Locoregional therapies using slow-release conjugates

Abstract

Provided herein are locoregional therapies using conjugates of therapeutic agents that demonstrate extended release of the native therapeutic agents, as well as methods for the manufacture of such conjugates. These conjugates may be useful in the treatment of various conditions and diseases that respond to the extended exposure to the therapeutic agents, or for delivery of therapeutic agents that suffer from undesired systemic toxicities. In certain embodiments, the locoregional therapy is intratumoral therapy, which may be combined with a systemic or local therapy for enhanced therapeutic efficacy and reduced toxicity of the combined agents.

Description

Docket No.: 67057-20030.40 LOCOREGIONAL THERAPIES USING SLOW-RELEASE CONJUGATES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 552,588, filed February 12, 2024, the entire contents of which are incorporated by reference herein. FIELD

[0002] The present disclosure relates to locoregional therapies using slow-release conjugates. BACKGROUND

[0003] For therapeutic agents to be efficacious, they must distribute from the site of administration to the location of their target of action. In many cases, the target of action is spatially confined to a relatively small location in the body of the patient, and if the site of administration is distal to the location of the target a significant amount of exposure to the therapeutic agent is to non-targeted tissues located in the intervening locations. This can lead to undesired toxicities at the non-targeted tissues. Further, delivery of therapeutic agents to the target location from a distal administration site can be compromised by poor transport resulting from systemic clearance or metabolism among other issues.

[0004] For the treatment of tumors, for example, an antitumor agent administered systemically via intravenous injection must travel through the circulation to reach the tumor, and then rely upon the tumor vasculature to reach the necessary sites on the tumor cells. For an agent administered orally, the agent must be absorbed from the gut into the circulation, adding an additional step. Cells in the blood and many untargeted tissues are exposed to relatively high concentrations of the therapeutic agents during transit to the tumor, increasing the risk of undesired toxicities. Tumors often have poorly developed vasculature, decreasing their exposure to therapeutic agents carried by the circulation. Absorption of therapeutic agents from the circulation at the tumor can also be problematic, especially for large proteins such as antibodies where convective transfer is inefficient, and absorption is primarily via diffusion across pores in the blood vessels. It is thus typically observed that tumor concentrations of systemically- administered therapeutic antibodies are typically much lower than the concentrations in the circulation. This problem is being addressed in one method through the use of antibody-drug conjugates (ADCs), which are designed to target tumor tissues using antibody binding to tumor- specific antigens followed by selective release inside tumor cells once the ADC is internalized and degraded. Systemically-administered ADCs still suffer from the generally poor tumorny-29043151Docket No.: 67057-20030.40 penetration discussed above. Further, ADCs may have difficulty penetrating deeply into the tumor from the supplying blood vessels due to a “binding-site barrier” wherein there is sufficient antigen present on the cells adjacent to the blood vessels to soak up the ADC and thus prevent them from diffusing further into the tumor tissue.

[0005] Locoregional administration of a therapeutic agent, i.e. direct administration of the agent either into or immediately adjacent to the target location, has been proposed as a means of ameliorating these concerns. Locoregional administration of a therapeutic agent to a particular site will necessarily provide very high concentrations at that site immediately upon administration, followed by a decrease in concentration as the agent distributes throughout the body. Various versions of locoregional administration have been disclosed, for example intratumoral injections for cancer therapy; intravitreal, subconjunctival, or intracameral injections for treatment of diseases and disorders of the eye, and intra-lymphatic injections into lymph nodes for treatment of autoimmune disorders. It is apparent that locoregional injection of a therapeutic agent will provide initially high concentrations of the agent at the target site that will subsequently decrease over time due to distribution from the injection site into the much greater systemic volume, unlike systemic administration where blood levels are initially highest and the concentration in the target tissues slowly increases as the agent distributes from the circulation into the tumor tissue.

[0006] For example, it has thus been recognized that intratumoral injection could provide relatively high levels of therapeutic agents in the tumor as compared with non-tumor tissues, thus potentially improving antitumor efficacy relative to systemic toxicity (Melero et al., 2021). This has been demonstrated, for example, with antibodies and ADCs (Chang et al., 2023). This advantage is limited by the residence time of the therapeutic agent in the tumor, however, as distribution away from the injection site will eventually occur, and further by dose restrictions resulting from the limited capacity of a tumor to hold injected liquids due to the typically small interstitial volume (the “holdup volume”). Dose volumes greater than the holdup volume of the tumor will be rapidly released into the circulation. It has been shown that increasing the intratumoral residence time of the cytokine IL-2 by increasing the molecular weight through fusion with antibodies and by imparting matrix-binding activity through fusion with a collagen- binding peptide improve the efficacy of intratumorally-administered IL-2 (Momin et al., 2022). In these cases, however, the intratumoral half-life was still at most 7.9 hours. It has further been reported that intratumoral injections may minimize the formation of neutralizing anti-drug antibodies (Baniel et al., 2020).ny-29043152Docket No.: 67057-20030.40

[0007] Intratumoral injections have been disclosed for a variety of therapeutic agents, including small molecules like the toll-like receptor agonist resiquimod (Lessmann et al., 2023), and doxorubicin / 5-fluorouracil (Kim et al., 2016); cytokines (Wittrup et al., 2022); monoclonal antibodies (Blanco et al., 2023); and ADCs (Chang et al., 2023). Various formulations have been investigated to try and prolong the tumor residence time, such as viscosity enhancement (Zaharoff et al. 2010), polymer encapsulation (Kim et al., 2016; Brachi et al., 2020; Lee et al., 2010 Cancer Res (2010) 70 (8_Supplement): 3591), Xie et al., 2006), nanoparticles (Al- Ghananeem et al., 2009) and conjugation (Lessmann et al., 2023; Srinivasan et al., 2023). However, in each of these cases the residence time of the therapeutic agent in the tumor may be difficult to predict and control, and is limited by the available diffusion rates from the encapsulating polymer or nanoparticle or the uncontrolled hydrolysis rate of a chemical linker. The therapeutic agent may be unstable in encapsulating polymers such as PLGA, especially when they are proteins (Nguyen et al., 2020; Christian et al., 2012). Structural modification to the therapeutic agent to add elements with affinity for the tumor microenvironment have been disclosed, however these represent profound changes to the therapeutic agent itself (Wittrup et al., 2022; Agarwal et al., 2022; Danielli et al., 2015).

[0008] Intravitreal injections for treatment of diseases and disorders of the eye with corresponding low systemic exposure are common for administration of therapeutic proteins and antibodies which can have relatively long local residence times in the eye due to their high molecular weight and subsequent slow diffusion through the vitreous, but this has not been generally used for low-molecular weight therapeutic agents due to their rapid diffusion and corresponding low residence time.

[0009] Intra-lymphatic injections have been disclosed for the administration of antigenic peptides for development of immune tolerance in the treatment of autoimmune disorders and diseases (Yousefpour et al 2023 Nature Reviews Bioengineering 1: 107-24; Senti and Kuendig 2015 World Allergy Organ J 8(1):9; Johansen et al. 2005 Eur. J. Immunol.35: 568; Gammon et al 2023 Nature Comm. 14: 681; Jewell et al. 2011 Proc Natl Acad Sci USA 108(38): 15745-50). It has been shown that lymph nodes are the primary site for T- and B-cell priming, and that the local residence time of vaccines in the lymph nodes correlates with efficacy. Controlled-release may provide enhanced efficacy after intra-lymphatic injection. Further, it has been shown that intra-tumoral injection of microspheres may be followed by draining and subsequent accumulation of the microspheres in tumor-draining lymph nodes (Han et al. 2020 Immunol Invest 49(7): 808-23) such that immunotherapy agents delivered by intratumoral injection may also eventually reach the lymph nodes and have efficacy resulting from that location.ny-29043153Docket No.: 67057-20030.40

[0010] Intra-articular injections of antibodies have been disclosed for the treatment of arthritis, joint pain, and other conditions (Lam et al. 2023, Mol. Pharmaceutics 20(4): 2053-66; Alvarado-Vasquez et al. 2015 Neurosci Letters 584:39-44; DeMaudave et al. 2022, J. Controlled Release 341: 578-90).

[0011] There remains an unmet need for a general, highly-controlled method for sustained delivery of therapeutic agents to the tumor microenvironment. Conjugation using beta- eliminative linkers in which the release rate of the therapeutic agent is highly controlled and tunable over a period of hours to months has been disclosed (US Patents 8,680,315; 8,754,190; and in PCT Publication WO2020 / 206358A1). These linkers have been combined with biodegradable hydrogels and hydrogel microspheres (US Patents 9,649,385; 10,398,779; 11,179,470; 11,181,803; and 11,454,861) to provide a general, highly-controlled extended- release system for small molecules, peptides, and proteins. Large proteins, especially antibodies, are structurally complex and do not readily lend themselves to site-specific derivatization without potentially undesirable modifications. To date, a method for the conjugation of these linkers to very large, complex proteins such as antibodies that releases the protein in unmodified form has not been available.

[0012] Thus, what is needed in the art are therapeutic agents with short local residence time. What is desired are slow-release depots of therapeutic agents that will provide increased local residence time and thereby increase local concentrations of therapeutic agents while minimizing systemic concentrations and associated undesired toxicities. The subject matter described herein addresses this unmet need. BRIEF SUMMARY

[0013] In some aspects, provided herein are locoregional therapies using conjugates of therapeutic agents that demonstrate extended release of the native therapeutic agents, as well as methods for the manufacture of such conjugates. These conjugates may be useful in the treatment of various conditions and diseases that respond to the extended exposure to the therapeutic agents, or for delivery of therapeutic agents that suffer from undesired systemic toxicities.

[0014] In one aspect, therapeutic methods are provided wherein a releasable conjugate is administered by locoregional injection. In various embodiments of the invention, these methods involve intratumoral, peritumoral, intravitreal, intraarticular, intralymphatic, or perilymphatic administration. In various embodiments of the invention, the releasable conjugate is between anny-29043154Docket No.: 67057-20030.40 insoluble carrier and a small molecule, prodrug of a small molecule, peptide, nucleic acid, protein, antibody, bispecific or trispecific antibody, PROTAC, peptide-drug conjugate, or antibody-drug conjugate. In specific embodiments of the invention, the insoluble carrier is a hydrogel. In some embodiments, the hydrogel is a hydrogel microsphere (MS) such as a compound of formula (VII), described below. In other specific embodiments of the invention, the carrier is a suspension of aluminum hydroxide particles. Such methods provide extended exposure to high local concentrations of the drug with minimal systemic concentrations, thus ameliorating systemic toxicities while providing enhanced efficacy against the target tissue.

[0015] In a second aspect, therapeutic methods are provided wherein a releasable conjugate is administered by locoregional injection together with systemic administration of a second therapeutic agent. Such methods are particularly advantageous in cases where the therapeutic agent attached to the conjugate provided by locoregional injection and the second therapeutic agent provided by systemic administration show overlapping toxicities. In certain embodiments of the invention, a conjugate with a small molecule or prodrug of a small molecule is administered by locoregional injection while an antibody is administered systemically. In certain other embodiments of the invention, a conjugate with a small molecule or prodrug of a small molecule is administered by locoregional injection while a second small molecule or small molecule prodrug is administered systemically. In certain other embodiments of the invention, a conjugate with a peptide, protein, antibody, or ADC is administered by locoregional injection while a small molecule is administered systemically. In certain other embodiments of the invention, a conjugate with a peptide, protein, antibody, or ADC is administered by locoregional injection while a second peptide, protein, antibody, or ADC is administered systemically. In certain other embodiments of the invention, a conjugate with a therapeutic agent is administered by locoregional injection while a second dose of the same therapeutic agent is administered systemically. In various embodiments of the invention, the administered conjugate is based on a hydrogel.

[0016] In a third aspect, methods for the preparation of conjugates are provided. In one embodiment, amine-reactive activated hydrogels are used to prepare conjugates upon reaction with amine-containing therapeutic agents or amine-containing prodrugs of therapeutic agents. Present methods wherein a complex therapeutic agent having multiple attachment sites is first reacted with a releasable linker and the resulting linker-agent is conjugated to a carrier produce complex products; conjugates produced by such a method may release linker-modified agents upon cleavage of a single linker if all linkers are not conjugated to the carrier. The present invention provides methods for producing hydrogel conjugates of complex therapeutic agentsny-29043155Docket No.: 67057-20030.40 that can only release the free agent with no secondary linkers. In various specific embodiments of the invention, such complex therapeutic agents are peptides, proteins, antibodies, or ADCs. In another embodiment of the invention, methods for preparation of releasable conjugates based on aluminum hydroxide particles as carrier are provided.

[0017] In a fourth aspect, the disclosure provides methods of treating cancer in a subject in need thereof, comprising intratumorally and / or peritumorally administering to the subject a pharmaceutically acceptable amount of a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI).

[0018] In a fifth aspect, the disclosure provides methods of treating cancer in a subject in need thereof, comprising intratumorally and / or peritumorally administering to the subject a pharmaceutically acceptable amount of a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI) in combination with a second agent, wherein the second agent is administered systemically or subcutaneously. DESCRIPTION OF THE FIGURES

[0019] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0020] Figure 1 shows a schematic illustration of the preparation of activated hydrogels followed by preparation of a releasable conjugate with an amine-containing therapeutic agent. The releasable conjugates release free therapeutic agent in a pH-dependent process.

[0021] Figure 2 shows plots of drug release and hydrogel dissolution kinetics of hydrogel- SN38 conjugates of Example 4 run under accelerated conditions (pH 9.4, 37oC). The graphs illustrate in vitro release of SN38 and hydrogel dissolution kinetics under accelerated conditions.

[0022] Figure 3 shows a plot of in vivo concentrations of SN38 released from and SN38 remaining at the depot after s.c. administration of the conjugate 3A of Example 3 in mice as described in Example 5. MS~SN-38 conjugate 3A and free SN-38 was recovered from microsphere (MS) containing skin plugs over time. Data were normalized to the fluorescein marker for MS particles. Error bars represent one standard deviation of the mean (n=4 / point).

[0023] Figure 4A shows a plot of tumor volume versus days post treatment. In vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Specifically,ny-29043156Docket No.: 67057-20030.40 the graph depicts the efficacy of MS~SN-38 in a xenograft model of prostate cancer (22Rv1 ATM-deficient).

[0024] Figure 4B (top image) shows a plot of the % survival versus days in accordance with the in vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Specifically, the graph depicts the efficacy of MS~SN-38 in xenograft model of prostate cancer (22Rv1 ATM-deficient). The bottom image depicts a table of the data (median survival).

[0025] Figure 5A shows a graph of tumor volume versus days post treatment. The study was undertaken to examine the in vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Anti-tumor effects of MS~SN-38 in mice bearing 22Rv1 ATM(- / -)tumors. Control (^) showing estimated exponential growth after 1,000 mm3(– –); IT MS~SN- 38 doses were 0.2- (▲), 0.6- (★), 2- (^) or 7.5 µmol / kg (^). Data show median tumor volume ± median absolute difference from the median (n=5 / group).

[0026] Figure 5B shows a Kaplan Meier plot of survival for animals treated IT with 0.2- to 7.5 µmol / kg MS~SN-38. The study was undertaken to examine the in vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Anti-tumor effects of MS~SN-38 in mice bearing 22Rv1 ATM(- / -)tumors. Survival times of the controls (– –) are based on the estimated times for the tumor volume to reach 2,000 mm3. The estimated median survival time of the control group was 19 days.

[0027] Figure 5C shows a plot of tumor volume versus days post treatment. The study was undertaken to examine the in vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Anti-tumor effects of MS~SN-38 in mice bearing 22Rv1 ATM(- / -)tumors. Data show median tumor volume ± median absolute difference from the median (n=5 / group).

[0028] Figure 5D shows a Kaplan Meier plot of overall survival following SC 0.2- (^) and 2 (▽) µmol / kg MS~SN-38. The study was undertaken to examine the in vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Anti-tumor effects of MS~SN-38 in mice bearing 22Rv1 ATM(- / -)tumors. Survival times of the controls (– –) are based on the estimated times for the tumor volume to reach 2,000 mm3. The estimated median survival time of the control group was 19 days.ny-29043157Docket No.: 67057-20030.40

[0029] Figure 5E a plot of tumor volume vs time post treatment. The study was undertaken to examine the in vivo efficacy of intratumoral hydrogel-SN38 in a 22Rv1 xenografts as described in Example 6. Anti-tumor effects of MS~SN-38 in mice bearing 22Rv1 ATM(- / -)tumors. Data show median tumor volume ± median absolute difference from the median (n=5 / group).

[0030] Figure 5F shows a Kaplan Meier plot of survival of mice with 1,000 mm3tumors treated with 2 - (^) or 20 µmol / kg (^) IT MS~SN-38. Survival times of the controls (– –) are based on the estimated times for the tumor volume to reach 2,000 mm3. The estimated median survival time of the control group was 19 days.

[0031] Figure 6A shows a plot of tumor volume vs days post treatment. The study was undertaken to examine in vivo efficacy of intratumoral hydrogel-SN38 in a syngeneic CT26 xenograft model as described in Example 6. MS~SN-38 was administered IT or SC to CT26 tumor bearing mice (n=5 / group) and compared to the control group treated IT with empty / sham MSs (n=8). Statistical significance differences were calculated using two-away analysis of variance and log-rank (Mantel-Cox). *P <0.05.

[0032] Figure 6B shows -a plot of the probability of survival versus days. 6B. The study was undertaken to examine in vivo efficacy of intratumoral hydrogel-SN38 in a syngeneic CT26 xenograft model as described in Example 6. MS~SN-38 was administered IT or SC to CT26 tumor bearing mice (n=5 / group) and compared to the control group treated IT with empty / sham MSs (n=8). Statistical significance differences were calculated using two-away analysis of variance and log-rank (Mantel-Cox). *P <0.05.

[0033] Figure 7 shows a plot of the in vitro release and gel dissolution kinetics of the hydrogel-talazoparib conjugate of Example 7 under accelerated conditions (pH 9.4, 37oC). The results predict a drug release t1 / 2= 680 h and a gel dissolution time of 2600 h at pH 7.4, 37oC.

[0034] Figure 8 shows a plot of the pharmacokinetics of IgG released from releasable hydrogel conjugates of Example 3 injected subcutaneously, as described in Example 9. The hydrogel conjugates of Example 3 and Example 12 comprising 50 µg of antibody were injected s.c. into mice, and IgG levels in plasma (squares) were subsequently followed by ELISA. Curves are calculated for a release t1 / 2 of 4.2 and 35 days, for linkers GDM ClPhSO2 and GDM MeSO2, respectively.ny-29043158Docket No.: 67057-20030.40

[0035] Figure 9 show a plot of modeled intratumoral concentrations of antibody after intratumoral injection of a hydrogel conjugate of Example 3. The dashed curve shows observed plasma levels; the solid curve shows predicted tissue concentrations in 300 uL surrounding the injection site as a model for levels expected for a 300 mm3tumor.

[0036] Figure 10A shows results of in vitro characterization of the hydrogel- immunomodulator (TLR7 / 8 agonist DV1001) conjugate of Example 10. The plot shows the results of accelerated release conditions at pH 9.4, 37oC, indicating a drug release t1 / 2 = 2.14 h and gel dissolution time >30 h. These data predict a drug release rate of 214 h and a gel dissolution time >3000 h at pH 7.4, 37oC.

[0037] Figure 10B shows in vitro characterization of the hydrogel-immunomodulator (TLR7 / 8 agonist DV1001) conjugate of Example 10. The plot shows the results of purity of DV1001 released from the conjugate. The expected total release of DV1001 was 300 nmol and the total amount of PEG in the reaction was expected to be 1.5 mg. Data points collected after 30 h resulted in a saturated UV / Vis reading at 535 nm.

[0038] Figure 11A shows a plot of mean tumor volume mm3from the results of Example 10 (efficacy of single dose administration of TLR agonists in CT26 tumor bearing mice).

[0039] Figure 11B shows a plot of % survival versus days from the result of Example 10 (efficacy of single dose administration of TLR agonists in CT26 tumor bearing mice).

[0040] Figure 12 shows spider plots of tumor volume vs time after intratumoral injection of hydrogel-DV1001 agonist of Example 10.

[0041] Figure 13 shows a graph of the pharmacokinetics of ENHERTU released from a releasable hydrogel conjugate of Example 11 injected SC or IT, as described in Example 13. The hydrogel conjugate of Example 11 comprising 50 µg of antibody was injected subcutaneously or intratumorally into NCI-N87 tumor bearing mice, and ENHERTU levels in plasma were measured by ELISA. Curves were calculated for a release t1 / 2 of 4.2 days and 6.2 days, after subcutaneous or intratumoral administration, respectively.

[0042] Figure 14 shows a graph of the pharmacokinetics of IgG released from a releasable hydrogel conjugate of Example 12 injected subcutaneously or intratumorally, as described in Example 14. The hydrogel conjugate of Example 12 comprising 50 µg of antibody was injected subcutaneously or intratumorally into DU-145 tumor bearing mice, and IgG levels in plasmany-29043159Docket No.: 67057-20030.40 were subsequently followed by ELISA. Curves are calculated for a release t1 / 2 of 4.2 days and 10.4 days, after subcutaneous or intratumoral administration, respectively.

[0043] Figure 15 shows the results of tumor growth and mouse survival of 22Rv1 ATM- / -xenografts in mice (n=6) treated with either IT or SC 0.07 μmol / kg MS~SN-38, 0.4 μmol (0.15 mg) / kg of TLZ PO QDx21, and a combination of MS~SN-38 IT or SC and oral TLZ.

[0044] Figure 16A shows a general synthetic scheme of double-labeled microspheres with stable-linked fluorescein (FLS) as a quantitative marker for MS particles and releasable rhodamine (RPR).

[0045] Figure 16B shows graphs depicting fluorescence measurements with variance in recovery of MS from tissue and a normalized florescence ratio (RPR / FLS).

[0046] Figure 17 shows a graph depicting release of RP from FLs-MS~RPR at pH 8.4, 37oC with calculated half-lives for materials 2A, 2B and 2C.

[0047] Figure 18 shows bar graphs demonstrating separation of FLS-MSs (A) from free RP (B) after blending with a tissue homogenizer.

[0048] Figure 19 shows plots of free RP and FLs-MS remaining in injection sites following SC injection of a mixture of RP and FLS-MS-Ac into rats. The left-hand plot shows raw data and the right-hand plot shows normalized data obtained using the ratiometric method, described herein.

[0049] Figure 20A shows the results of the separation of MSs and the released drug surrogate 24 hours after IT injection and in vivo IT pharmacokinetics. A bar graph is depicted, showing normalized fluorescence of FLS-MS~RPR and free RP 24 hours after injection into tumors.

[0050] Figure 20B shows results of the separation of MSs and the released drug surrogate 24 hours after IT injection and in vivo IT pharmacokinetics. Normalized fluorescence of C vs t plots of 2A, 2B and 2C after IT injections of FLS-MS~RPR are depicted. Normalization was performed as follows: RP = Ln[(RP / FLs) / (RP / FLs)t0)] and FLs= Ln(FLs / FLs).

[0051] Figure 21 shows a plot of in vitro release and dissolution results of MS~SN-38 at pH 9.4, 37°C. The release t1 / 2 for SN-38 was measured to be 1.6 h at pH 9.4 or 160 h at pH 7.4 based on t1 / 2-7.4= t1 / 2-9.4*10(9.4-7.4)and MS dissolution tRGwas 25 h at pH 9.4.ny-290431510Docket No.: 67057-20030.40

[0052] Figure 22 shows body weight measurements of MS~SN-38 in mice bearing 22Rv1 ATM- / -xenografts. Left-hand plot: body weight of mice bearing ~125 mm3tumors treated with 20 μmol / kg MS~SN-38 IT (▲) or SC (^). Right-hand plot: body weight of mice bearing ~1,000 mm3tumors treated with 2 μmol / kg MS~SN-38 IT (^) or 20 μmol / kg MS~SN-38 IT (^).

