Protein-Macromolecule Conjugates and Methods of Use Thereof

Abstract

The present disclosure provides protein-macromolecule conjugates, releasable linkers, and macromolecules, as defined herein. The disclosed conjugates offer unique properties based at least on the nature of the linker and the number of linker-macromolecule moieties. Also provided herein are methods for the synthesis and use of the conjugates in the treatment of diseases and disorders.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 908,435, filed September 30, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application has been submitted electronically via EFS-Web and includes a Sequence Listing in .txt format. The .txt file contains a Sequence Listing entitled "CSPL_008_01WO_SeqList_ST25.txt," which was created on September 29, 2020, and has a size of approximately 3.71 kilobytes. The Sequence Listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to methods for preparing protein-macromolecule conjugates via the use of bifunctional linkers. Additionally, the present disclosure relates to novel conjugates designed to control the pharmacokinetics in the delivery of biologically functional proteins. In particular, the present disclosure relates to protein-macromolecule conjugates with desired protein release rates. More specifically, the present disclosure relates to conjugates having a macromolecule comprising an IL-2 moiety (i.e., a moiety having at least some activity similar to human IL-2) and one or more linkers. Additionally, the present disclosure relates to conjugate compositions, methods for preparing the conjugates, methods for administering the conjugates, and methods for using the conjugates in the field of cancer therapy. [Background technology]

[0004] Many drugs suffer from unfavorable pharmacokinetic parameters that limit their efficacy. Such drugs are rapidly eliminated from physiological compartments either through metabolism or excretion, resulting in short lifespans and reduced target exposure. For example, therapeutic agonists based on natural proteins are attractive immunomodulators that can help establish effective and durable antitumor responses. However, poor pharmacokinetics (PK), poor tolerability, and pleiotropic activities that can be exacerbated by frequent administration make them less than ideal.

[0005] The cytokine interleukin-2 (IL-2) is the endogenous agonist of the IL-2 pathway and mediates CD8 + Aldesleukin is well known as a stimulator of T cells (CD8 T) and NK cells. High-dose IL-2 regimens administered every 8 hours in hospital settings using an IL-2 variant known as "aldesleukin" were approved by the U.S. Food and Drug Administration in the 1990s for the treatment of metastatic melanoma and renal cell carcinoma, resulting in durable responses in up to 25% of patients. High doses of IL-2 are required to activate CD8 T cells and NK cells, but these cells tend to express the low-affinity IL-2 receptor beta-gamma subunit (IL-2Rβγ). Further complicating the need for high doses of IL-2 is the poor PK profile of this protein. High-dose aldesleukin is not widely used due to severe toxicity associated with overactivation of the immune system. In addition to these toxicities, IL-2 also stimulates the proliferation and activation of regulatory T cells (Tregs). These cells constitutively express the high-affinity heterotrimeric IL-2 receptor alpha-beta-gamma subunit (IL-2Rαβγ). Treg activation can exacerbate immunosuppression and impair the intended antitumor response.

[0006] Polymeric prodrugs and polymer-drug conjugates can improve the efficacy of drugs in therapeutic applications. Polymeric conjugate drugs generally exhibit long half-lives, high stability, water solubility, low immunogenicity and antigenicity, and specific targeting to tissues or cells. Polymers are used as carriers of polymeric / macromolecular prodrugs to deliver drugs, proteins, targeting moieties, and imaging agents. Polymeric prodrugs can be considered as therapeutically active drug delivery systems because they release smaller therapeutic drug molecules from polymer chain molecules over a prolonged period, resulting in improved pharmacokinetic behavior due to extended half-life, increased bioavailability, and consequently, prolonged pharmacological action.

[0007] In an attempt to address concerns regarding the toxicity and poor PK properties of IL-2, certain conjugates of IL-2 have been proposed (see, e.g., U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, 4,902,502, 9,861,705, and WO2019 / 028419).

[0008] In addition to extending plasma half-life and reducing immunogenicity, PEGylation offers the opportunity to control protein binding selectivity. As an example, NKTR-214, a PEGylated IL-2 clinical candidate, exhibits reduced binding to the IL-2 receptor α-subunit (IL-2Rα) through site-specific PEGylation of lysine residues at the IL-2-IL-2Rα interface using a releasable linker. Binding to the IL-2 receptor β-subunit (IL-2Rβ) is only minimally affected. As a result, in preclinical evaluations, NKTR-214 can increase the proliferation of CD8+ tumor-killing memory effector T cells and decrease the proliferation of immunosuppressive regulatory T cells, improving antitumor efficacy compared to IL-2. See, e.g., US 9,861,705, Clin. Cancer Res. 22, 680-690 (2016); PLOS ONE 12, e0179431 (2017).

[0009] The selection of linker chemistry is important in the design of polymer-drug conjugate therapeutics because it provides spatiotemporal control over the cleavage and subsequent release of the active agent. Insufficient linker stability can lead to premature release of the conjugated drug, eliminating the benefits of the macromolecular carrier. On the other hand, inactive polymeric prodrugs can result in insufficient drug release, leading to subtherapeutic drug levels and, consequently, suboptimal therapeutic efficacy. Therefore, a sustained drug release profile that provides long-term therapeutic efficacy is highly desirable.

[0010] Some prodrug molecules release active drugs under physiological conditions by pH-dependent beta-elimination. This approach utilizes the spontaneous first-order cleavage rate of the drug from the PEG carrier, which is initiated when the conjugate is exposed to physiological pH, and the cleavage rate is predetermined by the acidity of the C-H bond on the linker, which is controlled by the electron-withdrawing group attached to the ionic C-H bond. See, for example, U.S. Patent Nos. 6,504,005, 8,680,315, and WO2004 / 089279.

[0011] Despite its widespread use, a major limitation of PEG and its subsequent practical utility in therapeutics is its non-biodegradability. Currently, approved PEGylated protein therapeutics employ PEG with molecular weights of 40 kDa or less, which is close to the glomerular filtration threshold of approximately 50 kDa. While increasing molecular weight generally extends circulation time, concerns about the accumulation of non-biodegradable PEG limit optimization of the polymer's molecular weight and the resulting pharmacokinetics. Summary of the Invention

[0012] Described herein are general designs for protein-[macromolecule]z conjugates containing multiple linkers. The unique linkers of the present disclosure enable the construction of drug conjugates with predictable and tunable release kinetics. In addition, the molecular weight of each macromolecule can be controlled within a mass desirable for renal clearance, in some embodiments, less than 40-50 kDa. Increasing the number of macromolecules (z) on the protein can increase the total molecular weight of the conjugate and thus extend the circulation time of the conjugate. In addition to using tunable electron-withdrawing groups on the releasable linker, varying the number of macromolecules (z) on the protein can further control and optimize the release rate of the active protein.

[0013] Generally, it is difficult and inefficient to conjugate multiple macromolecules to one protein. The present inventors have envisioned a general approach in which a protein is conjugated with multiple bifunctional linkers, and then the linkers are reacted with the macromolecule to obtain a protein-[macromolecule]z conjugate. This technique offers the advantage of minimizing steric hindrance, thereby improving reaction efficiency. Furthermore, the synthesis and purification steps are simplified and less expensive. Therefore, this technique offers great advantages for the large-scale production and manufacturing of macromolecular-protein therapeutics.

[0014] The present disclosure describes this general strategy for providing protein-[macromolecule]z conjugates with releasable linkers that have predictable and controllable release rates. These conjugates with controllable release rates can provide useful therapeutic tools for treating diseases. In some embodiments, the present disclosure describes protein-[macromolecule]z conjugates with non-releasable and releasable linkers. Accordingly, embodiments of the present disclosure relate to methods for preparing such conjugates, compositions comprising the conjugates, and methods of use thereof, which are novel and not previously proposed in the art.

[0015] Thus, in one or more embodiments of the present disclosure, the present disclosure relates to a conjugation method for preparing a conjugate having a protein with a related biological function and multiple macromolecules connected by a linker. In some embodiments, the conjugation method involves functionalizing the protein with a bifunctional linker and subsequently conjugating it to the macromolecule. In some embodiments, the protein includes, but is not limited to, a cytokine, a chemokine, an antibody, and a peptide. In some embodiments, the macromolecule includes, but is not limited to, a water-soluble polymer, PEG, a lipid, polysialic acid, albumin, and Fc.

[0016] The present disclosure relates to novel bifunctional releasable linkers and their compositions, their use in therapeutic applications, and methods for their preparation. Among the advantages of the disclosed technology is the ability to efficiently functionalize proteins with multiple bifunctional releasable linkers provided herein. Conjugation to macromolecules can then be used to improve the pharmacokinetic properties of highly functionalized proteins.

[0017] In one or more embodiments of the present disclosure, a conjugate is provided, the conjugate comprising a residue of an IL-2 moiety covalently attached to one or more water-soluble polymers via a releasable linker.

[0018] In one or more embodiments of the present disclosure, a conjugate is provided, the conjugate comprising a residue of an IL-2 moiety covalently attached to one or more water-soluble polymers via a non-releasable linker.

[0019] In one or more embodiments of the present disclosure, a conjugate is provided, the conjugate comprising a residue of an IL-2 moiety covalently attached to one or more water-soluble polymers via a non-releasable linker and a releasable linker.

[0020] In one or more embodiments of the present disclosure, a method for delivering a conjugate is provided, the method comprising administering intravenously or subcutaneously to a patient a composition comprising a conjugate of a residue of IL-2 and a water-soluble polymer.

[0021] In one or more embodiments of the present disclosure, a method for delivering a conjugate is provided, the method comprising administering to a cancer patient (a) a composition comprising a conjugate of a residue of IL-2 and one or more water-soluble polymers, and (b) an effective amount of an anti-CTLA-4 antibody or an effective amount of an anti-PD-1 / PD-L1 antibody. In some embodiments, the effective amount of the anti-CTLA-4 antibody is an amount that inhibits the CTLA-4 pathway. In some embodiments, the effective amount of the anti-PD-1 / PD-L1 antibody is an amount that inhibits the PD-1 / PD-L1 pathway. For clarity, with respect to the order of steps according to the method, unless otherwise specified, the method is not limited in order of steps, and step (a) can be performed before, after, or simultaneously with step (b).

[0022] The present disclosure provides protein-macromolecule conjugates, releasable linkers, and macromolecules, as defined herein. The disclosed conjugates offer unique properties based at least on the nature of the linker and the number of linker-macromolecule moieties. Also provided herein are unique methods for the synthesis and use of the conjugates in the treatment of diseases and disorders.

[0023] Additional embodiments of the present disclosure are described in the following detailed description and claims. [Brief explanation of the drawings]

[0024] [Figure 1] The nucleotide and amino acid sequences of rIL-2 (SEQ ID NOs: 1 to 3) are shown. [Figure 2]1 shows the distribution of IL-2-(N3)z determined by LC-MS for Examples 14, 16, 18, and 22. [Figure 3] For Examples 15, 17, 19, and 22, SDS-PAGE (Tris acetate) analysis of the click-PEGylated product rIL-2-(PEG)z is shown. [Figure 4A] 1 shows dose-response curves comparing CTLL-2 cell proliferation assays of IL-2, unreleased conjugate, and released conjugate in Example 15. Y-axis is labeled A450-A630. [Figure 4B] 1 shows dose-response curves comparing CTLL-2 cell proliferation assays of IL-2, unreleased conjugate, and released conjugate in Example 17. Y-axis is labeled A450-A630. [Figure 4C] 1 shows dose-response curves comparing CTLL-2 cell proliferation assays of IL-2, unreleased conjugate, and released conjugate in Example 19. Y-axis is labeled A450-A630. [Figure 4D] 1 shows dose-response curves comparing CTLL-2 cell proliferation assays of IL-2, unreleased conjugate, and released conjugate in Example 22. Y-axis is labeled A450-A630. [Figure 4E] 1 shows dose-response curves comparing CTLL-2 cell proliferation assays of IL-2, unreleased conjugate, and released conjugate in Example 27. Y-axis is labeled A450-A630. [Figure 5] Figure 1 shows tumor growth inhibition after administration of rIL-2 and rIL-2-polymer conjugates in different administration schemes. [Figure 6] Figure 1 shows tumor growth inhibition after administration of rIL-2 and rIL-2-polymer conjugates in different administration schemes. [Figure 7] Figure 1 shows tumor growth inhibition after administration of rIL-2 and rIL-2-polymer conjugates in different administration schemes. [Figure 8] Figure 1 shows tumor growth inhibition after administration of rIL-2 and rIL-2-polymer conjugates in different administration schemes. [Figure 9] Figure 1 shows tumor growth inhibition after administration of rIL-2 and rIL-2-polymer conjugates in different administration schemes. DETAILED DESCRIPTION OF THE INVENTION

[0025] Definition: In describing and claiming one or more embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.

[0026] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, representative methods and materials are described herein below.

[0028] Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a carrier" includes mixtures of one or more carriers, two or more carriers, etc.

[0029] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations and may vary depending upon the desired properties sought to be obtained in the application.

[0030] The term "compound(s) of the present disclosure" or "compound(s) of the present disclosure" refers to a compound of the formula disclosed herein or any subgenus thereof, or a pharmaceutically acceptable salt, stereoisomer, solvate, or hydrate thereof disclosed herein. In certain embodiments, intermediates are contemplated as compounds of the present disclosure.

[0031] The compounds of the present disclosure, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomers, which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or, in the case of amino acids, (D)- or (L)-. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms, whether or not specifically indicated herein. Optically active (+)- and (-), (R)- and (S)-, or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomeric forms.

[0032] "Stereoisomers" refer to compounds consisting of the same atoms connected by the same bonds, but having different three-dimensional structures and are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof. In some embodiments, "stereoisomer," as used herein, refers to an enantiomer, a mixture of enantiomers, a diastereomer, or a mixture of two or more diastereomers.

[0033] "Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of one another. A mixture of such isomers can be called an enantiomeric mixture.

[0034] A 50:50 mixture of enantiomers, also referred to as a racemic mixture or racemate, may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity. The present disclosure includes all stereoisomers of the compounds described herein.

[0035] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers differ in physical properties, such as melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography.

[0036] The term "regioisomer" is art-recognized and refers to compounds that have the same molecular formula but differ in the degree of bonding of the atoms. Thus, a "regioselective process" is one in which the formation of a particular regioisomer is favored over other regioisomers, e.g., the reaction significantly increases the yield of a particular regioisomer. As used herein, "regioisomer" can refer to a single regioisomer or a mixture of two or more regioisomers.

[0037] "Tautomer" refers to a migration of a proton from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any such compounds.

[0038] As used herein, the term "drug combination," "therapeutic combination," or "combination" refers to a single dosage form containing at least two therapeutically active agents, or separate dosage forms containing at least two therapeutically active agents together or separately for use in combination therapy.For example, one therapeutically active agent can be formulated into one dosage form, and the other therapeutically active agent can be formulated into a single or different dosage form.For example, one therapeutically active agent can be formulated into a solid oral dosage form, while the second therapeutically active agent can be formulated into a solution dosage form for parenteral administration.

[0039] The chemical naming protocols and structural diagrams used herein are modified IUPAC nomenclature using the ACD / Name Version 9.07 software program, ChemDraw Ultra Version 11.0.1, and / or ChemDraw Ultra Version 14.0 software naming program (CambridgeSoft). For example, in complex chemical names used herein, a substituent is named before the group to which it is attached. For example, cyclopropylethyl comprises an ethyl skeleton with a cyclopropyl substituent. Except as noted below, all bonds are depicted in the chemical structural diagrams herein, but it is assumed that some carbon atoms are attached to sufficient hydrogen atoms to satisfy valences.

[0040] The term "composition" or "formulation" refers to one or more substances in a physical form such as a solid, liquid, gas, or mixtures thereof. One example of a composition is a pharmaceutical composition, i.e., a composition related to, prepared for, or used for a medical treatment.

[0041] As used herein, "pharmaceutically acceptable" means suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, and the like, and is effective for its intended use at a reasonable benefit / risk ratio and within the scope of sound medical judgment.

[0042] "Salts" include derivatives of active agents, in which the active agent is modified by making an acid or base addition salt. Preferably, the salt is a pharmaceutically acceptable salt. Such salts include, but are not limited to, pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, and the like. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, sulfate ester, nitrate ester, phosphate ester, perchlorate ester, borate ester, acetate ester, benzoate ester, hydroxynaphthoate ester, glycerophosphate ester, ketoglutaric acid ester, and the like. Base addition salts include, but are not limited to, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris-(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids such as lysine and arginine dicyclohexylamine, etc. Examples of metal salts include lithium, sodium, potassium, magnesium salts, etc.Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts, etc. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline, etc. Standard methods for preparing pharmaceutically acceptable salts and their formulations are well known in the art and are disclosed in various references, including, for example, "Remington's Preparations: The Science and Practice of Pharmacy," A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, PA.

[0043] As used herein, "solvate" refers to a complex formed by solvation (a combination of a solvent molecule and a molecule or ion of an active agent of the present disclosure) or an aggregate consisting of one or more solvent molecules and a solute ion or molecule (an active agent of the present disclosure). In the present disclosure, a preferred solvate is a hydrate. Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, hexahydrate, etc. One of ordinary skill in the art will understand that pharmaceutically acceptable salts of the compounds of the present invention may also exist in solvated form. Solvates are typically formed through hydration as part of the preparation of the compounds or through the natural absorption of water by anhydrous compounds of the present disclosure. Solvates, including hydrates, may be composed of stoichiometric ratios, e.g., 2, 3, or 4 salt molecules per solvate or hydrate molecule. Another possibility is, for example, two salt molecules stoichiometrically associated with three, five, or seven solvent or hydrate molecules. Solvents used in crystallization, such as alcohols, especially methanol and ethanol, aldehydes, ketones, especially acetone, esters, such as ethyl acetate, etc., may be embedded within the crystal lattice. Preferred are pharmaceutically acceptable solvents.

[0044] The terms "excipient," "carrier," and "vehicle" are used interchangeably throughout this application to refer to a substance with which a compound of the present disclosure is administered.

[0045] "Therapeutically effective amount" means the amount of a compound or therapeutically active agent that, when administered to a patient for treating a disease or other undesirable medical condition, is sufficient to have a beneficial effect with respect to the disease or condition. The therapeutically effective amount may vary depending on the type of compound or therapeutically active agent selected, the disease or condition and its severity, and the age, weight, etc., of the patient being treated. Determining the therapeutically effective amount of a given compound or therapeutically active agent is within the ordinary skill of one in the art and requires no more than routine experimentation.

[0046] As used herein, "treating" or "treatment" encompasses the treatment of a disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes preventing the disease or condition from occurring in the mammal, particularly when the mammal is predisposed to but has not yet been diagnosed with the condition; inhibiting the disease or condition, i.e., preventing its onset; alleviating the disease or condition, i.e., causing the disease or condition to regress; or alleviating the symptoms resulting from the disease or condition, i.e., alleviating pain without addressing the underlying disease or condition.

[0047] As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular illness or condition may not have a known causative agent (i.e., the etiology has not yet been elucidated) and therefore is not yet recognized as a disease, but only as an undesirable state or syndrome in which a more or less specific set of symptoms has been identified by clinicians.

[0048] The present disclosure is also intended to encompass in vivo metabolic products of the compounds of the present disclosure. Such products may result primarily from enzymatic processes, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound. Thus, the present disclosure includes compounds produced by a process comprising administering a compound of the present disclosure to a mammal for a period of time sufficient to produce a metabolic product thereof. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the present disclosure to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur, and isolating the conversion product from urine, blood, or other biological sample.

[0049] As used herein, a "subject" may be a human, non-human primate, mammal, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The terms "subject" and "patient" are used interchangeably herein in reference to a mammalian subject, such as, for example, a human subject.

[0050] The subject may be suspected of or at risk of having cancer, such as prostate cancer, breast cancer, ovarian cancer, salivary gland cancer, or endometrial cancer, or may be suspected of or at risk of having acne, hirsutism, alopecia, benign prostatic hyperplasia, ovarian cysts, polycystic ovarian disease, precocious puberty, spinal and bulbar muscular atrophy, or age-related macular degeneration. Methods for diagnosing various cancers, such as prostate cancer, breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, salivary gland cancer, or endometrial cancer, and methods for diagnosing acne, hirsutism, alopecia, benign prostatic hyperplasia, ovarian cysts, polycystic ovarian disease, precocious puberty, spinal and bulbar muscular atrophy, or age-related macular degeneration, as well as clinical profiles of cancers, such as prostate cancer, breast cancer, ovarian cancer, bladder cancer, pancreatic cancer, hepatocellular carcinoma, salivary gland cancer, or endometrial cancer, and diagnostic and clinical profiles of acne, hirsutism, alopecia, benign prostatic hyperplasia, ovarian cysts, polycystic ovarian disease, precocious puberty, spinal and bulbar muscular atrophy, or age-related macular degeneration, are known to those skilled in the art.

[0051] "Mammal" includes humans and both domestic animals, such as laboratory animals and household pets (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domestic animals, such as wild animals.

[0052] "Optionally" or "optionally" means that the subsequently described circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where the event or circumstance does not occur. For example, "optionally substituted aryl" means that the aryl radical may or may not be substituted, and that the description includes both substituted and unsubstituted aryl radicals.

[0053] As used herein, "PEG," "polyethylene glycol," and "poly(ethylene glycol)" are synonymous and include any non-peptidic, water-soluble poly(ethylene oxide). Typically, PEG used in accordance with the present disclosure has the following structure: "-(OCHCH) n -" (where (n) is 2 to 4000). As used herein, PEG also includes "-CH2CH2-O(CH2CHO)" depending on whether the terminal oxygen has been substituted, for example, during synthetic transformations. n -CH2CH2-" and "-(OCH2CH2) n It should be noted that throughout this specification and claims, the term "PEG" includes structures having various terminal or "end-cap" groups, etc. The term "PEG" also refers to a polymer that contains a majority, i.e., greater than 50%, of -OCH2CH2- repeating subunits. With regard to specific forms, PEG can take any number of different molecular weights and structures or geometries, such as "branched," "linear," "forked," "multifunctional," etc., as described in more detail below.

[0054] The terms "end-capped" and "terminally capped" are used interchangeably herein to refer to a polymer having an end-cap moiety at its terminus or end. Typically, but not necessarily, the end-cap moiety is a hydroxy or C 1-20 Alkoxy groups, more preferably C 1-10 Alkoxy groups, even more preferably C 1-5 and alkoxy groups. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy, and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. Note that the end-capping moiety can include one or more atoms of the terminal monomer in the polymer [e.g., CHO(CHCHO)]. n - and CH3(OCH2CH2) n-"methoxy" end-capping moiety in the formula (I). Additionally, saturated, unsaturated, substituted, and unsubstituted forms of each of the foregoing are contemplated. Furthermore, the end-capping group may be a silane. The end-capping group may also advantageously comprise a detectable label. When a polymer has an end-capping group containing a detectable label, the amount or location of the polymer and / or the moiety (e.g., active agent) to which it is attached can be determined using a suitable detector. Such labels include, but are not limited to, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors include photometers, films, spectrometers, and the like. The end-capping group may also advantageously comprise a phospholipid. When a polymer has an end-capping group containing a phospholipid, unique properties are imparted to the polymer and the resulting conjugate. Exemplary phospholipids include, but are not limited to, those selected from the class of phospholipids known as phosphatidylcholines. Specific phospholipids include, but are not limited to, those selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin. The end-capping group may also include a targeting moiety so that the polymer (and anything attached to the polymer, e.g., the IL-2 moiety) can be preferentially localized to the area of ​​interest.

[0055] With respect to the polymers described herein, "non-natural" means a polymer that does not exist in nature in its intact state. However, a non-natural polymer may contain one or more monomers or segments of monomers that are natural, so long as the entire polymer structure does not occur in nature.

[0056] The term "water-soluble" polymer, as in "water-soluble polymer," refers to any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer transmits at least about 75%, more preferably at least about 95%, of the light transmitted by the same solution after filtration. The water-soluble polymer is preferably at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, even more preferably about 70% (by weight) soluble in water, and even more preferably about 85% (by weight) soluble in water. However, most preferably, the water-soluble polymer is about 95% (by weight) soluble in water, or completely soluble in water.

[0057] Molecular weight in the context of water-soluble polymers such as PEG can be expressed as either number-average molecular weight or weight-average molecular weight. Unless otherwise specified, all references to molecular weight herein refer to weight-average molecular weight. Both number-average and weight-average molecular weight measurements can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods can also be used to measure molecular weight values, such as end-group analysis or measurements of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine number-average molecular weight, or light scattering, ultracentrifugation, or viscometry to determine weight-average molecular weight. The polymers of the present invention are typically polydisperse (i.e., the number-average molecular weight and weight-average molecular weight of the polymer are not equal) and preferably have a low polydispersity value of less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

[0058] The terms "active," "reactive," or "activated," when used with certain functional groups, refer to a reactive functional group that readily reacts with an electrophile or nucleophile on another molecule. This is in contrast to groups that require strong catalysts or extremely impractical reaction conditions in order to react (i.e., "nonreactive" or "inert" groups).

[0059] As used herein, the term "functional group" or any synonym thereof is intended to encompass protected and unprotected forms thereof.

[0060] As used herein, the term "electron modifying group" is meant to include any atom or functional group that alters the electron density of the moiety to which it is attached. Electron modifying groups include electron donating groups (e.g., amine, hydroxy, alkoxyl, alkyl) that provide electron density and electron withdrawing groups (e.g., nitro, cyano, trifluoromethyl) that withdraw electron density.

[0061] The terms "spacer moiety," "linkage," and "linker" are used herein to refer to a bond or an atom or collection of atoms, optionally used to connect the ends of a macromolecule segment and an interconnecting moiety, such as a protein or an electrophile or nucleophile of a protein. A spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly indicates otherwise, a spacer moiety is optionally present between any two elements of a compound (e.g., provided conjugates comprising a residue of a protein and a macromolecule may be linked directly or indirectly via a spacer moiety).

[0062] Suitable spacers of the present disclosure include spacers comprising linkers that may include one or more of carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof. Suitable spacer moieties may include amides, secondary amines, carbamates, thioethers, phosphates, phosphorothioates, disulfide groups, and / or click chemistry product groups. Non-limiting examples of specific spacer moieties include -O-, -S-, -SS-, -C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -OC(O)-NH-, -OP(O)(OH)-, -OP(S)(OH)-, -C(S)-, -CH-, -CH-CH-, -CH-CH-CH-, -CH-CH-CH-CH-, -CH-CH-CH-CH-CH-, -CH-CH-CH-CH-CH-, O-CH-, -CH-O-, -O-CH-CH-, -C H2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2-CH2-O-, -O-CH2-CH2-CH2-CH2 -, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-CH2-CH2-O-, -C(O)-NH-CH2-, -C(O)-NH -CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)- NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-C H2-, -CH2-CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -C(O)-O-CH2-, -CH2- C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2-, -C(O)-O-CH2-CH2-, -NH-C(O)-CH2-, -CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-,-NH-C(O)-CH2-CH2-, -CH2-NH-C(O)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -OC(O)-NH-CH2-, - OC(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-NH-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH 2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH 2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-, -OC(O)-NH-[CH2], l -(OCH2CH2) m -, divalent cycloalkyl group, divalent aryl, -O-, -S-, divalent amino acid residue, -N(R 3 )—and combinations of any two or more of the foregoing (wherein R 3 is H or an organic radical selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, and substituted or unsubstituted aryl; (l) is 0 to 6; and (m) is 0 to 20. Other specific spacer moieties include those selected from the group consisting of: -C(O)-NH-(CH2) 1-6 -NH-C(O)-, -NH-C(O)-NH-(CH2) 1-6 -NH-C(O)- and -OC(O)-NH-(CH2) 1-6 -NH-C(O)-, where the subscript value after each methylene indicates the number of methylenes contained in the structure, e.g., (CH) 1-6 means that the structure can contain 1, 2, 3, 4, 5, or 6 methylenes.

[0063] The term "bifunctional linker" refers to a linker as defined above that has two reactive atoms or functional groups. In certain embodiments, the two reactive groups are orthogonal functional groups with different modes of reactivity, so that each functional group can react in a specific order independently of the other, as desired. As will be understood by those skilled in the art, the bifunctional linkers disclosed herein can be used to carry out site-specific reactions for assembling protein-macromolecule conjugates.

[0064] "Acyl" refers to a -C(=O)-alkyl radical.

[0065] "Amino" refers to the -NH2 radical.

[0066] "Cyano" refers to the -CN radical.

[0067] "Halo," "halide," or "halogen" refers to a bromo, chloro, fluoro, or iodo radical.

[0068] "Hydroxy" or "hydroxyl" refers to the --OH radical.

[0069] "Imino" refers to the =NH substituent.

[0070] "Nitro" refers to the -NO2 radical.

[0071] "Oxo" refers to the =O substituent.

[0072] "Thioxo" is the =S substituent.

[0073] "Sulfhydryl" and "mercapto" refer to the --SH radical.

[0074] Hydrogen is H or D.

[0075] "Alkyl" or "alkyl group" refers to a fully saturated, straight (linear) or branched hydrocarbon chain radical having from 1 to 20 carbon atoms attached to the rest of the molecule by a single bond. Alkyl groups containing any number of carbon atoms from 1 to 20 are included. Alkyl groups containing up to 20 carbon atoms include C1-C 20 Alkyl, containing up to 10 carbon atoms, is C1-C 10 An alkyl having up to 6 carbon atoms is a C1-C6 alkyl, and an alkyl having up to 5 carbon atoms is a C1-C5 alkyl. C1-C5 alkyl includes C5 alkyl, C4 alkyl, C3 alkyl, C2 alkyl, and C1 alkyl (i.e., methyl). C1-C6 alkyl includes all of the moieties described above for C1-C5 alkyl, including C6 alkyl. C1-C 10 Alkyl includes all of the moieties described above for C1-C5 alkyl and C1-C6 alkyl, including C7, C8, C9 and C 10 Also includes alkyl. Similarly, C1-C 12 Alkyl includes all of the above moieties, including C 11 and C 12 Includes alkyl. C1-C 12 Non-limiting examples of alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless otherwise specified herein, alkyl groups can be optionally substituted. The term "lower alkyl" refers to C1-C6 alkyl, which can be linear or branched, including, for example, branched C3-C6 alkyl. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl" includes cycloalkyl as well as cycloalkylene-containing alkyl.

[0076] "Alkylene", "-alkyl-" or "alkylene chain" refers to a divalent, straight or branched, fully saturated hydrocarbon chain radical having from 1 to 20 carbon atoms. C1-C 20 Non-limiting examples of alkylene include methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted.

