Extended-release microsphere drug conjugates

Abstract

Disclosed herein are microsphere (MS)-drug conjugates that provide prolonged drug release following administration to a subject in need thereof. The drug component of the (MS)-drug conjugates comprises a moiety that extends the half-life of the drug such as a lipid, polyethylene glycol or Fc fragment of an IgG.

Description

EXTENDED-RELEASE MICROSPHERE DRUG CONJUGATES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 699,070, filed on September 25, 2024, the contents of which are hereby incorporated herein by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 670572003340SeqList.xml, created on September 24, 2025, which is 50,280 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety. FIELD

[0003] The present disclosure relates generally to the production of extended-release microsphere drug conjugates, and more specifically to the use of such conjugates for administration to patients in need thereof. BACKGROUND

[0004] Several major technologies currently used for half-life extension of peptides. PEGylation permanently connects a high molecular weight PEG polymer – optimal at about 40 kDa – to the peptide to retard renal filtration. However, 40 kDa PEG itself has an elimination half-life of about 6 days, that limits half-life extension so PEGylated peptides and proteins require at least once weekly (QWk) administration. Lipidation permanently connects a fatty acid to a peptide. The fatty acid tightly and reversibly binds to serum albumin, and converts the parent peptide's half-life from approximately one hour to approximately one week by piggybacking on the long-lived albumin However, the price paid for half-life extension is the high dose of lipidated peptide needed to saturate the albumin sink and yet provide a small but sufficient amount of active free lipidated peptide to exert its intended activity. Moreover, the intense efforts of pharmaceutical companies to optimize the lipidation approach has likely achieved the practical upper limit of half-life extension to allow once weekly dosing.

[0005] Fc fusions of peptides to the Fc fragment of an IgG show a longer half-life because of the ~50 kDa MW increase and Fc recycling. Scientists who are unfamiliar with specifics of the technology assume Fc fusions have half-lives similar to the 20-day half-life of IgG or the approximately 14 day half-life of therapeutic antibodies, and are surprised to learn that the average half-life of Fc fusions are just 4- to 5 days and only support once weekly administration.

[0006] In summary, the three most frequently used technologies for half-life extension of peptides require once weekly administration, and there are no obvious approaches to decrease dosing frequencies with these other than by excessive dosing and consequent risk of toxicity.

[0007] It has not been possible so far to extend these systemic half-lives further using these techniques so as to provide peptide drugs such as GLP-1R agonists or protein drugs that are suitable for less frequent administration, for example monthly or quarterly. 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 various diseases. BRIEF SUMMARY

[0008] Provided herein is an approach for half-life extension of therapeutic drugs that allows achievement of any desirable half-life. The approach provides significantly increased half-lives of therapeutic drugs that can be administered at efficacious dosses without concomitant toxicological concerns.

[0009] In some aspects, the disclosure provides microsphere (MS)-drug conjugates that provide prolonged drug release following administration to a subject in need thereof. In the approaches described herein, the drug component of the (MS)-drug conjugates comprises a moiety that extends the half-life of the drug. In particular, the therapeutic drug comprising the half-life extending moiety has a longer half-life than an identical drug without the half-life extending moiety. In some embodiments, the half-life extending moiety is a lipid. In some embodiments, the half-life extending moiety is a polyethylene glycol (PEG) chain. In some embodiments, the half-life extending moiety is an Fc fragment of an IgG.

[0010] In some embodiments, the microsphere (MS) component of the MS-drug conjugates is a hydrogel. By hydrogel is meant a three-dimensional, predominantly hydrophilic polymeric network comprising a large quantity of water, formed by chemical or physical crosslinking of natural or synthetic homopolymers, copolymers, or oligomers. Hydrogels may be formed through crosslinking polyethylene glycols (considered to be synonymous with polyethylene oxides), polypropylene glycols, poly(N-vinylpyrrolidone), polymethacrylates, polyphosphazenes, polylactides, polyacrylamides, polyglycolates, polyethylene imines, poly-amino acids, agarose, dextran, gelatin, collagen, polylysine, chitosans, alginates, hyaluronans, pectin, carrageenan. In certain embodiments, the drug is covalently tethered to a hydrogel carrier.

[0011] In some embodiments, the MS component is a polyethylene glycol (PEG)-based hydrogel carrier, as set forth herein. In some embodiments, the hydrogel is a mesoporous PEG hydrogel. By mesoporous hydrogel is meant a hydrogel having pores between approximately 1 nm and approximately 100 nm in diameter. The pores in mesoporous hydrogels are sufficiently large to allow for free diffusion of biological molecules such as proteins.

[0012] In some such embodiments, the drug is tethered to the hydrogel carrier by a labile linker this is susceptible to cleavage following administration of the drug via the parenteral route. For instance, in some embodiments, the drug is cleaved by a β-elimination mechanism in a slow base-catalyzed rate-determining step. In one embodiment, biodegradable hydrogels are formed by reaction of a single reactive polymer and a cleavable crosslinker compound wherein said cleavable crosslinker compound comprises a functional group that reacts with the reactive polymer and a moiety also including a functional group that reacts with a reactive polymer that cleaves by elimination under physiological conditions. In this embodiment, the reactive polymer will be multi-valent, so as to allow formation of nodes in the three-dimensional hydrogel matrix.

[0013] In another embodiment, the disclosure provides methods for the formation of biodegradable hydrogels through reaction of a first reactive polymer, a second reactive polymer, and a cleavable crosslinker compound that comprises a first functional group that reacts with the first reactive polymer, a second functional group that reacts with the second polymer, and a moiety that cleaves by elimination under physiological conditions. The first and second functional groups may be the same or different. For the formation of a three-dimensional gelnetwork the reactive components (first reactive polymer, second reactive polymer if any) will be multi-armed and thus serve to form nodes in the gel matrix. In some embodiments of the invention, this node-forming reactive component comprises at least 3 arms and more preferably at least 4 arms.

[0014] In accordance with the disclosure, using a combination of slowly releasable linkers to increase drug half-life and decreasing of the clearance of the released drug to increase concentration achieves ultralong-acting drugs with dosing intervals that could not be previously approached. There could be huge practical impact of using potent drugs (e.g., peptides / proteins drugs) with long half-lives and low clearance. As described herein, MS-drug conjugates of potent PEGylated drugs, lipidated drugs and Fc fusions with moderate half-lives of ~5- to 7 days, could provide ultralong-acting peptides that could maintain therapeutic levels for up to 3 or even 6 months or more. Notably, there are over 100 PEGylated drugs that are approved or in clinical trials, and a similar number of Fc fusions. It is likely that some significant number would be suitable for this approach to developing ultralong-acting therapeutics.

[0015] In some embodiments, the therapeutic drug is a peptide. In some embodiments, the peptide is a GLP-1 receptor agonist. In some such embodiments, the peptide is semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, MET- 097i, MET233i, VK2735, , GUBamy, eloralintide, or survodutide. In some embodiments, the peptide drug is an amylin analog. In some embodiments, the peptide drug is a calcitonin analog. In some embodiments, the analog is an apelin analog.

[0016] In some embodiments, the therapeutic drug is a protein. In some embodiments, the therapeutic drug is a PEGylated protein. In some embodiments, the PEGylated protein is Pegasys (PEGylated interferon alfa-2a), Neulasta (pegfilgrastim, PEG-GCSF), Macugen (pegaptanib, PEGylated RNA), Omontys (peginesatide) or Plegridy (pegintereron beta-1a).

[0017] In some embodiments, the half-life of the therapeutic agent (e.g., peptide or protein) can be increased from minutes / hours to over two weeks. In some embodiments, the half-life of the therapeutic agent (e.g., peptide or peptide) can be increased from minutes / hours to over three weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year.

[0018] In some aspects, provided herein are extended-release (MS)-drug 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 (MS)-drug conjugates are administered by the parenteral route. In some embodiments the (MS)-drug conjugates are administered subcutaneously. In some embodiments the (MS)-drug conjugates are administered intravenously. In some embodiments the (MS)-drug conjugates are administered intramuscularly. In some embodiments the (MS)-drug conjugates are administered intradermally.

[0019] In some embodiments, the (MS)-drug 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 (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 3 mL. In some embodiments, the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 2 mL.

[0020] In some embodiments, the (MS)-drug 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 (MS)-drug 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 (MS)-drug 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 (MS)-drug 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 (MS)-drug 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 (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 6 μmol drug / mL.

[0021] In some embodiments, the (MS)-drug 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 sixmonths, or once every twelve months. In some embodiments, the (MS)-drug 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 (MS)-drug 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.

[0022] In one aspect, provided is a releasable hydrogel conjugate 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 drug comprising a half-life extending moiety. In some embodiments, the half-life extending moiety is a lipid. In some embodiments, the half-life extending moiety is a polyethylene glycol (PEG) chain. In some embodiments, the half-life extending moiety is an Fc fragment of an IgG. In some embodiments, the half-life extending domain is an albumin-binding peptide.

[0023] In some embodiments, the (MS)-drug conjugates comprise linker-drug units of theformula (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; D is a drug comprising a half-life extending moiety; 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).

[0024] In some embodiments of the linker-drug units of formula (II), D is a lipidated drug, In some embodiments, D is a lipidated peptide. In some such embodiments, D is a lipidated peptide connected through its Na-amine or through the Ne-amine of a lysine or ornithine residue. In some embodiments, D is semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide, MET-097i, MET-233i, eloralintide, or GUBamy. In some embodiments, D is a lipidated protein.

[0025] In some embodiments of the linker-drug units of formula (II), D is a pegylated drug. In some embodiments, D is a pegylated peptide. In some embodiments, D is a pegylated protein. In some embodiments, D is polyethylene glycol loxenatide (PEG-Loxe), also referred to as PEX198.

[0026] In some embodiments of the linker-drug units of the formula (II), D is a peptide or protein fused to an Fc domain of an IgG. In some embodiments, the Fc-fusion is a monomeric Fc-fusion. In some embodiments, the Fc fusion is a polymeric Fc-fusion. In some embodiments, D is Dulaglutide.

[0027] In some embodiments of the linker-drug unit of formula (II), n is an integer from 0 to 6; R1is -CN, -NO2, -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; R2is H; each R4is independently 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 drug comprising a half-life extending moiety; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0028] 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 optionally substituted 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 drug comprising a half-life extending moiety; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0029] 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 drug comprising a half-life extending moiety; anddepicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0030] In some such embodiments of the linker-drug unit of formula (II), n is 1. In othersuch 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.

[0031] In some embodiments, the linker-drug unit of formula (II) can be covalently attachedto a macromolecular carrier through a particular attachment moiety. For instance, the conjugate of formula (III) depicts a single linker-drug unit attached to a macromolecular carrier (M) via attachment moiety Z*.wherein M is a macromolecular carrier and n, R1, R2, R4and D are defined as above. Various connecting groups (Z*) will be defined in the disclosure.

[0032] In some embodiments, the attachment moiety Z* in the conjugate of formula (III) canbe formed from a reactive functional group Z that is covalently attached to the linker-drug unit of formula (II) and a functional group Z’ on the macromolecular carrier (M).

