Extended-release conjugates of lipidated peptides, and uses thereof

Abstract

Provided herein are conjugates of lipidated peptides having release kinetics appropriate for at least monthly administration. Also provided are uses of such conjugates to treat metabolic or neurologic disease or disorder or associated symptoms in a subject in need thereof.

Description

67057-20032.40 EXTENDED-RELEASE CONJUGATES OF LIPIDATED PEPTIDES, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 673,637, filed July 19, 2024, and U.S. Provisional Application No. 63 / 699,053, filed September 25, 2024, the entire contents of which are herein incorporated by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (670572003240seqlist.xml; Size: 21,859 bytes; and Date of Creation: July 17, 2025) is herein incorporated by reference in its entirety. FIELD

[0003] The present disclosure relates generally to the use of extended-release conjugates of lipidated peptides, and more specifically to the use of conjugates of lipidated peptides having release kinetics appropriate for at least monthly administration. BACKGROUND

[0004] Agonists of the GLP-1 receptor (GLP-1R) have found use in the treatment of metabolic disorders including diabetes, obesity, and non-alcoholic steatohepatitis (NASH). The initial GLP-1R agonist peptide discovered, exenatide, was effective in the treatment of type 2 diabetes, but suffered from a short systemic half-life and required twice-daily injection. Subsequent work led to the development of peptides having longer systemic half- lives through sequence modifications to confer protease stability and through attachment of lipid domains that confer binding to serum proteins such as albumin. Such modifications have provided GLP-1R agonists with systemic half-lives up to ~7 days, making them suitable for weekly administration and thus much more convenient and effective. One such lipidated GLP-1R agonist, semaglutide, has a systemic half-life of 7 days and has found widespread use as Wegovy®(high dose) for obesity and Ozempic®(low dose) for diabetes. After 1MOFO-35807479567057-20032.40 extensive market use, the Wegovy®label was expanded to include use to reduce the risk of death, heart attack, and stroke in adults with cardiovascular disease and obesity.

[0005] It has further been noted that GLP-1R agonists appear to have beneficial effects on various neurological problems, in some cases perhaps related to their effects on glucose metabolism and obesity. Exenatide has been found to reduce cortical neuroinflammation in diet-induced obese rodents, and thus has been proposed for use in the treatment of neuroinflammation-related neurodegeneration (Lin et al. 2023 International Immunopharmacology 115: 109653). Positive effects of exenatide have also been reported in the treatment of Parkinson’s disease (Athauda et al., Lancet 2017390(10103): 1664-75).

[0006] It has not been possible so far to extend these systemic half-lives further using these techniques so as to provide GLP-1R agonists that are suitable for less frequent administration, for example monthly or quarterly. Conjugation of a GLP-1R agonist peptide to an anti-GIPR antibody has provided an agent (maridebart cafraglutide, MariTide) that is under investigation for monthly administration. However, the systemic half-life is only 14-25 days and thus the dose is extremely large (420 mg in 6 mL) (Veniant et al., Nat. Metab. 2024 6(2): 290-303).

[0007] Thus, there exists an ongoing need in the art to develop extended-release conjugates that could provide greatly enhanced convenience and potentially improved efficacy for patients suffering from the direct and indirect consequences of metabolic diseases such as type 2 diabetes, obesity, and NASH. BRIEF SUMMARY

[0008] In some aspects, provided herein are extended-release conjugates of lipidated peptides having release kinetics suitable for monthly to quarterly administration.

[0009] In one aspect, provided is a releasable hydrogel conjugate of a lipidated peptide having the formula (I): M-[Z*-L-D] (I) wherein M is a biodegradable hydrogel, Z* is a connecting group, L is a β-eliminative linker, and D is a lipidated peptide. The units L-D will also be referred to herein as linker-drug units. 2MOFO-35807479567057-20032.40

[0010] In one aspect, provided is a releasable hydrogel conjugate of a lipidated peptide having the formula (IA): M-[Z*-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D]s. (IA) wherein M is a biodegradable hydrogel, Z* is a connecting group, n = 0-6, R1is an electron- withdrawing group, R2is H or C1-3 alkyl, each R4is independently H or C1-C3 alkyl, or taken together form a 4-7 membered ring, D is a lipidated peptide attached through its Na-amine (i.e. N-terminal amine of peptide), and s is the number of moles of D per milligram of biodegradable hydrogel (M). In particular embodiments, the lipidated peptide is selected from the group consisting of semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. Releasable hydrogel conjugate of lipidated peptides will also be referred to herein as hydrogel conjugates or conjugates.

[0011] In some embodiments, the hydrogel conjugates comprise a macromolecular carrier and linker-drug units (L-D) of the formula (II):wherein: n is an integer from 0 to 6; R1is an electron-withdrawing group; R2is H or C1-3alkyl; each R4is independently H or C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; 3MOFO-35807479567057-20032.40 D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to the macromolecular carrier (e.g., via connecting group Z* in the releasable hydrogel conjugate of formula (I) or formula (IA)).

[0012] In some embodiments of the linker-drug unit of formula (II), n is an integer from 0 to 6; R1is -CN, -NO2, -COR5, -SOR5, -SO2R5, or SR9, wherein R5is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR6or -NR62, wherein each R6is independently H or optionally substituted alkyl, or both R6groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring, and wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; C1-C6 alkyl, aryl, heteroaryl, or NR62, wherein each R6is independently optionally substituted C1-C6alkyl, aryl, or heteroaryl; R2is H or alkyl; each R4is independently H or C1-C3 alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0013] In some embodiments of the linker-drug unit of formula (II), n is an integer from 0 to 6; 4MOFO-35807479567057-20032.40 R1is -CN, -NO2, -COR5, -SOR5, or -SO2R5, wherein R5is C1-C6alkyl, aryl, heteroaryl, or NR62, wherein each R6is independently optionally substituted C1-C6 alkyl, aryl, or heteroaryl; R2is H or alkyl; each R4is independently H or C1-C3 alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0014] In some embodiments of the linker-drug unit of formula (II), n is an integer from 2 to 4; R1is -SOR5, or -SO2R5, wherein R5is C1-C6alkyl (e.g. CH3or CH2CH3); R2is H; each R4is CH3; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0015] In some such embodiments, n is 1. In other such embodiments, n is 2. In other such embodiments, n is 3. In other such embodiments, n is 4. In other such embodiments, n is 5. In other such embodiments, n is 6.

[0016] In some embodiments, the linker-drug unit of formula (II) can be covalently attached to a macromolecular carrier through a particular functional group. For instance, the conjugate of formula (III) depicts a single linker-drug unit attached to a macromolecular carrier (M) via functional group Z*. 5MOFO-35807479567057-20032.40wherein M, n, R1, R2, R4and D are defined as above. Various connecting groups (Z*) will be defined in the disclosure.

[0017] In some embodiments, the hydrogels of the disclosure are comprised of repeating crosslinked polymeric units. Each polymeric unit may have multiple arms, which allows it to be crosslinked to multiple polymeric units in the hydrogel. The individual crosslinked units comprising the hydrogel may include one or more drug units. In some embodiments, the individual polymeric units comprising the hydrogel include, without limitation, polyethylene glycol (PEG) or other synthetic polymer, dextran, antibody, antibody fragment, albumin or other protein, of sufficient molecular size to inhibit efficient renal filtration as is understood in the art. For polyethylene glycols, M can be single-chain, multiple-chain, or multiple-arm of average molecular weight between 1,000 and 100,000 Daltons, preferably between 1,000 and 40,000 Daltons.

[0018] In some embodiments, the individual polymeric units comprising the hydrogel conjugates are crosslinked through a cleavable crosslinker. In some embodiments, the cleavable crosslinker has the formula (V):wherein: 6MOFO-35807479567057-20032.40 A* and B* are independently connecting groups that connect the cleavable linker to polymeric units (e.g., r-armed polymers); q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; R11is an electron-withdrawing group; R12is H or C1-C3 alkyl; each R14is independently H, C1-C3alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; and shows the point of attachment to a linker-drug (e.g., a linker-drug unit of formula (II) or a connecting group that connects the cleavable crosslinker to a linker-drug unit of formula (II)).

[0019] In some embodiments of a hydrogel conjugate of formula (V), A* and B* independently comprise an amide group, a carbonate group, a 1,2,3-triazole group, an oxime, a thiosuccinimidyl group, or a thioether group. In some embodiments, A* or B* comprises a 1,2,3-triazole group. In some embodiments of a hydrogel conjugate of formula (V), A* or B* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group.

[0020] The cleavable linker of formula (V) can be covalently bonded to a to a linker-drug unit of formula (II) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (V), hence forming a cleavable linker of formula (Va), depicted below.wherein A*, B*, q, R11, R12, R14, x, y and z are defined as above. 7MOFO-35807479567057-20032.40

[0021] A cleavable linker of formula (Va) can be reacted with a linker-drug unit formula (IV) to yield a conjugate of formula (VI), as depicted in Scheme 2.Scheme 2

[0022] In scheme 2, Z and Z’ are reactive groups or comprise reactive groups. The reaction between Z and Z’ yields connecting group Z*. In some embodiments of Scheme 2, when Z is amine, Z’ is or comprises a carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (VI) wherein Z* is or comprises an amide or carbamate group. In other embodiments of Scheme 2, when Z is azide, Z’ is or comprises an alkynyl moiety, bicyclononynyl moiety, or cyclooctynyl moiety to yield a conjugate of formula (VI) wherein Z* is or comprises a 1,2,3-triazole moiety. In other embodiments of Scheme 2, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (VI) wherein Z* is oxime. In other embodiments of Scheme 2, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (VI) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles 8MOFO-35807479567057-20032.40 of Z and Z’ can be reversed to yield Z* of opposing orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0023] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable crosslinker having the formula (VII):wherein A*, q, R11, R12, and R14are defined as above.

[0024] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker. In some embodiments, the cleavable crosslinker has the formula (IX):wherein A*, q, R11, R12, and R14are defined as above.

[0025] In some embodiments, cleavable crosslinkers with covalently attached linker-drug units, such as formula (V), can be covalently bonded to individual polymeric units as set forth herein. For instance, the cleavable linker of formula (V) can be bonded to two individual polymeric units through connecting groups A* and B*. Formula (XI) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (V). 9MOFO-35807479567057-20032.40

[0026] In some embodiments, a crosslinked unit of the hydrogels of the disclosure is shown in formula (XII), formula (XIII), formula (XIV), formula (XV), or formula (XVI):10MOFO-35807479567057-20032.40(XVI), wherein P1, P2, A*, B*, q, R1, R2, R4, Y, R11, R12, R14, x, y, z, Z*, n, and D are defined as above.

[0027] In some embodiments, formula (XI), formula (XII), formula (XIII), formula (IV), formula (XV) or formula (XVI) represents a single repeating unit of a hydrogel. In such embodiments, each polymeric unit (P1or P2) may have multiple arms, which allows it to be 11MOFO-35807479567057-20032.40 crosslinked to multiple polymeric units in the hydrogel. For instance, if P1is a 4-armed polymer, it can be crosslinked to 4 polymeric units (e.g., 4 P2units). Likewise, if P2is a 4- armed polymer, it can be crosslinked to 4 polymeric units (e.g., 4 P1units).

[0028] In one aspect, the disclosure provides hydrogel conjugates comprising polymeric units (P1-P2) crosslinked by a cleavable crosslinker and further comprising a linker-drug (L- D), wherein said hydrogel conjugates have the formula (XXX):wherein: P1and P2are independently r-armed pegylated polymers, wherein r is an integer from 2 to 8; A* and B* are independently connecting groups (also referred to herein as attachment moieties) that connect the cleavable crosslinker to the r-armed pegylated polymers; q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; R11is -CN, -NO2, -COR13, -SOR13, -SO2R13, or SR19, wherein R13is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring, and wherein R19is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R12is H; 12MOFO-35807479567057-20032.40 each R14is independently H, C1-C3alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; Z* is a connecting group (also referred to herein as an attachment moiety) for attaching the cleavable crosslinker to the linker-drug (L-D); and L-D has the formula:wherein: n is an integer from 0 to 6; R1is -CN, -NO2, -COR3, -SOR3, or -SO2R3, wherein R3is , optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R2is H; each R4is independently H, C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug (L-D) to Z*.

[0029] In some embodiments, a hydrogel of the disclosure has one of the following structures: 13MOFO-35807479567057-20032.40

[0030] In other aspects, provided are methods for preparing these conjugate hydrogels and intermediates thereto.

[0031] In some aspects, provided herein are hydrogel conjugates having release kinetics suitable for biweekly, monthly, quarterly (i.e., once every 3 months), three times yearly, twice yearly or once yearly administration. In particular embodiments, the hydrogel conjugates of the disclosure are administered by the parenteral route. In some embodiments the hydrogel conjugates of the disclosure are administered subcutaneously. In some embodiments the hydrogel conjugates of the disclosure are administered intravenously. In some embodiments the hydrogel conjugates of the disclosure are administered intramuscularly. In some embodiments the hydrogel conjugates of the disclosure are administered intradermally. In 14MOFO-35807479567057-20032.40 any of the foregoing embodiments, the drug (D)is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide.

[0032] In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 0.5 mL to about 3 mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 3 mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 2 mL. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide.

[0033] In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of from about 2 μmol drug / mL to about 10 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel from about 3 μmol drug / mL to about 8 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel from about 4 μmol drug / mL to about 6 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 4 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 5 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 6 μmol drug / mL. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide.

[0034] In some embodiments, the hydrogel conjugates are administered at a dose of from about 1 μmol to about 20 μmol once every 3 months, once every four months, once every six 15MOFO-35807479567057-20032.40 months, or once every twelve months. In some embodiments, the hydrogel conjugates are administered at a dose of from about 2 μmol to about 10 μmol once every three months, once every four months, once every six months, or once every twelve months. In some embodiments, the hydrogel conjugates are administered at a dose of from about 2 μmol to about 5 μmol once every three months, once every four months, once every six months, or once every twelve months. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide.

[0035] In some aspects, provided are methods for using these conjugates in the treatment of metabolic diseases, such as type 2 diabetes, obesity, and NASH. In some embodiments, the conjugates are administered monthly. In some embodiments, the conjugates are administered quarterly. In some embodiments, the conjugates are administered three times yearly. In some embodiments, the conjugates are administered twice yearly. In some embodiments, the conjugates are administered once yearly. DESCRIPTION OF THE FIGURES

[0036] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0037] Fig. 1 shows the structure of linker semaglutide of Example 1, wherein Z = N3, n = 2, each R4= Me, and R1= MeSO2.

[0038] Fig. 2 shows the structures of specific hydrogels designed to release lipidated peptides.

[0039] Figs. 3A and 3B show the in vitro release of semaglutide from the conjugates of Example 2 and dissolution of the biodegradable hydrogel matrix under accelerated conditions (pH 9.4, 37ºC) according to Example 3. Panel A: release of 50% of conjugated semaglutide from the 10x10 con jugate was observed at 12.8 h, and complete dissolution of the hydrogel matrix was observed at 35 h. Panel B: release of 50% of conjugated semaglutide from the 20x20 conjugate was observed at 12.8 h, and complete dissolution of the hydrogel matrix was observed at 35 h. The indicated curves were generated using a semaglutide linker cleavage 16MOFO-35807479567057-20032.40 t1 / 2= 720 h at pH 7.4 for both conjugates and a hydrogel crosslink cleavage t1 / 2= 1200 h at pH 7.4 for the 10x10 conjugate and 2000 h for the 20x20 conjugate.

[0040] Fig. 4 shows the mouse pharmacokinetics of the conjugates of Example 2. Plasma levels of semaglutide measured by ELISA after a single dose comprising 10 or 50 nmol (400 or 2000 nmol / kg) of semaglutide on the 10x10 and 20x20 conjugates of Example 2. To compare data between doses, the plasma concentrations were dose-normalized. Data are fit to a slow-release model with release t1 / 2= 632 h (26 days), semaglutide elimination t1 / 2= 7.5 h, and Vd / Z = 0.41 L / kg. Good pharmacokinetics of plasma semaglutide were observed out to 3 months.

[0041] Fig. 5 shows weight loss in diet-induced obese (DIO) mice after treatment with a single dose of the conjugates of Example 3. DIO mice having initial body weight of ~47 g were treated with (Fig. 5A) the 10x10 and 20x20 conjugate s each comprising 2000 nmol / kg of semaglutide; (Fig. 5B) the 20x20 conjugate comprising 200, 660, or 2000 nmol / kg of semaglutide. The single dose gave body weight loss comparable to twice-daily free semaglutide at 10 nmol / kg per dose, or 600 nmol / kg / month.