[0077] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain radical having 2 to 20 carbon atoms and one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl groups containing any number of carbon atoms from 2 to 20 are included. Alkenyl groups containing up to 20 carbon atoms include C2-C 20 Alkenyl containing up to 10 carbon atoms is C2-C 10 An alkenyl group containing up to 6 carbon atoms is C2-C6 alkenyl, and an alkenyl containing up to 5 carbon atoms is C2-C5 alkenyl. C2-C5 alkenyl includes C5 alkenyl, C4 alkenyl, C3 alkenyl, and C2 alkenyl. C2-C6 alkenyl includes all of the moieties described above for C2-C5 alkenyl, including C6 alkenyl. C2-C 10 Alkenyl includes all of the moieties described above for C2-C5 alkenyl and C2-C6 alkenyl, including C7, C8, C9 and C 10 Also includes alkenyl. Similarly, C2-C 12 Alkenyl includes all of the above moieties, C 11 and C 12 Includes alkenyl. C2-C 12Non-limiting examples of alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, and 3-nonenyl. , 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, and 11-dodecenyl. Unless stated otherwise in the specification, an alkyl group may be optionally substituted.

[0078] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain radical having 2 to 20 carbon atoms and having one or more carbon-carbon double bonds. 20 Non-limiting examples of alkenylenes include ethene, propene, butene, etc. The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkenylene chain can be optionally substituted.

[0079] "Alkynyl" or "alkynyl group" refers to a straight or branched hydrocarbon chain radical having from 2 to 20 carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl groups containing any number of carbon atoms from 2 to 20 are included. Alkynyl groups containing up to 20 carbon atoms include C2-C 20 Alkynyl containing up to 10 carbon atoms is C2-C 10 An alkynyl group containing up to 6 carbon atoms is C2-C6 alkynyl, and an alkynyl containing up to 5 carbon atoms is C2-C5 alkynyl. C2-C5 alkynyl includes C5 alkynyl, C4 alkynyl, C3 alkynyl, and C2 alkynyl. C2-C6 alkynyl includes all of the moieties described above for C2-C5 alkynyl, including C6 alkynyl. C2-C 10 Alkynyl includes all of the moieties described above for C2-C5 alkynyl and C2-C6 alkynyl, including C7, C8, C9 and C 10 Also includes alkynyl. Similarly, C2-C 12 Alkynyl includes all of the above moieties, C 11 and C 12 Alkynyl is also included. C2-C 12 Non-limiting examples of alkenyl include ethynyl, propynyl, butynyl, pentynyl, etc. Unless stated otherwise in the specification, an alkyl group can be optionally substituted.

[0080] "Alkynylene" or "alkynylene chain" refers to a straight or branched divalent hydrocarbon chain radical having 2 to 20 carbon atoms and one or more carbon-carbon triple bonds. 20 Non-limiting examples of alkynylene include ethynylene, propargylene, and the like. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkynylene chain can be optionally substituted.

[0081] "Alkoxy" or "-O-alkyl" refers to an alkyl group of the formula -OR a where R a is an alkyl, alkenyl, or alkynyl radical as defined above containing 1 to 20 carbon atoms. Unless stated otherwise in the specification, an alkoxy group can be optionally substituted.

[0082] "Alkylamino" refers to a group of the formula -NHR a or -NR a R a where each R a is independently an alkyl, alkenyl, or alkynyl radical as defined above containing 1 to 20 carbon atoms. Unless stated otherwise in the specification, an alkylamino group can be optionally substituted.

[0083] "Alkylcarbonyl" is -C(=O)R a refers to the part, where R a is an alkyl, alkenyl, or alkynyl radical as defined above. A non-limiting example of an alkylcarbonyl is a methylcarbonyl ("acetal") moiety. An alkylcarbonyl group may also be referred to as a "Cw-Cz acyl," where w and z are R as defined above. a Indicates the range of carbon numbers in the molecule. For example, "C1-C 10 "Acyl" refers to an alkylcarbonyl group as defined above, where R a is C1-C as defined above 10 Alkyl, C1-C 10 Alkenyl, or C1-C 10 Unless stated otherwise in the specification, an alkylcarbonyl group may be optionally substituted.

[0084] The term "aminoalkyl" refers to an alkyl group substituted with one or more -NH groups. In certain embodiments, the aminoalkyl group is substituted with 1, 2, 3, 4, 5, or more -NH groups. The aminoalkyl group can be optionally substituted with one or more additional substituents described herein.

[0085] "Aryl" refers to a hydrocarbon ring system radical containing hydrogen, 6 to 18 carbon atoms, and at least one aromatic ring. For purposes of this disclosure, aryl radicals can be monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, which may include fused or bridged ring systems. Aryl radicals include, but are not limited to, aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specified herein, the term "aryl" is meant to include optionally substituted aryl radicals. Aryl can include multiple aryl rings, which may be fused, such as naphthyl, or non-fused, such as biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[0086] An "aralkyl", "arylalkyl" or "-alkylaryl" is an alkyl group of the formula -R b -R c where R b is an alkylene, alkenylene, or alkynylene group as defined above, and R c is one or more aryl radicals as defined above, e.g., benzyl, diphenylmethyl, etc. Unless stated otherwise in the specification, an aralkyl group may be optionally substituted.

[0087] "Alkoxy" refers to the group -OR, where R is alkyl or substituted alkyl, preferably C 1-6 alkyl (for example, methoxy, ethoxy, propyloxy, etc.);

[0088] "Carbocyclyl," "carbocyclic ring," or "carbocycle" refers to a ring structure in which each atom forming the ring is carbon. A carbocyclic ring can contain from 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryl and cycloalkyl. Cycloalkenyl and cycloalkynyl are defined herein. Unless stated otherwise in the specification, a carbocyclyl group can be optionally substituted.

[0089] "Cycloalkyl" refers to a stable, non-aromatic, monocyclic or polycyclic, fully saturated hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from 3 to 20 carbon atoms, preferably from 3 to about 12 carbon atoms, and more preferably from 3 to about 8 carbon atoms, attached to the remainder of the molecule by a single bond. Monocyclic cycloalkyl radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, bicyclo[3.1.0]hexane, octahydropentalene, bicyclo[1.1.1]pentane, cubane, and the like. Unless stated otherwise in the specification, cycloalkyl groups can be optionally substituted. "Cycloalkylene" refers to a cycloalkyl group inserted into an alkyl chain, with the chain attached at any two carbons of the cyclic ring system.

[0090] "Cycloalkenyl" refers to a stable, non-aromatic, monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon double bonds, which may include fused or bridged ring systems, having 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms, and attached to the remainder of the molecule by a single bond. Monocyclic cycloalkenyl radicals include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Polycyclic cycloalkenyl radicals include, for example, bicyclo[2.2.1]hept-2-enyl, and the like. Unless stated otherwise in the specification, cycloalkenyl groups can be optionally substituted.

[0091] "Cycloalkynyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon triple bonds, which may include fused or bridged ring systems, having from 3 to 20 carbon atoms, preferably from 3 to 10 carbon atoms, and attached to the remainder of the molecule by a single bond. Monocyclic cycloalkynyl radicals include, for example, cycloheptynyl, cyclooctynyl, and the like. Unless stated otherwise in the specification, a cycloalkynyl group can be optionally substituted.

[0092] A "cycloalkylalkyl" or "-alkylcycloalkyl" is a group of the formula -R b -R d where R b is an alkylene, alkenylene, or alkynylene group as defined above, and R d is a cycloalkyl, cycloalkenyl, cycloalkynyl radical as defined above. Unless stated otherwise in the specification, a cycloalkylalkyl group may be optionally substituted.

[0093] "Haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one, two, three, four, five, six, or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. Unless stated otherwise in the specification, a haloalkyl group can be optionally substituted.

[0094] "Haloalkenyl" refers to an alkenyl radical, as defined above, that is substituted by one, two, three, four, five, six, or more halo radicals, as defined above, e.g., 1-fluoropropenyl, 1,1-difluorobutenyl, etc. Unless stated otherwise in the specification, a haloalkenyl group can be optionally substituted.

[0095] "Haloalkynyl" refers to an alkynyl radical, as defined above, that is substituted with one, two, three, four, five, six, or more halo radicals, as defined above, e.g., 1-fluoropropynyl, 1-fluorobutynyl, etc. Unless stated otherwise in the specification, a haloalkenyl group can be optionally substituted.

[0096] The term "substituted," as in, for example, "substituted alkyl," refers to a moiety (e.g., alkyl group) substituted with one or more non-interfering substituents, including, but not limited to, alkyl, C 3-8 Cycloalkyl, such as cyclopropyl, cyclobutyl, and the like; halo, such as fluoro, chloro, bromo, and iodo; cyano; nitro; alkoxy, lower phenyl; substituted phenyl, and the like. "Substituted aryl" is an aryl having one or more non-interfering groups as substituents. In the case of substitution on the phenyl ring, the substituents can be in any orientation (i.e., ortho, meta, or para).

[0097] "Non-interfering substituents," when present in a molecule, are typically non-reactive with other functional groups contained within the molecule. Non-limiting examples include halogen (F, Br, Cl, I), alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, pentyl, neopentyl, hexyl, isoamyl, etc.), haloalkyl (e.g., CF, CHF, CHF, etc.), cycloalkyl (cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.), alkoxy (-OR), haloalkoxy (e.g., -OCF, -OCHF, -OCHF, etc.), amino, (e.g., —N(H)alkyl, —N(alkyl), —NH(cycloalkyl), —NH(aryl), etc.), amido (e.g., —NH(COR), sulfonyl (e.g., —SOR), acyl (e.g., —C(O)R, cyano, nitro, phenyl, and heteroaryl (e.g., oxazolyl, thiazolyl, imidazolyl, pyridyl, pyrimidinyl, etc.), where R is independently H, alkyl, alkyoxy, amino, or aryl (e.g., phenyl).

[0098] "Heterocyclyl," "heterocyclic ring," or "heterocycle" refers to a stable 3- to 20-membered non-aromatic ring radical consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms, preferably selected from the group consisting of nitrogen, oxygen, and sulfur. A heterocyclyl or heterocyclic ring includes heteroaryl, as defined below. Unless stated otherwise in the specification, a heterocyclyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems; the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise in the specification, heterocyclyl groups may be optionally substituted. In some embodiments, a "substituted heterocycle" is a heterocycle having one or more side chains formed from non-interfering substituents.

[0099] The term "hydroxyalkyl" or "hydroxylalkyl" refers to an alkyl group substituted with one or more hydroxyl (-OH) groups. In certain embodiments, a hydroxyalkyl group is substituted with 1, 2, 3, 4, 5, or more -OH groups. A hydroxyalkyl group can be optionally substituted with one or more additional substituents described herein.

[0100] The term "hydrocarbyl" refers to a monovalent hydrocarbon radical that is either aliphatic, partially or fully unsaturated, acyclic, cyclic, or aromatic, or any combination of the foregoing. In certain embodiments, the hydrocarbyl group has 1 to 40 or more, 1 to 30 or more, 1 to 20 or more, or 1 to 10 or more carbon atoms. The term "hydrocarbylene" refers to a divalent hydrocarbyl group. The hydrocarbyl or hydrocarbylene group can be optionally substituted with one or more substituents described herein.

[0101] The term "heterohydrocarbyl" refers to a hydrocarbyl group in which one or more of the carbon atoms are each independently replaced by a heteroatom selected from oxygen, sulfur, nitrogen, and phosphorus. In certain embodiments, a heterohydrocarbyl group has 1 to 40 or more, 1 to 30 or more, 1 to 20 or more, or 1 to 10 or more carbon atoms and 1 to 10 or more, or 1 to 5 or more heteroatoms. The term "heterohydrocarbylene" refers to a divalent hydrocarbyl group. Examples of heterohydrocarbyl and heterohydrocarbylene groups include, but are not limited to, ethylene glycol and polyethylene glycol moieties, e.g., (-CHCHO-). n H (monovalent heterohydrocarbyl group) and (-CH2CH2O-) n (divalent heterohydrocarbylene groups) where n is an integer from 1 to 12 or greater, and propylene glycol and polypropylene glycol moieties, such as (—CHCHCHO—) n H and (-CH2CH(CH3)O-) n H (monovalent heterohydrocarbyl group) and (-CH2CH2CH2O-) n and (-CH2CH(CH3)O-) n (divalent heterohydrocarbylene group) where n is an integer from 1 to 12 or greater. The heterohydrocarbyl or heterohydrocarbylene group can be optionally substituted with one or more substituents described herein.

[0102] "N-heterocyclyl" refers to a heterocyclyl radical as defined above containing at least one nitrogen, and the point of attachment of the heterocyclyl radical to the rest of the molecule is through a nitrogen atom in the heterocyclyl radical. Unless stated otherwise in the specification, an N-heterocyclyl group can be optionally substituted.

[0103] A "heterocyclylalkyl" or "-alkylheterocyclyl" is a heterocyclyl group of the formula -R b -R e where R b is an alkylene, alkenylene, or alkynylene chain as defined above, and R e is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to an alkyl, alkenyl, or alkynyl radical at the nitrogen atom. Unless stated otherwise in the specification, a heterocyclylalkyl group may be optionally substituted.

[0104] "Heteroaryl" refers to a 5- to 20-membered ring system radical containing a hydrogen atom, 1 to 13 carbon atoms, preferably 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, and at least one aromatic ring. For purposes of this disclosure, a heteroaryl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems, and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical can be optionally oxidized, and the nitrogen atom can be optionally quaternized. Examples include azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indophenyl, and indophenyl. Examples include, but are not limited to, dolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise in the specification, a heteroaryl group may be optionally substituted.In some embodiments, a "substituted heteroaryl" is a heteroaryl having one or more non-interfering groups as substituents.

[0105] "N-heteroaryl" refers to a heteroaryl radical, as defined above, containing at least one nitrogen, and the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical. Unless stated otherwise in the specification, an N-heteroaryl group can be optionally substituted.

[0106] A "heteroarylalkyl" or "-alkylheteroaryl" is a heteroaryl group of the formula -R b -R f where R b is an alkylene, alkenylene, or alkynylene chain as defined above, and R f is a heteroaryl radical, as defined above. Unless stated otherwise in the specification, a heteroarylalkyl group may be optionally substituted.

[0107] The term "substituted," as used herein, means any of the above groups (i.e., alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, alkoxy, alkylamino, alkylcarbonyl, thioalkyl, aryl, aralkyl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl) in which at least one hydrogen atom is replaced by a bond to a non-hydrogen atom from the list provided herein. If no list of substituents is included, the substituents may be, but are not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl, alkoxy, and ester groups; sulfur atoms in groups such as thiol, thioalkyl, sulfone, sulfonyl, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also refers to any of the above groups in which one or more hydrogen atoms are replaced by heteroatoms such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and higher bonds (e.g., double or triple bonds) to nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" refers to any of the above groups in which one or more hydrogen atoms have been replaced with a halide, cyano, nitro, hydroxyl, sulfhydryl, amino, -OR g , -SR g , -NR h R i, alkyl, alkenyl, alkynyl, haloalkyl, hydroxyalkyl, aminoalkyl, -alkylcycloalkyl, -alkylheterocyclyl, -alkylaryl, -alkylheteroaryl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(=O)R g , -C(=NR j )R g , -S(=O)R g , -S(=O)2R g , -S(=O)2OR k , -C(=O)OR k , -OC(=O)R g , -C(=O)NR h R i , -NR g C(=O)R g , -S(=O)2NR h R i , -NR g S(=O)2R g , -OC(=O)OR g , -OC(=O)NR h R i , -NR g C(=O)OR g , -NR g C(=O)NR h R i , -NR g C(=NR j )NR h R i , -P(=O)(R g )2, -P(=O)(OR k )R g , -P(=O)(OR k )2, -OP(=O)(R g )2, -OP(=O)(OR k )R g , and -OP(=O)(OR k )2(wherein, R g each occurrence of is independently selected from hydrogen, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, -alkylcycloalkyl, -alkylheterocyclyl, -alkylaryl, -alkylheteroaryl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R h and R ieach occurrence of is independently selected from hydrogen, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, -alkylcycloalkyl, -alkylheterocyclyl, -alkylaryl, -alkylheteroaryl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R h and R i together with the nitrogen atom to which they are attached form a heterocyclic or heteroaryl ring; R j Each occurrence of is independently hydrogen, -OR g , alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, -alkylcycloalkyl, -alkylheterocyclyl, -alkylaryl, -alkylheteroaryl, cycloalkyl, heterocyclyl, aryl or heteroaryl; R k each occurrence of is independently hydrogen, W, alkyl, haloalkyl, hydroxyalkyl, aminoalkyl, -alkylcycloalkyl, -alkylheterocyclyl, -alkylaryl, -alkylheteroaryl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; and each occurrence of W is independently H + , Li + , Na + , K. + , Cs + , Mg +2 , Ca +2 ,or- + N(R g )2R h R i This includes those substituted with (wherein

[0108] A "thioalkyl" is a group of the formula -SR a where R a is an alkyl, alkenyl, or alkynyl radical as defined above containing 1 to 12 carbon atoms. Unless stated otherwise in the specification, a thioalkyl group may be optionally substituted.

[0109] As used herein, "organic radical" is intended to include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0110] As used herein, " [ka] The symbol " (which may hereinafter be referred to as a "point of attachment") indicates a bond that is a point of attachment between two chemical entities, one of which is shown as attached to the point of attachment and the other of which is shown as not attached to the point of attachment. For example, " [ka] " indicates that the chemical entity "XY" is attached to another chemical entity through a point of attachment. Furthermore, a particular point of attachment to an unillustrated chemical entity can be identified by inference. For example, R 3 is H or " [ka] " is the compound CH3-R 3 is R 3 If "XY" is the connection point, R 3 is assumed to be the same bond as that shown attached to CH3.

[0111] "Fused" refers to any ring structure described herein that is fused to an existing ring structure in a compound of the present disclosure. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that becomes part of the fused heterocyclyl ring or fused heteroaryl ring can be replaced with a nitrogen atom.

[0112] "Electrophile" and "electrophilic group" refer to an ion or atom or group of atoms, which may be ionic, that has an electrophilic center, i.e., an electron-seeking center, and is capable of reacting with a nucleophile.

[0113] "Nucleophile" and "nucleophilic group" refer to an ion or atom or group of atoms, which may be ionic, that has a nucleophilic center, i.e., a center that seeks or uses an electrophilic center.

[0114] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is one that reacts with water (i.e., is hydrolyzed) under physiological conditions. The susceptibility of a bond to hydrolysis in water can depend not only on the general type of linkage connecting the two central atoms, but also on the substituents attached to those central atoms. Suitable hydrolytically unstable or hydrolytically vulnerable linkages include, but are not limited to, carbamates, carboxylate esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, and oligonucleotides.

[0115] A "releasable linker" refers to a linker that connects a protein and a macromolecule. The macromolecule is released, either through hydrolysis, an enzymatic process, a catalytic process, or otherwise, thereby resulting in an unconjugated protein moiety. In certain embodiments, the releasable linker releases the macromolecule by the aforementioned processes that occur in vivo.

[0116] An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.

[0117] A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a covalent bond, that is substantially stable in water, i.e., does not undergo significant hydrolysis under physiological conditions over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, carbon-carbon bonds (e.g., in aliphatic chains), carbon-sulfur bonds, ethers, amides, urethanes, and the like. Generally, a hydrolytically stable linkage is one that exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. Many standard chemistry textbooks can be consulted for hydrolysis rates of representative chemical bonds.

[0118] A "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the present disclosure and that does not cause any significant toxic effects in patients. The terms "pharmacologically effective amount," "physiologically effective amount," and "therapeutically effective amount" are used interchangeably herein to refer to the amount of protein-macromolecule conjugate required to achieve a desired level of conjugate (or the corresponding unconjugated protein) in the bloodstream or target tissue. The exact amount will depend on numerous factors, such as the particular protein, the components and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, etc., and can be readily determined by one of ordinary skill in the art based on the information provided herein.

[0119] The term "IL-2 moiety," as used herein, refers to a moiety having human IL-2 activity. An IL-2 moiety also has at least one electrophilic or nucleophilic group suitable for reaction with a polymeric reagent. Additionally, the term "IL-2 moiety" encompasses both the IL-2 moiety prior to conjugation and the IL-2 moiety residue after conjugation. As described in further detail below, one of ordinary skill in the art can determine whether any given moiety has IL-2 activity. Proteins containing an amino acid sequence corresponding to the sequence in FIG. 1 and any protein or polypeptide substantially homologous thereto are IL-2 moieties. As used herein, the term "IL-2 moiety" includes such proteins modified intentionally, e.g., by site-directed mutagenesis, or accidentally through mutation. These terms also include analogs having one to six additional glycosylation sites, analogs having at least one additional amino acid at the carboxy terminus of the protein, where the additional amino acid(s) comprises at least one glycosylation site, and analogs having an amino acid sequence that includes at least one glycosylation site. The term includes both naturally occurring and recombinantly produced moieties.

[0120] The term "substantially homologous" means that a particular subject sequence, e.g., a mutant sequence, differs from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is no adverse functional difference between the reference and subject sequences. For purposes of this disclosure, sequences having greater than 80 percent homology (more preferably, greater than 85 percent, even more preferably, greater than 90 percent, and most preferably, greater than 95 percent), equivalent biological activity (although not necessarily, equivalent strength of biological activity), and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncations of the mature sequence shall be disregarded.

[0121] The term "fragment" refers to any protein or polypeptide that has the amino acid sequence of a portion or fragment of an IL-2 moiety and that has the biological activity of IL-2. Fragments include proteins or polypeptides produced by proteolysis of the IL-2 moiety and proteins or polypeptides produced by chemical synthesis using methods conventional in the art.

[0122] The term "patient" refers to a living organism suffering from or susceptible to a condition that can be prevented or treated by administration of an active agent (eg, a conjugate), and includes both humans and animals.

[0123] "Optional" or "optionally" means that the circumstance described below may or may not occur, and thus the description includes cases where the circumstance occurs and cases where the circumstance does not occur.

[0124] "Substantially" means almost entirely or completely, for example, meeting one or more of the following conditions: more than 50%, 51% or more, 75% or more, 80% or more, 90% or more, and 95% or more.

[0125] Amino acid residues in peptides are abbreviated as follows: phenylalanine is Phe or F, leucine is Leu or L, isoleucine is Ie or I, methionine is Met or M, valine is Val or V, serine is Ser or S, proline is Pro or P, threonine is Thr or T, alanine is Ala or A, tyrosine is Tyr or Y, histidine is His or H, glutamine is Gln or Q, asparagine is Asn or N, lysine is Lys or K, aspartic acid is Asp or D, glutamic acid is Glu or E, cysteine ​​is Cys or C, tryptophan is Trp or W, arginine is Arg or R, and glycine is Gly or G.

[0126] The present disclosure includes all pharmaceutically acceptable isotopically labeled compounds of the present disclosure in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from that usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the present disclosure include: 2 H and 3 Isotopes of hydrogen such as H, 11 C. 13 C and 14 carbon isotopes such as C, 36 chlorine isotopes such as Cl, 18 fluorine isotopes such as F, 123 I and 125 isotopes of iodine, such as I, 13 N and 15 nitrogen isotopes such as N, 15 O. 17 O and 18 isotopes of oxygen, such as O 32 Isotopes of phosphorus such as P, and 35 Examples include sulfur isotopes such as S.

[0127] Certain isotopically labeled compounds of the present disclosure, for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. 3 H, and carbon-14, i.e., 14 C is particularly useful for this purpose because of its ease of incorporation and ease of detection means.

[0128] Deuterium, i.e., 2 Substitution with heavier isotopes, such as H, may be preferable in some circumstances because they may confer certain therapeutic advantages due to increased metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0129] 11 C. 18 F, 15 O and 13Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

[0130] Isotopically labeled compounds of the present disclosure may generally be prepared by conventional techniques known to those of ordinary skill in the art.

[0131] The phrase "an enantiomer, mixture of enantiomers, mixture of two or more diastereomers, tautomer, mixture of two or more tautomers, positional isomer, mixture of two or more positional isomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof" means "(i) an enantiomer, mixture of enantiomers, mixture of two or more diastereomers, tautomer, mixture of two or more tautomers, positional isomer, mixture of two or more positional isomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof" (ii) a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of a compound referred to therein; or (iii) an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, a positional isomer, a mixture of two or more positional isomers, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of an isotopic variant of a compound referred to therein. Preparation method

[0132] The present disclosure provides methods for preparing protein-[macromolecule]z conjugates for controlling the rate of delivery of therapeutic protein drugs when administered to a patient in need of therapeutic treatment. The conjugates prepared by the methods of the present disclosure provide a means for delivering therapeutic drugs over a sustained period of time, controlled by the releasability rate of the linker and the number of macromolecules.

[0133] In one aspect, the present disclosure relates to a method for preparing a protein-macromolecule conjugate using Scheme (I): [ka] In the formula, x is an integer of 1 to 25, y is an integer from 0 to 24, z is an integer from 1 to 25, x=y+z, L is a linker, FG 0 is a functional group capable of reacting with a nucleophilic group of an active protein drug to form a linkage, including a carbamate linkage, a thiol bridge, etc. FG 2 via click chemistry 3 functional groups capable of reacting with, for example, but not limited to, azido, alkynyl, and cycloalkynyl groups (e.g., dibenzocyclooctyne (DBCO)); FG 3 via click chemistry 2 functional groups capable of reacting with, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups; the protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide; Cytokines include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, or TNF-β.

[0134] In certain embodiments, the cytokine is IL-2.

[0135] In certain embodiments, the IL-2 comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1.

[0136] Chemokines include MCP-1, MCP-2, MCP-3, MCP-24, MCP-5, CXCL76, I-309 (CCL1), BCA1 (CXCL13), MIG, SDF-1 / PBSF, IP-10, I-TAC, MIP-1α, MIP-1β, RANTES, eotaxin-1, eotaxin-2, GCP-2, Gro-α, Gro-β, Gro-γ, LARC (CCL20), ELC (CCL19), SLC (CCL21), ENA-78, PBP, TECK (CCL25), CTACK (CCL27), MEC, XCL1, XCL2, HCC-1, HCC-2, HCC-3, or HCC-4.

[0137] Antibodies include angiopoietin 2, AXL, ACVR2B, angiopoietin 3, activin receptor-like kinase 1, amyloid A protein, β-amyloid, AOC3, BAFF, BAFF-R, B7-H3, BCMAC, A-125 (mimetic), C5, CA-125, CCL11 (eotaxin-1), CEA, CSF1R, CD2, CD3, CD4, CD6, CD15, CD19, CD20, CD22, CD23, CD25, CD28, and CD3. 0, CD33, CD37, CD38, CD40, CD41, CD44, CD51, CD52, CD54, CD56, CD70, CD74, CD97B, CD125, D134, CD147, CD152, CD154, CD279, CD221, C242 antigen, CD276, CD278, CD319, Clostridium difficile, claudin-18 isoform 2, CSF1R, CEACAM5, CSF2, carbonic anhydrase 9, CLDN18.2, cardiac myosin, CCR4, CGRP, coagulation factor III, c-Met, CTLA-4, DPP4, DR5, DLL3, DLL4, dabigatran, EpCAM, Ebola virus glycoprotein, endoglin, episialin, EPHA3, c-Met, FGFR2, fibrin II beta chain, FGF23, folate receptor 1, GMCSF, GD2 ganglioside, GDF-8, GCGR, gelatinase B, glypican 3, GPNMB, GMCSF receptor α-chain, kallikrein, KIR2D, ICAM-1, ICOS, IGF1, IGF2, IGF-1 receptor, IL-1α, IL-1β, IL-2, IL-4Rα, IL-5, IL-6, IL-6R, IL-9, IL-12, IL-13, IL17A, IL17F, IL-2 0, IL-22, IL-23, IL-31, IFN-α, IFN-β, IFN-γ, integrin α4β7, interferon α / β receptor, influenza A hemagglutinin, ILGF2, HER1, HER2, HER3, HHGFR, HGF, HLA-DR, hepatitis B surface antigen, HNGF, Hsp90, HGFR, L-selectin, Lewis-Y antigen, LYPD3, LOXL2, LIV-1, MUC1, MCP-1, MSLN, mesothelin, MIF, MCAM, NCA-90, NCA-90Notch1, nectin-4, PCDP1, PD-L1, PD-1, PCSK9, PTK7, PCDC1, phosphatidylserine, RANKL, RTN4, Rh factor, ROR1, SLAMF7, Staphylococcus aureus alpha toxin, Staphylococcus aureus two-component leukocidin, SOST, selectin P, SLITRK6, SDC1, TFPI, TRAIL-R2, tumor antigen CTAA16.88, TNF-α, TWEAK receptor, TNFRSF8, TYRP1, tau protein, TAG-72, TSLP, TRAIL-R1, TRAIL-R2, TGF-β, TAG-72, TRAP, TIGIT, tenascin-C, OX-40, VEGF-A, VWF, VEGFR1, or VEGFR2.