[0033] In some embodiments, the (MS)-drug conjugates of the disclosure are comprised ofrepeating crosslinked polymeric units. Each polymeric unit may have multiple arms, which allows it to be crosslinked to multiple polymeric units, hence forming a 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, poly(amino acid),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.

[0034] In some embodiments, the individual polymeric units comprising the hydrogel arecrosslinked through a cleavable crosslinker. In some embodiments, the cleavable crosslinker has the formula (V)wherein: A* and B* are independently attachment moieties 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-C3alkyl; 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)).

[0035] 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, a thioether group, or a dihydropyridazine 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. The cleavable crosslinker 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 cleavable crosslinker and the linker-drug unit of formula (II). For instance, the reactive group Z’ can be covalently bonded to the cleavable crosslinker of formula (V), hence forming a cleavable crosslinker of formula (Va), depicted belowwherein A*, B*, q, R11, R12, R14, x, y and z are defined as above.

[0036] Likewise, reactive group Z can be covalently bonded to a linker-drug unit to form areactive linker-drug unit of formula (IV).

[0037] 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 1.Scheme 1

[0038] It will be understood that the linker-drug units can be attached to the cleavablecrosslinker prior to or after attachment of the crosslinker to the polymeric units.

[0039] In scheme 1, Z and Z’ are reactive groups or comprise reactive groups. The reactionbetween Z and Z’ yields connecting group Z*. In some embodiments of Scheme 1, 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 1, 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 1, when Z is NH2O, Z’ is ketone or aldehydeto yield a conjugate of formula (VI) wherein Z* is oxime. In other embodiments of Scheme 1, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (VI) wherein Z* is thiosuccinimidyl or thioether. In other embodiments of Scheme 1, when Z is a 2,3,5,6- tetrazine, Z’ is a trans-cyclooctene to yield a conjugate of formula (VI) wherein Z* is dihydropyridazine. 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, dihydropyridazine, or ether.

[0040] In some embodiments, the individual polymeric units comprising the hydrogel arecrosslinked through a cleavable crosslinker having the formula (VII):wherein A*, q, R11, R12, and R14are defined as above.

[0041] In some embodiments, the individual polymeric units comprising the hydrogel arecrosslinked through a cleavable crosslinker having the formula (VIII):wherein A*, q, R11, R12, and R14are defined as above.

[0042] In accordance with the disclosure, cleavable linkers 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 polymers (P1and P2) crosslinked through the cleavable linker of formula (V).

[0043] In some embodiments, P1 and P2 are r-armed pegylated polymers, wherein r is aninteger from 2 to 8. In some embodiments, r is 4. In some embodiments, r is 6.

[0044] In one aspect, the disclosure provides (MS)-drug conjugates comprising polymericunits (P1-P2) crosslinked by a cleavable crosslinker and further comprising a linker-drug (L-D), wherein said (MS)-drug conjugates have the formula (XVII):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 - is -CN, -NO2, -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 SR19, 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-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 drug comprising a half-life extending moiety and depicts the point of attachment of the linker-drug (L-D) to Z*.

[0045] In some embodiments, a (MS)-drug conjugates of the disclosure has one of the following structures:

[0046] In other aspects, provided are methods for preparing these conjugate hydrogels andintermediates thereto.

[0047] In some aspects, provided are methods for using these conjugates in the treatment ofmetabolic 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 twice yearly. In some embodiments, the conjugates are administered once yearly.DESCRIPTION OF THE FIGURES

[0048] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0049] FIG. 1 shows β-elimanitive release of a drug from linkers bound to hydrogels of the disclosure.

[0050] FIG. 2 depicts the cleavage, release and clearance of peptides from microsphere carriers. The top panel shows an example where the peptide is not covalently linked to a half-life extension moiety. While the release rate (k1) is slow, the clearance (k2) is fast. The middle and bottom panels show examples where the peptide is covalently linked to a half-life extension moiety. comprising half-life extension moiety The release rate (k1) and the clearance (k2) are slow.

[0051] FIG.3 shows a simulation of C vs t curves for exenatide and loxenatide released from the same doses of MS~conjugates dosed at 2 μmol / 100kg person.

[0052] FIG.4 shows the structure of linker semaglutide of Example 1, wherein Z = N3, n = 2, each R4= Me, and R1= MeSO2.

[0053] FIG.5 shows the structures of specific hydrogels designed to release lipidated peptides.

[0054] FIGS.6A and 6B 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, 37 °C) 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 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.

[0055] FIG.7 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 or2000 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.

[0056] FIG.8 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.8A) the 10x10 and 20x20 conjugate s each comprising 2000 nmol / kg of semaglutide; (FIG. 8B) 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.

[0057] FIG.9A 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.9B shows that the composition of weight lost after 30 days treatment with a single dose of MS~semaglutide by DEXA scan.

[0058] FIG.10 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.

[0059] FIGS.11A, 11B, and11C show in vitro semaglutide release for conjugate A, conjugate B, and conjugate C, respectively. Specifically, FIGS. 11A, 11B, and 11C show semaglutide release (solid, •) and PEG dissolved (dashed, triangles) in vitro at pH 9.4, 37 °C.

[0060] FIGS.12A, 12B, and 12C show the amount of PEG at the injection site remaining after subcutaneous dosing in rats for conjugate A, conjugate B, and conjugate C, respectively.

[0061] FIGS.13A and 13B show the plasma concentration of semaglutide released from conjugates B and C, respectively, after SC administration in rats.

[0062] FIG 14 depicts an exemplary hydrogel of the disclosure.

[0063] FIG.15 shows the in vitro release and subsequent polymer dissolution of a releasable conjugate of the Fc-fusion Dulaglutide according to Example 12 under accelerated conditions. DETAILED DESCRIPTION

[0064] 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. I. Conjugates for Prolonged Release of Drugs

[0065] In some aspects, the disclosure provides microsphere (MS)-drug conjugates that provide prolonged drug release following administration to a subject in need thereof. In the approaches described in the drug component of the (MS)-drug conjugates comprises a moiety that extends the half-life of the drug. In particular, the therapeutic drug comprising the half-life extending moiety has a longer half-life than an identical drug without the half-life extending moiety. In some embodiments, the half-life extending moiety is a lipid. In some embodiments, the half-life extending moiety is a polyethylene glycol (PEG) chain. In some embodiments, the half-life extending moiety is an Fc fragment of an IgG.

[0066] In some embodiments, the microsphere (MS) component is MS-drug conjugates is a hydrogel. Examples of hydrogels that can be used in accordance with the disclosure are described in Section II, below. In certain embodiments, the drug is covalently tethered to a hydrogel carrier. In some such embodiments, the drug is tethered to the hydrogel carrier by a labile linker this is susceptible to cleavage following administration of the drug via the parenteral route. For instance, in some embodiments, the drug is cleaved by a β-elimination mechanism in a slow base-catalyzed rate-determining step (FIG. 1). All subsequent steps are faster, so the rate of drug release directly reflects that of this first step. In certain embodiments, the cleavage rate of the linker is controlled by the nature of an electron-withdrawing “modulator” (Mod) which regulates the acidity of an adjacent carbon-hydrogen bond. Since the in vitro and in vivo cleavage rates follow a tight structure activity relationship with the electron-withdrawing ability of the modules, cleavage rates are quite predictable and tunable, and the in vitro-in vivo correlation is high. Indeed, achieving a desired half-life is not an issue; instead, the limitation ofthe technology is whether in an acceptable injection volume, the carrier can hold sufficient drug to last the dosing interval

[0067] With this system, the pharmacokinetics can be accurately simulated and modeled. There are two relevant processes that define the pharmacokinetics: k1, the slow cleavage rate of the linker, and k2, the faster elimination rate of the released drug. Since the rate determining step is the linker cleavage reaction, it accurately manifests as the in vivo half-life of the released drug, t1 / 2,1, but the elimination half-life, t1 / 2,2, of the released drug has no effect on the in vivo t1 / 2,1. After release of the active drug, the drug is cleared from the system by elimination rate k2,which by design is faster than cleavage k1.

[0068] An important differentiating feature of this half-life extension technology from others is that drug release is chemically controlled so t1 / 2,1values are species-independent; they can be determined in the mouse and be translated to man. In contrast, half-lives of PEGylated, lipidated peptides and Fc fusions, are species-dependent and have shorter half-lives in rodents than humans; translation from preclinical models to humans requires oft-risky allometric scaling. Also, the releasable linkers are not affected by enzymes and are extraordinarily stable when stored at lower pH and temperature.

[0069] Although the MS technology has provided peptides suitable for once monthly dosing, delivering the amount of drug needed for longer periods – “ultralong-acting” – in a suitable dosing volume is a major impediment. The optimal half-life to keep a peptide above its minimal therapeutic level, or Cmin, is one that is about equal to the dosing interval. With peptides having a shorter half-life than dosing interval, it is a common practice to retain the peptide above Cminby increasing its dose and risking high Cmaxtoxicity levels. For example, AMG133 – a GLP-1 agonist / GIP antagonist mAb – has a half-life of 14 d, yet is dosed every month; here, administering drug to maintain it above Cmin for two half-lives appears not to cause significant toxicity. Another more extreme example is EXTEN-GLP-1 which has a half-life of 6 days, but was administered once monthly in a Ph 1 trial. This was achieved by dosing sufficient drug to maintain it above Cmin for ~5 half-lives, giving a Cmax 16-fold higher than the optimal dose; the results were not reported and trials discontinued. Although our technology can achieve a half- life similar to any dosing interval, as currently implemented, we cannot practically dosesufficient peptide in an acceptable volume to achieve ultralong-acting intervals unless we use the “overdosing” approach. In short, it remains a significant challenge to achieve dosing intervals of a peptide exceeding ~one month.

[0070] The microsphere (MS)-drug conjugates of the disclosure override this problem, and also mitigate problematic dose limitations, is to decrease the drug clearance rate. The C vs t profile for a drug released from a slow-release depot is described by eq 1 when the release rate (k1) is significantly slower than the clearance (CL) of the drug from the plasma: Ct= Dose*k1 / CL * e-k1t(1)

[0071] While the half-life is governed by the release rate, k1, the plasma concentration of the released drug is governed by the balance between the rates at which peptide is released into (k1) and cleared from the circulation (k2); so, all else constant, the CL is inversely proportional to the drug concentration. Maintaining the plasma concentrations above a critical threshold Cmin for the dosing interval can become increasingly difficult as the dosing intervals increase and the amount of drug needed increases. If the CL of the released peptide can be lowered, however, it is possible to alleviate this problem since, as in eq.1, lowering the CL has the effect of raising the plasma concentration.

[0072] Accordingly, provided herein is an approach for half-life extension of therapeutic drugs that allows achievement of any desirable half-life. The approach provides significantly increased half-lives of therapeutic drugs that can be administered at efficacious dosses without concomitant toxicological concerns.