[0042] Fig. 6A shows that Body weight of DIO mice treated with BID semaglutide or single dose of MS~semaglutide. DIO mice 25 wks old and ~50g were treated with vehicle (▼), MS~semaglutide 200 nmol / kg (■) MS~semaglutide 660 nmol / kg (▲), MS~semaglutide 2000 nmol / kg (●) or 10 μmol / kg BID semaglutide (◆ with dashed line). Data points are mean values ± SD. Fig. 6B shows that the composition of weight lost after 30 days treatment with a single dose of MS~semaglutide by DEXA scan.

[0043] Fig. 7 shows that the semaglutide conjugate of Example 2 showed superior weight loss compared to a hydrogel microsphere conjugate that releases exenatide-[N28Q] with a half-life of one month dosed at 2000 nmol peptide / kg.

[0044] Figs. 8A, 8B, and 8C show in vitro semaglutide release for conjugate A, conjugate B, and conjugate C, respectively. Specifically, Figs. 8A, 8B, and 8C show semaglutide release (solid, •) and PEG dissolved (dashed, triangles) in vitro at pH 9.4, 37ºC.

[0045] Figs. 9A, 9B, and 9C show the amount of PEG at the injection site remaining after subcutaneous dosing in rats for conjugate A, conjugate B, and conjugate C, respectively. 17MOFO-35807479567057-20032.40

[0046] Figs. 10A and 10B show the plasma concentration of semaglutide released from conjugates B and C, respectively, after SC administration in rats according to Example 7.

[0047] Fig. 11 depicts an exemplary hydrogel of the disclosure. DETAILED DESCRIPTION

[0048] The following description sets forth exemplary compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0049] In some embodiments, the lipidated peptide used in conjugates of the disclosure are GLP-1 receptor agonists used for the treatment of metabolic disorders including type 2 diabetes, obesity, and nonalcoholic steatohepatitis (NASH), and consequences of these metabolic disorders such as increased risk of death and stroke resulting from obesity.

[0050] In some embodiments, the lipidated peptide is semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide. These lipidated peptides have sequences depicted below: SEQ ID No: 1 His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-Lys(AEEAc-AEEAc-γ-Glu-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly-OH. (semaglutide) SEQ ID No: 2 His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln- Ala-Ala-Lys(γ-Glu-palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly- OH. (Liraglutide) SEQ ID No: 3 Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile- Ala-Gln-Lys(([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO—(CH2)18—CO2H)-Ala- Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- NH2. (Tirzepatide) 18MOFO-35807479567057-20032.40 SEQ ID No: 4 eicosanedioic acid-γ-Glu-Lys-Cys*-Asn-Thr-Ala-Thr-Cys*-Ala-Thr-Gln- Arg-Leu-Ala-Glu-Phe-Leu-Arg-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr- Asn-Val-Gly-Ser-Asn-Thr-Pro-NH2 [Cys2-Cys7disfulfide] (Cagrilintide). SEQ ID No: 5 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu*- Lys(1-O-(17-carboxyheptadecyl)-D-glucopyranuronoyl)-Ala-Ala-Lys*-Glu-Phe-Ile-Gln- Trp-Leu-Leu-Gln-Thr-NH2 [Glu16-Lys20cyclic lactam] (Pemvidutide) SEQ ID No: 6 Tyr-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-αMeLeu-Leu-Asp- Lys-Lys(([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO—(CH2)18—CO2H)-Ala-Gln- Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- NH2. (Retratrutide) SEQ ID No: 7 His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu- Lys-Lys-Ala-Lys([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γGlu)2-CO—(CH2)18—CO2H)- Glu-Phe-Val-Glu-Trp-Leu-Leu-Glu-Gly-Gly-Pro-Ser-Ser-Gly (Mazdutide) SEQ ID No: 8 His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln- Ala-Ala-Arg-Glu-Phe-Ile-Lys[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(s)- carboxybutyrylamino]-ethoxy)ethoxy]-acetylamino)ethoxy]ethoxy)acetyl]]-Trp-Leu-Val- Arg-Gly-Arg-Gly (Ecnoglutide) SEQ ID No: 9 Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile- Ala-Gln-Lys(acyl)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-NH2. (VK2735) SEQ ID No: 10 His-Ac4c-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu- Arg-Ala-Ala-Lys-Asp-Phe-Ile-Lys(acyl)-Trp-Leu-Glu-Ser-Ala-NH2. (survodutide).

[0051] Lipidated peptides used as GLP-1 receptor agonists are generally administered weekly, which according to their circulating half-life is appropriate to maintain agonism of the GLP-1 receptor. It is desirable, however, to be able to administer the GLP-1 receptor agonists less frequently while maintaining the same biological response. Patient compliance improves with less frequent dosing, as there is less chance for missing an administration. 19MOFO-35807479567057-20032.40 Further, in cases where the treatment is aimed at a neurodegenerative disease such as dementia where the treatment may be administered by a physician, longer-term dosing such as quarterly dosing may coincide with regular quarterly physician visits. There is at present no known method for extending the half-life of GLP-1 receptor agonists to support monthly to quarterly administrations.

[0052] Thus, there is thus an unmet need for a means of extending the circulating half-life of lipidated peptides (e.g., GLP-1 receptor agonists) beyond the limits possible from lipidation and albumin binding. The present invention meets this need by providing extended- release hydrogel conjugates of lipidated peptides having release kinetics suitable for monthly or quarterly administration.

[0053] In one aspect, provided is a releasable hydrogel conjugate of a lipidated peptide having the formula (I): M-[Z*-L-D] (I) wherein M is a biodegradable hydrogel, Z* is a connecting group, L is a β-eliminative linker, and D is a lipidated peptide. The units L-D will also be referred to herein as linker-drug units. In some embodiments, connecting group Z* is the product of a reactive moiety (Z) covalently attached to L-D and a reactive moiety (Z’) on the macromolecular carrier (M). For instance, in some embodiments, Z* is a click product formed from the click reaction between a first click handle (Z) attached to L-D and a second click handle (Z’) attached to the macromolecular carrier (M). In some embodiments, the click reaction include copper- catalyzed Click reactions such as Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. Other approaches rely on using copper-free Click chemistry, for example, the Click product can be formed between an azide and dibenzocyclooctene (DBCO), the Click product formed using an inverse electron demand Diels-alder cycloaddition (IEDDA) between a trans-cyclooctene (TCO) moiety and a tetrazine ring, or a Click product formed in a Staudinger reaction between an azide and a phosphine.

[0054] In some embodiments, the releasable hydrogel conjugate of formula (I) has the formula (IA): M-[Z*-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D]s. (IA) 20MOFO-35807479567057-20032.40 wherein M is a biodegradable hydrogel, Z* is a connecting group, n = 0-6, R1is an electron- withdrawing group, R2is H or C1-3 alkyl, each R4is independently H or C1-C3 alkyl, or taken together form a 4-7 membered ring, D is a lipidated peptide attached through its Na-amine, and s is the number of moles of D per milligram of biodegradable hydrogel (M). In particular embodiments, the lipidated peptide is selected from the group consisting of semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide.

[0055] In some variations, “electron-withdrawing group” is a functional group having a Hammett sigma constant greater than 0. In certain variations, examples of such electron- withdrawing groups include CN, NO2, carbonyl, ester, carboxamide, alkyl sulfone, aryl sulfone, heteroaryl sulfone, sulfonamide, N-alkyl sulfonamide, N-aryl sulfonamide, N-alkyl- N-aryl sulfonamide, aryl, and heteroaryl; each electron-withdrawing group may be optionally substituted so as to fine-tune their electron-withdrawing ability. Suitable substitutions include alkyl, halogen, alkoxy, haloalkyl, aryl, and heteroaryl groups. In certain embodiments, electron-withdrawing groups include CN, optionally substituted phenylsulfone, optionally substituted alkylsulfone, N-alkyl sulfonamide, N-alkyl-N-aryl sulfonamide, aryl, and heteroaryl. In particular embodiments of the invention, electron-withdrawing groups include CN, chlorophenylsulfone, (trifluoromethyl)phenylsulfone, phenylsulfone, methylphenylsulfone, methoxyphenylsulfone, methylsulfone, ethylsulfone, isopropylsulfone, N,N-dimethylsulfonamide, N-methyl-N-phenylsulfonamide, N-methyl-N-ethylsulfonamide, N-methyl-N-(2-methoxyethyl)sulfonamide, N-ethyl-N-(2-methoxyethyl)sulfonamide, N- methyl-N-(3-methoxypropyl)sulfonamide, morpholino-SO2, piperidinyl-SO2, 4- methylpiperidinyl-SO2, and fluorenyl.

[0056] The electron-withdrawing groups alter the acidity (pKa) of the hydrogen on the adjacent carbon (R1R2C-H) thereby making it more or less susceptible to removal by hydroxide ion, the concentration of which is determined by the pH of the medium. Once this hydrogen is removed, the linker undergoes an elimination of the beta-leaving group to cleave the linker. Thus, the electron-withdrawing groups control the rate at which a linker cleaves under given conditions of pH and temperature, and appropriate choice of these groups provides conjugates having the appropriate rates of lipidated peptide release and hydrogel degradation. 21MOFO-35807479567057-20032.40

[0057] In some variations, “connecting group” is a functional group that stably connects two sections of the conjugate and is not cleaved during the normal functioning of the releasable biodegradable hydrogel conjugate. In certain variations, examples of such connecting groups include carboxamides, thioethers, dihydropyridazines, and triazoles. Each connecting group is formed by the reaction of two cognate precursor functional groups (e.g., Z and Z’). Thus, a carboxamide is formed by the reaction of an amine with a carboxylate or active ester group. A dihydropyridazine is formed by the reaction of a tetrazine with a trans- cyclooctene. A thioether is formed by the reaction of a thiol with a maleimide, -halocarbonyl, or activated alkene. An oxime is formed by the reaction of an aminoether and a carbonyl. A triazole is formed by the 1,3-dipolar cycloaddition of an azide with an alkyne or cycloalkyne. Thus, Z and Z’ may be or comprise amine, carboxylate, active ester, tetrazine, trans- cyclooctene, thiol. maleimide, a-halocarbonyl, activated alkene, aminoether, carbonyl, azide, alkyne, or cycloalkyne groups.

[0058] In some variations, “optionally substituted” refers to having one or more H atoms replaced by a substituent, including halogen (F, Cl, Br, I); linear or branched C1-C6 alkyl; C3- C8 cycloalkyl; C1-C6 alkoxy; C1-C6 haloalkyl; alkylamino; dialkylamino; carboxy; carboxamido; carbonyl; nitro; CN; azido; thiol; C1-C6alkylthio; alkylsulfonyl; arylsulfonyl; sulfonamide; alkylsulfonamide; arylsulfonamide; C1-C10 aryl; and C1-C10 heteroaryl. In particular embodiments of the invention, substituents include trifluoromethyl; chloro; fluoro; methyl; methoxy; 2-methoxyethyl; phenyl; methylsulfonyl; sulfonamide; and N,N- dimethylsulfonamide.

[0059] In some embodiments of a compound of formula (IA), s is between 10 and 1,000. In some embodiments, s is between 50 and 1,000. In some embodiments, s is between 100 and 1,000. In some embodiments, s is between 200 and 1,000. In some embodiments, s is between 200 and 800. In some embodiments, s is between 200 and 600. In some embodiments, s is between 200 and 800. In some embodiments, s is between 50 and 200. In some embodiments, s is between 50 and 100. In some embodiments, s is between 100 and 200.

[0060] In some embodiments, “biodegradable hydrogel” is a highly hydrated insoluble crosslinked polymer matrix comprising cleavable linkers in the crosslinks. The insoluble matrix slowly degrades as the linkers cleave and break the crosslinks, thus returning the polymer matrix to soluble monomers that are subsequently cleared from the system. 22MOFO-35807479567057-20032.40 Examples of such biodegradable hydrogels include polymers such as polyethylene glycols (PEGs), hyaluronic acids, and polyacrylamides that are crosslinked with groups comprising either hydrolytically-sensitive functionalities such as esters, carbonates, or acetals; enzymatically-sensitive functionalities such as polypeptides; or pH-sensitive functionalities such as carbamates having electron-withdrawing groups positioned 2 atoms away and so are sensitive to base-mediated beta-elimination reactions. In certain embodiments of the invention, M is a hydrogel comprising crosslinkers that cleave by beta-elimination.

[0061] In some embodiments, the biodegradable polymer is a crosslinked PEG comprised of pegylated polymers. In some embodiments, the individual pegylated polymers comprising the hydrogel have a size of from about 5kDa to about 50 kDa. In some embodiments, the individual pegylated polymers comprising the hydrogel have a size of from about 10 kDa to about 50 kDa. In some embodiments, the individual pegylated polymers comprising the hydrogel have a size of from about 10 kDa to about 20 kDa. In some of the foregoing embodiments, s is between 10 and 400, 50 and 300, 50 and 200, or 100 and 200. Linker-Drug Units of Conjugates

[0062] In some embodiments, the hydrogel conjugates comprise linker-drug (L-D) units of the formula (II):wherein: n is an integer from 0 to 6; R1is an electron-withdrawing group; R2is H or C1-3alkyl; each R4is independently H or C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; 23MOFO-35807479567057-20032.40 D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier (e.g., via connecting group Z* in the releasable hydrogel conjugate of formula (I) or formula (IA)).

[0063] In some embodiments of the linker-drug units of the formula (II), n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 5. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is an integer from 0 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0064] In some embodiments of the linker-drug units of the formula (II), each R4group is independently C1-C3alkyl. In some embodiments, both R4groups are CH3.

[0065] In some embodiments of the linker-drug units of the formula (II), each R4group is independently H. In some embodiments, both R4groups are H.

[0066] In some embodiments of the linker-drug units of the formula (II), one R4group is CH3and one R4group is H.

[0067] In some embodiments of the linker-drug unit of formula (II), the electron- withdrawing group R1is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR5, -SOR5, or -SO2R5, 24MOFO-35807479567057-20032.40 wherein R5is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring; or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0068] In some embodiments of a linker-drug units of formula (II), the electron- withdrawing group R1is -CN, -SOR5or -SO2R5. In other embodiments, R1is –CN or -SO2R5. In other embodiments, R1is –CN or -SO2R5, wherein R5is optionally substituted alkyl, optionally substituted aryl, or -NR82. In other embodiments, R1is –CN, -SO2N(CH3)2, - SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0069] In some embodiments of the linker-drug unit of formula (II), the electron- withdrawing group R1is -CN. In some embodiments, the electron-withdrawing group of R1is -NO2. In some embodiments, the electron-withdrawing group R1is optionally substituted aryl containing 6-10 carbons. For instance, in some embodiments, the electron-withdrawing group of R1is optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, the electron-withdrawing group of R1is optionally substituted heteroaryl comprising 3-7 carbons and containing at least one N, O, or S atom. For instance, in some embodiments, the electron- withdrawing group of R1is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. In some embodiments, the electron-withdrawing group R1is optionally substituted alkenyl containing 2-20 carbon atoms. In some embodiments, the electron-withdrawing group R1is optionally substituted alkynyl containing 2-20 carbon atoms. In some embodiments, the electron- withdrawing group of R1is -COR5, -SOR5, or -SO2R5, wherein R5is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or - NR82, wherein each R8is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R8groups are taken together with the nitrogen to which they are 25MOFO-35807479567057-20032.40 attached to form a heterocyclic ring. In some embodiments, the electron-withdrawing group of R1is -SR9, wherein R9is optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0070] In some embodiments of the linker-drug unit of formula (II), R2is H.

[0071] In some embodiments of the linker-drug unit of formula (II), n is an integer from 0 to 6; R1is -CN, -NO2, -COR5, -SOR5, or -SO2R5, wherein R5is C1-C6 alkyl, aryl, heteroaryl, or NR62, wherein each R6is independently C1-C6 alkyl, aryl, or heteroaryl; R2is H or alkyl; each R4is independently H or C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735,or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0072] In some embodiments of the linker-drug unit of formula (II), n is an integer from 2 to 4; R1is -SOR5, or -SO2R5, wherein R5is C1-C6 alkyl; R2is H; each R4is independently H or C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735,or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier. 26MOFO-35807479567057-20032.40

[0073] In some embodiments of the linker-drug unit of formula (II), n is an integer from 2 to 4; R1is -SOR5, or -SO2R5, wherein R5is C1-C6 alkyl (e.g. CH3 or CH2CH3); R2is H; each R4is CH3; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through tits Na-amine.; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0074] some embodiments of the linker-drug unit of formula (II), n is 2; R1is -SO2R5, wherein R5is C1-C6 alkyl (e.g. CH3 or CH2CH3); R2is H; each R4is CH3; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through tits Na-amine.; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0075] The linker-drug unit of formula (II) can be covalently attached to a macromolecular carrier through a particular functional group. For instance, the conjugate of formula (III) depicts a single linker-drug unit attached to a macromolecular carrier (M) via functional group Z*. 27MOFO-35807479567057-20032.40(III), wherein M, n, R1, R2, R4and D are defined as above.

