector of type (IIa) is provided:

[0166]

[0167] Where n, Z, S, R 1 R 2 and R 4 As disclosed in formula (Ia) herein; X is a halogen, an active ester (e.g., N-succinimideoxy, nitrobenoxy, or pentahalophenoxy) or NH(CH2CH2O). p (CH2) (m-1) CHO, where m is an integer from 2 to 6 and p is an integer from 0 to 1000. In some embodiments, X is a halogen. In some embodiments, X is an active ester, such as succinimideoxy. In some embodiments, X is a halide, succinimideoxy, or nitrobenzoxy. In some embodiments, X is NH(CH2CH2O). p (CH2) (m-1) CHO. X is NH(CH2CH2O). p (CH2) (m-1)The CHO linker can be attached to cytokines via reductive alkylation, wherein the aldehyde group of the linker forms an imine with the amino group of the cytokine, and this imine is reduced to an amine in the presence of a reducing agent (e.g., sodium cyanoborohydride). This method is generally selective for the attachment of the linker to the N-terminal α-amine group of the cytokine. In this embodiment, this is achieved by adding NH2 (CH2CH2O). p (CH2) m The N-terminal α-amine is modified to release cytokines from the conjugate upon cleavage of the linker. These linkers are prepared as described by Schneider et al. (2016) Bioconjugate Chem 27:2534-9 (incorporated herein by reference). In some embodiments, p is 0, and this is achieved by adding NH2 (CH2). m The N-terminal α-amine is modified to release cytokines from the conjugate upon linker cleavage.

[0168] In some embodiments of the connector of formula (IIa), n = 1-6, R 1 and R 2 Independently an electron-withdrawing group, alkyl group, or H, wherein R 1 and R 2 At least one of them is an electron-withdrawing group; each R 4 Independently, it is H or a C1-C3 alkyl group, or together they can form 3-6 membered rings; Z is a group used to connect the linker to the macromolecular support; S is absent or is (CH2CH2O). h (CH2) g CONH, where g = 1-6, h = 0-1000; X is a halide, succinimide, or nitrobenzoxy.

[0169] The preparation of these connector 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.

[0170] These linkers are attached to cytokines or cytokine variants by methods known in the art, for example, by reacting with a protein buffer solution at a pH between 6 and 9, preferably between 7 and 8, such that the amino groups on the protein are aminoacylated to form a linker-protein of formula (I). Multiple linkers can be attached to each protein when more than one amino group is available for reaction. The selectivity of the number of linkers attached to the protein can be controlled using the stoichiometric ratio of the linker reagent to the protein. When only one linker is attached, the protein released from the conjugate upon linker cleavage has no additional modification.

[0171] Coupled

[0172] On the other hand, a coupling of formula (III) is provided:

[0173] M-[Z*-LD] q (III)

[0174] Wherein, M is a macromolecular carrier, Z* is a linker functional group, L is a cleavable linker, and D is a cytokine or a cytokine variant protein. When M is a soluble macromolecular carrier, q is an integer from 1 to 10; when M is an insoluble macromolecular carrier, q is the multiplicity. It should be understood that when M is an insoluble macromolecular carrier, such as an insoluble matrix or support, a multiplicity linker-drug can be attached to M. For example, in some embodiments, when M is a hydrogel of formula (IV), where P... 1 and P 2 All are four-armed polymers, capable of attaching 1, 2, 3, or 4 connectors-drugs to each P. 1 -P 2 Unit. Therefore, the desired weight number can be achieved by reacting the linker-drug with M in a suitable ratio. Thus, a suitable drug concentration can be obtained within the matrix volume.

[0175] In some embodiments, the coupling of formula (III) is formula (IIIa):

[0176]

[0177] Where M, Z*, S, n, R 1 R 2 and R 4 Y and D are defined in detail in equations (I), (Ia) or (IIa) of this paper.

[0178] In some embodiments, M is a soluble macromolecular carrier, such as polyethylene glycol, dextran, protein, or antibody; Z* is a linking group; q = 1 to 10. In each case, M includes a reactive group Z', which reacts with group Z on the compound of formula (I) to form the linking group Z*. When Z / Z' is an amine / carboxylate or an active ester, the linking group Z* is a carboxamide; when Z / Z' is an aminooxy / ketone or aldehyde, it is an oxime; when Z / Z' is a thiol / maleimide or a halocarbonyl, it is a thioether; or when Z / Z' is an azide / cyclooctyne, it is a triazole. In some embodiments, Z* comprises an amide, formamide, oxime, triazole, thioether, thiosuccinimide, or ether. In some embodiments,

[0179] In some embodiments, M is polyethylene glycol with an average molecular weight between 1,000 and 100,000 Daltons, preferably between 10,000 and 60,000 Daltons, and most preferably between 20,000 and 40,000 Daltons. M can be single-chain, branched, or multi-armed. M includes one or more functional groups Z' for attachment to the linker-drug. Z' can be attached to a commercially available polymer M using methods known in the art; for example, when M contains an amino group, it can be further derivatized by: reacting with (Boc-aminooxy)acetic acid and then deprotecting to introduce Z'=aminooxy to achieve acylation; introducing Z'=cyclooctyne to achieve acylation by reacting with an active ester or carbonate of cyclooctyne (e.g., 4-cyclooctyneyl succinimide carbonate or (1R,8S,9S)-bicyclo[6.1.0]non-4-ynyne-9-ylmethoxysuccinimide carbonate (BCN-OSu) or its (1R,8S,9r) diastereomer); or introducing maleimide to achieve acylation by reacting with 3-maleimide propionic acid to achieve maleimide group.

[0180] In some embodiments, M is an insoluble macromolecular carrier, such as a hydrogel or surgical device. In these embodiments, q is the multiplicity determined by the number of reactive groups Z' attached to the insoluble carrier. In some embodiments, M is a degradable crosslinked hydrogel of formula (IV):

[0181]

[0182] Where P 1 and P 2 It is an independent r-arm polymer, where r is an integer between 2 and 8;

[0183] n is an integer between 0 and 6;

[0184] x, y, and z are each independent integers between 0 and 6;

[0185] B is a group containing Z′;

[0186] A* and C* are each independently a linking group, such as carboxamide, oxime, ether, thioether or triazole;

[0187] R 11 and R 12 Independently, it is H, C1-C4 alkyl, or an electron-withdrawing group, wherein R 11 and R 12 At least one of them is an electron-withdrawing group; and

[0188] Each R 14 Independently C1-C3 alkyl or two R 14 Together with the carbon atoms they are attached to, they form a 3-6 membered ring;

[0189] R11 and R 12 A description of the electron-withdrawing group can be found in U.S. Patent No. 8,680,315, which is incorporated herein by reference. In some embodiments of the formula (IV) hydrogel, R 11 and R 12 The electron-withdrawing group is

[0190] -CN;

[0191] -NO2;

[0192] Optionally substituted aryl groups;

[0193] Optionally substituted heteroaryl groups;

[0194] Optional substituted alkenyl groups;

[0195] Optionally substituted alkynyl groups;

[0196] -COR 15 ,-SOR 15 , or -SO2R 15 ,

[0197] Where R 15 H, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaryl, -OR 16 or -NR 16 2, where each R 16 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 heterocycles; or

[0198] SR 17 , where R 17 It can be an alkyl group, an aryl group, an aralkyl group, a heteroaryl group, or a heteroaryl group that has been optionally substituted.

[0199] In some embodiments of the formula (IV) hydrogel, R 11 and R 12 The electron-withdrawing group is -CN. In some embodiments, R 11 and R 12 The electron-withdrawing group is -NO2. In some embodiments, R 11 and R 12 The electron-withdrawing group is an optionally substituted aryl group containing 6-10 carbons. For example, in some embodiments, R 11 and R 12 The electron-withdrawing group is optionally substituted with phenyl, naphthyl, or anthracene. In some embodiments, R 11 and R12 The electron-withdrawing group is an optionally substituted heteroaryl group containing 3-7 carbons and at least one N, O, or S atom. For example, in some embodiments, R 11 and R 12 The electron-withdrawing group is pyrrole, pyridinyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, indole, or indenyl, each of which may be optionally substituted. In some embodiments, R 11 and R 12 The electron-withdrawing group is an optionally substituted alkenyl group containing 2-20 carbons. In some embodiments, R 11 and R 12 The electron-withdrawing group is an optionally substituted alkynyl group containing 2-20 carbons. In some embodiments, R 11 and R 12 The electron-withdrawing group is -COR 15 -SOR 15 or -SO2R 15 , where R 15 H, optionally substituted alkyl groups containing 1-20 carbon atoms, optionally substituted aryl groups, optionally substituted aralkyl groups, optionally substituted heteroaryl groups, optionally substituted heteroaralkyl groups, -OR 16 or -NR 16 2, where each R 16 Independently H or an alkyl group with 1-20 carbon atoms, or two R 16 The groups, together with the nitrogen atoms they are attached to, form heterocycles. In some embodiments, R 11 and R 12 The electron-withdrawing group is -SR 17 , where R 17 It is an optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl or optionally substituted heteroaryl containing 1-20 carbon atoms.

[0200] In some embodiments of the formula (IV) hydrogel, R 11 and R 12 At least one of them is -CN or -SOR 15 or -SO2R 15 In some implementations, R 11 and R 12 At least one of them is -CN or -SO2R 15 In some implementations, R 11 and R 12 At least one of them is –CN or -SO2R 15 , where R 15 It can be an optionally substituted alkyl group, an optionally substituted aryl group, or... In some embodiments, R...11 and R 12 At least one of them is -CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2HCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3) or -SO2N(CH2CH2OCH3)2.

[0201] In some embodiments of the formula (IV) hydrogel, each R 14 Independently, it is a C1-C3 alkyl group. In some embodiments, at least one R 14 It is a methyl group. In some embodiments, the two R groups are... 14 They are all methyl groups.

[0202] In some embodiments of the hydrogel of formula (IV), R 11 For CN or -SO2R 15 , where R 15 It is a C1-C6 alkyl, aryl, heteroaryl or -NR 16 2, where R 16 Independently C1-C6 alkyl, aryl, or heteroaryl, and R 12 =H, where R 15 and R 16 Each of them is independently and arbitrarily replaced.

[0203] The general formula for the linker protein attached to this type of hydrogel is as follows: Figure 1 As shown.

[0204] In a particular embodiment, M is a hydrogel of formula (V) or formula (VI).

[0205]

[0206]

[0207] Where P 1 ,P 2 ,r,R 11 ,R 12 and R 14 As detailed in equation (IV) in this article; and

[0208] Z' contains a cyclooctyne group. In a particular embodiment, Z' is 4-cyclooctyneoxycarbonyl or (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl.

[0209] The preparation of hydrogel carriers of these general formulas are disclosed in U.S. Patent 9,649,385 and PCT / US2020 / 026726 (filed April 3, 2020), each of which is incorporated herein by reference.