[0073] In some aspects, the disclosure provides microsphere (MS)-drug conjugates that provide prolonged drug release following administration to a subject in need thereof. In the approaches described herein, the drug component of the (MS)-drug conjugates comprises a moiety that extends the half-life of the drug. In particular, the therapeutic drug comprising the half-life extending moiety has a longer half-life than an identical drug without the half-life extending moiety. In some embodiments, the half-life extending moiety is a lipid. In some embodiments, the half-life extending moiety is a polyethylene glycol (PEG) chain. In some embodiments, the half-life extending moiety is an Fc fragment of an IgG.

[0074] In some embodiments, the microsphere (MS) component is MS-drug conjugates is a hydrogel. By hydrogel is meant a three-dimensional, predominantly hydrophilic polymeric network comprising a large quantity of water, formed by chemical or physical crosslinking of natural or synthetic homopolymers, copolymers, or oligomers. Hydrogels may be formed through crosslinking polyethylene glycols (considered to be synonymous with polyethylene oxides), polypropylene glycols, poly(N-vinylpyrrolidone), polymethacrylates, polyphosphazenes, polylactides, polyacrylamides, polyglycolates, polyethylene imines, agarose, dextran, gelatin, collagen, polylysine, chitosans, alginates, hyaluronans, pectin, carrageenan. In certain embodiments, the drug is covalently tethered to a hydrogel carrier.

[0075] In some embodiments, the MS component is a polyethylene glycol (PEG)-based hydrogel carrier, as set forth herein. In some embodiments, the hydrogel is a mesoporous PEG hydrogel. By mesoporous hydrogel is meant a hydrogel having pores between approximately 1 nm and approximately 100 nm in diameter. The pores in mesoporous hydrogels are sufficiently large to allow for free diffusion of biological molecules such as proteins.

[0076] In some such embodiments, the drug is tethered to the hydrogel carrier by a labile linker this is susceptible to cleavage following administration of the drug via the parenteral route. For instance, in some embodiments, the drug is cleaved by a β-elimination mechanism in a slow base-catalyzed rate-determining step. In one embodiment of the invention, biodegradable hydrogels are formed by reaction of a single reactive polymer and a cleavable crosslinker compound wherein said cleavable crosslinker compound comprises a functional group that reacts with the reactive polymer and a moiety also including a functional group that reacts with a reactive polymer that cleaves by elimination under physiological conditions. In this embodiment, the reactive polymer will be multi-valent, so as to allow formation of nodes in the three- dimensional hydrogel matrix. By biodegradable hydrogel is meant a hydrogel that loses its structural integrity through the cleavage of component chemical bonds under physiological conditions of pH and temperature. Biodegradation may be enzymatically catalyzed or may be solely dependent upon environmental factors such as pH and temperature. Biodegradation results in formation of fragments of the polymeric network that are sufficiently small to be soluble and thus undergo clearance from the system through the usual physiological pathways.

[0077] In another embodiment the invention provides methods for the formation of biodegradable hydrogels through reaction of a first reactive polymer, a second reactive polymer, and a cleavable crosslinker compound that comprises a first functional group that reacts with the first reactive polymer, a second functional group that reacts with the second polymer, and a moiety that cleaves by elimination under physiological conditions. The first and second functional groups may be the same or different. For the formation of a three-dimensional gel network the reactive components (first reactive polymer, second reactive polymer if any) will be multi-armed and thus serve to form nodes in the gel matrix. In some embodiments of the invention, this node-forming reactive component comprises at least 3 arms and more preferably at least 4 arms.

[0078] In accordance with the disclosure, using a combination of slowly releasable linkers to increase drug half-life and decreasing of the clearance of the released drug to increase concentration achieves ultralong-acting drugs with dosing intervals that could not be previously approached. There could be huge practical impact of using potent drugs (e.g., peptides / proteins drugs) with long half-lives and low clearance. As described herein, MS-drug conjugates of potent PEGylated drugs, lipidated drugs and Fc fusions with moderate half-lives of ~5- to 7 days, could provide ultralong-acting peptides that could maintain therapeutic levels for up to 3 or even 6 months or 1 year or more. Notably, there are over 100 PEGylated drugs that are approved or in clinical trials, and a similar number of Fc fusions. It is likely that some significant number would be suitable for this approach to developing ultralong-acting therapeutics.

[0079] In some embodiments, the therapeutic drug is a peptide. In some embodiments, the peptide is a GLP-1 receptor agonist. In some such embodiments, the peptide is semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide. In some embodiments, the peptide drug is an amylin analog. In some embodiments, the peptide is GUBamy, eloralintide, or MET233i.In some embodiments, the peptide drug is a calcitonin analog. In some embodiments, the analog is an apelin analog.

[0080] In some embodiments, the therapeutic drug is a protein. In some embodiments, the therapeutic drug is an Fc-fusion protein. In some embodiments, the drug is Dulaglutide. In some embodiments, the therapeutic drug is a PEGylated protein. In some embodiments, thePEGylated protein is Pegasys (PEGylated interferon alfa-2a), Neulasta (pegfilgrastim, PEG- GCSF), Macugen (pegaptanib, PEGylated RNA), Omontys (peginesatide) or Plegridy (pegintereron beta-1a).

[0081] In some aspects, provided herein are extended-release (MS)-drug conjugates having release kinetics suitable for biweekly, monthly, quarterly (i.e., once every 3 months), twice yearly or once yearly administration. In particular embodiments, the (MS)-drug conjugates are administered by the parenteral route. In some embodiments the (MS)-drug conjugates are administered subcutaneously. In some embodiments the (MS)-drug conjugates are administered intravenously. In some embodiments the (MS)-drug conjugates are administered intramuscularly. In some embodiments the (MS)-drug conjugates are administered intradermally.

[0082] In some embodiments, the (MS)-drug 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 months, or once every 12 months. In some embodiments, the (MS)-drug 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 (MS)-drug 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.

[0083] As an example of an approach to obtain an ultralong-acting peptide, we compare the pharmacokinetics of MS conjugates to an unmodified GLP-1 peptide vs a long-acting PEGylated version of the peptide with a lower CL (FIG.2). PLX039 (top panel of FIG. 2) is a MS~exenatide conjugate with a linker that provides a 30-day half-life to the released peptide, and free exenatide has an elimination t1 / 2,2 of 2.5 h and CL = 9.1 L / h in humans. Pegloxenatide (middle panel of FIG.2) is an anti-obesity PEGylated exenatide with an elimination t1 / 2,2 of 120 h and CL = 0.014 L / h (21). According to eq 1, and illustrated in FIG. 3, this difference in CL values means that, for the same linker release rate k1 and dose, the plasma concentrations with Pegloxenatide will be 650-fold higher than those of exenatide (from CLEX / CLPeglox). Thus, for a given dose it should be possible to produce a much longer acting conjugate of Pegloxenatidethan with exenatide. Indeed, pharmacokinetic simulations indicate a MS~Pegloxenatide conjugate having a linker with a t1 / 2,1 similar to the dosing interval would allow Q3Mo, and potentially even Q6Mo dosing in an injection volume of less than 1 mL – in contrast, achieving the same with exenatide would require dosing over 100 mL. Linking of Fc fragment of an IgG (bottom panel of FIG.2) or a lipid to the peptide would also result in significant half-life extension with significantly lower dosing volumes.

[0084] Modifications of CL can also optimize efficiency of MS conjugates to alleviate problematic dose limitations. For example, MS conjugates of the same peptide, e.g. exenatide, with half-lives optimized to 1 and 3 months, could be problematic in a dose-limiting situation since the Q3M (once every three months) conjugate would require a ~3-fold increase in dose. From eq 1, when the release half-life is adjusted to equal to the dosing interval !, the dose required to maintain plasma levels above Cminis given as eq 2; and, the comparative doses to reach the same Cminof two conjugates, A and B, having different k1and CL parameters is givenAnd eq 3 expands eq 4 to allow dose estimations when Cmin of the peptides are different.Using eq 3, an optimized Q3M conjugate releasing Pegloxenatide would require only 0.5% (CLEx / CLPEGlox* !Ex / !PEGlox= 3 / 650) of the dose of the optimized QM exenatide to achieve the same Cminat the longer dosing interval, thus alleviating problematic dose limitation.

[0085] Similar ultra-long increases in duration could be achieved with other peptide / protein drugs that are sufficiently potent and have low CL. For example, Fc fusions of peptides typically have half-lives of 4- to 5 d (5), and when attached to MSs should provide higher concentrations of the fused than the non-fused peptide. Although they may have somewhat reduced potencies, this can be overcome if the decrease in clearance is sufficient (eq. 4). With a potent Fc fusion of GLP-1 such as Dulaglutide (t1 / 2,2 =5 d, CL = 0.11 L / h, Cmin ~1 nM) that has a Cmin about 10-foldhigher than exenatide, a MS~Dulaglutide with a release rate of 30 d should give a ~80-fold higher concentration than the same dose of the analogous MS conjugate of exenatide and, with an appropriate linker, can be translated to a much longer-acting GLP-1 agonist.

[0086] In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to over two weeks. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to over three weeks. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to over three weeks. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to over four weeks. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to one month or over one month. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to two months or over two months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to three months or over three months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to four months or over four months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to five months or over five months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to six months or over six months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to twelve months or over 12 months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 2 weeks to about 6 months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 2 weeks to about 3 months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 2 weeks to about 2 months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 1 month to about 3 months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 1 month to about 2 months. In some embodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 3 months to about 6 months. In someembodiments, the half-life of the therapeutic agent (e.g., peptide) can be increased from minutes / hours to from about 6 months to about 12 months.

[0087] In some aspects, provided herein are extended-release (MS)-drug conjugates having release kinetics suitable for biweekly, monthly, quarterly (i.e., once every 3 months), twice yearly or once yearly administration. In particular embodiments, the (MS)-drug conjugates are administered by the parenteral route. In some embodiments the (MS)-drug conjugates are administered subcutaneously. In some embodiments the (MS)-drug conjugates are administered intravenously. In some embodiments the (MS)-drug conjugates are administered intramuscularly. In some embodiments the (MS)-drug conjugates are administered intradermally.

[0088] In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 0.5 mL to about 3 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 3 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1 mL to about 2 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of from about 1.5 mL to about 2 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of less than 1 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of about 1 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of about 1.5 mL. In some embodiments where the (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at an injection volume of about 2 mL.

[0089] In some embodiments, the (MS)-drug 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 (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel from about 3 μmoldrug / mL to about 8 μmol drug / mL. In some embodiments, the (MS)-drug 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 (MS)-drug 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 (MS)-drug 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 (MS)-drug conjugates are administered parenterally (e.g., subcutaneously) at a concentration of drug in the hydrogel of about 6 μmol drug / mL. II. Microsphere-Drug Conjugates

[0090] In some embodiments, the microsphere (MS) component of the MS-drug conjugate is a hydrogel. In one aspect, provided is a releasable hydrogel conjugate 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 drug comprising a half-life extending moiety. 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.

[0091] In some embodiments, the half-life extending moiety is a lipid. In some embodiments, the half-life extending moiety is a polyethylene glycol (PEG) chain. In some embodiments, the half-life extending moiety is an Fc fragment of an IgG.

[0092] 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.

[0093] 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 semaglutide release and hydrogel degradation.

[0094] 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 groupsinclude 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.

[0095] 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-C6 alkylthio; 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.

[0096] 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. 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.