[0076] It will be understood that in accordance with the present disclosure, M in formula (III) is a biodegradable hydrogel. In particular embodiments, as set forth herein, M is comprised of multiple crosslinked polymers. The individual crosslinked polymers can each be attached to at least one linker-drug unit (e.g., a compound of formula (II)). Accordingly, a biodegradable hydrogel of the disclosure includes a plurality of drug units. In some embodiments, a biodegradable hydrogel of the disclosure includes between 1 to 20 micromoles of D per mL of hydrogel. In some embodiments, a biodegradable hydrogel of the disclosure includes between 2 to 20 micromoles of D per mL of hydrogel. In some embodiments, a biodegradable hydrogel of the disclosure includes between 2 to 15 micromoles of D per mL of hydrogel. In some embodiments, a biodegradable hydrogel of the disclosure includes between 2 to 10 micromoles of D per mL of hydrogel. In some embodiments, a biodegradable hydrogel of the disclosure includes between 5 to 10 micromoles of D per mL of hydrogel.

[0077] In some embodiments, the functional group Z* in the conjugate of formula (III) can be formed from a reactive functional group Z that is covalently attached to the linker- drug unit of formula (II) (which is referred to as a linker-drug unit of formula (IV) in Scheme 1 below) and a functional group Z’ on the macromolecular carrier (M). The reaction is depicted in Scheme 1. 28MOFO-35807479567057-20032.40Scheme 1

[0078] In some embodiments of Scheme 1, connecting group Z* is the product of a reactive moiety (Z) on a linker drug unit if formula (IV) and a reactive moiety (Z’) on the macromolecular carrier (M). For instance, in some embodiments, Z* is a click product formed from the reaction between a first click handle (Z) on a linker drug unit if formula (IV) and a second click handle (Z’) attached to the macromolecular carrier (M).

[0079] In some embodiments, when Z is or comprises an amine, Z’ is or comprises a carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (III) wherein Z* is or comprises amide or carbamate group. In other embodiments, when Z is or comprises an azide, Z’ is or comprises an alkynyl group, bicyclononynyl group, or cyclooctynyl group to yield a conjugate of formula (III) wherein Z* is or comprises a 1,2,3-triazole group. In other embodiments, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (III) wherein Z* is oxime. In other embodiments, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (III) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ can be reversed to yield Z* of opposing orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0080] In some embodiments, Z* comprises a carboxylic amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide. In some embodiments, Z* is or comprises a 1,2,3-triazole.

[0081] In some embodiments, Z* has the following structure: 29MOFO-35807479567057-20032.40, wherein the on top is covalently bonded to the linker drug unit (L-D) and the on the bottom is covalently bonded to the macromolecular carrier.

[0082] In some embodiments, Z* has the following structure:, wherein the on top is covalently bonded to the linker drug unit (L-D) and the on the bottom is covalently bonded to the macromolecular carrier.

[0083] In some embodiments. the disclosure provides methods of reacting the linker-drug unit of formula (IV), which comprises functional group Z, with a macromolecular carrier comprising cognate reactive groups Z’ under conditions whereby groups Z and Z’ react to form a residual connecting functional group Z*. Crosslinked Polymeric Units

[0084] In some embodiments, the hydrogels of the disclosure are comprised of repeating crosslinked polymeric units. Each polymeric unit may have multiple arms, which allows it to be crosslinked to multiple polymeric units in the hydrogel. As set forth above, the individual crosslinked units comprising the hydrogel may include one or more drug units. In some embodiments, the individual polymeric units comprising the hydrogel include, without limitation, polyethylene glycol (PEG) or other synthetic polymer, dextran, antibody, antibody fragment, albumin or other protein, of sufficient molecular size to inhibit efficient renal 30MOFO-35807479567057-20032.40 filtration as is understood in the art. For polyethylene glycols, M can be single-chain, multiple-chain, or multiple-arm of average molecular weight between 1,000 and 100,000 daltons, preferably between 1,000 and 40,000 daltons.

[0085] In some embodiments, the individual polymeric units of the hydrogel is a polypeptide selected from poly(amino acid)s such as poly(lysine) and poly(valine) and mixed-sequence polypeptides, or polypeptoids such as poly(sarcosine) and poly(oxazolines).

[0086] In some embodiments, the individual polymeric units of the hydrogel is a synthetic polymer including poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(ethylene imine) (PEI), and co-polymers thereof.

[0087] In certain embodiments of the invention, the individual polymeric units of the hydrogel are polyethylene glycols (PEGs). The polyethylene glycol may be linear or branched, with one end terminated with a functional group suitable for conjugation and the other end or ends terminated by a capping group (for example, methyl), or may comprise multiple arms each arm terminating in a functional group suitable for conjugation. In some embodiments, the polyethylene glycol is a linear, branched, or multiple-arm polymer having an average molecular weight between 20,000 and 200,000 Daltons, or between 20,000 and 100,000 Daltons, or between 10,000 and 40,000 Daltons, or between 20,000 and 40,000 Daltons. In some embodiments, P1and / or P2are approximately 10,000 Daltons. In some embodiments, P1and / or P2are approximately 20,000 Daltons. In some embodiments, P1and / or P2are approximately 40,000 Daltons. Examples of such polyethylene glycols are known in the art and are commercially available, for example from NOF Corporation (Tokyo, Japan).

[0088] In some embodiments, the individual polymeric units of the hydrogel is a polysaccharide such as a dextran.

[0089] In some embodiments, the macromolecules comprise at least one functional group suitable for conjugation, either natively or after chemical transformation, such as an amine, carboxylic acid, alcohol, thiol, alkyne, azide, or maleimide group as described above.

[0090] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker. In some embodiments, the cleavable linker has the formula (V): 31MOFO-35807479567057-20032.40wherein: A* and B* are independently connecting groups that connect the cleavable linker to the polymeric units (e.g., r-armed polymers); q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; R11is an electron-withdrawing group; R12is H or C1-C3 alkyl; each R14is independently H, C1-C3alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; and shows the point of attachment to a linker-drug unit (e.g., a linker-drug unit of formula (II) a connecting group that connects the cleavable crosslinker to a linker-drug unit of formula (II)).

[0091] In certain embodiments, A* is or comprises a carboxamide, oxime, or triazole moiety. In some embodiments of a hydrogel conjugate of formula (V), A* and B* independently comprise an amide group, a carbonate group, a 1,2,3-triazole group, an oxime, a thiosuccinimidyl group, or a thioether group. In some embodiments, A* and / or B* comprises a 1,2,3-triazole group. In some embodiments of a hydrogel conjugate of formula (V), A* and / or B* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group. In some embodiments, A* and / or B is produced via a click reaction between a first click handle on the polymeric units(s) and a second click handle on the cleavable linker.

[0092] In some embodiments of the cleavable linker of formula (V), q is an integer from 1 to 5. In some embodiments, q is an integer from 1 to 3. In some embodiments, q is an 32MOFO-35807479567057-20032.40 integer from 0 to 3. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4.

[0093] In some embodiments of the cleavable linker of formula (V), x is 0. In some embodiments of the cleavable linker of formula (V), x is 4.

[0094] In some embodiments of the cleavable linker of formula (V), y is 0. In some embodiments of the cleavable linker of formula (V), y is 4.

[0095] In some embodiments of the cleavable linker of formula (V), y is 0. In some embodiments of the cleavable linker of formula (V), z is 0.

[0096] In some embodiments of the cleavable linker of formula (V), x is 0, y is 4 and z is 0.

[0097] In some embodiments of the cleavable linker of formula (V), x is 4, y is 0 and z is 0.

[0098] In some embodiments of the cleavable linker of formula (V), x is 4, y is 0 and z is 1.

[0099] In some embodiments of the cleavable linker of formula (V), x is 0, y is 0 and z is 0.

[0100] In some embodiments of the cleavable linker of the formula (V), each R14group is independently C1-C3 alkyl. In some embodiments, both R4groups are CH3. In some embodiments, both R4groups are H. In some embodiments, one R4group is CH3 and one R4group is H.

[0101] In some embodiments of the cleavable linker of formula (V), the electron- withdrawing group R11is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; 33MOFO-35807479567057-20032.40 optionally substituted alkynyl; -COR15, -SOR15, or -SO2R15, wherein R15is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring; or SR19, wherein R19is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0102] In some embodiments of the cleavable linker of the formula (V), the electron- withdrawing group R11is -CN, -SOR15or -SO2R15. In other embodiments, R11is –CN or - SO2R15. In other embodiments, R11is –CN or -SO2R15, wherein R15is optionally substituted alkyl, optionally substituted aryl, or -NR182. In other embodiments, R11is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0103] In some embodiments of the cleavable linker of formula (V), the electron- withdrawing group R11is -CN. In some embodiments, the electron-withdrawing group of R11is -NO2. In some embodiments, the electron-withdrawing group R11is optionally substituted aryl containing 6-10 carbons. For instance, in some embodiments, the electron-withdrawing group of R11is optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, the electron-withdrawing group of R11is optionally substituted heteroaryl comprising 3-7 carbons and containing at least one N, O, or S atom. For instance, in some embodiments, the electron-withdrawing group of R11is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. In some embodiments, the electron-withdrawing group R11is optionally substituted alkenyl containing 2-20 carbon atoms. In some embodiments, the electron-withdrawing group R11is optionally substituted alkynyl containing 2-20 carbon atoms. In some embodiments, the electron- withdrawing group of R11is -COR15, -SOR15, or -SO2R15, wherein R15is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, 34MOFO-35807479567057-20032.40 -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring. In some embodiments, the electron- withdrawing group of R11is -SR19, wherein R19is optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0104] In some embodiments of a cleavable linker of formula (V), R12is H. In some such embodiments, R11is CN, -NO2, -COR15, -SOR15, or -SO2R15, wherein R15is C1-C6 alkyl, aryl, heteroaryl, or NR162, wherein each R16is independently C1-C6 alkyl, aryl, or heteroaryl.

[0105] In some embodiments of a cleavable linker of formula (V), R11is -SOR15, or - SO2R15, wherein R15is C1-C6alkyl (e.g. CH3or CH2CH3) and R12is H.

[0106] In some embodiments, the cleavable linker of formula (V) is covalently bonded to a linker-drug unit of formula (II) through a carboxamide, oxime, thioether, or triazole moiety.

[0107] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is CN or R15SO2 wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4 and z=0.

[0108] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 0-4; y = 0-4 and z=0.

[0109] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 4; y = 0 and z=0.

[0110] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 4; y = 0 and z=0.

[0111] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 0; y = 4 and z=0.

[0112] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is (CH3)2NSO2; R12is H; x = 4; y = 0 and z=0.

[0113] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is (CH3)2NSO2; R12is H; x = 0; y = 4 and z=0. 35MOFO-35807479567057-20032.40 Conjugates

[0114] The cleavable linker of formula (V) can be covalently bonded to a to a linker-drug unit of formula (II) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (V), hence forming a cleavable linker of formula (Va), depicted below.(Va), wherein A*, B*, q, R11, R12, R14, x, y and z are defined as above.

[0115] A cleavable linker of formula (Va) can be reacted with a linker-drug unit formula (IV) to yield a conjugate of formula (VI), as depicted in Scheme 2. In scheme 2, Z* is formed via a reaction between Z and Z’. In some embodiments, the reaction between Z and Z’ is a click reaction. 36MOFO-35807479567057-20032.40Scheme 2

[0116] In some embodiments of Scheme 2, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (VI) wherein Z* is amide or carbamate. In other embodiments of Scheme 2, when Z is or comprises an azide, Z’ is or comprises an alkynyl group, bicyclononynyl group, or cyclooctynyl group to yield a conjugate of formula (VI) wherein Z* is or comprises a 1,2,3-triazole group. In other embodiments of Scheme 2, when Z is or comprises a NH2O group, Z’ is or comprises a ketone or aldehyde group to yield a conjugate of formula (VI) wherein Z* is oxime. In other embodiments of Scheme 2, when Z is or comprises an SH group, Z’ is or comprises a maleimide or halocarbonyl group to yield a conjugate of formula (VI) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ can be reversed to yield Z* of opposing orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0117] In some embodiments of Scheme 2, the cleavable linker of formula (Va) has a - cyclooctynyloxycarbonyl or (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl as the 37MOFO-35807479567057-20032.40 Z’ group and the linker-drug unit of formula (IV) has an azide moiety as the Z group. In such embodiments, Z* is formed via a click reaction between the cyclooctyne moiety and the azide moiety.

[0118] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker having the formula (VII):wherein A*, q, R11, R12, and R14are defined as above.

[0119] The cleavable linker of formula (VII) can be covalently bonded to a to a linker- drug unit of formula (II) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (VII), hence forming a cleavable linker of formula (VIIa), depicted below.

[0120] A cleavable linker of formula (VIIa) can be reacted with a linker-drug unit formula (IV) to yield a conjugate of formula (VIII), as shown in Scheme 3. 38MOFO-35807479567057-20032.40Scheme 3

[0121] In some embodiments of Scheme 3, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (VI) wherein Z* is amide or carbamate. In other embodiments of Scheme 3, when Z is or comprises an azide, Z’ is or comprises an alkynyl group, bicyclononynyl group, or cyclooctynyl group to yield a conjugate of formula (VI) wherein Z* is or comprises a 1,2,3-triazole group. In other embodiments of Scheme 3, when Z is or comprises a NH2O group, Z’ is or comprises a ketone or aldehyde group to yield a conjugate of formula (VI) wherein Z* is oxime. In other embodiments of Scheme 3, when Z is or comprises an SH group, Z’ is or comprises a maleimide or halocarbonyl group to yield a conjugate of formula (VI) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ can be reversed to yield Z* of opposing orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0122] In some embodiments of scheme 3, the cleavable linker of formula (VIIa) has a - cyclooctynyloxycarbonyl or (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl as the 39MOFO-35807479567057-20032.40 Z’ group and the linker-drug unit of formula (IV) has an azide moiety as the Z group. In such embodiments, Z* is formed via a click reaction between the cyclooctyne moiety and the azide moiety.

[0123] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker. In some embodiments, the cleavable linker has the formula (IX):(IX), wherein A*, q, R11, R12, and R14are defined as above.

[0124] The cleavable linker of formula (IX) can be covalently bonded to a to a linker- drug unit of formula (II) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (IX), hence forming a cleavable linker of formula (IXa), depicted below.

[0125] A cleavable linker of formula (IXa) can be reacted with a linker-drug unit formula (IV) to yield a conjugate of formula (X), as shown in Scheme 4. 40MOFO-35807479567057-20032.40Scheme 4

[0126] In some embodiments of Scheme 4, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (VI) wherein Z* is amide or carbamate. In other embodiments of Scheme 4, when Z is or comprises an azide, Z’ is or comprises an alkynyl group, bicyclononynyl group, or cyclooctynyl group to yield a conjugate of formula (VI) wherein Z* is or comprises a 1,2,3-triazole group. In other embodiments of Scheme 4, when Z is or comprises a NH2O group, Z’ is or comprises a ketone or aldehyde group to yield a conjugate of formula (VI) wherein Z* is oxime. In other embodiments of Scheme 4, when Z is or comprises an SH group, Z’ is or comprises a maleimide or halocarbonyl group to yield a conjugate of formula (VI) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ can be reversed to yield Z* of opposing orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether. 41MOFO-35807479567057-20032.40

[0127] In some embodiments of scheme 4, the cleavable linker of formula (IXa) has a - cyclooctynyloxycarbonyl or (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxycarbonyl as the Z’ group and the linker-drug unit of formula (IV) has an azide moiety as the Z group. In such embodiments, Z* is formed via a click reaction between the cyclooctyne moiety and the azide moiety. Crosslinked Polymers

[0128] Cleavable linkers with covalently attached linker-drug units, such as formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), and formula (X), can be covalently bonded to individual polymeric units as set forth herein. For instance, the cleavable linker of formula (V) can be bonded to two individual polymeric units through connecting groups A* and B*. Formula (XI) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (V). As discussed herein, in particular embodiments, hydrogels of the disclosure comprise an array of repeating crosslinked units of formula (XI).