[0138] Peptides include glucagon-like peptide 1 (GLP-1), exendin-2, exendin-3, exendin-4, atrial natriuretic factor (ANF), ghrelin, vasopressin, growth hormone, growth hormone-releasing hormone (GHRH), RC-3095, somatostatin, bombesin, PCK-3145, Phe-His-Ser-Cys-Asn (PHSCN), IGF1, B-type natriuretic peptide, peptide YY (PYY), and interferon. Serone, thrombospondin, angiopoietin, calcitonin, gonadotropin-releasing hormone, hirudin, glucagon, anti-TNF-alpha, fibroblast growth factor, granulocyte colony-stimulating factor, obienepitide, parathyroid hormone (PTH), leuprolide, sermorelin, pramorelin, nesiritide, rotigaptide, cilengitide, MBP-8298, AL-108, enfuvirtide, thymalfasin, daptamycin, HLFI-II, lactoferrin, dextromethorphan Lumitide, glutathione, T cell epitope PR1, protease-3 peptide 1-11, B cell epitope P3, luteinizing hormone-releasing hormone (LHRH), substance P, neurokinin A, neurokinin B, CCK-8, enkephalins such as leucine enkephalin and methionine enkephalin, dermaseptin, [des-Ala20,Gln34]-dermaseptin, surfactant-related antibacterial anionic peptide, apidecin IA; apide Syn IB; OV-2; 1025, acetyl-adhesin peptide (1025-1044) amide; telomere-cin (49-63); pexiganan (MSI-78); indolicidin; apelin-15 (63-77); CFP1O (71-85); anthrax-associated lethal factor (LF) inhibitor; bactenecin; hepatitis C virus NS3 protease inhibitor 2; hepatitis C virus NS3 protease inhibitor 3; hepatitis virus NS3 protease inhibitor 4; NS4A-NS4B Hepatitis C virus (NS3 protease inhibitor I); HIV-1, HIV-2 protease substrate; anti-FM peptide; Bak-BH3; Bax BH3 peptide (55-74) (wild type); Bid BH3-r8; CTT (gelatinase inhibitor); E75 (Her-2 / neu) (369-377); GRP78-binding chimeric peptide motif; p53 (17-26);EGFR2 / KDR antagonist; Colivelin AGA-(C8R)HNGl7 (humanin derivative); Activity-dependent neurotrophic factor (ADNF); Beta-secretase inhibitor I; Beta-secretase inhibitor 2; ch[beta]-amyloid(30-16); Humanun (HN)sHNG, [Glyl4]-HN, [Glyl4-Humanin; Angiotensin-converting enzyme inhibitor (BPP); Renin inhibitor III; Annexin I (ANXA-I; Ac2-12); Anti-inflammatory peptide I; Anti-inflammatory peptide 2; Anti-inflammatory apelin 12; [D-Phel2,Leu4]-Bombesin; Antennapedia peptide (acid) (penetratin); Antennapedia leader peptide (CT); Mastoparan; [Thr28,N [le31]-Cholecystokinin (25-33) sulfated; Nociceptin (1-13) (amide); Fibrinolysis inhibitor; Gamma-Fibrinogen (377-395); Xenin; Obestatin (human); [Hisl,Lys6]-GHRP (GHRP-6); [Ala5,[beta]-Ala8]-Neurokinin A (4-10); Neuromedin B; Neuromedin C; Neuromedin N; Activity-Dependent Neurotrophic Factor (ADNF-14); Acetarin I (opioid receptor antagonist I); Acetarin 2 (opioid receptor antagonist 2); Acetarin 3 (opioid receptor antagonist 3); ACTH (1-39) (human); ACTH (7-38) (human); Sauvagin; Adipokinetic hormone (Locusta Migratoria); myristoylated ADP-ribosylation factor 6, myr-ARF6(2-13); PAMP(1-20) (proadrenomedullin(1-20) human); AGRP(25-51); amylin(8-37) (human); angiotensin I (human); angiotensin II (human); apstatin (aminopeptidase P inhibitor); brevinin-I; magainin I; RL-37; LL-37 (antimicrobial peptide) (human); cecropin A; antioxidant peptide A; antioxidant peptide B; L-camocin; BcI9-2; NPVF; neuropeptide AF (hNPAF) (human); Bax BH3 peptide(55-74); bFGF inhibitory peptide; bFGF inhibitory peptide II; bradykinin; [Des-Argl OJ-HOE140; caspase I inhibitor II;Caspase I inhibitor VIII; Smac N7 protein (MEK1-derived peptide inhibitor I); hBD-1 ([beta]-defensin-1) (human); hBD-3 ([beta]-defensin-3) (human); hBD-4 ([beta]-defensin-4) (human); HNP-I (defensin human neutrophil peptide I); HNP-2 (defensin human neutrophil peptide-2 dynorphin A(1-17)); endomorphin-I; [beta]-endorphin (human porcine); endothelin 2 (human); fibrinogen binding inhibitor peptide; cyclo(-GRGDSP); TP508 (thrombin-derived peptide); galanin (human); GIP (human); gastrin-releasing peptide (human); gastrin-1 (human); ghrelin (human); PDGF-BB peptide; [D-Lys3]-GHRP-6; HCV core protein (1-20); a3Bl integrin peptide fragment (325) (amide); laminin pentapeptide (amide) Mel -Anotropin-potentiating factor (MPF); VA-[beta]-MSH, lipotropin-Y (derived from proopiomelanocortin); atrial natriuretic peptide (1-28) (human); vasonatriuretic peptide (1-27); [Ala5,B-Ala8]-neurokinin A (4-10); neuromedin L (NKA); Ac-(Leu28,31)-neuropeptide Y (24-26); allitesin; brain neuropeptide II; [D-tyrll]-neurotensin; IKKy NEMO-binding domain (NBD) inhibitory peptide; PTD-p50 (NLS) inhibitory peptide; orexin A (bovine, human, mouse, rat); orexin B (human); aquaporin-2 (254-267) (human pancreastatin) (37-52); pancreatic polypeptide (human); neuropeptides; peptide YY (3-36) (human); hydroxymethyl-phytochelatin 2; PACAP (I-27) (amide, human, bovine, rat); prolactin-releasing peptide (1-31) (human); sarcin-alpha; sarcin-beta; saposin C22; secretin (human); L-selectin; endokinin A / B; endokinin C (human); endokinin D (human); thrombin receptor (42-48) agonist (human); LSKL (inhibitor of thrombospondin); thyrotropin-releasing hormone (TRH);P55-TNFR fragment; urotensin II (human); VIP (human, porcine, rat); VIP antagonist; helodermin; exenatide; ZPlO (AVEOOIOO); pramlintide; AC162352 (PYY)(3-36); PYY; obienepitide; glucagon; GRP; ghrelin (GHRP6); leuprolide; histrelin; oxytocin; atosiban (RWJ22164); sermorelin; nesiritide; bivalirudin (Hirulog); icatibant; abinet Putadine; Rotigaptide (ZP123, GAP486); Cilengitide (EMD-121924, RGD peptide); AlbuBNP; BN-054; Angiotensin II; MBP-8298; Leucine-Arginine Peptide; Ziconotide; AL-208; AL-108; Carbeticone; Tripeptide; SAL; Colliven; Humanin; ADNF-14; VIP (Vasoactive Intestinal Peptide); Thymalfasin; Bacitracin; Gramidicin; Pexiganan (MSI-78); Pl 13;PAC-113;SCV-07;HLFl-Il (lactoferrin);DAPTA;TRI-1144;tritorpticin;anti-flamin 2;Gatex (teduglutide, ALX-0600);Stimuvax (L-BLP25);Crysalin (TP508);Melanonan II;Spantide II;Ceruletide;Sincalide;Pentagastin;Secretin;Endostatin peptide;E-selectin;HER2;IL-6;IL-8;IL-10;PDGF;Thrombospondin;uPA(I);uPA(2);VEGF;VEGF(2);Pentapeptide-3;XXLRR;beta-amyloid fibril formation;Endomol Fin-2; TIP39 (infundibulohypophysial neuropeptide); PACAP (1-38) (amide, human, bovine, rat); TGFB-activating peptide; insulin-sensitizing factor (ISF402); transforming growth factor B1 peptide (TGF-B1); caerulein-releasing factor; IELLQAR (8-branched MAPS); tigapotide PK3145; goserelin; abarelix; cetrorelix; ganirelix; degarelix (triptorelin); barsiban (FE200440); pralmorelin; octreotide; eptifibatide; netamiftide (INN-00835); daptamycin; spantide II; dermitide (RDP-58);Examples of suitable proteins for use in the present disclosure include, but are not limited to, AL-209, enfuvirtide, IDR-I, hexapeptide-6, insulin A chain, lanreotide, hexa[rho]eptide-3, insulin B chain, glargine A chain, glargine B chain, insulin-LisPro B chain analog, insulin-aspart B chain analog, insulin-gululisine B chain analog, insulin-detemir B chain analog, somatostatin tumor inhibitor analog, pancreastatin (37-52), vasoactive intestinal peptide fragment (KKYL-NH2), and dynorphin A. Examples of suitable proteins for use in the present disclosure include, but are not limited to, immunotoxin SSlP, adenosine deaminase, argininase, and the like.

[0139] The macromolecule can be a water-soluble polymer, lipid, protein, or polypeptide.In some embodiments, the macromolecule is a fatty acid containing from about 6 to about 26 carbon atoms, 2-methacryloyl-oxyethylphosphoylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(N-acryloylmorpholine), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacryloyl). acrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(α-hydroxy acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxy methacrylate), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(hydroxypropyl oxazoline), poly(imino carbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(oxyethylated polyol), poly(olefin alcohol), polyphosphazene, poly(propylene glycol), poly(saccharide), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinyl pyrrolide) The polymers include polymers selected from the group consisting of: cellulose, hydroxypropyl methylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid (HA) and derivatives, functionalized hyaluronic acid, mannan, pectin, heparin, heparan sulfate (HS), rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch (HES), polysialic acid (PSA) and other carbohydrate-based polymers, xylan, and copolymers.

[0140] The macromolecule can also be a protein or polypeptide selected from the group consisting of albumin, transferrin, transthyretin, immunoglobulin, XTEN peptide, glycine-rich homoamino acid polymer (HAP), PAS polypeptide, elastin-like polypeptide (ELP), CTP peptide, or gelatin-like protein (GLK) polymer.

[0141] In certain embodiments, the linker L is a residue of a releasable linker (RL).

[0142] In certain embodiments, x or z is 2 or greater. In certain embodiments, x or z is 3 or greater. In certain embodiments, x or z is 4 or greater. In certain embodiments, x or z is 5 or greater. In certain embodiments, x or z is 6 or greater. In certain embodiments, x or z is greater than 6.

[0143] In certain embodiments, the preparation methods described herein involve a first step involving the conjugation of a protein with multiple bifunctional linkers. Because of the small size of the linkers, the conjugation process is expected to be more efficient and achieve a higher conjugation yield compared to direct conjugation of a protein and a macromolecule. Also, as described herein, the second step of the disclosed method may involve click chemistry, designed to connect the linker and the macromolecule with high efficiency. Without being bound by any particular theory, this method is believed to offer the advantage of minimizing steric hindrance and thus improving reaction efficiency. Furthermore, due to the simplified and less expensive synthesis and purification steps, this method offers significant advantages for the large-scale production and manufacturing of polymer-protein therapeutics.

[0144] Bifunctional Releasable Linkers The conjugates of the present disclosure can be derived from bifunctional releasable linkers.

[0145] In some embodiments, the present disclosure provides a compound of formula (I): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, X 1 is the first spacer moiety, X 2 is a second spacer moiety, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R e is an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; a is an integer from 0 to 4, b is an integer from 1 to 3, c is an integer from 0 to 1, FG 1 is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as a carbamate linkage; FG 2 are independently functional groups capable of reacting via click chemistry, such as, but not limited to, azide, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups. The present invention relates to a bifunctional releasable linker of the formula:

[0146] In some embodiments of Formula (I), R 1 and R 2 are each independently 1-5 Alkyl, substituted C 1-5 Alkyl, C 2-6 Alkenyl, substituted C 2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, phenyl, or substituted phenyl. In certain embodiments, R 1 and R 2 are each independently 1-5 Alkyl or substituted C 1-5 It is alkyl.

[0147] In some embodiments of Formula (I), R e is nitro, cyano, halogen, -CONH(C 1-5 alkyl) or -CONH(phenyl), substituted -CONH(C 1-5 alkyl) or -CONH(phenyl), -SONH(C 1-5 alkyl) or -SO2NH(phenyl), substituted -SO2NH(C 1-5 alkyl) or -SO2NH(phenyl), -SO2(C 1-5 alkyl) or -SO2(phenyl), substituted -SO2(C 1-5 alkyl) or -SO2 (phenyl), C 1-5 Alkoxy, substituted C 1-5 Alkoxy, C 1-5 Alkyl or C 3-6 Cycloalkyl, substituted C 1-5 Alkyl or C 3-6 It is cycloalkyl, phenyl or 5- to 6-membered heteroaryl, or substituted phenyl or 5- to 6-membered heteroaryl.

[0148] In some embodiments of Formula (I), a is an integer from 0 to 3. In some embodiments, a is an integer from 0 to 2. In some embodiments, a is 0. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4.

[0149] In some embodiments of Formula (I), b is an integer of 1 or 2. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3.

[0150] In some embodiments of Formula (I), c is 0. In some embodiments, c is 1.

[0151] In some embodiments of Formula (I), X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2 are the same spacer moiety. 1 and X 2 are different spacer moieties.

[0152] Within formula (I), bifunctional releasable linkers are provided having a more defined structure: [ka] (In the formula, X 1 is a first spacer moiety, and each of X 2 is a second spacer moiety, and R 1 , R 2 , [R e ] a , F.G. 1 and FG 2 is as defined above).

[0153] In certain embodiments of Formula (I), (IB), or (IC), a is an integer from 0 to 2, and R 1 and R 2 are each independently H, Me, or Et, and R e is nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0154] In certain embodiments of Formula (I), (IB), or (IC), the bifunctional releasable linker has the following structure: [ka]

[0155] In another aspect, the present disclosure provides a compound of formula (XVIII): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, X 1 is a spacer moiety, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R e is an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; a is an integer from 0 to 4, c is 2, FG 1 is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage; FG 2 is a functional group capable of reacting via click chemistry) The present invention relates to a bifunctional releasable linker of the formula:

[0156] In certain embodiments of Formula (XVIII), a is an integer from 0 to 2, and R 1 and R 2 are each independently hydrogen, Me, or Et; R e is nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0157] In certain embodiments of Formula (XVIII), the bifunctional releasable linker has one of the following structures: [ka]

[0158] In another aspect, the present disclosure provides a compound of formula (II): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, X 2 is a second spacer moiety, if present, X 3 is a third spacer moiety, if present, FG 1 is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as a carbamate linkage; FG 2 are independently functional groups capable of reacting via click chemistry, such as, but not limited to, azide, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups. The present invention relates to a bifunctional releasable linker of the formula:

[0159] In some embodiments of Formula (II), R 1 and R 2 are each independently 1-5 Alkyl, substituted C 1-5 Alkyl, C 2-6 Alkenyl, substituted C 2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, phenyl, or substituted phenyl. In certain embodiments, R 1 and R 2 are each independently 1-5 Alkyl or substituted C 1-5 It is alkyl.

[0160] In some embodiments of Formula (II), R e1 and R e2 are each independently nitro, cyano, halogen, haloalkyl (e.g., -CF3, -CHF2, -CH2F, -CH2F), -OC 1-5 Alkyl, -O-haloalkyl (e.g., -OCF3, -OCHF2, -OCH2F, -OCH2F), -NH(C 1-5alkyl), -NHCO(C 1-5 alkyl), -NHSO2(C 1-5 alkyl), -CONH(C 1-5 alkyl), or -SO2NH(C 1-5 In certain embodiments, R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0161] In some embodiments of Formula (II), X 2 and X 3 are each independently selected from spacer moieties described herein. 2 and X 3 are the same spacer moiety. 2 and X 3 are different spacer moieties.

[0162] In certain embodiments of Formula (II), a1 and a2 are each independently an integer from 0 to 2, and R 1 and R 2 are each independently H, Me, or Et, and R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0163] Further exemplary bifunctional linkers are within the scope of formula (II-A) or (II-B) below: [ka] (In the formula, R eis hydrogen or an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl). e is hydrogen or fluoro. [ka]

[0164] Reagents that provide these releasable linkages can be prepared according to the procedures described in US20060293499A1.

[0165] In another aspect, the present disclosure provides a compound of formula (III): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R p is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, FG 2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups; FG 4 is a functional group capable of reacting with an amino group of an active agent to form an amide linkage) The present invention relates to a bifunctional releasable linker of the formula:

[0166] In some embodiments of Formula (III), R 1 , R 2 and R p are each independently 1-5 Alkyl, substituted C 1-5 Alkyl, C 2-6 Alkenyl, substituted C 2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, phenyl, or substituted phenyl. In certain embodiments, R 1 and R 2 are each independently 1-5 Alkyl or substituted C 1-5 It is alkyl.

[0167] In some embodiments of Formula (III), R e1 and R e2 are each independently nitro, cyano, halogen, haloalkyl (e.g., -CF3, -CHF2, -CH2F, -CH2F), -OC 1-5Alkyl, -O-haloalkyl (e.g., -OCF3, -OCHF2, -OCH2F, -OCH2F), -NH(C 1-5 alkyl), -NHCO(C 1-5 alkyl), -NHSO2(C 1-5 alkyl), -CONH(C 1-5 alkyl), or -SO2NH(C 1-5 In certain embodiments, R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0168] In some embodiments of Formula (III), X 2 and X 3 are each independently selected from spacer moieties described herein. 2 and X 3 are the same spacer moiety. 2 and X 3 are different spacer moieties.

[0169] In certain embodiments of Formula (III), a1 and a2 are each independently an integer from 0 to 2, and R 1 and R 2 are each independently H, Me, or Et, and R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0170] Exemplary bifunctional releasable linkers are within the scope of formula (III-A) below: [ka]

[0171] In another aspect, the present disclosure provides a compound of formula (IV): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 4 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, c is an integer from 0 to 4, R el is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R d is nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl or cycloalkyl, substituted alkyl or cycloalkyl, aryl or heteroaryl, or substituted aryl or heteroaryl; X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, Y1 is O or S, Y 2 is O or S, FG 1 is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as a carbamate linkage; FG 2 are independently functional groups capable of reacting via click chemistry, such as, but not limited to, azide, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups. The present invention relates to a bifunctional releasable linker of the formula:

[0172] In some embodiments of Formula (IV), R 1 , R 2 , R 3 and R 4 are each independently 1-5 Alkyl, substituted C 1-5 Alkyl, C 2-6 Alkenyl, substituted C 2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, phenyl, or substituted phenyl. In certain embodiments, R 1 , R 2 , R 3 and R 4 are each independently 1-5 Alkyl or substituted C 1-5 It is alkyl.

[0173] In some embodiments of Formula (IV), R e1 and R e2 are each independently nitro, cyano, halogen, haloalkyl (e.g., -CF3, -CHF2, -CH2F, -CH2F), -OC 1-5 Alkyl, -O-haloalkyl (e.g., -OCF3, -OCHF2, -OCH2F, -OCH2F), -NH(C 1-5 alkyl), -NHCO(C 1-5 alkyl), -NHSO2(C 1-5 alkyl), -CONH(C1-5 alkyl), or -SO2NH(C 1-5 In certain embodiments, R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0174] In some embodiments of Formula (IV), R d is nitro, cyano, halogen, -CONH(C 1-5 alkyl) or -CONH(phenyl), substituted -CONH(C 1-5 alkyl) or -CONH(phenyl), -SONH(C 1-5 alkyl) or -SO2NH(phenyl), substituted -SO2NH(C 1-5 alkyl) or -SO2NH(phenyl), -SO2(C 1-5 alkyl) or -SO2(phenyl), substituted -SO2(C 1-5 alkyl) or -SO2 (phenyl), C 1-5 Alkoxy, substituted C 1-5 Alkoxy, C 1-5 Alkyl or C 3-6 Cycloalkyl, substituted C 1-5 Alkyl or C 3-6 It is cycloalkyl, phenyl or 5- to 6-membered heteroaryl, or substituted phenyl or 5- to 6-membered heteroaryl.

[0175] In some embodiments of Formula (IV), X 2 and X 3 are each independently selected from spacer moieties described herein. 2 and X 3 are the same spacer moiety. 2 and X 3 are different spacer moieties.

[0176] An advantage of using releasable linkers such as those of Formula (III) and Formula (IV) is the potential for improved stability, allowing for sustained drug release and ultimately leading to long-term therapeutic efficacy. Thus, the linkers of the present disclosure offer advantages over the prior art regarding the stability and storage of polymer-protein therapeutics.

[0177] Polymeric Reagents Containing Releasable Linkers The present disclosure also relates to conjugates that can be derived from polymeric reagents that include releasable linkers.

[0178] In some embodiments, the present disclosure provides a compound of formula (V): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, POLY 1 is a first water-soluble polymer, POLY 2 is a second water-soluble polymer, X 1 is the first spacer moiety, X 2 is a second spacer moiety, Y 1 is O or S, Y 2 is O or S, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 4is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 is an integer of 0 to 3, a2 is an integer of 0 to 3, c is an integer from 0 to 4, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R d is nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl or cycloalkyl, substituted alkyl or cycloalkyl, aryl or heteroaryl, substituted aryl or heteroaryl; FG 1 is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as a carbamate linkage. The present invention relates to a polymeric reagent comprising a releasable linker of the formula:

[0179] In some embodiments, R 1 , R 2 , R 3 , R 4 , R e1 , R e2 and R d is as defined above in formula (IV).

[0180] In some embodiments of Formula (V), R e1 and R e2 are the same electron-modifying group. In some embodiments, R e1 and R e2 are different electron-modifying groups.

[0181] In some embodiments of Formula (V), POLY 1 and POLY 2are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 1 and POLY 2 are the same water-soluble polymer. 1 and POLY 2 are different water-soluble polymers.

[0182] In some embodiments of Formula (V), X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2 are the same spacer moiety. 1 and X 2 are different spacer moieties. Exemplary polymeric reagents are within the scope of the following formula (VA): [ka] wherein n is independently an integer between 4 and 1500, e.g., 4, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, including all ranges and values ​​therebetween.

[0183] Other polymeric reagents containing two releasable linkages have the following formula (VI): [ka] or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, POLY 1 is a first water-soluble polymer, POLY 2 is a second water-soluble polymer, X 1 is the first spacer moiety, X 2 is a second spacer moiety, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 is an integer of 0 to 3, a2 is an integer of 0 to 3, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R p is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; FG 4 is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as an amide linkage. Includes.

[0184] In some embodiments of Formula (VI), R 1 , R 2 and R p are each independently 1-5 Alkyl, substituted C 1-5 Alkyl, C 2-6 Alkenyl, substituted C 2-6 alkenyl, C2-6 alkynyl, substituted C2-6 alkynyl, phenyl, or substituted phenyl. In certain embodiments, R 1 , R 2 , R 3 and R 4 are each independently 1-5 Alkyl or substituted C 1-5It is alkyl.

[0185] In some embodiments of Formula (VI), R e1 and R e2 are each independently nitro, cyano, halogen, -CONH(C 1-5 alkyl) or -CONH(phenyl), substituted -CONH(C 1-5 alkyl) or -CONH(phenyl), -SONH(C 1-5 alkyl) or -SO2NH(phenyl), substituted -SO2NH(C 1-5 alkyl) or -SO2NH(phenyl), -SO2(C 1-5 alkyl) or -SO2(phenyl), substituted -SO2(C 1-5 alkyl) or -SO2 (phenyl), C 1-5 Alkoxy, substituted C 1-5 Alkoxy, C 1-5 Alkyl or C 3-6 Cycloalkyl, substituted C 1-5 Alkyl or C 3-6 It is cycloalkyl, phenyl or 5- to 6-membered heteroaryl, or substituted phenyl or 5- to 6-membered heteroaryl.

[0186] In some embodiments of Formula (VI), POLY 1 and POLY 2 are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 1 and POLY 2 are the same water-soluble polymer. 1 and POLY 2 are different water-soluble polymers.

[0187] In some embodiments of Formula (VI), X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2are the same spacer moiety. 1 and X 2 are different spacer moieties.

[0188] Exemplary polymeric reagents are within the scope of formula (VI-A) below: [ka] wherein n is independently an integer between 4 and 1500, e.g., 4, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, including all ranges and values ​​therebetween.

[0189] Protein-linker conjugates In some embodiments, the present disclosure provides a conjugate comprising a residue of a protein covalently linked with one or more linkers, the residue having a structure according to formula (XIX): Protein-(L) z (XIX) or a stereoisomer, regioisomer, tautomer or mixture thereof, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. (In the formula, z is an integer from 1 to 25, L is a linker, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide. The present invention provides a conjugate comprising:

[0190] The conjugates described herein are the product of synthesis in Step 1 of Scheme (I). In certain embodiments, the linker is a non-releasable linker. In certain embodiments, the linker is a releasable linker. In some embodiments, the releasable linker is a derivative of a bifunctional releasable linker disclosed herein (e.g., a linker of Formula (I), Formula (II), Formula (III), or Formula (IV)).

[0191] In certain embodiments, the linker is covalently attached to an amine group of a residue within the protein. In certain embodiments, the residue is lysine. In certain embodiments, a composition is provided that includes a mixture of conjugates that include different numbers of linkers attached to the protein.

[0192] Exemplary conjugates formed using reagents that provide bifunctional releasable linkages conjugated to proteins include those of formula (VII): [ka] (In the formula, X 1 is the first spacer moiety, X 2 is a second spacer moiety, if present, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R eis an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; a is an integer from 0 to 5, b is an integer from 0 to 3, c is an integer from 0 to 2, z is an integer from 1 to 25, Y 1 is O or S, Y 2 is O or S, FG 2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups; -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0193] In some embodiments, R 1 , R 2 , and R e is as defined above in formula (I).

[0194] In some embodiments of Formula (VII), a is an integer from 0 to 4. In some embodiments, a is an integer from 0 to 3. In some embodiments, a is an integer from 0 to 2. In some embodiments, a is 0. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5.

[0195] In some embodiments of Formula (VII), b is an integer from 0 to 2. In some embodiments, b is 0. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3.

[0196] In some embodiments of Formula (VII), c is 0 or 1. In some embodiments, c is 0. In some embodiments, c is 1. In some embodiments, c is 2.

[0197] In some embodiments of Formula (VII), z is an integer from 1 to 20. In some embodiments, z is an integer from 1 to 15. In some embodiments, z is an integer from 1 to 10. In some embodiments, z is an integer from 1 to 8. In some embodiments, z is an integer from 1 to 5.

[0198] Those skilled in the art will recognize that the values ​​and ranges of a, b, c, and z described herein can be combined in any way to obtain a conjugate of the present disclosure. For example, in some embodiments, a is an integer from 0 to 2, b is 0 or 1, c is 0 or 1, and z is an integer from 1 to 25. In some embodiments, a is 1, b is 1, c is 1, and z is an integer from 1 to 25. In some embodiments, a is 1, b is 0, c is 1, and z is an integer from 1 to 25. In some embodiments, a is 1, b is 1, c is 0, and z is an integer from 1 to 25. These and many other combinations are contemplated by the present disclosure. In some embodiments, X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2 are the same spacer moiety. 1 and X 2are different spacer moieties.

[0199] Conjugates of formula (VII) having a more defined structure are contemplated as formula (VII-A), (VII-B), (VII-C), or (VII-D): [ka] (In the formula, X 1 is the first spacer moiety, and X 2 is a second spacer moiety, and R 1 , R 2 , R e , a, z, Y 1 , Y 2 , F.G. 2 and the protein is as defined above in formula (VII).

[0200] In certain embodiments of Formula (VII), (VII-A), (VII-B), (VII-C), or (VII-D), a is an integer from 0 to 2, and R 1 and R 2 are each independently hydrogen, Me, or Et; R e is nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0201] A further exemplary conjugate has the following structure (VII-A1): [ka] (In the formula, R eis an electron modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; z is an integer from 1 to 25; and "-NH-" represents one or more linkers individually attached to the protein moiety. In certain embodiments, a is an integer from 1 to 2; R e is 4-F, 4-Cl, 4-CF3, 2,4-difluoro, or 2-CF3-4-F substituted.

[0202] Further exemplary conjugates have the following structures: [ka] [ka]

[0203] Other exemplary conjugates formed using reagents that provide bifunctional releasable linkages include those of formula (VIII): [ka] (In the formula, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, Y 1 is O or S, Y 2 is O or S, FG 2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups; -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0204] In some embodiments, R 1 , R 2 , R e1 , and R e2 is as defined above in formula (VI).

[0205] In certain embodiments of Formula (VIII), a1 and a2 are each independently an integer from 0 to 2, and R 1 and R 2 are each independently hydrogen, Me, or Et; R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0206] A conjugate of formula (VIII) having a more defined structure is contemplated as formula (VIII-A). [ka]

[0207] Other exemplary conjugates formed using reagents that provide two releasable linkages include those of formula (IX): [ka] (In the formula, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R p is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, FG 2are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups; -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0208] In some embodiments, R 1 , R 2 , R p , R e1 , and R e2 is as defined above in formula (VI).

[0209] In certain embodiments of Formula (IX), a1 and a2 are each independently an integer from 0 to 2, and R 1 and R 2 are each independently hydrogen, Me, or Et; R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0210] A more defined structure of the conjugate of formula (IX) is shown below in formula (IX-A). [ka]

[0211] Other exemplary conjugates formed using reagents that provide two releasable linkages include those of formula (X): [ka] (In the formula, R 1is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 4 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, c is an integer from 0 to 4, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R d is nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl or cycloalkyl, substituted alkyl or cycloalkyl, aryl or heteroaryl, substituted aryl or heteroaryl; X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, Y 4 is O or S, FG2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups; -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0212] In some embodiments, R 1 , R 2 , R 3 , R 4 , R d , R e1 , and R e2 is as defined above in formula (IV).

[0213] In certain embodiments of the formulas disclosed herein, z is an integer from 1 to 22, 1 to 20, 1 to 18, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, or 1 to 3, where z represents the number of releasable linkers conjugated to the protein.

[0214] Protein-Macromolecule Conjugates

[0215] In one or more embodiments of the present disclosure, a protein-macromolecule conjugate is provided, comprising a protein, at least one linker, and at least one water-soluble polymer, wherein the protein is covalently attached to each water-soluble polymer via a linker, and the macromolecule is a linear or branched water-soluble polymer. In certain embodiments, the at least one linker is two or more linkers. In certain embodiments, the two or more linkers include at least one non-releasable linker. In certain embodiments, the two or more linkers include at least one releasable linker. In certain embodiments, the two or more linkers include at least one non-releasable linker and one releasable linker. In certain embodiments, the two or more linkers include at least one non-releasable linker and one to eight releasable linkers.

[0216] In certain embodiments, at least one linker is a non-releasable linker. In certain embodiments, at least one linker is a releasable linker. In certain embodiments, each of the linkers is a releasable linker. In certain embodiments, one or more macromolecules are covalently bound to the protein via one or more linkers. In certain embodiments, eight or more macromolecules are covalently bound to the protein via eight or more linkers.

[0217] In certain embodiments, the macromolecule is covalently attached via a linker to an amine group of a residue within the protein. In certain embodiments, the residue is lysine. In certain embodiments, the conjugate is a mixture of conjugates comprising different numbers of macromolecules attached to the protein.

[0218] In various embodiments, the macromolecule is a water-soluble polymer, lipid, protein, or polypeptide. The macromolecule may be any of the following: a fatty acid containing from about 6 to about 26 carbon atoms, 2-methacryloyl-oxyethylphosphoylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(N-acryloylmorpholine), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyano). acrylate), poly(dimethylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyl oxazoline), poly(glycolic acid), poly(α-hydroxy acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxy methacrylate), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(hydroxypropyl oxazoline) ), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(oxyethylated polyol), poly(olefin alcohol), polyphosphazene, poly(propylene glycol), poly(saccharide), poly(siloxane), poly(urethane), poly(vinyl alcohol), poly(vinylamine), poly(vinylmethyl ether) ester), poly(vinylpyrrolidone), silicone, amylose, cellulose, carbomethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid (HA) and derivatives, functionalized hyaluronic acid, mannan, pectin, heparin, heparan sulfate (HS), rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch (HES), polysialic acid (PSA) and other carbohydrate-based polymers, xylan, and copolymers.The polymer may comprise one selected from the group consisting of albumin, transferrin, transthyretin, immunoglobulin, XTEN peptide, glycine-rich homoamino acid polymer (HAP), PAS polypeptide, elastin-like polypeptide (ELP), CTP peptide, or gelatin-like protein (GLK) polymer.

[0219] In certain embodiments, the macromolecule is a water-soluble polymer. In certain embodiments, the water-soluble polymer is a polymer of poly(ethylene glycol). In certain embodiments, the poly(ethylene glycol) is end-capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy, and substituted aryloxy.

[0220] With respect to water-soluble polymers, the water-soluble polymer is non-toxic, non-naturally occurring, and biocompatible. Regarding biocompatibility, a substance is considered biocompatible if the beneficial effects associated with its use alone or with another substance (e.g., an active agent such as an IL-2 moiety) in connection with biological tissue (e.g., administration to a patient) outweigh any adverse effects as assessed by a clinician, e.g., a physician. Regarding non-immunogenicity, a substance is considered non-immunogenic if its intended use in vivo does not result in an undesirable immune response (e.g., antibody formation), or if an immune response does occur, such a response is deemed clinically insignificant or inconsequential as assessed by a clinician. It is particularly preferred that non-peptidic water-soluble polymers be biocompatible and non-immunogenic.