[0097] In some embodiments, the biodegradable polymer is a crosslinked PEG comprised of pegylated polymers. In some embodiments, the individual pegylated polymers comprising thehydrogel 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. Linker-Drug Units of Conjugates

[0098] In some embodiments, the MS-drug conjugates comprise linker-drug (L-D) units ofthe 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; D is a drug comprising a half-life extending moiety; 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).

[0099] In some embodiments of formula (II), D is a lipidated drug. In some suchembodiments, D is a lipidated peptide connected through its Na-amine. In some embodiments, Dis semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, or survodutide. In some embodiments, D is a lipidated protein.

[0100] In some embodiments of the linker-drug units of the formula (I) or formula (II), D is a pegylated drug. In some embodiments, D is a pegylated peptide. In some embodiments, D is polyethylene glycol loxenatide (PEG-Loxe), also referred to as PEX198. In some embodiments, D is a pegylated protein.

[0101] In some embodiments of the linker-drug units of the formula (II), D is a peptide or protein fused to an Fc domain of an IgG. In some embodiments, the Fc-fusion is a monomeric Fc-fusion. In some embodiments, the Fc fusion is a polymeric Fc-fusion. In some embodiments, D is Dulaglutide.

[0102] 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.

[0103] 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.

[0104] In some embodiments of the linker-drug units of the formula (II), one R4group is H. In some embodiments, both R4groups are H.

[0105] In some embodiments of the linker-drug units of the formula (II), one R4group is CH3and one R4group is H.

[0106] 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, 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.

[0107] 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 -SO2R3. In other embodiments, R1is –CN or -SO2R5, wherein R5is optionally substituted alkyl, optionallysubstituted aryl, or -NR82. In other embodiments, R1 is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0108] 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 1-20carbon 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 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.

[0109] In some embodiments of the linker-drug unit of formula (II), R2is H.

[0110] 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 drug comprising a half-life extension moiety; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0111] 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; 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 drug comprising a half-life extension moiety; anddepicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0112] 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. CH3 or CH2CH3); R2is H; each R4is CH3; D is a drug comprising a half-life extension moiety; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0113] 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.

[0114] In some embodiments of the linker-drug unit of formula (II), n is 2; R1is -SO2R5, wherein R5is C1-C6alkyl (e.g. CH3or CH2CH3); R2is H; each R4is CH3.

[0115] In some embodiments of the linker-drug unit of formula (II), n is 2; R1is -SO2R5, wherein R5is N(R6)2, wherein each R6is independently C1-C6alkyl, aryl, or heteroaryl; R2is H; and each R4is CH3.

[0116] In some embodiments, the MS-drug conjugates comprise linker-drug units of theformula (II-A)wherein: n is an integer from 0 to 6; R1is -CN, -NO2, -COR5, -SOR5, or -SO2R5, wherein R5is C1-C6alkyl, 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; S is absent or (CH2CH2O)h(CH2)gCONH wherein g = 1-6 and h = 0-1000; Y is NH(CH2CH2O)p(CH2)m wherein m = 2-6 and p = 0-1000; D is an IL-15 or an IL-15•IL-15RaSu fusion protein; and depicts the point of attachment of the linker drug units to a macromolecular carrier.

[0117] In some embodiments of a linker-drug unit of formula (II-A), linker drug units areattached to the macromolecular carrier through a carboxamide, amide, oxime, triazole, thioether, thiosuccinimide, or ether moiety.

[0118] In some embodiments of a linker-drug unit of formula (II-A), R1 is -CN or -SO2R5,wherein R5is C1-C6 alkyl, aryl, heteroaryl, or NR62, wherein each R6is independently C1-C6alkyl, aryl, or heteroaryl, and R2is H, and wherein each of R5and R6is independently optionally substituted. In some embodiments, R1is -CN, SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0119] In some embodiments of a linker-drug unit of formula (II-A), R2 is H.

[0120] In some embodiments of a linker-drug unit of formula (II-A), n is 4, each R4 is H, R1is -SO2R5wherein R5is methyl, R2is H, S is (CH2CH2O)h(CH2)gCONH wherein g = 2 and h = 4, and Y is NH(CH2CH2O)p(CH2)m wherein m = 3 and p = 0.

[0121] In some embodiments of a linker-drug unit of formula (II-A), M comprises polymershaving multi-armed chains.

[0122] In some embodiments of a linker-drug unit of formula (II-A), M comprises r-armedpolymers, wherein r is an integer from 2 to 8. In some embodiments, r is 4. In some embodiments, the r-armed polymers are pegylated polymers. In some embodiments, the r-armed polymers are linked through a cleavable crosslinker. In some embodiments, the cleavable crosslinker has the formula (V).

[0123] The linker-drug unit of formula (II) or formula (II-A) can be covalently attached to amacromolecular 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*.wherein M is a macromolecular carrier, and n, R1, R2, R4and D are defined as above.

[0124] 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.

[0125] In some embodiments, the functional group Z* in the conjugate of formula (III) canbe 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 2.Scheme 2

[0126] In some embodiments of Scheme 2, connecting group Z* is the product of a reactivemoiety (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).

[0127] In some embodiments, when Z is or comprises an amine, Z’ is or comprises acarboxylic acid, active ester, or active carbonate to yield a conjugate of formula (III) wherein Z* is or comprises an 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. In other embodiments, when Z is a 2,3,5,6-tetrazine, Z’ is a trans-cyclooctene to yield a conjugate of formula (II) wherein Z* is dihydropyridazine. 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, dihydropyridazine, or ether.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] In some embodiments. the disclosure provides methods of reacting the linker-drugunit 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

[0132] In some embodiments, the hydrogels of the disclosure are comprised of repeatingcrosslinked 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, poly(amino acid), 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.

[0133] In some embodiments, the individual polymeric units of the hydrogel is a polypeptideselected 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).

[0134] In some embodiments, the individual polymeric units of the hydrogel is a synthetic polymers including poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(ethylene imine) (PEI), and co-polymers thereof.

[0135] 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).

[0136] In some embodiments, the individual polymeric units of the hydrogel is a polysaccharide such as a dextran.

[0137] 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.

[0138] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable crosslinker. In some embodiments, the cleavable crosslinker has the formula (V):wherein: 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-C3alkyl; 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) or a connecting group that connects the cleavable crosslinker to a linker-dug unit of formula (II)).

[0139] 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, , a dihydropyridazine, 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, A* comprises a 1,2,3-triazole and B* comprises a carboxamide. 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.

[0140] In some embodiments, A* or B* is an amide group. In some embodiments, A* or B* is a click product formed from a first click handle on the polymeric unit and a second click handle on the cleavable linker.

[0141] 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 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] In some embodiments of the cleavable linker of formula (V), x is 0, y is 4 and z is 0.

[0146] In some embodiments of the cleavable linker of formula (V), x is 4, y is 0 and z is 0.

[0147] In some embodiments of the cleavable linker of formula (V), x is 4, y is 0 and z is 1.

[0148] In some embodiments of the cleavable linker of formula (V), x is 0, y is 0 and z is 0.

[0149] In some embodiments of the cleavable linker of the formula (V), each R14group is independently C1-C3alkyl. 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.

[0150] 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; 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.

[0151] 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.

[0152] 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, -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.

[0153] 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 .

[0154] In some embodiments of a cleavable linker of formula (V), R11is -SOR15, or -SO2R15, wherein R15is C1-C6 alkyl (e.g. CH3 or CH2CH3) and R12is H.

[0155] 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.

[0156] In some embodiments of a cleavable linker of formula (V), q = 1-2; each R14is methyl; R11is CN or R15SO2wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4 and z=0.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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. Conjugates

[0163] The cleavable linker of formula (V) can be covalently bonded to a to a linker-drug unit of formula (II) or formula (II-A) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II) or formula (II-A). For instance, the reactive group Z’ can be covalently bonded to the cleavable crosslinker 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.

[0164] A cleavable crosslinker 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.Scheme 1

[0165] In some embodiments of Scheme 1, when Z is amine, Z’ is carboxylic acid, activeester, or active carbonate to yield a conjugate of formula (VI) wherein Z* is amide or carbamate. In other embodiments of Scheme 1, 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 1, when Z is or comprises an 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 1, when Z is or comprises an SH group, Z’ is or comprises a maleimide or halocarbonyl group to yield aconjugate 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.

[0166] In some embodiments of Scheme 1, the cleavable crosslinker of formula (Va) 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.

[0167] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker having the formula (VII):(VII), wherein A*, q, R11, R12, and R14are defined as above.

[0168] The cleavable crosslinker of formula (VII) can be covalently bonded to a to a linker- drug unit of formula (II) or formula (II-A) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II) or formula (II-A). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (VII), hence forming a cleavable linker of formula (VII-A), depicted below.

[0169] A cleavable linker of formula (VII-A) can be reacted with a linker-drug unit formula(IV) to yield a conjugate of formula (XXX), as shown in Scheme 3. Scheme 3 is shown without the polymeric units (e.g., P1and P2). It will be understood that the drug-linker units can be reacted with the crosslinker prior to or after the polymeric units are bonded to the crosslinker.Scheme 3

[0170] In some embodiments of Scheme 3, when Z is amine, Z’ is carboxylic acid, activeester, or active carbonate to yield a conjugate of formula (XXX) 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 (XXX) 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 (XXX) 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 (XXX) 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.

[0171] In some embodiments of scheme 3, the cleavable linker of formula (VII-A) 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.

[0172] In some embodiments, the individual polymeric units comprising the hydrogel arecrosslinked through a cleavable linker. In some embodiments, the cleavable linker has the formula (VIII):wherein A*, q, R11, R12, and R14are defined as above.

[0173] The cleavable linker of formula (VIII) can be covalently bonded to a to a linker-drugunit of formula (II) or formula (II-A) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (II) or formula (II-A). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (VIII), hence forming a cleavable linker of formula (IX), depicted below.

[0174] A cleavable linker of formula (IX) can be reacted with a linker-drug unit formula (IV)to yield a conjugate of formula (X), as shown in Scheme 4.Scheme 4

[0175] In some embodiments of Scheme 4, when Z is amine, Z’ is carboxylic acid, activeester, or active carbonate to yield a conjugate of formula (X) 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 (X) 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 (X) 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 (X) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ canbe reversed to yield Z* of opposing orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0176] In some embodiments of scheme 4, the cleavable linker of formula (IX) 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

[0177] 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).

[0178] 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*.

[0179] 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 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.

[0180] 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.

[0181] 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 bereversed to yield B* of opposing orientation. In some embodiments, B* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0182] 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.

[0183] In some embodiments, A* has the following structure:, 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.

[0184] 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.

[0185] 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, and 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.

[0186] In some embodiments of formula (XI), q is an integer from 1 to 5. In someembodiments, 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.

[0187] In some embodiments of formula (XI), x is 0. In some embodiments of formula (XI),x is 4.

[0188] In some embodiments of formula (V), y is 0. In some embodiments of formula (XI),y is 4.

[0189] In some embodiments of formula (V), y is 0. In some embodiments of formula (XI), z is 0.

[0190] In some embodiments of formula (XI), x is 0, y is 4 and z is 0.