[0129] In certain embodiments, connecting groups A* and B* can be formed via a reaction between a functional group on the polymeric unit (P1or P2) and a cognate functional group on the crosslinker. For instance, a functional group A on the crosslinker can be reacted with a cognate functional group A’ on polymeric unit P1to form connecting group A*. Likewise, a functional group B on the crosslinker can be reacted with a cognate functional group B’ on polymeric unit P2to form connecting group B*.

[0130] In certain variations, examples of connecting groups A* and B* include carboxamides, thioethers, dihydropyridazines, and triazoles. Each connecting group is formed by the reaction of two cognate precursor functional groups A and A’ or B and B’. Thus, a carboxamide is formed by the reaction of an amine with a carboxylate or active ester group. A dihydropyridazine is formed by the reaction of a tetrazine with a trans-cyclooctene. A 42MOFO-35807479567057-20032.40 thioether is formed by the reaction of a thiol with a maleimide, a-halocarbonyl, or activated alkene. An oxime is formed by the reaction of an aminoether and a carbonyl. A triazole is formed by the 1,3-dipolar cycloaddition of an azide with an alkyne or cycloalkyne. Accordingly, A and A’ (or B and B’) may be or comprise amine, carboxylate, active ester, tetrazine, trans-cyclooctene, thiol, maleimide, a-halocarbonyl, activated alkene, aminoether, carbonyl, azide, alkyne, or cycloalkyne groups.

[0131] In some embodiments, when A is amine, A’ is carboxylic acid, active ester, or active carbonate to yield a conjugate wherein A* is amide or carbamate. In other embodiments, when A is or comprises an azide, A’ is or comprises an alkynyl group, bicyclononynyl group, or cyclooctynyl group to yield a conjugate wherein A* is or comprises a 1,2,3-triazole group. In other embodiments, when A is or comprises a NH2O group, A’ is or comprises a ketone or aldehyde group to yield a conjugate wherein A* is oxime. In other embodiments, when A is or comprises an SH group, A’ is or comprises a maleimide or halocarbonyl group to yield a conjugate wherein A* is thiosuccinimidyl or thioether. Similarly, these roles of A and A’ can be reversed to yield A* of opposing orientation. In some embodiments, A* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0132] In some embodiments, when B is amine, B’ is carboxylic acid, active ester, or active carbonate to yield a conjugate wherein B* is amide or carbamate. In other embodiments, when B is or comprises an azide, B’ is or comprises an alkynyl group, bicyclononynyl group, or cyclooctynyl group to yield a conjugate wherein B* is or comprises a 1,2,3-triazole group. In other embodiments, when B is or comprises a NH2O group, B’ is or comprises a ketone or aldehyde group to yield a conjugate wherein B* is oxime. In other embodiments, when B is or comprises an SH group, B’ is or comprises a maleimide or halocarbonyl group to yield a conjugate wherein B* is thiosuccinimidyl or thioether. Similarly, these roles of B and B’ can be reversed to yield B* of opposing orientation. In some embodiments, B* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0133] In some embodiments, A* has the following structure: 43MOFO-35807479567057-20032.40, wherein the on top is covalently bound to (CH2)qof the cleavable linker and the on the bottom is covalently bound to polymeric unit P1.

[0134] In some embodiments, A* has the following structure:, wherein the on top is covalently bound to (CH2)qof the cleavable linker and the on the bottom is covalently bound to polymeric unit P1.

[0135] In some embodiments, B* has the following structure:, wherein the attached to the carbonyl group is covalently bound to (CH2)z of the cleavable linker and the attached to the N-H group covalently bound to polymeric unit P2.

[0136] In some embodiments, A* has the following structure: 44MOFO-35807479567057-20032.40, wherein the on top is covalently bound to (CH2)q of the cleavable linker and the on the bottom is covalently bound to polymeric unit P1, and B* has the following structure:, wherein the attached to the carbonyl group is covalently bound to (CH2)zof the cleavable linker and the attached to the N-H group covalently bound to polymeric unit P2.

[0137] In some embodiments of formula (XI), q is an integer from 1 to 5. In some embodiments, q is an integer from 1 to 3. In some embodiments, q is an integer from 0 to 3. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, q is 4.

[0138] In some embodiments of formula (XI), x is 0. In some embodiments of formula (XI), x is 4.

[0139] In some embodiments of formula (XI), y is 0. In some embodiments of formula (XI), y is 4.

[0140] In some embodiments of formula (XI), y is 0. In some embodiments of formula (XI), z is 0.

[0141] In some embodiments of formula (XI), x is 0, y is 4 and z is 0.

[0142] In some embodiments of formula (XI), x is 4, y is 0 and z is 0.

[0143] In some embodiments of formula (XI), x is 4, y is 0 and z is 1. 45MOFO-35807479567057-20032.40

[0144] In some embodiments of formula (XI), x is 0, y is 0 and z is 0.

[0145] In some embodiments of the formula (XI), each R14group is independently C1-C3 alkyl. In some embodiments, both R4groups are CH3. In some embodiments, both R4groups are H. In some embodiments, one R4group is CH3and one R4group is H.

[0146] In some embodiments of formula (XI), the electron-withdrawing group R11is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR15, -SOR15, or -SO2R15, wherein R15is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring; or SR19, wherein R19is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0147] In some embodiments of formula (XI), the electron-withdrawing group R11is - CN, -SOR15or -SO2R15. In other embodiments, R11is –CN or -SO2R15. In other embodiments, R11is –CN or -SO2R15, wherein R15is optionally substituted alkyl, optionally substituted aryl, or -NR182. In other embodiments, R11is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0148] In some embodiments of formula (XI), the electron-withdrawing group R11is - CN. In some embodiments, the electron-withdrawing group of R11is -NO2. In some 46MOFO-35807479567057-20032.40 embodiments, the electron-withdrawing group R11is optionally substituted aryl containing 6- 10 carbons. For instance, in some embodiments, the electron-withdrawing group of R11is optionally substituted phenyl, naphthyl, or anthracenyl. In some embodiments, the electron- withdrawing group of R11is optionally substituted heteroaryl comprising 3-7 carbons and containing at least one N, O, or S atom. For instance, in some embodiments, the electron- withdrawing group of R11is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. In some embodiments, the electron-withdrawing group R11is optionally substituted alkenyl containing 2-20 carbon atoms. In some embodiments, the electron-withdrawing group R11is optionally substituted alkynyl containing 2-20 carbon atoms. In some embodiments, the electron- withdrawing group of R11is -COR15, -SOR15, or -SO2R15, wherein R15is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring. In some embodiments, the electron- withdrawing group of R11is -SR19, wherein R19is optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0149] In some embodiments of formula (XI), R12is H. In some such embodiments, R11is CN, -NO2, -COR15, -SOR15, or -SO2R15, wherein R15is C1-C6alkyl, aryl, heteroaryl, or NR162, wherein each R16is independently C1-C6 alkyl, aryl, or heteroaryl.

[0150] In some embodiments of formula (XI), R11is -SOR15, or -SO2R15, wherein R15is C1-C6alkyl (e.g. CH3or CH2CH3) and R12is H.

[0151] In some embodiments, the crosslinked unit of formula (XI) is covalently bonded to a linker-drug unit of formula (II) through a carboxamide, oxime, thioether, or triazole moiety.

[0152] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is CN or R15SO2 wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4 and z=0. 47MOFO-35807479567057-20032.40

[0153] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 0-4; y = 0-4 and z=0.

[0154] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 4; y = 0 and z=0.

[0155] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 4; y = 0 and z=0.

[0156] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 0; y = 4 and z=0.

[0157] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is (CH3)2NSO2; R12is H; x = 4; y = 0 and z=0.

[0158] In some embodiments of formula (XI), q = 1-2; each R14is methyl; R11is (CH3)2NSO2; R12is H; x = 0; y = 4 and z=0.

[0159] Formula (XII) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker.

[0160] In some embodiments of formula (XII), P1and P2are each independently 4- armed PEGs of average molecular weight between 5,000 and 60,000 daltons; A* comprises a triazole or oxime group; q = 0-6; each R4and R14is C1-C3 alkyl; R11is CN or R15SO2 48MOFO-35807479567057-20032.40 wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 0-6; R1is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through the Na- amine.

[0161] In some embodiments of formula (XII), P1and P2are each independently 4- armed PEGs of average molecular weight of between 10,000 and 40,000 daltons; A* comprises a triazole group; q = 0-2; each R4is methyl; each R14is methyl; R11is CN or R15SO2wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 0-3; R1is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0162] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 or 20,000 daltons; A* comprises a triazole group; q = 0-2; each R4is methyl; each R14is methyl; R11is R15SO2 wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4; z = 0; B* is carboxamide; Z* comprises a triazole group; n = 0-3; R1is CN or R3SO2wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na-amine.

[0163] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 1; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine. 49MOFO-35807479567057-20032.40

[0164] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; q = 1; each R4is methyl; each R14is methyl, R11is Me2NSO2; R12is H; x = 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its amine.

[0165] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 1; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x = 4; y = 0; z = 0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0166] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; q = 1; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0167] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 2; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0168] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; q = 2; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x= 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a 50MOFO-35807479567057-20032.40 lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0169] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 2; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x= 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0170] In some embodiments of formula (XII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; q = 2; each R4is methyl; each R14is methyl; R11is Me2NSO2; R12is H; x = 0; y = 4; z=0’ B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) attached through its Na- amine.

[0171] In any of the foregoing embodiments of formula (XII), Z* can have one of the following structures:, wherein the on top is covalently bonded to (CH2)nand the on the bottom is covalently bonded to (CH2)y.

[0172] In any of the foregoing embodiments of formula (XII), Z* can have one of the following structures: 51MOFO-35807479567057-20032.40, wherein the on top is covalently bound to (CH2)qof the cleavable linker and the on the bottom is covalently bound to polymeric unit P1,

[0173] Formula (XIII) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (VIII).

[0174] Formula (XIV) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (VIII).

[0175] Formula (XV) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (IX). 52MOFO-35807479567057-20032.40

[0176] Formula (XVI) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (X).Hydrogels

[0177] In some embodiments, formula (XI), formula (XII), formula (XIII). formula (IV), formula (XV) or formula (XVI) represents a single repeating unit of a hydrogel. In such embodiments, each polymeric unit may have multiple arms, which allows it to be crosslinked to multiple polymeric units in the hydrogel. For instance, if P1is a 4-armed polymer, it can be crosslinked to 4 polymeric units (e.g., 4 P2units). Likewise, if P2is a 4-armed polymer, it can be crosslinked to 4 polymeric units (e.g., 4 P1units).

[0178] Representative hydrogels of the disclosure are depicted below: 53MOFO-35807479567057-20032.4054MOFO-35807479567057-20032.4055MOFO-35807479567057-20032.40wherein P1and P2are each independently r-armed polymers, where r = 2-8. In some embodiments, r is 3. In some embodiments, r is 4. In some embodiments, r is 5. In some embodiments, r is 6.

[0179] In some embodiments of a hydrogel of formula (XVII), formula (XVIII), formula (XIX), formula (XX), formula (XXI) or formula (XXII), P1and / or P2are r-armed PEGs. In some embodiments, P1and / or P2are 4-armed PEGs. In some embodiments, P1and / or P2are 5-armed PEGs. In some embodiments, P1and / or P2are 6-armed PEGs. In some embodiments, P1and / or P2are 7-armed PEGs. In some embodiments, P1and / or P2are 8- armed PEGs. In some embodiments, both P1and P2are r-armed PEGs. In some embodiments, both P1and P2are 4-armed PEGs. In some embodiments, both P1and P2are 5- armed PEGs. In some embodiments, both P1and P2are 6-armed PEGs. In some embodiments, both P1and P2are 7-armed PEGs. In some embodiments, both P1and P2are 8- armed PEGs. 56MOFO-35807479567057-20032.40

[0180] The polyethylene glycol (P1and / or P2) may be linear or branched, with one end terminated with a functional group suitable for conjugation and the other end or ends terminated by a capping group (for example, methyl), or may comprise multiple arms each arm terminating in a functional group suitable for conjugation. In some embodiments, the polyethylene glycol is a linear, branched, or multiple-arm polymer having an average molecular weight between 20,000 and 200,000 Daltons, or between 20,000 and 100,000 Daltons, or between 10,000 and 40,000 Daltons, or between 20,000 and 40,000 Daltons. In some embodiments, P1and / or P2are approximately 10,000 Daltons. In some embodiments, P1and / or P2are approximately 20,000 Daltons. In some embodiments, P1and / or P2are approximately 40,000 Daltons. Examples of such polyethylene glycols are known in the art and are commercially available, for example from NOF Corporation (Tokyo, Japan).

[0181] The disclosure also provides hydrogel conjugates comprising polymeric units (P1- P2) crosslinked by a cleavable crosslinker and further comprising a linker-drug (L-D), wherein said crosslinked polymeric units have the formula (XXX):wherein: P1and P2are independently r-armed pegylated polymers, wherein r is an integer from 2 to 8; A* and B* are independently connecting groups (also referred to herein as attachment moieties) that connect the cleavable crosslinker to the r-armed pegylated polymers; q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; 57MOFO-35807479567057-20032.40 R11is -CN, -NO2, -COR13, -SOR13, -SO2R13, or SR19, wherein R13is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring, and wherein R19is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R12is H; each R14is independently H, C1-C3 alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; Z* is a connecting group (also referred to herein as an attachment moiety) for attaching the cleavable crosslinker to the linker-drug (L-D); and L-D has the formula:wherein: n is an integer from 0 to 6; R1is -CN, -NO2, -COR3, -SOR3, or -SO2R3, wherein R3is , optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R2is H; each R4is independently H, C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; 58MOFO-35807479567057-20032.40 D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug (L-D) to Z*.

[0182] In some embodiments of the crosslinked polymeric units of formula (XXX), each R4is independently C1-C3 alkyl. In some embodiments, each R4is methyl.

[0183] In some embodiments of the crosslinked polymeric units of formula (XXX), R1is CN or -SO2R3. In some embodiments, R1is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, - SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0184] In some embodiments the crosslinked polymeric units of formula (XXX), n is an integer from 1 to 3.

[0185] In some embodiments of the crosslinked polymeric units of formula (XXX), D is semaglutide. In some embodiments of a hydrogel conjugate of formula (XXX), D is tirzepatide

[0186] In some embodiments of the crosslinked polymeric units of formula (I), Z* comprises a carbonate, amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether group.

[0187] In some embodiments the crosslinked polymeric units of formula (XXX), each R14is independently C1-C3 alkyl. In some embodiments, both R14are methyl. In some embodiments, one R14is methyl and one R14is H. In some embodiments, both R14are H.

[0188] In some embodiments of the crosslinked polymeric units of formula (XXX), each R4is independently C1-C3 alkyl and each R14is independently C1-C3 alkyl. In some embodiments, both R4are methyl and both R14are methyl.

[0189] In some embodiments of the crosslinked polymeric units of formula (XXX), each R4is independently C1-C3 alkyl and both R14are H. In some embodiments, both R4are methyl and both R14are H. 59MOFO-35807479567057-20032.40

[0190] In some embodiments of the crosslinked polymeric units formula (XXX), R11is - CN or -SO2R15, wherein R15is defined above. In some embodiments, R11is -SO2CH3.

[0191] In some embodiments the crosslinked polymeric units of formula (XXX), wherein A* and B* independently comprise an amide group, carbonate group, 1,2,3-triazole group, an oxime, a thiosuccinimidyl group, or a thioether group. In some embodiments, Z* comprises a 1,2,3-triazole group.

[0192] In some embodiments of the crosslinked polymeric units formula (XXX), A* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group.

[0193] In some embodiments of the crosslinked polymeric units of formula (XXX), A* comprises an amide group. In some embodiments of a hydrogel conjugate of formula (XXX), A* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group. In some embodiments, A* the second moiety is bicyclononynyl or cyclooctynyl. In some embodiments of a hydrogel conjugate of formula (XXX), B* comprises an amide group.

[0194] In some embodiments, the crosslinked polymeric units of formula (XXX) are crosslinked polymeric units of formula (XXXI):60MOFO-35807479567057-20032.40

[0195] In some embodiments of formula (XXXI), q is 1. In some embodiments of formula (XXXI), q is 2. In some embodiments of formula (XXXI), q is 3.

[0196] In some embodiments, the crosslinked polymeric units of formula (XXXI) has the following structure:.