[0053] Figure 23 shows a synthetic scheme of MS~RLI and RLIAPrelease. Note that upon β- elimination the aminopropyl moiety of the linker is transferred to the protein to give aminopropyl-RLI (RLIAP) as the final product.

[0054] Figure 24 shows the results of reductive alkylation of RLI. In the left-hand image, SDS-PAGE of RLI was performed with varying amounts of N3-PEG4-L(MeSO2)-CHO showing free-, mono- and multi-alkylated RLI. Alkylated RLI was reacted with DBCO-PEG5K to slow migration of alkylated RLI species. L1, Novex Sharp Pre-Stained Protein Ladder; L2, RLI; L3, RLI + PEG5KDa; L4, N3-linker:RLIRM = 1.5; L5, N3-linker:RLIRM = 2.0; L6, N3-linker:RLI RM = 3.0; L7, N3-linker:RLI RM = 5.0. In the right-hand image, the percent of RLI modified, as determined from the SDS PAGE gel. Bands were quantified using ImageJ software. RLI (10 nmol) was treated with 1.5 – 5 equivalents N3-linker-CHO (Mod = MeSO2) and NaCNBH3(10 mM) in 25 mM Citrate 500 mM NaCl and 0.05% tween-20 for 20 hours at room temperature in the dark.

[0055] Figure 25 shows a plot of in vitro release kinetics for MS~RLI. Data for the release of RLIAP (t1 / 2,pH9.4= 7 h) and degelation (tRG,pH 9.4= 28 h) of MS~RLI are shown.

[0056] Figure 26 shows a plot of the purity of RLI bound to the microspheres. The released proteins from the microspheres were monitored by HPLC. The analysis shows >93% of the protein on the MSs was RLI. Approximately 45% of the RLI loaded to the MSs was glycosylated.

[0057] Figure 27 shows a plot of the bioactivity of RLIAP. RLI and RLIAP were assayed for IL-2 / IL-15Rβγ binding via an U2OS cell-based assay. The EC50for RLI induced dimerization of IL-2 / IL-15Rβγ by RLI (EC50= 160 pM) and RLIAP(EC50= 200 pM) was determined by fitting data to a four-parameter logistic model; data points represent mean ± SD.

[0058] Figure 28 shows a log-linear plasma C vs t plot of RLIAP released from MS~RLI in C57BL / 6J and NSG mice. NSG mice (●) and C57BL / 6J mice (■) were administered MS~RLI 10ny-290431511Docket No.: 67057-20030.40 μg on Day 0 and RLIAP plasma concentrations were determined by ELISA. Data are represented as mean ± SD.

[0059] Figure 29 shows data plots demonstrating how MS~RLI elicits expansion of target immune cells. The top row of plots show the frequency of NK, CD8+, and CD44hiCD8+T cell expansion in PBMCs. The bottom row of plots show the percentage of proliferating NK, CD8+, and CD44hiCD8+T cells. Mice were administered a single SC dose of MS~RLI (1 – 10 μg) or MS~IL-15 (50 μg). Error bars represent one SD of the mean (n=5 / group).

[0060] Figure 30A shows how MS~RLI10μg treatment increases the number of CD44hiCD8+T cells and NK cells compared to free RLI. In the plots, absolute cell number of proliferating CD44hiCD8+and NK cells are shown. Mice (n=5 / group) were administered a single IP dose RLI (10 μg), a single SC dose of MS~IL-1550μg, or MS~RLI10μg. Data represented as mean ± SD (n=5 / group).

[0061] Figure 30B shows how MS~RLI10μgtreatment increases the number of CD44hiCD8+T cells and NK cells compared to free RLI. In the plots, percentages of proliferating CD44hiCD8+, and NK cells are shown. Mice (n=5 / group) were administered a single IP dose RLI (10 μg), a single SC dose of MS~IL-1550μg, or MS~RLI10μg. Data represented as mean ± SD (n=5 / group).

[0062] Figure 31 shows a plot of how the body weight of naive C57BL / 6J mice is affected by SC administration of MS~IL-15 SC. Mice were administered a single SC dose 10 μg MS~RLI or 50 μg MS~IL-15. Data represented as mean + / - SD (n=5 / group).

[0063] Figure 32 shows bar graphs of PD response following two doses of MS~RLI10μg. The left-hand graph shows absolute cell number of proliferating immune cells 5 days after dosing on D 0 (Dose 1) and D 35 (Dose 2). Mice (n=4-5 / group) were administered MS~RLI10μg, RLI10μg, or two doses of 2 μg RLI separated by 48h. After 35 days, mice were treated again. Data represented as mean + / - SD. The statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, **p<0.01.

[0064] Figure 33A shows data demonstrating anti-tumor effects of MS~RLI in CT26 tumor bearing mice. The plot shows mean tumor volume vs. time post treatment; mice were administered empty MSs IT, 10 μg MS~RLI IT, or empty MSs IT plus 10 μg MS~RLI SC. Data was pooled from 2 independent experiments (n=11 / group). The median survival times for the control (18 d), SC MS~RLI (20 d), and IT MS~RLI (27 d) are based on the tumor volumeny-290431512Docket No.: 67057-20030.40 reaching 2,000 mm3or animal death. Tumor growth volumes were analyzed by two-way ANOVA, ***p<0.001.

[0065] Figure 33B shows data demonstrating anti-tumor effects of MS~RLI in CT26 tumor bearing mice. The Kaplan Meier plot shows overall animal survival. The median survival times for the control (18 d), SC MS~RLI (20 d), and IT MS~RLI (27 d) are based on the tumor volume reaching 2,000 mm3or animal death. The Kaplan Meier plot was analyzed by Mantel-Cox test, *p<0.05.

[0066] Figure 34 shows a plot of the results of body weights of CT26 tumor bearing mice following treatment. Mice were administered empty MSs IT, 10 μg MS~RLI IT, or empty MSs IT plus 10 μg MS~RLI SC (n=7-8 / group).

[0067] Figure 35A shows immunophenotyping of tumors of CT26 tumor bearing mice on D5 following treatment with 10 μg MS~RLI administered SC or IT. Empty MS administered IT were used as untreated control. The graph shows the number of NK cells and CD8+T cells normalized to grams of tissue (tumor) and the percentage of proliferative cells (Ki67+) of every cell subtype. First bar (left) is empty MS; center bar graph is SC administration; right bar graph is IT administration. Results are expressed as mean + / - SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05; **p<0.01; ****p<0.0001. n=3-4 / group.

[0068] Figure 35B shows immunophenotyping of spleens of CT26 tumor bearing mice on D5 following treatment with 10 μg MS~RLI administered SC or IT. Empty MS administered IT were used as untreated control. The graph shows the number of NK cells and CD8+T cells normalized to grams of tissue (spleen) and the percentage of proliferative cells (Ki67+) of every cell subtype. First bar (left) is empty MS; center bar graph is SC administration; right bar graph is IT administration. Results are expressed as mean + / - SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05; **p<0.01; ****p<0.0001. n=3-4 / group.

[0069] Figure 35C shows immunophenotyping of blood of CT26 tumor bearing mice on D5 following treatment with 10 μg MS~RLI administered SC or IT. Empty MS administered IT were used as untreated control. The graph shows the number of NK cells and CD8+T cells normalized to μL of blood and the percentage of proliferative cells (Ki67+) of every cell subtype. First bar (left) is empty MS; center bar graph is SC administration; right bar graph is IT administration. Results are expressed as mean + / - SD. Statistical significance was determined byny-290431513Docket No.: 67057-20030.40 one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05; **p<0.01; ****p<0.0001. n=3-4 / group.

[0070] Figure 36 shows a treatment schedule for EO771 tumor bearing mice. On day -7, C57BL / 6 mice were inoculated with 3x105EO771 cells orthotopically in the fourth mammary pad. Mice with palpable tumors were distributed in different groups 7 days later and treated with MS~IL-15, MS~RLI or empty microspheres.

[0071] Figure 37 shows a plot of the body weights of EO771 tumor bearing mice following treatment. Mice were treated IT with empty microspheres, 1.2 μg MS~IL-15, 6 μg MS~IL-15, 2 μg MS~RLI (s), or 10 μg MS~RLI (n=5 / group).

[0072] Figure 38A shows tumor growth curves for anti-tumor effects of MS~RLI and MS~IL-15 in EO771 tumor-bearing C57BL / 6 mice. Mice (n=5 / group) were treated with empty MSs, MS~RLI (2 or 10 μg), or equimolar MS~IL-15 (1.2 μg or 6 μg). Tumor volumes are represented as mean ± SEM and statistical significance was calculated by 2 way mixed-effects analysis, *p<0.05.

[0073] Figure 38B shows a plot of the histologic analysis of metastatic lung lesions on D15 (bar indicates mean) for the anti-tumor effects of MS~RLI and MS~IL-15 in EO771 tumor- bearing C57BL / 6 mice. Mice (n=5 / group) were treated with empty MSs, MS~RLI (2 or 10 μg), or equimolar MS~IL-15 (1.2 μg or 6 μg). The statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparisons test. All MS~RLI and MS~IL-15 treated groups differ from the empty MS control (****p<0.0001). *p<0.05.

[0074] Figure 39 shows a bar graph disclosing the final weight of EO771 tumors as described in the examples herein.

[0075] Figure 40 shows a flow cytometric analysis of immune cells in tumors and spleen in EO771 tumor bearing mice. A) CD8+T cells, B) CD8+GrB+T cells, C) NK and D) NK GrB+cells in tumors, and E) CD8+T cells and F) NK cells in the spleen on day 15. Data in graphs are given as percentage of total live cells and represented as mean + / - SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05.

[0076] Figure 41 shows a complete blood count analysis following IT MS~RLI or MS~IL- 15.The total number of whole white blood cells (A), lymphocytes (B), neutrophiles (C), and monocytes (D) are shown.ny-290431514Docket No.: 67057-20030.40

[0077] Figure 42 shows pictures of H&E staining of lungs. Representative images from H&E analysis of metastatic lesions in lungs of treated and control animals. The arrows indicate the metastatic lesions; scale bar = 1000 μm.

[0078] Figure 43 shows a representative gating strategy in accordance with methods disclosed herein.

[0079] Figure 44 shows deglycosylation results of RLI with PNGase F. A) SDS-PAGE analysis of RLI. Lane 1: Molecular weight marker; Lane 2) Untreated RLI sample; Lane 3: RLI treated with PNGase F; Lane 4: heat denatured RLI was treated with PNGase F. B) HPLC chromatogram of RLI before and after treatment with PNGaseF. Native RLI or heat denatured RLI was treated with PNGase F (0.4 mU / µg RLI) at 37°C for 48 hours.

[0080] Figure 45 shows C18-HPLC analysis of reaction mixtures, showing RP, and Azido- linker-RP A, B, and C.

[0081] Figure 46 shows dosing syringes containing 0.025 mL single doses of materials 2A, 2B and 2C with purged and capped needles (left-hand image) and a bar graph showing average absorbance of FLS and RP from multiple (N = 4) 0.025 mL doses of 2A, 2B, and 2C into 0.1M NaOH (right-hand image). Error bars are standard deviation.

[0082] Figure 47 shows a small-scale PEEK dissolution cell. A) Cell in 5 mL conical tube with stirrer. (B) Top view of the assembly shown in part A. C) Exploded view of cell showing body, caps and nylon mesh membranes. D) 12 position stirring heat block for operation of cells on a liquid handling robot.

[0083] Figure 48 shows anti-tumor effects of MS~SN-38 and PLX038A in mice bearing 22Rv1 ATM- / -xenografts. A) Tumor volume vs days post treatment; control with estimated exponential tumor growth after 1,000 mm3(dashed line); IT 7.5 μmol / kg MS~SN-38, and IP 7.5 μmol / kg PLX038A. B) Kaplan Meier plot of overall survival for animals treated in A. Tumor volume data represent median tumor volume ± interquartile range (n=5 / group). DETAILED DESCRIPTION

[0084] The present invention provides locoregional therapies using conjugates of therapeutic agents that demonstrate extended release of the native therapeutic agents, as well as methods for the manufacture of such conjugates. These conjugates may be useful in the treatment of variousny-290431515Docket No.: 67057-20030.40 conditions and diseases that respond to the extended exposure to the therapeutic agents, or for delivery of therapeutic agents that suffer from undesired systemic toxicities. DEFINITIONS

[0085] It is understood that the term “electron-withdrawing group” includes functional groups having Hammett sigma constants greater than 0 as described, for example, in Hansch et al. (1991) Chem. Rev. 91: 165-195, including but not limited to CN, NO2, fluoroalkyl, carbonyl, aryl, heteroaryl, carboxylate, carboxamide, sulfone, or sulfonamide. In particular embodiments of the invention, groups R1, R1and R2are selected from the group consisting of H, 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, -OR6or –NR62, wherein each R6is independently H, optionally substituted alkyl, optionally substituted aryl, or optionally substituted heteroaryl, or both R6groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring. Due to the requirement for at least one electron- withdrawing group to control the rate of the beta-elimination release mechanism, at least one of R1and R2cannot be H and must be an electron-withdrawing group. In specific embodiments of the invention, R1and R2are independently H or CN or SO2R5. Preferred electron-withdrawing groups include CN, PhSO2, CF3-PhSO2, Cl-PhSO2, MeO-PhSO2, CH3-PhSO2, MeSO2, (CH3)2CHSO2, CH2(CH2CH2)2NSO2, (CH3)CH(CH2CH2)2NSO2, O(CH2CH2)2NSO2, (CH3)2NSO2, and (CH3CH2)2NSO2. Each R4is independently H or C1-C3 alkyl, or taken together form a ring of 3-7 carbon atoms. Z is a connecting functionality that allows for conjugation of the linker to the support described below, and in certain embodiments may be azide, cyclooctyne, bicyclononyne, trans-cyclooctene, tetrazine, carbonyl, carboxylate, thiol, or maleimide. In a particular embodiment, Z comprises a peptide sequence that binds aluminum hydroxide. The integer n = 0-6.

[0086] It is understood that the term “alkyl” includes linear, branched, or cyclic saturated hydrocarbon groups of 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms. In some embodiment, an alkyl is linear or branched. Examples of linear or branched alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n- heptyl, n-octyl, n-nonyl, n-decyl, and the like. In some embodiments, an alkyl is cyclic. Examples of cyclic alkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, and the like.ny-290431516Docket No.: 67057-20030.40

[0087] It is understood that the term “alkoxy” includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and the like.

[0088] It is understood that the term “alkenyl” includes non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds and 2-20, 2-12, 2-8, 2-6, or 2-4 carbon atoms.

[0089] It is understood that the term “alkynyl” includes non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds and 2-20, 2-12, 2-8, 2-6, or 2-4 carbon atoms.

[0090] It is understood that the term “aryl” includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term “heteroaryl” includes aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and the like.

[0091] In some instances, alkenyl, alkynyl, aryl or heteroaryl moieties may be coupled to the remainder of the molecule through an alkyl linkage. Under those circumstances, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.

[0092] It is understood that the term “halogen” or “halo” includes bromo, fluoro, chloro and iodo.

[0093] It is understood that the term “heterocyclic ring” or “heterocyclyl” refers to a 3-15 membered aromatic or non-aromatic ring comprising at least one N, O, or S atom. Examples include, without limitation, piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, morpholino, and tetrahydrofuranyl, as well as the exemplary groups provided for the term “heteroaryl” above. In some embodiments, a heterocyclic ring or heterocyclyl is non-aromatic. In some embodiments, a heterocyclic ring or heterocyclyl is aromatic.

[0094] It is understood that “optionally substituted,” unless otherwise specified, means that a group may be unsubstituted or substituted by one or more (e.g., 1, 2, 3, 4 or 5) of the substituents which may be same or different. Examples of substituents include, without limitation, alkyl, alkenyl, alkynyl, halogen, -CN, -ORaa, -SRaa, -NRaaRbb, -NO2, -C=NH(ORaa), -C(O)Raa, -OC(O)Raa, -C(O)ORaa, -C(O)NRaaRbb, -OC(O)NRaaRbb, -NRaaC(O)Rbb, -NRaaC(O)ORbb, -S(O)Raa, -S(O)2Raa, -NRaaS(O)Rbb, -C(O)NRaaS(O)Rbb, -NRaaS(O)2Rbb, -C(O)NRaaS(O)2Rbb,ny-290431517Docket No.: 67057-20030.40 -S(O)NRaaRbb, -S(O)2NRaaRbb, -P(O)(ORaa) (ORbb), heterocyclyl, heteroaryl, or aryl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heteroaryl, and aryl are each independently optionally substituted by Rcc, wherein Raaand Rbbare each independently H, alkyl, alkenyl, alkynyl, heterocyclyl, heteroaryl, or aryl, or Raaand Rbbare taken together with the nitrogen atom to which they attach to form a heterocyclyl, which is optionally substituted by alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, or -CN, and wherein: each Rccis independently alkyl, alkenyl, alkynyl, halogen, heterocyclyl, heteroaryl, aryl, -CN, or -NO2.

[0095] As used herein, “SC” refers to subcutaneous delivery.

[0096] As used herein, “IT” refers to intratumoral delivery.

[0097] As used herein, “intratumoral” refers to the region within a solid tumor. Intratumoral injections deliver therapeutic agents directly to the tumor tissue See, for example, Hong et al., Clin Cancer Res 26(13):3091-9 (2020).

[0098] As used herein, “peritumoral” refers to the region immediately surrounding a tumor and includes adjacent normal tissue as well as regions potentially influenced by the tumor. For example, as discussed in Koca et al., British J Cancer 131: 1111-5 (2024), peritumoral injections can deliver therapeutic agents directly to tumors as well as tumor-draining lymph nodes that receive the lymphatic drainage from tumors and thus may be particularly effective means for administration of immunomodulatory agents.

[0099] For use herein, unless clearly indicated otherwise, use of the terms “a”, “an” and the like refers to one or more. CONJUGATES

[0100] The present invention uses therapeutic agents conjugated to carriers through releasable (also known as cleavable) linkers. The releasable linkers used in the conjugates of the invention are of the formulany-290431518Docket No.: 67057-20030.40and have been disclosed for example in Santi et al. 2011 Proc Natl Acad Sci USA 109(16): 6211- 6; US Patents 8,680,315; 8,754,190; U.S. Publication No. US20220280654; U.S. Publication No. US20220193253; and in PCT Publication WO2020 / 206358A1 (each of which is incorporated herein by reference). In the Compound of Formula (I), X is a leaving group, Z is a functional group for connecting the linker to a macromolecular carrier, each R4is independently hydrogen, C1-3 alkyl or the two R4are taken together with the carbon atom which they are attached to form a 3-6 membered ring, and R1and R2are independently an electron-withdrawing group, alkyl or H, wherein at least one of R1and R2is an electron-withdrawing group.

[0101] In some embodiments of a linker of formula (I), X is halogen, active ester (e.g., N- succinimidyloxy, nitrophenoxy, or pentahalophenoxy), optionally substituted heteroaryl (e.g., imidazolyl, triazolyl, or tetrazolyl), or -N(R6)CH2Cl wherein R6is optionally substituted C1-C6alkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, X is halogen. In some embodiments, X is an active ester such as succinimidyloxy. In some embodiments, X is -N(R6)CH2Cl, wherein R6is optionally substituted aryl.

[0102] For a linker of formula (I), Z can be any functional group known in the art for conjugation. Examples of such functional groups include, without limitation, amine, aminooxy, ketone, aldehyde, maleimidyl, thiol, alcohol, azide, 1,2,4,5-tetrazinyl, trans-cyclooctenyl, bicyclononynyl, cyclooctynyl, and protected variants thereof. In some embodiments, Z is protected amine, protected aminooxy, ketone or protected ketone, aldehyde or protected aldehyde, maleimidyl, protected thiol, protected alcohol, azide, 1,2,4,5-tetrazinyl, trans- cyclooctenyl, bicyclononynyl, or cyclooctynyl. In some embodiments, Z is azide, ketone, or protected ketone.

[0103] The linker of formula (I) can be reacted with a moiety that comprises a drug (D), thereby forming a compound of formula (II),ny-290431519Docket No.: 67057-20030.40wherein n, R1, R2, R4, and Z are as disclosed herein for formula (I); D is a drug; Y is absent when D is a drug connected through an amine, or Y is -N(R6)CH2- when D is a drug connected through a phenol, alcohol, thiol, thiophenol, imidazole, or non-basic amine; wherein R6is optionally substituted C1-C6alkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, the compound of formula (II) is a linker-drug prepared by combining the linker of formula (I) with a drug such as a small molecule, peptide, or protein therapeutic.

[0104] In some embodiments of compound of formula (II), Y is absent. In some embodiments, Y is -N(R6)CH2-.

[0105] In some embodiments of compound of formula (II), suitable drugs include, without limitation, small-molecules, peptides, proteins, and nucleic acids. Examples of suitable drugs include, without limitation, antidiabetic drugs, growth promoters, antibacterials including aminoglycosides, penicillins, cephalosporins, macrolides and peptides, trimethoprim, piromidic acid, and sulfamethazine; analgesic and anti-inflammatory drugs, antiallergic and antiasthmatic drugs, antihypercholesterolemic drugs, beta-adrenergic blockers and antihypertensive drugs, antineoplastic drugs, and antiviral drugs.

[0106] Further examples of such drugs include alcohols such as paclitaxel and analogues, epothilones and analogues, camptothecin and analogues such as irinotecan, and nucleosides such as 5-fluorouracil and capecitabine. In another embodiment, the drug is a peptide comprising a serine residue. In another embodiment, the drug is a small molecule comprising an arylol group; examples of such drugs include SN-38, etilefrine, prenalterol, and estradiol. In another embodiment, the drug is a peptide comprising a tyrosine residue. If coupling is through S, the drug may be a small molecule comprising a thiol group. Examples of such drugs include penicillamine, captopril, and enalapril. The drug may be a small molecule comprising a thioaryl or thioheteroaryl group; examples of such drugs include 6-mercaptopurine. If coupling is through a non-basic N, the drug may be a small molecule or peptide comprising a primary or secondary amide (such as a pyroglutamate residue or other amide) or sulfonamide, or a heteroaryl group such as an indole (e.g., tryptophan) or purine. Examples include thyrotropin-releasing hormone,ny-290431520Docket No.: 67057-20030.40 bombesin, luteinizing hormone-releasing hormone, follicle-stimulating releasing hormone, octreotide, 5-fluorouracil and allopurinol.

[0107] Examples of nucleic acid-based drugs include the sense strand and antisense strand of any gene from an animal, and particularly from a mammal. Such genes can be those that are already the subjects of antisense DNAs or RNAs, or small interfering RNAs that have been provided with the purpose of treating various diseases, for example genes for protein kinase C-alpha, BCL-2, ICAM-1, tumor necrosis factor alpha and the like. Also included are CpG oligonucleotide agonists of toll-like receptors. Nucleic acids may be coupled directly to the linkers or through a modified group on the nucleic acid, for example an oligonucleotide comprising a 5’- or 3’-amine modification or comprising an amine-containing base.

[0108] In some embodiments of a compound of formula (II), D is a peptide. Examples of suitable peptides include, without limitation, octreotide, exenatide and variants including [N28Q]exenatide, insulin lispro, or Teduglutide ([Gly2]GLP-2) and sequence variants thereof.