[0191] In some embodiments of formula (V), x is 4, y is 0 and z is 0.

[0192] In some embodiments of formula (XI), x is 4, y is 0 and z is 1.

[0193] In some embodiments of formula (XI), x is 0, y is 0 and z is 0.

[0194] In some embodiments of the formula (XI), each R14group is independently C1-C3alkyl. 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.

[0195] 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.

[0196] 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.

[0197] In some embodiments of formula (XI), 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, -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.

[0198] 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.

[0199] In some embodiments of formula (XI), R11is -SOR15, or -SO2R15, wherein R15is C1- C6 alkyl (e.g. CH3 or CH2CH3) and R12is H.

[0200] 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.

[0201] In some embodiments of the crosslinked unit formula (XI), q = 1-2; each R14is methyl; R11is CN or R15SO2wherein R15is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4 and z=0.

[0202] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 0-4; y = 0-4 and z=0.

[0203] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 4; y = 0 and z=0.

[0204] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 4; y = 0 and z=0.

[0205] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is CH3SO2; R12is H; x = 0; y = 4 and z=0.

[0206] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is (CH3)2NSO2; R12is H; x = 4; y = 0 and z=0.

[0207] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is (CH3)2NSO2; R12is H; x = 0; y = 4 and z=0.

[0208] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is (CH3CH2)2NSO2; R12is H; x = 4; y = 0 and z=0.

[0209] In some embodiments of the crosslinked unit of formula (XI), q = 1-2; each R14is methyl; R11is (CH3CH2)2NSO2; R12is H; x = 0; y = 4 and z=0.

[0210] Formula (XII) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker.

[0211] In some embodiments of a compound 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-C3alkyl; R11is CN or R15SO2wherein R15is methyl, isopropyl, Me2N, MeEtN, Et2N, piperidinyl, or (MeOCH2CH2)2N; R12is H; w = 0-4; y = 0-4; z=0; B* is carboxamide; Z* is comprises a triazole group; n = 0-6; R1is CN or R3SO2wherein R3is methyl, isopropyl, Me2N, Et2N, piperidinyl, MeEtN, or (MeOCH2CH2)2N; R2is H; and D is a drug comprising a half-life extending moiety.

[0212] In some embodiments of a compound 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, Et2N, piperidinyl, 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, Et2N, piperidinyl, or (MeOCH2CH2)2N; R2is H; and D is a drug comprising a half-life extending moiety.

[0213] In some embodiments of a compound 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 R15SO2wherein R15is methyl, isopropyl, Me2N, MeEtN, Et2N, piperidinyl, or (MeOCH2CH2)2N; R12is H; w = 0- 4; y = 0-4; B* is carboxamide; Z* comprises a triazole group; n = 0-3; R1is CN or R3SO2wherein R3is methyl, isopropyl, Me2N, MeEtN, Et2N, piperidinyl, or (MeOCH2CH2)2N; R2is H; and D is a drug comprising a half-life extending moiety.

[0214] In some embodiments of a compound 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; R12is H; and D is a drug comprising a half-life extending moiety.

[0215] In some embodiments of a compound 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 drug comprising a half-life extending moiety.

[0216] In some embodiments of a compound 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; w = 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0217] In some embodiments of a compound 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 drug comprising a half-life extending moiety.

[0218] In some embodiments of a compound 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 drug comprising a half-life extending moiety.

[0219] In some embodiments of a compound 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 drug comprising a half-life extending moiety.

[0220] In some embodiments of a compound 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 drug comprising a half-life extending moiety.

[0221] In some embodiments of a compound 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* is triazole; n = 2; R1is CH3SO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0222] In some embodiments of a compound 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 Et2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R12is H; and D is a drug comprising a half-life extending moiety.

[0223] In some embodiments of a compound 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 Et2NSO2; 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 drug comprising a half-life extending moiety.

[0224] In some embodiments of a compound 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 Et2NSO2; R12is H; w = 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0225] In some embodiments of a compound 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 Et2NSO2; 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 drug comprising a half-life extending moiety.

[0226] In some embodiments of a compound 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 Et2NSO2; 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 drug comprising a half-life extending moiety.

[0227] In some embodiments of a compound 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 Et2NSO2; 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 drug comprising a half-life extending moiety.

[0228] In some embodiments of a compound 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 Et2NSO2; 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 drug comprising a half-life extending moiety.

[0229] In some embodiments of a compound 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 Et2NSO2; R12is H; x = 0; y =4; z=0’ B* is carboxamide; Z* is triazole; n = 2; R1is CH3SO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0230] In some embodiments of a compound 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 Et2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R12is H; and D is a drug comprising a half-life extending moiety.

[0231] In some embodiments of a compound 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 Et2NSO2; R12is H; x = 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0232] In some embodiments of a compound 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 Et2NSO2; R12is H; w = 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0233] In some embodiments of a compound 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 Et2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0234] In some embodiments of a compound 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 Et2NSO2; R12is H; x = 0; y = 4; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0235] In some embodiments of a compound of formula (XII), P1 and P2 are eachindependently 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 Et2NSO2; R12is H; x= 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0236] In some embodiments of a compound of formula (XII), P1 and P2 are eachindependently 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 Et2NSO2; R12is H; x= 4; y = 0; z=0; B* is carboxamide; Z* comprises a triazole group; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0237] In some embodiments of a compound of formula (XII), P1 and P2 are eachindependently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises atriazole group; q = 2; each R4 is methyl; each R14 is methyl; R11 is Et2NSO2; R12 is H; x = 0; y =4; z=0’ B* is carboxamide; Z* is triazole; n = 2; R1is Me2NSO2; R2is H; and D is a drug comprising a half-life extending moiety.

[0238] In any of the foregoing embodiments of the compound of formula (XII), Z* can haveone of the following structure:, wherein the on top is covalently bonded to (CH2)n and the on the bottom is covalently bonded to (CH2)y.

[0239] In any of the foregoing embodiments of the compound of formula (XII), Z* can haveone of the following structures:, 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.

[0240] Formula (XIII) depicts two individual polymeric units (P1 and P2) crosslinked throughthe cleavable linker of formula (VIII).

[0241] Formula (XIV) depicts two individual polymeric units (P1 and P2) crosslinkedthrough the cleavable linker of formula (VIII).

[0242] Formula (XV) depicts two individual polymeric units (P1 and P2) crosslinked throughthe cleavable linker of formula (IX).

[0243] Formula (XVI) depicts two individual polymeric units (P1 and P2) crosslinkedthrough the cleavable linker of formula (X).

[0244] The disclosure also provides (MS)-drug 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 (XVII):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 - is -CN, -NO2, -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 SR19, 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-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 drug comprising a half-life extending moiety; and depicts the point of attachment of the linker-drug (L-D) to Z*.

[0245] In some embodiments of the crosslinked polymeric units of formula (XVII), eachR4is independently C1-C3alkyl. In some embodiments, each R4is methyl.

[0246] In some embodiments of the crosslinked polymeric units of formula (XVII), R1 is CNor -SO2R3. In some embodiments, R1is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), -SO2-N(CH2)5, - SO2N(CH2CH2)2CHMe, or -SO2N(CH2CH2OCH3)2.

[0247] In some embodiments the crosslinked polymeric units of formula (XVII), n is aninteger from 1 to 3.

[0248] In some embodiments of the crosslinked polymeric units of formula (I), Z* comprises a carbonate, amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, , dihydropyridazine or ether group.

[0249] In some embodiments the crosslinked polymeric units of formula (XVII), each R14is independently C1-C3alkyl. In some embodiments, both R14are methyl. In some embodiments, one R14is methyl and one R14is H. In some embodiments, both R14are H.

[0250] In some embodiments of the crosslinked polymeric units of formula (XVII), each R4is independently C1-C3alkyl and each R14is independently C1-C3alkyl. In some embodiments, both R4are methyl and both R14are methyl.

[0251] In some embodiments of the crosslinked polymeric units of formula (XVII), each R4is independently C1-C3alkyl and both R14are H. In some embodiments, both R4are methyl and both R14are H.

[0252] In some embodiments of the crosslinked polymeric units formula (XVII), R11is -CN or -SO2R15, wherein R15is defined above. In some embodiments, R11is -SO2CH3. In some embodiments, R11is -SO2(R6)2. In some embodiments, R11is -SO2(Me)2or -SO2(Et)2.

[0253] In some embodiments the crosslinked polymeric units of formula (XVII), 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.

[0254] In some embodiments of the crosslinked polymeric units formula (XVII), A* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group.

[0255] In some embodiments of the crosslinked polymeric units of formula (XVII), A* comprises an amide group. In some embodiments of a polymeric unit of formula (XVII), A* is produced by a reaction between a first moiety comprising an acid group and a second moiety comprising an amine group. In some embodiments of a polymeric unit of formula (XVII), B* comprises an amide group.

[0256] In some embodiments, the crosslinked polymeric units of formula (XVII) arecrosslinked polymeric units of formula (XVIII):XVIII

[0257] In some embodiments of formula (XVIII), q is 1. In some embodiments of formula(XVIII), q is 2. In some embodiments of formula (XVIII), q is 3.

[0258] In some embodiments, the crosslinked polymeric units of formula (XVII) arecrosslinked polymeric units of formula (XVIII-A):

[0259] In some embodiments of formula (XVIII) or formula (XVIII-A), P1 and / or P2 are 4-armed PEGs between 10-kDa and 20-kDa. In some embodiments of formula (XVIII) or formula (XVIII-A), both R14groups are methyl. In some embodiments of formula (XVIII) or formula (XVIII-A), both R14groups are hydrogen. In some embodiments of formula (XVIII) or formula (XVIII-A), both R4groups are methyl. In some embodiments of formula (XVIII) or formula (XVIII-A), both R4groups are hydrogen. In some embodiments of formula (XVIII) or formula (XVIII-A), both R4groups are methyl and both R14groups are methyl.

[0260] In some embodiments, the crosslinked polymeric units of formula (XVII) arecrosslinked polymeric units of formula (XIX).

[0261] In some embodiments of formula (XIX), q is 1. In some embodiments of formula(XIX), q is 2. In some embodiments of formula (XIX), q is 3.

[0262] In some embodiments, the crosslinked polymeric units of formula (XVII) have one ofthe following structures:

[0263] In some embodiments of formula (XX) or formula (XX-A), P1 and / or P2 are 4-armedPEGs between 10-kDa and 20-kDa. In some embodiments of formula (XX) or formula (XX-A),both R14groups are methyl. In some embodiments of formula (XX) or formula (XX-A), both R14groups are hydrogen. In some embodiments of formula (XX) or formula (XX-A). both R4groups are methyl. In some embodiments of formula (XX0 or formula (XX-A), both R4groups are hydrogen. In some embodiments of formula (XX) or formula (XX-A), both R4groups are methyl and both R14groups are methyl.

[0264] In some embodiments, the crosslinked polymeric units, such as the crosslinkedpolymeric units of formula (XVII), 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. 14.