[0210] The conjugates described above can be used to deliver low, continuous doses of cytokines to subjects suffering from diseases or conditions treatable with this regimen. Specific diseases and conditions treatable with low, continuous doses of cytokines include those related to CD4 tolerance. + CD25 + FOXP3 + Chronic graft-versus-host disease (cGVHD) associated with insufficient regulatory T cell remodeling (Koreth et al., Blood 128:130-7 (2016)); systemic lupus erythematosus; sarcoidosis; hepatitis C-related vasculitis; alopecia areata; rheumatoid arthritis; inflammatory bowel disease; multiple sclerosis; and type 1 diabetes (Koreth et al., Oncology & Hematology Review 10:157-63 (2014)). Enhancing immunity through exogenous cytokines may be beneficial in the treatment of cancer and immunodeficiency.

[0211] The conjugates of the present invention can be formulated using standard buffer solutions and excipients known in the art. The buffer solution used is preferably between pH 3 and pH 7, more preferably between pH 4 and pH 6. For soluble conjugates, administration can be by intravenous, subcutaneous, intravitreal, or intramuscular injection; for insoluble conjugates, administration can be by subcutaneous, intravitreal, or intramuscular injection. Intratumoral injection may also be used.

[0212] Pharmaceutical Composition

[0213] In another aspect, this document provides pharmaceutical compositions comprising a macromolecular carrier-drug conjugate or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable buffers and / or excipients. The choice of buffer should ensure the stability of the connector during storage and, if necessary, during reconstitution, and generally has a pH between 2 and 7, preferably between 2 and 6, and more preferably between 2 and 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-ketoglutarate, etc. Excipients may include tonic and isotonic agents such as sodium chloride; preservatives such as citric acid or citrates and parabens; antimicrobial agents such as phenol and cresol; antioxidants such as butylated hydroxytoluene, vitamins A, C, or E, cysteine, and methionine; and density modifiers such as sucrose, polyols, hyaluronic acid, and carboxymethyl cellulose. These formulations can be prepared using conventional methods known to those skilled in the art, such as those described in "Remington's Pharmaceutical Science," ARGennaro, ed., 17th edition, 1985, Mack Publishing Company, Easton, PA, USA. Pharmaceutical compositions may be provided in liquid solution or suspension form, or in solid form, such as by freeze-drying a liquid composition. Such freeze-dried products may also contain fillers to ensure rapid and efficient reconstitution before use.

[0214] How to use

[0215] On the other hand, the macromolecular carrier-drug conjugates and pharmaceutical compositions comprising them described herein can be used to treat or prevent diseases or conditions in an individual. In some embodiments, a method of treating a disease or condition is provided, comprising administering to a subject in need the macromolecular carrier-drug conjugate described herein or a pharmaceutical composition comprising the macromolecular carrier-drug conjugate described herein. "Individual" can be a human or an animal, such as a cat, dog, cow, rat, mouse, horse, rabbit, or other domesticated animal.

[0216] Compositions containing the macromolecular carrier-drug conjugates described herein are also provided for the treatment of diseases or conditions. The use of the macromolecular carrier-drug conjugates described herein in the manufacture of medicaments for the treatment of diseases or conditions is also provided herein.

[0217] Those skilled in the art will know from the nature of the conjugate drug the applicable disease or condition that needs to be treated.

[0218] The following provides some representative implementation methods.

[0219] Implementation method 1: A coupling having the following formula:

[0220] M-[Z*-LD] q

[0221] Where M is a macromolecular carrier;

[0222] Z* is a linker functional group;

[0223] L is a pyrolytic connector; and

[0224] D is an amine residue of a cytokine or a variant thereof; and

[0225] Wherein, when M is a soluble carrier, q = 1-10, and when M is an insoluble carrier, q is the multiplicity.

[0226] Implementation Method 2: The coupling of Implementation Method 1, wherein Z* is a carboxamide, oxime, thioether, or triazole; and L has the following formula

[0227]

[0228] in,

[0229] n = 0-6 or 1-6;

[0230] R 1 and R 2 Independently an electron-withdrawing group, alkyl group, or H, wherein R 1 and R 2 At least one of them is an electron-withdrawing group;

[0231] Each R 4 Independently H or C1-C3 alkyl or two R 4 Together they can form a 3-6 elemental ring;

[0232] S is absent or (CH2CH2O) h (CH2) g CONH, where g = 1-6 and h = 0-1000;

[0233] Y is either absent or NH(CH2CH2O). p (CH2) m Where m = 2 - 6 and p = 0 - 1000.

[0234] Implementation method 3: The coupling of implementation method 2, wherein R 1 For CN or R 5 SO2, where R 5 It is a C1-C6 alkyl, aryl, heteroaryl or (R 6 )2N, where R 6 It is a C1-C6 alkyl, aryl, or heteroaryl group, and R 2 =H, and where R 4 –R 6Each of the terms can be optionally replaced.

[0235] Implementation Method 4: Any of the couplings described in Implementation Methods 1-3, wherein M is a soluble polyethylene glycol with an average molecular weight between 1,000 and 100,000 Daltons, and q = 1-10.

[0236] Implementation Method 5: Any of the couplings described in Implementation Methods 1-3, wherein M is an insoluble hydrogel or surgical device, and q is the weight number.

[0237] Implementation method 6: Any of the couplings described in implementation methods 1-3, wherein D is IL-2, IL-7, IL-9, IL-10, IL-15, IL-21 or a variant thereof.

[0238] Implementation 7: The conjugate of Implementation 6, wherein D is an IL-2 variant that selectively binds to a trimeric αβγ receptor compared to a dimeric βγ receptor, or an IL-2 variant that selectively binds to a dimeric βγ receptor compared to a trimeric αβγ receptor.

[0239] Embodiment 8: Any of the couplings described in Embodiments 1-3, wherein D is an IL-15 variant that is stable to deamidation.

[0240] Implementation Method 9: A protein-linked adapter of the following form:

[0241]

[0242] Where n = 0-6 or 1-6, R 1 and R 2 Independently an electron-withdrawing group, alkyl group, or H, and wherein R 1 and R 2 At least one of them is an electron-withdrawing group; each R 4 Independently, it is H or a C1-C3 alkyl group, or together they can form 3-6 membered rings; Z is a functional group used to connect the linker to the macromolecular support; S is absent or is (CH2CH2O). h (CH2) g CONH, where g = 1-6 and h = 0-1000; Y is absent or is NH(CH2CH2O). p (CH2) m , where m = 2-6 and p = 0-1000; and D are amine residues of cytokines or their variants.

[0243] Implementation Method 10: The adapter-protein of Implementation Method 9, wherein R 1 For CN or R 5 SO2, where R 5 It is a C1-C6 alkyl, aryl, heteroaryl or (R 6)2N, where R 6 It is a C1-C6 alkyl, aryl, or heteroaryl group, and R 2 =H, and where R 4 –R 6 Each of the terms can be optionally replaced.

[0244] Embodiment 11: The adapter 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.

[0245] Implementation 12: The adapter protein of Implementation 11, wherein D is an IL-2 variant that selectively binds to a trimeric αβγ receptor compared to a dimeric βγ receptor, or an IL-2 variant that selectively binds to a dimeric βγ receptor compared to a trimeric αβγ receptor.

[0246] Implementation method 13: The adapter protein of implementation method 12, wherein D is selected from the group consisting of IL-2, IL-2N88R, IL-2N88D, IL-2N88R, C125S and IL-2N88D, C125S.

[0247] Implementation method 14: The adapter protein of implementation method 9 or 10, wherein D is selected from the group consisting of IL-15, IL-15N77A and IL-15-[N71S, N72A, N77A].

[0248] Implementation Method 15: The linker protein of Implementation Method 9 or 10, wherein D is selected from IL-2, IL-7, IL-9, IL-10, IL-15, IL-21 or variants thereof, wherein the N-α amino group is obtained by adding NH2(CH2CH2O). p (CH2) m Modify the expression, where m = 2-6 and p = 0-1000.

[0249] Implementation Method 16: A selective amplification target in T reg The cellular method includes treating the object with any of the conjugates described in embodiments 1-3, wherein D is IL-2 or an IL-2 variant.

[0250] Implementation Method 17: A method for selectively amplifying CD8+ effector T cells in a target, comprising treating the target with any of the conjugates described in Implementation Methods 1-3, wherein D is IL-15 or an IL-15 variant.

[0251] Implementation 18: A method for treating a disease or ailment of a person requiring such treatment, comprising administering the conjugate described in any of Implementations 1-8.

[0252] Implementation Method 19: The method described in Implementation Method 18, wherein the disease or condition is an autoimmune disease, and is associated with CD4 tolerance. + CD25 + FOXP3 + Chronic graft-versus-host disease (cGVHD) associated with inadequate recombination of regulatory T cells; systemic lupus erythematosus; sarcoidosis; vasculitis caused by hepatitis C; alopecia; rheumatoid arthritis; inflammatory bowel disease; multiple sclerosis; or type 1 diabetes.

[0253] Implementation 20: A method for enhancing immunotherapy in a subject receiving such treatment, comprising administering the conjugate described in any of Implementations 1-8.

[0254] The following examples are intended to illustrate, and not limit, the scope of this disclosure. All references cited herein are incorporated by way of reference, including references cited for specific aspects of the disclosure, and in particular references cited for those aspects and in general.

[0255] Preparation of A

[0256] The connector of formula (IIa), wherein S is absent

[0257]

[0258] The (IIa) connector without S is prepared according to the following general procedure. In one method, groups Z and R are included. 4 The ester reacts with R in the presence of a base (usually potassium tert-butoxide or potassium tert-pentoxide). 1 R 2 CH2 condensation forms an intermediate ketone, which is reduced to an alcohol by sodium borohydride. This is then activated by reaction with triphosgene and pyridine to obtain the linker of formula (IIa), where X = Cl. It can be further converted to X = succinimideoxy by reaction of chloroformate and N-hydroxysuccinimide. In another method, by first making R... 1 R 2 CH2 reacts with a strong base (e.g., butyllithium, lithium diisopropylamide, or metallized hexamethyldisilazane), and the resulting R is then treated with an ester. 1 R 2 CH - The carbanion is used to obtain the same ketone intermediate for the initial condensation. Some specific examples are as follows:

[0259] (1) 4-Azide-1-cyano-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =CN,R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0260] A 1 M potassium tert-butoxide THF solution (3.5 mL, 3.5 mmol) was added to a 7 mL THF 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) at -30 °C. The mixture was stirred at -30 °C for 30 min, then heated to ambient temperature over 1 hour and stirred for another 30 min. The mixture was cooled on ice and quenched by adding 6 N HCl (0.62 mL, 3.7 mmol), then partitioned between EtOAc and water. The aqueous phase was extracted twice with EtOAc, the combined organic matter was washed with brine, dried over MgSO4, filtered, and concentrated to provide crude ketones.