[0109] To form a conjugate, a compound of formula (II) can be reacted with a macromolecular carrier (M), thereby forming a compound of formula (III),wherein n, R1, R2, R4, D, and Y are as disclosed herein for formula (I) or (II); M is a macromolecular carrier; q is an integer from 1 to 10 when M is a soluble macromolecule, or q is a multiplicity when M is an insoluble matrix (e.g., an MS hydrogel); Z* indicates coupling to M. In some embodiments of a conjugate of formula (III), the molecular carrier M comprises at least one functional group Z’ cognate to Z that allows for conjugation. For example, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (III) wherein Z* is amide or carbamate. As another example, when Z is azide, Z’ is alkynyl, bicyclononynyl, or cyclooctynyl to yield a conjugate of formula (III) wherein Z* is 1,2,3- triazole. As another example, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (III) wherein Z* is oxime. As another example, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (III) wherein Z* is thiosuccinimidyl or thioether.ny-290431521Docket No.: 67057-20030.40 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.

[0110] In some embodiments, the compound of formula (III) is a conjugate of drug D releasably linked to the macromolecular carrier M through a linker of formula (I). It is understood that, when M is an insoluble matrix, a multiplicity of linker-drugs can be attached to M. For example, in some embodiments, when M is a hydrogel of formula (IV) wherein both P1and P2are 4-armed polymers, 1, 2, 3, or 4 linker-drugs can be attached to each P1-P2unit. Thus, the desired multiplicity can be achieved by reacting the linker-drug with M in a suitable ratio. As such, suitable drug concentration in the volume of the matrix can be achieved. Additional aspects of the hydrogels are described below.

[0111] In some embodiments of a conjugate of formula (III), molecular carrier M is a soluble macromolecule and q is an integer from 1 to 10. In some embodiments, M is an insoluble matrix and q is a multiplicity. In some embodiments, when M is an insoluble matrix, q is a multiplicity such that suitable drug concentration in the volume of the matrix can be achieved. Examples of soluble macromolecules include, without limitation, polyethylene glycol or other synthetic polymer, dextran, antibody, antibody fragment, albumin or other protein, of sufficient molecular size to inhibit efficient renal filtration as is understood in the art. For polyethylene glycols, M can be single-chain, multiple-chain, or multiple-arm of average molecular weight between 1,000 and 100,000 daltons, preferably between 1,000 and 40,000 daltons. Examples of insoluble matrices include, without limitation, hydrogel, implant, or surgical device, either in bulk or as microparticles or nanoparticles. In some embodiments, M is a soluble macromolecule. In some embodiments, M is an insoluble matrix. In some embodiments, M is a hydrogel of formula (VII) as disclosed herein.

[0112] In some embodiments of a compound of formula (I), (II) or (III), n = 1-6, R1and R2are independently electron-withdrawing groups, alkyl, or H, and wherein at least one of R1and R2is an electron-withdrawing group; each R4is independently C1-C3 alkyl or taken together may form a 3-6 membered ring; X is halogen, active ester such as N-succinimidyloxy, nitrophenoxy, or pentahalophenoxy, or imidazolyl, triazolyl, tetrazolyl, or N(R6)CH2Cl wherein R6is optionally substituted C1-C6alkyl, optionally substituted aryl , or optionally substituted heteroaryl; and Z is a functional group for connecting the linker to a macromolecular carrier.

[0113] In some embodiments of a compound of formula (I), (II) or (III),, the electron- withdrawing group of R1and R2isny-290431522Docket No.: 67057-20030.40 -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.

[0114] In some embodiments, the electron-withdrawing group of R1and R2is -CN. In some embodiments, the electron-withdrawing group of R1and R2is -NO2. In some embodiments, the electron-withdrawing group of R1and R2is optionally substituted aryl containing 6-10 carbons. For instance, in some embodiments, the electron-withdrawing group of R1and R2is optionally substituted phenyl, naphthyl,or anthracenyl. In some embodiments, the electron-withdrawing group of R1and R2is 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 R1and R2is optionally substituted pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, or indenyl. In some embodiments, the electron-withdrawing group of R1and R2is optionally substituted alkenyl containing 2-20 carbon atoms. In some embodiments, the electron-withdrawing group of R1and R2is optionally substituted alkynyl containing 2-20 carbon atoms. In some embodiments, the electron- withdrawing group of R1and R2is -COR3, -SOR3, or -SO2R3, wherein R3is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring. In some embodiments, the electron-withdrawing group of R1and R2is -SR9,ny-290431523Docket No.: 67057-20030.40 wherein R9is optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0115] In some embodiments of a compound of formula (I), (II) or (III), at least one of R1and R2is -CN, -SOR3or -SO2R3. In some embodiments, at least one of R1and R2is –CN or - SO2R3. In some embodiments, at least one of R1and R2is –CN or -SO2R3, wherein R3is optionally substituted alkyl, optionally substituted aryl, or -NR82. In some embodiments, at least one of R1and R2is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, -SO2N(CH2CH2)2O, - SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0116] In some embodiments of a compound of formula (I), (II) or (III), each R4is independently C1-C3 alkyl. In some embodiments, both R4are methyl.

[0117] In some embodiments of a compound of formula (I), (II) or (III), n is an integer from 1 to 6. In some embodiments, n is an integer from 1 to 3. In some embodiments, n is an integer from 0 to 3. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0118] These cleavable linkers of formula (III) cleave under appropriate conditions of pH and temperature through a beta-elimination mechanism, with the rate of cleavage being controlled primarily by the presence of one or more electron-withdrawing groups R1and R2. Scheme 1 shows an exemplary beta-eliminative drug release from a linker. In accordance with Scheme 1, a drug is covalently tethered to a long-lived carrier by a linker that slowly cleaves by beta-elimination to release the native drug (“drug” in scheme 1). The cleavage rate of the linker is controlled by the nature of an electron-withdrawing “modulator” (Mod in scheme 1), which regulates the acidity of an adjacent carbon-hydrogen bond. These linkers can control drug release rates over long periods, are not affected by enzymes, and are stable when stored at low pH and temperature (D. V. Santi, E. L. Schneider, R. Reid, L. Robinson, G. W. Ashley, Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates. Proc Natl Acad Sci U S A 109, 6211-6216 (2012); J. Henise et al., High-throughput, aseptic production of injectable Tetra-PEG hydrogel microspheres for delivery of releasable covalently bound drugs. Engineering Reports 2:e12213, 1-13 (2020)).ny-290431524Docket No.: 67057-20030.40 Scheme 1 MS DRUG CONJUGATES

[0119] In some embodiments, the microsphere (MS) component is MS-drug conjugates is a hydrogel. In one aspect, provided is a releasable hydrogel conjugate of a drug (D) having the formula (IV) M-[Z*-(CH2)nC(R4)2CH(CHR1R2)-O-CO-D]s. (IV) wherein M is a biodegradable hydrogel, Z* is a connecting group, n = 0-6, R1is an electron- withdrawing group, R2is H or C1-3alkyl, each R4is independently H or C1-C3alkyl, or taken together form a 4-7 membered ring, D is a drug, and s is the number of moles of D per milligram of biodegradable hydrogel (M). 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.

[0120] 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.ny-290431525Docket No.: 67057-20030.40

[0121] 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.

[0122] In some variations, “connecting group” is a functional group that stably connects two sections of the conjugate and is not cleaved during the normal functioning of the releasable biodegradable hydrogel conjugate. In certain variations, examples of such connecting groups include carboxamides, thioethers, dihydropyridazines, and triazoles. Each connecting group is formed by the reaction of two cognate precursor functional groups 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, a-halocarbonyl, or activated alkene; an oxime is formed by the reaction of an aminoether and a carbonyl; and a triazole is formed by the 1,3- dipolar cycloaddition of an azide with an alkyne or cycloalkyne. Thus Z and Z’ may be amine, carboxylate, active ester, tetrazine, trans-cyclooctene, thiol. Maleimide, a-halocarbonyl, activated alkene, aminoether, carbonyl, azide, alkyne, or cycloalkyne.

[0123] In some variations, “optionally substituted” refers to having one or more H atoms replaced by a substituent, including halogen (F, Cl, Br, I); linear or branched C1-C6 alkyl; C3-C8 cycloalkyl; C1-C6 alkoxy; C1-C6 haloalkyl; alkylamino; dialkylamino; carboxy; carboxamido; carbonyl; nitro; CN; azido; thiol; C1-C6alkylthio; alkylsulfonyl; arylsulfonyl; sulfonamide; alkylsulfonamide; arylsulfonamide; C1-C10 aryl; and C1-C10 heteroaryl. In particular embodiments of the invention, substituents include trifluoromethyl; chloro; fluoro; methyl; methoxy; 2-methoxyethyl; phenyl; methylsulfonyl; sulfonamide; and N,N-dimethylsulfonamide.

[0124] In some embodiments, s is between 10 and 1,000. In some embodiments, s is between 50 and 1,000. In some embodiments, s is between 100 and 1,000. In some embodiments, s is between 200 and 1,000. In some embodiments, s is between 200 and 800. In some embodiments, s is between 200 and 600. In some embodiments, s is between 200 and 800. In some embodiments, s is between 50 and 200. In some embodiments, s is between 50 and 100. In some embodiments, s is between 100 and 200.ny-290431526Docket No.: 67057-20030.40

[0125] 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.

[0126] In some embodiments, the biodegradable polymer is a crosslinked PEG comprised of pegylated polymers. In some embodiments, the individual pegylated polymers comprising the hydrogel have a size of from about 5kDa to about 50 kDa. In some embodiments, the individual pegylated polymers comprising the hydrogel have a size of from about 10 kDa to about 50 kDa. In some embodiments, the individual pegylated polymers comprising the hydrogel have a size of from about 10 kDa to about 20 kDa. In some of the foregoing embodiments, s is between 10 and 400, 50 and 300, 50 and 200. or 100 and 200. Linker-Drug Units of Conjugates

[0127] In some embodiments, the MS-drug conjugates comprise linker-drug units of the formula (V)wherein: n is an integer from 0 to 6; R1is an electron-withdrawing group; R2is H or C1-3alkyl;ny-290431527Docket No.: 67057-20030.40 each R4is independently H or C1-C3 alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a drug; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0128] In some embodiments of a compound of the formula (IV) or formula (V), D is a small molecule, protein or antibody.

[0129] In some embodiments of the linker-drug units of the formula (V), 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.

[0130] In some embodiments of the linker-drug units of the formula (V), each R4group is independently C1-C3alkyl. In some embodiments, both R4groups are CH3.

[0131] In some embodiments of the linker-drug unit of formula (V), the electron- withdrawing group 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.ny-290431528Docket No.: 67057-20030.40

[0132] In some embodiments of a linker-drug units of formula (V), the electron-withdrawing group 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.

[0133] In some embodiments of the linker-drug unit of formula (V), 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 -COR3, -SOR3, or -SO2R3, wherein R3is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R8groups are taken together with the nitrogen to which they are 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.

[0134] In some embodiments of the linker-drug unit of formula (V), R2is H.

[0135] In some embodiments of the linker-drug unit of formula (V), 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;ny-290431529Docket No.: 67057-20030.40 each R4is independently H or C1-C3 alkyl or the two R4are taken together with the carbon atom to which they attach to form a 3-6 membered ring; D is a drug; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0136] In some embodiments of the linker-drug unit of formula (V), 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; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0137] In some embodiments of the linker-drug unit of formula (V), 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; and depicts the point of attachment of the linker-drug units to a macromolecular carrier.

[0138] 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.

[0139] In some embodiments, the MS-drug conjugates comprise linker-drug units of the formula (VI)ny-290431530Docket No.: 67057-20030.40wherein: 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 a drug; and depicts the point of attachment of the linker drug units to a macromolecular carrier.

[0140] In some embodiments of a compound of formula (VI), D is a protein. In some embodiments of a compound of formula (VI), D is an antibody or antibody fragment. In some embodiments of a compound of formula (VI), D is a cytokine or cytokine variant. In some embodiments, the N-terminal alpha amine of the antibody, antibody fragment, or protein (e.g., cytokine) is bonded to the linker of the linker-drug units. Following administration of a hydrogel comprising such linker-drug units, the N-terminal of the released drug (antibody, antibody fragment, or protein) would be modified. For instance, in some embodiments, the N-terminal end of the antibody, antibody fragment, or protein is modified with NH(CH2CH2O)p(CH2)m.In some embodiments p is 0. In some embodiments p is 0. In some embodiments m is 2. In some embodiments m is 3. In some embodiments p is 0 and m is 2. In some embodiments p is 0 and m is 3.ny-290431531Docket No.: 67057-20030.40 .

[0141] In some embodiments of a linker-drug of formula (VI), the cytokine D is IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-15, IL-21, or a cytokine variant thereof. D also encompasses a cytokine with certain chemical modifications to the cytokine, such as NH(CH2CH2O)p(CH2)m, wherein m is a integer from 2 to 6 and p is an integer from 0 to 1000, attached to an amine group resulting from reductive amination to attach the linker L. In certain embodiments, this modification is attached to the N-terminal alpha-amino group of the protein sequence.

[0142] By “cytokine variant” is meant a protein of altered sequence (“mutein”) having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the native cytokine. In some embodiments, the cytokine variant has at least 90% sequence identity to the native cytokine. In some embodiments, the cytokine variant comprises between 1 and 10 altered amino acids from the native sequence, and is selected based on improvements in protein stability and / or receptor binding affinity or selectivity. Depending on the expression system used to produce recombinant cytokines, the sequence may or may not include the initiating methionine residue. For example, IL-2 variants useful in the disclosure may be selected from those having increased binding affinity for the trimeric αβγ-receptor over the dimeric βγ receptor. In some embodiments, the IL-2 variant has a mutation at asparagine-88, for example N88R or N88D, which can be combined with other mutations such as C125S, to confer added stability or selectivity. Other IL-2 muteins suitable for use in the disclosure are disclosed, for example muteins with alterations at aspartate-20 such as IL-2 D20T, or muteins having reduced affinity for the trimeric receptor as disclosed in US Patent No. 9,206,243.

[0143] In some embodiments of a compound of formula (VI), D is an IL-15 or an IL-15•IL- 15RαSu fusion protein.

[0144] In some embodiments of a linker-drug unit of formula (VI), linker drug units are attached to the macromolecular carrier through a carboxamide, amide, oxime, triazole, thioether, thiosuccinimide, or ether moiety.

[0145] In some embodiments of a linker-drug unit of formula (VI), R1is -CN or -SO2R5, wherein R5is C1-C6 alkyl, aryl, heteroaryl, or NR62, wherein each R6is independently C1-C6 alkyl, 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.

[0146] In some embodiments of a linker-drug unit of formula (VI), R2is H.ny-290431532Docket No.: 67057-20030.40

[0147] In some embodiments of a linker-drug unit of formula (VI), n is 4, each R4is 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)mwherein m = 3 and p = 0.

[0148] In some embodiments of a linker-drug unit of formula (VI), M comprises polymers having multi-armed chains.

[0149] In some embodiments of a linker-drug unit of formula (VI), M comprises r-armed polymers, 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.

[0150] The linker-drug unit of formula (V) or formula (VI) can be covalently attached to a macromolecular carrier through a particular functional group. For instance, the conjugate of formula (III) depicts a single linker-drug unit attached to a macromolecular carrier (M) via functional group Z*.(VII), wherein M is a macromolecular carrier, and n, R1, R2, R4and D are defined as above.

[0151] It will be understood that in accordance with the present disclosure, M in formula (VII) 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 (V)). 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 2ny-290431533Docket No.: 67057-20030.40 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.

[0152] In some embodiments, the functional group Z* in the conjugate of formula (VII) can be formed from a reactive functional group Z that is covalently attached to the linker-drug unit of formula (V) and a functional group Z’ on the macromolecular carrier (M). The reaction is depicted in Scheme 2.Scheme 2

[0153] In some embodiments, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (VII) wherein Z* is amide or carbamate. In other embodiments, when Z is azide, Z’ is alkynyl, bicyclononynyl, or cyclooctynyl to yield a conjugate of formula (VII) wherein Z* is 1,2,3-triazole. In other embodiments, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (VII) wherein Z* is oxime. In other embodiments, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (VII) 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.

[0154] In some embodiments, Z* comprises a carboxylic amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide. In some embodiments, Z* is a 1,2,3-triazole.

[0155] In some embodiments. the disclosure provides methods of reacting the linker-drug unit, 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*.

[0156] In some embodiments of a compound of formula (VII), D is a protein. In some embodiments of a compound of formula (VII), D is an antibody or antibody fragment. In someny-290431534Docket No.: 67057-20030.40 embodiments of a compound of formula (VII), D is a cytokine or cytokine variant. In some embodiments, the N-terminal alpha amine of the antibody, antibody fragment, or protein (e.g., cytokine) is bonded to the linker of the linker-drug units. Following administration of a hydrogel comprising such linker-drug units, the N-terminal of the released drug (antibody, antibody fragment, or protein) would be modified. For instance, in some embodiments, the N-terminal end of the antibody, antibody fragment, or protein is modified with NH(CH2CH2O)p(CH2)m.In some embodiments p is 0. In some embodiments p is 0. In some embodiments m is 2. In some embodiments m is 3. In some embodiments p is 0 and m is 2. In some embodiments p is 0 and m is 3. .

[0157] In some embodiments of a linker-drug of formula (VII), the cytokine D is IL-2, IL-4, IL-7, IL-9, IL-10, IL-12, IL-15, IL-21, or a cytokine variant thereof. D also encompasses a cytokine with certain chemical modifications to the cytokine, such as NH(CH2CH2O)p(CH2)m, wherein m is a integer from 2 to 6 and p is an integer from 0 to 1000, attached to an amine group resulting from reductive amination to attach the linker L. In certain embodiments, this modification is attached to the N-terminal alpha-amino group of the protein sequence.

[0158] In some embodiments of a compound of formula (VII), D is an IL-15 or an IL-15•IL- 15RαSu fusion protein.

[0159] In some embodiments of a compound of formula (VII), D is a small molecule. For instance, D may be an small-molecule antitumor agent, for example a topoisomerase inhibitor such as a camptothecin, SN38, and exatecan; a PARP inhibitor, such as talazoparib, rucaparib, nirabarib, and olaparib; an immunomodulator, such as rapamycin or a toll-like receptor agonist like resiquimod, DV-1001, and gardiquimod; a kinase inhibitor, such as adavosertib, axitinib, cobimetinib, crizotinib, dasatinib, erdafitinib, lapatinib, pazopanib, and pegaptanib; an anti- microtubule agent, such as eribulin, docetaxel, paclitaxel, and vinblastine; alkylating agent such as cisplatin or carboplatin; cytotoxin such as doxorubicin, daunorubicin; or antimetabolite such as methotrexate. Crosslinked Polymeric Units

[0160] In some embodiments, the hydrogels of the disclosure are comprised of repeating crosslinked polymeric units. Each polymeric unit may have multiple arms, which allows it to be crosslinked to multiple polymeric units in the hydrogel. As set forth above, the individual crosslinked units comprising the hydrogel may include one or more drug units. In someny-290431535Docket No.: 67057-20030.40 embodiments, the individual polymeric units comprising the hydrogel include, without limitation, polyethylene glycol (PEG) or other synthetic polymer, dextran, antibody, antibody fragment, albumin or other protein, of sufficient molecular size to inhibit efficient renal filtration as is understood in the art. For polyethylene glycols, M can be single-chain, multiple-chain, or multiple-arm of average molecular weight between 1,000 and 100,000 daltons, preferably between 1,000 and 40,000 daltons.

[0161] In some embodiments, the individual polymeric units of the hydrogel is a polypeptide selected from poly(amino acid)s such as poly(lysine) and poly(valine) and mixed-sequence polypeptides.

[0162] 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.

[0163] 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).

[0164] In some embodiments, the individual polymeric units of the hydrogel is a polysaccharide such as a dextran.

[0165] 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.

[0166] 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 (VIII)ny-290431536Docket No.: 67057-20030.40wherein: A* and C* are independently connecting groups that connect the cleavable linker to the polymeric units (e.g., r-armed polymers); q is an integer from 0 to 6; x, y, and z are independently an integer from 0 to 6; R11is an electron-withdrawing group; R12is H or C1-C3 alkyl; each R14is independently H, C1-C3alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; and shows the point of attachment to a linker-drug (e.g., a linker-drug unit of formula (V)).

[0167] In certain embodiments, A* is carboxamide, oxime, or triazole; C* is carboxamide; and Z’ is amine or cycloalkyne.

[0168] In some embodiments, A* or C* is an amide group. In some embodiments, A* or C* is a click product formed from a first click handle on the polymeric unit and a second click handle on the cleavable linker.

[0169] In some embodiments of the cleavable linker of formula (VIII), 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.

[0170] In some embodiments of the cleavable linker of the formula (VIII), each R14group is independently C1-C3alkyl. In some embodiments, both R4groups are CH3.

[0171] In some embodiments of the cleavable linker of formula (VIII), the electron- withdrawing group R11isny-290431537Docket No.: 67057-20030.40 -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.

[0172] In some embodiments of the cleavable linker of the formula (VIII), the electron- withdrawing group R11is -CN, -SOR3or -SO2R3. In other embodiments, R1is –CN or -SO2R3. In other embodiments, R11is –CN or -SO2R3, wherein R3is optionally substituted alkyl, optionally substituted aryl, or -NR82. In other embodiments, R11is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, - SO2PhCl, -SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or - SO2N(CH2CH2OCH3)2.

[0173] In some embodiments of the cleavable linker of formula (VIII), 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 ofny-290431538Docket No.: 67057-20030.40 R1is -COR3, -SOR3, or -SO2R3, wherein R3is H, optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl containing 1-20 carbon atoms, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring. In some embodiments, the electron-withdrawing group of R11is -SR9, wherein R9is optionally substituted alkyl containing 1-20 carbon atoms, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl.

[0174] In some embodiments of a cleavable linker of formula (VIII), R12is H.

[0175] In some embodiments of a cleavable linker of formula (VIII), R11is 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 and R12is H.

[0176] In some embodiments of a cleavable linker of formula (VIII), R11is -SOR5, or - SO2R5, wherein R5is C1-C6 alkyl (e.g. CH3 or CH2CH3) and R12is H.

[0177] In some embodiments, the cleavable linker of formula (VIII) is covalently bonded to a linker-drug unit of formula (II) through a carboxamide, oxime, thioether, or triazole moiety.

[0178] In some embodiments of a cleavable linker of formula (VIII), q = 1-2; each R14is methyl; R11is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4 and z=0. Conjugates

[0179] The cleavable linker of formula (VIII) can be covalently bonded to a to a linker-drug unit of formula (V) or formula (VI) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (V) or formula (VI). For instance, the reactive group Z’ can be covalently bonded to the cleavable crosslinker of formula (VIII), hence forming a cleavable linker of formula (VIIIa), depicted below.ny-290431539Docket No.: 67057-20030.40 (VIIIa), wherein A*, C*, q, R11, R12, R14, x, y and z are defined as above.

[0180] A cleavable crosslinker of formula (Va) can be reacted with a linker-drug unit formula to yield a conjugate of formula (IX), as depicted in Scheme 3.Scheme 3

[0181] In some embodiments of Scheme 3, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (IX) wherein Z* is amide or carbamate. In other embodiments of Scheme 3, when Z is azide, Z’ is alkynyl, bicyclononynyl, or cyclooctynyl to yield a conjugate of formula (IX) wherein Z* is 1,2,3-triazole. In other embodiments of Scheme 3, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (IX) wherein Z* is oxime. In other embodiments of Scheme 3, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (IX) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ can be reversed to yield Z* of opposingny-290431540Docket No.: 67057-20030.40 orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0182] In some embodiments of Scheme 3, the cleavable crosslinker of formula (VIIIa) 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.

[0183] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker having the formula (X):(X), wherein A*, q, R11, R12, and R14are defined as above.