[0265] The disclosure also provides (MS)-drug conjugates comprising polymeric units (P1-P2) crosslinked by a cleavable crosslinker and further comprising a linker-drug (L-D), wherein said (MS)-drug conjugates have the formula (XVII-A):(XVII-A) 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, 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 SR19, 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-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 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, -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; S is absent or (CH2CH2O)h(CH2)gCONH wherein g = 1-6 and h = 0-1000; Y is NH(CH2CH2O)p(CH2)mwherein m = 2-6 and p = 0-1000;D is drug comprising a half-life extending moiety; and depicts the point of attachment of the linker drug units to a macromolecular carrier.

[0266] In some embodiments of an (MS)-drug conjugate of formula (XVII-A), D is a peptide drug. In some embodiments of an (MS)-drug conjugate of formula (XVII-A), D is a protein. In some embodiments of an (MS)-drug conjugate of formula (XVII-A), D is an antibody or fragment thereof. In some embodiments of an (MS)-drug conjugate of formula (XVII-A), D is an Fc-fusion protein. in some embodiments D is a pegylated protein or pegylated antibody (or fragment thereof). In some embodiments, D is a cytokine. For instance, in particular embodiments D is a pegylated cytokine. In some embodiments, the cytokine (e.g., pegylated cytokine), is Interleukin-2 (IL-2) or Interleukin-15 (IL-15). n some embodiments, the cytokine (e.g., pegylated cytokine) comprises an IL-15 (e.g., pegylated cytokine) and the sushi domain of the IL-15Ra (IL-15RaSu). Hydrogels Comprising Muli-Polymeric Units

[0267] In some embodiments, formula (XI), formula (XII), formula (XIII), formula (IV), formula (XV) or formula (XVI) represent 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).

[0268] Representative hydrogels of the disclosure are depicted below:wherein 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.

[0269] In some embodiments of a hydrogel of formula (XXI), formula (XXII), formula (XXIII), formula (XXIV) formula (XXV), formula (XXVI), formula (XXVII), or formula (XXVIII), 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.

[0270] 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 5,000 and 200,000 Daltons, between 20,000 and 200,000 Daltons, between 10,000 and 100,000 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).Methods of Making Hydrogels

[0271] 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 (IV) M-(Z’)s+ Z-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D -> (IV) 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.

[0272] 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

[0273] In some embodiments, Z’ is a cyclooctyne such as 5-hydroxycyclooctyne attached through an amine group on the hydrogel such that M-(Z’)shas the structureand Z is an azide such that conjugate (I) has the more detailed structure

[0274] In some embodiments, the intermediate linker-drug of formula (IV) is prepared by reaction of a drug with a linker reagent of formula (XXXI) Z-(CH2)nC(R4)2CH(CHR1R2)-O-CO-X (XXXI) 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 (XXXI) 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, unprotected drug is reacted with a linker reagent of formula (XXXI) in the presence of a mild base such as a teriary amine. In embodiments where the N- terminal amino acid residue is histidine, such as the case of semaglutide, both the N1-amine and the imidazole of His(1) are carbamoylated. This intermediate is then reacted with hydroxylamine to selectively remove the carbamoyl-imidazole.

[0275] In an alternative embodiment, protected drug on resin is prepared by standard solid- phase peptide synthesis. The terminal FMOC group is then removed and a final coupling step isperformed using the linker reagent of formula (XXXI). The protected linker-drug is then cleaved from the resin and deblocked under standard conditions and purified to provide linker-drug. In a third embodiment of the invention, the linker-drug is prepared by a fragment coupling process, where the fragments may be chemically or biologically produced.

[0276] The activated hydrogels M-(Z’)s may be prepared according to known methods, for example the methods disclosed in PCT Publication WO2013 / 036847; WO2020 / 206358; Henise et al. 2021 Engineering Reports, e12412. https: / / doi.org / 10.1002 / eng2.12412. 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’)swherein Z’ comprises a cyclooctyne moiety.

[0277] In one embodiment, the hydrogels are prepared as injectable microspheres having Z’ = NH2by 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. III. Lipidated Drugs

[0278] In certain aspects, the half-life extending moiety covalently attached to the drug is a lipid. In some embodiments, the drug is a lipidated peptide drug. In particular embodiments,lipidated peptide drugs 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.

[0279] In some embodiments, the lipidated peptide is semaglutide, liraglutide, tirzepatide, cagrilintide, pemvidutide, retratrutide, mazdutide, ecnoglutide, VK2735, MET-097i, MET233i, eloralintide, GUBamy, NN1213, CT-868, CT-388, SAR441255, NN1213, UBT251, 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) SEQ ID No: 4 eicosanedioic acid-g-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-Cys7disulfide] (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-aMeLeu-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) SEQ ID No: 11 gGlu-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gly-Orn-Leu-Ala-Glu-aMeF-Leu- Val-Arg-Ser-Ser-Asn-NMeAsn-Pge-Gly-Pro-Lys(acyl)-LeuPro-Pro-Thr-Glu-ValGly-Ser-Asn- Thr-Tyr-NH22methylene bridged disulfide (eloralintide) SEQ ID No: 12 lipid-Lys-Cys-Leu-Thr-Ala-Thr-Cys-Thr-Val-Ala-Arg-Leu-Ala-Glu-Gln-Ile- Ala-Gln-phe-Thr-Asp-Lys-Asp-Lys-Glu-Asn-Val-Ala-Pro-Pro-Thr-Ala-Val-Gly-Ser-Ala-Gly- Hyp methylene bridged cysteines (GUBamy) SEQ ID No: 13 Lipid- gGlu -Lys-Cys-Ser-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Glu- Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Pro-Val-Gly-Ser-Asn-Thr-Pro-NH2(MET233i)SEQ ID No: 14 Lys(acyl)-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asp-phe- Leu-Arg-His-Ser-Ser-Pro-Asn-Phe-Gly-Ala-Ile-Pro-Ser-Ser-Thr-Asn-Val-Gly-Ser-Arg-Thr- Tyr-NH2 (NN1213) SEQ ID No: 15 N-2-((2-oxo-2-((2-(2-oxopiperidin-1-yl)ethyl)amino)ethyl)thio)acetyl-Glu-Gly- Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Tyr-Leu-Asp-Lys-Gln-Ala-Ala-Aib-Glu-Phe-Val-Asn-Trp- Leu-Leu-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(acyl) (CT-868) SEQ ID No: 16 His-Aib-His-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Leu-Lys(acyl)-Glu-Glu- Gln-Arg-Aib-Glu-Phe-Ile-Glu-Trp-Leu-Lys-Ala-(d-Ala)-Gly-Pro-Pro-Ser-Aib-Lys-Pro-Pro-Pro- Lys-NH22(SAR441255) SEQ ID No: 17 Tyr-Aib-Gln-Gly-Thr-aMeF-Thr-Ser-Asp-Tyr-Ser-Ile-aMeL-Leu-Asp-Lys- Lys(acyl)-Ala-Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu-Leu-Glu-Ser-His-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Ac3c-Ser-NH2

[0280] 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. 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 or twice- yearly administrations. IV. PEGylated Drugs

[0281] In certain aspects, the half-life extending moiety covalently attached to the drug is a polyethylene glycol (PEG). In some embodiments, the PEG is attached to the drug through a cleavable linker. In some embodiments, the PEGylated drugs are enzymes, monoclonal antibodies or cytokines. In some embodiments, the drug is a fragment of an antibody such as aFab fragment or single-chain variable fragment (scFv). In some embodiments, the PEGylated drug is a peptide having a sequence listed below: SEQ ID No: 18 His-(d-Ala)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Nle-Glu-Glu- Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Gln-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Cys{PEG}-NH2 (Pegloxenatide) SEQ ID No: 19 His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu- Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Cys{PEG}-NH2(visepegenatide)

[0282] In some embodiments, the PEGylated drug is selected from SylatronTM, Pegasys®, Neulasta®, Oncaspar, Yorvipath, Izervay, Elfabrio, Syfovre, Rolvedon, Syimufend, FyIntera, Besremi, Sktrofa, Empaveli, Nyvepria, Esperoct, Ziextenzo, Udenyca, Palinziq, Revcovi, Filphilia, Asparlas, Jivi, Rebinyn, Adynovate, Plegridy, Omontys, Sylatron, Krystexxa, Cimzia, Micera, Macugen, Somavert, Pegintron, Adagen, Movantik, Asclera, Doxil, Onivyde, Onpattro, Comirnaty and Spikevax®. Fc Fusion Drugs

[0283] In certain aspects, the half-life extending moiety covalently attached to the drug is an Fc fragment of an IgG. In some embodiments, the drug is a peptide. In some embodiments, the drug is a protein. In some embodiments, the drug is an RNase.

[0284] In some embodiments, the Fc fusion drug is selected from Enbrel®, Ontak®, Orencia®, Amevive®, Arcalyst®, Nplate®, Nulojix® and Eylea®. In some embodiments, the Fc fusion drug is Dulaglutide (SEQ ID No: 20) or efinopegdutide (SEQ ID No: 21). HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID No: 20) PSCPAPEFLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN WYVDGVEVHNAKTKPREEQF NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK GLPSSIEKTI SKAKGQPREP QVYTLPPSQE EMTKNQVSLT CLVKGFYPSDIAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSRLTVDKSR WQEGNVFSCS VMHEALHNHY TQKSLSLSLG K (SEQ ID No: 21)

[0285] In some embodiments, the Fc fusion drug is Dulaglutide. V. Methods of Treatment

[0286] 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.

[0287] 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.EXAMPLES

[0288] 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

[0289] 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.

[0290] (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 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 chlorideas 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)

[0291] (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)

[0292] (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 extractedwith 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.

[0293] 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).

[0294] (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 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

[0295] Hydrogel microspheres were prepared, sterilized, and activated as described in PCT Publication WO2021 / 026494, US Published Application No, 20220265873, and Henise et al. Engineering Reports, e12412. https: / / doi.org / 10.1002 / eng2.12412. (2021), each of which 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).

[0296] 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-cyclooctynylsuccinimidyl 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.

[0297] (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) thendried 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.

[0298] C18 HPLC, purity was determined by ELSD: 99.1% (RV = 9.29 mL).

[0299] LC-MS (m / z): calc, 521.2; obsd, 521.3 [M-H]-.

[0300] (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.

[0301] C18 HPLC, purity was determined by ELSD: 99.7% (RV = 10.23 mL).

[0302] LC-MS (m / z): calc, 520.2; obsd, 520.2 [M+H-Boc]+.

[0303] (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 10min, 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.

[0304] C18 HPLC, purity was determined by ELSD: 89.1% (RV = 10.38 mL) with a 10.6% impurity (RV = 10.08).

[0305] (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 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.

[0306] C18 HPLC, purity was determined by ELSD: 99.2% (RV = 9.34 mL).

[0307] 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. Theresulting 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-N3 with DBCO-CO2H. 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.

[0308] (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 C18HPLC (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]-.

[0309] (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]+.

[0310] (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 10min, 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).

[0311] (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 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.

[0312] (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 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.

[0313] (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.

[0314] (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.

[0315] (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 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.

[0316] (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) 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.

[0317] (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 monitoredby 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.

[0318] (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.

[0319] (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 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 wasdried 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.