[0221] Furthermore, the polymer is typically characterized as having from 2 to about 300 termini. Examples of such polymers include, but are not limited to, poly(alkylene glycols), such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), polyvinylpyrrolidone), poly(hydroxyalkyl methacrylamides), poly(hydroxyalkyl methacrylates), polysaccharides, poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines ("POZ") (as described in WO 2008 / 106186), poly(N-acyloylmorpholines), and combinations of any of the foregoing.

[0222] The water-soluble polymer is not limited to a particular structure and may be linear (e.g., end-capped, e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), dendritic (or star) structure, each of which may or may not have one or more degradable linkages. Furthermore, the internal structure of the water-soluble polymer may be organized in any number of different repeating patterns and may be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating tripolymers, random tripolymers, and block tripolymers.

[0223] Activated PEG and other activated water-soluble polymers (i.e., polymeric reagents) are activated with a suitable activating group suitable for coupling to a desired site on a protein. Thus, the polymeric reagent has a reactive group for reacting with a protein moiety. Representative polymeric reagents and methods for conjugating these polymers to active moieties are known in the art and are further described in Zalipsky, S., et al., "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides," Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J.M. Harris, Plenus Press, New York (1992), and Zalipsky (1995) Advanced Drug Reviews 16:157-182. Exemplary active groups suitable for coupling to a protein moiety include, among others, hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol, vinyl sulfone, and hydrazine.

[0224] Typically, the weight average molecular weight of the water-soluble polymer of the conjugate is from about 100 daltons to about 150,000 daltons. However, exemplary ranges include from about 500 daltons to less than 20,000 daltons, from about 20,000 daltons to less than 85,000 daltons, from about 85,000 daltons to about 100,000 daltons, from greater than 5,000 daltons to about 100,000 daltons, from about 6,000 daltons to about 90,000 daltons, from about 10,000 daltons to about 85,000 daltons, and from greater than 10,000 daltons to about 85,000 daltons. Examples of weight average molecular weights include the range of about 20,000 daltons to about 85,000 daltons, the range of about 53,000 daltons to about 85,000 daltons, the range of about 25,000 daltons to about 120,000 daltons, the range of about 29,000 daltons to about 120,000 daltons, the range of about 35,000 daltons to about 120,000 daltons, and the range of about 40,000 daltons to about 120,000 daltons. For any given water-soluble polymer, PEGs having molecular weights in one or more of these ranges are preferred.

[0225] Exemplary weight average molecular weights of water-soluble polymers include about 100 daltons, about 200 daltons, about 300 daltons, about 400 daltons, about 500 daltons, about 600 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 900 daltons, about 1,000 daltons, about 1,500 daltons, about 2,000 daltons, about 2,200 daltons, about 2,500 daltons, about 3,000 daltons, about 4,000 daltons, about 4,400 daltons, about 4,500 daltons, about 5,000 daltons, about 5,500 daltons, about 6,000 daltons, about 7,000 daltons, about 7,500 daltons, and about 8,000 daltons. daltons, about 9,000 daltons, about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, about 16,000 daltons, about 18,000 daltons, about 20,000 daltons, about 22,500 daltons, about 25,000 daltons, about 30,000 daltons, about 35,000 daltons, about 40,000 daltons, about 45,000 daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. Branched water-soluble polymers having any of the above total molecular weights (eg, a 40,000 dalton branched water-soluble polymer composed of two 20,000 dalton polymers) can also be used.

[0226] When used as a polymer, PEG typically comprises multiple (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how PEG is defined]. As used throughout this description, the number of repeat units is referred to as "(OCH2CH2) n". Thus, the value of (n) typically falls within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, about 450 to about 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer whose molecular weight is known, the number of repeat units (i.e., "n") can be determined by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0227] One particularly preferred polymer for use in the present disclosure is an end-capped polymer, i.e., at least one terminus is a lower C, although a hydroxyl group may also be used. 1-6 It is a polymer capped with a relatively inert group such as an alkoxy group. For example, when the polymer is PEG, it is preferable to use methoxy-PEG (commonly referred to as mPEG), which is a linear PEG in which one end of the polymer is a methoxy (-OCH3) group and the other end is a hydroxyl or other functional group that can be optionally chemically modified.

[0228] In one form useful in one or more embodiments of the present disclosure, the free or unconjugated PEG is a linear polymer terminated at each end with a hydroxyl group: HO-CH2CH2O-(CH2CH2O) n -CH2CH2-OH (wherein (n) typically ranges from 0 to about 4,000).

[0229] The above polymer, alpha-, omega-dihydroxyl poly(ethylene glycol), can be represented in shorthand form as HO-PEG-OH, where it is understood that the -PEG- symbol can represent the following structural unit: -CH2CH2O-(CH2CH2O) n -CH2CH2- where (n) is as defined above.

[0230] Another type of PEG useful in one or more embodiments of the present disclosure is methoxy-PEG-OH, or simply mPEG-OH, which has a relatively inert methoxy group at one end and a hydroxyl group at the other end. The structure of mPEG-OH is shown below: CH3O-CH2CH2O-(CH2CH2O) n -CH2CH2-OH where (n) is as described above.

[0231] Another type of PEG useful in one or more embodiments of the present disclosure is methoxy-PEG-NH2, or simply mPEG-NH2, which has a relatively inert methoxy group at one terminus and an amino group at the other. The structure of mPEG-NH2 is shown below. CH3O-CH2CH2O-(CH2CH2O) n -CH2CH2-NH2 where (n) is as described above.

[0232] Another type of PEG useful in one or more embodiments of the present disclosure is methoxy-PEG-CO2H, or simply mPEG-CO2H, which has a relatively inert methoxy group at one end and a carboxylic acid group at the other end. The structure of mPEG-CO2H is set forth below: CH3O-CH2CH2O-(CH2CH2O) n -CH2CH2-CO2H where (n) is as described above.

[0233] Another type of PEG useful in one or more embodiments of the present disclosure is methoxy-PEG-N3, or simply mPEG-N3, which has a relatively inert methoxy group at one end and an azide group at the other end. The structure of mPEG-N3 is shown below. CH3O-CH2CH2O-(CH2CH2O) n -CH2CH2-N3 where (n) is as described above.

[0234] Another type of PEG useful in one or more embodiments of the present disclosure is methoxy-PEG-DBCO, or simply mPEG-DBCO, which has a relatively inert methoxy group at one end and a dibenzocyclooctyne (DBCO) group at the other end. An example of the structure of mPEG-DBCO is shown below: [ka] where (n) is as described above.

[0235] Multiarmed or branched PEG molecules, such as those described in U.S. Patent No. 5,932,462, can also be used as the PEG polymer. For example, the PEG can have the following structure: [ka] (In the formula, poly a and poly b is a PEG backbone (either the same or different) such as methoxypoly(ethylene glycol), R' is a non-reactive moiety such as H, methyl or a PEG backbone, and P and Q are non-reactive linkages).

[0236] In addition, PEG can include forked PEG. An example of a forked PEG is represented by the following structure: [ka] (wherein X is a spacer moiety of one or more atoms, and each Z is an activated end group linked to CH by a chain of atoms of defined length. International Patent Application Publication No. WO 99 / 45964 discloses various forked PEG structures that can be used in one or more embodiments of the present disclosure. The chain of atoms connecting the Z functional group to the branching carbon atom serves as a tether group and can include, for example, alkyl chains, ether chains, ester chains, amide chains, and combinations thereof.

[0237] The PEG polymer may include pendant PEG molecules having reactive groups, such as carboxyl, that are covalently attached along the length of the PEG rather than at the end of the PEG chain. The pendant reactive groups may be attached to the PEG directly or via a spacer moiety, such as an alkylene group.

[0238] Some of the hydrolytically degradable linkages useful as degradable linkages within the polymer backbone and / or as degradable linkages to protein moieties include ester linkages, carbonate linkages; imine linkages, e.g., obtained from the reaction of an amine with an aldehyde (e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphate ester linkages, for example, formed by reacting an alcohol with a phosphate group; hydrazone linkages, typically formed by the reaction of a hydrazide with an aldehyde; acetal linkages, typically formed by the reaction between an aldehyde and an alcohol; orthoester linkages, for example, formed by the reaction between formic acid and an alcohol; amide linkages, for example, formed between an amine group at the terminus of a polymer, such as PEG, and a carboxyl group of another PEG chain; urethane linkages, for example, formed from the reaction of PEG having a terminal isocyanate group with a PEG alcohol; peptide linkages, for example, formed between an amine group at the terminus of a polymer, such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages, for example, formed between a phosphoramidite group at the terminus of a polymer and a 5' hydroxyl group of an oligonucleotide.

[0239] Such optional features of the conjugate, i.e., the introduction of one or more degradable linkages into the polymer chain or protein moiety, can provide further control over the final desired pharmacological properties of the conjugate upon administration. For example, a large, relatively inert conjugate (i.e., one to which one or more high-molecular-weight PEG chains, e.g., one or more PEG chains having a molecular weight greater than about 10,000, are attached, and the conjugate has essentially no biological activity) can be administered, which, upon release, yields a biologically active conjugate having some of the original PEG chains. In this way, the properties of the conjugate can be more effectively tailored to balance the biological activity of the conjugate over time.

[0240] A water-soluble polymer associated with a conjugate can be "releasable." That is, the water-soluble polymer can be released (either via hydrolysis, an enzymatic process, a catalytic process, or otherwise) to yield an unconjugated protein moiety. In some cases, the releasable polymer separates from the protein moiety in vivo without leaving any fragment of the water-soluble polymer. In other cases, the releasable polymer separates from the protein moiety in vivo, leaving behind a relatively small fragment (e.g., a succinate tag) derived from the water-soluble polymer. Exemplary cleavable polymers include those attached to the protein moiety via a carbamate linkage.

[0241] Those skilled in the art will recognize that the foregoing discussion of water-soluble polymers is by no means exhaustive but merely exemplary, and that any polymeric material having the qualities described above is contemplated. As used herein, the term "polymeric reagent" generally refers to an entire molecule that may include a water-soluble polymer segment and a functional group.

[0242] As described above, the conjugates of the present disclosure may include multiple water-soluble polymers covalently attached to the protein moiety. In some embodiments, the multiple water-soluble polymers covalently attached to the protein moiety are the same. In some embodiments, at least one of the multiple water-soluble polymers covalently attached to the protein moiety is different. Typically, for any given conjugate, there will be one or more water-soluble polymers covalently attached to one or more moieties having protein activity. In some cases, the conjugate may have 1, 2, 3, 4, 5, 6, 7, 8, or more water-soluble polymers individually attached to the protein moiety. Any given water-soluble polymer may be covalently attached to an amino acid of the protein moiety or, if the protein moiety is (for example) a glycoprotein, to a carbohydrate of the protein moiety. Conjugation to carbohydrates can be performed using other suitable approaches, such as metabolic functionalization employing sialic acid-azide chemistry [Luchansky et al. (2004) Biochemistry 43(38):12358-123661] or using glycidol to facilitate the introduction of aldehyde groups [Heldt et al. (2007) European Journal of Organic Chemistry 32:5429-5433].

[0243] The particular linkage between the protein moiety and the polymer will depend on a number of factors, including, for example, the particular linking chemistry employed, the particular protein moiety, available functional groups within the protein moiety (either linker, attachment to the polymer, or conversion into a suitable attachment site), the presence of additional reactive functional groups within the protein moiety, etc.

[0244] The conjugates of the present disclosure may be prodrugs, meaning that the linkage between the polymer and the protein moiety is releasable to allow release of the parent moiety. In addition to the releasable linkers described in this disclosure, other exemplary releasable linkages include carboxylate esters, phosphate esters, thiol esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, and oligonucleotides. Such linkages can be readily prepared by appropriately modifying either the protein moiety (e.g., the carboxyl C-terminus of the protein, or the side chain hydroxyl groups of amino acids such as serine or threonine contained within the protein, or similar functional groups within carbohydrates) and / or the polymeric reagent using coupling methods commonly employed in the art. However, most preferred are releasable linkages readily formed by reacting a suitably activated polymer with an unmodified functional group contained within the protein moiety.

[0245] Alternatively, hydrolytically stable linkages such as amide, urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide) linkages can also be employed as linkages for coupling protein moieties. A preferred hydrolytically stable linkage is amide. In one approach, an amide linkage can be obtained by reacting a water-soluble polymer having an activated ester with an amine group on the protein moiety. Another preferred hydrolytically stable linkage is a thiol crosslink.

[0246] The conjugate (as opposed to the unconjugated protein moiety) may or may not have a measurable degree of protein activity. That is, a polymer-protein conjugate according to the present disclosure has anywhere from about 0.1% to about 100% of the biological activity of the unmodified parent protein moiety, e.g., about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 55%, or about 100%. In some cases, the polymer-protein conjugate may have greater than 100% of the biological activity of the unmodified parent protein moiety. Preferably, conjugates with little or no protein activity contain a hydrolyzable linkage connecting the polymer to the protein such that water-induced cleavage of the hydrolyzable linkage releases the active parent molecule (or a derivative thereof), despite the lack (or relative lack) of activity of the conjugate. Such activity can be determined using a suitable in vivo or in vitro model, depending on the known activity of the particular protein.

[0247] For conjugates having a hydrolytically stable linkage coupling the protein to the polymer, the conjugate typically has a measurable degree of biological activity. For example, such conjugates are typically characterized by having a biological activity that meets one or more of the following percentages compared to the unconjugated protein: at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 100%, and greater than 105% (as measured by a suitable model, such as one well known in the art). Preferably, conjugates having a hydrolytically stable linkage (e.g., an amide linkage, a thiol bridge) have at least some of the biological activity of the unmodified parent protein.

[0248] The bond between the protein and the water-soluble polymer via a linker may be direct, with no intervening atoms between the linker and the polymer, or indirect, with one or more atoms between the linker and the polymer. For indirect bonds, a "spacer moiety" may function as a linker between the residue of the linker and the water-soluble polymer. The one or more atoms constituting the spacer moiety may include one or more of a carbon atom, a nitrogen atom, a sulfur atom, an oxygen atom, and combinations thereof. The spacer moiety may include an amide, a secondary amine, a carbamate, a thioether, a disulfide group, and / or a click chemistry product group. Non-limiting examples of specific spacer moieties include -O-, -S-, -SS-, -C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -OC(O)-NH-, -C(S)-, -CH-, -CH-CH-, -CH-CH-CH-, -CH-CH-CH-CH-, -CH-CH-CH-CH-CH-, -CH-CH-CH-CH-CH-, O-CH-, -CH-O-, and -O-CH 2-CH2-, -CH2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2 -CH2-O-, -O-CH2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-CH2-CH2-O-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-C(O)-NH- , -C(O)-NH-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH -, -C(O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH 2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -C(O)-O-CH2-,-CH2-C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2-, -C(O)-O-CH2-CH2-, -NH-C(O)-CH2-, - CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -NH-C(O)-CH2-CH2-, -CH2-NH-C(O)-C H2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -OC(O)-NH-CH2-, -OC(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH 2-NH-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-, -OC(O)-NH-[CH2], l -(OCH2CH2) m -, divalent cycloalkyl group, -O-, -S-, amino acid, -N(R 3 )—and combinations of any two or more of the foregoing (wherein R 3 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl; (l) is 0 to 6; and (m) is 0 to 20. Another specific spacer moiety is selected from the group consisting of the following structure: -C(O)-NH-(CH2) 1-6 -NH-C(O)-, -NH-C(O)-NH-(CH2) 1-6 -NH-C(O)- and -OC(O)-NH-(CH2) 1-6-NH-C(O)-, where the subscript value after each methylene indicates the number of methylenes contained in the structure, e.g., (CH) 1-6 means that the structure may contain 1, 2, 3, 4, 5, or 6 methylenes. Additionally, any of the above spacer moieties may contain 1 to 20 ethylene oxide monomer units [i.e., -(CH2CH2O) 1-20

[0039] The spacer moiety may further comprise an ethylene oxide oligomer chain comprising:

[0040] , i.e., the ethylene oxide oligomer chain may be present before or after the spacer moiety, and optionally between any two atoms of a spacer moiety consisting of two or more atoms. Also, an oligomer chain is not considered part of the spacer moiety if the oligomer is adjacent to the polymer segment and is simply an extension of the polymer segment.

[0249] A typical protein-macromolecule conjugate has a structure according to formula (XX): Proteins (L-macromolecules) z (XX) or a stereoisomer, regioisomer, tautomer or mixture thereof, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. (In the formula, z is an integer from 1 to 25, L is a linker, the protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide; Macromolecules include water-soluble polymers, lipids, proteins, or polypeptides.

[0250] In some embodiments, the linker, L, is a linker of the present disclosure. In some embodiments, L is one or more non-releasable linkers and / or one or more releasable linkers. In some embodiments, one or more releasable linkers are derived from a bifunctional releasable linker of the present disclosure (e.g., a linker of Formula (I), Formula (II), Formula (III), or Formula (IV)) and / or a polymeric reagent comprising a releasable linker (e.g., Formula (V) or Formula (VI)).

[0251] In some embodiments, z is an integer from 1 to 20. In some embodiments, z is an integer from 1 to 15. In some embodiments, z is an integer from 1 to 10. In some embodiments, z is an integer from 1 to 8. In some embodiments, z is an integer from 1 to 5.

[0252] In some embodiments, when z is 2 or greater, each L-macromolecule attached to the protein is the same. In some embodiments, when z is 2 or greater, at least one L-macromolecule attached to the protein is different. In some embodiments, when z is 2 or greater, each L-macromolecule attached to the protein is different.

[0253] An exemplary protein-macromolecule conjugate of formula XX is encompassed within the following structure: [ka] (In the formula, n is an integer from 2 to 4000, X is a spacer moiety, RL is a releasable linker, z is an integer from 1 to 25, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0254] In some embodiments, R is a releasable linker of the present disclosure. In some embodiments, the releasable linker is derived from a bifunctional releasable linker (e.g., a linker of Formula (I), Formula (II), Formula (III), or Formula (IV)) or a polymeric reagent comprising a releasable linker (e.g., Formula (V) or Formula (VI)) disclosed herein.

[0255] In another embodiment, the exemplary protein-macromolecule conjugate of formula XX is encompassed within the following structure: [ka] (In the formula, n is an integer from 2 to 4000, X is a spacer moiety, RL 1 is the first releasable linker, RL 2 is a second releasable linker, z is an integer from 1 to 25, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide). Exemplary conjugates of the present disclosure in which the water-soluble polymer is in branched form include those in which the water-soluble polymer is encompassed within the following structure: [ka] wherein Y=O and NH, and each (n) is independently an integer having a value between 2 and 4000, e.g., 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 4000, including all values ​​and ranges therebetween.

[0256] Exemplary conjugates of the present disclosure in which the water-soluble polymer is in branched form include those in which the water-soluble polymer is encompassed within the following structure: [ka] wherein each (n) is independently an integer having a value between 2 and 4000, e.g., 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, or 4000, including all values ​​and ranges therebetween.

[0257] Exemplary protein-macromolecule conjugates formed using polymeric reagents that provide two releasable linkages include those of formula (XI): [ka] (In the formula, POLY 1 is a first water-soluble polymer, POLY 2 is a second water-soluble polymer, X 1 is the first spacer moiety, X 2 is a second spacer moiety, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, Y 4 is O or S, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 4 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 is an integer of 0 to 3, a2 is an integer of 0 to 3, c is an integer from 0 to 4, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R d is nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl or cycloalkyl, substituted alkyl or cycloalkyl, aryl or heteroaryl, substituted aryl or heteroaryl; -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0258] In some embodiments, R 1 , R 2 , R 3 , R 4 , R e1 , R e2 and R d is as defined above in formula (IV).

[0259] In some embodiments of Formula (XI), POLY 1 and POLY 2 are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY1 and POLY 2 are the same water-soluble polymer. 1 and POLY 2 are different water-soluble polymers.

[0260] In some embodiments of Formula (XI), X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2 are the same spacer moiety. 1 and X 2 are different spacer moieties.

[0261] An exemplary conjugate has the following structure (XI-A): [ka] (wherein n is independently an integer of 4 to 1500, and z is an integer of 1 to 25).

[0262] Other exemplary conjugates formed using polymeric reagents that provide two releasable linkages include those of formula (XII): [ka] (In the formula, POLY 1 is a first water-soluble polymer, POLY 2 is a second water-soluble polymer, X 1 is the first spacer moiety, X 2 is a second spacer moiety, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 is an integer of 0 to 3, a2 is an integer of 0 to 3, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is a second electron-modifying group, if present, R p is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0263] In some embodiments, R 1 , R 2 , R e1 , R e2 , and R p is as defined above in formula (VI).

[0264] In some embodiments of Formula (XII), POLY 1 and POLY 2 are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 1 and POLY 2 are the same water-soluble polymer. 1 and POLY 2 are different water-soluble polymers.

[0265] In some embodiments of Formula (XII), X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2 are the same spacer moiety. 1 and X 2 are different spacer moieties.

[0266] An exemplary conjugate has the following structure (XII-A): [ka] (wherein n is independently an integer of 4 to 1500, and z is an integer of 1 to 25).

[0267] Exemplary conjugates formed using click chemistry with suitable polymeric reagents include those of formula (XIII): [ka] (In the formula, POLY 1 is a first linear or branched water-soluble polymer; POLY 2 is a second linear or branched water-soluble polymer, X 1 is the first spacer moiety or -X-FG 2 and X 2 is a second spacer moiety, if present, T 1 is a first triazole functional group, T 2 is a second triazole functional group, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R e is an electron modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, and -X-FG 2 (In the formula, X is a spacer moiety, FG 2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups); a is an integer from 0 to 5, b is an integer from 0 to 3, c is an integer from 0 to 2, z is an integer from 1 to 25, Y 1 is O or S, Y 2 is O or S, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0268] In some embodiments, R 1 , R 2 , R e , a, b, c, and z are as defined above in formula (I).

[0269] In some embodiments of Formula (XIII), POLY 1 and POLY 2are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 1 and POLY 2 are the same water-soluble polymer. 1 and POLY 2 are different water-soluble polymers.

[0270] In some embodiments of Formula (XIII), X 1 and X 2 are each independently selected from spacer moieties described herein. 1 and X 2 are the same spacer moiety. 1 and X 2 are different spacer moieties.

[0271] Conjugates of formula (XIII) having a more defined structure are contemplated as formula (XIII-A), (XIII-B), (XIII-C), or (XIII-D): [ka] (In the formula, X 1 is a first spacer moiety, and each of X 2 is the second spacer moiety, and POLY 1 , POLY 2 , T 1 , T 2 , R 1 , R 2 , R e , a, z, Y 1 , Y 2 , and protein is as defined above).

[0272] In certain embodiments of Formula (XIII), (XIII-A), (XIII-B), (XIII-C), or (XIII-D), a is an integer from 0 to 2, and R 1 and R 2are each independently hydrogen, Me, or Et; R e is nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0273] A further exemplary conjugate has the following structure (XIII-A1): [ka] (In the formula, R e is an electron modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; n is independently an integer from 4 to 1500; z is an integer from 1 to 25; and "-NH-" is an amine group of a residue in the protein, representing one or more polymers individually attached to the protein. In certain embodiments, a is an integer from 1 to 2, and R e is 4-F, 4-Cl, 4-CF3, 2,4-difluoro, or 2-CF3-4-F substituted.

[0274] Further exemplary conjugates have the following structure of (XIII-B1), (XIII-C1), (XIII-D1), or (XIII-D2): [ka] [ka] [ka] (In the formula, n is independently an integer from 4 to 1500; z is an integer from 1 to 25, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0275] Other exemplary conjugates formed using click chemistry with suitable polymeric reagents include those of formula (XIV): [ka] (In the formula, POLY 2 is a linear or branched water-soluble polymer, POLY 3 is a linear or branched water-soluble polymer, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is the second electron-modifying group, if present, or -X-FG 2 (In the formula, X is a spacer moiety, FG 2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups); X 2 is a spacer moiety, if present, X3 is a spacer moiety, if present, T 2 is a triazole functional group, T 3 is a triazole functional group, Y 1 is O or S, Y 2 is O or S, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0276] In some embodiments, R 1 , R 2 , R e1 , and R e2 is as defined above in formula (VI).

[0277] In some embodiments of Formula (XIV), POLY 2 and POLY 3 are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 2 and POLY 3 are the same water-soluble polymer. 2 and POLY 3 are different water-soluble polymers.

[0278] In some embodiments of Formula (XIV), X 2 and X 3 are each independently selected from spacer moieties described herein. 2 and X 3 are the same spacer moiety. 2 and X 3 are different spacer moieties.

[0279] In certain embodiments of Formula (XIV), a1 and a2 are each independently an integer from 0 to 2, and R 1 and R 2 are each independently hydrogen, Me, or Et; R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF.

[0280] A conjugate of formula (XIV) having a more defined structure is contemplated as formula (XIV-A): [ka] (wherein n is independently an integer from 4 to 1500, z is an integer from 1 to 25, and -NH- is an amine group of a residue in a protein).

[0281] Other exemplary conjugates formed using click chemistry with suitable polymeric reagents include those of formula (XV): [ka] (In the formula, POLY 2 is a linear or branched water-soluble polymer, POLY 3 is a linear or branched water-soluble polymer, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is the second electron-modifying group, if present, or -X-FG 2 (In the formula, X is a spacer moiety, FG 2 are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups); R p is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, T 2 is a triazole functional group, T 3 is a triazole functional group, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0282] In some embodiments, R 1 , R 2 , R p , R e1 , and R e2 is as defined above in formula (VI).

[0283] In some embodiments of Formula (XV), POLY 2 and POLY 3 are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 2 and POLY 3 are the same water-soluble polymer. 2 and POLY 3 are different water-soluble polymers.

[0284] In some embodiments of Formula (XV), X 2 and X 3 are each independently selected from spacer moieties described herein. 2 and X 3 are the same spacer moiety. 2 and X 3 are different spacer moieties.

[0285] In certain embodiments of Formula (XV), a1 and a2 are each independently an integer from 0 to 2, and R 1 and R 2 are each independently hydrogen, Me, or Et; R e1 and R e2 are each independently nitro, cyano, halogen, —CF3, —CONHMe, —SO2NHMe, —OMe, —NHMe, —NHAc, —NHSO2Me, or —OCF3.

[0286] A more defined structure of the conjugate of formula (XV) is shown below in formula (XV-A): [ka] (wherein n is independently an integer from 4 to 1500, z is an integer from 1 to 25, and -NH- is an amine group of a residue in a protein).

[0287] Other exemplary conjugates formed using click chemistry with suitable polymeric reagents include those of formula (XVI): [ka] (In the formula, POLY 2 is a linear or branched water-soluble polymer, POLY 3 is a linear or branched water-soluble polymer, R 1 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 3 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R 4 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; a1 and a2 each independently represent an integer of 0 to 4, b1 is 1, b2 is an integer from 0 to 1, c is an integer from 0 to 4, z is an integer from 1 to 25, R e1 is the first electron modifying group, if present; R e2 is the second electron-modifying group, if present, or -X-FG 2 (In the formula, X is a spacer moiety, FG 2are functional groups capable of reacting via click chemistry, independently, for example, but not limited to, azido, alkynyl, and cycloalkynyl (e.g., dibenzocyclooctyne (DBCO)) groups); R d is nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl or cycloalkyl, substituted alkyl or cycloalkyl, aryl or heteroaryl, substituted aryl or heteroaryl; X 2 is a spacer moiety, if present, X 3 is a spacer moiety, if present, T 2 is a triazole functional group, T 3 is a triazole functional group, Y 1 is O or S, Y 2 is O or S, Y 3 is O or S, Y 4 is O or S, -NH- is the amine group of a residue in a protein, The protein is a chemokine, a chemokine antagonist, a cytokine, a cytokine antagonist, an antibody, or a therapeutic peptide).

[0288] In some embodiments, R 1 , R 2 , R 3 , R 4 , R d , R e1 , and R e2 is as defined above in formula (IV).

[0289] In some embodiments of Formula (XVI), POLY 2 and POLY 3are each independently selected from the water-soluble polymers described herein. In some embodiments, POLY 2 and POLY 3 are the same water-soluble polymer. 2 and POLY 3 are different water-soluble polymers.

[0290] In some embodiments of Formula (XVI), X 2 and X 3 are each independently selected from spacer moieties described herein. 2 and X 3 are the same spacer moiety. 2 and X 3 are different spacer moieties. In some embodiments, the protein is a cytokine. Cytokines include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, or TNF-β. In certain embodiments, the cytokine is IL-2. In certain embodiments, the IL-2 comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0291] In some embodiments, the protein is a chemokine, including MCP-1, MCP-2, MCP-3, MCP-24, MCP-5, CXCL76, I-309 (CCL1), BCA1 (CXCL13), MIG, SDF-1 / PBSF, IP-10, I-TAC, MIP-1α, MIP-1β, RANTES, eotaxin-1, eotaxin-2, GCP-2, Gro-α, Gro-β, Gro-γ, LARC (CCL20), ELC (CCL19), SLC (CCL21), ENA-78, PBP, TECK (CCL25), CTACK (CCL27), MEC, XCL1, XCL2, HCC-1, HCC-2, HCC-3, or HCC-4.

[0292] In some embodiments, the protein is an antibody. The antibody may be an antibody to angiopoietin 2, AXL, ACVR2B, angiopoietin 3, activin receptor-like kinase 1, amyloid A protein, β-amyloid, AOC3, BAFF, BAFF-R, B7-H3, BCMAC, A-125 (mimetic), C5, CA-125, CCL11 (eotaxin-1), CEA, CSF1R, CD2, CD3, CD4, CD6, CD15, CD19, CD20, CD22, CD23, CD25, CD28, CD3 0, CD33, CD37, CD38, CD40, CD41, CD44, CD51, CD52, CD54, CD56, CD70, CD74, CD97B, CD125, D134, CD147, CD152, CD154, CD279, CD221, C242 antigen, CD276, CD278, CD319, Clostridium difficile, claudin-18 isoform 2, CSF1R, CEACAM5, CSF2, carbonic anhydrase 9, CLDN18.2, cardiac myosin, CCR4, CGRP, coagulation factor III, c-Met, CTLA-4, DPP4, DR5, DLL3, DLL4, dabigatran, EpCAM, Ebola virus glycoprotein, endoglin, episialin, EPHA3, c-Met, FGFR2, fibrin II beta chain, FGF23, folate receptor 1, GMCSF, GD2 ganglioside, GDF-8, GCGR, gelatinase B, glypican 3, GPNMB, GMCSF receptor α-chain, kallikrein, KIR2D, ICAM-1, ICOS, IGF1, IGF2, IGF-1 receptor, IL-1α, IL-1β, IL-2, IL-4Rα, IL-5, IL-6, IL-6R, IL-9, IL-12, IL-13, IL17A, IL17F, IL-2 0, IL-22, IL-23, IL-31, IFN-α, IFN-β, IFN-γ, integrin α4β7, interferon α / β receptor, influenza A hemagglutinin, ILGF2, HER1, HER2, HER3, HHGFR, HGF, HLA-DR, hepatitis B surface antigen, HNGF, Hsp90, HGFR, L-selectin, Lewis-Y antigen, LYPD3, LOXL2, LIV-1, MUC1, MCP-1, MSLN, mesothelin, MIF, MCAM, NCA-90, NCA-90Notch1, nectin-4, PCDP1, PD-L1, PD-1, PCSK9, PTK7, PCDC1, phosphatidylserine, RANKL, RTN4, Rh factor, ROR1, SLAMF7, Staphylococcus The therapeutic agent can target one or more of Staphylococcus aureus alpha toxin, Staphylococcus aureus two-component leukocidin, SOST, selectin P, SLITRK6, SDC1, TFPI, TRAIL-R2, tumor antigen CTAA16.88, TNF-α, TWEAK receptor, TNFRSF8, TYRP1, tau protein, TAG-72, TSLP, TRAIL-R1, TRAIL-R2, TGF-β, TAG-72, TRAP, TIGIT, tenascin-C, OX-40, VEGF-A, VWF, VEGFR1, or VEGFR2.