[0261] Sodium borohydride (33 mg, 0.88 mmol) was added to a solution of crude ketone (300 mg, approximately 1.75 mmol) in 7 mL of methanol. The mixture was stirred for 15 minutes and quenched by adding 6N HCl (0.7 mL), then partitioned between EtOAc and water. The aqueous phase was extracted twice with EtOAc, the combined organic matter was washed with brine, dried over MgSO4, filtered, and concentrated to provide crude alcohol. Purification on SiO2 (20–40% ethyl acetate / hexane) provided 4-azido-1-cyano-3,3-dimethyl-2-butanol (142 mg, 0.85 mmol). 1 H-NMR (CDCl3, 300MHz) 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), and 0.96 (s, 3H).

[0262] Pyridine (136 μL, 1.7 mmol) was added dropwise to a 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 ice-cooled THF. The resulting suspension was heated to ambient temperature and stirred for 15 min, then filtered and concentrated to provide crude chloroformate. It 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 heated to ambient temperature and stirred for 15 min, then partitioned between EtOAc and 5% KHSO4. The aqueous phase was extracted twice with EtOAc, the combined organic matter was washed with brine, dried over MgSO4, filtered, and concentrated to provide crude succinimide carbonate. Purification on SiO2 (20-40% ethyl acetate / hexane) yielded 4-azido-1-cyano-3,3-dimethyl-2-butylsuccinimide carbonate (174 mg, 0.56 mmol). 1 H-NMR (CDCl3, 300MHz) 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), and 0.96 (s, 3H).

[0263] (2) 4-Azide-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate Ester (formula (I), where n = 1, R) 1 =SO2N(CH3)2,R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0264] A 1.43 M solution of n-butyllithium in hexane (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 maintained at -50 °C under an inert atmosphere. The mixture was warmed to -20 °C over 1 hour and then recooled to -50 °C before the addition of methyl 3-azido-2,2-dimethylpropionate (prepared according to Kim, Synthetic Communications; 7.70 g, 50 mmol). The mixture was warmed to +10 °C over 2 hours and then quenched with 20 mL of 6N HCl. The mixture was diluted with methyl tert-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. Chromatography was performed on SiO2 (220 g) using step gradients of 0, 20, 30, 40 and 50% EtOAc / hexane to obtain purified crystalline solid of 10.65 g, 86% 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanone.

[0265] The ketone was dissolved in 200 mL of methanol, cooled on ice, and treated with sodium borohydride (0.96 g, 25 mmol) for 15 min. The solution was then quenched with 4 mL of 6N HCl and concentrated. The resulting slurry was diluted with methyl tert-butyl ether (MTBE, 200 mL), washed once with 100 mL of water, washed once with 100 mL of brine, dried over MgSO4, filtered, and concentrated to obtain 10.0 g of 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butanol crystals.

[0266] After 10 minutes, 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 dichloromethane cooled on ice. The mixture was stirred on ice for 15 minutes, then heated to ambient temperature for 30 minutes. 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 stirred again at ambient temperature for 1 hour. After cooling on ice, the mixture was treated with 100 mL of water and the phase was separated. The organic phase was washed twice with water, once with 5% KHSO4, once with brine, dried over MgSO4, filtered, and concentrated. The crude product was crystallized from 100 mL of 30% EtOAc / hexane to give a white crystalline solid (11.1 g, 71%) of 4-azido-1-((N,N-dimethylamino)sulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate.

[0267] (3) Other compounds of formula (I) prepared according to these methods include:

[0268] 4-Azide-1-(methylsulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2CH3,R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0269] 4-Azide-1-((4-methylpiperidinyl)sulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2N(CH2CH2)2CHCH3, R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy). LC / MS shows [M+H]. + =446.15.

[0270] 4-Azide-1-(phenylsulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2Ph,R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0271] 4-Azide-1-(4-chlorophenylsulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2PhCl, R2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0272] 4-Azide-1-(4-morpholinosulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2N(CH2CH2)2O, R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0273] 4-Azide-1-(isopropylsulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2CH(CH3)2, R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0274] 4-Azide-1-((N-ethyl-N-4-methylamino)sulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2N(CH3)(CH2CH3), R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0275] 4-Azide-1-((N,N-Di(2-methoxyethyl)aminosulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula I, where n = 1, R 1 =SO2N(CH2CH2OCH3)2, R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0276] 4-Azide-1-(4-methylphenylsulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula I, where n = 1, R 1 =SO2PhCH3,R 2 =H,R 4 =CH3, Z=N3, and X=succinimideoxy).

[0277] 4-(tert-Butoxycarbonyl)amino-1-(methylsulfonyl)-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2CH3,R 2 =H,R 4=CH3, Z=NH-Boc, and X=succinimideoxy).

[0278] 4-(tert-Butoxycarbonyl)amino-1-cyano-3,3-dimethyl-2-butylsuccinimide carbonate (Formula (I), where n = 1, R 1 =SO2CH3,R 2 =H,R 4 =CH3, Z=NH-Boc, and X=succinimideoxy).

[0279] Compounds of formula (I) were also prepared according to Santi et al., Proc. Natl. Acad. Sci. USA 2012, 109(16): 6211-6, wherein S was absent and each R 4 It's H.

[0280] Preparation of B

[0281] Type (IIa) connector

[0282] Where S = (CH2CH2O) h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) CHO

[0283]

[0284] The connector of formula (IIa) is prepared as follows, where S = (CH2CH2O). h (CH2) g C(O)NH and X=NH(CH2CH2O) p (CH2) (m-1) CHO. In one method, the linker of formula (IIa) (where Z is an azide, S is absent, and X = succinimideoxy) reacts with an amine-acetal H2N-(CH2). m-1 The reaction of CH(OR)₂, where R is an alkyl group, yields an azide carbamate acetal. The azide group is then reduced to an amine via catalytic hydrogenolysis over a palladium catalyst or via Staudinger reduction of trimethylphosphine in the presence of water, followed by the addition of a spacer-succinimide ester Z-(CH₂CH₂O). h (CH2) g C(O)OSu is used to obtain a linker in the acetal-protected form. Hydrolysis of the acetal under acidic conditions then provides a linker of formula (IIa), where S=(CH2CH2O). h (CH2) g C(O)NH, X=NH(CH2CH2O) p (CH2) (m-1) CHO. Specific examples are as follows:

[0285] 7-(15-azido-4,7,10,13-tetraoxopentadecanoamide)-1-(4-benzenesulfonyl)-2-heptylN-(3-oxopropyl)carbamate (Formula IIa, where 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):

[0286]

[0287] (1) 7-Azide-1-(4-methylbenzenesulfonyl)-2-heptyl N-(3,3-diethoxypropyl)carbamate. 7-Azide-1-(4-methylbenzenesulfonyl)-2-heptylsuccinimide carbonate (125 mg, 277 μmol, 50 mM final concentration) (Santi et al., Proc. Natl. Acad. Sci. USA 2012, 109(16): 6211-6) was dissolved in 5.5 mL MeCN and 1-amino-3,3-diethoxypropane (54 μL, 0.33 mmol, 60 mM final concentration) was added. The reaction mixture was stirred overnight at ambient temperature. The starting carbonate was completely consumed within 15 minutes according to TLC. The reaction mixture was partitioned between 100 mL of 1:1 EtOAc:NaHCO3 (saturated aqueous solution). The aqueous layer was extracted with 40 mL of EtOAc. The combined organic layers were washed sequentially with water, KHSO4 (5% aqueous solution), water, and brine (1 x 30 mL each). The organic phase was separated, dried on MgSO4, filtered, and concentrated to provide 109 mg (81% crude product) of the title compound as a colorless oil, which was used entirely in the next step without further purification. 1 H NMR (300MHz, CDCl3) δ7.76(d,J=8.1Hz,2H),7.33(d,J=8.1Hz,2H),5.04(quin,J=6.8Hz,1H),4.91(t,J=5.4Hz,1H),4 .49(t,J=5.2Hz,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=5.8Hz,2H),1.63(br q,J=5.7Hz,2H),1.52(m,2H),1.30(br m,4H),1.17(td,J=7.0,2.1Hz,6H).

[0288] LC-MS (m / z): Calculated value 529.2; Observed value 529.6 M + HCO2- .

[0289]

[0290] (2) 7-(15-azido-4,7,10,13-tetraoxopentadecanoylamino)-1-(4-methylbenzenesulfonyl)-2-heptylN-(3,3-diethoxypropyl)carbamate. 7-azido-1-(4-methylbenzenesulfonyl)-2-heptylN-(3,3-diethoxypropyl)carbamate (109 mg, 225 μmol, 0.1 M final concentration) 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 purged and backfilled with hydrogen (3x). The reaction mixture was vigorously stirred at ambient temperature under a H2 (balloon) atmosphere. The starting material was completely consumed within 90 minutes, as determined by TLC. The reaction mixture was filtered through a short pipette stopper made of diatomaceous earth and the stopper was washed with 10 mL of ethanol. Concentrate the filtrate to dryness to provide 90 mg of a colorless, oily intermediate amine, which is used entirely in the next step without further purification.

[0291] The crude product 7-amino-1-(4-methylbenzenesulfonyl)-2-heptylN-(3,3-diethoxypropyl)carbamate (90 mg, maximum 0.20 mmol, 0.1 M final concentration) was dissolved in 2.0 mL of MeCN. 15-azido-4,7,10,13-tetraoxopentadecanoic acid succinimide ester (93 mg, 0.24 mmol, 0.12 M final concentration) and DIPEA (42 μL, 0.22 mmol) were added, and the reaction was stirred at ambient temperature and monitored by TLC. After 1 hour, the reaction mixture was partitioned between 60 mL of 1:1 EtOAc:NaHCO3 (saturated aqueous solution). The organic layer was washed sequentially with water, citric acid (10% aqueous solution), water, and brine (1 x 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 in acetone in CH2Cl2 solution with the following gradients: 0%, 10%, 20%, 30%, 40%, and 50% (30 mL each). The clear fractions containing the product were combined and concentrated to provide a colorless, oily title compound (68 mg, 93 μmol, 41% two-step). 1H NMR (300MHz, CDCl3) δ7.76(d,J=8.3Hz,2H),7.32(d,J=8.1Hz,2H),6.60(br t,J=5.8Hz,1H),4.95-5.08(m,2H),4.50(br t,J=4.9Hz,1H),3.56-3.74(m,18H),3.40-3.52(m,3H),3.36(t,J=5.1Hz,2H),3.10- 3.26(m,5H),2.44(t,J=5.8Hz,2H,fuzzy),2.42(s,3H),1.74(q,J=6.0Hz,2H),1.62(br s,2H),1.43(br m,2H),1.26(br s, 4H), 1.17 (td, J = 7.0, 2.3Hz, 6H).