[0320] 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 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

[0321] 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 MicrospheresExample 1 Preparation of linker-semaglutide

[0322] 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).

[0323] 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 1).

[0324] Other linker-semaglutides were prepared similarly using the appropriate linker reagent (Table 2). Table 2. Linker-semaglutides prepared according to the method of Example 1Example 2 Preparation of semaglutide conjugates

[0325] A slurry of amino microspheres wherein P1and P2are each 10-kDa 4-armed PEG, A* = triazole, 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 2020, Engineering Reports). 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 A280 in 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.

[0326] 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, 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.

[0327] Structures of the conjugates are shown in FIG. 5. Example 3 In vitro release and hydrogel degradation

[0328] In vitro peptide release kinetics and hydrogel degradation were measured simultaneously under accelerated conditions (pH 9.4, 37oC) using the previously described automated method (Henise 2020, Engineering Reports). The peptide release and PEG solubilization curves can be seen in FIGS. 6A and 6B for each conjugate from Example 2. Forboth 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. Example 4 Mouse pharmacokinetics

[0329] 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.7. Example 5 Weight loss in DIO mice

[0330] 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. 8A and 8B), comparable to twice-daily administration of free semaglutide at 10 nmol / kg / dose, or a total of 600 nmol / kg per month.

[0331] Body composition was determined by DEXA scan (FIGS. 9A and 9B), and indicated that treatment with the conjugates of Example 2 gave rise to selective loss of fat. FIG.9A 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.9B shows that thecomposition of weight lost after 30 days treatment with a single dose of MS~semaglutide by DEXA scan.

[0332] 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. 10, The semaglutide conjugate of Example 2 showed superior weight loss in this model (25% vs 10%) over a one-month period. Example 6 Linker-Pegloxenatide Formula (IV) wherein D = Na-linked Pegloxenatide

[0333] Linker-Pegloxenatide (H-{dAla}-EGTFTSDLSKQ-{Nle}- EEEAVRLFIEWLKQGGPSSGAPPPC{PEG}-NH2, SEQ ID NO.18, wherein dAla = (D)- alanine, Nle = norleucine) is prepared using a modification of the method for preparing PEX168 given in PCT Publication WO2012 / 155780A1. The protected amino acid sequence is constructed on Rink Amide AM resin using standard Fmoc chemistry as described. After removal of the last Fmoc group from the N-terminal Histidine, a linker reagent of formula XXXI is added and coupled to the peptide chain. The crude linker-peptide is then cleaved from the resin and deprotected using TFA / TIS / H2O and if necessary purified by HPLC. The linker-peptide is thenreacted with a branched mPEG2-Lys-MAL (40kDa) pegylation reagent in 0.1 M phosphate, pH 7.7, as described, and the linker-pegloxenatide is purified by HPLC.

[0334] When the linker reagent of formula XXXI is 5-azido-3,3-dimethyl-1- (methylsulfonyl)-2-pentyl succinimidyl carbonate (Example A), the product is formula (IV) wherein Z = N3, n = 2, R1= CH3SO2, R2= H, each R4= CH3, and D = Na-linked Pegloxenatide. Similar linker-pegloxenatides are produced using the appropriate linker reagent of formula XXXI. Analogously, use of different pegylation reagents other than the mPEG2-Lys-MAL (40kDa) will provide related pegylated peptide analogs, for example ones having PEG chains of different molecular weights (such as 5, 10, 20, or 60 kDa).

[0335] The linker-pegloxenatide is conjugated to hydrogels according to the procedures described in Example 2. Example 7 Gel dissolution profile of hydrogels following subcutaneous injection

[0336] 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 C

[0337] 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. Table 3: Loss of Hydrogels from the injection siteExample 8 Linker-Pegylated peptides Formula (IV) wherein D = Na-linked Ne-pegylated semaglutide peptide

[0338] The present invention contemplates replacement of an albumin-binding lipid protracting group of a lipidated peptide with a non-lipid group such as a linear or branched polyethylene glycol as a means of reducing the peptide clearance without an associated decrease of active unbound fraction in the plasma due to albumin binding.

[0339] As an example, the lipid protractor in semaglutide is replaced by a linear or branched polyethylene glycol. The amino acid sequence of semaglutide, H-{Aib}- EGTFTSDVSSYLEGQAAKEFIAWLVRGRG, SEQ ID No: B wherein Aib = aminoisobutyric acid, is prepared by solid phase peptide synthesis as described using Fmoc / t-Bu chemistry on a RAPP AM-Rink Amide resin with the lysine at position 20 being incorporated as Lys(ivDde). After removal of the last Fmoc group from the N-terminal tyrosine, a linker reagent of formula XXXI is added and coupled to provide resin with protected linker-peptide. The ivDde group is removed using 2% hydrazine / DMF and the resulting free amine is acylated using a PEG- succinimidyl ester, for example a linear 5, 10, 20, or 40 kDa methoxy PEG-OSu or a branched 5, 10, 20, or 40-kDa mPEG2-Lys-OSu reagent. The crude linker-peptide is then cleaved from the resin and deprotected using TFA / TIS / H2O and purified by HPLC.

[0340] When the linker reagent of formula XXXI is 5-azido-3,3-dimethyl-1- (methylsulfonyl)-2-pentyl succinimidyl carbonate (Example A), the product is formula (IV) wherein Z = N3, n = 2, R1= CH3SO2, R2= H, each R4= CH3, and D = Na-linked Lys20-pegylatedsemaglutide peptide. Similar linker-pegylated semaglutides are produced using the appropriate linker reagent of formula XXXI.

[0341] Other pegylated analogs of lipidated peptides may be similarly prepared, for example analogs of tirzepatide, retatrutide, mazdutide, survodutide, ecnoglutide, pemvidutide, VK2735, HRS-9531, CT-388, SAR441255, utreglutide, gubamy, olatorepatide, amycretin, dapiglutide, cagrilintide, KBP-336, NN1213, GUBamy, eloralintide, MET-097i, MET233i, and GZR18.

[0342] The linker-pegylated peptide is conjugated to hydrogels according to the procedures described in Example 2. Example 9 Linker-Fc Fusion peptides Formula (IV) wherein D = Dulaglutide

[0343] The present invention contemplates use of Fc fusion proteins as a means of reducing the clearance of the peptide released from hydrogel microspheres. Fc fusion proteins are peptides linked to the immunoglobulin Fc domain; the Fc domain reduces clearance through interaction with the salvage neonatal Fc receptor as well as through reducing renal filtration due to increased molecular size. Dulaglutide (SEQ ID No: 20) is an Fc fusion of GLP-1 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPA PEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0344] As further detailed in Example 11, reaction of Dulaglutide with a linker reagent of formula XXXI is expected to randomly acylate the various lysine residues; this is inconsequential, however, as subsequent cleavage of the linker will release native Dulaglutide regardless of the position of linker attachment. Thus, linker-Dulaglutide is prepared by reacting a linker reagent of formula XXXI with Dulaglutide in aqueous medium buffered at a pH between 7 and 9, preferably between 8 and 9, using a stoichiometric amount of linker reagent to controlattachment of multiple linkers. The resulting mixture may be separated by ion exchange chromatography, as each linker addition results in loss of a positive charge.

[0345] The linker-Dulaglutide is conjugated to hydrogels according to the procedures described in Example 2. Example 10 In vitro semaglutide release and degelation and in vivo depot residence

[0346] 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.

[0347] 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 37°C 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.11A, 11B, and 11C show in vitro semaglutide release for conjugate A, conjugate B, and conjugate C, respectively. Specifically, Figs.11A, 11B, and 11C show semaglutide release (solid, •) and PEG dissolved (dashed, triangles) in vitro at pH 9.4, 37°C.

[0348] 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.12A, 12B, and 12C show the amount of PEG at the injection site remaining after subcutaneous dosing in rats for conjugate A, conjugate B, and conjugate C, respectively.

[0349] Figs.13A and 13B show the plasma concentration of semaglutide released from conjugates B and C, respectively, after SC administration in rats. Example 11 Linker-Drugs of Fc-fusion proteins Formula (IV) wherein D = dulaglutide

[0350] Linker-Dulaglutides (formula (IV) wherein D = Seq ID No: 20) were prepared by reaction of a linker reagent of formula (XXXI) wherein Z = N3and X = O-succinimidyl (Example A; PCT Publication WO2020 / 206358; Hearn et al., Bioconjugate Chem.32, 794-800 (2021)) with dulaglutide (Seq ID No: 20) as follows.

[0351] In a total volume of 6450 μL, the reaction contained: 2.15 μmol (130 mg, 330 μM) dulaglutide in PBS pH 7.4 and 1.08 μmol of linker reagent of formula (XXXI) (0.5 molar equivalents). After 16 hours incubation at ambient temperature, a 1 μL sample of the reaction was mixed with 1 μL of 1 mM PEG40kDa-BCN and incubated for 4 hours at 37 ºC and later analyzed by SDS PAGE shift assay to determine the extent and distribution of acylation (Table 4). Due to the random nature of the linker attachment to reactive groups on the protein, a mixture of linker-proteins comprising, 0, 1, 2, or 3 attached linkers results. The rest of the reaction mixture was buffer exchanged and concentrated to 500 μL in Amicon 3k MWCO filters, pre- equilibrated with PBST (10mM PBS pH7.4 containing 0.05% Tween 20), to remove unbound linker.

[0352] The following linker-dulaglutides of formula (IV) were prepared according to this method: (A) Z = N3; n = 1; R1= SO2CH3; R2= H; each R4= Me; (B) n = 2; R1= SO2CH3; R2= H; each R4= Me; (C) n = 1; R1= SO2N(CH3)2; R2= H; each R4= Me.Table 4: Extent and distribution of acylation for each linker-dulaglutide conjugateExample 12 Releasable Conjugates of Fc-fusion proteins

[0353] Five releasable hydrogel conjugates of dulaglutide were prepared by reaction of linker-dulaglutides 8A, 8B, and 8C (Example 11) with activated hydrogel microspheres comprising cleavable crosslinks of Formula (IX) and based on either 10-kDa or 20-kDa 4-armed PEGs as follows.

[0354] Construct A: 100 μL of MS~BCN slurry (2 μmol / mL BCN) (Formula (IX) wherein A* = triazole; q = 1; R11= SO2NMe2; R12= H; each R14= Me; Z’ = O-bicyclo[6.1.0]non-4-yn-9- ylmethyl (“BCN”) and the PEGs are 4-armed 20-kDa) in PBST (PBS , pH 7.5, 0.05% Tween 20) was mixed with 100 μL of linker-dulaglutide 8A (103 mg / mL, 1.72 μmol / mL N3-linker- dulaglutide) in PBST giving 1 mM BCN and 0.86 mM N3-linker-dulaglutide in the reaction mixture. The reaction was gently mixed at ambient temperature for 150 hours. After incubation, the reaction was washed five times using 5 volumes of PBST to remove unreacted protein. One molar equivalent 1 μmol of O-2-(azidoethyl)heptaethylene glycol (“N3-PEG7”) was added to the slurry to cap unreacted BCN sites and the slurry was gently mixed for 20 hours at ambient temperature. The capped MS~dulaglutide microsphere slurry was washed nine times using 3- volumes of PBST to remove excess azido-PEG. The suspension of conjugated microspheres was buffer exchanged into 16.7mM Tris-HCl, pH 8.4, containing 0.05% Tween20 and held at room temperature for 23 h to remove linkers attached to the imidazole of histidines. The microspheres were then washed with 10 volumes of PBST.