[0197] In some embodiments of formula (XXXI), P1and / or P2are 4-armed PEGs between 10-kDa and 20-kDa. In some embodiments of formula (XXXI), both R14groups are methyl. In some embodiments of formula (XXXI), both R14groups are hydrogen. In some embodiments of formula (XXXI), both R4groups are methyl. In some embodiments of formula (XXXI), both R4groups are hydrogen. In some embodiments of formula (XXXI), both R4groups are methyl and both R14groups are methyl.

[0198] In some embodiments, the crosslinked polymeric units of formula (XXX) are crosslinked polymeric units of formula (XXXII). 61MOFO-35807479567057-20032.40(XXXII)

[0199] In some embodiments of formula (XXXII), q is 1. In some embodiments of formula (XXXII), q is 2. In some embodiments of formula (XXXII), q is 3.

[0200] In some embodiments, the crosslinked polymeric units of formula (XXXII) have one of the following structures:62MOFO-35807479567057-20032.40(XXXIIB)

[0201] In some embodiments of formula (XXXII), P1and / or P2are 4-armed PEGs between 10-kDa and 20-kDa. In some embodiments of formula (XXXII), both R14groups are methyl. In some embodiments of formula (XXXII), both R14groups are hydrogen. In some embodiments of formula (XXXII), both R4groups are methyl. In some embodiments of formula (XXXII), both R4groups are hydrogen. In some embodiments of formula (XXXII), both R4groups are methyl and both R14groups are methyl.

[0202] In some embodiments, the crosslinked polymeric units, such as the crosslinked polymeric units of formula (XXX), formula (XXXI) or formula (XXXII), are the fundamental units of the hydrogels of the disclosure. Each repeating polymeric has multiple arms, which allows it to be crosslinked to multiple polymeric units, thus forming an array of repeating crosslinked units that constitute the hydrogel. An exemplary schematic of a hydrogel of the disclosure is depicted in FIG. 11. 63MOFO-35807479567057-20032.40 Methods of Making Hydrogels

[0203] In another aspect, provided are methods for the production of conjugates of formula (I). These conjugates are prepared by the reaction of an activated degradable hydrogen, M-Z’, with a linker-drug of formula (XXIII) M-(Z’)s + Z-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D -> (I) (XXIII) wherein Z and Z’ are cognate functional groups that react to form connecting group Z* as described above. In certain embodiments of the invention, Z’ and Z are an azide / cyclooctyne pair such that Z* comprises a triazole moiety.

[0204] In particular embodiments, Z’ is a cyclooctyne such as bicyclo[6.1.0]non-4-yn-9- ylmethyl (BCN) attached through an amine group on the hydrogel such that M-(Z’)s has the structureand Z is an azide such that conjugate (I) has the more detailed structure.

[0205] In some embodiments, Z’ is a cyclooctyne such as 5-hydroxycyclooctyne attached through an amine group on the hydrogel such that M-(Z’)s has the structure 64MOFO-35807479567057-20032.40and Z is an azide such that conjugate (I) has the more detailed structure

[0206] In some embodiments, the intermediate linker-drug of formula (XXIII) is prepared by reaction of the lipidated peptide or a protected form thereof with a linker reagent of formula (XXIV) Z-(CH2)nC(R4)2CH(CHR1R2)-O-CO-X (XXIV) wherein X is an activating group suitable for carbamoylation of an amine such as succinimidyloxy, chloride, fluoride, optionally substituted phenoxy, and the like. Linker reagents of formula (XXIV) may be prepared as described in PCT Publication WO2020 / 206358 and US published Application No. US20220280654, which is incorporated herein by reference in its entirety. In one embodiment of the invention, unprotected lipidated peptide is reacted with a linker reagent of formula (XXIV) in the presence of a mild base such as a tertiary amine to prepare an intermediate wherein both the Na-amine group and the imidazole of His(1) are carbamoylated. This intermediate is then reacted with hydroxylamine to selectively remove the carbamoyl-imidazole and provide linker lipidated peptide of formula (XXIII) wherein the linker is attached selectively to the Na-amine group of the lipidated drug (Fig. 1).

[0207] In an alternative embodiment, protected lipidated peptide on resin is prepared by standard solid-phase peptide synthesis. The terminal FMOC group is then removed and a 65MOFO-35807479567057-20032.40 final coupling step is performed using the linker reagent of formula (XXIV). The protected linker-peptide is then cleaved from the resin and deblocked under standard conditions and purified to provide linker-peptide of formula (XXIII). In a third embodiment of the invention, the linker-peptide of formula (XXIII) is prepared by a fragment coupling process, where the fragments may be chemically or biologically produced. In this embodiment, the N-terminal fragment is chemically prepared with the linker reagent attached to the Na-amine group of His(1), then the fragments are assembled as set forth above.

[0208] The activated hydrogels M-(Z’)s may be prepared according to known methods, for example the methods disclosed in PCT Publication WO2013 / 036847; US Patent No. 11179470, WO2020 / 206358; Henise et al. 2021 Engineering Reports, e12412 , each of which is incorporated by reference in theirentireties. The hydrogels may be prepared by in situ gelation, or more preferably as injectable microspheres. These microspheres may, in some embodiments, range in size between 10-100 microns, preferably 20-80 microns, and more preferably 40-70 microns in diameter. In one embodiment of the disclosure, the hydrogels are prepared as amine-substituted hydrogel microspheres (i.e., wherein Z’ = NH2) which are subsequently steam-sterilized as disclosed in PCT Publication WO2021 / 026494. The sterile microspheres are then activated by reaction with a cyclooctyne reagent, for example the succinimidyl carbonate of BCN or 5- hydroxycyclooctyne to give activated hydrogels M-(Z’)s wherein Z’ comprises a cyclooctyne moiety.

[0209] In one embodiment, the hydrogels are prepared as injectable microspheres having Z’ = NH2 by the polymerization of two prepolymers. A first prepolymer comprising an x- armed polymer P1wherein each arm is terminated with a functional group B, P1-(B)x, is mixed with a second prepolymer comprising an x-armed polymer P2wherein each arm is terminated with a branched adapter comprising two orthogonal functional groups B’ and Z’ and a cleavable linker and connected via group B*, under conditions such that cognate functional groups B and B’ react to form a connecting group B* and thereby forming the three-dimensional crosslinked hydrogel structure having pendant functional groups Z’. The resulting hydrogel can be sterilized by autoclaving. Subsequently, Z’ = NH2 may be further converted to other functional groups, for example Z’ comprises a cyclooctyne moiety, through reaction with an appropriate reagent such as a succinimidyl cyclooctynyl carbonate as described in the examples below. 66MOFO-35807479567057-20032.40 Methods of Treatment

[0210] In other aspects, provided are methods for the use of the conjugates of formula (I) as described herein in the treatment of metabolic diseases. In some embodiments, the metabolic diseases include diabetes (such as type 2 diabetes), obesity, and NASH. The conjugates described herein may be formulated in a pharmaceutically acceptable buffer having a pH value between 4 and 7, preferably between 5 and 6, which optionally comprises excipients to stabilize the conjugate such as antioxidants, isotonicity agents, detergents, and viscosity / suspending agents such as hyaluronic acid. In an embodiment, the conjugate of formula (I) is suspended in isotonic acetate or citrate buffer further comprising sodium chloride, methionine, and hyaluronic acid. In a particular embodiment, the conjugate of formula (I) is suspended in 10 mM sodium acetate, pH 5.0, 143 mM NaCl, 0.05% Tween 20, 10 mM methionine, and 1.2% 40kDa hyaluronic acid.

[0211] In some embodiments, the conjugates of formula (I) may be injected subcutaneously to patients in need to treatment for a metabolic disorder, such as type 2 diabetes, obesity, or NASH. When prepared as microspheres, the conjugates of formula (I) may be injected via a narrow-gauge needle, or example using an insulin syringe or autoinjector.

[0212] In some aspects, provided herein are hydrogel conjugates having release kinetics suitable for biweekly, monthly, quarterly (i.e., once every 3 months), three times yearly, twice yearly or once yearly administration. In particular embodiments, the hydrogel conjugates of the disclosure are administered by the parenteral route. In some embodiments the hydrogel conjugates of the disclosure are administered subcutaneously. In some embodiments the hydrogel conjugates of the disclosure are administered intravenously. In some embodiments the hydrogel conjugates of the disclosure are administered intramuscularly. In some embodiments the hydrogel conjugates of the disclosure are administered intradermally. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide. In any of the foregoing embodiments, the hydrogel conjugates are administered to a patient in need thereof to treat a particular disease or disorder. In some embodiments, the disease is obesity. 67MOFO-35807479567057-20032.40

[0213] In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 0.5 mL to about 3 mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 3 mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 2 mL. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide. In any of the foregoing embodiments, the hydrogel conjugates are administered to a patient in need thereof to treat a particular disease or disorder. In some embodiments, the disease is obesity.

[0214] In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of from about 2 μmol drug / mL to about 10 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel from about 3 μmol drug / mL to about 8 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel from about 4 μmol drug / mL to about 6 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 4 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 5 μmol drug / mL. In some embodiments, the hydrogel conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 6 μmol drug / mL. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide. In any of the foregoing embodiments, the hydrogel conjugates are administered to a patient in need thereof to treat a particular disease or disorder. In some embodiments, the disease is obesity.

[0215] In some embodiments, the hydrogel conjugates are administered at a dose of from about 1 μmol to about 20 μmol once every 3 months, once every four months, once every six 68MOFO-35807479567057-20032.40 months, or once every twelve months. In some embodiments, the hydrogel conjugates are administered at a dose of from about 2 μmol to about 10 μmol once every three months, once every four months, once every six months, or once every twelve months. In some embodiments, the hydrogel conjugates are administered at a dose of from about 2 μmol to about 5 μmol once every three months, once every four months, once every six months, or once every twelve months. In any of the foregoing embodiments, the drug (D) is a lipidated peptide selected from semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide. In some embodiments, the lipidated peptide is semaglutide. In some embodiments, the lipidated peptide is tirzepatide. In any of the foregoing embodiments, the hydrogel conjugates are administered to a patient in need thereof to treat a particular disease or disorder. In some embodiments, the disease is obesity. EXAMPLES

[0216] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation. Example A: Preparation A Synthesis of linker reagents

[0217] Linker reagents were prepared according to the procedures described in PCT Publication WO2020 / 206358 and Hearn et al., Bioconjugate Chem.32, 794-800 (2021) (each incorporated herein by reference). As an example, 5-azido-1-(methylsulfonyl)-3,3- dimethylpentyl succinimidyl carbonate was prepared as follows.

[0218] (a) Ethyl 4-chloro-2,2-dimethylbutanoate. A heat-gun dried, 500-mL, round- bottom flask equipped with a stir bar, rubber septum, nitrogen inlet, and thermocouple probe was charged with iPr2NH (5.30 mL, 37.4 mmol, 1.1 equiv, 0.27 M final concentration) and THF (100 mL). The reaction mixture was cooled at 0 °C while a solution of nBuLi (1.28 M in hexanes, 27.8 mL, 35.7 mmol, 1.05 equiv, 0.26 M final concentration) was added dropwise via syringe at a rate such that the internal temperature did not exceed +10 °C (~10 min). The reaction mixture was stirred at 0 °C for 15 min, cooled to -78 °C and a solution of ethyl 69MOFO-35807479567057-20032.40 isobutyrate (4.6 mL, 4.0 g, 34 mmol, 1.0 equiv, 0.24 M final concentration) in THF (5 mL) was added dropwise via syringe at a rate such that the internal temperature did not exceed - 65 °C (~5 min). The reaction mixture was stirred at – 78 °C for 45 min then a solution 1- bromo-2-chloro ethane (2.8 mL, 34 mmol, 1.0 equiv, 0.24 M final concentration) in THF (5 mL) was added at a rate such that the internal temperature did not exceed -68 °C. The reaction mixture was stirred at -78 °C for 15 min, allowed to warm to 0 °C, and stirred at 0 °C for 15 min. The reaction mixture was diluted with EtOAc (100 mL) and 5% KHSO4(100 mL). The aqueous phase was separated and extracted with EtOAc (3 x 50 mL). The aqueous phase was separated and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered, and concentrated from toluene (10 mL x 2) to afford 4.85 g (27 mmol, 79%) of desired chloride as a pale yellow oil.1H NMR (CDCl3, 300 MHz) δ 4.14 (q, J=7.2 Hz, 2 H), 3.43 - 3.57 (m, 2 H), 1.94 - 2.19 (m, 2 H), 1.27 (t, J=7.1 Hz, 3 H), 1.22 (s, 6 H)

[0219] (b) Ethyl 4-azido-2,2-dimethylbutanoate. A 100-mL, round-bottomed flask equipped stir bar, rubber septum, and nitrogen inlet was charged with ethyl 4-chloro-2,2- dimethylbutanoate (2-1) (4.85 g, 27 mmol, 1.0 equiv, 0.54 M final concentration), DMSO (50 mL), and sodium azide (2.28 g, 35 mmol, 1.3 equiv, 0.70 M). The reaction mixture was stirred behind a blast shield at 70 °C for 18 h. The reaction mixture was cooled to ambient temperature and was diluted with EtOAc (200 mL) and H2O (100 mL). The organic phase was separated, washed with H2O (3 x 100 mL) and brine (100 mL), dried over MgSO4, filtered, and concentrated. Purification via column chromatography (40 g silica gel cartridge; stepwise gradient elution: 0%, 5%, 10%, 20% EtOAc / hexanes) afforded 4.33 g (23.3 mmol, 87%) the desired azide 2-3a as a pale yellow oil.1H NMR (CDCl3, 300 MHz) δ 4.15 (q, J=7.1 Hz, 2 H), 3.22 - 3.35 (m, 2 H), 1.81 - 1.96 (m, 2 H), 1.27 (t, J=7.2 Hz, 3 H), 1.15 - 1.24 (m, 6 H)70MOFO-35807479567057-20032.40

[0220] (c) 5-azido-1-(methylsulfonyl)-3,3-dimethyl-2-pentanol. A 50 mL heat-gun dried, round-bottomed flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with dimethyl sulfone (1.6 g, 17 mmol, 2.1 equiv, 0.57 M final concentration), DMSO (6.8 mL), and THF (11 mL). The mixture was stirred at 0 °C while a solution of nBuLi (1.3 M in hexanes, 12.5 mL, 16.3 mmol, 2.0 equiv, 0.54 M final concentration) was added dropwise via syringe over 10 min. The reaction mixture was stirred at 0 °C for 1h. A solution of the ester (1.5 g, 8.10 mmol, 1.0 equiv, 0.27 M final concentration) in THF (4 mL) was added dropwise via syringe over 5 min. The reaction mixture was stirred at 0 °C for 30 min, allowed to warm to ambient temperature over 30 min, and was stirred at ambient temperature for 30 min. The reaction mixture was poured onto 50 mL of 1N HCl and 25 g ice. The resulting mixture was extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine, dried over MgSO4, filtered, and concentrated to afford 1.4 g of a yellow oil that was used in the next step without further purification.

[0221] A 50-mL round-bottomed flask was charged with the crude ketone (1.4 g, ~6 mmol, 1.0 equiv, 0.3 M final concentration) prepared above and MeOH (20 mL) and cool to 0 °C. NaBH4 (113 mg, 3 mmol, 0.5 equiv, 0.15 M final concentration) was added in a single portion (gas evolution observed) and the resulting mixture was stirred at 0 °C for 30 min. The reaction mixture was diluted with EtOAc (50 mL) and 5% aq KHSO4 (50 mL). The aqueous phase was separated and extracted with EtOAc (3x50 mL). The combined organics were washed with brine (50 mL), dried over MgSO4, filtered, and concentrated. Purification via column chromatography (80 g silica gel cartridge; 30%, 40%, 50%, 75%, 100% EtOAc / hexanes) afforded 204 mg (0.87 mmol, 11% yield) has an off white semi-solid that crystallized from ethyl acetate / hexane.1H NMR (CDCl3, 300 MHz) δ 3.97 - 4.05 (m, 1 H), 3.33 - 3.51 (m, 2 H), 3.12 (d, J=6.0 Hz, 2 H), 2.98 - 3.07 (m, 4 H), 1.77 (dt, J=14.4, 7.4 Hz, 1 H), 1.44 - 1.56 (m, 1 H), 0.97 (s, 3 H), 0.95 (s, 3 H).