[0292] LC-MS (m / z): Calculated value 776.4; Observed value 776.7 [M+HCO2] - .

[0293]

[0294] (3) 7-(15-azido-4,7,10,13-tetraoxopentadecanoylamino)-1-(4-methylbenzenesulfonyl)-2-heptylN-(3-oxypropyl)carbamate. 7-(15-azido-4,7,10,13-tetraoxopentadecanoylamino)-1-(4-methylbenzenesulfonyl)-2-heptylN-(3,3-diethoxypropyl)carbamate (68 mg, 93 μmol, 0.1 M final concentration) was dissolved in 0.62 mL of CHCl3. Water and TFA (0.16 mL each) were added sequentially. The reaction mixture was stirred vigorously overnight at ambient temperature. After 2 hours, the starting material was completely consumed according to TLC. The reaction mixture was concentrated to dryness and then purified on a 4 g SiliaSep column, eluting with the following stepwise gradient of acetone in CH2Cl2: 0%, 15%, 30%, 45%, 60%, and 75% (30 mL each). The clear fractions containing the product were combined and concentrated to provide a colorless oily title compound (26 mg, 40 μmol, 43%). 1¹H NMR (300MHz, CDCl₃) δ 9.78 (s, 1H), 7.78 (d, J = 8.3Hz, 2H), 7.36 (d, J = 8.0Hz, 2H), 6.60 (br s, 1H), 5.09 (m, 1H), 4.98 (t, J = 6.0Hz, 1H), 3.62–3.75 (m, 16H), 3.36–3.44 (m, 5H), 3.13–3.26 (m, 3H), 2.70 (t, J = 5.7Hz, 2H), 2.47 (t, J = 5.7Hz, 2H, blurred), 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): calculated value 656.3; observed value 656.6 [MH] - ; Calculated value 702.3; Observed value 702.6 [M+HCO2] - Calculated value: 734.3; Observed value: 734.7 [M+CH3OH+HCO2] - .

[0295] In the second method, the linker of formula (IIa) is carried out through a similar sequence of steps, where Z = Boc amino, S = absent, and X = OH, but the Boc group is first removed under acidic treatment, and the spacer-succinimide ester Z-(CH2CH2O) is attached. h (CH2) g C(O)OSu.

[0296] The alcohol is then activated by reaction with triphosgene and pyridine, resulting in a chloroformate reacting with an amine-acetal (H₂N-(CH₂)). m-1 The reaction CH(OR)₂, where R is an alkyl group, yields an acetal-protected linker. Hydrolysis of the acetal under acidic conditions then provides a linker of formula (IIa), where S = (CH₂CH₂O). h (CH2) g C(O)NH, X=NH(CH2CH2O) p (CH2) (m-1) CHO. Specific examples are as follows:

[0297]

[0298] 1-Azide-18,18-dimethyl-20-benzenesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosanol-9-yl(3-oxopropyl)carbamate (Formula IIa, where Z = N3, S = (CH2CH2O)4(CH2)2C(O)NH, n = 1, R 1 =PhenylSO2,R 2=H, each R 4 =Methyl, and X=NH(CH2)2CHO):

[0299] Step 1 and 21-azido-18,18-dimethyl-20-benzenesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-aza-19-eicosanol. Trifluoroacetic acid (1 mL) was added to a solution of 4-[(tert-butoxycarbonyl)amino]-1-benzenesulfonyl-3,3-dimethyl-2-butanol (124 mg 58% w / w mixture; 72 mg, 0.20 mmol, 0.1 M final concentration) in 1 mL CH2Cl2. The reaction was stirred at ambient temperature and monitored by TLC (40% EtOAc in hexane solution, cerium molybdate staining). 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 provide a white film-like intermediate amine. The intermediate was dissolved in 1.8 mL MeCN, and DIPEA (0.17 mL, 1.0 mmol) was added. Pure azido-PEG4-OSu (78 mg, 0.2 mmol) was added. The reaction was stirred at ambient temperature and monitored by C18 HPLC (ELSD). Azido-PEG4-OSu completely converted to a single, rapidly moving HPLC peak within 5 minutes. The reactants were 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, 30 mL per step). The clear, product-containing fractions determined by C18 HPLC were combined and concentrated to dryness. Residual volatiles were removed under high vacuum, providing a colorless, oily title compound (85 mg, 0.16 mmol, 80% two-step yield). Purity determined by C18 HPLC and ELSD: 98.2% (RV = 9.12 mL).

[0300] Step 3. 1-Azide-18,18-dimethyl-20-benzenesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosano-19-yl(3,3-dioxypropyl)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, immediately forming a white precipitate. The suspension was stirred at ambient temperature for 15 minutes 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-benzenesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-aza-19-eicosanol (86 mg, 0.16 mmol, 0.1 M) in 1 mL of anhydrous THF was added. The reaction mixture was stirred at ambient temperature and monitored by C18 HPLC (ELSD). After 1 hour, the starting alcohol was consumed. The reaction mixture was partitioned between 50 mL of 1:1 EtOAc:KHSO4 (5% aqueous solution). The layers were separated and the organic phase was washed sequentially with KHSO4 (5% aqueous solution), water, NaHCO3 (saturated aqueous solution), and brine (25 mL each). The washed organic phase was dried over MgSO4, filtered, and concentrated by rotary evaporation. Crude succinimide 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 minutes, succinimide carbonate converted to two slower elution peaks. The reaction mixture was partitioned between 30 mL of 1:1 EtOAc:sodium acetate (0.2 M, pH 5.0). The layers were separated, and the organic phase was washed sequentially 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 provide a yellow, oily crude title compound (105 mg, 0.15 mmol, 94% crude product in two-step yield).

[0301] Step 4. 1-Azide-18,18-dimethyl-20-benzenesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosaecan-19-yl(3-oxypropyl)carbamate. Water (0.21 mL) and TFA (0.21 mL) were added sequentially to 1.1 mL of CH₂Cl₂ containing a solution of 1-azido-18,18-dimethyl-20-benzenesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosaecan-19-yl(3,3-diethoxypropyl)carbamate (105 mg, 0.15 mmol, 0.1 M final concentration). The reaction was stirred at ambient temperature and monitored by C18 HPLC (ELSD). The reaction was deemed complete after 10 minutes. The mixture was concentrated to dryness. The concentrate was loaded onto a SiliaSep 4g silica gel column and eluted using a stepwise gradient of acetone in CH2Cl2 (0%, 20%, 40%, 60% acetone; 30 mL per step). Fractions were analyzed by TLC (cerium molybdate staining). The clear fractions containing the product were combined and concentrated to dryness. Residual volatiles were removed under high vacuum, providing a colorless oily title compound (34 mg, 54 μmol, 36% yield). The product was dissolved in 5.0 mL Gibco H2O (0.01 M mass). Purity was determined by C18 HPLC and ELSD: 99.0% (RV = 8.76 mL).

[0302] 1-Azide-18,18-dimethyl-20-methanesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosano-19-yl(3-oxopropyl)carbamate (Formula IIa, where 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.

[0303] Step 3. 1-Azide-18,18-dimethyl-20-methanesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosaecan-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, immediately forming a white precipitate. The suspension was stirred at ambient temperature for 15 minutes 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-methanesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-aza-19-eicosanol (80 mg, 0.17 mmol, 0.06 M) in 1 mL of anhydrous THF was added. The reaction mixture was stirred at ambient temperature and monitored by C18 HPLC (ELSD). After 2 hours, the starting alcohol was consumed. The reaction mixture was partitioned between 50 mL of 1:1 EtOAc:KHSO4 (5% aqueous solution). The layers were separated, and the washed organic phase was washed sequentially with KHSO4 (5% aqueous solution), water, NaHCO3 (saturated aqueous solution), and brine (25 mL each). The organic phase was dried over MgSO4, filtered, and concentrated by rotary evaporation. Crude succinimide 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, succinimide carbonate converted to a slower elution peak. The reaction mixture was partitioned between 30 mL of 1:1 EtOAc:sodium acetate (0.2 M, pH 5.0). The layers were separated, and the organic phase was washed sequentially with water and brine (15 mL each). The washed organic phase was dried on MgSO4, filtered, and concentrated by rotary evaporation. Residual volatiles were removed under high vacuum, providing a yellow oily crude title compound (61 mg, 95 μmol, 56% crude product in two-step yield).

[0304] Step 4. 1-Azide-18,18-dimethyl-20-methanesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosaecan-19-yl(3-oxypropyl)carbamate. Water (135 μL) and TFA (135 μL) were added sequentially to a solution of 1-azido-18,18-dimethyl-20-methanesulfonyl-15-oxo-3,6,9,12-tetraoxa-16-azaeicosaecan-19-yl(3,3-diethoxypropyl)carbamate (61 mg, 95 μmol, 0.1 M final concentration) in 0.68 mL of CH₂Cl₂. The reaction was stirred at ambient temperature and monitored by C18 HPLC (ELSD). The reaction was deemed complete after 25 minutes. The mixture was concentrated to dryness. The concentrate was loaded onto a SiliaSep 4g silica gel column and eluted using a stepwise gradient of acetone in CH2Cl2 (0%, 20%, 40%, 60%, 80%, 100% acetone; 30 mL per step). Each fraction was analyzed by TLC (cerium molybdate staining) and C18 HPLC. The clear fractions containing the product were combined and concentrated to dryness. Residual volatiles were removed under high vacuum, providing a colorless oily title compound (12 mg, 21 μmol, 22% yield). After qualitative analysis, the product was dissolved in 2.0 mL Gibco H2O (0.01 M mass). Purity was determined by C18 HPLC and ELSD: 91.3% (RV = 5.60 mL).

[0305] (4) Other compounds of formula (I) prepared according to these methods include:

[0306] 7-(15-azido-4,7,10,13-tetraoxapentadecanylamino)-1-(4-benzenesulfonyl)-2-heptylN-(3-oxopropyl)carbamate (Formula IIa, where 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).

[0307] 7-(15-azido-4,7,10,13-tetraoxapentadecanylamino)-1-(methanesulfonyl)-2-heptylN-(3-oxopropyl)carbamate (Formula IIa, where 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).

[0308] 7-(15-azido-4,7,10,13-tetraoxapentadecanylamino)-1-(morpholinosulfonyl)-2-heptyl N-(3-oxopropyl)carbamate (Formula IIa, where 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).

[0309] 5-(15-azido-4,7,10,13-tetraoxapentadecanylamino)-3,3-dimethyl-1-(thiomorpholinosulfonyl)-2-pentyl N-(3-oxopropyl)carbamate (Formula IIa, where 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).