[0184] The cleavable crosslinker of formula (X) can be covalently bonded to a to a linker- drug unit of formula (V) or formula (VI) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (V) or formula (VI). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (X), hence forming a cleavable linker of formula (XI), depicted below.ny-290431541Docket No.: 67057-20030.40

[0185] A cleavable linker of formula (XI) can be reacted with a linker-drug unit to yield a conjugate of formula (XII), as shown in Scheme 4. Scheme 4 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 4

[0186] In some embodiments of Scheme 4, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (XII) wherein Z* is amide or carbamate. In other embodiments of Scheme 4, when Z is azide, Z’ is alkynyl, bicyclononynyl, or cyclooctynyl to yield a conjugate of formula (XII) wherein Z* is 1,2,3-triazole. In other embodiments of Scheme 4, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (XII) wherein Z* is oxime. In other embodiments of Scheme 4, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (XII) wherein Z* is thiosuccinimidyl or thioether. Similarly, these roles of Z and Z’ can be reversed to yield Z* of opposingny-290431542Docket No.: 67057-20030.40 orientation. In some embodiments, Z* comprises an amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether.

[0187] In some embodiments of scheme 4, the cleavable linker of formula (XI) 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 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.

[0188] In some embodiments, the individual polymeric units comprising the hydrogel are crosslinked through a cleavable linker. In some embodiments, the cleavable linker has the formula (XIII):(XIII), wherein A*, q, R11, R12, and R14are defined as above.

[0189] The cleavable linker of formula (XIII) can be covalently bonded to a to a linker-drug unit of formula (V) or formula (VI) through the addition of appropriate reactive functional groups on both the linker and the linker-drug unit of formula (VI) or formula (VII). For instance, the reactive group Z’ can be covalently bonded to the cleavable linker of formula (XIII), hence forming a cleavable linker of formula (XIV), depicted below.ny-290431543Docket No.: 67057-20030.40

[0190] A cleavable linker of formula (XIV) can be reacted with a linker-drug unit formula (IV) to yield a conjugate of formula (XV), as shown in Scheme 5.Scheme 5

[0191] In some embodiments of Scheme 5, when Z is amine, Z’ is carboxylic acid, active ester, or active carbonate to yield a conjugate of formula (XV) wherein Z* is amide or carbamate. In other embodiments of Scheme 5, when Z is azide, Z’ is alkynyl, bicyclononynyl, or cyclooctynyl to yield a conjugate of formula (XV) wherein Z* is 1,2,3-triazole. In other embodiments of Scheme 5, when Z is NH2O, Z’ is ketone or aldehyde to yield a conjugate of formula (XV) wherein Z* is oxime. In other embodiments of Scheme 5, when Z is SH, Z’ is maleimide or halocarbonyl to yield a conjugate of formula (XV) 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.

[0192] In some embodiments of scheme 5, the cleavable linker of formula (XIV) 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 has an azide moiety as the Z group. In suchny-290431544Docket No.: 67057-20030.40 embodiments, Z* is formed via a click reaction between the cyclooctyne moiety and the azide moiety. Crosslinked Polymers

[0193] Cleavable linkers with covalently attached linker-drug units, such as formula (VIII), formula (IX), formula (X), formula (XIV), and formula (XV), can be covalently bonded to individual polymeric units as set forth herein. For instance, the cleavable linker of formula (VIII) can be bonded to two individual polymeric units through connecting groups A* and C*. Formula (XVI) depicts two individual polymeric units (P1and P2) crosslinked through a cleavable linker.

[0194] Formula (XVII) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (IX).ny-290431545Docket No.: 67057-20030.40

[0195] In some embodiments of a compound of formula (XVII), 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 R4ais C1-C3alkyl, R11is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; w = 0-4; y = 0-4; z=0; C* is carboxamide; Z* is comprises a triazole group; n = 0-6; R1is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; and D is a drug.

[0196] In some embodiments of a compound of formula (XVII), 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 R14is methyl, R11is CN or R3SO2wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; x = 0-4; y = 0-4; z=0; C* is carboxamide; Z* comprises a triazole group; n = 0-3; R1is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; and D is a drug.

[0197] In some embodiments of a compound of formula (XVII), 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 R14is methyl, R11is R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; R12is H; w = 0-4; y = 0-4; C* is carboxamide; Z* comprises a triazole group; n = 0-3; R1is CN or R3SO2 wherein R3is methyl, isopropyl, Me2N, MeEtN, or (MeOCH2CH2)2N; and D is a drug.

[0198] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 1; each R14is methyl, R22is Me2NSO2; x = 0; y = 4; z=0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0199] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; q = 1; each R14is methyl, R11is Me2NSO2; x = 4; y = 0; z=0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0200] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* is triazole; q = 1; each R14is methyl, R11is Me2NSO2; w = 4; y = 0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0201] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises any-290431546Docket No.: 67057-20030.40 triazole group; q = 1; each R14is methyl, R11is Me2NSO2; w = 0; y = 4; z=0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0202] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 2; each R14is methyl, R11is Me2NSO2; w = 0; y = 4; z=0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0203] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; q = 2; each R14is methyl, R11is Me2NSO2; x= 4; y = 0; z=0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0204] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 10,000 daltons; A* comprises a triazole group; q = 2; each R14is methyl, R11is Me2NSO2; wx= 4; y = 0; z=0; C* is carboxamide; Z* comprises a triazole group; n = 2; R1is CH3SO2; and D is a drug.

[0205] In some embodiments of a compound of formula (XVII), P1and P2are each independently 4-armed PEGs of average molecular weight of 20,000 daltons; A* comprises a triazole group; triazole; q = 2; each R14is methyl, R11is Me2NSO2; w = 0; y = 4; z=0’ C* is carboxamide; Z* is triazole; n = 2; R1is CH3SO2; and D is a drug.

[0206] Formula (XVIII) depicts two individual polymeric units (P1and P2) crosslinked through a cleavable linker.(XVIII)

[0207] Formula (XIX) depicts two individual polymeric units (P1and P2) crosslinked through a cleavable linker.ny-290431547Docket No.: 67057-20030.40

[0208] Formula (XX) depicts two individual polymeric units (P1and P2) crosslinked through a cleavable linker.

[0209] Formula (XXI) depicts two individual polymeric units (P1and P2) crosslinked through the cleavable linker of formula (XV).

[0210] 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 (XXII):ny-290431548Docket No.: 67057-20030.40wherein: P1and P2are independently r-armed pegylated polymers, wherein r is an integer from 2 to 8; A* and C* 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:ny-290431549Docket No.: 67057-20030.40wherein: n is an integer from 0 to 6; R1is -CN, -NO2, -COR3, -SOR3, or -SO2R3, wherein R3is , optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR8or -NR82, wherein each R8is independently H or optionally substituted alkyl, or both R8groups are taken together with the nitrogen to which they are attached to form a heterocyclic ring or SR9, wherein R9is optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, or optionally substituted heteroarylalkyl; R2is H; each R4is independently H, C1-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; and depicts the point of attachment of the linker-drug (L-D) to Z*.

[0211] In some embodiments of an (MS)-drug conjugate of formula (XXII), each R4is independently C1-C3 alkyl. In some embodiments, each R4is methyl.

[0212] In some embodiments of an (MS)-drug conjugate of formula (XXII), R1is CN or - SO2R3. In some embodiments, R1is –CN, -SO2N(CH3)2, -SO2CH3, -SO2Ph, -SO2PhCl, - SO2N(CH2CH2)2O, -SO2CH(CH3)2, -SO2N(CH3)(CH2CH3), or -SO2N(CH2CH2OCH3)2.

[0213] In some embodiments of an (MS)-drug conjugate of formula (XXII), n is an integer from 1 to 3.

[0214] In some embodiments of an (MS)-drug conjugate of formula (XXII), D is a peptide drug. In some such embodiments, peptide drug is octreotide, exenatide, [N28Q]exenatide.ny-290431550Docket No.: 67057-20030.40 insulin lispro, or teduglutide. In some embodiments of an (MS)-drug conjugate of formula (XVII), D is a protein.

[0215] In some embodiments of an (MS)-drug conjugate of formula (XXII), D is a protein. In some embodiments of an (MS)-drug conjugate of formula (XXII), D is a cytokine

[0216] In some embodiments of an (MS)-drug conjugate of formula (XXII), Z* comprises a carbonate, amide, oxime, 1,2,3-triazole, thioether, thiosuccinimide, or ether group.

[0217] In some embodiments of an (MS)-drug conjugate of formula (XXII), each R14is independently C1-C3 alkyl. In some embodiments, both R14are methyl. In some embodiments, one R14 is methyl and one R14is H. In some embodiments, both R14are H.

[0218] In some embodiments of an (MS)-drug conjugate of formula (XXII), each R4is independently C1-C3 alkyl and each R14is independently C1-C3 alkyl. In some embodiments, both R4are methyl and both R14are methyl.

[0219] In some embodiments of an (MS)-drug conjugate of formula (XXII), each R4is independently C1-C3 alkyl and both R14are H. In some embodiments, both R4are methyl and both R14are H.

[0220] In some embodiments of an (MS)-drug conjugate of formula (XXII), R11is -CN or - SO2R13. In some embodiments, R11is -SO2CH3.

[0221] In some embodiments of an (MS)-drug conjugate of formula (XXII), wherein A* and C* 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* is a 1,2,3-triazole group.

[0222] In some embodiments of an (MS)-drug conjugate of formula (XXII), A* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group.

[0223] In some embodiments of an (MS)-drug conjugate of formula (XXII), A* comprises an amide group.

[0224] In some embodiments of an (MS)-drug conjugate of formula (XXII), A* is produced by a reaction between a first moiety comprising an azide group and a second moiety comprising a cyclooctyne group. In some embodiments, A* the second moiety is bicyclonynyl or cyclooctynyl.ny-290431551Docket No.: 67057-20030.40

[0225] In some embodiments of an (MS)-drug conjugate of formula (XXII), C* comprises an amide group.

[0226] In some embodiments of an (MS)-drug conjugate of formula (XXII), x is 0 and z is 0.

[0227] 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 (XXIII):(XXIII) wherein: P1and P2are independently r-armed pegylated polymers, wherein r is an integer from 2 to 8; A* and C* 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;ny-290431552Docket No.: 67057-20030.40 each R14is independently H, C1-C3 alkyl or the two R14are taken together with the carbon atom to which they attach to form a 3-6 membered ring; Z* is a connecting group for attaching the cleavable crosslinker to the linker-drug (L-D); and L-D has the formula: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)mwherein m = 2-6 and p = 0-1000; D is drug; and depicts the point of attachment of the linker drug units to a macromolecular carrier.

[0228] In some embodiments of an (MS)-drug conjugate of formula (XXIII), D is a peptide drug. In some embodiments of an (MS)-drug conjugate of formula (XXIII), D is a protein. In some embodiments of an (MS)-drug conjugate of formula (XXIII), D is an antibody or fragment thereof. For instance, 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 or pegylated cytokine is Interleukin-2 (IL-2) or Interleukin-15 (IL-15). In some embodiments, the cytokine or pegylated cytokine comprises an IL-15 and the sushi domain of the IL-15 receptor.ny-290431553Docket No.: 67057-20030.40 Hydrogels Comprising Multi-Polymeric Units

[0229] In some embodiments, formula (XVI), formula (XVII), formula (XVIII). formula (XIX), formula (XX) or formula (XXI) 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).

[0230] Representative hydrogels of the disclosure are depicted below:(XXV)ny-290431554Docket No.: 67057-20030.40ny-290431555Docket No.: 67057-20030.40ny-290431556Docket No.: 67057-20030.40ny-290431557Docket No.: 67057-20030.40(XXXI), 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.

[0231] In some embodiments of a hydrogel of the formulas in the preceding paragraph, 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.

[0232] 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 terminatingny-290431558Docket No.: 67057-20030.40 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).

[0233] In another embodiment, the support is aluminum hydroxide (alum), and Z is a phospho-serine alum-binding peptide, for example as described in Moyer et al (2020) Proc Natl Acad Sci USA 26: 430-40. Suspensions of alum particles are commonly used as vaccine adjuvants, and conjugation of antigens to alum nanoparticles via phosphor-serine peptides has been shown to enhance humoral immunity presumably through extending the local residence time after systemic administration and eventual accumulation of the nanoparticles in draining lymph nodes. It is known, however, that nanoparticles tend to accumulate in many different organs after systemic administration, for example in lung and spleen, and so may be associated with toxicities at non-target tissues. Further, the disclosed approach relies on physical desorption of the phosphoserine-peptide-antigen from the particles and so is not subject to control. In the present invention, control over antigen release is provided by the cleavable linker. In specific embodiments, Z comprises an oligomer of phospho-serine, W-[Ser(OPO3=)]q-C(O)NH- where q = 1-10, W = NH2 or AcNH, and the alum-binding peptide is connected to the cleavable linker via the carboxamide, giving a linker-drug conjugate of formula (XXXII).

[0234] Attachment of peptides and proteins to hydrogels through releasable linkers has been disclosed, for example in Schneider et al. (2022) PNAS, 119(30), e2201067119. doi.org / 10.1073 / pnas.2201067119; Hangasky et al. (2022) J Immunother Cancer, 10, e004104. doi:10.1136 / jitc-2021-004104; Schneider et al (2017) ACS Chemical Biology, 12, 2107-2116; Schneider et al. (2016) Bioconjugate Chemistry, 27, 2534-2539; Schneider et al. (2016)ny-290431559Docket No.: 67057-20030.40 Bioconjugate Chemistry, 27, 1638-1644; and in PCT Publications WO2017 / 161174A1; WO2020 / 219943A1; and WO2022 / 115563A1; each of which is incorporated herein by reference. In each of these cases, the conjugate was prepared by first attaching the releasable linker to the peptide or protein, and then conjugating the linker-peptide or linker-protein to the hydrogel. While advantageous for small peptides where the linker can be attached during chemical synthesis or for peptides comprising a single amine group, the presence of multiple linker attachment sites in larger and more complex peptides and proteins can be problematic due to the highly similar reactivity of the various amine groups. One method to provide site-specific attachment is to use a reductive amination reaction between a linker-aldehyde and the N-terminal amine of the peptide or protein; this method often provides modest yields of the linker-peptide or linker-protein, and separation of linker-protein from unreacted protein can be difficult. Further, the agent released from the conjugate typically comprises a remnant tag resulting from the reductive amination process and thus is not the native agent. For example, conjugates of a single- chain antibody fragment prepared by reductive amination released a protein having an N- terminal 3-aminopropyl remnant (Schneider et al. (2016) Bioconjugate Chemistry, 27, 2534- 2539). Such remnants may be avoided by using direct attachment to an amine group, for example by acylation with a linker, however most complex peptides and proteins comprise several equivalently reactive amines (typically epsilon-amines of lysine residues) and it is extremely difficult to obtain acylated proteins comprising a single attached linker in acceptable chemical yields. Controlling the stoichiometry of linker reagent to protein can minimize the attachment of multiple linkers to a protein, but at the expense of overall conversion and the subsequent difficulty of separating linker-protein from unreacted protein. The presence of multiple-linkers on a protein may complicate conjugation, since it is not certain that all linkers will be able to conjugate (for example, conjugation through a first linker may preclude the second linker from reaching a conjugation site on the hydrogel matrix); in such cases it is conceivable that the protein subsequently released from the conjugate may be modified by additional, unconjugated linkers. To obviate these issues, methods for the preparation of conjugates are provided wherein activated hydrogels comprising amine-reactive acylating groups are used to prepare conjugates upon reaction with amine-containing therapeutic agents or amine-containing prodrugs of therapeutic agents. The acylating groups on the activated hydrogels react with amines on the peptide or protein to form a releasable conjugate; while multiple linkers may under certain conditions be able to conjugate to the peptide or protein, since all linkers are necessarily attached to the hydrogel matrix, there is no possibility for release of peptide or protein modified by additional, unconjugated linkers; released peptide or protein will comprise no unnatural remnants from the conjugation process.ny-290431560Docket No.: 67057-20030.40 THERAPEUTIC DRUG

[0235] Therapeutic agent D may be a small molecule, prodrug of a small molecule, peptide, protein, antibody, antibody-drug conjugate, or nucleic acid. Hydrogel conjugates (V) release D by a beta-elimination reaction under appropriate conditions of pH and temperature, with the rate being primarily determined by the identity of the electron-withdrawing groups R1and R2and to a lesser degree by the spacing relative to connecting group Z* determined by integer n. The crosslinks in the hydrogel matrix are also cleaved through a similar beta-elimination reaction, with the rate being primarily determined by the identity of the electron-withdrawing groups R1aand R2aand to a lesser degree by the spacing relative to connecting group B* determined by integer q. The rates of these two processes are independently controlled by the selection of the appropriate groups, such that release of D may be separated from degradation of the hydrogel matrix.

[0236] Depending on the required use, therapeutic agent D may be an small-molecule antitumor agent, for example a topoisomerase inhibitor such as a camptothecin, SN38, and exatecan; a PARP inhibitor, such as talazoparib, rucaparib, nirabarib, and olaparib; an immunomodulator, such as rapamycin or a toll-like receptor agonist like resiquimod, DV-1001, and gardiquimod; a kinase inhibitor, such as adavosertib, axitinib, cobimetinib, crizotinib, dasatinib, erdafitinib, lapatinib, pazopanib, and pegaptanib; an anti-microtubule agent, such as eribulin, docetaxel, paclitaxel, and vinblastine; alkylating agent such as cisplatin or carboplatin; cytotoxin such as doxorubicin, daunorubicin; or antimetabolite such as methotrexate. Prodrugs of small-molecule agents may also be used, for example irinotecan and other prodrugs of SN38 as described in Fontaine et al. 2020 Cancer Chemother Pharmacol 85:225-9; and prodrugs of PARP inhibitors such as described in Fontaine et al. 2021 Cancer Res 81: 1076-86.

[0237] D may also be a peptide, for example an anti-cancer peptide inhibiting tumor cell proliferation or migration or suppress formation of blood vessels (anti-angiogenic peptides) such as those described in Xie et al. 2020 Open Biol 10(7): 200004; or a peptide-drug conjugate such as described in Chavda et al 2022 Molecules 27(21): 7232.

[0238] D may also be a protein, for example a cytokine such as an interleukin like IL-12 or IL-15; an antibody, such as an anti-CD40 like mitazalimab, an anti-HER2 like trastuzumab, an anti-PD1 like nivolumab or pembrolizumab, an anti-VEGF like ramucirumab or bevacizumab, an anti-CTLA4 like ipilimumab or tremelimumab, an anti-PD-L1 like atezolizumab, an antipCD274 like durvalumab, and an anti-CD20 like rituximab; a bispecific antibody such asny-290431561Docket No.: 67057-20030.40 mosunetuzumab (CD20xCD3E), faricimab (VEGFAxANGPT2), and amivantamab (EGFRxMET); a T-cell engager like blinatumomab which binds both antigen on the target cell and to an antigen on the surface of T-cells and so brings T-cells together with target cancer cells, or the related BiKEs (bispecific killer-cell engagers) and TriKEs (trispecific killer-cell engagers); or an antibody-drug conjugate, wherein an anti-cancer agent is covalently attached to an antibody that targets antigen on the surface of the cancer cell, such as trastuzumab deruxtecan, sacituzumab govitecan, ado-trastuzumab emtansine, trastuzumab deruxtecan, enfortumab- vedotin, tisotumab vedotin, and mirvetuximab soravtansine.

[0239] D may be a proteolysis-targeting chimera (PROTAC) comprising a ligand for a targeted protein and a ligand for an E3 ubiquitin ligase.

[0240] D may also be a nucleic acid, for example a CpG toll-like receptor agonist such as SD-101; an anti-sense oligonucleotide; a small interfering (siRNA); or a microRNA (miRNA). LOCOREGIONAL INJECTION

[0241] Therapeutic methods are provided wherein a releasable conjugate is administered by locoregional injection. In various embodiments of the invention, these methods involve intratumoral, peritumoral, intravitreal, intra-articular, intralymphatic, or peri-lymphatic administration. In various embodiments of the invention, the releasable conjugate is between an insoluble carrier and a small molecule, prodrug of a small molecule, peptide, nucleic acid, protein, antibody, or ADCs. In specific embodiments of the invention, the insoluble carrier is a hydrogel as described herein. In other specific embodiments of the invention, the carrier is a suspension of aluminum hydroxide particles. Such methods provide extended exposure to high local concentrations of the drug with minimal systemic concentrations, thus ameliorating systemic toxicities while providing enhanced efficacy against the target tissue. The locoregional administration may involve either injection directly into the target tissue (intra-target administration), or it may be adjacent to the target site (peri-target administration).

[0242] In one aspect, therapeutic methods are provided wherein a releasable conjugate is administered by locoregional injection. In various embodiments of the invention, these methods involve intratumoral, peritumoral, intravitreal, intraarticular, intralymphatic, or perilymphatic administration. In various embodiments of the invention, the releasable conjugate is between an insoluble carrier and a small molecule, prodrug of a small molecule, peptide, nucleic acid, protein, antibody, bispecific or trispecific antibody, PROTAC, peptide-drug conjugate, or antibody-drug conjugate. In specific embodiments of the invention, the insoluble carrier is any-290431562Docket No.: 67057-20030.40 hydrogel. In some embodiments, the hydrogel is a hydrogel microsphere (MS) such as a compound of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI), described below. In other specific embodiments of the invention, the carrier is a suspension of aluminum hydroxide particles. Such methods provide extended exposure to high local concentrations of the drug with minimal systemic concentrations, thus ameliorating systemic toxicities while providing enhanced efficacy against the target tissue.

[0243] In a second aspect, therapeutic methods are provided wherein a releasable conjugate is administered by locoregional injection together with systemic administration of a second therapeutic agent. Such methods are particularly advantageous in cases where the therapeutic agent attached to the conjugate provided by locoregional injection and the second therapeutic agent provided by systemic administration show overlapping toxicities. In certain embodiments of the invention, a conjugate with a small molecule or prodrug of a small molecule is administered by locoregional injection while an antibody is administered systemically. In certain other embodiments of the invention, a conjugate with a small molecule or prodrug of a small molecule is administered by locoregional injection while a second small molecule or small molecule prodrug is administered systemically. In certain other embodiments of the invention, a conjugate with a peptide, protein, antibody, or ADC is administered by locoregional injection while a small molecule is administered systemically. In certain other embodiments of the invention, a conjugate with a peptide, protein, antibody, or ADC is administered by locoregional injection while a second peptide, protein, antibody, or ADC is administered systemically. In certain other embodiments of the invention, a conjugate with a therapeutic agent is administered by locoregional injection while a second dose of the same therapeutic agent is administered systemically. In various embodiments of the invention, the administered conjugate is based on a hydrogel.

[0244] In a third aspect, methods for the preparation of conjugates are provided. In one embodiment, amine-reactive activated hydrogels are used to prepare conjugates upon reaction with amine-containing therapeutic agents or amine-containing prodrugs of therapeutic agents. Present methods wherein a complex therapeutic agent having multiple attachment sites is first reacted with a releasable linker and the resulting linker-agent is conjugated to a carrier produce complex products; conjugates produced by such a method may release linker-modified agents upon cleavage of a single linker if all linkers are not conjugated to the carrier. The present invention provides methods for producing hydrogel conjugates of complex therapeutic agents that can only release the free agent with no secondary linkers. In various specific embodiments of the invention, such complex therapeutic agents are peptides, proteins, antibodies, or ADCs. Inny-290431563Docket No.: 67057-20030.40 another embodiment of the invention, methods for preparation of releasable conjugates based on aluminum hydroxide particles as carrier are provided. Intratumoral Administration

[0245] Although drugs administered intratumorally (IT) ultimately enter the systemic circulation, they initially transit through the tumor at rates governed by tumor transport and drug properties (A. Huang et al., J Control Release 326, 203-221 (2020)). An underappreciated problem with IT injections is the rapidity with which drugs leak out of a tumor, leading to reduced exposure and a need for impractical frequent injections (N. Momin et al., Nat Commun 13, 109 (2022); W. X. Hong et al., Clin Cancer Res 26, 3091-3099 (2020)). For example, IT residence of a protein therapeutic is a function of size (N. Momin et al., Sci Transl Med 11 (2019)), but even large mAbs have an IT t1 / 2 of only ~6 hours (D. M. Francis et al., Sci Transl Med 12 (2020); H. P. Chang, et al., Pharmaceutics 15 (2023)), which is likely insufficient to achieve high exposures for maximal efficacy.