[0222] (d) 5-azido-3,3-dimethyl-1-(methylsulfonyl)pentan-2-yl (2,5-dioxopyrrolidin-1-yl) carbonate. A 25-mL round-bottomed flask equipped with a stir bar, rubber septum, and nitrogen inlet was charged with NHS (150 mg, 1.30 mmol, 1.5 equiv, 0.22 M final concentration), DCM (6 mL), and triphosgene (129 mg, 0.433 mmol, 0.5 equiv, 0.11 M final 71MOFO-35807479567057-20032.40 concentration) and cooled at 0 °C. Pyridine (0.23 mL, 2.9 mmol, 3.3 equiv, 0.48 M final concentration) was added and the reaction mixture was allowed to warm and stir at ambient temperature for 30 min. A solution of linker alcohol 2-3a (204 mg, 0.867 mmol, 1.0 equiv, 0.15 M final concentration) in DCM (1 mL) was added and the reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was cooled at 0 °C while H2O (5 mL) was added. The reaction mixture was further diluted with EtOAc (50 mL), and the organic phase was separated and washed with water, 5% aq KHSO4, and brine. The organic phase was dried over MgSO4, filter, and concentrated. Purification via column chromatography (25 g silica gel cartridge; stepwise gradient elution: 20%, 30%, 40%, 50%, 60% acetone / hexanes) afforded 197 mg (0.523 mmol, 60% yield) of the desired product as a colorless sticky residue that crystallized from ethyl acetate / hexane.1H NMR (CDCl3, 300 MHz) δ 5.29 (dd, J=9.4, 1.7 Hz, 1 H), 3.34 - 3.51 (m, 3 H), 3.22 - 3.31 (m, 1 H), 3.03 (s, 3 H), 2.85 (s, 4 H), 2.18 (s, 3 H), 1.68 (dd, J=8.4, 6.1 Hz, 1 H), 1.51 - 1.64 (m, 2 H), 1.07 (s, 3 H), 1.06 (s, 3 H). Example B: Preparation B Preparation of activated hydrogel microspheres

[0223] Hydrogel microspheres were prepared, sterilized, and activated as described in PCT Publication WO2021 / 026494, US published Application No. US20220265873, and Henise et al. Engineering Reports, e12412. https: / / doi.org / 10.1002 / eng2.12412. (2021), each of which is incorporated herein by reference). As examples, amino-hydrogel microspheres of formula (XVII) were prepared wherein P1and P2are each 10-kDa 4-armed polyethylene glycols, Z* is triazole, q = 1, each R14= Me, R11= SO2NMe2, R12= H, x = 0, y = 4, z=0, Z’ = NH2, and B* = carboxamide by crossflow-emulsification of the prepolymer monomers. Similarly, microspheres of formula (XVII) were prepared wherein P1and P2are each 20- kDa 4-armed polyethylene glycols, Z* is triazole, q = 1, each R14= Me, R11= SO2NMe2, R12= H, x = 4, y = 0, z=0, Z’ = NH2, and B* = carboxamide. After sterilization by autoclaving (Henise et al., Engineering Reports, https: / / doi.org / 10.1002 / eng2.12091 (2019), incorporated herein by reference), the amino-hydrogel microspheres were activated by reaction with BCN-OSu (Engineering Reports, https: / / onlinelibrary.wiley.com / doi / full / 10.1002 / eng2.12213 (2020), incorporated herein by reference). 72MOFO-35807479567057-20032.40

[0224] As an illustrative example, a first prepolymer was prepared as follows. H- Lys(Boc)-OH was acylated with a linker of Formula (I) wherein Z = azide to give an adapter unit where A = COOH, B = Boc-protected NH2, and C = azide. This was coupled to 20-kDa 4-armed PEG-tetraamine, and the Boc group was removed to provide a first prepolymer wherein A* = amide, B = NH2, and C = azide and wherein a cleavable linker of formula (I) is incorporated into the linkage between each arm and group C of the first prepolymer. The corresponding second prepolymer was prepared by acylation of 20-kDa 4-armed PEG- tetraamine with 5-cyclooctynyl succinimidyl carbonate to give a second prepolymer wherein C’ = cyclooctyne. Upon mixing of the first and second prepolymers, reaction of the C = azide and C’ = cyclooctyne groups form corresponding triazole groups and thereby crosslink the two prepolymers into a 3-dimensional network, with each crosslink comprising a cleavage linker resulting from incorporation of the compound of Formula (I), and wherein each node resulting from incorporation of a first prepolymer comprises a remaining functional group B = NH2which can be derivatized for attachment of further linkers, drugs, fluorophores, metal chelators, and the like. 73MOFO-35807479567057-20032.40

[0225] (1) Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys-OH. A solution of Boc-Lys-OH (2.96 g, 12.0 mmol) in 28 mL of H2O was successively treated with 1 M aq NaOH (12.0 mL, 12.0 mmol), 1 M aq NaHCO3 (10.0 mL, 10.0 mmol), and a solution of O-{4-azido-3,3-dimethyl-1-[(N,N- dimethyl)aminosulfonyl]-2-butyl}-O’-succinimidyl carbonate (3.91 g, 10.0 mmol, 0.1 M final concentration) in 50 mL of MeCN. After stirring for 2 h at ambient temperature, the reaction was judged to be complete by C18 HPLC (ELSD). The reaction was quenched with 30 mL of 1 M KHSO4(aq). The mixture was partitioned between 500 mL of 1:1 EtOAc:H2O. The aqueous phase was extracted with 100 mL of EtOAc. The combined organic phase was washed with H2O and brine (100 mL each) then dried over MgSO4, filtered, and concentrated by rotary evaporation to provide the crude title compound (5.22 g, 9.99 mmol, 99.9% crude yield) as a white foam.

[0226] C18 HPLC, purity was determined by ELSD: 99.1% (RV = 9.29 mL).

[0227] LC-MS (m / z): calc, 521.2; obsd, 521.3 [M-H]-.

[0228] (2) Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys-OSu. Dicyclohexylcarbodiimide (60% in xylenes, 2.6 M, 4.90 mL, 12.7 mmol) was added to a solution of Nα-Boc-Nε-{4-azido-3,3-dimethyl-1-[(N,N- 74MOFO-35807479567057-20032.40 dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys -OH (5.11 g, 9.79 mmol, 0.1 M final concentration) and N-hydroxysuccinimide (1.46 g, 12.7 mmol) in 98 mL of CH2Cl2. The reaction suspension was stirred at ambient temperature and monitored by C18 HPLC (ELSD). After 2.5 h, the reaction mixture was filtered, and the filtrate was loaded onto a SiliaSep 120 g column. Product was eluted with a step-wise gradient of acetone in hexane (0%, 20%, 30%, 40%, 50%, 60%, 240 mL each). Clean product-containing fractions were combined and concentrated to provide the title compound (4.95 g, 7.99 mmol, 81.6% yield) as a white foam.

[0229] C18 HPLC, purity was determined by ELSD: 99.7% (RV = 10.23 mL).

[0230] LC-MS (m / z): calc, 520.2; obsd, 520.2 [M+H-Boc]+.

[0231] (3) (Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys)4-PEG20kDa. PEG20kDa-(NH)4 (20.08 g, 0.9996 mmol, 3.998 mmol NH2, 0.02 M NH2 final concentration) was dissolved in 145 mL of MeCN. A solution of Nα- Boc-Nε-{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys- OSu (2.976 g, 4.798 mmol) in 50 mL of MeCN was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three slower eluting intermediate peaks. After 1 h, Ac2O (0.37 mL, 4.0 mmol) was added. The reaction mixture was stirred 30 min more than concentrated to ~50 mL by rotary evaporation. The reaction concentrate was added to 400 mL of stirred MTBE. The mixture was stirred at ambient temperature for 30 min then decanted. MTBE (400 mL) was added to the wet solid, and the suspension was stirred for 5 min and decanted. The solid was transferred to a vacuum filter, and washed / triturated with 3x 100 mL of MTBE. After drying on the filter for 10 min, the solid was transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum until the weight stabilized to provide the title compound (21.23 g, 0.9602 mmol, 96.1% yield) as a white solid.

[0232] C18 HPLC, purity was determined by ELSD: 89.1% (RV = 10.38 mL) with a 10.6% impurity (RV = 10.08).

[0233] (4) (Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys)4-PEG20kDa. (Nε-{4-Azido-3,3-dimethyl-1-[(N,N- dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4-PEG20kDa(19.00 g, 0.8594 mmol, 3.438 75MOFO-35807479567057-20032.40 mmol Boc, 0.02 M Boc final concentration) was dissolved in 86 mL of 1,4-dioxane. After stirring for 5 min to fully dissolve the PEG, 4 M HCl in dioxane (86 mL, 344 mmol HCl) was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three faster eluting intermediate peaks. After 2 h, the reaction mixture was concentrated to ~40 mL. THF (10 mL) was added to the concentrate, and the solution was again concentrated to ~40 mL. The viscous oil was poured into 400 mL of stirred Et2O. After stirring at ambient temperature for 20 min, the supernatant was decanted from the precipitate. The wet solid was transferred to a vacuum filter with the aid of 200 mL Et2O and washed with Et2O (3x 75 mL). The solid was dried on the filter for 10 min then transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum overnight to provide the title compound (17.52 g, 0.8019 mmol, 93.3% yield @ 4 HCl) as a white solid.

[0234] C18 HPLC, purity was determined by ELSD: 99.2% (RV = 9.34 mL).

[0235] Prepolymer B wherein C’ = cyclooctynyl. A 4-mL, screw top vial was charged with PEG20kDa-[NH2]4 (SunBright PTE-200PA; 150 mg, 7.6 μmol PEG, 30.2 μmol NH2, 1.0 equiv, 20 mM final amine concentration), MeCN (1.5 mL), and iPr2NEt (7 μL, 40 μmol, 1.3 equiv, 27 mM final concentration). A solution of the activated ester cyclooctyne (39 μmol, 1.3 equiv, 27 mM final concentration) was added and the reaction mixture was stirred at ambient temperature. Reactions were monitored by C18 HPLC (20-80%B over 11 min) by ELSD. When complete, Ac2O (3 μL, 30 μmol, 1 equiv per starting NH2) was added to the reaction mixture and the mixture was stirred for 30 min. The reaction mixture was then concentrated to a thick oil and suspended in MTBE (20 mL). The resulting suspension as vigorously stirred for 10 min. The resulting solids were triturated three times with MTBE (20 mL) by vigorously mixing, pelleting in a centrifuge (2800 rpm, 4 °C, 10 min), and removal of the supernatant by pipette. The resulting solids were dried under vacuum at ambient temperature for no more than 30 min. Stock solutions were prepared in 20 mM NaOAc (pH 5) with a target amine concentration of 20 mM. Cyclooctyne concentration was then verified by treatment with PEG7-N3(2 equiv) and back-titration of the unreacted PEG7-N3with DBCO-CO2H. 76MOFO-35807479567057-20032.40 Example C: Preparation C Preparation of prepolymers for hydrogels Prepolymer A wherein q = 1 and R11= Me2NSO2 (Nε-{4-Azido-3,3-dimethyl-1-[(N,N- dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4-PEG20kDa.

[0236] (1) Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys-OH. A solution of Boc-Lys-OH (2.96 g, 12.0 mmol) in 28 mL of H2O was successively treated with 1 M aq NaOH (12.0 mL, 12.0 mmol), 1 M aq NaHCO3 (10.0 mL, 10.0 mmol), and a solution of O-{4-azido-3,3-dimethyl-1-[(N,N- dimethyl)aminosulfonyl]-2-butyl}-O’-succinimidyl carbonate (3.91 g, 10.0 mmol, 0.1 M final concentration) in 50 mL of MeCN. After stirring for 2 h at ambient temperature, the reaction was judged to be complete by C18 HPLC (ELSD). The reaction was quenched with 30 mL of 1 M KHSO4(aq). The mixture was partitioned between 500 mL of 1:1 EtOAc:H2O. The aqueous phase was extracted with 100 mL of EtOAc. The combined organic phase was washed with H2O and brine (100 mL each) then dried over MgSO4, filtered, and concentrated by rotary evaporation to provide the crude title compound (5.22 g, 9.99 mmol, 99.9% crude yield) as a white foam. C18 HPLC, purity was determined by ELSD: 99.1% (RV = 9.29 mL). LC-MS (m / z): calc, 521.2; obsd, 521.3 [M-H]-. 77MOFO-35807479567057-20032.40

[0237] (2) Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys-OSu. Dicyclohexylcarbodiimide (60% in xylenes, 2.6 M, 4.90 mL, 12.7 mmol) was added to a solution of Nα-Boc-Nε-{4-azido-3,3-dimethyl-1-[(N,N- dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys -OH (5.11 g, 9.79 mmol, 0.1 M final concentration) and N-hydroxysuccinimide (1.46 g, 12.7 mmol) in 98 mL of CH2Cl2. The reaction suspension was stirred at ambient temperature and monitored by C18 HPLC (ELSD). After 2.5 h, the reaction mixture was filtered, and the filtrate was loaded onto a SiliaSep 120 g column. Product was eluted with a step-wise gradient of acetone in hexane (0%, 20%, 30%, 40%, 50%, 60%, 240 mL each). Clean product-containing fractions were combined and concentrated to provide the title compound (4.95 g, 7.99 mmol, 81.6% yield) as a white foam. C18 HPLC, purity was determined by ELSD: 99.7% (RV = 10.23 mL). LC- MS (m / z): calc, 520.2; obsd, 520.2 [M+H-Boc]+.

[0238] (3) (Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys)4-PEG20kDa. PEG20kDa-(NH)4(20.08 g, 0.9996 mmol, 3.998 mmol NH2, 0.02 M NH2 final concentration) was dissolved in 145 mL of MeCN. A solution of Nα- Boc-Nε-{4-azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys- OSu (2.976 g, 4.798 mmol) in 50 mL of MeCN was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three slower eluting intermediate peaks. After 1 h, Ac2O (0.37 mL, 4.0 mmol) was added. The reaction mixture was stirred 30 min more than concentrated to ~50 mL by rotary evaporation. The reaction concentrate was added to 400 mL of stirred MTBE. The mixture was stirred at ambient temperature for 30 min then decanted. MTBE (400 mL) was added to the wet solid, and the suspension was stirred for 5 min and decanted. The solid was transferred to a vacuum filter, and washed / triturated with 3x 100 mL of MTBE. After drying on the filter for 10 min, the solid was transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum until the weight stabilized to provide the title compound (21.23 g, 0.9602 mmol, 96.1% yield) as a white solid. C18 HPLC, purity was determined by ELSD: 89.1% (RV = 10.38 mL) with a 10.6% impurity (RV = 10.08). (4)(Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys)4- PEG20kDa(19.00 g, 0.8594 mmol, 3.438 mmol Boc, 0.02 M Boc final concentration) was dissolved in 86 mL of 1,4-dioxane. After stirring for 5 min to fully dissolve the PEG, 4 M 78MOFO-35807479567057-20032.40 HCl in dioxane (86 mL, 344 mmol HCl) was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three faster eluting intermediate peaks. After 2 h, the reaction mixture was concentrated to ~40 mL. THF (10 mL) was added to the concentrate, and the solution was again concentrated to ~40 mL. The viscous oil was poured into 400 mL of stirred Et2O. After stirring at ambient temperature for 20 min, the supernatant was decanted from the precipitate. The wet solid was transferred to a vacuum filter with the aid of 200 mL Et2O and washed with Et2O (3x 75 mL). The solid was dried on the filter for 10 min then transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum overnight to provide the title compound (17.52 g, 0.8019 mmol, 93.3% yield @ 4 HCl) as a white solid. C18 HPLC, purity was determined by ELSD: 99.2% (RV = 9.34 mL). Prepolymer A wherein q = 1 and R11= MeSO2 (Nε-{4-Azido-3,3-dimethyl-1-[methylsulfonyl]- 2-butyloxycarbonyl}-Lys)4-PEG20kDa.

[0239] (1) Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(methylsulfonyl]-2-butyloxycarbonyl}- Lys-OH. A solution of Boc-Lys-OH (2.96 g, 12.0 mmol) in 28 mL of H2O was successively treated with 1 M aq NaOH (12.0 mL, 12.0 mmol), 1 M aq NaHCO3(10.0 mL, 10.0 mmol), and a solution of O-{4-azido-3,3-dimethyl-1-[methylsulfonyl]-2-butyl}-O’-succinimidyl carbonate (3.91 g, 10.0 mmol, 0.1 M final concentration) in 50 mL of MeCN. After stirring for 2 h at ambient temperature, the reaction was judged to be complete by C18 HPLC (ELSD). The reaction was quenched with 30 mL of 1 M KHSO4(aq). The mixture was partitioned between 500 mL of 1:1 EtOAc:H2O. The aqueous phase was extracted with 100 79MOFO-35807479567057-20032.40 mL of EtOAc. The combined organic phase was washed with H2O and brine (100 mL each) then dried over MgSO4, filtered, and concentrated by rotary evaporation to provide the crude title compound (5.22 g, 9.99 mmol, 99.9% crude yield) as a white foam.