[0310] Example 1

[0311] Preparation and activity of IL-2 [N88R, C125S]

[0312] IL-2 [N88R, C125S] was prepared by expression in HEK cells. Cell-based receptor binding assays were performed to evaluate the high affinity of the mutant protein for the IL-2 receptor αβγ trimer (T). reg ) and moderate affinity βγ dimer (T eff The activity of the mutant protein in the form of IL-2Rαβγ is shown in Table 1. The binding strength of the mutant protein to IL-2Rαβγ is only 6-fold lower than that to IL-2, while the binding strength to IL-2Rβγ is about 900-fold lower than that to IL-2. Importantly, the selectivity of the mutant protein for IL-2Rαβγ is more than 3000-fold higher than that for IL-2Rβγ.

[0313] Following the manufacturer's instructions (DiscoverX, part #93-1003E3CP0), the IL-2Rαβγ binding assay kit based on U2OS cells was used. Cells were seeded at a concentration of 100 μL (~10,000 cells / well) in 96-well assay plates and cultured at 37°C and 5% CO2 for 24 h. Cells were then treated for 6 h at 37°C and 5% CO2 with serially diluted WT IL-2, IL-2N88R, C125S, or IL-2N88R, C125S released from microspheres at pH 9.4. The concentrations of 11 WT IL-2 compounds were determined between 2 pg / mL and 100 ng / mL (0.1 pM–6 nM). The concentrations of 11 IL-2N88R, C125S, and released IL-2N88R, C125S were determined within the range of 200 pg / mL to 10 μg / mL (10 pM to 600 nM). After treatment, cells were incubated with the chemiluminescent substrate in the dark at room temperature for 1 hour, and then the luminescence was read using a Spectramax i3 plate reader with an integration time of 250 ms.

[0314] The IL-2Rβγ binding assay kit based on U2OS cells was performed according to the manufacturer's instructions (DiscoverX, part #93-0998E3CP5). Cells were seeded at a concentration of 50 μL (~5,000 cells / well) in 96-well assay plates and cultured at 37°C and 5% CO2 for 48 h. Cells were then treated for 6 h at 37°C and 5% CO2 with serially diluted WT IL-2, IL-2N88R, C125S, or IL-2N88R, C125S released from microspheres at pH 9.4. The concentrations of 11 WT IL-2 compounds were determined between 17 pg / mL and 1 μg / mL (1 pM–61 nM). The concentrations of 11 WT IL-2N88R, C125S compounds were determined between 1.7 ng / mL and 100 μg / mL (100 pM–6 μM). The concentrations of 11 released residues, WT IL-2N88R and C125S, were determined between 170 pg / mL and 10 μg / mL (10 pM to 600 nM). The treated cells were incubated with the chemiluminescent substrate in the dark at room temperature for 1 hour, and the luminescence was then read using a Spectramax i3 plate reader with an integration time of 250 ms.

[0315] These measurement results are as follows Figure 2 As shown in Table 1.

[0316] Table 1. IL-2 and IL-2N88R,C125S bind to cells containing αβγ and βγ receptors.

[0317]

[0318] Example 2

[0319] Optimization of cytokine reductive alkylation

[0320]

[0321] Attachment of the linker is achieved via reductive alkylation of the N-terminal amino group of IL-2. In the presence of 10 mM NaCNBH3, a series of concentrations of the linker reagent of formula (IIb) (i.e., the linker of formula (IIa)) are used, where 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) CHO, where h = 4, g = 2, p = 0, and m = 3) was used to treat 200 μM IL-2N88R, C125S. The N3 group was then reacted with PEG-cyclooctynyl DBCO-PEG. 5kDa After the reaction, the reaction was analyzed by SDS-PAGE to induce gel displacement caused by PEG adhesion. A 1.5:1 linker:protein ratio was found to be optimal, resulting in a mixture of unmodified protein: single-linker protein: double-linker protein: triple-linker protein of 58:34:5:3 (Table 2). Figure 3 The resulting gel bands, quantified using ImageJ, are shown. C125S (200 μM) was treated at ambient temperature with 1, 1.5, 2, or 3 equivalents of adapter-CHO (Mod = MeSO2) and 10 mM NaCNBH3 in 50 mM MES, 150 mM NaCl, pH 6.0 for 20 hours.

[0322] Table 2. Linker-protein product distribution of IL-2N88R, C125S reductive alkylation products

[0323] Joint equivalent Unmodified single connector Dual connector Three-connector 1 66 30 4 0 1.5 58 34 5 3 2 52 35 9 4 3 43 38 15 4

[0324] Example 3

[0325] Preparation of linker-cytokines

[0326] The IL-2 [N88R, C125S] is attached to the releasable connector using one of two methods.

[0327] (1) Random acylation: A mixture of cytokines (3.4 mL, 4.81 mg / mL, 1.00 μmol) and 1.44 mL of 100 mM HEPES at pH 7.0 was mixed with 4-azido-3,3-dimethyl-1-(isopropylsulfonyl)-2-butylsuccinimide carbonate [Formula (II), wherein R 1 = i PrSO2,R 2 =H,R 4 =Me,Z=N3,n=1;S=None;X=Succinimideoxy] (156uL, 10mg / mL in acetonitrile, 4umol) was mixed and maintained at 4°C for 20 hours. Hydroxylamine (0.55mL, 1M, pH 7.0) was added and maintained at 4°C for another 23 hours. The mixture was then added to a PD-10 column using 50mM MES, pH 6.0, 0.05% Tween-20 to provide OD. 280 The recovered protein was 1 μmol. SDS-PAGE analysis showed that it formed a mixture of unmodified:1 adapter:2 adapter:3+ adapter in a ratio of 57:31:6:6.

[0328] (2) Reductive alkylation was performed using the method described in Schneider et al., Bioconjugate Chem (2016) 27:2534-9 (incorporated hereby by reference). O-7-[(15-azido-13,10,7,4-tetraoxapentadecanoyl)amino]-1-(methanesulfonyl)-2-heptylN-3-oxapropylcarbamate [formula (II), where R] was added to a solution of IL-2N88R, C125S (250 μM final concentration, 1.25 μmol, 20.5 mg) in 4.25 mL of 50 mM MES, 150 mM NaCl pH 6.0 (reaction buffer) at 0 °C. 1 =MeSO2,R 2 =H,R 4=H,Z=N3,S=(CH2CH2O)4CH2CH2CONH);n=4; andX=(CH2)2CHO] (375 μM final concentration, 1.9 μmol, 0.9 mg, 0.27 mL) in a solution of 20 mM NaOAC pH 5.0 and NaCNBH3 (10 mM final concentration, 1 μmol, 0.5 μL) in reaction buffer. The reaction was carried out at ambient temperature and in the dark for 22 h. Excess reagents were removed using a PD-10 column equilibrated in 20 mM MES, 150 mM NaCl, 0.05% Tween-20, pH 6.0. After concentration using an Amicon Ultra 10000MW retardation concentrator, the linker-N-terminal aminopropyl-IL-2 [N88R, C125S] was recovered to 600 μM (A 280 1.85 mL – 1.1 μmol, 89% – total peptides.

[0329] Example 4

[0330] Preparation of hydrogel microspheres that release IL2- and [aminopropyl]-IL2

[0331] Microsphere activation: PEG hydrogel microspheres of formula (IV) were used (according to Henise et al., Engineering Reports (2020)). https: / / doi.org / 10.1002 / eng2.1209 ), where P 1 and P 2 20 kDa 4-arm PEG; Z* is a triazole derived from Z=N3 and Z'=5-hydroxycyclooctylene; n=4; R 11 =CN;R 12 =H; each R 14 =H; B=NH2; x=4; y=0; z=0; and r=4. Activate it to formula (IV), where B=NH-CO-O-(4-cyclooctynyl), as shown below. Add a solution of 4-cyclooctynyl succinimide carbonate (5 μmol, 1.2 equivalents) in 1 mL MeCN and a solution of N,N-diisopropylethylamine (17 μmol, 4 equivalents) in 1 mL MeCN to a suspension of 1.3 g microspheres in 1 mL MeCN in a 15 mL conical tube, where B=NH2 (4.2 μmol NH2). Invert the reaction at ambient temperature for 6 hours. Wash the slurry with 4 x 12 mL MeCN, then with 4 x 12 mL of 20 mM MES, 150 mM NaCl, 0.05% Tween-20, pH 6.0.

[0332] Using the same method, microspheres of formula (IV) (where B = NH2) were activated to formula (IV) (where B = (1R, 8S, 9S)-bicyclo[6.1.0]non-4-yn-9-ylmethylsuccinimide carbonate replaced 4-cycloacynylsuccinimide carbamate) by reacting with BCN-OSu((1R, 8S, 9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy-CO-NH).

[0333] To attach the adapter-cytokine, a suspension of 2 g of activated microsphere slurry (4.2 μmol 5HCO3) (20 mM MES, 150 mM NaCl, 0.05% Tween-20, pH 6.0) was placed in a 15 mL conical tube and mixed with 18.3 mg (1.1 nmol) of adapter-AP-IL-2N88R, C125S (37% adapter-IL-2 according to gel displacement analysis, Example 3) in 1.9 mL of the same buffer. The mixture was incubated at 37 °C for 23 h and subjected to orbital shaking at 250 rpm. The slurry was washed with 8 x 12 mL of the above buffer, followed by 4 x 6 mL of 20 mM MES, 250 mM NaCl, 0.05% Tween-20, pH 6.0. The total loading of the microspheres was 102 nmol IL-2N88R, C125S gm in the slurry. -1 AP-IL-2 A280(ε) released from 29-32 mg of slurry dissolved in 9 volumes of 50 mM NaOH 280 =10095M -1 cm -1 ) Measurement.

[0334] The linker-IL-2 (Example 3) prepared by random acylation was added to PEG hydrogel microspheres in the same manner, so that the insoluble conjugate was loaded at 0.11 mM and the protein had SEQ ID NO:3.

[0335] Example 5

[0336] In vitro release kinetics:

[0337] Under accelerated release conditions, β-elimination kinetics were determined using 257 mg of the microsphere-IL-2 mutant protein slurry from Example 4 in Eppendorf tubes at 37°C, in 257 μL of 250 mmHg sodium borate, 0.05% (v / v) Tween-20, and pH 9.4. Samples were removed from the 37°C water bath at intervals, centrifuged at 21000 x g for 1 min, and the A0 of 100 μL of the supernatant was measured using a cuvette-based UV / Vis spectrophotometer. 280After measurement, the analyzed supernatant was returned to the tube containing the microspheres and incubated further at 37°C. The release rate was measured by measuring the released A... 280 The rate was calculated by fitting a first-order rate equation to Graphpad Prism over time. Knowing that β-elimination is first-order in hydroxide ions, the calculated rate at pH 7.4 was k. pH 7.4 =k pH x 10 (pH-7.4) The release profile of IL-2 [N88R, C125S] from the random acylated conjugate of Example 2 was biphasic, with half-lives of 0.4 and 41 hours at pH 9.4, corresponding to 40 and 4100 hours at pH 7.4. The release profile of AP-IL-2 [N88R, C125S] from the reductive alkylated conjugate of Example 2 was monophasic, with a half-life of 11 hours at pH 9.0, corresponding to 440 hours at pH 7.4.