[0246] Half-life extension of IT therapeutics greatly increases their anti-tumor efficacy and favorable effects on immune responses (D. M. Francis et al., Sci Transl Med 12 (2020); N. Momin et al., Sci Transl Med 11 (2019); N. Momin et al., Maximizing response to intratumoral immunotherapy in mice by tuning local retention. Nat Commun 13, 109 (2022); F. Wang et al., Supramolecular Filament Hydrogel as a Universal Immunomodulator Carrier for Immunotherapy Combinations. ACS Nano 17, 10651-10664 (2023)). For example, Wang et al. (ACS Nano 17, 10651-10664 (2023)) showed much superior efficacy of IT IL-15, PD-1 and combinations of the two when they were administered as a long-acting hydrogel with a ~7 day t1 / 2. Likewise, Momin et al. (Nat Commun 13, 109 (2022)) showed that extending the IT t1 / 2 of locally-injected IL-2 by increasing molecular size and improving matrix-affinity increases therapeutic efficacy in mice. Of the existing methods for achieving IT t1 / 2 extension, the most efficacious ones involve some form of “anchoring” to the site of injection, usually through binding of the therapeutic to ECM components (N. Momin et al., Sci Transl Med 11 (2019); N. Momin et al., Nat Commun 13, 109 (2022); K. D. Wittrup, et al., Expert Opin Drug Deliv 19, 725-732 (2022)).

[0247] A technology for long-acting SC injections has been previously reported (E. L. Schneider et al., ACS Chem Biol 12, 2107-2116 (2017); J. A. Hangasky et al., J Immunother Cancer 10 (2022)). In accordance with the technology, a drug is covalently tethered to long-lived hydrogel MSs by a cleavable linker. After SC injection, the particulate MSs deposit locally and the linker slowly cleaves to release the native drug, which then enters the systemic circulation. Without wishing to be bound by theory, it is understood that the t1 / 2of the drug should be similarny-290431564Docket No.: 67057-20030.40 in different tissues – SC, peritumoral and IT – unless there is a direct effect on the rate- determining linker cleavage. The determinants of in vivo linker cleavage rates are the modulator used, the pH and the temperature (D. V. Santi, et al., Proc Natl Acad Sci U S A 109, 6211-6216 (2012). As such, without wishing to be bound by theory, peritumoral and IT cleavage rates should only differ from SC rates because of lower pH. Although the lowered tumor pH may reduce the rate of cleavage, as demonstrated herein, the slower rate may be beneficial because it provides longer drug exposure. As shown herein in the examples, the particulate conjugates and hydrogels of the disclosure deposited IT using slowly release the attached drug to “bathe” the tumor for long periods before it diffuses to the systemic circulation.

[0248] The approach described herein has several advantages over other technologies for IT half-life extension. A major advantage, for example, is the ability to tune the rate of drug release by modifying the linker. Thus, tunable IT pharmacokinetics can be used to optimize exposure of the targeted tumor. Further, unlike other anchoring technologies, MS carriers (e.g., hydrogels of the disclosure) could be used with a wide variety of drugs, such as peptide, proteins or small molecules, and provide similar pharmacokinetic behavior.

[0249] Moreover, because the MS particles (e.g., hydrogels of the disclosure) are physically deposited in or around the tumor before drug release, they have advantages over approaches for IT delivery that utilize drug solutions. First, unlike most anchoring approaches, the MS technique described herein does not require a tumor-localization domain or an ECM-binding target protein. Second, MS particles avoid the problem of drug “spillover” that occurs with volumes of soluble drugs over the 25- to 50% “hold up” volume of solid tumors (N. Momin et al., Nat Commun 13, 109 (2022); A. Som, et al., Adv Drug Deliv Rev 189, 114505 (2022)). Third, MS particles are not affected by biophysical barriers within the tumor (H. T. Nia, et al., Science 370 (2020)) that can result in drug leakage with consequent dose variability and systemic toxicity.

[0250] The examples described herein demonstrate the many benefits of utilizing MS particles in IT delivery.

[0251] Approaches were developed for direct determination of IT pharmacokinetics in tissue that, in principle, could be adapted for human biopsy specimens. After showing that MSs and free drug in tissue biopsies could be cleanly separated, methods were developed that allow quantitation of the free drug, as well as the drug remaining on recovered MSs. Here, MSs were labeled with fluorescein by a stable linker to serve as a quantitative marker for particles; MSs were also prepared in which rhodamine was attached by a releasable linker to serve as any-290431565Docket No.: 67057-20030.40 surrogate for a releasable drug. When combined, the rhodamine / fluorescein ratio on the MSs measure the amount of rhodamine remaining on the MSs, and normalizes the measurement independent of the efficiency of particle recovery. With this approach, it was confirmed that the IT release rate of RP from MS~RPR conjugates with different linkers correlates to their in vitro release rates. Using the same fluorescein quantitative marker for particles, the ratio-metric method can be adapted to determine the IT pharmacokinetics of MSs attached to non-fluorescent drugs.

[0252] The local pharmacokinetics and anti-tumor effects of IT MS~SN-38 conjugates were then determined. SN-38 is a potent inhibitor of topoisomerase 1 (TOP1) that causes single- and double stranded DNA breaks which initiate the DNA damage response (DDR) (Y. Pommier, Chem Rev 109, 2894-2902 (2009).; A. Thomas, Y. Pommier, Clin Cancer Res 25, 6581-6589 (2019)). TOP1i also have immune modulatory effects including activation of the STING pathway (J. Marinello et al., Br J Cancer 127, 1214-1225 (2022)), sensitization of tumors to checkpoint inhibitors (J. A. McKenzie et al., J Natl Cancer Inst 110, 777-786 (2018)), and immunogenic cell death (X. Liu et al., Adv Sci (Weinh) 8, 2002147 (2021); Z. Wang et al., J Control Release 349, 929-939 (2022)). In BRCA- and ATM-deficient tumors, the DDR is lacking important repair enzymes, which makes the tumors particularly sensitive to DNA damage – as by a TOP1i – as well as inhibitors of DDR enzymes – such as PARPi (A. Thomas, Y. Pommier, Clin Cancer Res 25, 6581-6589 (2019); S. P. Chowdhuri, B. B. Das, NAR Cancer 3, zcab003 (2021)). The effects of MS~SN-38 on BRCA and ATM deficient, TOP1i-sensitive 22Rv1 xenografts were investigated herein.

[0253] SN-38 was attached to MSs using a linker with a MeSO2 modulator to provide a MS~SN-38 conjugate with an in vitro release t1 / 2, pH 7.4 of 150 h. When MS~SN-38 containing 10 nmol SN-38 was administered SC, the normalized tissue C vs t plots showed that in vivo release of SN-38 from the MSs occurred with a t1 / 2 of ~6.5 d, which is close to the in vitro t1 / 2 of ~6 d. Further, the released free SN-38 reached a Cmaxof ~1 μM, and remained above 10 nM – a concentration sufficient for TOP1 inhibition – for ~3 Wk. After correcting for the slower t1 / 2 of linker cleavage in the acidic tumor vs neutral SC tissue, the IT t1 / 2 in CT26 tumors was estimated as ~10 d.

[0254] To determine antitumor effects of IT vs systemic delivery of SN-38, tumor growth inhibition and host survival of TOP1i-sensitive (A. Thomas et al., Mol Cancer Ther 21, 1722- 1728 (2022) 22Rv1ATM- xenografts were determined after treatment with various concentrations of IT and SC MS~SN-38. Both routes of administration ultimately deliver SN-38ny-290431566Docket No.: 67057-20030.40 systemically to the same extent, but after IT injection high concentrations of the released SN-38 transit through the tumor tissue before entering the systemic circulation. Without wishing to be bound by theory, it was hypothesized that if the efficacy of IT SN-38 was due to systemic exposure, the IT and SC dosing required for efficacy should be the same; however, if the efficacy of IT administration was due to a local effect, the IT injection would be more potent. However, IT administration of MS~SN-38 required a ten-fold lower dose than SC administration to achieve the same tumor growth inhibition and host survival. Without wishing to be bound by theory, these results demonstrate that the anti-tumor effect of IT MS~SN-38 is likely due to a local vs systemic effect, and IT administration of MS~SN-38 is ~10-fold more effective than systemic administration.

[0255] The ability to localize a drug in a tumor for prolonged periods is also demonstrated herein in the examples. This approach allows for the safe use of a combination of drugs that have overlapping toxicities, which is common in cancer chemotherapy. In this approach, the long- acting IT drug is injected into the tumor along with systemic treatment with the second drug. The tumor is exposed to the combined effects of both drugs, but all other tissues are exposed only to the single systemic drug. As such, the drugs would show additive or synergistic effects in tumor inhibition, but the tissues would only have toxic effects due to the single systemically administered drug.

[0256] TOP1i and PARPi are highly synergistic in growth inhibition of tumors but have overlapping myelosuppression that prevents safe use of the combination (M. A. Bjornsti, S. H. Kaufmann, F1000Res 8 (2019); A. Thomas, Y., Clin Cancer Res 25, 6581-6589 (2019)). The examples provided herein assess whether SN-38 localized in a tumor by IT MS~SN-38 would have synergistic anti-tumor effects in combination with systemic administration of a PARPi. When a single low dose of MS~SN-38 was administered IT to 22Rv1ATM- xenografts and oral TLZ was administered QD, high anti-tumor synergy of the combination was observed. Without wishing to be bound by theory, because the same amount of single-agent SN-38 administered systemically has little effect, it is understood that there is no overlapping systemic toxicity.

[0257] There are many potential uses of this technology for safely administering combinations of immuno-oncology agents. For example, immune checkpoint inhibitors (ICI) such as ^CTLA4 and ^PD-1 and their combination have shown remarkable activity against multiple tumor types, and can lead to durable remissions (A. Rotte, J Exp Clin Cancer Res 38, 255 (2019)). However, the success of ICI has been limited by inflammatory immune-related adverse events (irAEs). Combination of both ^CTLA4 and ^PD-1 – which has been approvedny-290431567Docket No.: 67057-20030.40 for several cancers – is more effective than either single agent, but it is also considerably more toxic (S. J. Wang, S. K. Dougan, M. Dougan, Immune mechanisms of toxicity from checkpoint inhibitors. Trends Cancer 9, 543-553 (2023)). A possible approach to treat a tumor with both agents and avoid irAE-associated toxicities would be to treat the tumor with a long-acting IT ^CTLA4 along with systemic ^PD-1. The tumor would be exposed to the synergistic effects of both drugs but the irAEs in normal organs would be limited to those of single agent systemic ^PD-1.

[0258] Also demonstrated herein are investigations with long-lasting IL-15 agonists for intratumoral and / or peritumoral delivery and enhanced antimetastatic activity. Interleukin-15 (IL-15) has emerged as a promising immunotherapeutic agent for the treatment of cancer, and other immune-related disorders (T.O. Robinson, K. D. Schluns, Immunol Lett 190, 159-168 (2017); T. A. Waldmann et al., J Exp Med 217 (2020); Y. Yang, A. Lundqvist, Cancers (Basel) 12 (2020)). IL-15 plays an important role in facilitating the survival, differentiation, as well as activation and expansion of NK cells, cytotoxic CD8+T cells, and γδ T cells without expanding immunosuppressive Tregs or mediating activation-induced cell death (C. C. Ku et al., Science 288, 675-678 (2000); T. A. Waldmann et al., Front Immunol 11, 868 (2020)).

[0259] The biologically active form of IL-15 is the non-covalent heterodimeric IL-15 / IL- 15Rα complex (C. Bergamaschi et al., Blood 120, e1-8 (2012)). Numerous IL-15 superagonists, consisting of the soluble IL-15Rα domain transiently or covalently fused to IL-15, have been reported to have superior pharmacokinetic and biological activity than IL-15 (M. P. Rubinstein et al., Proc Natl Acad Sci U S A 103, 9166-9171 (2006); T. A. Stoklasek et al., J Immunol 177, 6072-6080 (2006); E. Chertova et al. J Biol Chem 288, 18093-18103 (2013); E. Mortier et al., J Biol Chem 281, 1612-1619 (2006); H. Perdreau et al., Eur Cytokine Netw 21, 297-307 (2010)). Several IL-15 superagonists have shown promising results in preclinical and early-phase clinical trials, and one, N-803 (Anktiva), has recently been approved for treatment of BCG-unresponsive non muscle invasive bladder cancer. However, a common problem among all IL-15 agonists are their short half-lives leading to frequent and / or inconvenient dosing schedules. For example, receptor-linked IL-15 (RLI, SOT101) (Z. Antosova et al., Front Immunol 13, 989895 (2022); A. Bessard et al., Mol Cancer Ther 8, 2736-2745 (2009)) and the heterodimeric IL-15 / IL-15Rα complex, hetIL-15 (NIZ985) (E. Chertova et al., J Biol Chem 288, 18093-18103 (2013)), and N- 803 (Romee R., et al., Blood. (2018) 121:2515-27; Liu B, et al., Cytokine. (2018) 107:105-12) have apparent half-lives of 8 h or less in mice, ~12 h in nonhuman primates (NHP), and up to 30 h in humans (J. A. Hangasky et al., Front Immunol 11, 1813 (2020)). Therefore, these IL-15 agonists require frequent and inconvenient dosing schedules.ny-290431568Docket No.: 67057-20030.40

[0260] As described herein, a general approach to half-live extension through the covalent attachment of drugs through a releasable linker to non-circulating ~50 µm diameter tetra-PEG hydrogel microspheres (MS) has been developed. Drug release occurs via a β-elimination reaction, and the release rate is controlled by a rate modulating (Mod) electron-withdrawing group. MS conjugates can be injected subcutaneously or intratumorally through a 29G needle to serve as a localized drug depot that can sustain drug release for weeks to months (E. L. Schneider et al., Bioconjugate chemistry 27, 2534-2539 (2016); E. L. Schneider et al., Bioconjugate chemistry 27, 1638-1644 (2016)). In addition, β-eliminative linkers are engineered into the polymers of the MS hydrogels to enable gel dissolution subsequent to drug release (J. Henise et al., International Journal of Polymer Science 2019, Article ID 9483127 (2019)).

[0261] A MS hydrogel conjugate of IL-15 was previously reported with the longest reported half-life of any IL-15 agonist in mice (J. A. Hangasky et al., J Immunother Cancer 10 (2022)). The MS depot provided continuous release of IL-15 with a half-life of ~5 days, and improved PK, pharmacodynamics (PD) and efficacy. As described below, an analogous MS conjugate of the IL-15 superagonist – RLI (E. Mortier et al., J Biol Chem 281, 1612-1619 (2006))– could also extend the half-life and improve pharmacodynamics over the free drug. As shown in the examples, the sustained release facilitated by the conjugation to the MSs is an attractive approach for loco-regional delivery of IL-15 agonists. The prolonged IT release of an MS~IL-15 agonist in combination with a second agent, administered systemically or locally, should improve the antitumor and anti-metastatic effects, minimizing systemic toxic effects of the IL-15 agonist.

[0262] As described above, a long-acting delivery system was originally investigated for IL- 15 that was composed of hydrogel microspheres tethered to IL-15 by a releasable linker, designated as MS~IL-15 (J. A. Hangasky et al., J Immunother Cancer 10 (2022)). The aminopropyl-appended IL-15 released from SC MS~IL-15, IL-15AP, showed a long initial t1 / 2 of ~168 h and extraordinary effects on expansion of NK and CD44hiCD8+cells. The increased half- life and efficacy provided by MS~IL-15 prompted analogous studies of MS conjugates containing IL-15 super-agonists.

[0263] As described in the examples herein, the MS~RLI conjugate was prepared by the two-step procedure used for MS~IL-15. First, an azido-linker-aldehyde was attached to the N- terminus of RLI via reductive alkylation. Then, the azido-linker-RLI was attached to cyclooctyne-activated MSs by SPAAC. The purity of RLI on the MSs was >90% and MS~RLIny-290431569Docket No.: 67057-20030.40 released RLIAP with a half-life of ~700 h that maintained its functionality to bind IL-2 / IL- 15Rβγc.

[0264] After SC administration of MS~RLI in immunocompetent mice, the released RLIAP showed a half-life of 30 h, some 6-fold lower than its in vitro half-life or the in vivo half-life of IL-15APreleased from MS~IL-15. Unexpectedly, SC administration of MS~RLI in immunodeficient mice resulted in an in vivo half-life of released RLIAP of ~700 h, in agreement with the half-life for in vitro release. The high release rate of MS~RLI in immune-competent mice could be closely simulated by a TMDD model, similar to that reported for the IL-15 agonist XmAb24306 (D. Lu et al., Eur J Pharm Sci 186, 106450 (2023)), where the amount of target immune cells increases exponentially over time due to RLI-stimulated NK and T cell expansion – the nebulous “cytokine sink.”

[0265] When administered SC, MS~RLI was ~10- and 20-fold more effective at increasing CD44hiCD8+T cells and NK cells, respectively, than MS~IL-15 – in accord with the higher potency of RLI. However, after 1- to 2-weeks, most mice treated with ≥20 μg SC MS~RLI unexpectedly showed ulcerating lesions at injection sites – histologically confirmed as severe transmural coagulative necrosis – reminiscent of the injection-site necrosis reported with a slow- release IL-2 in dextran microspheres (J. W. Koten et al., Cytokine 24, 57-66 (2003)). Without wishing to be bound by theory, because the skin lesions were not seen in immunodeficient NSG mice, it was surmised that they were due to local immune-activation caused by the continuous high local exposure to RLIAPslowly released from the depot. Interestingly, skin reactions did not occur with free RLI injections, or with 40-fold higher doses of MS~IL-15 (J. A. Hangasky et al., J Immunother Cancer 10 (2022)). Regardless of the mechanism, the toxicity of MS~RLI occurred at a precarious 2-fold higher concentration than its most effective safe dose.

[0266] In view of the skin necrosis and low therapeutic index for SC MS~RLI, investigations were then focused on studying the utility of MS~RLI for long-acting IT therapy, where some degree of necrosis might be beneficial. A significant advantage of IT administration is that low doses can achieve high local concentrations in the setting of low systemic exposure. However, the rapid escape of most locally injected therapies requires the use of multiple sequential doses for efficacy, and undermines potential advantages of IT therapy; for example, the IT t1 / 2 of a protein of ~13 kDa such as IL-15 is only ~1 h (N. Momin et al., Sci Transl Med 11 (2019); F. Wang et al., ACS Nano 17, 10651-10664 (2023)).

[0267] It has been shown that multiple IT injections of IL-15 in MC38 colon carcinoma tumors suppress tumor growth during the period of IL-15 administration, after which the tumorsny-290431570Docket No.: 67057-20030.40 rapidly grew (H. Fukushima et al., Mol Cancer Ther 22, 1215-1227 (2023)). It has also been shown that multiple peri-tumoral injections of hetIL-15 in EO771 TNBC tumors gave significant tumor regression, increased host survival and prevention or elimination of metastasis (D. Stellas et al., Cell Rep 42, 112501 (2023)). It was hypothesized that instead of multiple IT injections of IL-15 agonists over time, a single injection of long-acting MS~RLI or MS~IL-15 conjugates would provide prolonged exposure to the released cytokine to give enhanced pharmacologic and immunologic responses.

[0268] The effect of single-agent IL-15 agonists is not uniform in their growth inhibition of preclinical model tumors – varying from little or none, to very potent (T. Miyazaki et al., J Immunother Cancer 9 (2021); M. Desbois et al., J Immunol 197, 168-178 (2016); M. Zhang et al., Proc Natl Acad Sci U S A 115, E10915-E10924 (2018); P. R. Rhode et al., Cancer Immunol Res 4, 49-60 (2016); P. Yu et al., Proc Natl Acad Sci U S A 109, 6187-6192 (2012); C. Bergamaschi et al., J Immunother Cancer 8 (2020)). Likewise, single agent IL-15 agonists have not shown to be very successful in treating human tumors, and the high doses required to induce immune changes have resulted in dose-limiting toxicities with no clinical response (K. C. Conlon et al., J Clin Oncol 33, 74-82 (2015)). Given this background, the general consensus is that maximal efficacy of IL-15 will require combinations with tumor specific monoclonal antibodies such as CTLA-4 and PD-L1 (T. A. Waldmann et al., Front Immunol 11, 868 (2020)).

[0269] Nevertheless, an important potential utility of IT IL-15 agonists is as anti-metastatic agents. It has been established that systemically administered IL-15 agonists – IL-15 (P. Yu et al., Clin Cancer Res 16, 6019-6028 (2010); F. Tang et al., Cell Mol Immunol 5, 189-196 (2008)) hetIL-15 (V. Stravokefalou et al., Front Immunol 13, 1014802 (2022)), N-803 (P. S. Kim et al., Oncotarget 7, 16130-16145 (2016)), and RLI (A. Bessard et al., Mol Cancer Ther 8, 2736-2745 (2009); M. Desbois et al., J Immunother Cancer 8 (2020))– all have potent anti-metastatic effects. Further, it was reported that multiple IT injections of hetIL-15 showed impressive anti- metastatic activity and a long-lasting specific anti-tumor immunity (D. Stellas et al., Cell Rep 42, 112501 (2023)). It is demonstrated herein that a single IT injection of MS~RLI or IT MS~IL-15 results in moderate tumor growth inhibition but complete reduction of lung metastases. The antimetastatic effects of IL-15 agonists have been proposed to involve NK (46-48) and CD8 cells (L. Cheng et al., J Hepatol 61, 1297-1303 (2014)). Without wishing to be bound by theory, at low SC doses MS~RLI and MS~IL-15 do not affect immune cells, so the anti-metastatic effects of these IT IL-15 agonists likely originate in the tumor before the agonists dissipate systemically. As such, use of IT MS~RLI or MS~IL-15, along with other agents, can advantageously both treat the primary tumor and prevent remote metastatic lesions.ny-290431571Docket No.: 67057-20030.40

[0270] In some embodiments, the disclosure provides methods of treating cancer by intratumorally and / or peritumorally administering a hydrogel described above to a subject in need thereof. In some embodiments, the subject is a human subject. For instance, the disclosure provides methods of treating cancer by intratumorally and / or peritumorally administering a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI). In some embodiments the hydrogel is administered once weekly. In some embodiments, the hydrogel is administered every two weeks. In some embodiments, the hydrogel is administered every three weeks. In some embodiments, the hydrogel is administered once monthly. In some embodiments, the hydrogel is administered once every two months. In some embodiments, the hydrogel is administered once every three months. In some embodiments, the hydrogel is administered once every four months. In some embodiments, the hydrogel is administered once every six months. In some embodiments, the hydrogel is administered once yearly.