[0240] (2) Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[(N,N-dimethyl)aminosulfonyl]-2- butyloxycarbonyl}-Lys-OSu. Dicyclohexylcarbodiimide (60% in xylenes, 2.6 M, 4.90 mL, 12.7 mmol) was added to a solution of Nα-Boc-Nε-{4-azido-3,3-dimethyl-1-[(N,N- dimethyl)aminosulfonyl]-2-butyloxycarbonyl}-Lys -OH (5.11 g, 9.79 mmol, 0.1 M final concentration) and N-hydroxysuccinimide (1.46 g, 12.7 mmol) in 98 mL of CH2Cl2. The reaction suspension was stirred at ambient temperature and monitored by C18 HPLC (ELSD). After 2.5 h, the reaction mixture was filtered, and the filtrate was loaded onto a SiliaSep 120 g column. Product was eluted with a step-wise gradient of acetone in hexane (0%, 20%, 30%, 40%, 50%, 60%, 240 mL each). Clean product-containing fractions were combined and concentrated to provide the title compound (4.95 g, 7.99 mmol, 81.6% yield) as a white foam.

[0241] (3) (Nα-Boc-Nε-{4-Azido-3,3-dimethyl-1-[methylsulfonyl]-2-butyloxycarbonyl}- Lys)4-PEG20kDa. PEG20kDa-(NH)4 (20.08 g, 0.9996 mmol, 3.998 mmol NH2, 0.02 M NH2 final concentration) was dissolved in 145 mL of MeCN. A solution of Nα-Boc-Nε-{4-azido-3,3- dimethyl-1-[methylsulfonyl]-2-butyloxycarbonyl}-Lys-OSu (2.976 g, 4.798 mmol) in 50 mL of MeCN was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three slower eluting intermediate peaks. After 1 h, Ac2O (0.37 mL, 4.0 mmol) was added. The reaction mixture was stirred 30 min more than concentrated to ~50 mL by rotary evaporation. The reaction concentrate was added to 400 mL of stirred MTBE. The mixture was stirred at ambient temperature for 30 min then decanted. MTBE (400 mL) was added to the wet solid, and the suspension was stirred for 5 min and decanted. The solid was transferred to a vacuum filter, and washed / triturated with 3x 100 mL of MTBE. After drying on the filter for 10 min, the solid was transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum until the weight stabilized to provide the title compound (21.23 g, 0.9602 mmol, 96.1% yield) as a white solid.

[0242] (Nε-{4-Azido-3,3-dimethyl-1-[methylsulfonyl]-2-butyloxycarbonyl}-Lys)4- PEG20kDa(19.00 g, 0.8594 mmol, 3.438 mmol Boc, 0.02 M Boc final concentration) was dissolved in 86 mL of 1,4-dioxane. After stirring for 5 min to fully dissolve the PEG, 4 M 80MOFO-35807479567057-20032.40 HCl in dioxane (86 mL, 344 mmol HCl) was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three faster eluting intermediate peaks. After 2 h, the reaction mixture was concentrated to ~40 mL. THF (10 mL) was added to the concentrate, and the solution was again concentrated to ~40 mL. The viscous oil was poured into 400 mL of stirred Et2O. After stirring at ambient temperature for 20 min, the supernatant was decanted from the precipitate. The wet solid was transferred to a vacuum filter with the aid of 200 mL Et2O and washed with Et2O (3x 75 mL). The solid was dried on the filter for 10 min then transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum overnight to provide the title compound (17.52 g, 0.8019 mmol, 93.3% yield @ 4 HCl) as a white solid. Prepolymer A wherein q = 2 and R11= MeSO2 (Nε-{5-Azido-3,3-dimethyl-1-[methylsulfonyl]- 2-butyloxycarbonyl}-Lys)4-PEG20kDa.

[0243] (1) Nα-Boc-Nε-{5-Azido-3,3-dimethyl-1-[methylsulfonyl]-2-pentyloxycarbonyl}- Lys-OH. A solution of Boc-Lys-OH (2.96 g, 12.0 mmol) in 28 mL of H2O was successively treated with 1 M aq NaOH (12.0 mL, 12.0 mmol), 1 M aq NaHCO3 (10.0 mL, 10.0 mmol), and a solution of O-{5-azido-3,3-dimethyl-1-[methylsulfonyl]-2-pentyl}-O’-succinimidyl carbonate (3.91 g, 10.0 mmol, 0.1 M final concentration) in 50 mL of MeCN. After stirring for 2 h at ambient temperature, the reaction was judged to be complete by C18 HPLC (ELSD). The reaction was quenched with 30 mL of 1 M KHSO4(aq). The mixture was partitioned between 500 mL of 1:1 EtOAc:H2O. The aqueous phase was extracted with 100 mL of EtOAc. The combined organic phase was washed with H2O and brine (100 mL each) 81MOFO-35807479567057-20032.40 then dried over MgSO4, filtered, and concentrated by rotary evaporation to provide the crude title compound (5.22 g, 9.99 mmol, 99.9% crude yield) as a white foam.

[0244] (2) Nα-Boc-Nε-{5-Azido-3,3-dimethyl-1-[methylsulfonyl]-2-pentyloxycarbonyl}- Lys-OSu. Dicyclohexylcarbodiimide (60% in xylenes, 2.6 M, 4.90 mL, 12.7 mmol) was added to a solution of Nα-Boc-Nε-{5-azido-3,3-dimethyl-1-[methylsulfonyl]-2- pentyloxycarbonyl}-Lys -OH (5.11 g, 9.79 mmol, 0.1 M final concentration) and N- hydroxysuccinimide (1.46 g, 12.7 mmol) in 98 mL of CH2Cl2. The reaction suspension was stirred at ambient temperature and monitored by C18 HPLC (ELSD). After 2.5 h, the reaction mixture was filtered, and the filtrate was loaded onto a SiliaSep 120 g column. Product was eluted with a step-wise gradient of acetone in hexane (0%, 20%, 30%, 40%, 50%, 60%, 240 mL each). Clean product-containing fractions were combined and concentrated to provide the title compound (4.95 g, 7.99 mmol, 81.6% yield) as a white foam.

[0245] (3) (Nα-Boc-Nε-{5-Azido-3,3-dimethyl-1-[methylsulfonyl]-2-butyloxycarbonyl}- Lys)4-PEG20kDa. PEG20kDa-(NH)4(20.08 g, 0.9996 mmol, 3.998 mmol NH2, 0.02 M NH2final concentration) was dissolved in 145 mL of MeCN. A solution of Nα-Boc-Nε-{5-azido-3,3- dimethyl-1-[methylsulfonyl]-2-butyloxycarbonyl}-Lys-OSu (2.976 g, 4.798 mmol) in 50 mL of MeCN was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a single product peak via three slower eluting intermediate peaks. After 1 h, Ac2O (0.37 mL, 4.0 mmol) was added. The reaction mixture was stirred 30 min more than concentrated to ~50 mL by rotary evaporation. The reaction concentrate was added to 400 mL of stirred MTBE. The mixture was stirred at ambient temperature for 30 min then decanted. MTBE (400 mL) was added to the wet solid, and the suspension was stirred for 5 min and decanted. The solid was transferred to a vacuum filter, and washed / triturated with 3x 100 mL of MTBE. After drying on the filter for 10 min, the solid was transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum until the weight stabilized to provide the title compound (21.23 g, 0.9602 mmol, 96.1% yield) as a white solid.

[0246] (Nε-{5-Azido-3,3-dimethyl-1-[methylsulfonyl]-2-butyloxycarbonyl}-Lys)4- PEG20kDa (19.00 g, 0.8594 mmol, 3.438 mmol Boc, 0.02 M Boc final concentration) was dissolved in 86 mL of 1,4-dioxane. After stirring for 5 min to fully dissolve the PEG, 4 M HCl in dioxane (86 mL, 344 mmol HCl) was added. The reaction was stirred at ambient temperature and analyzed by C18 HPLC (ELSD). The starting material was converted to a 82MOFO-35807479567057-20032.40 single product peak via three faster eluting intermediate peaks. After 2 h, the reaction mixture was concentrated to ~40 mL. THF (10 mL) was added to the concentrate, and the solution was again concentrated to ~40 mL. The viscous oil was poured into 400 mL of stirred Et2O. After stirring at ambient temperature for 20 min, the supernatant was decanted from the precipitate. The wet solid was transferred to a vacuum filter with the aid of 200 mL Et2O and washed with Et2O (3x 75 mL). The solid was dried on the filter for 10 min then transferred to a tared 250 mL HDPE packaging bottle. Residual volatiles were removed under high vacuum overnight to provide the title compound as the HCl salt (17.52 g, 0.8019 mmol, 93.3% yield) as a white solid.

[0247] Prepolymer B wherein A’ = cyclooctynyl. A 4-mL, screw top vial was charged with PEG20kDa-[NH2]4(SunBright PTE-200PA; 150 mg, 7.6 μmol PEG, 30.2 μmol NH2, 1.0 equiv, 20 mM final amine concentration), MeCN (1.5 mL), and iPr2NEt (7 μL, 40 μmol, 1.3 equiv, 27 mM final concentration). A solution of the activated ester cyclooctyne (39 μmol, 1.3 equiv, 27 mM final concentration) was added and the reaction mixture was stirred at ambient temperature. Reactions were monitored by C18 HPLC (20-80%B over 11 min) by ELSD. When complete, Ac2O (3 μL, 30 μmol, 1 equiv per starting NH2) was added to the reaction mixture and the mixture was stirred for 30 min. The reaction mixture was then concentrated to a thick oil and suspended in MTBE (20 mL). The resulting suspension as vigorously stirred for 10 min. The resulting solids were triturated three times with MTBE (20 mL) by vigorously mixing, pelleting in a centrifuge (2800 rpm, 4 °C, 10 min), and removal of the supernatant by pipette. The resulting solids were dried under vacuum at ambient temperature for no more than 30 min. Stock solutions were prepared in 20 mM NaOAc (pH 5) with a target amine concentration of 20 mM. Cyclooctyne concentration was then verified 83MOFO-35807479567057-20032.40 by treatment with PEG7-N3(2 equiv) and back-titration of the unreacted PEG7-N3with DBCO-CO2H. Example D: Preparation D Preparation of amino hydrogel microspheres

[0248] Hydrogel microspheres were prepared, sterilized, and activated as described in PCT Publication WO2021 / 026494 and Henise et al. Engineering Reports, e12412. https: / / doi.org / 10.1002 / eng2.12412. (2021), incorporated herein by reference). Hydrogel microspheres prepared according to this procedure are shown in Table 1. Table 1. Exemplary Hydrogel Microspheres84MOFO-35807479567057-20032.40 Example 1 Preparation of linker-semaglutide

[0249] Semaglutide sodium salt (43 mg, 10.5 mmol, 1.0 Eq) was dissolved in 1 mL of 9:1 DMF / H2O, and N,N-diisopropylethylamine (9 mL, 52 mmol, 5.0 Eq) followed by 5-azido- 3,3-dimethyl-1-(methylsulfonyl)-2-pentyl succinimidyl carbonate (11.8 mg, 31.4 mmol, 3.0 Eq) were added. After 3 h at ambient temperature, HPLC indicated conversion to a mixture of mono- and di-linker species acylated on the Naand imidazole of histidine (2% semaglutide, 30% Na-acylated, 9% imidazole-acylated, 59% diacylated). A 0.5 M solution of hydroxylamine hydrochloride, pH 7, (200 mL) was added and the mixture kept for an additional 16 h, at which time HPLC indicated conversion of the mixture to predominantly mono-linker-semaglutide (8% semaglutide, 88% Na-acylated, 4% diacylated). Preparative HPLC (Phenomenex Jupiter 5 um 300Å C18250 x 21.4 mm column, 20-100% MeCN in H2O + 0.05% TFA) gave the purified linker-semaglutide (36 mg, 8.2 mmol, 78%). One peak by HPLC (280 nm). MS gives [M+3H]3+1458.4379 (calc. 1458.3979).

[0250] A sample was attached to 40-kDa MeO-PEG-BCN and analyzed for the rate of release of free semaglutide by HPLC under accelerated conditions (0.1 M borate, pH 9.4, 37ºC) where rates are 100x-faster than at pH 7.4, 37ºC. A release t1 / 2(pH 9.4) = 7.2 h was observed, corresponding to a release half-life of 720 h at pH 7.4 (Table 2).

[0251] Other linker-semaglutides were prepared similarly using the appropriate linker reagent (Table 2). Table 2. Linker-semaglutides prepared according to the method of Example 185MOFO-35807479567057-20032.40 Example 2 Preparation of semaglutide conjugates

[0252] A slurry of amino microspheres wherein P1and P2are each 10-kDa 4-armed PEG, A* comprises a triazole group, q = 1, each R14= Me, R11= (Me2N)SO2, R12= H, x = 0, y = 4, z=0. Z’ = NH2, and B* = carboxamide was sterilized by autoclave following the previously described method (Henise, Engineering Reports 2020;e12091). To the sterile slurry of amino microspheres in acetonitrile (2.64 µmol NH2 / gram of slurry, 5.9 µmol NH2, ~0.75 mL), 1.5 equivalents of bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN-HSC, 11.7 µmol, 3.4 mg in 250 μL acetonitrile) and 4 equivalents of triethylamine (23.5 µmol, 3.3 µL in 250 μL acetonitrile) was added and the reaction proceeded for 2 hours at room temperature with rotation. Reaction completion was determined by no color change with a trinitrobenzesulfonate (TNBS) test. Excess reagents were washed from the slurry with 3 washes of 4 mL acetonitrile followed by 4 washes with 4 mL methanol. Linker-semaglutide C from Example 1, wherein Z = N3, n = 2, each R4= Me, R1= MeSO2, D = semaglutide (1.2 equivalents, 7.1 µmol, 31 mg) in 500 µL of DMF was added to the BCN-microsphere slurry for a final volume of ~1.2 mL and the reaction proceeded for 42 hours at 37ºC with mixing at 225 rpm. The reaction progress was followed by loss of A280in the supernatant. Unreacted linker-semaglutide was removed by washing the slurry 6 times with ~4 mL DMF, followed by 4 x ~4 mL 10 mM acetate 143 mM NaCl 0.05% Tween 20 pH 5.0, 10 mM methionine with a final exchange into 10 mM acetate 143 mM NaCl 0.05% Tween 20 pH 5.0, 10 mM methionine, 1.2% 40kD hyaluronic acid. To quantify semaglutide loading, 20 mg aliquots of slurry were dissolved with 180 µL 50 mM NaOH at room temperature for 60 minutes (in triplicate) then assayed for peptide by A280 using e280 = 6790 M-1cm-1(after neutralization with 50 μL sample to 150 μL 125 μL HEPES pH 7.4) and PEG content. For this “10x10” conjugate, semaglutide was loaded at 5.3 μmol / g of slurry, with a peptide to PEG ratio of 183 nmol / mg.

[0253] A second conjugate (“20x20”) was similarly prepared wherein P1and P2are each 20-kDa 4-armed PEG, A* comprises a triazole group, q = 1, each R14= Me, R11= (Me2N)SO2, R12= H, x = 4, y = 0, z=0, B* = carboxamide. A slurry of amino microspheres was sterilized by autoclave following the previously described method (Henise 2020, 86MOFO-35807479567057-20032.40 Engineering Reports). To the sterile slurry of amino microspheres in acetonitrile (1.4 µmol NH2 / gram of slurry, 5.8 µmol NH2, ~2.7 mL), 1.5 equivalents BCN-HSC (8.6 µmol, 2.5 mg in 500 μL acetonitrile) and 4 equivalents triethylamine (23.0 µmol, 3.2 µL in 500 μL acetonitrile) was added and the reaction proceeded for 2 hours at room temperature with rotation. Reaction completion was determined by no color change with a TNBS test. Excess reagents were washed from the slurry with 3 washes of 4 mL acetonitrile followed by 4 washes with 4 mL methanol. Linker-semaglutide from Example 1 (1.2 equivalents, 7.1 µmol, 31 mg) in 500 µL of DMF was added to the BCN-microsphere slurry along with 1 mL of DMF for a final volume of ~3.4 mL and the reaction proceeded for 42 hours at 37ºC with mixing at 225 rpm. The reaction progress was followed by loss of A280 in the supernatant. At the final timepoint the slurry would not pellet, so an additional 2 mL DMF was added to get a sample of the supernatant. Unreacted linker-semaglutide was removed by washing the slurry 6 times with ~10 mL DMF, followed by 4 x ~10 mL 10 mM acetate, 143 mM NaCl, 0.05% Tween 20, pH 5.0, 10 mM methionine with a final exchange into 10 mM acetate, 143 mM NaCl, 0.05% Tween 20, pH 5.0, 10 mM methionine, 1.2% 40k hyaluronic acid. Semaglutide loading was assayed as described above. Semaglutide was loaded at 1.4 μmol / g of slurry, with a peptide to PEG ratio of 65 nmol / mg.