[0338] Example 6

[0339] Pharmacokinetics of IL-2 [N88R, C125S] released from hydrogel microspheres in rats

[0340] Under aseptic conditions, syringes (0.5 mL, 29 gauge length, fixed needle, BD) were filled with an average of 50 mg or 300 mg of the microsphere-IL-2 slurry from Example 4 (5 nmol or 30 nmol IL-2 [N88R, C125S]) 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 subcutaneously injected into the flank of four male Sprague-Dawley rats with an average weight of 250 g. Blood samples (200 μL) were collected 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. Using the microsphere conjugate of Example 2, IL-2 (or NH2(CH2)3-IL-2, "AP-IL2") was observed in plasma 96 hours after administration. Figure 4 As shown.

[0341] Example 7

[0342] Pharmacokinetics of IL2 and IL2[N88R,C125S] in mice

[0343] In NOD (non-obese diabetic) mice, the pharmacodynamics of free IL-2 [N88R, C125S] and native IL-2 were compared. Three NOD mice in three groups were injected daily for five consecutive days with either a PBS carrier, adenoleukin (25,000 units, 63 μg), or IL-2 [N88R, C125S] (25,000 units, 63 μg). Two hours after the last injection, the mice were sacrificed, and the spleen and pancreas were collected for flow cytometry analysis to measure changes in the total number and differentiation of T cells in the spleen and pancreatic islets.

[0344] IL-2 [N88R, C125S] affects CD4+ in the spleen + and CD8 + Effector / memory T cells were barely affected, while natural IL-2 increased with the increase of both T cell populations. Figure 5 ).

[0345] NOD mice were injected daily with PBS carrier, adeleukin (25,000 units), or IL-2 [N88R, C125S] (25,000 units), and were sacrificed 2 hours after the last injection on the fifth day. Figure 5 The pharmacokinetics of IL-2 [N88R, C125S] in the spleen were shown.

[0346] Compared with PBS carriers, natural IL-2 and IL-2 [N88R, C125S] had almost no effect on T cells in the pancreas. Figure 6 It is noteworthy that when mice were treated with IL-2 [N88R, C125S], the total number of pancreatic islets decreased.

[0347] NOD mice were injected daily with PBS carrier, adeleukin (25,000 units), or IL-2 [N88R, C125S] (25,000 units), and were sacrificed 2 hours after the last injection on the fifth day. Figure 6 The pharmacokinetics of IL-2 [N88R, C125S] in the pancreas were shown.

[0348] Example 8

[0349] Pharmacokinetics of [aminopropyl]-IL2[N88R,C125S] released from microsphere-IL-2[N88R,C125S] in mice kinetics / pharmacodynamics

[0350] The PK / PD of [aminopropyl]-IL-2[N88R,C125S] released from the microsphere-IL-2[N88R,C125S] conjugate was determined using three groups of six NOD mice. Group 1 received five daily injections of free IL-2[N88R,C125S] (25,000 units, 63 μg). Group 2 received subcutaneous injections of empty microspheres capped with N3(CH2CH2O)7H. Group 3 received a single subcutaneous injection of the microsphere-IL-2[N88R,C125S] from 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 illustration. Lymphocytes were analyzed by flow cytometry to monitor changes in the T cell population. Spleen, lymph nodes, and pancreatic islets were isolated, and single-cell suspensions were prepared. Surface staining was performed following standard flow cytometry immunofluorescence staining of cell surfaces. Fixation and intracellular staining followed the protocol of eBioscience Foxp3 / transcription factor staining buffer kit (ThermoFisher Scientific). Antibodies used targeted CD3, CD4, CD8, CD25, CD44, CD45, and FoxP3; all were from commercial suppliers. Stained single-cell suspensions were analyzed using an LSRII flow cytometer (BD Biosciences).

[0351] Figure 7 The pharmacokinetics of [aminopropyl]-IL-2[N88R,C125S] released from microspheres-IL-2[N88R,C125S] (“MS-IL-2 mutant protein”) are shown in mice. Figure A: BALB / c mice (n=6) received a single subcutaneous injection on their flanks containing either 28 nmol (19 mg / kg) or 9.9 nmol (6.5 mg / kg) of microspheres-IL-2[N88R,C125S]. The t-value was determined at 31 hours. 1 / 2 Figure B: NOD mice (n=6) were administered microsphere-IL-2 [N88R, C125S] ventrally. In both cases, plasma concentrations of IL-2 [N88R, C125S] were analyzed using a Thermofisher ELISA.

[0352] Treatment with microspheres-IL-2 [N88R, C125S] led to the reduction of Foxp3 in the spleen and PBMCs. + CD4 + T cell proliferation was significant. Nearly 70% and 55% of the T cells in the spleen and PBMCs, respectively, were Foxp3. + CD4 + T cells ( Figure 8 Compared with the control group, CD8 + The percentage of T cells also increased. CD8 in the spleen+ Cell percentage increased from 11% to 25%, CD8 in PBMCs + The cell percentage increased from 15% to 60%.

[0353] Figure 8 A shows Foxp3 in the spleen and PBMCs. + CD4 + T cell expansion. Figure 8 B shows CD8 in the spleen and PBMCs. + T cell expansion. CD8 cells were found in the spleen and PBMCs. + The cell percentages were approximately 11% and 19%, respectively. When treated with microspheres-IL-2 [N88R, C125S], these percentages increased to approximately 25% and 60%, respectively. NOD mice were given a single injection of IL-2 mutant protein (QDx5, 25,000 units) or microspheres-IL-2 [N88R, C125S] (18 mg / kg). Mice were sacrificed 2 hours after the last administration on day 5.

[0354] To determine inactive CD8 + Expanding Foxp3 in cellular conditions + CD4 + A dose-titer study of microsphere-IL-2 [N88R, C125S] was conducted to determine the effective dose for T cell populations. 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 was monitored over two weeks using PBMCs. Foxp3 was observed in PBMCs following a single injection of microsphere-IL-2 [N88R, C125S]. + CD4 + T cell dose-dependent expansion. All doses of Foxp3 + CD4 + T cell expansion peaked on day 4 and returned to baseline levels on day 14. Figure 9 A). Importantly, at any dosage, CD8 + The percentage of cells did not increase ( Figure 9 B).

[0355] Figure 9 A shows the microsphere-IL-2 [N88R, C125S] preferential amplification of Foxp3. + CD4 + T cells, Figure 9 B shows that they avoid activating CD8 in NOD mice (n=3 / dose group). + Cells (right). As shown, all doses of Foxp3 + CD4+ T cell expansion peaked on day 4 and returned to baseline levels on day 14.

[0356] Example 9

[0357] Preparation of connector-IL-15

[0358] As described above for IL-2, the linker from Example 2 was coupled to the N-terminus of IL-15 using reductive alkylation with NaCNBH3. 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 at ambient temperature and in the dark for 24 hours. Excess reagent was removed using a PD-10 column equilibrated in 20 mM sodium citrate, 500 mM NaCl, 0.05% Tween-20, pH 5.86. The desalted reaction mixture was concentrated using an Amicon Ultra 3500MW retardant concentrator.

[0359] Small-scale (2.25 nmol, 75 μL) reductive alkylation reactions were performed using IL-15, varying adapter equivalents to determine optimal reaction conditions. Initial reactions were conducted using 1, 1.5, and 2 equivalents of N3-PEG4-L(MeSO2)-CHO adapters, showing one adapter added to the protein at a 1:1 ratio (data not shown). Subsequent reactions were conducted using 1.5, 3, and 5 equivalents of adapter to increase the conversion of unmodified protein. With 3 equivalents of adapter, the reaction produced approximately 52% of IL-15 with only one adapter attached to the protein, and approximately 5% of IL-15 with two adapters attached. Increasing the adapter concentration to 5 equivalents resulted in only a small increase in single-adaptor-protein, but approximately 27% of the total protein had two or more adapters attached.

[0360] like Figure 10 As shown, via SDS-PAGE DBCO-PEG 5k Gel displacement analysis was used to determine the reaction progress. Table 3 shows the percentage of IL-15 modification. 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 and 500 mM NaCl for 20 h at ambient temperature in the dark.

[0361] Table 3. IL-15 Modification %

[0362]

[0363] Optimized reaction conditions with a 3-equivalent linker were used in the large-scale (0.93 μmol–1.08 μmol) reaction. The large-scale reaction was carried out twice.

[0364] Example 10

[0365] Preparation of microspheres-IL-15

[0366] In a sterile syringe, a BCN-activated microsphere slurry (2.6 μmol BCN, Example 4) was washed five times (~35 mL) with 20 mM sodium citrate, 500 mM NaCl, 0.05% Tween-20, and pH 5.86. The adapter-IL-15 (Example 9) (1 μmol total protein containing approximately 50% alkylated IL-15) was added to the syringe through a sterile filter (0.22 μM). The mixture was inverted and rotated at ambient temperature for 18 hours. The slurry mixture was then washed five times with 20 mM sodium citrate, 500 mM NaCl, 0.05% Tween-20, and pH 5.86. Unreacted BCN-activated microspheres were capped with N3(CH2CH2O)7H, followed by six more washes. The A280(ε) of IL-15 released from 5 mg aliquots of the slurry dissolved in 4 volumes of 50 mM NaOH was used to determine the IL-15 concentration. 280 =7240M –1 cm –1 The concentrations of IL-15 loaded on the microspheres were determined (216–336 μM). The MS-IL-15 concentrations for three different loadings were determined to be 336 nmol / mL, 216 nmol / mL, and 232 nmol / mL, respectively.

[0367] Example 11

[0368] Pharmacokinetics of IL-15 release from microspheres-IL-15

[0369] The microsphere-IL-15 slurry (275 nmol protein / mL) from Example 10 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-IL-15, serial dilutions were used to obtain the desired concentrations. In all cases, the microsphere conjugates were processed and prepared under aseptic conditions. 100 μL of the conjugate was backfilled into syringes with a fixed needle (27G). The syringe contents were administered subcutaneously or intraperitoneally to normal male C57BL / 6J mice. Blood samples were collected from alternating groups of three mice at -24, 4, 8, 24, 48, 96, 168, and 240 hours. All plasma samples were added to a HALT protease inhibitor mixture (ThermoFisher Scientific) and frozen at -80°C until analysis.