[0271] In some embodiments according to any one of the methods described above, the cancer is colon cancer, colorectal cancer, liver cancer, bone cancer, lung cancer, brain cancer, heart cancer, bladder cancer, kidney cancer, a hematological cancer (e.g., leukemia), skin cancer, breast cancer, pancreatic cancer, thyroid cancer, a head and / or neck cancer, an eye-related cancer, prostate cancer, testicular cancer, cecum cancer, ovarian cancer, uterine cancer, or cervical cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is advanced-stage cancer. In some embodiments, the cancer is colorectal cancer. In another embodiment, cancer includes but is not limited to melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), or urothelial carcinoma. Combination Therapies

[0272] In another aspect of the invention, therapeutic methods are provided wherein a releasable conjugate is administered by locoregional injection together with systemic administration or subcutaneous administration of a second therapeutic agent. Such methods may be particularly advantageous in cases where the therapeutic agent attached to the conjugate provided by locoregional injection and the second therapeutic agent provided by systemic administration or subcutaneous administration show overlapping toxicities. In certain embodiments of the invention, a conjugate with a small molecule or prodrug of a small moleculeny-290431572Docket No.: 67057-20030.40 is administered by locoregional injection while an antibody is administered systemically or systemically. In certain other embodiments of the invention, a conjugate with a small molecule or prodrug of a small molecule is administered by locoregional injection while a second small molecule or small molecule prodrug is administered systemically. In certain other embodiments of the invention, a conjugate with a peptide, protein, antibody, or ADC is administered by locoregional injection while a small molecule is administered systemically. In certain other embodiments of the invention, a conjugate with a peptide, protein, antibody, or ADC is administered by locoregional injection while a second peptide, protein, antibody, or ADC is administered systemically. In certain other embodiments of the invention, a conjugate with a therapeutic agent is administered by locoregional injection while a second dose of the same therapeutic agent is administered systemically. In various embodiments of the invention, the administered conjugate is based on a hydrogel. In any of the foregoing embodiments, systemic administration may be through an oral, intravenous, rectal or transdermal route.

[0273] In some embodiments, the hydrogel administered intratumorally and / or peritumorally is administered on the same dosing schedule as the drug that is administered systemically or subcutaneously. In some embodiments, the hydrogel administered intratumorally and / or peritumorally is administered on a different dosing schedule as the drug that is administered systemically or subcutaneously. For instance, in one embodiment the hydrogel is administered once weekly and the systemic drug is administered once daily.

[0274] In some embodiments, the disclosure provides methods of treating cancer by intratumorally and / or peritumorally administering a hydrogel described above to a subject in need thereof in combination with a second drug administered systemically. In some embodiments, the subject is a human subject. For instance, the disclosure provides methods of treating cancer by intratumorally and / or peritumorally administering a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI) in combination with a second drug administered systemically.

[0275] IT therapy has assumed an important role in delivery of immuno- and chemotherapeutics (A. Marabelle et al. Ann Oncol 29, 2163-2174 (2018); I. Melero, et al., Nat Rev Clin Oncol 18, 558-576 (2021); S. Champiat et al., Clin Cancer Res 27, 665-679 (2021)). Between January 2018 and June 2021, there were 153 IT clinical trials initiated (Humeau, J. et al., Oncoimmunology 10, 1984677 (2021)) and a single institution performed over 1,500 image- guided IT immunotherapy injections over a few years (N. M. Munoz et al., J Immunother Cancer 9 (2021)). As described herein, a significant advantage of IT injection is that low dosesny-290431573Docket No.: 67057-20030.40 can achieve extraordinarily high IT drug concentrations in the setting of very low systemic exposure, resulting in high efficacy and low toxicity.

[0276] Indeed, a potential advantage of localized administration is that, due to only the local concentrations of therapeutic agent being high, combination therapy between two agents having overlapping or synergistic toxicities may be facilitated. For two agents both given intratumorally, this has been disclosed for example with a combination depot of doxorubicin and 5-fluorouracil encapsulated in a microcapsule or hydrogel (Kim et al., 2016). For one agent given intratumorally and a second agent given systemically, however, the tumor may be the only tissue in which efficacious / toxic concentrations of both agents occurs.

[0277] Although drugs administered IT ultimately enter the systemic circulation, they initially transit through the tumor at rates governed by tumor transport and drug properties (A. Huang et al., J Control Release 326, 203-221 (2020)). An underappreciated problem with IT injections is the rapidity with which drugs leak out of a tumor, leading to reduced exposure and a need for impractical frequent injections (N. Momin et al., Nat Commun 13, 109 (2022); W. X. Hong et al., Clin Cancer Res 26, 3091-3099 (2020)). For example, IT residence of a protein therapeutic is a function of size (N. Momin et al., Sci Transl Med 11 (2019)), but even large mAbs have an IT t1 / 2 of only ~6 hours (D. M. Francis et al., Sci Transl Med 12 (2020); H. P. Chang, et al., Pharmaceutics 15 (2023)), which is likely insufficient to achieve high exposures for maximal efficacy.

[0278] Half-life extension of IT therapeutics greatly increases their anti-tumor efficacy and favorable effects on immune responses (D. M. Francis et al., Sci Transl Med 12 (2020); N. Momin et al., Sci Transl Med 11 (2019); N. Momin et al., Maximizing response to intratumoral immunotherapy in mice by tuning local retention. Nat Commun 13, 109 (2022); F. Wang et al., Supramolecular Filament Hydrogel as a Universal Immunomodulator Carrier for Immunotherapy Combinations. ACS Nano 17, 10651-10664 (2023)). For example, Wang et al. (ACS Nano 17, 10651-10664 (2023)) showed much superior efficacy of IT IL-15, PD-1 and combinations of the two when they were administered as a long-acting hydrogel with a ~7 day t1 / 2. Likewise, Momin et al. (Nat Commun 13, 109 (2022)) showed that extending the IT t1 / 2 of locally-injected IL-2 by increasing molecular size and improving matrix-affinity increases therapeutic efficacy in mice. Of the existing methods for achieving IT t1 / 2extension, the most efficacious ones involve some form of “anchoring” to the site of injection, usually through binding of the therapeutic to ECM components (N. Momin et al., Sci Transl Med 11 (2019); N. Momin et al., Nat Commun 13, 109 (2022); K. D. Wittrup, et al., Expert Opin Drug Deliv 19, 725-732 (2022)).ny-290431574Docket No.: 67057-20030.40

[0279] For example, intratumoral and / or peritumoral injection of a conjugated topoisomerase inhibitor such as SN38 may be combined with systemic administration of a PARP inhibitor. This combination suffers from severe bone marrow toxicity when both agents are administered systemically. When one agent is provided by locoregional administration, the target tissue is the only location where both agents are present in toxic levels at the same time. Therefore, in one embodiment, the disclosure provides methods of treating cancer by administering intratumorally and / or peritumorally a conjugate comprising SN38 (e.g., a conjugate of formula (III), wherein D is SN38), in combination with a PARP inhibitor administered systemically. In some such embodiments, the conjugate of formula (III) is a hydrogel of the disclosure (e.g., a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI)). In some embodiments, the PARP inhibitor is selected from the group consisting of talazoparib, olaparib, niraparib, rucaparib, and iniparib.

[0280] Similarly, coadministration of antibodies can lead to undesired toxicities. For example, combinations of ipilimumab and nivolumab show severe toxicity even though they are efficacious in treating metastatic melanoma (Lindardou et al. 2016 Annals Transl Med 4(14):272). The combination of anti-CTLA4 antibody and anti-PD1 antibody has shown intestinal inflammation in mouse models (Bauche et al 2020, J. Immunotherapy Cancer 8e:001584). Locoregional administration (e.g., intratumoral or peritumoral administration) of one antibody could be combined with systemic administration of a second antibody as a means to avoid toxicity in non-target locations. For instance, in one embodiment, the disclosure provides methods of treating cancer by administering intratumorally and / or peritumorally a conjugate comprising an anti-CTLA4 antibody (e.g., a conjugate of formula (III), wherein D is an anti-CTLA4 antibody), in combination with an anti-PD1 antibody. In other embodiments, the disclosure provides methods of treating cancer by administering intratumorally and / or peritumorally a conjugate comprising an anti-PD1 antibody (e.g., a conjugate of formula (III), wherein D is an anti-PD1 antibody), in combination with an anti-CTLA4 antibody. In some of the foregoing embodiments, the conjugate of formula (III) is a hydrogel of the disclosure (e.g., a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI)). In some of the foregoing embodiments, the anti-PD1 antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, pembrolizumab, nivolumab, cemiplimab, retifanlimab, tislelizumab, and toripalimab. In some of the foregoing embodiments, the anti-CTLA4 antibody is selected from the group consisting of ipilimumab, quavonlimab, and tremelimumab.ny-290431575Docket No.: 67057-20030.40

[0281] Furthermore, there are multiple circumstances where IT administration of MS~IL-15 agonists in combination with systemic administration of other agents could provide dual benefits of anti-tumor and anti-metastatic effects without the on-target off-tumor toxicities associated with systemic IL-15. First, therapies that require frequent IT doses of IL-15 would be simplified to a single dose. For example, the optimal efficacy of near-infrared photoimmunotherapy (NIR- PIT) requires at least four injections of IT IL-15 over 6 days to achieve the needed exposure (H. Fukushima et al., Mol Cancer Ther 22, 1215-1227 (2023)). Second, local MS~IL-15 agonists could be a safe alternative for potentially toxic systemic IL-15 when used in combination with synergistic agents such as checkpoint inhibitors (M. Desbois et al., J Immunol 197, 168-178 (2016)); indeed, the higher IT concentrations compared to systemic administration could also confer higher efficacy. Last, IT MS~IL-15 agonists could enable safe use with a second agent that has overlapping toxicities. For example, the combination of IT MS~IL-15 with synergistic agents like IL-12 (J. K. Cini et al., Front Immunol 14, 1326927 (2023)), CAR T and NK cells (S. E. Franks et al., Cancers (Basel) 12 (2020)), S. Zhang et al., Int Immunopharmacol 91, 107318 (2021)), and T cell engagers (J. Li et al., Mol Cancer Ther 10.1158 / 1535-7163.MCT-23-0910 (2024)), could enhance antitumor activity without potential for severe, systemic overlapping toxicities such as cytokine release syndrome. To allow such combinations, MS~IL-15 agonists would be deposited into the tumor along with concurrent systemic treatment with the second drug; the tumor would be exposed to both agents whereas other tissues are exposed only to the systemically administered agent (J. Henise et al., Releasable hydrogel microsphere-drug conjugates as generic prodrugs for long-acting intra-tumoral therapy. bioRxiv (2024).

[0282] In some embodiments, the MS~IL-15 agonist is administered intratumorally and / or peritumorally and a checkpoint inhibitor is administered systemically. In some embodiments, the checkpoint inhibitor administered systemically is a PD-1 inhibitor or PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor administered systemically is an anti-PD1 antibody selected from the group consisting of atezolizumab, durvalumab, avelumab, pembrolizumab, nivolumab, cemiplimab, retifanlimab, tislelizumab, and toripalimab. In some embodiments, the checkpoint inhibitor administered systemically is a CTLA-4 inhibitor. In some embodiments, the CTLA-4 inhibitor administered systemically is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA4 antibody is selected from the group consisting of ipilimumab, quavonlimab, and tremelimumab.

[0283] In some embodiments, the MS~IL-15 agonist is administered intratumorally and / or peritumorally and another cytokine is administered systemically. In some embodiments, the cytokine is IL-12. In other embodiments, the cytokine is IFN-γ.ny-290431576Docket No.: 67057-20030.40

[0284] In some embodiments, the intratumorally and / or peritumorally administered MS~IL- 15 agonist is administered in combination with Near-Infrared Photoimmunotherapy (NIR-PIT). Combining intratumoral IL-15 with NIR-PIT can significantly enhance tumor control by inducing a strong immune response and promoting abscopal effects.

[0285] In some embodiments, the intratumorally and / or peritumorally administered MS~IL- 15 agonist is administered in combination with a CAR-T Cell Therapy.

[0286] In some embodiments, the intratumorally and / or peritumorally administered MS~IL- 15 agonist is administered in combination with an oncolytic virus. These viruses selectively infect and kill cancer cells, releasing tumor antigens and stimulating an immune response.

[0287] In some embodiments, the intratumorally and / or peritumorally administered MS~IL- 15 agonist is administered in combination an anti-VEGF agent. In some embodiments, the anti- VEGF agent is bevacizumab. EMBODIMENTS

[0288] The invention is further described by the following embodiments.

[0289] Embodiment 1: A method of treatment, comprising: locoregionally injecting to a subject in need thereof an effective dose of a conjugate of formula (XXXIII) comprising a therapeutic agent releasably connected to a degradable hydrogelwherein P1and P2are each independently x-armed polymers of 1-40 kDa average molecular weight; x= 2-8; B* and C*are each independently connecting groups; n and q are each independently 0-6; R1, R1a, R2, and R2aare each independently H or an electron-withdrawing group, with the proviso that at least one of R1and R2and at least one of R1aand R2aare electron- withdrawing groups; each R4and R4aare independently H, C1-C3 alkyl, or both R4taken together or both R4ataken together independently for a 3-6 member ring; w and y are each independentlyny-290431577Docket No.: 67057-20030.40 0-4; and D is a therapeutic agent or prodrug of a therapeutic agent conjugated through an amine group.

[0290] Embodiment 2: The method of embodiment 1, wherein D is a small molecule, peptide, peptide-drug conjugate, protein, protein-drug conjugate, antibody, bispecific or trispecific antibody, PROTAC, or antibody-drug conjugate.

[0291] Embodiment 3: The method of embodiment 1, wherein D is a toll-like receptor agonist, exatecan or a prodrug having the formula HN(R7)CH2Q wherein R7is H, optionally substituted (C1-C6) alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and Q is.

[0292] Embodiment 4: The method of embodiment 1, wherein D is an antibody or antibody- drug conjugate.

[0293] Embodiment 5: The method of embodiment 1, wherein the injection is intratumoral, peritumoral, intravitreal, intra-articular, intralymphatic, or perilymphatic.

[0294] Embodiment 6: The method of embodiment 1, wherein the method further comprises systemic administration of a second agent.

[0295] Embodiment 7: The method of embodiment 6, wherein the second agent is a small molecule, peptide, protein, antibody, or antibody-drug conjugate.

[0296] Embodiment 8: The method of embodiment 6, wherein the first agent comprises a conjugated topoisomerase I inhibitor and the second agent is a PARP inhibitor or an antibody.ny-290431578Docket No.: 67057-20030.40

[0297] Embodiment 9: The method of embodiment 6, wherein both agents are antibodies.

[0298] Embodiment 10: The method of embodiment 1, wherein the electron-withdrawing groups are selected from the group consisting of CN and SO2R3, wherein R3= optionally substituted C1-C6alkyl or cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or N(R5)2 wherein each R5is independently H or optionally substituted C1-C6 alkyl or cycloalkyl.

[0299] Embodiment 11: A method for producing a releasable hydrogel-drug conjugate of formula (XXXIV),comprising reacting an amine-containing drug or prodrug D with an activated hydrogel of formula (XXXV) under conditions where an amine of D displaces leaving group X to form a carbamate group, wherein D is a therapeutic agent or prodrug of a therapeutic agent conjugated through an amine group,wherein P1and P2are each independently r-armed polymers of 1-40 kDa average molecular weight; r = 2-8; B*, C*, and Z* are each independently connecting groups; n and q are eachny-290431579Docket No.: 67057-20030.40 independently 0-6; R1, R1a, R2, and R2aare each independently H or an electron-withdrawing group, with the proviso that at least one of R1and R2and at least one of R1aand R2aare electron- withdrawing groups; each R4and R4aare independently H, C1-C3alkyl, or both R4taken together or both R4ataken together independently for a 3-6 member ring; w and y are each independently 0-4.

[0300] Embodiment 12: The method of embodiment 11, wherein P1and P2are each independently r-armed polyethylene glycols of average molecular weight 1-40 kDa; r = 2-8; R1, R2, R1a, and R2aare each independently H, CN, or SO2R3, wherein R3= optionally substituted C1-C6alkyl or cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or N(R5)2wherein each R5is independently H or optionally substituted C1-C6 alkyl or cycloalkyl, and wherein at least one of R1and R2and at least one of R1aand R2aare not H; each R4and R4aare independently H or C1-C3alkyl; connecting groups B*, C*, and Z* each independently comprise a carboxamide or 1,2,3-triazole; and X is N-hydroxysuccinimidyl, 4-nitrophenyl, pentafluorophenyl, pentachlorophenyl, 2,6-dichloro-4-sulfophenyl, 2,3,5,6-tetrafluoro-4- sulfophenyl, 1-benzotriazolyl, or 1-imidazolyl.

[0301] Embodiment 13: The method of embodiment 11, wherein the hydrogel is in the form of a particulate suspension.

[0302] Embodiment 14: A releasable hydrogel conjugate of formula (XXXVI) wherein D is a prodrug having the formula H¬N(R7)CH2Qwherein : R7is H, optionally substituted (C1-C6) alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and Q isny-290431580Docket No.: 67057-20030.40

[0303] Embodiment 15: A releasable conjugate of formula (XXXVII)wherein W is NH2 or AcNH; q = 1-6; n = 0-6; R1and R2are each independently H or an electron-withdrawing group, with the proviso that at least one of R1and R2is an electron- withdrawing group; each R4is independently H, C1-C3alkyl, or both R4taken together independently form a 3-7 member ring; and D is a therapeutic agent or prodrug of a therapeutic agent conjugated through an amine group.

[0304] Embodiment 16: The releasable conjugate of embodiment 15, wherein D is an antigenic peptide.

[0305] Embodiment 17: A method of treatment, comprising: a) mixing a conjugate of embodiment 15 with a suspension of alum particles to form an alum / conjugate mixture; and b) intra-lymphatically or peri-lymphatically injecting to a subject in need thereof the alum / conjugate mixture. EXAMPLES

[0306] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary, and not by way of limitation.ny-290431581Docket No.: 67057-20030.40 Example 1 Preparation of an activated hydrogel of Formula (XXXV) wherein X is N-succinimidyloxy

[0307] Activated hydrogels of Formula (XXXV) wherein P1and P2are each independently 4-armed polymers of 20 kDa average molecular weight; r = 4; B* and Z* comprise a 1,2,3- triazole; C* is carboxamide; q =1; R1ais SO2NMe2; R2ais H; each R4ais Me; w = 4; y = 0; n = 4; R1is SO2Me; R2is H; each R4is H; and X is N-succinimidyloxy were prepared as follows. Bicyclononyne-hydrogel microspheres (BCN-microspheres) of formula (VIIIa) wherein Z comprises a bicyclononyne; w = 0; y = 4; C* = carboxamide; R1a= SO2NMe2’ R2a= H; each R41= Me; q = 1; B* = 1,2,3-triazole; P1and P2= 20 kDa 4-armed PEG; and r = 4 were prepared as previously described (Schneider et al., 2016 Bioconj. Chem. 27: 1210-5) by reaction of the corresponding hydrogel amino-microspheres wherein Z = NH2 with (1R,8S,9s)- bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN-OSu). The amino- microsphere slurry (22 g) stored in 100 mM NaOAc pH 4.0 was dispensed into a 100 mL glass bottle and placed in an autoclave for sterilization. The sterilized amino microspheres were transferred to a 50 mL conical tube and washed by suspension followed by centrifugation in 2x35 mL of sterile water, 2x35 mL of 50%v / v sterile water / acetonitrile, and finally 5x40mL of sterile acetonitrile (sterile filtered using a 0.2 µm PTFE PureFlo 50 mm Disc filter. The packed amino microsphere slurry in acetonitrile (13.2 g) containing 34.9 μmol amine was combined with triethylamine (4 eq., 141 μmol) and BCN-HSC (1.25 eq., 43.7 μmol). The reaction was mixed end-over-end for 2.5 hr and a qualitative color test using trinitrobenzenesulfonate confirmed loading of the amines. Acetic anhydride was added (1 eq, 34.9 μmol) to cap any remaining free amines and after a 0.5 hr reaction the slurry was washed 6 times with 40 mL of sterile acetonitrile to provide the BCN-microspheres.

[0308] A solution of the Z-linker-X of formula (I) wherein Z = N3; R1= SO2Me; R2= H; each R4= H; n = 4; and X = N-succinimidyloxy (1.1 eq, 0.525 mL, 42 µmol) in sterile acetonitrile was added to the BCN-microspheres. The reaction solution was mixed end over end at 37°C for 8 hr. The activated microspheres were washed twice with 40 mL of acetonitrile to remove excess reagent before a 4 hr incubation with 1-azidoheptaethylene glycol to cap any unreacted BCN groups. Finally, the microspheres were washed 5x35mL of sterile MeCN and stored at -20oC.

[0309] Other activated hydrogels of Formula (XXXIV) may be prepared similarly using the appropriate amino-hydrogel together with the appropriate linker of formula (I).ny-290431582Docket No.: 67057-20030.40 Example 2 Preparation of a hydrogel-antibody conjugate of formula (V) wherein D = human IgG

[0310] A suspension of the activated hydrogel of Formula (XXXV) from Example 1 was washed 10-fold, twice with phosphate buffered saline (PBS, 9.8 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.3). An aliquot (40 uL) was mixed with 250 µl of 16.9 mg / ml human IgG solution in PBS (28 nmol, 4.21 mg IgG) and incubated for 16 hours at ambient temperature with mixing. Then, unreacted IgG was removed by washing 3 times with 10 volumes of PBS.

[0311] A 10 µl sample of resulting MS~IgG slurry was mixed with 50 µl of 50 mM NaOH and incubated 30 minutes at ambient temperature. After incubation, 10 µl of 1 M Tris, pH 7.5 were added to the mixture. IgG concentration was obtained by UV spectrophotometry. PEG content was obtained by BaCl2 / I2 spectrophotometry. MS~IgG prepared slurry contained 2.89 mg / ml IgG and 14 mg / ml PEG, implying 206 µg IgG per mg of PEG. Example 3 Preparation of a hydrogel-antibody conjugate of formula (V) wherein D = (3- aminopropyl)-human IgG

[0312] Preparation of Azido-linker-IgG: In a total volume of 40 µL, reactions contained 1.9 nmol (288 µg, 48 µM, 1 equivalent) human IgG in 25 mM Citrate, pH 6.0, 500 mM NaCl and 0.05% Tween-20 (Buffer Cit), 1; 2; 4; 8 equivalents of a linker of formula (I) wherein R1= MeSO2; R2= H; each R4= H; n = 4; Z = N3; and X = NH(CH2)2CHO (48 µM to 384 µM) and 416 nmol (25 µg, 10 mM) of NaCNBH3. After 14 hours, 4 ºC, mixtures were analyzed by gel- shift assay as follows. Samples were treated with 10 equivalents of BCN-PEG40kDa for 4 hours at 37 ºC, and analyzed by non-reducing SDS-PAGE. Here, BCN-PEG40kDa reacts with the azide of linker-protein Lnk-IgG by SPAAC and slows migration of the pegylated protein on SDS-PAGE; multiple PEG addition slows migration proportionally such that the band for Lnk2-IgG moves more slowly than the band for Lnk1-IgG, etc. The bands were quantitated by densitometry after staining with Coomassie Blue. Analysis of the reaction mixture that used 1 molar equivalents of linker showed a mixture comprising 58.9% unreacted IgG + 29% Lnk1-IgG + 10.1% Lnk2-IgG + 2.0 % Lnk3-IgG. Analysis of the reaction mixture that used 2 molar equivalents of linker showed a mixture comprising 52.2% unreacted IgG + 29.3% Lnk1-IgG + 15.8% Lnk2-IgG + 2.7 % Lnk3- IgG. Analysis of the reaction mixture that used 4 molar equivalents of linker showed a mixture comprising 41.3% unreacted IgG + 27.8% Lnk1-IgG + 21.6% Lnk2-IgG + 7.9% Lnk3-IgG + 1.4% Lnk4-IgG. Analysis of the reaction mixture that used 8 molar equivalents of linker showedny-290431583Docket No.: 67057-20030.40 a mixture comprising 29.9% unreacted IgG + 25.2% Lnk1-IgG + 24.5% Lnk2-IgG + 15.6% Lnk3- IgG + 3.9% Lnk4-IgG + 0.9% Lnk5-IgG. Table 1. Distribution of linkers resulting from reductive alkylation of IgG

[0313] Preparation of Lnk1-IgG. In 1 mL of Buffer Cit, reaction mixture contained 67 nmol (10.05 mg, 67 µM) IgG, 67 nmol (42 µg, 67 µM, 1 Eq) of linker reagent of formula (I) wherein R1= PhSO2; R2= H; each R4= Me; n = 1; Z = N3; and X = NH(CH2)2CHO (“gdm1PhSO2linker”) and 10 µmol NaCNBH3 (628.4 µg, 10 mM). The reaction was allowed to proceed 14 hours at 4 ºC in the dark. Excess reagents were removed using a 3.5 mL PD-10 column (Cytiva) previously equilibrated in Buffer Cit with 10 mM L-Methionine. Protein concentration was determined by A280(ε280= 210,000 M–1cm-1) using a NanoDrop spectrophotometer. Gel-shift analysis determined this Lnkx-IgG to comprise 67% unreacted IgG + 25% Lnk1-IgG + 8% Lnk2- IgG. Preparations of Lnk1-IgG were similarly performed with linkers of formula (I) wherein R1= MeSO2; R2= H; each R4= H; n = 4; Z = N3; and X = NH(CH2)2CHO (“std MeSO2linker”); and wherein R1= MeSO2; R2= H; each R4= Me; n = 1; Z = N3; and X = NH(CH2)2CHO (“gdm1MeSO2 linker”).