[0293] In some embodiments, the protein is a therapeutic peptide. Peptides include glucagon-like peptide 1 (GLP-1), exendin-2, exendin-3, exendin-4, atrial natriuretic factor (ANF), ghrelin, vasopressin, growth hormone, growth hormone-releasing hormone (GHRH), RC-3095, somatostatin, bombesin, PCK-3145, Phe-His-Ser-Cys-Asn (PHSCN), IGF1, B-type natriuretic peptide, peptide YY (PYY), interferon, and the like. Serone, thrombospondin, angiopoietin, calcitonin, gonadotropin-releasing hormone, hirudin, glucagon, anti-TNF-alpha, fibroblast growth factor, granulocyte colony-stimulating factor, obienepitide, parathyroid hormone (PTH), leuprolide, sermorelin, pramorelin, nesiritide, rotigaptide, cilengitide, MBP-8298, AL-108, enfuvirtide, thymalfasin, daptamycin, HLFI-II, lactoferrin, dextromethorphan Lumitide, glutathione, T cell epitope PR1, protease-3 peptide 1-11, B cell epitope P3, luteinizing hormone-releasing hormone (LHRH), substance P, neurokinin A, neurokinin B, CCK-8, enkephalins such as leucine enkephalin and methionine enkephalin, dermaseptin, [des-Ala20,Gln34]-dermaseptin, surfactant-related antibacterial anionic peptide, apidecin IA; apide Syn-IB; OV-2; 1025, acetyl-adhesin peptide (1025-1044) amide; telome-cin (49-63); pexiganan (MSI-78); indolicidin; apelin-15 (63-77); CFP1O (71-85); anthrax-associated lethal factor (LF) inhibitor; bactenecin; hepatitis C virus NS3 protease inhibitor 2; hepatitis C virus NS3 protease inhibitor 3; hepatitis virus NS3 protease inhibitor 4; NS4A-NS4B hepatitis C virus (NS3 protease inhibitor I); HIV-1, HIV-2 protease substrate; anti-FM peptide; Bak-BH3; Bax BH3 peptide (55-74) (wild type); Bid BH3-r8; CTT (gelatinase inhibitor); E75 (Her-2 / neu) (369-377);GRP78-binding chimeric peptide motif; p53(17-26); EGFR2 / KDR antagonist; Colivelin AGA-(C8R)HNGl7 (humanin derivative); activity-dependent neurotrophic factor (ADNF); beta-secretase inhibitor I; beta-secretase inhibitor 2; ch[beta]-amyloid(30-16); humanun (HN)sHNG, [Glyl4]-HN, [Glyl4]-humanin; angiotensin-converting enzyme inhibitor (BPP); renin inhibitor III; annexin I (ANXA-I; Ac2-12); anti-inflammatory peptide I; anti-inflammatory peptide 2; anti-inflammatory apelin 12; [D-Phel2,Leul4]-bombesin; antennapedia peptide (acid) (penetratin); antennapedia leader peptide (CT) ;Mastoparan;[Thr28,Nle31]-Cholecystokinin(25-33) Sulfated;Nociceptin(1-13)(Amide);Fibrinolysis Inhibitor;Gamma-Fibrinogen(377-395);Xenin;Obestatin(Human);[Hisl,Lys6]-GHRP(GHRP-6);[Ala5,[beta]-Ala8]-Neurokinin A(4-10);Neuromedin B;Neuromedin C;Neuromedin N;Activity-Dependent Neurotrophic Factor (ADNF-14);Acetarin I (Opioid Receptor Antagonist I);Acetarin 2 (Opioid Receptor Antagonist 2);Acetarin 3 (Opioid Receptor Antagonist 3);ACTH(1-39)(Human);ACTH(7-38)(Human);Sauvagine;Adipokinetic Hormone(Locusta Migratoria); myristoylated ADP-ribosylation factor 6, myr-ARF6(2-13); PAMP(1-20) (proadrenomedullin(1-20) human); AGRP(25-51); amylin(8-37) (human); angiotensin I (human); angiotensin II (human); apstatin (aminopeptidase P inhibitor); brevinin-I; magainin I; RL-37; LL-37 (antimicrobial peptide) (human); cecropin A; antioxidant peptide A; antioxidant peptide B; L-camocin; BcI9-2; NPVF; neuropeptide AF (hNPAF) (human); Bax BH3 peptide(55-74); bFGF inhibitory peptide; bFGF inhibitory peptide II; bradykinin;[Des-Argl OJ-HOE140; caspase I inhibitor II; caspase I inhibitor VIII; Smac N7 protein (MEKl-derived peptide inhibitor I); hBD-1 ([beta]-defensin-1) (human); hBD-3 ([beta]-defensin-3) (human); hBD-4 ([beta]-defensin-4) (human); HNP-I (defensin human neutrophil peptide I); HNP-2 (defensin human neutrophil peptide 2 dynorphin A(1-17)); endomorphin-I; [beta]-endorphin (human porcine); endothelin 2 (human); fibrinogen binding inhibitor peptide; cyclo(-GRGDSP); TP508 (thrombin-derived peptide); galanin (human); GIP (human); gastrin-releasing peptide (human); gastrin-1 (human); ghrelin (human); PDGF-BB peptide; [D-Lys3]-GHRP-6; HCV core protein (1-20); a3Bl integrin peptide fragment (325) (amide); laminin pentapeptide (amide) Mel -Anotropin-potentiating factor (MPF); VA-[beta]-MSH, lipotropin-Y (derived from proopiomelanocortin); atrial natriuretic peptide (1-28) (human); vasonatriuretic peptide (1-27); [Ala5,B-Ala8]-neurokinin A (4-10); neuromedin L (NKA); Ac-(Leu28,31)-neuropeptide Y (24-26); allitesin; brain neuropeptide II; [D-tyrll]-neurotensin; IKKy NEMO-binding domain (NBD) inhibitory peptide; PTD-p50 (NLS) inhibitory peptide; orexin A (bovine, human, mouse, rat); orexin B (human); aquaporin-2 (254-267) (human pancreastatin) (37-52); pancreatic polypeptide (human); neuropeptides; peptide YY (3-36) (human); hydroxymethyl-phytochelatin 2; PACAP (I-27) (amide, human, bovine, rat); prolactin-releasing peptide (1-31) (human); sarcin-alpha; sarcin-beta; saposin C22; secretin (human); L-selectin; endokinin A / B; endokinin C (human); endokinin D (human); thrombin receptor (42-48) agonist (human);LSKL (thrombospondin inhibitor); thyrotropin-releasing hormone (TRH); P55-TNFR fragment; urotensin II (human); VIP (human, porcine, rat); VIP antagonist; helodermin; exenatide; ZPlO (AVEOOIOO); pramlintide; AC162352 (PYY)(3-36); PYY; obienepitide; glucagon; GRP; ghrelin (GHRP6); leuprolide; histrelin; oxytocin; atosiban (RWJ22164); sermorelin; nesiritide; bivalirudin (Hirulog); Icatibant; Aviptadine; Rotigaptide (ZP123, GAP486); Cilengitide (EMD-121924, RGD peptide); AlbuBNP; BN-054; Angiotensin II; MBP-8298; Peptide leucine arginine; Ziconotide; AL-208; AL-108; Carbeticone; Tripeptide; SAL; Colliven; Humanin; ADNF-14; VIP (vasoactive intestinal peptide); Thymalfasin; Bacitracin; Gramidicin; Pexiganan (MSI-78); Pl 13;PAC-113;SCV-07;HLFl-Il (lactoferrin);DAPTA;TRI-1144;tritorpticin;antiflamin 2;Gatex (teduglutide, ALX-0600);Stimuvax (L-BLP25);Crysalin (TP508);Melanonan II;Spantide II;Ceruletide;Sincalide;Pentagastin;Secretin;Endostatin peptide;E-selectin;HER2;IL-6;IL-8;IL-10;PDGF;Thrombospondin;uPA(I);uPA(2);VEGF;VEGF(2);Pentapeptide-3;XX LRR; beta-amyloid fibril formation; endomorphin-2; TIP39 (infundibular pituitary neuropeptide); PACAP(1-38) (amide, human, bovine, rat); TGFB-activating peptide; insulin-sensitizing factor (ISF402); transforming growth factor B1 peptide (TGF-B1); caerulein-releasing factor; IELLQAR (8-branched MAPS); tigapotide PK3145; goserelin; abarelix; cetrorelix; ganirelix; degarelix (triptorelin); barsiban (FE200440); pralmorelin; octreotide; eptifibatide;Examples of suitable proteins for use in the present disclosure include, but are not limited to, netamiftide (INN-00835); daptamycin; spantide II; dermitide (RDP-58); AL-209; enfuvirtide; IDR-I; hexapeptide-6; insulin A chain; lanreotide; hexa[rho]eptide-3; insulin B chain; glargine A chain; glargine B chain; insulin-LisPro B chain analog; insulin-aspart B chain analog; insulin-gululisine B chain analog; insulin-detemir B chain analog; somatostatin tumor inhibitor analog; pancreastatin (37-52); vasoactive intestinal peptide fragment (KKYL-NH2); and dynorphin A. Examples of suitable proteins for use in the present disclosure include, but are not limited to, immunotoxin SSIP, adenosine deaminase, argininase, and the like.

[0294] IL-2-Macromolecule Conjugates Turning to one or more embodiments of the present disclosure, more specific protein-macromolecule conjugates are provided, which comprise residues of an IL-2 moiety covalently attached via linkers to a plurality of water-soluble polymers. The conjugates of the present disclosure have one or more of the following characteristics:

[0295] IL-2 part As previously mentioned, conjugates generally comprise a residue of an IL-2 moiety covalently attached to one or more water-soluble polymers via a releasable or non-releasable linker. As used herein, the term "IL-2 moiety" refers to the IL-2 moiety prior to conjugation and to the IL-2 moiety after attachment to a water-soluble polymer. However, it will be understood that when the native IL-2 moiety is attached to a water-soluble polymer, the IL-2 moiety is slightly altered due to the presence of one or more covalent bonds associated with attachment to the polymer(s). Often, the slightly altered IL-2 moiety attached to another molecule is referred to as a "residue" of the IL-2 moiety.

[0296] The IL-2 moiety can be derived from non-recombinant or recombinant methods, and the disclosure is not limited in this respect. In addition, the IL-2 moiety can be derived from human, animal, and plant sources.

[0297] Any IL-2 moiety obtained by non-recombinant and recombinant approaches can be used as the IL-2 moiety in preparing the conjugates described herein.

[0298] Depending on the system used to express the protein having IL-2 activity, the IL-2 moiety may be either unglycosylated or glycosylated, and either may be used. That is, the IL-2 moiety may be unglycosylated, or the IL-2 moiety may be glycosylated. In one or more embodiments of the present disclosure, the IL-2 moiety is unglycosylated.

[0299] The IL-2 moiety can be advantageously modified to include and / or substitute one or more amino acid residues, such as, for example, lysine, cysteine, histidine, and / or arginine, thereby facilitating attachment of the polymer to atoms in the side chain of the amino acid. An example of an IL-2 moiety substitution is described in U.S. Pat. No. 5,206,344. Additionally, the IL-2 moiety can be modified to include a non-natural amino acid residue. An example of an IL-2 moiety substitution with a non-natural amino acid residue is described in WO 2019 / 028419. Techniques for adding amino acid residues and non-natural amino acid residues are well known to those skilled in the art. See J. March, Advanced Organic IL-2 Ministry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).

[0300] In addition, the IL-2 moiety may advantageously be modified to include the attachment of a functional group (other than by the addition of an amino acid residue containing a functional group). For example, the IL-2 moiety may be modified to include a thiol group. In addition, the IL-2 moiety may be modified to include an N-terminal alpha carbon. In addition, the IL-2 moiety may be modified to include one or more carbohydrate moieties. In addition, the IL-2 moiety may be modified to include an aldehyde group. In addition, the IL-2 moiety may be modified to include a ketone group. In certain embodiments of the present disclosure, the IL-2 moiety is preferably modified to exclude one or more of a thiol group, an N-terminal alpha carbon, a carbohydrate, an aldehyde group, and a ketone group.

[0301] Exemplary IL-2 moieties are described in the literature, as well as in, for example, U.S. Patent Nos. 5,116,943, 5,153,310, 5,635,597, 7,101,965, and 7,567,215 and U.S. Patent Application Publication Nos. 2010 / 0036097 and 2004 / 0175337. A preferred IL-2 moiety has an amino acid sequence corresponding to FIG.

[0302] In some cases, the IL-2 moiety may be in the form of a "monomer," in which a single expression of the corresponding peptide is organized into a separate unit. In other cases, the IL-2 moiety may be in the form of a "dimer," in which two monomeric forms of the protein are associated with each other (e.g., by a disulfide bond) (e.g., a dimer of recombinant IL-2). For example, in the context of a dimer of recombinant human IL-2, the dimer may be in the form of two monomers associated with each other by a disulfide bond formed from the Cys125 residue of each monomer.

[0303] In addition, precursor forms of IL-2 can be used as IL-2 moieties. Truncated forms, hybrid variants, and peptidomimetics of any of the foregoing sequences can also function as IL-2 moieties. Biologically active fragments, deletion variants, substitution variants, or addition variants of any of the foregoing that maintain at least some IL-2 activity can also function as IL-2 moieties.

[0304] For any given peptide or protein moiety, it is possible to determine whether the moiety has IL-2 activity. Various methods for determining IL-2 activity in vitro have been described in the art. An exemplary approach is the CTLL-2 cell proliferation assay described in the following experiment. An exemplary approach is described in Moreau et al. (1995) Mol. Immunol. 32:1047-1056). Other methods known in the art can also be used to assess IL-2 function, including electrometric, spectrophotometric, chromatographic, and radiometric methods.

[0305] More specific exemplary conjugates according to the present disclosure are described below. Typically, such IL-2 moieties are expected to share (at least in part) a similar amino acid sequence to the sequence provided in Figure 1. Thus, when reference is made to a particular position or atom within the sequence of Figure 1, such reference is for convenience only, and one of ordinary skill in the art will be able to readily determine the corresponding position or atom in other moieties having IL-2 activity. In particular, the descriptions provided herein with respect to native human IL-2 often also apply to fragments, deletion variants, substitution variants, or addition variants of any of the foregoing.

[0306] Conjugate Assembly The amino group of the IL-2 moiety provides the point of attachment between the IL-2 moiety and the water-soluble polymer. Using the amino acid sequence provided in Figure 1, it is clear that there are several lysine residues, each with an ε-amino acid, that may be available for conjugation. Additionally, the N-terminal amine of any protein can also serve as a point of attachment.

[0307] There are numerous examples of suitable reagents useful for forming covalent, releasable linkages with available amines on an IL-2 moiety. Specific, non-limiting examples, along with the corresponding conjugates, are provided in Table 1 below. In the table, the variable "n" represents the number of repeating monomeric units, z is an integer between 1 and 25, and "-NH-IL-2" represents the residue of the IL-2 moiety after conjugation to a polymeric reagent or linker to form one or more water-soluble polymers individually attached to the IL-2 moiety, or one or more linkers individually attached to the IL-2 moiety. Each polymeric moiety shown in Table 1 [e.g., (OCH2CH2) n or (CH2CH2O) n ] terminates in a "CH3" group, although other groups (such as H and benzyl) may be substituted. [Table 1-1] [Table 1-2]

[0308] Conjugation of a reagent to the amino group of the IL-2 moiety can be achieved by a variety of techniques. In one approach, the IL-2 moiety can be conjugated to a coupling reagent functionalized with a succinimidyl derivative (or other activated ester group; in this case, a similar approach to that described for reagents containing such alternative activated ester groups can be used). In this approach, a reagent bearing a succinimidyl derivative can be coupled to the IL-2 moiety in aqueous media at pH 7-9.0; however, using different reaction conditions (e.g., a lower pH, such as 6-7, or a different temperature and / or below 15°C), the reagent can be coupled to a different position on the IL-2 moiety.

[0309] Because there are multiple amino sites on IL-2, more than one functionalization of the IL-2 moiety can be achieved with the coupling reagents of the present disclosure by using an excess of equivalents of the reagent. Conjugates with multiple amino groups on the IL-2 moiety require a very high equivalent weight of the polymeric reagent (e.g., 100 equivalents). High functionalization of the IL-2 moiety can be achieved more efficiently by using a bifunctional linker reagent.

[0310] The bifunctional linker reagent generally has a succinimidyl derivative and a reactive group suitable for click chemistry. Functionalization of the IL-2 moiety can be achieved by conjugation of the bifunctional reagent to the amino group of the IL-2 moiety via NHS coupling. Then, click chemistry with a suitable polymeric reagent can yield highly polymer-derivatized IL-2. Some non-limiting examples, along with the corresponding conjugates, are provided in Table 2 below. In the table, the variable (n) represents the number of repeating monomer units, z is an integer between 1 and 25, and "-NH-IL-2" represents a residue of IL-2 to which one or more water-soluble polymers are individually attached. Each polymeric moiety shown in Table 2 [e.g., (OCH2CH2) n or (CH2CH2O) n ] terminates in a "CH3" group, although other groups (such as H and benzyl) may be substituted. [Table 2-1] [Table 2-2]

[0311] Click chemistry is employed for site-specific PEGylation, which is achieved by incorporating an azide-containing unnatural amino acid, i.e., homoazidoalanine, into recombinant proteins, which allows for site-specific conjugation with alkyne-PEG molecules.

[0312] One of the major drawbacks of Cu-catalyzed click reactions is the need for highly toxic Cu(I) and Cu(II). Even small amounts of copper can damage proteins, especially fluorescent proteins such as GFP. In addition, the presence of reducing agents, ligands, and oxygen-free conditions may be required.

[0313] A method to achieve site-specific PEGylation with efficiency comparable to Cu-catalyzed click reactions while maintaining protein viability is the introduction of cyclooctynes, where the strain of the eight-membered ring allows the reaction with azides to occur at 4 °C or room temperature in the absence of a catalyst. Dibenzylcyclooctyne, or DBCO, belongs to this family of reactive cyclooctynes.

[0314] The DBCO-PEG molecule enables Cu-free PEGylation of azide-containing proteins under mild reaction conditions, and the covalent attachment of PEG molecules to azide residues is efficient and highly site-specific, inheriting the selectivity of click chemistry.

[0315] Using click-PEGylation, multiple azide-functionalized IL-2 (IL-2-linker conjugates) could be converted to multiple PEGylated conjugates (IL-2-polymer conjugates) with high efficiency. When the click reaction occurs between an azide and an unsymmetrical 1,2-disubstituted alkyne such as DBCO, those skilled in the art will understand that two regioisomeric compounds can be obtained as products. Regioisomers differ in the position of the C-N bond formed.

[0316] The thiol group contained within the IL-2 moiety can serve as an effective attachment site for a water-soluble polymer. The IL-2 moiety contains one solvent-exposed disulfide, which typically contributes to the stability of the protein, but not its structure or function. As reported in Bioconjugate Chem. 2007, 18, 61-76, mild reduction of the exposed native disulfide bond to liberate the cysteine ​​thiol can be followed by PEGylation with a bis(thiol)-specific reagent. This crosslinks the two cysteine ​​thiols to which PEG is attached.

[0317] A representative conjugate according to the present disclosure using thiol cross-linking PEGylation may comprise the following formula (XVII): [ka] or a stereoisomer, tautomer or mixture thereof, positional isomer or mixture thereof, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof. (wherein X is a spacer moiety, POLY is a linear or branched water-soluble polymer, and "-S-" is a sulfur group of a residue within the IL-2 moiety.) In certain embodiments, the water-soluble polymer is poly(ethylene glycol).

[0318] With respect to the polymeric reagents, those described herein and elsewhere can be purchased from commercial sources or prepared from commercially available starting materials. In addition, methods for preparing the polymeric reagents are described in the literature.

[0319] Click Chemistry Certain embodiments of the conjugates, linkers, and formulas disclosed herein include functional groups capable of reacting via click chemistry. As used herein, click chemistry refers to the 1,3-dipolar cycloaddition or [3 + 2] cycloaddition between an azide and an alkyne to form a 1,2,3-triazole. The terms "1,3-dipolar cycloaddition" and "[3 + 2] cycloaddition" also encompass the "copper-free" 1,3-dipolar cycloaddition between an azide and a cyclooctyne.

[0320] Thus, unless otherwise specified, the description of any triazole compound herein is meant to include positional isomers of the compound and mixtures thereof.

[0321] For example, the [3+2] cycloaddition of an azide with an alkyne can generate two regioisomeric triazoles as follows: [ka]

[0322] In certain embodiments, the alkyne is a strained cycloalkynyl or heterocycloalkynyl, and the cycloaddition reaction can be carried out in the presence or absence of a catalyst. In certain embodiments, for example, the cycloaddition reaction can occur spontaneously through a reaction called strain-promoted azide-alkyne cycloaddition (SPAAC), known in the art as "metal-free click chemistry." In certain embodiments, the strained cycloalkynyl or heterocycloalkynyl is as described herein.

[0323] Such catalyst-free [3 + 2] cycloadditions can be used in the methods described herein to form the conjugates of the present disclosure. Alkynes can be activated by ring strain, such as, by way of example only, an eight-membered ring structure, and an electron-withdrawing group can be added to the alkyne ring. Alternatively, alkynes can be activated by adding a Lewis acid, such as Au(l) or Au(lll). Ring-strain activated alkynes have been described, for example, cyclooctynes ​​and difluorocyclooctynes ​​described by Agard et al., J. Am. Chem. Soc., 2004, 126(46):15046-15047; dibenzocyclooctynes ​​described by Boon et al., WO 2009 / 067663 A1 (2009); and aza-dibenzocyclooctynes ​​described by Debets et al., Chem. Comm., 2010, 46:97-99.

[0324] In certain embodiments, the conjugates of the present disclosure can be obtained by reacting a functionalized macromolecule containing an alkyne group with a functionalized protein containing an azide group to form the conjugate, as described herein. In other embodiments, the functionalized protein can have an activated alkyne moiety and the functionalized macromolecule has an azide moiety.

[0325] In certain embodiments, the functionalized macromolecule is a functionalized PEG. In certain embodiments, the functionalized protein is a functionalized IL-2. In certain embodiments, an azide in the functionalized IL-2 reacts with an alkyne in the functionalized PEG to form a triazole moiety (e.g., via 1,3-dipolar cycloaddition). In certain embodiments, an azide in the functionalized PEG reacts with an alkyne in the functionalized IL-2 to form a triazole moiety.

[0326] In certain embodiments, the click chemistry product group of the present disclosure comprises a triazole group.

[0327] In certain embodiments, the click chemistry product group is [ka] is selected from the group consisting of:

[0328] In certain embodiments of the compounds, conjugates, and formulas disclosed herein, T is selected from the following: [ka]

[0329] In certain embodiments of the compounds, conjugates, and formulas disclosed herein that include a triazole functional group (T), the triazole functional group may exist as a mixture of positional isomers, such that the compound, or conjugate, exists as a mixture of positional isomers.

[0330] As used herein, [ka] The structure represents a mixture of positional isomers of the following structures: [ka]

[0331] When the conjugate provided herein contains an acidic or basic moiety, it can also be provided as a pharmaceutically acceptable salt.See Berge et al., J.Pharm.Sci.1977,66,1-19; Handbook of Pharmaceutical Salts: Properties, Selection, and Use, 2nd ed.; Stahl and Wermuth Eds.; John Wiley & Sons, 2011.In certain embodiments, the pharmaceutically acceptable salt of the compound provided herein is a solvate.In certain embodiments, the pharmaceutically acceptable salt of the compound provided herein is a hydrate.

[0332] Suitable salts to be used in the preparation of pharmaceutically acceptable salts of the compounds provided herein include acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, These include, but are not limited to, α-oxoglutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (−)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid.

[0333] Suitable bases used in preparing the pharmaceutically acceptable salts of the compounds provided herein include inorganic bases such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and primary, secondary, tertiary, and quaternary aliphatic and aromatic amines, including, but not limited to, L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, methyl ... and tromethamine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, 1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, triethanolamine, trimethylamine, triethylamine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, and the like.

[0334] The conjugates provided herein are functional derivatives of compounds and may also be provided as prodrugs that are readily convertible to the parent compound in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. For example, a prodrug may be orally bioavailable, whereas the parent compound is not. A prodrug may also have improved solubility in pharmaceutical compositions compared to the parent compound. A prodrug may be converted to the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis.

[0335] Pharmaceutical Composition A conjugate is typically part of a composition. Generally, a composition comprises multiple conjugates. In certain embodiments, each conjugate is composed of the same protein (i.e., only one type of protein is present in the overall composition). In addition, a composition may comprise multiple conjugates, where any given conjugate is composed of a moiety selected from the group consisting of two or more different proteins (i.e., two or more different proteins are present in the overall composition). In other embodiments, substantially all of the conjugates in the composition (e.g., 85% or more of the multiple conjugates in the composition) each comprise the same protein. More specifically, the protein is IL-2.

[0336] The composition can include a single conjugate species (e.g., a mono-PEGylated conjugate, in which a single polymer is attached to the same position for substantially all conjugates in the composition) or a mixture of conjugate species (e.g., a mixture of mono-PEGylated conjugates and / or a mixture of mono-, di-, tri-, and multi-PEGylated conjugates, in which the polymer attachment occurs at different sites). The composition can also include other conjugates having 4, 5, 6, 7, 8, or more polymers attached to any given protein. In addition, the present disclosure includes compositions comprising multiple conjugates, each conjugate comprising one water-soluble polymer covalently attached to a protein, as well as compositions comprising 2, 3, 4, 5, 6, 7, 8, or more water-soluble polymers covalently attached to a protein. More specifically, the protein is IL-2.

[0337] With respect to the conjugates in the composition, the composition generally meets one or more of the following characteristics: at least about 85% of the conjugates in the composition have 1-10 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-9 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-8 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-7 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-6 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-5 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-4 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-3 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-4 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1-3 polymers attached to the protein. at least about 95% of the conjugates in the composition have 1 to 2 polymers attached to the protein; at least about 85% of the conjugates in the composition have 1 polymer attached to the protein; at least about 95% of the conjugates in the composition have 1 to 10 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 9 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 8 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 7 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 6 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 5 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 4 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 to 3 polymers attached to the protein;At least about 95% of the conjugates in the composition have 1-2 polymers attached to the protein; at least about 95% of the conjugates in the composition have 1 polymer attached to the protein; at least about 99% of the conjugates in the composition have 1-10 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1-9 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1-8 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1-7 polymers attached to the protein; At least about 99% of the conjugates in the composition have 1 to 6 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1 to 5 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1 to 4 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1 to 3 polymers attached to the protein; at least about 99% of the conjugates in the composition have 1 to 2 polymers attached to the protein; and at least about 99% of the conjugates in the composition have 1 polymer attached to the protein. Reference to a range of polymers, e.g., "x to y polymers," is understood to contemplate the number of polymers from x to y inclusive (i.e., for example, "1 to 3 polymers" contemplates 1 polymer, 2 polymers, and 3 polymers, "1 to 2 polymers" contemplates 1 polymer and 2 polymers, etc.). More specifically, the protein is IL-2.

[0338] Control of the number of desired polymers for any given moiety can be achieved by selecting the appropriate polymeric reagent, polymeric reagent to protein ratio, temperature, pH conditions, and other aspects of the conjugation reaction. Additionally, reduction or removal of undesired conjugates can be achieved through purification means.

[0339] For example, polymer-protein moiety conjugates can be purified to obtain / isolate different conjugate species. Specifically, the product mixture can be purified to obtain an average of 1, 2, 3, 4, 5, or any number of PEGs per IL-2 moiety. The purification strategy for the final conjugation reaction mixture depends on a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the particular protein, the desired dosing regimen, and the residual activity and in vivo properties of the individual conjugate(s).

[0340] If necessary, conjugates with different molecular weights can be isolated using gel filtration chromatography and / or ion exchange chromatography. That is, by using gel filtration chromatography, conjugates with different ratios of polymer to protein moiety (e.g., 1-mer, 2-mer, 3-mer, etc., where "1-mer" refers to one polymer relative to the protein moiety and "2-mer" refers to two polymers relative to the protein moiety) can be fractionated based on their molecular weight differences (the difference essentially corresponds to the average molecular weight of the water-soluble polymer moiety). For example, in an exemplary reaction in which a 15,000 dalton protein is randomly conjugated to a polymeric reagent having a molecular weight of approximately 20,000 daltons, the resulting reaction mixture may contain unmodified protein (having a molecular weight of approximately 15,000 daltons), monoPEGylated protein (having a molecular weight of approximately 35,000 daltons), diPEGylated protein (having a molecular weight of approximately 55,000 daltons), etc.

[0341] While this approach can be used to separate PEG and other polymer-protein conjugates with different molecular weights, it is generally not effective at separating positional isoforms that have different polymer attachment sites within the protein. For example, gel filtration chromatography can be used to separate mixtures of PEG 1-mers, 2-mers, 3-mers, etc., from each other, but each of the collected conjugate compositions may contain PEG(s) attached to different reactive groups (e.g., lysine residues) within the protein.

[0342] The choice of a particular gel filtration column depends on the desired fraction range desired. Elution is generally carried out using a suitable buffer, such as phosphate or acetate. Collected fractions can be analyzed by a number of different methods, including (i) absorbance at 280 nm for protein content, (ii) dye-based protein analysis using bovine serum albumin (BSA) as a standard, (iii) iodine testing for PEG content (Sims et al. (1980) Anal. BioIL-2m, 107:60-63), (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with barium iodide, and (v) high-performance liquid chromatography (HPLC).

[0343] Separation of positional isoforms is accomplished by reversed-phase chromatography using a suitable column (e.g., a C18 column or a C3 column) using reversed-phase high-performance liquid chromatography (RP-HPLC), or by ion-exchange chromatography using an ion-exchange column. Either approach can separate polymer-active agent isomers with the same molecular weight (i.e., positional isoforms).