[0370] hIL-15 was determined by ELISA according to the manufacturer's instructions (R&D Systems, hIL-15Quantikine, catalog #D1500) to identify rhIL-15 in plasma. Plasma samples were thawed on ice before being diluted with the standard diluent provided by the manufacturer. Samples were diluted 50-fold at 4 hours and 8 hours, 25-fold at 24 hours, and 10-fold at 48, 96, 168, and 240 hours prior to collection. GraphPad Prism software was used to plot and fit the hIL-15 concentration over time.

[0371] For flow cytometry analysis, PMBCs were prepared and surface-stained to quantify the expression of NK1.1, CD3, CD8, and CD44. Commercially available FITC-, PE-, or allophycocyanin-conjugated antibodies were used. Sample data were acquired on a FACScan flow cytometer (BDBiosciences) and analyzed using FlowJo flow cytometry software (TreeStar, Ashland, Oregon).

[0372] The pharmacokinetics of [aminopropyl]-IL-15 released from the microsphere conjugate were measured in normal C57BL / 6J mice. Mice were injected with 2.4 nmol of the conjugated protein (200 μL injection solution). There were no significant changes in the initial mean mouse body weight (25.1 ± 1.3 g) and the final mean mouse body weight (25.1 ± 1.4 g). Approximately 120 hours later, a rapid decrease in concentration was observed. Figure 11 The half-life obtained by fitting a single-phase decay model to 120 hours of data is at least 200 hours. The half-life obtained by fitting a single-phase decay model to data points from 120 hours to 240 hours is t. 1 / 2The time was 27 hours. A second injection of MS-IL-15 (50 μg) increased the plasma IL-15 concentration at 248 hours to a level similar to the initial dose. From 264 hours to 360 hours, a t-value of 23 hours was observed. 1 / 2 .

[0373] Figure 11 The pharmacokinetics of [aminopropyl]-IL-15 released from MS-IL-15 in C57BL / 6J mice are shown. 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 human IL-15 Quantikine ELISA (R&D systems). Two distinct t=240-hour intervals were observed. 1 / 2 t was observed within 120 hours. 1 / 2 For at least 115 hours, then a second t was observed for 43 hours within 120 to 240 hours. 1 / 2 Immediately after a blood draw 240 hours later, a second injection of MS-IL15 (50 μg) was administered (blue data).

[0374] Figure 12 The dose-dependent pharmacokinetics of [aminopropyl]-IL-15 released from microspheres-IL-15 in C57BL / 6J mice were demonstrated. Normal male C57BL / 6J mice were administered MS-IL-15 (12.5, 25, or 50 μg). Plasma samples were prepared and analyzed using human IL-15 Quantikine ELISA (R&D systems).

[0375] The route of administration (i.e., subcutaneous (sc) and intraperitoneal (ip)) did not alter the release of [aminopropyl]-IL15. 1 / 2 ( Figure 13 However, AUC ip (25.2nM*h) ratio to AUC sc (14.9 nM*h) is nearly twice as high. This may indicate an increased bioavailability or absorption rate of IL-15 when it is absorbed from the intraperitoneal space.

[0376] Figure 13 The pharmacokinetics of [aminopropyl]-IL-15 released from microspheres-IL-15 in C57BL / 6J mice (subcutaneous vs. intraperitoneal) are shown. MS-IL-15 (50 μg) was administered subcutaneously (black, ●) or intraperitoneally (blue, ■) to normal male C57BL / 6J mice. Plasma samples were prepared and analyzed using the human IL-15 Quantikine ELISA (R&D systems). Similar t-response rates were observed between subcutaneous and intraperitoneal administrations over 120 hours.1 / 2 (115 hours (subcutaneous) and 129 hours (intraperitoneal)).

[0377] Example 12

[0378] Pharmacokinetics of microsphere-IL-15 release of [aminopropyl]-IL-15

[0379] The pharmacokinetics of [aminopropyl]-IL-15 released from the microspheres of Example 10 were measured in normal male C57BL / 6J mice (n=3 / group). Figure 14 Mice were administered the microspheres prepared in Example 10 containing IL-15 (2.5, 12.5, 25, or 50 μg of protein conjugated). PMBCs were prepared and surface stained for flow cytometry analysis of cells expressing NK1.1, CD3, CD8, and CD44. Commercially available FITC-, PE-, or allophycocyanin-conjugated antibodies were used. Sample data were obtained on a FACScan flow cytometer (BDBiosciences) and analyzed using FlowJo flow cytometry software (TreeStar, Ashland, Oregon). Clinical observations included no injection site reaction and no significant changes in initial mean body weight (25.1 ± 1.3 g) and final mean body weight (25.1 ± 1.4 g).

[0380] A single subcutaneous injection of MS-IL-15 conjugate (12.5 μg, 25 μg, or 50 μg) can lead to CD44 in PBMCs. hi CD8 + T cell dose-dependent expansion. CD44 hi CD8 + T cells reached a 2-4 fold increase peak 5 days after treatment. These cells remained higher than the control group for 21 days. Figure 14 A). On day 28, CD44 in mice administered the highest dose of MS-IL-15 (50 μg) hi CD8 + T cell levels remained twice that of the control group. No CD44 levels were observed during the experiment with a single dose of natural rhIL-15 (2.5 μg) or an equivalent dose of MS-IL-15 (2.5 μg). hi CD8 + T cell expansion.

[0381] A dose-dependent expansion of NK cells was also observed in PBMCs after a single subcutaneous injection of MS-IL-15 conjugate. Figure 14B). When MS-IL-15 (12.5 μg, 25 μg, or 50 μg) was administered, NK cells reached a peak expansion of approximately 2-3 times within 5 to 7 days post-treatment. NK cell proliferation continued for 14 to 21 days. No NK cell expansion was observed with a single dose of natural rhIL-15 (2.5 μg) or an equivalent dose of MS-IL-15 (2.5 μg).

[0382] Example 13

[0383] Preparation of connector-RLI and microsphere-RLI couplings

[0384] RLI (receptor-linked interleukin) is a fusion protein containing the sushi domain of IL-15 and the receptor α subunit, and it acts as a super agonist of the IL-15 receptor β / γ complex (Mortier et al., J. Biological Chem. 2006, 281:1612-9; US Patent 1,0358,488).

[0385] Small-scale reductive alkylation reactions were carried out with varying linker concentrations of RLI (10 nmol, 50 μL) to determine the optimal reaction conditions for stoichiometric linker addition. Initial reactions were performed using 1.5, 2, 3, and 5 equivalents of linker (IIb) from Example 2. Under the test conditions, when using 1.5 equivalents of linker, 44% of the RLI was modified with one linker, and 46% remained unmodified. It was determined that 2 equivalents of linker resulted in approximately 53% of the RLI having stoichiometric linker addition; 33% of the RLI was unmodified, and 14% of the RLI had multiple covalently bonded linkers. Increasing the linker equivalent to 3 equivalents resulted in an increase in the percentage of 2-linker addition (27%) and the formation of RLI containing 3 linkers (6%). These percentages increased even more in the presence of 5 equivalents of linker. Figure 15 ).

[0386] Table 4.

[0387] Unmodified +1 connector +2 connector +3 connector 1.5 equivalent 46％ 44％ 10％ – 2.0 equivalent 33％ 53％ 14％ – 3.0 equivalent 14％ 53％ 27％ 6％ 5.0 equivalent 5％ 48％ 28％ 19％

[0388] Use SDS-PAGE DBCO-PEG 5K The reductive alkylation reaction of RLI was determined by gel displacement analysis. Figure 15 The percentage of modified RLI, as determined by gel shift analysis, is shown. Bands were quantified using ImageJ software. RLI (10 nmol) was treated for 20 hours at room temperature in the dark with 1.5, 2, 3, or 5 equivalents of linker-CHO (Mod = MeSO2) and NaCNBH3 (10 mM) in 25 mM MES 500, 500 mM NaCl, and 0.05% Tween-20.

[0389] Large-scale reductive alkylation was performed using a 2-equivalent adapter (800 nmol, 4 mL). The reductively alkylated RLI was then coupled to BCN-activated microspheres (Example 4). To minimize oxidation, EDTA (1 mM) and methionine (30 mM) were added to the reaction. After coupling, the microspheres were thoroughly washed 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 sample (~25 mg) of the washed microspheres was digested in NaOH (50 mM) to determine the concentration of RLI covalently bound to the microspheres. The RLI concentration on the microspheres was determined to be 175 nmol / mL.

[0390] Example 14

[0391] Bioactivity of microsphere conjugates in releasing RLI

[0392] After RLI was released from the microsphere conjugate, the aminopropyl residue remained at the coupling site. To test the bioactivity of the released [aminopropyl]-RLI, cell-based assays were used to determine the ability of [aminopropyl]-RLI to induce receptor dimerization compared to native RLI. As evaluated in this bioactivity assay, the EC50 values ​​of native RLI and [aminopropyl]-RLI... 50 The curves overlap, indicating that the aminopropyl residue does not affect IL-15 activity. Figure 16 ).

[0393] Figure 16 The results of RLI analysis based on IL-2Rβγ receptor binding cells are shown. The assay was performed using U2OS cells at pH 7.4 (EC5). 50 [Aminopropyl]-RLI released from the coupling compound at 180 μM (=180 μM) and natural RLI (EC) 50 The binding activity compared to (=160μm).

[0394] Example 15

[0395] Pharmacokinetics of microsphere conjugate release of [aminopropyl]-RLI

[0396] The pharmacokinetics of [aminopropyl]-RLI released from the microsphere conjugate were measured in normal C57BL / 6J mice. Mice were subcutaneously injected with the conjugate (1.5 nmol protein, 100 μL). Blood was collected at predetermined time points over 10 days, and plasma was prepared. The concentration of [aminopropyl]-RLI in plasma was determined using an RLI-specific ELISA. Figure 17 Manually checking the data indicates that T 最大The half-life was 135 hours after the data was fitted to a single-phase decay model. The mice's body weight did not change (initial body weight: 21.5±1.1g; final body weight: 21.5±1.1g).

[0397] Figure 17 The pharmacokinetics of [aminopropyl]-PLI released from the microsphere conjugate in C57BL / 6J mice were demonstrated. Normal male C57BL / 6J mice were administered the microsphere-RLI conjugate (1.5 nmol). Plasma samples were prepared and analyzed using the R&D systems DuoSethIL15 / IL15Rα composite ELISA (DY6924). The data conformed to a single-phase decay model with a half-life of 135 hours.