[0254] Structures of the conjugates are shown in Fig. 2. Example 3 In vitro release and hydrogel degradation

[0255] In vitro peptide release kinetics and hydrogel degradation were measured simultaneously under accelerated conditions (pH 9.4, 37ºC) using the previously described automated method (Engineering Reports. 2020;e12213). The peptide release and PEG solubilization curves can be seen in Figs. 3A and 3B for each conjugate from Example 2. For both conjugates, 50% of the conjugated semaglutide was released by 12.8 h and the complete solubilization (tRG) of the hydrogel microspheres occurred at ~35 h. 87MOFO-35807479567057-20032.40 Example 4 Mouse pharmacokinetics

[0256] The pharmacokinetics of semaglutide released from the conjugates of Example 2(R11= MeSO2; R12= H; n = 2) were measured in normal C57BL / 6 mice. Mice were dosed with either 10 or 50 nmol of conjugated semaglutide and plasma samples were collected over a 3-month period and analyzed for semaglutide using ELISA. The conjugates of Example 2 demonstrated extended release of semaglutide for greater than 30 days after a single injection, with a half-life of ~900 hours. Data is shown in Fig. 4. Example 5 Weight loss in DIO mice

[0257] The conjugates of Example 2 were administered to diet-induced obese (DIO) mice. DIO mice having initial body weight of approximately 47 g were treated with single doses of the 10x10 conjugate of Example 2 comprising 200, 660, or 2000 nmol / kg of semaglutide, or a single dose of the 10x10 conjugate of Example 2 comprising 2000 nmol / kg of semaglutide, and weights were recorded over a 1-month period. The mice lost up to 25% of their initial body weight over the 1-month period (Figs. 5A and 5B), comparable to twice- daily administration of free semaglutide at 10 nmol / kg / dose, or a total of 600 nmol / kg per month.

[0258] Body composition was determined by DEXA scan (Figs. 6A and 6B) and indicated that treatment with the conjugates of Example 2 gave rise to selective loss of fat. Fig. 6A shows that Body weight of DIO mice treated with BID semaglutide or single dose of MS~semaglutide. DIO mice 25 wks old and ~50g were treated with vehicle (▼), MS~semaglutide 200 nmol / kg (■) MS~semaglutide 660 nmol / kg (▲), MS~semaglutide 2000 nmol / kg (●) or 10 μmol / kg BID semaglutide (◆ with dashed line). Data points are mean values ± SD. Fig. 6B shows that the composition of weight lost after 30 days treatment with a single dose of MS~semaglutide by DEXA scan. 88MOFO-35807479567057-20032.40

[0259] In a second experiment, weight loss in DIO mice from a single dose of the 20x20 conjugate of Example 2 was compared to that from PLX039, a hydrogel microsphere conjugate that releases exenatide-[N28Q] with a half-life of one month (see WO 2020 / 206358) dosed at 2000 nmol peptide / kg. As shown in Fig. 7, The semaglutide conjugate of Example 2 showed superior weight loss in this model (25% vs 10%) over a one- month period. Example 6 Gel dissolution profile of hydrogels following subcutaneous injection

[0260] Three hydrogel conjugates (Conjugate A, Conjugate B and Conjugate C) were prepared all have same drug release linker (R1= MeSO2; n = 2; each R4= Me) but on different microspheres. The structures of Conjugate A, Conjugate B and Conjugate C are depicted below.Conjugate A 89MOFO-35807479567057-20032.40Conjugate C

[0261] The conjugates were administered subcutaneously to rats and the loss of the hydrogel as a result of gel dissolution at the injection site was monitored. Table 3 shows the results of the study, where T80% is the time for 80% loss of hydrogel from the injection site. The results in Table 3 show that gel dissolution at the injection site can be controlled by the β-eliminative functionality on the crosslinks of the hydrogel. 90MOFO-35807479567057-20032.40 Table 3: Loss of Hydrogels from the injection siteExample 7 In vitro semaglutide release and degelation and in vivo depot residence

[0262] The hydrogel microsphere conjugates (Conjugate A, Conjugate B, and Conjugate C) of Example 6 were studied in vitro to measure the release of semaglutide and dissolution of the hydrogel matrix, and in vivo after SC administration to rats to determine the dissolution kinetics of the hydrogel.

[0263] For in vitro measurements, samples of the conjugates were placed into porous dissolution cells which were then suspended in 0.1 M borate buffer, pH 9.4, and kept at 37ºC as described in Henise et al (Engineering Reports 2020;e12213). Samples from outside the cells were periodically removed and assayed for released semaglutide by absorbance at 280 nm and dissolved PEG by the barium triiodide method as described. In vitro data were fit to a model for hydrogel dissolution and drug release (Reid et al., Macromolecules 2015, 48: 7359-69) adapted to account for variations in linker cleavage rates observed as the hydrogels disintegrate and variations in environment pH. Figs. 8A, 8B, and 8C show in vitro semaglutide release for conjugate A, conjugate B, and conjugate C, respectively. Specifically, Figs. 8A, 8B, and 8C show semaglutide release (solid, •) and PEG dissolved (dashed, triangles) in vitro at pH 9.4, 37ºC.

[0264] For in vivo measurements, aliquots of the conjugates were injected subcutaneously into rats, and the depots were periodically removed using a biopsy punch, dissolved in 50 mM NaOH, and assayed for PEG remaining at the injection site using the barium triiodide method. In vivo data were fit using the same model, but calculating the fraction of the initial PEG remaining at the injection site assuming that PEG fragments larger than 20 kDa had restricted diffusion from the subcutaneous space (Ishikawa et al., 2023 ACS Macro Lett 12(4): 510-7). Figs. 9A, 9B, and 9C show the amount of PEG at the injection site 91MOFO-35807479567057-20032.40 remaining after subcutaneous dosing in rats for conjugate A, conjugate B, and conjugate C, respectively.

[0265] Figs. 10A and 10B show the plasma concentration of semaglutide released from conjugates B and C, respectively, after SC administration in rats. 92MOFO-358074795

Claims

67057-20032.40 CLAIMS What is claimed is:

1. A controlled release conjugate of semaglutide having the formula (I) M-[Z*-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D]s. (I) wherein M is a biodegradable hydrogel, Z* is a connecting group, n = 0-6, R1is an electron- withdrawing group, R2is H or C1-3 alkyl, each R4is independently H or C1-C3 alkyl, or taken together form a 4-7 membered ring, D is a lipidated peptide attached through its Na-amine, and s is the number of moles of D per milligram of biodegradable hydrogel (M), wherein D is selected from the group consisting of semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide.

2. The conjugate of claim 1, wherein R1is selected from CN, NO2, carbonyl, ester, carboxamide, alkyl sulfone, aryl sulfone, heteroaryl sulfone, sulfonamide, N-alkyl sulfonamide, N-aryl sulfonamide, N-alkyl-N-aryl sulfonamide, aryl, and heteroaryl 3. A linker-drug units of the formula (II)wherein: n is an integer from 0 to 6; R1is an electron-withdrawing group; 93MOFO-35807479567057-20032.40 R2is H or C1-3alkyl; each R4is independently H or C1-C3 alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide selected from the group consisting of semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide connected through its Na-amine; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

4. The linker-drug unit of claim 3, wherein n is 2, 3, or 4.

5. The linker-drug unit of claim 3 or claim 4, wherein both R4groups are CH3.

6. The linker-drug unit of any one of claims 3-5, wherein R1is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR3, -SOR3, or -SO2R3, wherein R3is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring; or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl. 94MOFO-35807479567057-20032.40 7. The linker-drug unit of any one of claims 3-5, wherein R1is CN, -SOR3or -SO2R3. In other embodiments, R1is –CN or -SO2R3. In other embodiments, R1is –CN or -SO2R3, wherein R3is optionally substituted alkyl, optionally substituted aryl, or -NR82. In other embodiments, R1is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, - SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

8. The linker-drug unit of any one of claims 3-5, wherein R1is SOR5, or -SO2R5, wherein R5is C1-C6 alkyl.

9. The linker-drug unit of any one of claims 3-8, wherein R2is H.

10. A hydrogel comprising a linker-drug unit of any one of claims 2-9 and a macromolecular carrier.

11. A conjugate of the formula (II)(III), wherein: n is an integer from 0 to 6; R1is an electron-withdrawing group; R2is H or C1-3alkyl; each R4is independently H or C1-C3alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide selected from the group consisting of semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide connected through its Na-amine; 95MOFO-35807479567057-20032.40 M is a macromolecular carrier; and Z* is a connecting group.

12. The conjugate of claim 11, wherein n is 2, 3, or 4.

13. The conjugate of claim 11 or claim 12, wherein both R4groups are CH3.

14. The conjugate of any one of claims 11-13, wherein R1is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR3, -SOR3, or -SO2R3, wherein R3is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring; or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

15. The conjugate of ant one of claims 11-13, wherein R1is CN, -SOR3or -SO2R3. In other embodiments, R1is –CN or -SO2R3. In other embodiments, R1is –CN or -SO2R3, wherein R3is optionally substituted alkyl, optionally substituted aryl, or -NR82. In other embodiments, R1is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, - SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2. 96MOFO-35807479567057-20032.40 16. The conjugate of any one of claims 11-13, wherein R1is SOR5, or -SO2R5, wherein R5is C1-C6 alkyl.

17. The conjugate of any one of claims 11-16, wherein R2is H.

18. The conjugate of any one of claims 11-17, wherein M is comprised of crosslinked polymers.

19. The conjugate of claim 18, wherein the crosslinked polymers are pegylated polymers.

20. The conjugate of claim 19, wherein the pegylated polymers are comprised of multi- armed chains.

21. The conjugate of claim 20, wherein the multi-armed pegylated chains have a weight of between 1,000 and 40,000 Daltons.

22. A cleavable linker of formula (V)wherein: A* and B* are independently connecting groups that connect the cleavable linker to the polymeric units ; q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; R11is an electron-withdrawing group; R12is H or C1-C3 alkyl; 97MOFO-35807479567057-20032.40 each R14is independently H, C1-C3alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; and shows the point of attachment to a linker-drug comprising semaglutide.

23. The cleavable linker of claim 22, wherein R11is -CN; -NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; -COR13, -SOR13, or -SO2R13, wherein R13is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring; or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

24. The cleavable linker of claim 22 or claim 23, wherein both R4groups are methyl.

25. The cleavable linker of anyone of claims 22-24, having the formula (VII): 98MOFO-35807479567057-20032.

40.

26. The cleavable linker of anyone of claims 22-24, having the formula (VII):.

27. A conjugate hydrogel comprising a cleavable linker of any one of claims 22-26.

28. The conjugate hydrogel of claim 27, wherein the cleavable linker crosslinks pegylated polymers.

29. The conjugate of claim 28, wherein the pegylated polymers are r-armed polymers, where r is 2, 3, 4, 5, 6, 7, 0r 8.

30. The conjugate of claim 29, wherein r is 4.

31. The conjugate of any one of claims 27-30, wherein the cleavable linker is linked to a linker-drug unit of any one of claims 3-9 at the point of attachment .

32. A conjugate hydrogel that has one of the following structures: 99MOFO-35807479567057-20032.

40.

33. A method of treating a metabolic or neurologic disease or disorder or associated symptoms in a subject in need thereof, comprising administering to the subject a conjugate of any one of claims 1, 2, 10-21 or 27-32.

34. The method of Claim 33, wherein the metabolic disease or disorder is type 2 diabetes, obesity, or nonalcoholic steatohepatitis.

35. The method of Claim 33, wherein the associated symptoms are stroke and / or heart attack. 100MOFO-35807479567057-20032.40 36. The method of Claim 33, wherein the neurologic disease or disorder is Parkinson’s disease or dementia.

37. The method of any one of claims 33 to 36, wherein the conjugate is administered monthly or quarterly.

38. The method of any one of claims 33 to 36, wherein the conjugate is administered at a frequency between monthly and quarterly.

39. The method of any one of Claims 33 to 38, wherein the subject is a human.

40. A method of preparing a linker-semaglutide of formula (IV) Z-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D (IV), the method comprising: (a) reacting semaglutide with a linker reagent of formula (V) Z-(CH2)nC(R4)2CH(CHR1R2)-O-CO-X (V) wherein X is an activating group such as N-oxysuccinimidyl, chloride, or 4- nitrophenoxy, in the presence of a mild base such as a tertiary amine to prepare an intermediate wherein both the Na-amine group and the imidazole of His(1) are carbamoylated; (b) reaction of the intermediate with hydroxylamine to selectively remove the carbamoyl-imidazole and provide linker semaglutide of formula (IV) wherein the linker is attached selectively to the Na-amine group of semaglutide.

41. The method of Claim 40, wherein Z is N3; q = 1-2; each R4= methyl; R2= H; R1= MeSO2, Me2NSO2, or (MeOCH2CH2)2NSO2, and D is a lipidated peptide selected from the group consisting of semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, and survodutide connected through its Na- amine. 101MOFO-35807479567057-20032.40 42. A hydrogel conjugate having the formula (XXX):wherein: P1and P2are independently r-armed pegylated polymers, wherein r is an integer from 2 to 8; A* and B* are independently connecting groups that connect the cleavable crosslinker to the r-armed polymers; q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; R11is - is -CN, -NO2, -COR13, -SOR13, -SO2R13, or SR19,wherein R13is H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR18or -NR182, wherein each R18is independently H or optionally substituted alkyl, or both R18groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring or SR19, and wherein R19is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R12is H; each R14is independently H, C1-C3 alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; Z* is a connecting group for attaching the cleavable crosslinker to the linker-drug (L- D); and L-D has the formula: 102MOFO-35807479567057-20032.40wherein: n is an integer from 0 to 6; R1is -CN, -NO2, -COR3, -SOR3, or -SO2R3, wherein R3is , optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R2is H; each R4is independently H, C1-C3 alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a lipidated peptide (e.g., semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide) connected through its Na-amine; and depicts the point of attachment of the linker-drug (L-D) to Z*.

43. A method of treating a disease or disorder, said method comprising administering a pharmaceutical composition comprising a hydrogel conjugate of claim 42.

44. The method of claim 43, wherein the disease is obesity.

45. The method of claim 43 or 44, wherein the pharmaceutical composition is administered four times yearly. 103MOFO-35807479567057-20032.40 46. The method of claim 43 or 44, wherein the pharmaceutical composition is administered three times yearly.

47. The method of claim 43 or 44, wherein the pharmaceutical composition is administered two times yearly.

48. The method of claim 43 or 44, wherein the pharmaceutical composition is administered once yearly.

49. The method of any one of claims 43-48, wherein the pharmaceutical composition is administered at an injection volume of from about 0.5 mL to about 3 mL.

50. The method of any one of claims 43-48, wherein the pharmaceutical composition is administered at an injection volume of from about 1 mL to about 2 mL.

51. The method of any one of claims 43-50, wherein the pharmaceutical composition is administered parenterally at a concentration of drug in the hydrogel of from about 2 μmol drug / mL to about 10 μmol drug / mL.

52. The method of any one of claims 43-50, wherein the pharmaceutical composition is administered parenterally at a concentration of drug in the hydrogel of from about 3 μmol drug / mL to about 8 μmol drug / mL.

53. The method of any one of claims 43-50, wherein the pharmaceutical composition is administered parenterally at a concentration of drug in the hydrogel of from about 4 μmol drug / mL to about 6 μmol drug / mL.

54. The method of any one of claims 43-52, wherein the pharmaceutical composition is administered subcutaneously.

55. The method of any one of claims 43-54, wherein the hydrogel is administered at a dose of from about 1 μmol to about 20 μmol. 104MOFO-35807479567057-20032.40 56. The method of any one of claims 43-54, wherein the hydrogel is administered at a dose of from about 2 μmol to about 10 μmol.

57. The method of any one of claims 43-54, wherein the hydrogel is administered at a dose of from about 3 μmol to about 5 μmol.

58. The method of any one of claims 42-57, wherein the lipidated peptide is semaglutide.

59. The method of any one of claims 42-57, wherein the lipidated peptide is tirzepatide. 105MOFO-358074795

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