[0355] Protein content of the hydrogel microspheres was determined by A280in 10 mg aliquots of slurry after dissolution in 80 µL of 50 mM NaOH and neutralization in 1M Tris-HCl,pH 7.55. The PEG content of the conjugate was determined following dissolution through BaCl2 / I2 spectrophotometry. Results are shown in Table 5.

[0356] Construct B: A 500 μL MS~BCN slurry (2 μmol / mL BCN) (Formula (IX) wherein A* = triazole; q = 1; R11= SO2NEt2; R12= H; each R14= Me; Z’ = O-bicyclo[6.1.0]non-4-yn-9- ylmethyl (“BCN”) and the PEGs are 4-armed 20-kDa) in PBST was mixed with 500 μL of linker-dulaglutide 8B (87 mg / mL, 1.45 μmol / mL linker-dulaglutide) in PBST, giving 1 mM BCN and 0.73 mM N3-linker-dulaglutide in the reaction mixtures. The reaction was gently mixed at ambient temperature for 150 hours. After incubation, the reaction was washed five times using 5 volumes of PBST to remove unreacted protein. One molar equivalent 1 μmol of O-2- (azidoethyl)heptaethylene glycol (“N3-PEG7”) was added to the slurry to cap unreacted BCN sites and the slurry was gently mixed for 20 hours at ambient temperature. The capped MS~dulaglutide microsphere slurry was washed nine times using 3-volumes of PBST to remove excess azido-PEG. The suspension of conjugated microspheres was buffer exchanged into 16.7mM Tris-HCl, pH 8.4, containing 0.05% Tween20 and held at room temperature for 23 h to remove linkers attached to the imidazole of histidines. The microspheres were then washed with 10 volumes of PBST.

[0357] Protein content of the hydrogel microspheres was determined by A280in 10 mg aliquots of slurry after dissolution in 80 µL of 50 mM NaOH and neutralization in 1M Tris-HCl, pH 7.55. The PEG content of the conjugate was determined following dissolution through BaCl2 / I2 spectrophotometry. Results are shown in Table 5.

[0358] Construct C: A 60 μL MS~BCN slurry (2 μmol / mL BCN) (Formula (IX) wherein A* = triazole; q = 1; R11= SO2NEt2; R12= H; each R14= Me; Z’ = O-bicyclo[6.1.0]non-4-yn-9- ylmethyl (“BCN”) and the PEGs are 4-armed 10-kDa) was mixed with 60 μL N3-linker- dulaglutide 8B (87 mg / mL, 1.45 μmol / mL N3-linker-dulaglutide), giving 1 mM BCN and 0.73 mM N3-linker-dulaglutide in the reaction mixtures. The reaction was gently mixed at ambient temperature for 150 hours. After incubation, the reaction was washed five times using 5 volumes of PBST to remove unreacted protein. One molar equivalent 1 μmol of O-2- (azidoethyl)heptaethylene glycol (“N3-PEG7”) was added to the slurry to cap unreacted BCN sites and the slurry was gently mixed for 20 hours at ambient temperature. The cappedMS~dulaglutide microsphere slurry was washed nine times using 3-volumes of PBST to remove excess azido-PEG. The suspension of conjugated microspheres was buffer exchanged into 16.7mM Tris-HCl, pH 8.4, containing 0.05% Tween20 and held at room temperature for 23 h to remove linkers attached to the imidazole of histidines. The microspheres were then washed with 10 volumes of PBST.

[0359] Protein content of the hydrogel microspheres was determined by A280in 10 mg aliquots of slurry after dissolution in 80 µL of 50 mM NaOH and neutralization in 1M Tris-HCl, pH 7.55. The PEG content of the conjugate was determined following dissolution through BaCl2 / I2 spectrophotometry. Results are shown in Table 5.

[0360] Construct D: A 500 μL MS~BCN slurry (2 μmol / mL BCN) (Formula (IX) wherein A* = triazole; q = 1; R11= SO2NEt2; R12= H; each R14= Me; Z’ = O-bicyclo[6.1.0]non-4-yn-9- ylmethyl (“BCN”) and the PEGs are 4-armed 20-kDa) was mixed with 500 μL N3-linker- dulaglutide 8C (70 mg / mL, 1.17 μmol / mL N3-linker-dulaglutide), giving 1 mM BCN and 0.58 mM N3-linker-dulaglutide in the reaction mixtures. The reaction was gently mixed at ambient temperature for 150 hours. After incubation, the reaction was washed five times using 5 volumes of PBST to remove unreacted protein. One molar equivalent 1 μmol of O-2- (azidoethyl)heptaethylene glycol (“N3-PEG7”) was added to the slurry to cap unreacted BCN sites and the slurry was gently mixed for 20 hours at ambient temperature. The capped MS~dulaglutide microsphere slurry was washed nine times using 3-volumes of PBST to remove excess azido-PEG. The suspension of conjugated microspheres was buffer exchanged into 16.7mM Tris-HCl, pH 8.4, containing 0.05% Tween20 and held at room temperature for 23 h to remove linkers attached to the imidazole of histidines. The microspheres were then washed with 10 volumes of PBST.

[0361] Protein content of the hydrogel microspheres was determined by A280 in 10 mg aliquots of slurry after dissolution in 80 µL of 50 mM NaOH and neutralization in 1M Tris-HCl, pH 7.55. The PEG content of the conjugate was determined following dissolution through BaCl2 / I2spectrophotometry. Results are shown in Table 5.

[0362] Construct E: A 60 μL MS~BCN slurry (2 μmol / mL BCN) (Formula (IX) wherein A* = triazole; q = 1; R11= SO2NEt2; R12= H; each R14= Me; Z’ = O-bicyclo[6.1.0]non-4-yn-9-ylmethyl (“BCN”) and the PEGs are 4-armed 10-kDa) was mixed with 60 μL dulaglutide / N3- linker-dulaglutide 8C (70 mg / mL, 1.17 μmol / mL N3-linker-dulaglutide), giving 1 mM BCN and 0.58 mM N3-linker-dulaglutide in the reaction mixtures. The reaction was gently mixed at ambient temperature for 150 hours. After incubation, the reaction was washed five times using 5 volumes of PBST to remove unreacted protein. One molar equivalent 1 μmol of O-2- (azidoethyl)heptaethylene glycol (“N3-PEG7”) was added to the slurry to cap unreacted BCN sites and the slurry was gently mixed for 20 hours at ambient temperature. The capped MS~dulaglutide microsphere slurry was washed nine times using 3-volumes of PBST to remove excess azido-PEG. The suspension of conjugated microspheres was buffer exchanged into 16.7mM Tris-HCl, pH 8.4, containing 0.05% Tween20 and held at room temperature for 23 h to remove linkers attached to the imidazole of histidines. The microspheres were then washed with 10 volumes of PBST.

[0363] Protein content of the hydrogel microspheres was determined by A280in 10 mg aliquots of slurry after dissolution in 80 µL of 50 mM NaOH and neutralization in 1M Tris-HCl, pH 7.55. The PEG content of the conjugate was determined following dissolution through BaCl2 / I2 spectrophotometry. Results are shown in Table 5.

[0364] Characterizing MS~dulaglutide conjugates: 10uL of microsphere slurry from each construct was resuspended in 190 uL of 100mM CAPS buffer, pH 10.0. The suspensions were then pelleted, and 50 uL of the supernatant was removed. Construct A was expected to have a faster release rate at pH10 than the other constructs, so a duplicate incubation was done with 100mM Borate, pH 9.4, for this construct alongside its pH10 CAPS duplicate.

[0365] All constructs were incubated at 37C in a water bath, sealed with Parafilm to prevent evaporation. At selected timepoints, the incubation tubes were vortexed, pelleted (18,000 xg, 1.5 min), and 5 uL of supernatant was removed for later characterization by A280 and PEG quantification. The A280 values from each timepoint were measured and graphed against time to determine the release of dulaglutide. A regression curve was generated using GraphPad Prism 10, and the t1 / 2of release was determined. The PEG content at each timepoint was determined, following dissolution, through BaCl2 / I2spectrophotometry. The tRG, representing the time at which microspheres have fully solubilized, was determined for each construct. Results are shownin Table 5. FIG.15 shows the in vitro release and subsequent polymer dissolution of releasable conjugate (9A) of the Fc-fusion Dulaglutide according to Example 12 under accelerated conditions. Table 5. Characterization of Releasable Conjugates of Dulaglutide

Claims

CLAIMS What is claimed is:

1. A controlled release conjugate of a drug 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 drug comprising a half-life extending moiety 2. The conjugate of claim 1, wherein the half-life extending moiety is a lipid, polyethylene glycol or the Fc fragment of an IgG.

3. A linker-drug 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;D is a drug comprising a half-life extending moiety, wherein the half-life extending moiety is a lipid, polyethylene glycol or the Fc fragment of an IgG; 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.

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-C6alkyl.

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

10. A conjugate of the formula (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 drug comprising a half-life extending moiety, wherein the half-life extending moiety is a lipid, polyethylene glycol or the Fc fragment of an IgG; M is a macromolecular carrier; and Z* is a connecting group.

11. The conjugate of claim 10, wherein n is 2, 3, or 4.

12. The conjugate of claim 10 or claim 11, wherein both R4groups are CH3.

13. The conjugate of any one of claims 10-12, 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.

14. The conjugate of ant one of claims 10-12, 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.

15. The conjugate of any one of claims 10-12, wherein R1is SOR5, or -SO2R5, wherein R5is C1-C6 alkyl.

16. The conjugate of any one of claims 10-15, wherein R2is H.

17. The conjugate of any one of claims 10-16, wherein M is comprised of crosslinked polymers.

18. The conjugate of claim 17, wherein the crosslinked polymers are pegylated polymers.

19. The conjugate of claim 18, wherein the pegylated polymers are comprised of multi-armed chains.

20. The conjugate of claim 19, wherein the multi-armed pegylated chains have a weight of between 1,000 and 40,000 Daltons.

21. A hydrogel comprising a linker-drug of any one of claims 3-9 and 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-C3alkyl; 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 the linker-drug.

22. The hydrogel of claim 21, wherein R11is -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.

23. The hydrogel of claim 21 or claim 22, wherein both R4groups are methyl.

24. The hydrogel of anyone of claims 21-23, having the formula (VII):

25. The hydrogel of anyone of claims 21-23, having the formula (VIII):

26. An (MS)-drug conjugate comprising polymeric units (P1-P2) crosslinked by a cleavable crosslinker and further comprising a linker-drug (L-D), wherein said (MS)-drug conjugates have the formula (XVII):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, or -SO2R13, wherein R13is H. optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionallysubstituted 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; R12is H; 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 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 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 drug comprising a half-life extending moiety anddepicts the point of attachment of the linker-drug (L-D) to Z*.

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