[0344] For IL-2-polymer conjugates, the composition is preferably substantially free of proteins that do not have IL-2 activity. In addition, the composition is preferably substantially free of any other non-covalently attached water-soluble polymers. However, in some cases, the composition may contain a mixture of polymer-IL-2 moiety conjugates and unconjugated IL-2 moieties.

[0345] Optionally, the compositions of the present disclosure further comprise one or more pharmaceutically acceptable carriers or excipients. If necessary, a pharmaceutically acceptable excipient can be added to the conjugate to form the composition.

[0346] Exemplary excipients include, but are not limited to, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.

[0347] Carbohydrates such as sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and / or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, and starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, myo-inositol, and cyclodextrin.

[0348] The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0349] The compositions may also include an antimicrobial agent to prevent or inhibit the growth of microorganisms. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present disclosure include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimerosal, and combinations thereof.

[0350] Antioxidants can also be present in the composition.Antioxidants are used to prevent oxidation, thereby preventing the deterioration of conjugate or other components of preparation.Suitable antioxidants for use in one or more embodiments of the present disclosure include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0351] A surfactant may be present as an excipient. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88; sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids and fatty acid esters; steroids such as cholesterol; and IL-2 inhibitors such as EDTA, zinc, and other such suitable cations.

[0352] Acids or bases may be present in the composition as excipients.Non-limiting examples of acids that can be used include acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.Examples of suitable bases include, but are not limited to, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0353] One or more amino acids may be present in the compositions described herein as excipients. Exemplary amino acids in this regard include arginine, lysine, and glycine.

[0354] The amount of conjugate (i.e., the conjugate formed between the active agent and the polymeric reagent) in the composition can vary depending on many factors, but optimally, when the composition is stored in a unit-dose container (e.g., a vial), it can be a therapeutically effective dose. In addition, the pharmaceutical preparation can be contained in a syringe. The therapeutically effective dose can be experimentally determined by repeatedly administering increasing amounts of the conjugate to determine which amount produces the desired clinical endpoint.

[0355] The amount of any individual excipient in a composition can vary depending on the activity of the excipient and the particular needs of the composition. Typically, the optimal amount of any individual excipient is determined by routine experimentation, i.e., preparing compositions containing various amounts of the excipient (ranging from low to high amounts), examining stability and other parameters, and then determining the range in which optimal effect is obtained without significant adverse effects.

[0356] Generally, however, the excipient will be present in the composition in an amount of from about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations of less than 30% by weight being most preferred.

[0357] These aforementioned pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy," 19th ed., Williams & Williams, (1995), the "Physician's Desk Reference," 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3 rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

[0358] Treatment method The conjugates and compositions thereof can be used to treat any condition that can be alleviated or prevented by administration of the conjugate. Those skilled in the art will understand which conditions a particular conjugate can effectively treat. For example, the conjugates can be used alone or in combination with other drug therapies to treat cancer, infectious diseases (e.g., viral), and / or autoimmune diseases.

[0359] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is multiple myeloma, lymphoma, or leukemia. In some embodiments, the blood cancer is acute myeloid leukemia, Hodgkin's lymphoma, or cutaneous T-cell lymphoma. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is renal cell carcinoma, melanoma, breast cancer, or bladder cancer. In some embodiments, the melanoma is metastatic melanoma. In some embodiments, the cancer is selected from the group consisting of sarcoma, chordoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular cancer, gastric cancer, non-small cell lung cancer, The cancer that can be treated with IL-2 is selected from the group consisting of small cell lung cancer, bladder cancer, renal cell carcinoma, urothelial carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, acute myeloid leukemia, and leukemia.

[0360] In some embodiments, the present disclosure provides a method of treating an infectious disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein. In some embodiments, the infectious disease is a viral disease. In some embodiments, the viral disease is human immunodeficiency virus (HIV) or hepatitis C virus (HCV). In some embodiments, the infectious disease is HIV. In some embodiments, the infectious disease is HCV.

[0361] In some embodiments, the present disclosure provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein. In some embodiments, the autoimmune disease is rheumatoid arthritis, lupus erythematosus, inflammatory bowel disease (IBD), or atopic dermatitis. In some embodiments, the rheumatoid arthritis is juvenile rheumatoid arthritis.

[0362] In certain embodiments, the patient has a disease selected from the group consisting of renal cell carcinoma, metastatic melanoma, hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer.

[0363] Advantageously, the conjugate can be administered to a patient before, simultaneously with, or after the administration of another active agent. In some embodiments, the conjugate can be combined with an anti-tumor antigen antibody to produce a synergistic innate and adaptive immune response. In some embodiments, the conjugate can be combined with an anti-tumor antibody that has anti-tumor activity via antibody-dependent cell-mediated cytotoxicity (ADCC) function. The PEG-IL-2 conjugates described in this disclosure can stimulate CD8+ T cells. Stimulating CD8+ T cells not only offers the benefit of direct tumor killing, but also modulation of polymorphonuclear neutrophils (PMNs) for antibody-dependent cellular cytotoxicity (ADCC), such as through the release of cytokines like IFNγ, which are known to promote neutrophil activity (Pelletier et al., J. Leukoc. Biol. 2010;88:1163-1170). Combination therapy of a PEG-IL-2 conjugate with an anti-tumor antibody with ADCC function could potentially enhance the anti-tumor activity of these antibodies.

[0364] Formulation / Administration The conjugates and compositions disclosed herein that are administered to patients in need are intended to encompass all types of formulations, particularly those suitable for injection, such as powders or lyophilized powders that can be reconstituted, and liquids. Examples of diluents suitable for reconstituting solid compositions before injection include bacteriostatic water for injection, 5% dextrose in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. For liquid pharmaceutical compositions, solutions and suspensions are contemplated.

[0365] The compositions of one or more embodiments of the present disclosure are typically, but not necessarily, administered via injection, and therefore are generally liquid solutions or suspensions immediately before administration. Pharmaceutical preparations may also take other forms, such as syrups, creams, ointments, tablets, powders, etc. Other administration methods also include intrapulmonary, rectal, transdermal, transmucosal, oral, intrathecal, intratumoral, peritumoral, intraperitoneal, subcutaneous, intraarterial, etc.

[0366] The present disclosure also provides methods for administering the conjugates provided herein to patients suffering from conditions responsive to treatment with the conjugates. The methods generally involve administering a therapeutically effective amount of the conjugate to the patient via injection (preferably provided as part of a pharmaceutical composition). As previously described, the conjugates can be injected (e.g., intramuscularly, subcutaneously, and parenterally). Types of formulations suitable for parenteral administration include, among others, solutions ready for injection, dry powders to be mixed with a solvent before use, suspensions ready for injection, insoluble dry compositions to be mixed with a vehicle before use, and emulsions and liquid concentrates to be diluted before administration.

[0367] The method of administering the conjugate (preferably provided as part of a pharmaceutical composition) can optionally be performed to localize the conjugate to a specific area. For example, liquid, gel, and solid formulations containing the conjugate can be surgically implanted in the affected area (inside a tumor, near a tumor, in an area of ​​inflammation, and the like). Advantageously, organs and tissues can also be imaged to ensure the desired location is better exposed to the conjugate.

[0368] The actual dose administered may vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition being treated, the judgment of the healthcare professional, and the conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and / or are described in the relevant reference texts and literature. Generally, a therapeutically effective amount may range from about 0.001 mg to 100 mg, preferably in doses of 0.01 mg / day to 75 mg / day, and more preferably in doses of 0.10 mg / day to 50 mg / day. A given dose can be administered periodically, for example, until the symptoms of the disease are reduced and / or completely eliminated.

[0369] The unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules, depending on the judgment of the clinician, the needs of the patient, etc. Specific dosing schedules will be known to those of skill in the art or can be determined empirically using routine methods. Exemplary dosing schedules include, but are not limited to, administration once daily, three times a week, twice a week, once a week, once every three weeks, twice a month, once a month, and any combination thereof. Once the clinical endpoint is achieved, administration of the composition is discontinued.

[0370] While the present disclosure has been described with reference to preferred and specific embodiments thereof, it should be understood that the foregoing description and the examples that follow are intended to illustrate, but not limit, the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which this disclosure pertains.

[0371] All articles, books, patents and other publications referenced herein are hereby incorporated by reference in their entirety. [Example]

[0372] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of organic synthesis, biochemistry, protein purification, and the like, which are within the skill of one in the art. Such techniques are explained fully in the literature. See, e.g., J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992) (ibid.).

[0373] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near sea level. All reagents are commercially obtained from Sigma-Aldrich or Thermo Fisher Scientific unless otherwise indicated. All NMR generated is obtained from a 300 MHz or 400 MHz NMR spectrometer. All processing is performed in glass or glass-lined containers, and contact with metal-containing containers or equipment is avoided.

[0374] Materials: Unless otherwise stated, all organic solvents and reagents (anhydrous CHCl, 2-propanol, acetone, NMM, and DBCO-amine) were purchased from Sigma-Aldrich and used as received. PyClocK was purchased from Novabiochem®. 15 kDa, 17 kDa, and

[0375] 20 kDa Y-PEG-NHS reagent was purchased from JenKem Technology USA and used as received. 5 kDa, 10 kDa, and 20 kDa TheraPEG™ reagents were prepared using a method adapted from a published procedure (Brocchini et al., Nat. Protoc. 2006, 1:5, 2241-2252). DL-Dithiothreitol (DTT) was purchased from Melford, and a 0.1 M solution was prepared in cell culture-grade water (GE Healthcare) before use. Materials for preparing buffer solutions were supplied by Thermo Fisher Scientific, Merck, and Sigma-Aldrich and used as received. PBS (pH 7.4) was prepared from DPBS (Sigma-Aldrich) by adjusting the pH using 2 M NaOH (VWR). All other materials were purchased from VWR, Sigma-Aldrich, GE Healthcare, Thermo Fisher Scientific and Merck and used as received.

[0376] All precursor polymeric reagents referred to in these examples are commercially available unless otherwise indicated. Lyophilized powder of IL-2 ("rIL-2") corresponding to the amino acid sequence of Figure 1.

[0377] The mass and molar amount of the IL-2-PEG conjugate was calculated based on the amount of IL-2.

[0378] SDS-PAGE analysis Samples are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples are prepared, loaded onto gels, and electrophoresis is performed as described by the manufacturer.

[0379] Size exclusion chromatography The prepared PEG-rIL-2 conjugate was purified using size exclusion chromatography, and the details of the purification process are described below.

[0380] RP-HPLC analysis Samples were analyzed by reversed-phase chromatography (RP-HPLC) performed on an HPLC system. Analytical RP-HPLC analysis was performed on a Dionex 2 UPLC system equipped with an ACE Excel 2 super C18 column (dimensions: 75 x 2.1 mm i.d., 2 μm particle size). A 10 min linear gradient of 0 to 100% buffer B (99.95% MeCN, 0.05% TFA) in buffer A (94.95% HO, 5.0% MeCN, 0.05% TFA) was used, performed at a flow rate of 0.8 mL / min. Sample loading was 10 μg.

[0381] Example 1 7-Azido-1-((4-fluorophenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (8) [ka] Preparation of 6-azidohexan-1-ol (2): To a solution of 6-chlorohexan-1-ol (75 g, 0.549 mol, 1.0 equiv.) in HO (750 mL) was added NaN (97.5 g, 1.50 mol, 2.73 equiv.). The mixture was stirred at 105 °C for 16 h. LCMS analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then extracted with ethyl acetate. The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give crude compound 2 (75 g, 95%).

[0382] Preparation of 6-azidohexanal (3): To a solution of compound 2 (75 g, 0.523 mol, 1.0 equiv), TEMPO (817 mg, 5.23 mmol, 0.01 equiv), and NaHCO (52.7 g, 0.628 mol, 1.2 equiv) in DCM / HO (750 mL / 75 mL) was added TCCA (45 g, 0.194 mol, 0.37 equiv) in three portions at 0 °C. The mixture was stirred at 0 °C for 0.5 h. LCMS analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then filtered and diluted with water. The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure to give crude compound 3 (70 g, 94%).

[0383] Preparation of (4-fluorophenyl)(methyl)sulfane (5): To a solution of compound 4 (30 g, 0.234 mol, 1.0 equiv.) in DMF (250 mL) was added MeI (40 g, 0.281 mol, 1.2 equiv.) and K2CO3 (97 g, 0.702 mol, 3.0 equiv.) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 4 hours. TLC analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with 5% LiCl (aq.), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude compound 5 (45 g, 100%).

[0384] Preparation of 1-fluoro-4-(methylsulfonyl)benzene (6): To a solution of compound 5 (45 g, 0.317 mol, 1.0 equiv.) in THF / HO (450 mL / 450 mL) was added oxone (487 g, 0.792 mol, 2.5 equiv.) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 16 hours. LCMS analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then filtered, diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give crude compound 6 (35 g, 63%).

[0385] Preparation of 7-azido-1-((4-fluorophenyl)sulfonyl)heptan-2-ol (7): To a solution of compound 6 (20 g, 0.115 mol, 1.0 equiv.) in anhydrous THF (200 mL), n-BuLi (2.5 M in hexane, 60 mL, 0.149 mol, 1.3 equiv.) was added dropwise at −78° C. The cooling bath was removed, and the mixture was allowed to warm to 0° C. After stirring for 30 minutes, compound 3 (21 g, 0.149 mol, 1.3 equiv.) was added at −78° C. After stirring for 15 minutes, the mixture was allowed to warm. The mixture was then added to a saturated aqueous solution of NH4Cl (the mixture became clear) and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 7 (26 g, 71%).

[0386] Preparation of 7-azido-1-((4-fluorophenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (8) To a stirred solution of compound 7 (15 g, 47.62 mmol, 1.0 equiv.) and triphosgene (24 g, 80.95 mmol, 1.7 equiv.) in anhydrous THF (200 mL) was added pyridine (7.5 g, 95.24 mmol, 2.0 equiv.) dropwise at room temperature under a nitrogen atmosphere. After stirring for 10 min, the mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous THF (100 mL) and treated sequentially with NHS (16.4 g, 0.143 mol, 3.0 equiv.) and pyridine (11.3 g, 0.143 mmol, 3.0 equiv.). After stirring for 10 min, the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and washed with 0.1 N HCl, water, saturated aqueous NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 8 (12 g, 55%) as a solid. 1H NMR (400 MHz, d6-DMSO) δ 7.95-7.92 (m, 2H), 7.46 (t, J = 8.8 Hz, 2H), 5.10-5.09 (m, 1H), 4.04-3.97 (m, 1H), 3.84 (dd, J = 15.2, 2.0 Hz, 1H), 3.27-3.24 (m, 2H), 2.77 (s, 4H), 1.65-1.64 (m, 2H), 1.44-1.42 (m, 2H), 1.23-1.22 (m, 4H).

[0387] Example 2 7-Azido-1-((4-(trifluoromethyl)phenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (13) [ka] Preparation of methyl(4-(trifluoromethyl)phenyl)sulfane (10): To a solution of compound 9 (24.5 g, 0.138 mol, 1.0 equiv.) in DMF (200 mL) was added MeI (23.4 g, 0.165 mol, 1.2 equiv.) and K2CO3 (57 g, 0.413 mol, 3.0 equiv.) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 4 hours. TLC analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with 5% LiCl (aq.), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude compound 10 (24 g, 90%).

[0388] Preparation of 1-(methylsulfonyl)-4-(trifluoromethyl)benzene (11): To a solution of compound 10 (24 g, 0.125 mol, 1.0 equiv.) in THF / HO (200 mL / 200 mL) was added oxone (171 g, 0.264 mol, 2.1 equiv.) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 16 hours. LCMS analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then filtered, diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give crude compound 11 (30.6 g, 100%).

[0389] Preparation of 7-azido-1-((4-(trifluoromethyl)phenyl)sulfonyl)heptan-2-ol (12): To a solution of compound 11 (15 g, 66.96 mmol, 1.0 equiv.) in anhydrous THF (150 mL), n-BuLi (2.5 M in hexane, 35 mL, 87.05 mmol, 1.3 equiv.) was added dropwise at −78° C. The cooling bath was removed, and the mixture was allowed to warm to 0° C. After stirring for 30 min, compound 3 (12.5 g, 87.05 mmol, 1.3 equiv.) was added at −78° C. After stirring for 15 min, the mixture was allowed to warm. The mixture was then added to a saturated aqueous solution of NH4Cl (the mixture became clear) and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give impure compound 12 (19 g, 77%).

[0390] Preparation of 7-azido-1-((4-(trifluoromethyl)phenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (13) To a stirred solution of compound 12 (19 g, 52.05 mmol, 1.0 equiv.) and triphosgene (26.3 g, 88.49 mmol, 1.7 equiv.) in anhydrous THF (200 mL) was added pyridine (8 mL, 0.104 mol, 2.0 equiv.) dropwise at room temperature under a nitrogen atmosphere. After stirring for 10 minutes, the mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous THF (100 mL) and treated sequentially with NHS (17.95 g, 0.156 mol, 3.0 equiv.) and pyridine (12.5 mL, 0.156 mmol, 3.0 equiv.). After stirring for 10 minutes, the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL) and washed with 0.1 N HCl, water, saturated aqueous NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 13 (12.5 g, 47%) as a solid. 1 H NMR (400 MHz, d6-DMSO) δ 8.10 (d, J = 8.4 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 5.16-5.15 (m, 1H), 4.16-4.09 (m, 1H), 3.95-3.92 (m, 1H), 3.26 (t, J = 6.8 Hz, 2H), 2.77 (s, 4H), 1.66-1.65 (m, 2H), 1.44-1.42 (m, 2H), 1.24-1.23 (m, 4H).

[0391] Example 3 7-Azido-1-((4-chlorophenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (18) [ka] Preparation of (4-chlorophenyl)(methyl)sulfane (15): To a solution of compound 14 (30 g, 0.207 mol, 1.0 equiv.) in DMF (250 mL) was added MeI (35.3 g, 0.249 mol, 1.2 equiv.) and K2CO3 (85.8 g, 0.622 mol, 3.0 equiv.) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 4 h. TLC analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then diluted with water and extracted with ethyl acetate. The organic layer was washed with 5% LiCl (aq.), dried over anhydrous Na2SO4, and concentrated under reduced pressure to give crude compound 15 (44 g, 100%) as an orange oil. TLC: PE:EA = 10:1, Rf(14) = 0.5, Rf(15) = 0.7.

[0392] Preparation of 1-chloro-4-(methylsulfonyl)benzene (16): To a solution of compound 15 (60 g, 0.380 mol, 1.0 equiv.) in THF / HO (400 mL / 400 mL) was added oxone (583 g, 0.948 mol, 2.5 equiv.) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 16 hours. LCMS analysis of the reaction mixture showed complete conversion to the desired product. The mixture was then filtered, diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous NaSO, and concentrated under reduced pressure to give crude compound 16 (57.8 g, 80%) as a white solid.

[0393] Preparation of 7-azido-1-((4-chlorophenyl)sulfonyl)heptan-2-ol (17): To a solution of compound 16 (20 g, 0.105 mol, 1.0 equiv.) in anhydrous THF (300 mL), n-BuLi (2.5 M in hexane, 55 mL, 0.137 mol, 1.3 equiv.) was added dropwise at −78° C. The cooling bath was removed, and the mixture was allowed to warm to 0° C. After stirring for 30 minutes, compound 3 (19 g, 0.137 mol, 1.3 equiv.) was added at −78° C. After stirring for 15 minutes, the mixture was allowed to warm. The mixture was then added to a saturated aqueous solution of NH4Cl (the mixture became clear) and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 17 (26 g, 74%) as a yellow solid.

[0394] Preparation of 7-azido-1-((4-chlorophenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (18): To a stirred solution of compound 17 (31 g, 93.42 mmol, 1.0 equiv.) and triphosgene (47 g, 0.159 mol, 1.7 equiv.) in anhydrous THF (500 mL) was added pyridine (15 mL, 0.187 mol, 2.0 equiv.) dropwise at room temperature under a nitrogen atmosphere. After stirring for 10 min, the mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous THF (500 mL) and treated sequentially with NHS (32 g, 0.280 mol, 3.0 equiv.) and pyridine (22 mL, 0.280 mmol, 3.0 equiv.). After stirring for 10 min, the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 mL) and washed with 0.1 N HCl, water, saturated aqueous NaHCO3, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 18 (26 g, 59%) as a solid. 1 H NMR (400 MHz, d6-DMSO) δ 7.87 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz, 2H), 5.11-5.10 (m, 1H), 4.06-4.00 (m, 1H), 3.86 (dd, J = 15.6, 2.4 Hz, 1H), 3.26 (t, J = 6.8 Hz, 2H), 2.77 (s, 4H), 1.66-1.62 (m, 2H), 1.45-1.42 (m, 2H), 1.23-1.22 (m, 4H).

[0395] Example 4 7-Azido-1-((2,4-difluorophenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (19) [ka] Example 4 was prepared using the same preparative procedure as Example 1, but using 2,4-difluorobenzenethiol.1 H NMR (400 MHz, CDCl3) δ 8.01 - 7.94 (m, 1H), 7.12 - 7.05 (m, 1H), 7.05 - 6.97 (m, 1H), 5.24 (d, J = 6.6 Hz, 1H), 3.78 (dd, J = 15.2, 8.4 Hz, 1H), 3.46 (dd, J = 15.2, 3.4 Hz, 1H), 3.26 (t, J = 6.8 Hz, 2H), 2.80 (s, 4H), 1.79 (s, 2H), 1.63 - 1.56 (m, 2H), 1.43 - 1.33 (m, 4H).

[0396] Example 5 7-Azido-1-((4-fluoro-2-(trifluoromethyl)phenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (20) [ka] Example 5 was prepared using the same preparative procedure as Example 1, but using 4-fluoro-2-(trifluoromethyl)benzenethiol. 1 H NMR (400 MHz, CDCl3) δ 8.31 (dd, J = 8.8, 5.2 Hz, 1H), 7.60 (dd, J = 8.8, 2.6 Hz, 1H), 7.54 - 7.46 (m, 1H), 5.36 - 5.26 (m, 1H), 3.79 (dd, J = 15.2, 8.8 Hz, 1H), 3.47 (dd, J = 15.2, 3.2 Hz, 1H), 3.25 (t, J = 6.8 Hz, 2H), 2.81 (s, 4H), 1.83 - 1.70 (m, 2H), 1.61 - 1.52 (m, 2H), 1.45 - 1.34 (m, 4H).

[0397] Example 6 (2,7-bis((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (24) [ka] Preparation of N2,N7-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-9-(hydroxymethyl)-9H-fluorene-2,7-dicarboxamide (23): 9-(Hydroxymethyl)-9H-fluorene-2,7-dicarboxylic acid (82.5 mg, 0.24 mmol) was dissolved in anhydrous pyridine (1.0 mL), and to this solution was added HATU (273.8 mg, 0.72 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (117.1 mg, 0.54 mmol) at room temperature. The reaction was then stirred for 2 hours. The product was purified by HPLC using 0-70% MeCN / HO (containing 0.1% formic acid) to give compound 23 (47.4 mg, 30%). LCMS: m / z 685 (M+1). + .

[0398] Preparation of (2,7-bis((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (24) Compound 23 (47.4 mg, 0.069 mmol) was dissolved in DCM (0.2 mL) and treated with DSC (35.47 mg, 0.14 mmol) and pyridine (16.7 μL, 0.21 mmol) at room temperature under N. The reaction was stirred for 1.5 h, then diluted with DCM and washed with 1 N HCl and brine. The organic phase was dried over NaSO and concentrated. The residue was purified by HPLC in MeCN / HO (containing 0.1% TFA) to give the desired product 24 (31.7 mg, 56%, pale yellow oil). LCMS: m / z 826 (M+1) + .

[0399] Example 7 (2-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (31) [ka] Preparation of methyl 9H-fluorene-2-carboxylate (26): A mixture of 2-bromo-9H-fluorene (128 g, 522 mmol), triethylamine, TEA (106 g, 1.04 mol, 145 mL), and Pd(dppf)Cl (38.2 g, 52.2 mmol) in MeOH (890 mL) was degassed and purged with CO (50 Psi) three times. The mixture was then stirred at 80 °C under a N atmosphere for 5 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed the formation of a new spot (Rf = 0.42). The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give compound 26 (120 g, crude) as a white solid.

[0400] Preparation of 9H-fluorene-2-carboxylic acid (27): To a mixture of compound 26 (120 g, 535 mmol) in MeOH (840 mL) was added NaOH (2 M), and the mixture was stirred at 20 °C under a N atmosphere for 5 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed complete consumption of the starting material and the formation of a new spot (Rf = 0.01). Water (50 mL) was added to the solution, which was then extracted with EtOAc (100 mL). The aqueous phase was adjusted to pH 3 with 3 M HCl and then extracted with EtOAc (100 mL). The organic phase was concentrated under reduced pressure to give compound 27 (40.0 g, 190 mmol, 35.6% yield) as a yellow solid.

[0401] Preparation of 9-formyl-9H-fluorene-2-carboxylic acid (28): To a mixture of compound 27 (6.00 g, 28.5 mmol) in DMF (196 mL), ethyl formate (276 g, 3.73 mol) and t-BuOK (25.6 g, 228 mmol) were slowly added. The mixture was stirred at 45 °C for 0.5 h and then cooled to 25 °C for 2.5 h. TLC (petroleum ether / ethyl acetate = 0 / 1) showed complete consumption of the starting material and the formation of a new spot (Rf = 0.48). The solution was adjusted to pH 3 with 1 M HCl. The mixture was then extracted with EtOAc (50.0 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 28 (7.00 g, crude) as a brown solid.

[0402] Preparation of 9-(hydroxymethyl)-9H-fluorene-2-carboxylic acid (29): To a mixture of compound 28 (7.00 g, 29.4 mmol) in MeOH (42.0 mL) was added NaBH (2.78 g, 73.5 mmol). The reaction mixture was degassed and purged with N three times, and then the mixture was stirred under N at 25 °C for 16 h. LCMS (product: RT = 0.863 min) showed MS for the desired compound. Water (120 mL) was added to the solution, which was then extracted with EtOAc (100 mL). The aqueous phase was adjusted to pH 3 with 1 M HCl, which was then extracted with EtOAc (100 mL). The organic phase was separated, dried over NaSO, filtered, and concentrated under reduced pressure to give compound 29 (4.00 g, 16.7 mmol, 56.7% yield) as a yellow solid.

[0403] Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-9-(hydroxymethyl)-9H-fluorene-2-carboxamide (30): To a solution of compound 29 (1.00 g, 4.16 mmol) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (908 mg, 4.16 mmol) in DMF (7.00 mL) was added HOBt (619 mg, 4.58 mmol), EDCl (878 mg, 4.58 mmol), and DIPEA (1.24 g, 9.57 mmol) at 25 °C. The mixture was stirred at 25 °C for 12 h. LCMS (product: RT = 1.002 min) showed that the starting material was completely consumed. The reaction mixture was diluted with water (10.0 mL) and extracted with EtOAc (10.0 mL × 2). The combined organic phase was washed with water (10.0 mL × 2) and brine (10.0 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C18 250*50mm*10um; mobile phase: [water (10mM NH4HCO3)-ACN]; B%: 18%~48%, 26 min) to give compound 30 (1.40g, 3.17mmol, yield 76.2%, purity 99.8%) as a yellow oil. 1 H NMR (400 MHz, CDCl3): δ 8.10 (s, 1H), 7.88 - 7.76 (m, 3H), 7.63 (d, J = 7.2 Hz, 1H), 7.46 - 7.34 (m, 2H), 6.98 (s, 1H), 4.18 - 4.08 (m, 2H), 4.02 - 3.92 (m, 1H), 3.76 - 3.56 (m, 14H), 3.32 (t, J = 5.2 Hz, 2H), 2.37 (s, 1H);LC-MS: m / z 441.1 (M+1) + .

[0404] Preparation of (2-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (31) A solution of 1-hydroxypyrrolidine-2,5-dione (0.5 g, 1 equiv.) in DCM (5 mL) was cooled to −30° C. To this solution, trichloromethyl carbonochloridate (860 mg, 1 equiv.) was added dropwise, followed by DIPEA (561 mg, 1 equiv.) at −30° C. The mixture was warmed to 0° C. and stirred for 3 hours. It was warmed to 25° C. and continued stirring for 6 hours. TLC (petroleum ether / ethyl acetate=0 / 1, Rf=0.3) showed complete consumption of the starting material. The reaction mixture was filtered to give the filtrate (a DCM solution of 2,5-dioxopyrrolidin-1-yl carbonochloridate), which was used directly without further purification.

[0405] To a solution of compound 30 (0.1 g, 1 equiv.) and Py (17.96 mg, 1 equiv.) in DCM (1 mL) was added 2,5-dioxopyrrolidin-1-yl carbonochloridate (10 equiv., DCM solution from the previous step) at 0 °C. The mixture was stirred at 25 °C for 12 h. LCMS (starting material: RT = 0.992 min, product: RT = 1.059 min) showed 3.71% starting material remaining and 40.2% desired compound. The reaction was quenched with water (2.0 mL), and then the pH was adjusted to 6 with saturated aqueous citric acid. The mixture was extracted with DCM (2 mL × 2). The combined organic layers were washed with brine (5.0 mL), then dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Ultimate AQ-C18 150*30mm*5um; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-60%, 12 min). After preparative HPLC purification, the fractions were lyophilized to give compound 31 as a colorless oil. LC-MS: m / z 582.2 (M+1) + .

[0406] Example 8 (2-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-7-fluoro-9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (39) [ka] Preparation of 2-fluoro-7-iodo-9H-fluorene (33): A mixture of 2-fluoro-9H-fluorene 32 (24.4 g, 132 mmol), I2 (14.1 g, 55.6 mmol), and KIO3 (7.08 g, 33.1 mmol) in CH3COOH (408 mL), H2SO4 (9.60 mL), and HO (19.2 mL) was degassed and purged with N2 three times. The mixture was stirred at 80 °C under a N2 atmosphere for 5 h. HPLC (product: RT = 3.515 min) showed the detection of the desired compound. The aqueous solution was extracted with EtOAc (50.0 mL). The organic layer was washed with HO (20.0 mL), brine (10.0 mL), separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 33 (38.0 g, 123 mmol, 92.6% yield) as a brown solid. 1 H NMR (400 MHz, MeOD): 7.87 (s, 1H), 7.70-7.67 (m, 2H), 7.48-7.46 (m, 1H), 7.27-7.22 (m, 1H), 7.17-7.09 (m, 1H), 3.86 (s, 2H).