[0314] MS~BCN preparation: 1.31 g of sterile hydrogel microspheres of formula (VIIIa) wherein P1and P2are each 20-kDaPEG; B* = 1,2,3-triazole; r = 1; R1a= SO2NMe2; R2a= H; each R4a= Me; w = 0; y = 4; C* = carboxamide; and Z = NH2; comprising 2.1 µmol NH2 was washed twice with 0.05 %w / v Tween-20 in H2O (5-fold dilution each wash). Then it was washed 10-fold in Acetonitrile for 6 times and treated with 1.5 equivalents (3.15 µmol) of BCN-OSu and 4 equivalents (8.4 µmol) of triethylamine. Reaction was allowed to progress for three hours at ambient temperature and mixing. After that, 1 equivalent (2.1 µmol) of acetic anhydride was added to the reaction mixture and allowed to react for 1 hour at ambient temperature. Excess reagents were washed 3-fold, 8 times in Acetonitrile; 3-fold, 3 times in 0.05 %w / v Tween-20 in water and 5-fold once in Buffer Cit with 10 mM L-Methionine. All the reagent solutions and buffers used in the preparation were previously sterilized by 0.2 µm pore filtration and the preparation was performed aseptically in a biosafety cabinet.ny-290431584Docket No.: 67057-20030.40

[0315] Lnk~IgG loading to MS~BCN: Lnk1-IgG (R1= PhSO2; “gdm1 PhSO2 linker”) (10.05 mg, 67 nmol IgG: 67% unreacted IgG + 25% Lnk1-IgG + 8% Lnk2-IgG) was sterilized by 0.2 µm pore filtration and mixed with MS~BCN slurry preparation (2.1 µmol BCN) and 500 µl Buffer Cit with 10 mM L-Methionine were added to sum 3.5 ml reaction volume. SPAAC reaction was monitored by drop of A280 in supernatant and allowed to progress for 67 hours. Excess reagents were washed 5-fold, 3 times with Buffer Cit with 10 mM L-Mthionine. Then, 2.33 µmol PEG7-N3 (1.1 equivalents from initial NH2 in slurry) were added and second SPAAC reaction was allowed to progress for 72 hours at 4 ºC. Excess reagents were washed 5-fold, 6 times with Buffer Cit with 10 mM L-Methionine. A 10 µl sample of resulting MS~IgG slurry was mixed with 50 µl of 40 mM NaOH and incubated 30 minutes at ambient temperature. After incubation, 10 µl of 1 M Tris, pH 7.5 were added to the mixture. IgG concentration was obtained by UV spectrophotometry. PEG content was obtained by BaCl2 / I2 spectrophotometry. MS~IgG prepared slurry contained 0.85 mg / ml IgG and 20.15 mg / ml PEG, implying 42 µg IgG per mg of PEG. Preparations of MS~IgG were similarly performed by conjugating Lnk1-IgG Mod prepared using the “std MeSO2 linker” and the “gdm1 MeSO2 linker.” Example 4 In vitro release kinetics of hydrogel-antibody conjugates

[0316] The in vitro release rate of the antibody from the conjugates from Examples 2 and 3 was determined under accelerated release conditions, incubating the MS~IgG slurry in 125 mM Sodium Borate, 0.05% (v / v) tween-20, pH 9.4 at 37°C in an Eppendorf tube. At time intervals, samples were removed from the 37 ºC water bath, centrifuged at 21,000 x g for 1 minute, and A280 of 4 µl of supernatant sample was measured with a NanoDrop spectrophotometer. After sampling each timepoint, incubation at 37 ºC continued. The release rate was calculated by fitting the time points A280 vs time to the first-order rate equation in Graphpad Prism. Knowing that the β-elimination is first-order in hydroxide ion, rates were calculated at pH 7.4 as kpH 7.4= kpHx 10(pH-7.4). Table 2. Release rates for IgG from hydrogel conjugates, extrapolated to pH 7.4, 37oCny-290431585Docket No.: 67057-20030.40Example 5AA Materials and Methods for Examples 5A-8

[0317] General Methods

[0318] All reagents were reagent grade. HPLC analyses were performed on a Shimadzu LC- 20AD HPLC system equipped with an SPD-M20A diode array detector, a RF-10AXL fluorescence detector, and a Phenomenex Jupiter 5 μm C18 column (300 A, 150 x 4.6 mm). Unless otherwise noted, peaks were eluted with a 10-minute linear gradient of 20-100% acetonitrile in water (0.1% TFA) at 1 mL / min. Fluorescence measurements were made using a Molecular Devices, Spectramax i3 plate reader, and the following excitation and emission wavelengths: FLS ex 485 nm em 535 nm, RP ex 565 nm, em 589 nm. The following extinction coefficients were used for quantification by absorbance: FL A495(e = 80,000 M-1cm-1for pH > 9), RP A565(e= 96,100 M-1cm-1), and SN-38 A363 (e = 27,500 M-1cm-1) or A414(e = 27,500 M-

[0319] Fluorescein labeled microspheres (FLs-MS-Ac)

[0320] Stable linked fluorescein labeled tetra-PEG hydrogel MSs with acylated amines (FLs- MS-Ac) containing the –CN α-Lys crosslink cleavage rate modulator were produced from amino-MS and suspended in isotonic acetate tween buffer (IAT, 10 mM pH 5 acetate, 145 mM NaCl, % tween 20) as previously described (J. Henise et al., International Journal of Polymer Science 2019, 1-7 (2019)). The fluorescein concentration of the suspension (0.8 mM) was determined by absorbance by dissolving 0.050 mL of suspension into 0.450 mL of 50 mM NaOH then measuring absorbance at A495. These MS were used in experiments when FLs-MS were mixed with other MS-drug conjugates to serve as a tracer for MS at the injection site.

[0321] Dual labeled MSs with stable fluorescein and releasable RP (FLs-MS~RPR)

[0322] Rhodamine piperazine (RP). RP was synthesized from Rhodamine B as previously reported (T. Nguyen, M. B. Francis, Org Lett 5, 3245-3248 (2003), in accordance with Scheme A.

[0323] Scheme A. Synthesis of RP from rhodamine B.ny-290431586Docket No.: 67057-20030.40

[0324] Cleavable linkers (azido-linker(Mod)-HSCs). The following azido linker hydroxy succinimidyl carbonates (azido-linker(Mod)-HSCs) with cleavage rate modulating groups: A = ClPhSO2-, B = PhSO2-, and C = MePhSO2- were prepared as previously described (D. V. Santi et al., Proc Natl Acad Sci U S A 109, 6211-6216 (2012)): A: O-[1-(phenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidyl carbonate B: O-[1-(4-chlorophenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidyl carbonate C: O-[1-(4-methoxyphenylsulfonyl)-7-azido-2-heptyl]-O′-succinimidyl carbonate

[0325] Synthesis of azido-linker-RP conjugates.

[0326] Synthesis of azido-linker-RP conjugate A. The following procedure (Scheme B) was used to synthesize azido-linker-RP conjugates A, B, and C from azido-linker(Mod)-HSCs A, B, and C. Azido-linker(Mod)-HSC (0.0075 mmol, 1 equiv) was treated with solution of RP (3.8 mg, 0.0075 mmol, 1 equiv) and DIPEA (2.3 mg, 0.018 mmol, 2.4 equiv) in 1 mL of acetonitrile containing 20% (v / v) DMF. After 2 h the reaction mixtures were analyzed by reverse phase HPLC with detection at 261 nm. All reactions showed greater than a 95% yield of the azido- linker-RP conjugate, as well as the presence of some unreacted rhodamine B that was present in the starting material (Fig. 45). The reaction mixtures were then used as is for loading of the azido-linker-RP conjugates onto MSs as described below.

[0327] Scheme B.

[0328] Fluorescein labeled and cyclooctyne activated MSs (FLS-MS-CO). Synthesis of FLS- MS-CO and FLS-MS~RPR from amino-MS.

[0329] A slurry of amino-MS containing the (CH3CH2)2NSO2- α-Lys crosslink cleavage rate modulator (4) in acetonitrile (45 mL, 3.3 mM -NH2, 0.150 mmol -NH2) was treated with DIPEA (76.8 mg, 0.104 mL, 0.594 mmol, 4 equiv) in acetonitrile (10 mL) and a solution of (5-ny-290431587Docket No.: 67057-20030.40 (and-6)-carboxyfluorescein succinimidyl ester (Invitrogen C1311, 14 mg, 0.0297 mmol, 0.2 equiv) in 0.8 mL DMF. After 35 min at room temperature a solution of 5-HCO-OSu (5) (1 mL, 150 mM, 0.150 mmol, 1 equiv) in acetonitrile was added. After standing 1.5 h, the slurry was washed three times with acetonitrile (80 mL), using centrifugation at 4500 x G between washes to separate the MSs from supernatant. The slurry was then divided into five equal portions in 50 mL centrifuge tubes (Falcon). Each tube contained ~8 mL of packed FLS-MS-CO (Scheme C), containing 0.024 mmol of cyclooctyne, and was used as is for attachment to azido-linker-RP conjugates.

[0330] Scheme C.

[0331] Dual labeled microspheres (FLs-MS~RPR 2A, 2B and 2C)

[0332] The following procedure (Scheme C) was used to synthesize azido-linker-RP MS conjugates 2A, 2B, and 2C. A slurry of FLS-MS-CO (0.024 mmol CO, 1 equiv) in acetonitrile (8 mL) was treated with one of the azido-linker-RP containing reaction mixtures (A, B, or C, 1 mL, 0.0075 mmol, azido-linker-RP, 0.3 equiv), methanol (5 mL), and acetic acid (0.001 mL). The resulting slurries were kept at room temperature for 36 h then centrifuged at 4500 x G to separate the MSs from supernatant. The MSs were then washed with 1 x 40 mL of methanol, separated by centrifugation at 4500 x G then treated with a solution of O-(2-Azidoethyl)heptaethylene glycol (0.5 mL, 40 mM, 0.020 mmol, 0.8 equiv) in water, to cap unreacted cyclooctynes. After 18 h the MSs were washed with 3 x 40 mL methanol, then with 4 x 40 mL of IAT buffer centrifuging at 4500 x G in between washes. Finally, the FLs-MS~RPR slurries were transferred to 10 mL syringes and packed at 3000 x G for 5 min using a special fixture for centrifugation of the syringes (6) to provide ~5 mL of FLs-MS~RPR slurry for each material (2A, 2B, and 2C). Measurements of the RP release rates as well as the FLS and RP content for these materials are described below (Fig. 17, Fig. 46, and Table AA).

[0333] Table AA. Accuracy of dispensing 0.025 mL of MSs from a 250 μL syringe with a 30 g needleny-290431588Docket No.: 67057-20030.40

[0334] In vitro RP release rates from FLs-MS~RPR. Release of RP was measured using a previously reported assay (E. L. Schneider et al., Bioconjug Chem 27, 1210-1215 (2016)). In brief: samples (0.150 mL) of FLs-MS~RPR (2A, 2B, and 2C) slurry were placed into nylon mesh pouches and then the pouches were heat sealed. The pouches were placed into divided cuvettes containing 2.7 mL of pH 8.4 bicine buffer (0.1 M) at 37oC and a magnetic stir bar. The absorbance of the buffer was measured over 35 h using a heated and stirred auto sampler on a UV-VIS spectrophotometer (Hewlett Packard 8453). RP (565 nm) was released over the course of monitoring, and no release of the stably linked FLS (495 nm) was observed. The time to reverse gelation (tRG) for the fluorescein labeled gels is 200 h at pH 8.4(4) and should not occur over the duration of this experiment. Half-lives for RP release were determined using a first order exponential fit of the experimental data (Fig. 17). Half-lives measured at pH 8.4 were converted to pH 7.4 values based on T1 / 2_7.4 = T1 / 2_8.4*10(8.4-7.4)(D. V. Santi et al., Proc Natl Acad Sci U S A 109, 6211-6216 (2012).

[0335] Dosing syringes, dosing accuracy, and FLs / RP ratio for FLs-MS~RPR. Slurries of FLs-MS~RPR 2A, 2B, and 2C were passaged once through a 25 gauge tube (Cadence Micro- Emulsification tube, 7974) connected between two 10 mL Luer lock syringes to ensure they were fluid suspensions void of aggregates. Then 0.070 mL of slurry was transferred to a 0.250 mL glass syringe (Hamilton Gastight 1725) using a female-female Luer coupling (Cole Palmer). The glass syringe was fitted with a 30 g x 1 / 2” needle (BD 305106) then purged by expelling all but 0.025 mL of material through the needle. Four syringes containing 0.025 mL of MSs were prepared for each material (Fig. 46). The needle was capped with a small piece of platinum cure silicone cord (McMaster Carr 9808K21) to prevent leakage and evaporation prior to use. To assess the dosing accuracy, the material in the syringes (0.025 mL) was injected into a 1.5 mL centrifuge tube containing 1.0 mL of sodium hydroxide (0.1 N). After 1 hour the absorbance of the solution was measured to detect FLs (495 nm) and RP (565 nm). The molar ratio of FLS to RP was determined for each material using A495and A565(Table AA). Dosing syringes for use in intra-tumoral injections were prepared in the exact same manner in a laminar flow cabinet, and using syringes that had been disinfecting with 70% ethanol.

[0336] Assay development, and intra-tumoral injections of FLs-MS~RPRX

[0337] In vitro separation of RP from FLs-MS-Ac following blending

[0338] Mixture of free RP and FLS-MS-Ac: RP (0.122 mL, 5.9 mM, 0.72 nmol) in IAT buffer was mixed into a slurry (3.48 mL) of FLs-MS (0.8 mM FLS) then the material was loaded into insulin syringes (BD 324702) containing 0.100 mL of slurry each.ny-290431589Docket No.: 67057-20030.40

[0339] Separation of RP from FLS-MS-Ac: Two separation methods (centrifugation and filtration) were tested to isolate FLs-MS-Ac and free RP. Both methods began by injecting 0.10 mL of slurry into IAT buffer (1.0 mL), the resulting suspension was homogenized with a tissue blender (IKA T25 Ultra-Turrax homogenizer) for 1 minute at 8000 RPM.

[0340] Centrifugation Method: Homogenized suspensions were centrifuged 20000 x G for 2 min, then the supernatant extract was removed by pipette. The pellet of FLs-MS-Ac was washed with 5 x 1 mL of IAT, separating MSs by centrifugation at 20000 x G for 2 min between washes.

[0341] Filtration method: Homogenized suspensions were filtered using 0.2 μm PTFE spin filters (Millipore Ultrafree MC hydrophilic, Cat no. UFC30LG25) by centrifugation at 10000 x G for 5 min. The filtrate extract was collected and the retained pellet of FLs-MS-Ac was washed with 4 x 0.5 mL of IAT, by spinning at 10000 x G for 5 min for each wash.

[0342] Analysis of pellets and washes from both methods (Fig. 18): After the final wash the pellet was dissolved in 1.00 mL of 1N NaOH for 1 h. The supernatant (or filtrate) extracts and wash samples were diluted 1:1 with 1N NaOH and kept for 1 h. Next, all samples were diluted 1:10 with 1N NaOH and then a portion (0.200 mL) was transferred to a well of a black microtiter plate. Fluorescence was measured using a plate reader as described herein.

[0343] Clearance half-life of RP in tissue, following SC injection into rats

[0344] After shaving, sub cutaneous injections of the RP / FLS-MS-Ac mixture in insulin syringes described above (0.100 mL each) were made into the backs of 3 female Sprague Dawley rats (body weight ~250 g) according to the schedule and diagram in Table BB. After injection the implants were marked with a permanent marker (Sharpie) by drawing a 12 mm circle around the implant. After injection #5 the animals were euthanized with CO2 and then injection #6 was made immediately prior to harvesting tissue. Tissue was harvested to a depth down to the peritoneal membrane, using a 12 mm biopsy punch (Acuderm Acu.Punch P1250). Tissue samples were placed into 2 mL screw cap vials and stored at -80oC prior to analysis. Untreated tissue samples (X) were also collected to serve as background samples.

[0345] Table BB. Dosing schedule and injection map for implantation of RP / FLS-MS mixture into rats.ny-290431590Docket No.: 67057-20030.40

[0346] Determination of FLS and RP in tissue

[0347] Tissue samples were thawed and then placed into 5 mL snap-cap round bottom centrifuge tubes. Tissue samples were covered with 2.00 mL of IAT buffer, homogenized at 8000 RPM for 1 minute (IKA T25 Ultra-Turrax homogenizer), and then allowed to stand for 20 min prior to centrifugation at 5000 x G for 20 min. The supernatant was transferred to a 5 mL Eppendorf tube. The pellet was treated with an additional 2.00 mL of IAT, mixed, allowed to stand for 20 min, and then centrifuged at 5000 x G for 20 min. The supernatant was removed and combined with the first supernatant. The pellet was digested in 4.0 mL of 1 N NaOH for 1 h at room temperature. The pellet digest and supernatant (extract) were diluted to exactly 5.0 mL with water. For FLS detection, samples were further diluted 5-fold with water and for RP detection samples were measured as-is. Samples were added to a black 96-well microtiter plate (0.250 mL / well) and analyzed for fluorescence as described herein (Fig. 19).

[0348] Tumor xenografts in mice and intra-tumoral injection of FLs-MS~RPR

[0349] Female Balb / c mice weighing 21 + / - 1.2 g were implanted with 1x105CT26 Murine Colon Carcinoma cells, sc, in 0.100 mL of serum free media. One implant per animal was made. Tumors were allowed to grow to 251 + / - 90 mm3(12 days) as determined by measurement with calipers prior injections. Intra-tumoral injection of FLs-MS~RPR slurries (2A, 2B and 2C) were made from 0.250 mL glass syringes with 30 g needles described above (Fig. 46). The 0.025 mL payload of each syringe was injected into a single tumor in two portions (~0.0125 mL each) on either side of the tumor. IAT buffer was injected in the same manner to provide background samples.

[0350] Tissue harvesting: At the desired time (hours) after injections, animals were euthanized by cervical dislocation and then the tumors and surrounding tissue were harvested using a 12 mm biopsy punch (Acuderm Acu-Punch P1050). Tissue samples were placed into 10 mL round bottom snap cap centrifuge tubes (Green Bio Research MC01100) containing 4 mL ofny-290431591Docket No.: 67057-20030.40 pH 5.0 IAT buffer. Samples were stored at 4oC for 40 min prior to being frozen on dry ice and then stored at -80oC for 24 h prior to assay.

[0351] Determination of FLS and RP in excised tumors

[0352] Tissue samples were thawed to room temperature and immediately homogenized at 8000 RPM for 1 minute (IKA T25 Ultra-Turrax homogenizer). Samples were then centrifuged at 5000 x G for 30 min. The supernatant extract (~4 mL) was transferred to a 15 mL Falcon tube. The pellet was washed twice with IAT buffer by treating it with 2.00 mL of buffer, was allowed to rock for 45 min, and then centrifuged at 5000 x G for 20 min. The wash extracts were removed and combined with the original supernatant extract to give (~8 mL) of total extract. Extract solutions were treated with 1 mL of NaOH (1 N) and diluted to 10 mL with water. The pellets were treated with 1 mL of 1 N NaOH and then diluted to 10 mL with water. Standards were made by injecting 0.025 mL of each material (2A, 2B and 2C) from dosing syringes into 10 mL of NaOH (0.1 N). After standing for 1 hour, the samples were centrifuged at 5000 x G for 20 min, and then 0.150 mL of each was loaded into a black 96 well microtiter plate and scanned for fluorescence as described herein.

[0353] Release of SN-38 from subcutaneous microspheres

[0354] Preparation of microspheres with releasable SN-38 (MS~SN-38) (Scheme D)

[0355] Scheme D. Synthesis of microspheres with releasable SN-38A

[0356] Bicyclononyne-microspheres (BCN-microspheres, 4) were prepared from 10 kDa tetra-PEG amino-MS crosslinked with the a-LYS gem dimethyl (GDM) linker containing Mod = Me2SO2N-(7) using BCN-HSC (Synaffix, SX-A1028). A slurry of amino-MS (53.53 mL, 2.64 mM amine) in 100 mM NaOAc pH 4.0 was placed into a 100 mL GL45 glass bottle and sterilized in an autoclave (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020)). The sterilized amino-MSs were transferred to a 500 mL conical tube and washed with 2 x 300 mL of sterile aqueous 25% acetonitrile (%v / v), separating the amino-MS from the excess solvent byny-290431592Docket No.: 67057-20030.40 centrifugation (3700 x G, 10 min) between washes. Prior to washing, the acetonitrile was sterilized by filtration using a 0.2 μm PTFE 50 mm Disc filter (Saint Gobain PureFlo, D50CF0201N1N-1), whereas the water was sterilized in the autoclave. The amino-MS were then washed with 5 x 200 mL of acetonitrile as described above to give packed amino-MS slurry in acetonitrile (containing 141 μmol amine). This slurry was then treated with triethylamine (4 eq., 565 μmol) and BCN-HSC (1.2 eq., 170 μmol) in 8 mL acetonitrile. The reaction was mixed end- over-end at ambient temperature for 2 h. at which time a qualitative TNBS test (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020)) confirmed that the amines had been consumed. Acetic anhydride (1 eq, 141 μmol) was then added for 0.5 h to cap any residual free amines, and the slurry was washed with 6 x 250 mL acetonitrile as described above. The final packed slurry of BCN-microspheres was 50 mL and contained 141 μmol BCN and was used as is for loading with N3-linker(SO2Me)-SN-38 (below).

[0357] N3-linker(SO2Me)-SN-38 (3) was synthesized as previously described (D. V. Santi et al., Med Chem 57, 2303-2314 (2014)). A 17.3 mM solution of N3-linker(SO2Me)-SN-38 (8.95 mL, 155 μmol, 1.1 equiv) in acetonitrile was added to 53.5 g BCN loaded microspheres in acetonitrile (141 μmol BCN, 1.0 equiv). The reaction mixture was mixed by rocking end over end at 37°C for 22 hr. Progress was monitored by following loss of SN-38 in the supernatant by A363. The loaded microspheres were washed with 4 x 250 mL of acetonitrile followed by 6 x 250 mL of IAT-Met buffer (10 mM NaAcOH, 143 mM NaCl, 0.05% Tween 20, pH 5.0, 10 mM methionine), separating MS from excess buffer by centrifugation (3700 x G, 10 min) between washes. The resulting slurry of MS~SN-38 (5) was pelleted (3700 x G, 10 min), transferred to a 10 mL syringe, and stored at 4°C. The mass of the packed slurry was 10 g.