[0398] Example 16

[0399] Pharmacokinetics of microsphere conjugate release of [aminopropyl]-RLI

[0400] The pharmacokinetics of the MS-RLI conjugate (34 μg, 1.5 nmol) were compared with those of empty MS and free RLI (2.5 μg, QDx4) in C57Black mice (n=5 / group). Blood was collected within 13 days and the surface of PBMCs was stained using standard laboratory procedures. Fixation and intracellular staining followed the protocol of eBioscience Foxp3 / transcription factor staining buffer kit (ThermoFisher Scientific). Commercial antibodies used targeted cells expressing NK1.1, CD3, CD8, CD19, CD44, and Ki-67. Stained single-cell suspensions were analyzed using LSRII flow cytometry (BD Biosciences) and flow cytometry analysis software (TreeStar, Ashland, OR). The CD8 cell population was of particular interest. + Memory T cells (CD44) hi CD8 + ), natural killer cells (CD3) - NK1.1 + Cells, proliferating CD8 + Memory T cells (CD44) hi CD8 + Ki-67 + ) and proliferating natural killer cells (CD3) - NK1.1 + Ki - 67 + ).

[0401] Five days after treatment, CD44 levels in the MS-RLI group and the natural RLI group were... hi CD8+ T cells increased significantly ( Figure 18 A). The cell population failed to be maintained by natural RLI and recovered to baseline levels by day 7. This was due to the short half-life (t). 1 / 2 =3 hours) and rapid clearance of free RLI. MS-RLI conjugate maintains CD44 for 13 days after treatment. hi CD8 + T cell levels. All five mice given the MS-RLI conjugate developed injection site lesions and required euthanasia.

[0402] CD8 + T cell proliferation was measured using the proliferation marker Ki-67. Three days after injection, CD8+ was observed to increase compared to the control group. + Increased T cells ( Figure 18 B). CD8 of all groups + The percentage of T cell proliferation peaked on day 5 and then quickly returned to baseline levels.

[0403] Compared with the control group and natural RLI, the percentage of NK cells in mice given the MS-RLI conjugate was also increased. Figure 19 A). Free RLI injection and MS-RLI conjugate increased the percentage of NK cells in PBMCs by approximately 4-fold and 15-fold, respectively. NK cell levels in all groups returned to baseline levels 10 days post-treatment. NK cell proliferation significantly increased three days post-treatment and remained elevated for five days in each group. Figure 19 B). Seven days after treatment, NK cell proliferation returned to baseline levels.

[0404] Example 17

[0405] Preparation of biodegradable PEG-hydrogels

[0406]

[0407] The hydrogel of the present invention is prepared by polymerization of two prepolymers comprising groups C and C', which react to form a linking functional group C*. The prepolymer linked to one of C or C' further includes a pyrolytic connector, which is introduced by reacting with a pyrolytic connector, such as the connector of formula (IIa) described herein, so as to introduce the pyrolytic connector into each crosslink of the hydrogel.

[0408] In one embodiment, the first prepolymer comprises a four-armed PEG, wherein each arm is terminated with a linker unit having two mutually non-reactive (“orthogonal”) functional groups B and C. B and C may initially exist in a protected form to allow for selective chemical reactions in subsequent steps. In some embodiments, the linker unit is a derivative of an amino acid, particularly lysine, cysteine, aspartic acid, or glutamic acid, including derivatives in which the α-amino group has been converted to an azide, such as 2-azidoglutamate monoester. The linker unit is connected to each first prepolymer arm via a linking functional group a*, formed by the condensation of functional group A on each prepolymer arm with a homologous functional group A' on the linker unit. The second prepolymer comprises a four-armed PEG, wherein each arm is terminated with a functional group C' that is complementary to the group C of the first prepolymer, such that crosslinking occurs between the two prepolymers when C and C' react to form C*.

[0409] 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 obtain a linker subunit, wherein A = COOH, B = Boc-protected NH2, and C = azide. This was coupled to a 20 kDa four-arm PEG-tetraamine, and the Boc group was removed to provide a first prepolymer, wherein A* = amide, B = NH2, C = azide, and wherein the cleavable linker of formula (IIa) was incorporated into the connection between each arm of the first prepolymer and the C group. A corresponding second prepolymer was prepared by acylation of the 20 kDa four-arm PEG-tetraamine with 5-cyclooctyneyl succinimide carbonate to obtain a second prepolymer, wherein C' = cyclooctyne. When the first and second prepolymers are mixed, the reaction of C=azide and C'=cyclooctyne groups forms the corresponding triazole groups, thereby crosslinking the two prepolymers into a three-dimensional network, wherein each crosslink contains a cleavage linker generated by the incorporation of the (IIa) compound, and wherein each node generated by the incorporation of the first prepolymer includes the remaining functional group B=NH2, which can be derived to attach other linkers, drugs, fluorophores, metal chelators, etc.

[0410] All publications (including patents, patent applications, and scientific articles) mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication (including patents, patent applications, or scientific articles) were incorporated herein by reference separately and individually.

[0411] Although the invention has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain minor changes and modifications may be made. Therefore, the specification and embodiments should not be construed as limiting the scope of the invention. SEQUENCE LISTING <110> ProLynx LLC <120> Slow-release cytokine conjugates <130> 67057-20022.40 <140> not specified <141> Provided here <150> US 62 / 839,112 <151> 2019-04-26 <160> twenty one <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 132 <212> PRT <213> Homo sapiens <400> 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Leo Thr 130 <210> 2 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 2 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 3 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 3 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 4 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 4 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 5 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 5 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 6 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 6 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Lys Met Leu Thr Ile Lys Phe Asn Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Leu Leu Lys 50 55 60 Pro Leu Glu Val Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 7 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 7 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Lys Met Leu Thr Gln Lys Phe Glu Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Val Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 8 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 8 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ile Lys Phe Asn Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Leu Leu Lys 50 55 60 Pro Leu Glu Val Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 9 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 9 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Lys Met Leu Thr Lys Lys Phe Arg Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Leu Leu Lys 50 55 60 Pro Leu Glu Val Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 10 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 10 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Lys Met Leu Thr Ile Lys Phe Glu Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Val Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 11 <211> 133 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 11 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Ala Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 12 <211> 114 <212> PRT <213> Homo sapiens <400> 12 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Serum <210> 13 <211> 114 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 13 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Ala Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Serum <210> 14 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Ser Ala Ser Leu Ser Ser Ala Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 15 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Serum <210> 16 <211> 253 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 16 Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Asp Tyr 20 25 30 Lys Asp Asp Asp Asp Lys Ile Glu Gly Arg Ile Thr Cys Arg Arg Arg 35 40 45 Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr 50 55 60 Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly 65 70 75 80 Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala 85 90 95 His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp Pro Ala Leu Val 100 105 110 His Gln Arg Pro Ala Pro Pro Ser Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Gln Asn Trp Val Asn Val 130 135 140 Ile Ser Asp Leu Lys Lys Ile Gln Asp Leu Ile Gln Ser Met His Ile 145 150 155 160 Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val 165 170 175 Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu 180 185 190 Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu 195 200 205 Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys 210 215 220 Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln 225 230 235 240 Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser 245 250 <210> 17 <211> 212 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val 1 5 10 15 Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly 20 25 30 Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn 35 40 45 Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile 50 55 60 Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Ser Gly 65 70 75 80 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser 85 90 95 Leu Gln Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp 100 105 110 Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp 115 120 125 Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu 130 135 140 Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr 145 150 155 160 Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Ala Gly 165 170 175 Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys 180 185 190 Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe 195 200 205 Ile Asn Thr Ser 210 <210> 18 <211> 152 <212> PRT <213> Homo sapiens <400> 18 Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu 1 5 10 15 Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser 20 25 30 Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp 35 40 45 Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg 50 55 60 Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu 65 70 75 80 Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val 85 90 95 Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser 100 105 110 Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu 115 120 125 Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys 130 135 140 Ile Leu Met Gly Thr Lys Glu His 145 150 <210> 19 <211> 126 <212> PRT <213> Homo sapiens <400> 19 Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile 1 5 10 15 Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser Ala Asn 20 25 30 Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg 35 40 45 Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr Met Gln 50 55 60 Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val Glu Val 65 70 75 80 Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn 85 90 95 Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu 100 105 110 Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile 115 120 125 <210> 20 <211> 133 <212> PRT <213> Homo sapiens <400> 20 Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile 1 5 10 15 Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu 20 25 30 Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser 35 40 45 Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu 50 55 60 Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser 65 70 75 80 Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys 85 90 95 Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys 100 105 110 Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His 115 120 125 Gly Ser Glu Asp Ser 130 <210> 21 <211> 161 <212> PRT <213> Homo sapiens <400> 21 Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe 1 5 10 15 Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser 20 25 30 Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu 35 40 45 Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln 50 55 60 Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln 65 70 75 80 Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly 85 90 95 Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe 100 105 110 Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala 115 120 125 Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe 130 135 140 Asp With Phe With Asn Tyr With Glu Ala Tyr Met Thr Met With Lys Arg 145 150 155 160 Asn

Claims

1. A conjugate wherein a drug is attached to a hydrogel via a linker at an N-terminal α-amino group, the unit of the conjugate being shown in the following formula: ； Where S = (CH2CH2O) h (CH2) g CONH, g = 2 and h = 4, n = 4, R 4 =H,R 1 =-SO2R 5 R 5 =C1-C6 alkyl, R 2 =H, Y=NH(CH2CH2O) p (CH2) m m = 3 and p = 0, Z It is a triazole formed by the reaction of azide with the cyclooctyne of B on M, thereby forming a coupling unit. When M is a soluble hydrogel, q is an integer between 1 and 10; or when M is an insoluble hydrogel, q is the multiplicity. M is a hydrogel of formula (IV): (IV), in: A The linking group is selected from carboxamide, oxime, ether, thioether or triazole, n=1, R 14 =CH3,R 11 =SO2R 15 R 12 =H, x=0, y=4, Z is 0, B=(1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy-CO-NH; C It is a carboxylamide, and R 15 It is a C1-C6 alkyl group; D is IL-15 or IL-15 IL-15RαSu fusion protein; P 1 and P 2 are 4-arm PEG polymers, thus r is 4, where P 1 as follows: ， P 2 As shown below: ,and P represents the coupling unit. 1 With A Or P2 and C The connection point.

2. The conjugate of claim 1, wherein R 15 is -CH3.

3. The conjugate of claim 1, wherein R 15 is -CH(CH3)2.

4. The conjugate of claim 1, wherein R 5 is -CH3.

5. The coupling as claimed in claim 1, wherein R 5 It is -CH(CH3)2.

6. The conjugate according to any one of claims 1-5, wherein D is IL-15 of SEQ ID No:

12.

7. The conjugate according to any one of claims 1-5, wherein D is IL-15 of SEQ ID No:

13.

8. The conjugate according to any one of claims 1-5, wherein D is IL-15 of SEQ ID No:

14.

9. The conjugate according to any one of claims 1-5, wherein D is IL-15 of SEQ ID No:

15.

10. The conjugate according to any one of claims 1-5, wherein D is IL-15 of SEQ ID No:

16. IL-15RαSu fusion protein.

11. The conjugate according to any one of claims 1-5, wherein D is IL-15 of SEQ ID No:

17. IL-15RαSu fusion protein.

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