[0407] Preparation of methyl 7-fluoro-9H-fluorene-2-carboxylate (34): A mixture of compound 33 (38.0 g, 123 mmol), TEA (31.0 g, 306 mmol), and Pd(dppf)Cl (8.97 g, 12.3 mmol) in MeOH (200 mL) was degassed and purged with CO (50 Psi) three times. The mixture was stirred under CO atmosphere at 80 °C for 24 h. TLC (petroleum ether / ethyl acetate = 100 / 1) showed complete consumption of the starting material and the appearance of a new spot (R f =0.40). The solution was concentrated under reduced pressure to give compound 34 (40.0 g, crude) as a brown solid.

[0408] Preparation of 7-fluoro-9H-fluorene-2-carboxylic acid (35): To a mixture of compound 34 (40.0 g, 165 mmol) in MeOH (280 mL) was added aqueous NaOH (2 M, 206 mL, 2.5 equiv.). The reaction mixture was stirred at 100 °C under N atmosphere for 2 h. TLC (petroleum ether / ethyl acetate = 0 / 1) showed complete consumption of the starting material and the appearance of a new spot (R f =0.03). To the reaction solution was added HO (150 mL). It was then extracted with EtOAc (250 mL). The aqueous layer was separated and the pH was adjusted to 3 with 1 M HCl. It was extracted with EtOAc (200 mL). The organic layer was washed with brine (20.0 mL), separated, dried over NaSO, filtered, and concentrated under reduced pressure to give compound 35 (33.0 g, 145 mmol, 87.6% yield) as a brown solid.

[0409] Preparation of 7-fluoro-9-formyl-9H-fluorene-2-carboxylic acid (36): To a mixture of compound 35 (33.0 g, 145 mmol) in DMF (210 mL) was added ethyl formate (507 g, 6.84 mol). Then, t-BuOK (130 g, 1.16 mol) was slowly added. The mixture was stirred at 45 °C for 0.5 h, and then the mixture was cooled to 25 °C for 2.5 h. LCMS (product: RT = 0.889) showed the detection of the desired compound. Water (150 mL) was added to the reaction solution, and the mixture was extracted with EtOAc (500 mL). The aqueous phase was adjusted to pH 3 with 1 M HCl, and then extracted with EtOAc (500 mL). The organic layer was washed with brine (120 mL), separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 36 (30.0 g, crude) as a yellow solid.

[0410] Preparation of 7-fluoro-9-(hydroxymethyl)-9H-fluorene-2-carboxylic acid (37): To a mixture of compound 36 (30.0 g, 117 mmol) in MeOH (210 mL) was added NaBH (31.0 g, 820 mmol), and the mixture was then stirred at 25 °C under a N atmosphere for 24 hours. LCMS (product: RT = 0.906 min) showed the detection of the desired compound. Water (150 mL) was added to the reaction solution, and the mixture was extracted with EtOAc (450 mL). The aqueous phase was adjusted to pH 3 with 1 M HCl, and then extracted with EtOAc (300 mL). The organic layer was washed with brine (120 mL), separated, dried over NaSO, filtered, and concentrated under reduced pressure to give compound 37 (35.0 g, crude) as a yellow solid.

[0411] Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-7-fluoro-9-(hydroxymethyl)-9H-fluorene-2-carboxamide (38): A mixture of compound 37 (2.00 g, 7.74 mmol), HOBt (1.15 g, 8.52 mmol), EDCl (1.63 g, 8.52 mmol), and DIPEA (2.50 g, 19.4 mmol) in DMF (14.0 mL) was stirred at 25 °C for 0.5 h. Then, 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethanamine (1.86 g, 8.52 mmol) was added to the mixture. The reaction mixture was stirred at 25 °C for 3 h. LCMS (product: RT = 1.171 min) showed the detection of the desired compound. The reaction solution was diluted with water (20 mL) and extracted with EtOAc (20 mL). The organic layer was washed with brine (20.0 mL), separated, dried over Na SO , filtered, and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC (column: Phenomenex Luna C18 250mm*100mm*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%~53%, 25 min) to give compound 38 (1.00g, 2.12mmol, 48.7% yield, 97.4% purity) as a yellow oil. 1H NMR: (400 MHz CDCl3): δ 8.07 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.76 - 7.70 (m, 2H), 7.35 (d, J = 7.2 Hz, 1H), 7.39 - 7.31 (m, 1H), 7.18 - 7.08 (m, 1H), 7.02 (s, 1H), 4.16 - 3.96 (m, 3H), 3.76 - 3.56 (m, 14H), 3.33 (t, J = 4.8 Hz, 2H);LC-MS: m / z 459.1 (M+1) + .

[0412] Preparation of (2-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate (39) To a solution of compound 38 (0.1 g, 1 eq) in DCM (1 mL) was added compound 2,5-dioxopyrrolidin-1-yl carbonochloridate (10 eq, DCM solution) at 0° C. The reaction mixture was stirred at 25° C. for 12 hours. LCMS (starting material: RT = 1.026 min, product: RT = 1.084 min) showed 6.33% starting material remaining and 28.3% desired compound was detected. The reaction mixture was adjusted to pH 6 with saturated aqueous citric acid. The mixture was extracted with DCM (2 mL × 2). The combined organic layers were washed with brine (5.0 mL), separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 200*40mm*10um; mobile phase: [water (0.1% TFA)-ACN]; B%: 25%-55%, 10 min). After preparative HPLC purification, the solution was lyophilized to give compound 39 as a colorless oil. LC-MS: m / z 600.2 (M+1) + .

[0413] Example 9 2,5-Dioxopyrrolidin-1-yl N-(2-acetoxyethyl)-N-(2-(((2,7-bis((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)glycinate (44) [ka] Preparation of tert-butyl N-(2-acetoxyethyl)-N-(2-aminoethyl)glycinate (41): To a solution of tert-butyl N-(2-acetoxyethyl)-N-(2-(((benzyloxy)carbonyl)amino)ethyl)glycinate 40 (75.0 mg, 0.19 mmol, 1.0 equiv.) in ethyl acetate (0.6 mL) was added Pd / C (40 mg, 10%, dry) at room temperature. The reaction mixture was purged with H2 three times. The mixture was then stirred under H2 at room temperature for 2 hours. 1 The reaction mixture was monitored by HNMR and TLC. (PE:EA=1:1) Compound 40: Rf=0.3; Compound 41: Rf=0.05. The reaction solution was filtered through a pad of Celite. The organic layer was concentrated to give product 41 (48.4 mg, 98%) as a pale yellow oil.

[0414] Preparation of N-(2-acetoxyethyl)-N-(2-((((2,7-bis((2-(2-(2-(2-azidoethoxy)-ethoxy)ethoxy)-ethyl)carbamoyl)-9H-fluoren-9-yl)methoxy)carbonyl)amino)ethyl)glycine (43) Compound 41 prepared above was redissolved in EtOAc (0.6 mL). To this was added compound 24 (161.0 mg, 0.19 mmol) in DCM (1 mL), followed by pyridine (20 μL). The reaction was stirred at room temperature for 1 hour and monitored by LCMS. The reaction was taken up in EtOAc (5 mL), washed with 1N HCl (2 mL), and the organic phase was dried over NaSO and filtered. The solvent was then removed under vacuum.

[0415] To the crude product 42 was added HC0H (4 mL) and heated to 60 °C for 3 h. The product was purified by HPLC in 10–100% MeCN / HO (0.1% TFA) to give the desired compound 43 (31.4 mg, 18% over three steps).

[0416] Preparation of 2,5-dioxopyrrolidin-1-yl N-(2-acetoxyethyl)-N-(2-(((2,7-bis((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-fluoren-9-yl)methoxy)carbonyl)-amino)ethyl)glycinate (44) Compound 43 (9.7 mg, 0.011 mmol) was dissolved in DCM (0.037 mL) and treated with HOSu (2.56 mg, 0.022 mmol) and DCC (4.54 mg, 0.022 mmol) in DCM (0.04 mL) at 0 °C. The reaction was stirred overnight at room temperature. The reaction was filtered and concentrated. 3-5 volumes of EtO were added, causing the solution to become cloudy. This cloudy solution was centrifuged. The top clear solution was decanted, and the bottom oily solid was washed with EtO (2X) and dried under high vacuum to give compound 44 (7.2 mg, 65%). LCMS: 1012 (M+1) + ;HPLC 96% (UV254); 1 H NMR (300 MHz, chloroform-d) δ 8.09 (p, J = 0.7 Hz, 2H), 7.84 (td, J = 8.3, 1.1 Hz, 4H), 6.92 (s, 3H), 4.43 (d, J = 6.9 Hz, 2H), 4.31 (m, 1H), 4.16 (t, J = 5.3 Hz, 2H), 3.81 (s, 2H), 3.79 - 3.55 (m, 47H), 3.38 - 3.24 (m, 8H), 3.00 - 2.78 (m, 11H), 2.01 (s, 3H).

[0417] Example 10 20kDa Y-PEG-DBCO [ka] A dry round-bottom flask equipped with a Teflon-coated magnetic stir bar was charged with 20 kDa Y-PEG-NHS (1.08 g, 50.0 μmol, 1.0 equiv.) and PyClocK (0.033 g, 60.0 μmol, 1.2 equiv.). The flask was sealed with a rubber septum and placed under an inert atmosphere of argon. Anhydrous CHCl (5.0 mL) was added, followed by N-methylmorpholine (6.10 μL, 55.0 μmol, 1.1 equiv.), and the reaction solution was stirred at room temperature for 30 min. DBCO-amine (0.028 mg, 100 μmol, 2.0 equiv.) was added as a solid in one portion, and the reaction mixture was stirred at room temperature for an additional 3 h. The crude reaction mixture was transferred to a glass pipette and added dropwise to 2-propanol (100 mL) with vigorous stirring. A white precipitate was obtained (PEG material). The resulting suspension was cooled to 4°C, filtered (vacuum filtration), and washed with ice-cold 2-propanol (3 x 50 mL). The isolated precipitate was transferred to two pre-weighed Falcon tubes and dissolved in warm (40°C) acetone (90 mL). The solution was cooled in an ice bath for 15 minutes to induce precipitation of the PEG material. The suspension was pelleted by centrifugation (10,500 rpm, 20 minutes, 4°C), and the supernatant was carefully discarded. The pellet was redissolved in fresh warm acetone (40°C), cooled in an ice bath to induce precipitation, and another round of centrifugation / decantation was performed. This process was repeated four times in total. The pellet was dried under vacuum. Mass of isolated white solid = 1.08 g (99%). RP-HPLC retention time = 6.9 minutes.

[0418] Example 11 mPEG2-Fmoc-Bn-20K-NHS [ka] Example 11 mPEG2-Fmoc-Bn-20K-NHS was prepared according to a modification of the literature procedures in US20060293499A1 and Bioconjugate Chemistry 2003, 14, 395-403. 1H NMR (300 MHz, d6-DMSO) δ 9.14 (br, 1H), 8.56 (m, 2H), 8.25-8.17 (m, 2H), 8.04-7.97 (m, 4H), 7.44 (m, 2H), 7.33 (m, 2H), 5.77 (s, 2H), 4.69 (m, 2H), 4.46 (m, 1H), 3.51 (br, 1800H), 2.81 (s, 4H). HPLC: purity 94.7%; GPC: purity 91.2%; MALDI / GPC: 21048Da.

[0419] Example 12 mPEG2-Fmoc-Bi-20K-NHS [ka] Example 12 mPEG2-Fmoc-Bi-20K-NHS is prepared according to a modification of the literature procedures in US20060293499A1 and Bioconjugate Chemistry 2006, 17, 341-351.

[0420] Example 13 Preparation of rIL-2 The IL-2 gene encoding the polypeptide shown in Figure 1 was synthesized and cloned into the pET21a(+) expression vector as an Ndel / Xhol fragment. The sequences of the synthetic primers used for cloning were forward primer: 5'-aatcatatggcacctacttcaagttctacaaa-3' (SEQ ID NO: 4) and reverse primer: 5'-aatttatcaagttagtgttgagatgat-3' (SEQ ID NO: 5). Positive clones were identified by restriction enzyme digestion (Ndel and Xhol) and sequenced using standard sequencing protocols.

[0421] Positive clones were selected and transformed into E. coli cells (BL21 DE3). Standard procedures were followed for induction of IL-2 protein. Briefly, a single colony was inoculated into 5 ml of Luria Broth (LB) medium containing 100 μg / ml ampicillin and grown overnight at 37°C and 200 rpm. The overnight culture was diluted 100-fold into LB medium containing 100 μg / ml ampicillin and grown at 37°C and 200 rpm. When the absorbance at 600 nm reached approximately 0.8, the culture was induced with 1 mM IPTG. During the induction period, the culture temperature was increased to 42°C. Fermentation was terminated after 4 hours of induction.

[0422] After fermentation, cells were harvested by centrifugation. The cell mass pellet was stored at -80°C for further homogenization. The frozen cell mass pellet was resuspended in cell wash buffer (20 mM Tris, 1 mM EDTA, pH 8.0) to a concentration of 10% (w / v) and centrifuged at 15,600 × g for 30 minutes at 4°C. The supernatant was discarded. The washed pellet was resuspended in homogenization buffer (20 mM Tris, 0.1 M NaCl, 1 mM EDTA, 1 mM PMSF, 0.5% Trition-X100, pH 8.0) and homogenized three times at 4–15°C using a sonicator (SCIENTZ-IID, SCIENTZ, Ningbo, Zhejiang, PR). The homogenate was centrifuged at 15,600 × g for 30 minutes at 4°C. The supernatant was discarded. The inclusion body pellet was washed in buffer (20 mM Tris, 0.1 M NaCl, 2 M urea, 1 mM EDTA, pH 8.0) and centrifuged at 15,600 × g for 30 minutes at 4 °C. The supernatant was discarded. After centrifugation, crude IL-2 inclusion bodies were obtained.

[0423] Crude IL-2 inclusion bodies were dissolved in a buffer solution of 6 M guanidine, 100 mM Tris, 2 mM EDTA, and 5 mM dithiothreitol (DTT) (pH 8.0). The mixture was incubated at 50°C for 30 minutes. After reduction, water was added to the mixture to reduce the guanidine concentration to 4.8 M. After centrifugation at 15,600 × g for 1 hour, the resulting gel-like pellet was discarded. The guanidine concentration of the supernatant was further reduced to 3.5 M by adding water. The pH was adjusted to 5 by titration with 100% acetic acid. The mixture was incubated at room temperature for 60 minutes and then centrifuged at 15,600 × g for 1 hour. The resulting pellet was suspended in a buffer solution of 3.5 M guanidine, 20 mM acetic acid, and 5 mM DTT (pH 5) and centrifuged at 15,600 × g for 1 hour. The washing step was repeated once more.

[0424] The reduced and purified IL-2 inclusion bodies were dissolved in a buffer of 6 M guanidine, 100 mM Tris (pH 8). 100 mM CuCl2 stock was added to the solution. 2+ The final concentration of was 0.1 mM. The mixture was incubated overnight at 4°C.

[0425] The expressed IL-2 solution was placed in a dialysis bag (molecular weight pore size 3 kDa). The dialysis bag was placed in a reservoir containing 4.8 M guanidine, 0.1 M Tris (pH 8) buffer. After equilibration for 3 hours, the guanidine concentration in the reservoir was gradually reduced to 2 M by first pumping water into the reservoir over 15 hours, and then to less than 10 mM by pumping 20 mM PB (pH 6.0) buffer into the reservoir over 8 hours. The entire refolding process was completed at 4°C. The refolded IL-2 was confirmed by SEC-HPLC.

[0426] The refolded IL-2 was centrifuged at 15,600 xg for 60 minutes to remove the precipitate, and the supernatant was concentrated using a Mini Pellicon TFF membrane system (Millipore Corporation, USA).

[0427] The refolded concentrated IL-2 was loaded onto an XK column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) packed with SP Sepharose FF resin. The running buffer was 20 mM PB (pH 6.0) and the flow rate was 10 mL / min. Fractions below the IL-2 monomer peak were pooled.

[0428] The pooled SP Sepharsoe FF eluate was desalted by loading onto an XK column packed with Sephadex G25 resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The running buffer was 20 mM PB (pH 6.0) and the flow rate was 25 mL / min. Fractions below the IL-2 monomer peak were pooled.

[0429] The desalted IL-2 monomer pool was loaded onto an XK column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) packed with Q Sepharose FF resin. The running buffer was 20 mM PB (pH 6.0) and the flow rate was 25 mL / min. The flow below the peak was pooled. Note that other suitable purification methods, such as size exclusion chromatography and hydrophobic interaction chromatography (HIC chromatography), can also be employed.

[0430] The IL-2 monomer fraction pool was concentrated to approximately 1-2 mg / mL using a Mini Pellicon TFF membrane system (Millipore Corporation, USA) at 4°C and an operating pressure of 10-22 psi. The concentrated IL-2 monomer solution was dialyzed against the final formulation buffer (10 mM Na acetate, 5% trehalose, pH 4.5) at 4°C. The formulated IL-2 solution was sterilized by passing through a 0.22 μm filter and stored at -80°C for further use.

[0431] Preparation of lyophilized rIL-2 for conjugation: Sixteen vials of rIL-2 (16 x 5 mg) were warmed from -80 °C to room temperature. To each vial of lyophilized material, 21 mL of 0.1% aqueous SDS solution was added, and the contents of the vial were mixed until complete dissolution was achieved. The rIL-2 solution was buffer-exchanged into 100 mM sodium borate (pH 8) and concentrated via UF / DF (Vivaspin 20, 5 kDa MWCO PES). The buffer-exchanged protein solution was sterile-filtered (0.22 μm PVDF) and quantified by UV-A280 (3.19 mg / mL) using a Nanodrop 2000 spectrophotometer.

[0432] Preparation of solution-form IL-2 in pH 8.0 buffer for conjugation IL-2 (15 mg, 10 mL) was buffer-exchanged into 100 mM sodium borate (pH 8), 20 mM EDTA, 0.05% SDS using a P100 column according to the manufacturer's instructions. The IL-2 solution was concentrated via UF / DF (Vivaspin 20, 5 kDa MWCO PES). The buffer-exchanged protein solution was sterile filtered (0.22 μm PVDF) and quantified by UV-A280 using a Nanodrop 2000 spectrophotometer (2.67, 2.5, or 3.0 mg / mL, respectively).

[0433] Preparation of solution-form IL-2 in pH 9.0 buffer for conjugation IL-2 (15 mg, 10 mL) was buffer-exchanged into 100 mM sodium borate (pH 9), 20 mM EDTA, and 0.05% SDS using a P100 column according to the manufacturer's instructions. The IL-2 solution was concentrated via UF / DF (Vivaspin 20, 5 kDa MWCO PES). The buffer-exchanged protein solution was sterile-filtered (0.22 μm PVDF) and quantified by UV-A280 using a Nanodrop 2000 spectrophotometer (2.9 mg / mL).

[0434] Example 14 [ka] NHS conjugation of rIL-2 using Example 1 [rIL-2]-[F-Ph-SO2-N3] z Generation of Prior to conjugation, IL-2 was diluted to 3.09 mg / mL with 100 mM sodium borate (pH 8).

[0435] Compound 8 (4.4 mg) was dissolved in DMF (0.885 mL) to give a solution of 4.97 mg / mL of reagent. To a vial of rIL-2 (10 mg, 3.24 mL) was added compound 8 (1.79 mg, 360 μL, 6 equivalents), and the reaction was mixed and incubated at 22° C. for 1 hour. At the 1 hour time point, the reaction was analyzed by LC-MS to determine the identity of [rIL-2]-[F-Ph-SO2-N3]. z The distribution of functionalized IL-2 species was determined.

[0436] Figure 2 shows the 6-centered [rIL-2]-[F-Ph-SO2-N3] complex as determined by LC-MS. z The distribution of

[0437] Example 15 [ka] [rIL-2]-[F-Ph-SO2-N3] using 20kDa Y-PEG-DBCO z Click-PEGylation of 20 kDa Y-PEG-DBCO (143.7 mg) was dissolved in 100 mM sodium borate (pH 8) (1.419 mL). [rIL-2]-[F-Ph-SO2-N3] of Example 14 zTo a solution of (9.5 mg, 3.42 mL) was added 20 kDa Y-PEG-DBCO (134 mg, 1.33 mL, 10 equiv.). The reaction was mixed and incubated at 22 °C. After 2 h, the reaction mixture was analyzed by SDS-PAGE. The crude reaction mixture was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate, pH 4.5 (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the process were analyzed by SDS-PAGE, and high-purity fractions were pooled. The pooled fractions were concentrated / buffer-exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF).

[0438] The sample was quantified by IR using a DirectDetect instrument (8.8 mg, 92% yield). The PEG:IL-2 ratio was determined by SDS-PAGE.

[0439] Figure 3 shows the PEG:IL-2 ratio equal to 4.9 [20K mPEG-(F-Ph-SO2)] z Figure 1 shows an SDS-analysis of the -[rIL-2] conjugate.

[0440] Example 16 [ka] NHS conjugation of rIL-2 using Example 2 [rIL-2]-[CF3-Ph-SO2-N3] z Generate: Prior to conjugation, IL-2 was diluted to 3.09 mg / mL with 100 mM sodium borate (pH 8).

[0441] Compound 13 (7.5 mg) was dissolved in DMF (0.816 mL) to give a solution of 9.19 mg / mL of reagent. To a vial of rIL-2 (10 mg, 3.24 mL) was added compound 13 (3.31 mg, 360 μL, 10 equivalents), and the reaction was mixed and incubated at 22° C. for 1 hour. At the 1 hour time point, the reaction was analyzed by LC-MS to determine the identity of [rIL-2]-[CF3-Ph-SO2-N3]. z The distribution of functionalized IL-2 species was determined.

[0442] Figure 1 shows the 6-centered [rIL-2]-[CF3-Ph-SO2-N3] complex as determined by LC-MS. z The distribution of

[0443] Example 17 [ka] [rIL-2]-[CF3-Ph-SO2-N3] with 20kDa Y-PEG-DBCO z Click-PEGylation of 20 kDa Y-PEG-DBCO (210.9 mg) was dissolved in 100 mM sodium borate (pH 8) (1.388 mL). [rIL-2]-[CF3-Ph-SO2-N3] of Example 16 zTo a solution of (9.7 mg, 3.49 mL) was added 20 kDa Y-PEG-DBCO (207 mg, 1.36 mL, 15 equiv.). The reaction was mixed and incubated at 22 °C. After 2 h, the reaction mixture was analyzed by SDS-PAGE. The crude reaction mixture was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate, pH 4.5 (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the process were analyzed by SDS-PAGE, and high-purity fractions were pooled. The pooled fractions were concentrated / buffer-exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF).

[0444] The sample was quantified by IR using a DirectDetect instrument (7.9 mg, 81% yield). The PEG:IL-2 ratio was determined by SDS-PAGE.

[0445] Figure 3 shows the PEG:IL-2 ratio equal to 5.4 [20K mPEG-(CF3-Ph-SO2)] z Figure 1 shows an SDS-analysis of the -[rIL-2] conjugate.

[0446] Example 18 [ka] NHS conjugation of rIL-2 using Example 3 [rIL-2]-[Cl-Ph-SO2-N3] z Generate: Prior to conjugation, IL-2 was diluted to 3.09 mg / mL with 100 mM sodium borate (pH 8). Compound 18 (5.0 mg) was dissolved in DMF (0.971 mL) to yield a solution of 5.15 mg / mL of the reagent. To a vial of rIL-2 (10 mg, 3.24 mL) was added compound 18 (1.85 mg, 360 μL, 6 equivalents), and the reaction was mixed and incubated at 22°C for 1 hour. At the 1 hour time point, the reaction was analyzed by LC-MS to determine the identity of [rIL-2]-[Cl-Ph-SO2-N3]. z The distribution of functionalized IL-2 species was determined.

[0447] Figure 1 shows the [rIL-2]-[Cl-Ph-SO2-N3] complex with 5 as determined by LC-MS. z The distribution of

[0448] Example 19 [ka] [rIL-2]-[Cl-Ph-SO2-N3] using 20kDa Y-PEG-DBCO z Click-PEGylation of 20 kDa Y-PEG-DBCO (213.2 mg) was dissolved in 100 mM sodium borate (pH 8) (1.403 mL). [rIL-2]-[Cl-Ph-SO2-N3] from Example 18 zTo a solution of (9.7 mg, 3.49 mL) was added 20 kDa Y-PEG-DBCO (207 mg, 1.36 mL, 15 equiv.). The reaction was mixed and incubated at 22 °C. After 2 h, the reaction mixture was analyzed by SDS-PAGE. The crude reaction mixture was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate, pH 4.5 (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the process were analyzed by SDS-PAGE, and high-purity fractions were pooled. The pooled fractions were concentrated / buffer-exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF).

[0449] The sample was quantified (8.2 mg, 84%) by IR using a DirectDetect instrument. The PEG:IL-2 ratio was determined by SDS-PAGE.

[0450] Figure 3 shows the PEG:IL-2 ratio equal to 4.9 [20K mPEG-(Cl-Ph-SO2)] z Figure 1 shows an SDS-analysis of the -[rIL-2] conjugate.

[0451] Example 20 [ka] NHS conjugation of rIL-2 using Example 4 and click-PEGylation with 20kDa Y-PEG-DBCO Example 4 (5.8 mg) was dissolved in DMF (0.677 mL) to give a solution of 8.57 mg / mL of reagent. To a vial of IL-2 (7 mg, 0.458 μmol, 2.265 mL) was added Example 4 (2.16 mg, 4.55 μmol, 252 μL, 10 equiv.), the reaction was mixed, and incubated at 22° C. for 1 hour. After 1 hour, the reaction was analyzed by LC-MS to determine the average degree of IL-2 functionalization.

[0452] 20 kDa Y-PEG-DBCO (406.4 mg) was dissolved in 100 mM sodium borate (pH 8) (2.00 mL) to give a 203 mg / mL solution. [rIL-2]-[F,F-Ph-SO2-N3] z To the 20 kDa Y-PEG-DBCO (7.0 mg, 0.458 μmol, 2.52 mL) was added 20 kDa Y-PEG-DBCO (199 mg, 9.15 μmol, 0.98 mL, 20 equiv.). The reaction was mixed and incubated at 22 °C. After 2 h, the reaction mixture was analyzed by SDS-PAGE, and the crude reaction mixture was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate (pH 4.5) (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the procedure were analyzed by SDS-PAGE, and the purest fractions were pooled. The pooled fractions were concentrated / buffer exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF). Example 20 was purified by IR using a DirectDetect instrument to obtain [20K mPEG-(F,F-Ph-SO)] z -[rIL-2] (5.2 mg, 74% yield). SDS-PAGE analysis of the conjugate showed a PEG:IL-2 ratio equal to 4.8.

[0453] Example 21 [ka] NHS conjugation of rIL-2 using Example 5 and click-PEGylation with 20kDa Y-PEG-DBCO Example 5 (4.5 mg) was dissolved in DMF (0.528 mL) to give a solution of 8.52 mg / mL of reagent. To a vial of IL-2 (7 mg, 0.458 μmol, 2.265 mL) was added Example 5 (2.15 mg, 4.10 μmol, 252 μL, 9 equiv.), the reaction was mixed, and incubated at 22° C. for 1 hour. After 1 hour, the reaction was analyzed by LC-MS to determine the average degree of IL-2 functionalization.

[0454] 20 kDa Y-PEG-DBCO (406.4 mg) was dissolved in 100 mM sodium borate (pH 8) (2.00 mL) to give a 203 mg / mL solution. [rIL-2]-[F,CF3-Ph-SO2-N3] z To the 20 kDa Y-PEG-DBCO (7.0 mg, 0.458 μmol, 2.52 mL) was added 20 kDa Y-PEG-DBCO (199 mg, 9.15 μmol, 0.98 mL, 20 equiv.). The reaction was mixed and incubated at 22 °C. After 2 h, the reaction mixture was analyzed by SDS-PAGE, and the crude reaction mixture was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate (pH 4.5) (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the procedure were analyzed by SDS-PAGE, and the purest fractions were pooled. The pooled fractions were concentrated / buffer exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF). Example 21 was purified by IR using a DirectDetect instrument to identify [20K mPEG-(F,CF3-Ph-SO2)] z -[rIL-2] (2.9 mg, 41% yield). SDS-PAGE analysis of the conjugate showed a PEG:IL-2 ratio equal to 4.5.

[0455] Example 22 [ka] NHS conjugation of rIL-2 using Example 6 and click-PEGylation with 10 kDa PEG-DBCO Prior to conjugation, IL-2 was diluted to 3.09 mg / mL with 100 mM sodium borate (pH 8). Compound 24 (16.5 mg) was dissolved in DMF (1.107 mL) to yield a 14.9 mg / mL solution of the reagent. To a vial of IL-2 (10 mg, 3.24 mL) was added compound 24 (5.96 mg, 400 μL, 11 equivalents), and the reaction was mixed and incubated at 22° C. for 1 hour. At the 1 hour time point, the reaction was analyzed by LC-MS to determine the identity of [rIL-2]-[Fmoc-(N3)2]. z The distribution of functionalized IL-2 species was determined.

[0456] 10 kDa PEG-DBCO (Iris Biotech, 276.3 mg) was dissolved in 100 mM sodium borate (pH 8) (1.439 mL). [rIL-2]-[Fmoc-(N3)2] z To a solution of (10 mg, 3.64 mL) was added 10 kDa PEG-DBCO (262 mg, 1.36 mL, 40 equiv.). The reaction was mixed and incubated at 22 °C. After 2 h, the reaction mixture was analyzed by SDS-PAGE. The crude reaction mixture was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate, pH 4.5 (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the process were analyzed by SDS-PAGE, and high-purity fractions were pooled. The pooled fractions were concentrated / buffer exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF).

[0457] Samples were quantified by IR using a DirectDetect instrument. PEG:IL-2 ratios were determined by SDS-PAGE.

[0458] Figure 3 shows the conjugate [mPEG2-T2-Fmoc-20K] with a PEG:IL-2 ratio equal to 4.9. z -SDS-analysis of [rIL-2] is shown.

[0459] Example 23 [ka] NHS conjugation of rIL-2 using Example 9 and click-PEGylation with 10 kDa PEG-DBCO Example 23 was prepared using Example 9 in the same manner as in Examples 14 and 15, to give [mPEG2-T2-Fmoc-Bi-20K] z -Prepared as [rIL-2].

[0460] Example 24 [ka] PEGylation of rIL-2 using Example 11 Prior to conjugation, IL-2 is diluted to 1.5 mg / mL using 100 mM sodium borate (pH 8). mPEG2-Fmoc-Bn-20K-NHS from Example 11 is dissolved in 100 mM sodium borate (pH 8). This is added to rIL-2 (10 mg) in an amount sufficient to achieve a molar ratio of mPEG2-Fmoc-Bn-20K-NHS to rIL-2 of 100:1. The conjugation reaction is allowed to proceed for 1 hour at 22°C to obtain [mPEG2-Fmoc-Bn-20K] zThe -[rIL-2] conjugate is obtained. The crude reaction mixture is purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample is isocratically eluted with 50 mM sodium acetate, pH 4.5 (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the process are analyzed by SDS-PAGE, and highly pure fractions are pooled. The pooled fractions are concentrated / buffer exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF).

[0461] [mPEG2-Fmoc-Bn-20K] was detected by IR using a DirectDetect instrument. z - [rIL-2] is quantified. The PEG:IL-2 ratio is determined by SDS-PAGE.

[0462] Example 25 [ka] PEGylation of rIL-2 using Example 12 PEGylation of rIL-2 with mPEG2-Fmoc-Bi-20K-NHS from Example 12 using the same PEGylation and purification conditions as in Example 24 yielded [mPEG2-Fmoc-Bi-20K] z -[rIL-2] conjugates are produced.

[0463] Example 26 [ka] PEGylation of rIL-2 disulfide bonds using 10 kDa PEG bis(sulfone) 45 [ka] To a solution of r-IL-2 (4.2 mg, 0.25 mg / mL) in 100 mM sodium borate buffer (pH 8), 10 mM DTT was added. The solution was incubated at 22°C for 1 hour. Excess DTT was removed by gel filtration using 100 mM sodium borate buffer (pH 8) containing 20 mM EDTA. To the reduced protein solution, 1.3 equivalents of 10 kDa PEG bis(sulfone) 45 and 0.05% w / v SDS were added, and the solution was allowed to react for 16 hours at 22°C. The reaction solution was filtered through a Vivapure Q Mani H filter to remove SDS. The solution was then buffer exchanged into 50 mM sodium acetate (pH 4.0) by ultrafiltration using a 5 kDa MWCO spin filter. The solution was then loaded onto a 5 mL MacroCapSP resin column. The column was washed with a linear gradient of 0 to 1 M sodium chloride in 50 mM sodium acetate buffer (pH 4) to elute the conjugate. The conjugate was further isolated by size exclusion chromatography (SEC) to yield 1.4 mg of product. Purity by SDS-PAGE: 97%. Purity by analytical SEC: 87.3%.

[0464] Example 27 PEGylation of rIL-2 using PEG reagent 46 [ka] A 405 mg / mL solution of PEG reagent 46 (1.50 g) was prepared in 2 mM HCl (3.702 mL). To rIL-2 (10 mg, 3.135 mL) was added 405 mg / mL of PEG reagent 46 (1.43 g, 3.535 mL, 100 equiv.). The reaction was mixed and incubated at 22°C. After 1 hour, the crude reaction was analyzed by SDS-PAGE and purified by SEC. Crude IL-2-(PEG) zThe product was purified by SEC using a HiLoad 26 / 600 Superdex 200 pg column. The sample was isocratically eluted with 50 mM sodium acetate, pH 4.5 (150 mM NaCl) at a flow rate of 3 mL / min. Fractions collected throughout the process were analyzed by SDS-PAGE, and high-purity fractions were pooled. The pooled fractions were concentrated / buffer-exchanged into 50 mM sodium acetate, pH 4.5 (150 mM NaCl) by UF / DF (Vivaspin 20, 50 kDa MWCO PES) and finally sterile filtered (0.22 μm PVDF). Protein concentration was quantified by IR using a DirectDetect instrument (6.6 mg, 66%), and the PEG:IL-2 ratio was determined by SDS-PAGE. Conjugate [mPEG2-Fmoc-20K] z SDS-analysis of -[rIL-2] showed a PEG:IL-2 ratio equal to 5.1.

[0465] Example 28 1-((3-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)sulfonyl)-5-methoxypentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (52) [ka] Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(methylsulfonyl)benzamide (48) To a solution of compound 47 (1.0 g, 5.0 mmol) in DMF (15 mL) was added compound 22 (1.3 g, 6.0 mmol), HATU (2.47 g, 6.5 mmol), and TEA (1.01 g, 10.0 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated and dissolved in ethyl acetate and water. The mixture was extracted with ethyl acetate (3 × 20 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The resulting residue was purified by column chromatography to give compound 48 (900 mg) as a yellow oil.

[0466] Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-((2-hydroxy-5-methoxypentyl)sulfonyl)benzamide (50) To a solution of compound 48 (400 mg, 1 mmol) and compound 49 (560 mg, 5.5 mmol) in dry THF (30 mL) was added KHMDS (5.5 mL, 5.5 mmol) slowly at −78° C. under N. The reaction mixture was stirred at −78° C. for 2 h. The reaction mixture was quenched with saturated aqueous NH4Cl. The mixture was extracted with ethyl acetate (3 × 20 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The resulting residue was purified by column chromatography eluting with 2% CH3OH / CH2Cl2 to give compound 50 (168 mg) as a yellow oil. Preparation of 1-((3-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-carbamoyl)phenyl)sulfonyl)-5-methoxypentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (51)

[0467] To a solution of compound 50 (100 mg, 0.2 mmol) and triphosgene (89 mg, 0.3 mmol) in dry THF (5 mL) was slowly added pyridine (64 mg, 0.8 mmol). The reaction mixture was stirred at room temperature for 20 minutes. It was then filtered and concentrated by rotary evaporation. The resulting residue was used in the next step.

[0468] To a solution of the resulting residue (117 mg, 0.2 mmol) and HOSu (69 mg, 0.6 mmol) in dry THF (5 mL) was slowly added pyridine (64 mg, 0.8 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The mixture was extracted with ethyl acetate (3 × 10 mL). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated by rotary evaporation. The resulting residue was purified by preparative TLC (CH2Cl2:CH3OH = 30:1) to give compound 51 (55 mg) as a colorless oil. LCMS: m / z 644.25 [M+1]. 1 H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.18 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 7.7 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.37 (br s, 1H), 5.30 - 5.24 (m, 1H), 3.77 - 3.55 (m, 15H), 3.45 - 3.31 (m, 5H), 3.27 (s, 3H), 2.81 (s, 4H), 1.94 - 1.78 (m, 2H), 1.66 - 1.58 (m, 2H).

[0469] Example 29 1-((3-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-4-(trifluoromethyl)phenyl)sulfonyl)-5-methoxypentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (61) [ka] Preparation of methyl 5-((4-methoxybenzyl)thio)-2-(trifluoromethyl)benzoate (54) A solution of compound 52 (5.0 g, 17.66 mmol, 1.0 equiv.), compound 53 (4.09 g, 26.5 mmol, 1.5 equiv.), Pd(dba) (1.62 g, 1.76 mmol, 0.1 equiv.), Xantphos (2.04 g, 3.52 mmol, 0.2 equiv.), and DIEA (6.84 g, 52.99 mol, 3.0 equiv.) was stirred at 80 °C for 2 h. The resulting mixture was cooled to room temperature and filtered through a Celite pad. The filtrate was concentrated, and the residue was dissolved in EtOAc (100 mL). The mixture was washed with water (100 mL), extracted with EtOAc (100 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA=100 / 1 to 80 / 1 to 50 / 1) to give compound 54 (6.2 g, 98%) as a pale yellow oil. TLC: PE / EA=10 / 1, UV, R f (Compound 52)=0.80, R f (Compound 54)=0.60. LC-MS: 379.10 [M+23] + . Preparation of methyl 5-mercapto-2-(trifluoromethyl)benzoate (55):

[0470] Compound 54 (1.0 g, 2.80 mmol, 1.0 equiv) and TES (0.98 g, 8.42 mmol, 3.0 equiv) in TFA (15 mL) were treated with microwave at 120 °C for 1 h. The resulting mixture was concentrated under reduced pressure. The residue was poured into ice water (20 mL), and the mixture was adjusted to pH 7-8 with aqueous sodium bicarbonate. The mixture was extracted with EtOAc (30 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 55 (800 mg) as a gray oil, which was used directly in the next step without further purification. TLC:PE / EA=5:1, UV, R f (Compound 54)=0.80, R f (Compound 55)=0.30.

[0471] Preparation of methyl 5-(methylthio)-2-(trifluoromethyl)benzoate (56): To a solution of compound 55 (4.8 g, 20.32 mmol, 1.0 equiv.) in MeCN (50 mL) was added KCO (8.5 g, 60.96 mmol, 3.0 equiv.) and CHI (14.4 g, 101.6 mmol, 5.0 equiv.) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 16 h. Water was added to the resulting mixture, which was extracted with EtOAc (50 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE) to give compound 56 (4.0 g, 78%) as a yellow solid. TLC:PE / EA=5:1, UV, R f (Compound 55)=0.30, R f (Compound 56)=0.85. LC-MS: 251.00 [M+1] + .

[0472] Preparation of methyl 5-(methylsulfonyl)-2-(trifluoromethyl)benzoate (57):

[0473] To a solution of compound 56 (4.7 g, 18.78 mmol, 1.0 equiv.) in DCM (50 mL) was added m-CPBA (19.5 g, 112.68 mmol, 6.0 equiv.) in one portion at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with a solution of sodium bicarbonate. The mixture was extracted with DCM (50 mL × 3), washed with NaCl solution (100 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA = 100 / 1 to 50 / 1 to 20 / 1 to 10 / 1) to give compound 57 (2.97 g, 56%) as a white solid. TLC:PE / EA=5:1, UV, R f (Compound 56)=0.85, R f (Compound 57)=0.10.

[0474] Preparation of methyl 5-((2-hydroxy-5-methoxypentyl)sulfonyl)-2-(trifluoromethyl)benzoate (58) To a solution of compound 57 (0.9 g, 3.543 mmol, 1.0 equiv.) and 4-methoxybutanal (0.724 mg, 7.086 mmol, 2.0 equiv.) in THF (10 mL) was added KHMDS (5.4 mL, 5.315 mmol, 1.5 equiv.) dropwise at −78 °C, and the reaction mixture was stirred at −78 °C for 2 h. The reaction was quenched with aqueous NH4Cl solution at 0 °C and extracted with EtOAc (30 mL × 3). The organic phase was washed with saturated NaCl solution (100 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA = 20 / 1 to 5 / 1 to 2 / 1) to give compound 58 (520 mg, 40%) as a yellow oil. TLC: PE / EA = 2:1, UV, R f (Compound 57)=0.60, R f (Compound 58)=0.20. LC-MS: 385.10 [M+1] + .

[0475] Preparation of 5-((2-hydroxy-5-methoxypentyl)sulfonyl)-2-(trifluoromethyl)benzoic acid (59): To a solution of compound 58 (510 mg, 1.327 mmol, 1.0 equiv.) in MeOH / THF=1 / 1 (6 mL) was added 5% LiOH (63.6 mg, 2.654 mmol, 2.0 equiv.) dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was adjusted to pH 2 with 1 N HCl. The mixture was extracted with EtOAc (20 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure to give compound 59 (505 mg, crude, 100%) as a yellow oil. LC-MS: 393.10 [M+23] + . Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-((2-hydroxy-5-methoxypentyl)sulfonyl)-2-(trifluoromethyl)benzamide (60) A solution of compound 59 (1.0 g, 3.24 mmol, 1.0 equiv.), compound 22 (0.849 g, 3.89 mmol, 1.2 equiv.), HATU (1.6 g, 4.21 mmol, 1.3 equiv.), and TEA (0.982 g, 9.72 mol, 3.0 equiv.) in DMF (12 mL) was stirred at room temperature for 16 h. Water (50 mL) was added to the reaction mixture, which was then extracted with ethyl acetate (30 mL × 3). The organic phase was washed with an aqueous solution of NaCl (50 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA = 20 / 1-10 / 1-5 / 1-2 / 1-1 / 1) and preparative TLC to give compound 60 (520 mg, 34%) as a pale yellow oil. LC-MS: 571.35 [M+1] + .

[0476] Preparation of 1-((3-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-4-(trifluoromethyl)phenyl)sulfonyl)-5-methoxypentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (61) To a solution of compound 60 (0.3 g, 0.5258 mmol, 1.0 equiv) in THF (3 mL) was added pyridine (0.166 g, 2.103 mmol, 4.0 equiv) and triphosgene (0.39 g, 1.3145 mmol, 2.5 equiv) in one portion at 0 °C. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in THF (3 mL). To the mixture was added pyridine (0.166 g, 2.103 mmol, 4.0 equiv) and HOSU (0.182 g, 1.5774 mmol, 3.0 equiv) in one portion at 0 °C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with water at 0 °C and extracted with EtOAc (20 mL × 3). The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (0.1% HCOOH). The eluted solution was extracted with EtOAc. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 61 (150 mg, 40%) as a colorless oil. LC-MS: 712.35[M+1] + . 1 H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 9.5 Hz, 2H), 7.94 (d, J = 8.1 Hz, 1H), 6.94 (s, 1H), 5.31 (d, J = 6.9 Hz, 1H), 3.65 (d, J = 6.3 Hz, 8H), 3.59 (q, J = 5.0 Hz, 6H), 3.36 (dt, J = 18.4, 5.4 Hz, 4H), 3.29 (d, J = 1.0 Hz, 3H), 2.83 (s, 4H), 1.90 (q, J = 7.2 Hz, 2H), 1.65 (d, J = 8.5Hz, 2H).

[0477] Example 30 1-((3-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-4-chlorophenyl)sulfonyl)-5-methoxypentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (68) [ka] Preparation of methyl 2-chloro-5-(methylthio)benzoate (63) To a solution of compound 62 (10.0 g, 49.53 mmol, 1.0 equiv.) and CHCl (7.73 g, 54.48 mmol, 1.1 equiv.) was added KCO (7.5 g, 54.48 mmol, 1.1 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. Water (200 mL) and EtOAc (200 mL) were added to the resulting mixture. The organic layer was separated, washed five times with 5% aqueous LiCl, dried over NaSO, filtered, and concentrated under reduced pressure to give compound 63 (11.0 g, crude) as a yellow oil. TLC: PE / EA=3 / 1, UV, R f (Compound 62)=0.05, R f (Compound 63)=0.85. 1HNMR (400 MHz, CD3OD) δ 7.61 (d, J = 2.3 Hz, 1H), 7.44 - 7.32 (m, 2H), 3.88 (s, 3H), 2.48 (s, 3H).

[0478] Preparation of methyl 2-chloro-5-(methylsulfonyl)benzoate (64) To a solution of compound 63 (6.0 g, 27.78 mmol, 1.0 equiv.) in DCM (60 mL) was added m-CPBA (28.7 g, 166.67 mmol, 6.0 equiv.) in one portion at 0 °C. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with aqueous sodium bicarbonate, extracted with DCM (100 mL × 3), washed with aqueous NaCl (100 mL × 3), dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA = 40 / 1 to 20 / 1 to 3 / 1) to give compound 64 (5.6 g, 81%) as a white solid. TLC: PE / EA=3 / 1, UV, R f (Compound 63)=0.85, R f (Compound 64)=0.45. 1 HNMR (CD3OD, 400 MHz) δ 8.39 (d, J = 2.4 Hz, 1H), 7.96 (dd, J = 8.4, 2.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 3.96 (s, 3H), 3.07 (s, 3H).

[0479] Preparation of 2-chloro-5-(methylsulfonyl)benzoic acid (65) To a solution of compound 64 (2.5 g, 1.327 mmol, 1.0 equiv.) in MeOH / THF = 1 / 1 (6 mL) was added 5% aqueous LiOH solution (63.6 mg, 2.654 mmol, 2.0 equiv.) dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 h. The reaction was adjusted to pH = 3-4 with 1 N HCl (concentrated). The aqueous was extracted with EtOAc (20 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 65 (2.1 g, crude) as a pale yellow solid. TLC: PE / EA = 3:1, UV, R f (Compound 64)=0.45, R f (Compound 65)=0.05. 1 HNMR (CD3OD, 400 MHz) δ 8.36 (d, J = 2.3 Hz, 1H), 8.02 (dd, J = 8.4, 2.3 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 3.15 (s, 3H).

[0480] Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-2-chloro-5-(methylsulfonyl)benzamide (66) A suspension of compound 65 (879 mg, 3.74 mmol, 1.0 equiv.), compound 22 (900 mg, 4.12 mmol, 1.1 equiv.), HATU (1.85 g, 4.87 mmol, 1.3 equiv.), and TEA (1.14 g, 11.24 mol, 3.0 equiv.) in DMF (8 mL) was stirred at room temperature for 16 h. Water (20 mL) was added to the reaction mixture, which was extracted with ethyl acetate (30 mL × 3), washed with aqueous NaCl (50 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA = 100 / 1, 10 / 1, 5 / 1, 2 / 1, 1 / 1) to give compound 66 (995 mg, 61%) as a colorless oil. TLC:PE / EA=0:1, UV, R f (Compound 65)=0.25, R f (Compound 66)=0.55. 1 HNMR (CD3OD, 400 MHz) δ 8.04-7.96 (m, 2H), 7.73 (d, J = 8.3 Hz, 1H), 3.70-3.53 (m, 14H), 3.33 (s, 2H), 3.15 (s, 3H).

[0481] Preparation of N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-2-chloro-5-((2-hydroxy-5-methoxypentyl)sulfonyl)benzamide (67) Compound 66 (700 mg, 1.609 mmol, 1.0 equiv.) and 4-methoxybutanal in THF (7 mL)

[0482] To a solution of 657 mg (6.44 mmol, 4.0 equiv.) in HCl (5.4 mL, 5.315 mmol, 1.5 equiv.) was added dropwise at −78°C, and the reaction mixture was stirred at −78°C for 2 h. The reaction mixture was quenched with aqueous NH4Cl solution at 0°C, extracted with ethyl acetate (30 mL × 3), washed with aqueous NaCl solution (100 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE / EA = 20 / 1 to 5 / 1 to 2 / 1) to give compound 67 (205 mg, 25%) as a pale yellow oil. TLC:PE / EA=0:1, UV, R f (Compound 66)=0.55, R f (Compound 67)=0.50. 1 HNMR (CD3OD, 400 MHz) δ 8.01-7.92 (m, 2H), 7.71 (d, J = 8.4 Hz, 1H), 4.16-4.01 (m, 2H), 3.72-3.53 (m, 12H), 3.42-3.35 (m, 3H), 3.31-3.25 (m, 5H), 1.73-1.39 (m, 4H).

[0483] Preparation of 1-((3-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-4-chlorophenyl)sulfonyl)-5-methoxypentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (68) To a solution of compound 67 (200 mg, 0.372 mmol, 1.0 equiv) in THF (2 mL) was added pyridine (117.5 mg, 1.49 mmol, 4.0 equiv) and triphosgene (221 mg, 0.744 mmol, 2.0 equiv) in one portion at 0 °C. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in THF (3 mL). To the mixture was added pyridine (117.5 mg, 1.49 mmol, 4.0 equiv) and HOSU (128 mg, 1.12 mmol, 3.0 equiv) in one portion at 0 °C. The mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with water at 0 °C, extracted with ethyl acetate (20 mL × 3), dried over Na SO , filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (0.1% HCOOH), extracted with ethyl acetate, dried over Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure to give compound 68 (101 mg, 27%) as a pale yellow oil. TLC: PE / EA=0 / 1, UV, R f (Compound 67)=0.50, R f (Compound 68)=0.55. LC-MS: 678.25 [M+1] + . 1 HNMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.94-7.86 (m, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 5.29 (s, 1H), 3.74-3.52 (m, 15H), 3.44-3.31 (m, 5H), 3.28 (s, 3H), 2.82 (s, 4H), 1.87 (d, J = 7.4 Hz, 2H), 1.61 (s, 2H).

[0484] Example 31 7-((3-(2,7-bis((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)carbamoyl)-9H-carbazol-9-yl)propyl)amino)-7-oxo-1-((4-(trifluoromethyl)phenyl)sulfonyl)heptan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate (82) [ka] [ka] [ka] Preparation of tert-butyl 6-hydroxyhexanoate (70) A mixture of...

Claims

1. A conjugate comprising a protein covalently attached to at least one linker, the conjugate having a structure according to formula (XIX): 【Chemistry 1】 or a stereoisomer, regioisomer, tautomer or mixture thereof, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof, (In the formula, z is an integer from 1 to 25; L is a linker, wherein at least one linker is a releasable linker; The protein is IL-2. Including, The conjugate has a structure according to formula (VII): 【Chemistry 2】 (In the formula, X １ is a first spacer moiety, X ２ is a second spacer moiety, if present, the first spacer moiety and the second spacer moiety are each independently selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; R １ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ２ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ｅ is an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; a is an integer from 0 to 5, b is an integer from 0 to 3; c is an integer from 0 to 2; z is an integer from 1 to 25; Y １ is O or S, Y ２ is O or S, FG ２ is a functional group capable of reacting via click chemistry selected from the group consisting of azide, alkynyl, and cycloalkynyl groups; -NH- is the amine group of a lysine residue in the protein. Including, the conjugate is (a) a structure according to formula (VII-A): 【Transformation 3】 (b) a structure according to formula (VII-B): 【Chemistry 4】 (c) a structure according to formula (VII-C): 【Transformation 5】 or (d) a structure according to formula (VII-D): 【Transformation 6】 The conjugate comprising:

2. In any one of formula (VII), formula (VII-A), formula (VII-B), formula (VII-C) and formula (VII-D), a is an integer from 0 to 2, R １ and R ２ are each independently hydrogen, Me, or Et; R ｅ Nitro, cyano, halogen, -CF ３ , -CONHMe, -SO ２ NHMe, -OMe, -NHMe, -NHAc, -NHSO ２ Me, or -OCF ３ That is, The conjugate of claim 1.

3. The conjugate has one of the following structures: 【Transformation 7】 【Transformation 8】 (wherein a is an integer of 1 to 2, and R ｅ are 4-F, 4-Cl, and 4-CF ３ , 2,4-difluoro or 2-CF ３ -4-F substitution), 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 2. The conjugate of claim 1, having the formula:

4. A structure according to formula (XX): 【Chemistry 12】 or a stereoisomer, regioisomer, tautomer or mixture thereof, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, or hydrate thereof, (In the formula, z is an integer from 1 to 25; L is a linker, the protein is IL-2, the macromolecule is a water-soluble polymer, lipid, protein or polypeptide; The water-soluble polymer is poly(ethylene glycol). Including, The conjugate has a structure according to formula (XIII): 【Chemistry 13】 (In the formula, POLY １ is a first linear or branched poly(ethylene glycol), POLY ２ is a second linear or branched poly(ethylene glycol), X １ is a first spacer moiety; or -X-FG ２ and X ２ is a second spacer moiety, if present, the first spacer moiety and the second spacer moiety are each independently selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; T １ is a first triazole functional group; T ２ is a second triazole functional group; The first triazole functional group and the second triazole functional group are 【Chemistry 14】 are each independently selected from R １ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ２ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ｅ is an electron modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and -X-FG ２ (In the formula, X is a spacer moiety selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; FG ２ is a functional group capable of reacting via click chemistry selected from the group consisting of azide, alkynyl, and cycloalkynyl groups; a is an integer from 0 to 5, b is an integer from 0 to 3; c is an integer from 0 to 2; z is an integer from 1 to 25; Y １ is O or S, Y ２ is O or S, -NH- is the amine group of a lysine residue in the protein. Including, The conjugate is (a) a structure according to formula (XIII-A): 【Chemistry 15】 (b) a structure according to formula (XIII-B): 【Chemistry 16】 (c) a structure according to formula (XIII-C): 【Chemistry 17】 or (d) a structure according to formula (XIII-D): [Chemistry 18] , a conjugate comprising:

5. In any one of formula (XIII), formula (XIII-A), formula (XIII-B), formula (XIII-C) and formula (XIII-D), a is an integer from 0 to 2, R １ and R ２ are each independently hydrogen, Me, or Et; R ｅ Nitro, cyano, halogen, -CF ３ , -CONHMe, -SO ２ NHMe, -OMe, -NHMe, -NHAc, -NHSO ２ Me, or -OCF ３ That is, The conjugate of claim 4.

6. The conjugate has a structure according to formula (XIII-B1), (XIII-A1), (XIII-C1), (XIII-D1) or (XIII-D2): 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 (In the formula, a is an integer of 1 to 2, R ｅ is 4-F, 4-Cl, 4-CF ３ , 2,4-difluoro or 2-CF ３ -4-F substitution, n is independently an integer from 4 to 1500; z is an integer from 1 to 25; -NH- is the amine group of a lysine residue in the protein. The conjugate of claim 4, comprising:

7. Method for preparing protein-macromolecule conjugates according to scheme (I): 【Chemistry 24】 (In the formula, x is an integer from 1 to 25; y is an integer from 0 to 24; z is an integer from 1 to 25, where x=y+z; L is a linker, FG ０ is a functional group capable of reacting with a nucleophilic group of an active protein drug to form a linkage, including a carbamate linkage, a thiol bridge, etc. FG ２ is a functional group capable of reacting with FG3 via click chemistry selected from the group consisting of azide, alkynyl, and cycloalkynyl groups; FG ３ is a functional group capable of reacting with FG2 via click chemistry, selected from the group consisting of azide, alkynyl, and cycloalkynyl groups; the protein is IL-2, The macromolecule may be a water-soluble polymer, lipid, protein, or polypeptide, and may be any of the following: a fatty acid containing from about 6 to about 26 carbon atoms, 2-methacryloyl-oxyethylphosphoylcholine, poly(acrylic acid), poly(acrylate), poly(acrylamide), poly(N-acryloylmorpholine), poly(alkyloxy)polymer, poly(amide), poly(amidoamine), poly(amino acid), poly(anhydride), poly(aspartamide), poly(butyric acid), poly(glycolic acid), polybutylene terephthalate, poly(caprolactone), poly(carbonate), poly(cyanoacrylate), poly(dimethicone). methylacrylamide), poly(ester), poly(ethylene), poly(ethylene glycol), poly(ethylene oxide), poly(ethyl phosphate), poly(ethyloxazoline), poly(glycolic acid), poly(α-hydroxy acid), poly(hydroxyethyl acrylate), poly(hydroxyethyl oxazoline), poly(hydroxymethacrylate), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(hydroxypropyl oxazoline), poly(iminocarbonate), poly(lactic acid), poly(lactic acid-co-glycolic acid), poly(methacrylamide), poly(methacrylate), poly(methyloxazoline), poly(organophosphazene), poly(orthoester), poly(oxazoline), poly(oxyethylated polyol), poly(olefinic alcohol), polyphosphazene, poly(propylene glycol), poly(saccharide), poly(siloxane), poly(urethane ), poly(vinyl alcohol), poly(vinylamine), poly(vinyl methyl ether), poly(vinylpyrrolidone), silicone, amylose, cellulose, carbomethylcellulose, hydroxypropylmethylcellulose, chitin, chitosan, dextran, dextrin, gelatin, hyaluronic acid (HA) and derivatives, functionalized hyaluronic acid, mannan, pectin, heparin, heparan sulfate (HS), rhamnogalacturonan, starch, hydroxyalkyl starch, hydroxyethyl starch (HES), polysialic acid (PSA) and other carbohydrate-based polymers, xylan, and copolymers, albumin, transferrin, transthyretin, immunoglobulins, XTEN peptides, glycine-rich homoamino acid polymers (HAP), PAS polypeptides, elastin-like polypeptides (ELP), CTP peptides, or gelatin-like protein (GLK) polymers; The water-soluble polymer is poly(ethylene glycol). Protein-(L-FG ２ ) x is a structure according to formula (VII): 【Chemistry 25】 (In the formula, X １ is a first spacer moiety, X ２ is a second spacer moiety, if present, the first spacer moiety and the second spacer moiety are each independently selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; R １ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ２ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ｅ is an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; a is an integer from 0 to 5, b is an integer from 0 to 3; c is an integer from 0 to 2; z is an integer from 1 to 25; Y １ is O or S, Y ２ is O or S, FG ２ is a functional group capable of reacting via click chemistry selected from the group consisting of azide, alkynyl, and cycloalkynyl groups; -NH- is the amine group of a lysine residue in the protein. Including, (F.G. ２ -L)y-protein-(L-macromolecule)z has a structure according to formula (XIII): 【Chemistry 26】 (In the formula, POLY １ is a first linear or branched poly(ethylene glycol), POLY ２ is a second linear or branched poly(ethylene glycol), X １ is a first spacer moiety; or -X-FG ２ and X ２ is a second spacer moiety, if present, the first spacer moiety and the second spacer moiety are each independently selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; T １ is a first triazole functional group; T ２ is a second triazole functional group; The first triazole functional group and the second triazole functional group are 【Chemistry 27】 are each independently selected from R １ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ２ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ｅ is an electron modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and -X-FG ２ (In the formula, X is a spacer moiety selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; FG ２ is a functional group capable of reacting via click chemistry selected from the group consisting of azide, alkynyl, and cycloalkynyl groups; a is an integer from 0 to 5, b is an integer from 0 to 3; c is an integer from 0 to 2; z is an integer from 1 to 25; Y １ is O or S, Y ２ is O or S, -NH- is the amine group of a lysine residue in the protein. Including, Protein-(L-FG ２ ) x is (a) a structure according to formula (VII-A): 【Chemistry 28】 (b) a structure according to formula (VII-B): 【Chemistry 29】 (c) a structure according to formula (VII-C): 【Transformation 30】 Or (d) a structure according to formula (VII-D): 【Chemistry 31】 Including, In response, (FG ２ -L)y-protein-(L-macromolecule)z is (a) a structure according to formula (XIII-A): 【Chemistry 32】 (b) a structure according to formula (XIII-B): 【Transformation 33】 (c) a structure according to formula (XIII-C): 【Transformation 34】 or (d) a structure according to formula (XIII-D): 【Chemistry 35】 A method for preparing a protein-macromolecule conjugate, comprising:

8. A composition comprising a mixture of conjugates according to any one of claims 1 to 3 or a mixture of conjugates according to any one of claims 4 to 6.

9. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 6 and one or more pharmaceutically acceptable excipients.

10. 10. The pharmaceutical composition of claim 9 for use in administration for the treatment of cancer, an infectious disease, or an autoimmune disease.

11. A structure according to formula (I), formula (IB), formula (IC) or formula (XVIII): 【Transformation 36】 【Chemistry 37】 【Transformation 38】 【Chemistry 39】 or a stereoisomer, tautomer or mixture thereof, or an isotopic variant thereof (In the formula, X １ is a first spacer moiety, X ２ is a second spacer moiety, the first spacer moiety and the second spacer moiety are each independently selected from linkers containing one or more carbon atoms, nitrogen atoms, sulfur atoms, phosphorus atoms, oxygen atoms, and combinations thereof; R １ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ２ is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, or substituted aryl; R ｅ is an electron-modifying group selected from nitro, cyano, halogen, amido, substituted amido, sulfone, substituted sulfone, sulfonamido, substituted sulfonamido, alkoxy, substituted alkoxy, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; a is an integer from 0 to 4, b is an integer from 1 to 3; c is an integer from 0 to 1 in formula (I) and 2 in formula (XVIII); FG １ is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage; FG ２ is a functional group capable of reacting via click chemistry selected from the group consisting of azide, alkynyl, and cycloalkynyl groups. A releasable linker having:

12. a is an integer from 0 to 2, R １ and R ２ are each independently hydrogen, Me, or Et; R ｅ Nitro, cyano, halogen, -CF ３ , -CONHMe, -SO ２ NHMe, -OMe, -NHMe, -NHAc, -NHSO ２ Me, or -OCF ３ That is, 12. The releasable linker of claim 11.

13. The releasable linker has one of the following structures: 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 12. The releasable linker of claim 11, having the formula:

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