[0358] The concentration and loading efficiency of the MS~SN-38 was determined by dissolving 20 μL of the packed slurry (20 mg) in 50 mM NaOH (80 μL) for 1 hour at room temperature. The SN- 38 content was determined by absorbance of the SN-38 anion by A414. The PEG content in the solution was determined using a previously described colorimetric PEG assay (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020)). The percent loading of the microsphere slurry was determined to be 98%, by the ratio of SN-38 to PEG (found 196 + / - 10 nmol SN-38 / mg PEG, 200 theory).

[0359] In vitro release and dissolution of MS~SN-38

[0360] Samples of MS~SN-38 (60 μL) were placed in custom made dissolution cells with permeable nylon mesh membranes (Fig. 47). These cells are smaller scale versions of previously described dissolution cells (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020). Theny-290431593Docket No.: 67057-20030.40 cells were place in 5 mL tubes containing 4.1 mL of 100 mM Na borate buffer pH 9.4 pre- equilibrated to 37°C in a temperature-controlled stirred heat block (Fig. 47). A liquid handling robot (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020)) was programmed to remove 60 μL samples from the reaction buffer at t=0 and specified time points over 50 h. The concentration of SN-38 in the reaction supernatant was determined by absorbance at 414 nm based on previously described methods (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020). The A414 of the supernatant vs time were fit to a single exponential using Prism software to determine the release t1 / 2 for SN-38 was 1.6 h. Similarly, the PEG content of the supernatant was determined using a previously described PEG assay (J. Henise et al., Engineering Reports 2:e12213, 1-13 (2020) to determine the tRG= 25 h (Fig. 21).

[0361] In vivo release of SN-38 from microspheres

[0362] Dose formulation preparation and analysis. MS~SN-38 and FLs-MS-Ac described above were combined to generate a slurry containing 100 μM SN-38 and 32 μM fluorescein in IAT buffer containing 1% 40 kDa hyaluronic acid (J. Henise et al., Engineering Reports, 2:e12213, 1-13 (2020)). The SN-38 and FLs concentration of the mixture was determined by dissolving 25 μL samples of the slurry in 0.1 M NaOH (175 μL) for 1 h at room temperature. Fluorescein and SN-38 were quantitated spectrophotometrically at by A495 for FLs and A363 for SN-38. This material was loaded into insulin syringes (BD 324702) containing 0.100 mL of slurry each to administer SC injections.

[0363] Dose administration and recovery. On Day 0, seven male SD rats were weighed, anesthetized with isoflurane, and their backs shaved. Then, 100 μL of microsphere slurry (containing the mixture of MS~SN-38 and FLs-MSAc described above) was injected SC into 4 sites equally spaced on their backs. The location of each injection was marked with a circular tattoo ~12 mm in diameter prior to injection. On days 0, 1, 3, 6, 12, 18 and 24, one rat / day was euthanized with CO2, then the hair was removed by shaving and treatment with Nair (Church & Dwight), followed by cleaning with 70% isopropyl alcohol. Using the tattoos for reference, the tissue surrounding the SC injection site was excised down to the peritoneal membrane using a 12 mm diameter biopsy punch (Acuderm Acu-Punch P1250). For each rat, a control skin sample (no injection) was obtained from the flank at least 2 cm away from all injection sites. Each tissue sample was placed into a 1.5 mL Eppendorf tube and stored at -80 °C until analysis.

[0364] Analysis of free SN-38 and MS-bound SN-38 at injection sites. To assess free SN-38 present at the injection site, tissue samples were thawed in 1 mL 0.5% HOAc, and then mixed with a Dounce homogenizer. The pestle was rinsed with 1 mL of 0.5% HOAc which wasny-290431594Docket No.: 67057-20030.40 combined with the homogenized tissue sample. Samples were then clarified by centrifugation (15 min) and the supernatant was removed. The tissue sample was washed with 2 mL 0.5% HOAc, clarified by centrifugation (15 min) and the wash was combined with the previous supernatant to give ~4 mL 0.5% HOAc extract for analysis. A sample of each extract (0.050 mL) was analyzed by HPLC (below) to determine free SN-38 present in the tissue surrounding the injection site.

[0365] To assess the content of MS~SN-38 and MS-FLs-Ac at the injection site, the tissue pellets from the HOAc extracts (above) were treated with 0.25 M NaOH (4 mL) vortexed to mix then heated to 80 °C for 1 h to dissolve MS and release MS bound SN-38. The mixture was clarified by centrifugation (15 min), and the supernatant was removed for analysis. A sample of each supernatant (0.050 mL) was analyzed by HPLC to quantify SN-38 and FLs.

[0366] HPLC analysis. The acid and base extracts of tissue samples described above were analyzed by reverse phase HPLC using the following method: 0% ACN 0-1 min, 0-100% 1-30 min, 100% 30-31 min, 100-0, 31-32 min, 0% 32-33 min all with 0.1% TFA. Fluorescence detection: EX 360 nm EM 545 nm (0-14.5 min) for SN-38, and EX 442 nm EM 520 nm, (14.5- 30 min) for Fluorescein. SN-38 eluted at 13.2 min, and PEG-fluorescein eluted at 16.7 min. An SN-38 standard curve was generated using 50 μL 0.72 nM to 11.3 uM SN-38 quantitated by HPLC. The fluorescent SN-38 peak area of 50 μL injections was plotted vs. concentration to obtain the standard curve.

[0367] Anti-tumor efficacy of MS~SN-38

[0368] The 22Rv1 ATM- / -cell line (A. Thomas et al., Mol Cancer Ther 21, 1722-1728 (2022)) was tested negative for Mycoplasma contamination prior to the initiation of in vivo studies. Tumor xenograft studies were conducted at Murigenics. Male, 6-8 week old NSG mice were injected SC with 22Rv1 ATM- / -cells (1 x107in 100 μL of 1:1 PBS:Matrigel). When the average tumor volume reached ~125 mm3, MS~SN-38 (0.2 – 7.5 μmol / kg) was administered IT (50 μL injection) or SC (50 μL injection) and PLX038A (7.5 μmol / kg) was administered IP (Scheme 2, Fig 48). The control group was left untreated until the average tumor volume reached ~1,000 mm3. Then, mice were treated with a single IT dose of MS~SN-38 (2 or 20 μmol / kg, 100 μL injection). In combination experiments with TLZ, mice were treated with IT MS~SN-38 (0.2 μmol / kg), SC MS~SN-38 (2 μmol / kg), PO TLZ (0.4 μmol / kg) QDx21 or a combination of IT MS~SN-38 and PO TLZ. The tumor volume (caliper measurement, V=0.5x(length x width2) and body weights were measured twice weekly. Example 5A Preparation and Characterization of microspheres with fluorescent probesny-290431595Docket No.: 67057-20030.40

[0369] Technologies were developed using fluorescent probes that facilitate direct determination of the local pharmacokinetics of drugs released from microsphere prodrugs (Fig. 16A and Fig. 16B). First, amine-derivatized microspheres were coupled to fluorescein (FL) by a stable linker (FLs) to give FLS-MS (1); here, the FL serves as a quantitative marker for MS particles. Then, rhodamine piperazine (RP) – a fluorescent drug surrogate –was attached to FLS- MS via a releasable β-eliminative linker (RPR) to give FLS-MS~RPR (2). To measure the release rate of RP, the FLS-MS~RPR and free RP were separated at various times, and the free RP and MS-bound RPR and FLs were quantified. To avoid errors due to inconsistent recovery of MSs, the fluorescence signals of RPR remaining on MSs were normalized to the amount of MS recovered as determined by the FLs content; plots of RPR / FLS vs t were used for ratio-metric determinations of release rates of the drug surrogate from the MSs. Where a drug is used instead of the RPR surrogate (e.g. SN-38, vide infra), stable MS-FLs and releasable MS~drug conjugates were prepared separately and then mixed to give the desired proportions for in vivo experiments.

[0370] The releasable β-eliminative linkers used for RP in FLS-MS~RPR had modulators 4- ClPhSO2- (2A), PhSO2- (2B), and 4-MePhSO2- (2C) (D. V. Santi, E. L. Schneider, R. Reid, L. Robinson, G. W. Ashley, Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates. Proc Natl Acad Sci U S A 109, 6211-6216 (2012)). The in vitro rates of release of RP from these conjugates (Fig. 17) were determined at pH 8.4, 37oC, and the estimated t1 / 2values at pH 7.4, 37 °C (28) were 9 hours for 2A, 27 hours for 2B, and 40 hours for 2C. These MS also contained base-labile β-eliminative linkers (Mod = -(CH3CH2)2NSO2- α-Lys) within each crosslink to allow solubilization in base prior to analyses; the MSs had a time to reverse gelation (tRG) of ~1000 hours at pH 7.4, 37 °C (Henise, J. et al. High-throughput, aseptic production of injectable Tetra-PEG hydrogel microspheres for delivery of releasable covalently bound drugs. Eng. Rep. 2(e12213), 1–13 (2020); Henise, J., Yao, B., Ashley, G. W. & Santi, D. V. Facile preparation of tetra- polyethylene glycol hydrogel microspheres for drug delivery by cross-flow membrane emulsification. Eng. Rep. 3, e12412 (2021)). Example 5B Separation of intact FLS-MS from free RP under mock in vivo conditions

[0371] Methods were then developed to separate intact MS particles labeled with FL (1) from free RP under conditions simulating analyses of biopsies of local injections. In early experiments, it was discovered that MSs sheared upon sonification, but were stable to mechanical homogenization. When homogenized suspensions containing FLS-MS and spikedny-290431596Docket No.: 67057-20030.40 free RP were centrifuged and the pellet washed, the pellet retained >98% of the FLS-MS; the free RP was hardly detectable in the pellet, but present at high levels in the supernatants. In a similar experiment, when the mixture of FLS-MS and free RP was passed through a 0.2 μ PTFE spin filter and the retentate washed, the retentate contained >99% of the FLS-MS whereas the filtrate contained only released RP (Fig. 18). Hence, either centrifugation or filtration followed by washing effectively separates particulate MSs from free fluorescent drug surrogates. Example 5C Half‑life of free RP in tissue and normalization of data for ratio‑metric analysis

[0372] To estimate the t1 / 2 of diffusion of free RP in tissues, a mixture of stable FlS-MS marker and free RP was injected SC in temporal subcutaneous injections at six locations on a rat (J. Henise et al., In Vitro-In Vivo Correlation for the Degradation of Tetra-PEG Hydrogel Microspheres with Tunable β-Eliminative Crosslink Cleavage Rates. International Journal of Polymer Science 2019, 1-7 (2019)). After euthanasia, tissue samples of 150 ± 22 mg surrounding the injection sites were obtained using a 12 mm biopsy punch, homogenized and analyzed for free RP and FlS-MS. The raw data were normalized to the recovered FlS-MS (Fig. 19), and using the ratio-metric method of plotting RP / FLs vs t, a local t1 / 2of 1.5 ± 0.25 hour was calculated for RP in SC tissue. Example 5D Intra-tumoral t1 / 2 values of RP released from FLS-MS~RPR

[0373] In an exploratory experiment, three groups (n=2) of Balb / c mice were implanted SC with CT26 murine colon carcinoma cells. When tumors reached ~265 mm3, each was injected with 25 μL of FLS-MS~RPR 2A, 2B or 2C. After 24 hours, animals were euthanized, tumors and surrounding tissue (150 ± 22 mg) were harvested using a 12 mm biopsy punch. Samples were mechanically homogenized, pellets were obtained by the centrifugation method and thoroughly washed. After hydroxide dissolution of the MSs, fluorescence of FLS and RPR were quantified. Fig. 20A shows the data at 24 hours for each dye in conjugates 2A, 2B, and 2C normalized to the amount of FLSin the pellet. There was a high level of FLSin the pellets – a measure of the isolated microsphere depot – with <1.6% in the extracts. Most RP was also found in the pelleted FLS-MS~RPR, and the amounts track the t1 / 2 of the linkers in the order 2A<2B<2C, indicating that pelleted RP emanates from FLS-MS~RPRand not residual free RP.ny-290431597Docket No.: 67057-20030.40

[0374] A larger experiment was performed in which murine CT26 colon carcinoma tumors (n=15) were injected IT with MSs 2A, 2B and 2C. Three tumors containing each conjugate were harvested at 0, 8, 24, 32 and 48 hours and the isolated MSs were analyzed as above for FLSand RPR content over 48 hr. Fig. 20B shows the normalized ratio-metric plots of these experiments from which t1 / 2 values of 30 hour for 2A, 43 hour for 2B and 63 hour for 2C were determined. Together, these results show that the IT t1 / 2of the surrogate drug released from the MSs is affected by the linker used. Example 5E Release rates in subcutaneous / peritumoral tissues

[0375] Because drug release from the MSs is rate-determining the tumor t1 / 2of the drug should be similar in different tissues – SC, peritumoral and IT – unless there is a direct effect on the linker cleavage. When 2C (Mod PhSO2-) was injected SC rather than IT, the release t1 / 2 was 43 h, some 1.5-fold faster than the 63 hour t1 / 2when injected IT. Drug release from MS conjugates occurs by a first-order base catalyzed elimination, and the tumor pH is lower than the peritumoral / SC pH (D. Coman et al., Imaging the intratumoral-peritumoral extracellular pH gradient of gliomas. NMR Biomed 29, 309-319 (2016)). Hence, the slower release rate of IT vs SC injected MS~RP can be explained by the lower pH in tumors, which slows linker cleavage. Example 5 Preparation of releasable hydrogel microsphere-SN38 conjugates

[0376] Preparation of releasable hydrogel microsphere-SN38 conjugates of formula (IV) was undertaken wherein: R1= SO2Me or CN; R2= H; each R4= H or Me; n = 1,2, or 4; D = prodrug- SN38; Z* = 1,2,3-triazole; w = 0; y = 4; C* = carboxamide; R1a= SO2NMe2’ R2a= H; each R41= Me; q = 1; B* = 1,2,3-triazole; P1and P2= 10 kDa 4-armed PEG; r = 4.ny-290431598Docket No.: 67057-20030.40

[0377] Degradable hydrogel microspheres releasing the topoisomerase I inhibitor SN38 were prepared by methods in Examples 5A-5D, as well as adaptation of previously disclosed methods. Scheme 2 shows a general reaction scheme to prepare MS~SN-38, which will be discussed in further detail below.Scheme 2

[0378] Bicyclononyne-hydrogel microspheres (BCN-microspheres) of formula (VIIIa) wherein Z comprises a bicyclononyne; w = 0; y = 4; C* = carboxamide; R1a= SO2NMe2’ R2a= H; each R41= Me; q = 1; B* = 1,2,3-triazole; P1and P2= 10kDa 4-armed PEG; and r = 4 were prepared as previously described (Schneider et al., 2016 Bioconj. Chem. 27: 1210-5) by reaction of the corresponding hydrogel microspheres wherein Z = NH2 with (1R,8S,9s)- bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN-OSu). The amino microsphere slurry (13.2 g) stored in 100 mM NaOAc pH 4.0 was dispensed into a 100 mL glass bottle and placed in an autoclave for sterilization. The sterilized amino microspheres were transferred to a 50 mL conical tube and washed by suspension followed by centrifugation in 2x35 mL of sterile water, 2x35 mL of 50%v / v sterile water / acetonitrile, and finally 5x40mL of sterile acetonitrile (sterile filtered using a 0.2 µm PTFE PureFlo 50 mm Disc filter. The packed amino microsphere slurry in acetonitrile (13.2 g) containing 34.9 μmol amine was combined with triethylamine (4 eq., 141 μmol) and BCN-HSC (1.25 eq., 43.7 μmol). The reaction was mixed end-over-end for 2.5 hr and a qualitative color test using trinitrobenzenesulfonate confirmed loading of the amines. Acetic anhydride was added (1 eq, 34.9 μmol) to cap any remaining free amines and after a 0.5 hr reaction the slurry was washed 6 times with 40 mL of sterile acetonitrile to provide the BCN-microspheres.

[0379] A 80 mM solution of the N3-linker-prodrug-SN38 of formula (II) wherein Z = N3; R1= SO2Me; R2= H; each R4= H; n = 4; and D-NH = N-[4-(N,N-diethylcarboxamido)phenyl]-N- [7-ethylcamptothecin-10-oxy]methylamine (1.1 eq, 0.525 mL, 42 µmol) in sterile acetonitrile was added to the BCN-microspheres. The reaction solution was mixed end over end at 37°C for 8 hr. The loaded microspheres were washed twice with 40 mL of acetonitrile to remove excess reagent before a 4 hr incubation with 1-azidoheptaethylene glycol to cap any unreacted BCNny-290431599Docket No.: 67057-20030.40 groups. Finally, the microspheres were washed 5x35mL of sterile MeCN followed by 7x35 mL of 10 mM sodium acetate, 143 mM NaCl, 0.05% Tween 20, pH 5.0, 10 mM methionine. The resulting slurry of conjugate 3A was pelleted and stored at 4°C. The mass of the packed slurry was 4.7 g.

[0380] The concentration and loading efficiency of the hydrogel microspheres was determined by solubilizing 20 μL of the packed slurry (20 mg) in 4 volumes of 50 mM NaOH (80 μL) for 1 hour at room temperature, in triplicate. The SN-38 content (6.2 mM) was determined by absorbance of the SN-38 anion at 414 nm (ε414nm = 27,500 M-1cm-1). A total of 29.2 µmol SN-38 was loaded onto the microspheres. The PEG content (29.2 mg / mL) was determined using the previously described PEG assay (Henise et al, Engineering Reports. 2020;e12091). The percent loading of the microsphere slurry, determined from the ratio of SN- 38 concentration to the calculated PEG reactive end-groups, was 196 nmol SN-38 / mg PEG or ~5 μmol / mL slurry, 98% of the maximum.

[0381] Similar releasable MS~SN38 conjugates were also prepared in similar yields: (3B) R1= MeSO2; R2= H; each R4= Me; and n = 2; (3C) R1= CN; R2= H; each R4= Me; and n = 2; and (3D) R1= CN; R2= H; each R4= Me; and n = 1. A non-releasable conjugate was similarly prepared wherein R1R2CH is missing; each R4= H; and n = 4. In vitro characterization

[0382] Samples of the loaded microspheres (60 μL) were placed in a custom made disk sample holders with permeable mesh sides. The disks were place in 5 mL tubes containing 4.1 mL of 100 mM Na borate buffer pH 9.4 pre-equilibrated to 37°C in a temperature controlled heat block with stirring. A liquid handling robot was programmed to pull 60 μL samples from the reaction buffer at t=0 and various timepoints over 60 hours and placed in sealed HPLC vials for later analysis. The concentration of SN-38 in the reaction supernatant was determined by absorbance at 414nm on an HP8450 UV-Vis. The A414nm of the supernatant timepoints were plotted and fit to a single exponential using Prism software to determine the release rate of SN- 38. Similarly, the PEG content in each aliquot was determined using the previously described PEG assay to determine the tRG. In addition, samples collected over the first 10 hours were analyzed by HPLC to determine the purity of the SN-38 on the MSs. Half-lives were extrapolated to pH 7.4, 37oC according to the relationship t1 / 2(pH y) = t1 / 2(pH x)•10(x-y)based on the known first-order relationship between rate and [OH-]. The data are shown in Figures 2A-2B, and results are given in Table 3. The in vitro t1 / 2 of release of SN-38 from MS~SN-38 conjugates was ~160 hour at pH 7.4, 37°C (Fig. 21) and the SN-38 released at pH 8.4 was >95% pure. Forny-2904315100Docket No.: 67057-20030.40 background information on these calculations, see D. V. Santi, E. L. Schneider, R. Reid, L. Robinson, G. W. Ashley, Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates. Proc Natl Acad Sci U S A 109, 6211-6216 (2012).

[0383] To quantitate residual unbound SN-38 in the packed MS~SN-38 slurry, equal volumes of slurry and MeCN (50 µL) were mixed and incubated for 30 minutes at ambient temperature. Following centrifugation of the reaction mixture (5 minutes, 15,000xg), the 10 µL of the supernatant was injected onto the HPLC. Table 3. In vitro release rates and gel dissolution timesExample 6 In vivo local release of SN38 from releasable hydrogel microsphere-SN38

[0384] The in vivo local release of SN38 from the hydrogel microspheres of Example 3A (R1= SO2Me; R2= H; each R4= H; n = 4) was measured after subcutaneous injection into rats by analysis of conjugated SN38 and free SN38 in a tissue plug surrounding the injection site. The same hydrogel microspheres containing stably-linked fluorescein in place of releasable SN38 (“MS-FL”) were used to control for recovery of the conjugate from the skin plugs. The stable MS-FL and 3A were made separately and mixed prior to injection to provide FLs-MS~SN-38 as described above in examples 5A-5D. As discussed in the above examples, FLs serves as a quantitative marker of the MSs to allow ratio-metric analysis from normalized measurements of SN-38 / FLS. A 100 uL mixture containing 100 uM SN38 (MS~SN-38) and 32 uM fluorescein (MS-FLs) was administered SC to four locations on the backs of seven Sprague-Dawley rats. At specified times each rat was euthanized and samples containing the conjugates and surrounding tissue were excised with a 12 mm diameter biopsy punch. Each of the tissue samples was dounced in 0.5% acetic acid, clarified by centrifugation, and washed with 0.5% acetic acid. Theny-2904315101Docket No.: 67057-20030.40 pellets were then treated with 0.25 M NaOH to dissolve the conjugates (MS~SN-38). The acetic acid extracts containing SN38 that had been released from conjugate 3A in vivo and the NaOH- digested pellets containing SN38 and fluorescein that had remained conjugated were separately analyzed by HPLC for SN38 and fluorescein.

[0385] A plot of the free SN38 in the AcOH extracts and NaOH tissue pellets normalized to the recovered fluorescein content of the MSs (i.e. SN-38 / FLS) vs t gave a t1 / 2of 6.6 days (R2of 0.974) for the released SN38 in the acid extract, and 6.1 days (R20.910) for the conjugated SN38 in the dissolved pellet (Figure 3). Correcting for the 1.5 slower t1 / 2 of linker cleavage in tumor vs SC tissue (discussed above), the IT t1 / 2in CT26 tumors is estimated to be ~10 d. Example 7 In vivo efficacy of intratumoral releasable hydrogel microsphere-SN38 in mouse xenograft models

[0386] The efficacy of the hydrogel microsphere conjugate of Example 3A (R1= SO2Me; R2= H; each R4= H; n = 4) after intratumoral injection was examined in a mouse xenograft models. This was compared with the efficacy of the same hydrogel microsphere conjugate after subcutaneous injection distal to the tumor, as well as a systemically-administered soluble conjugate PLX038A (Fontaine et al., 2019, Cancer Chemother. Pharmacol. 84: 729-38).

[0387] Efficacy of MS~SN-38 in xenograft model of prostate cancer (22Rv1 ATM-deficient): Male NSG mice were injected SC with 22Rv1 ATM- / -cells (1 x107in 100 μL of 1:1 PBS:Matrigel). When the average tumor volume reached ~125 mm3, the releasable conjugate 3A (0.2 – 7.5 μmol / kg) was administered intratumorally (50 μL IT injection) or subcutaneously (50 μL SC injection) and soluble PEG-conjugate PLX038A (7.5 μmol / kg IP) was administered intraperitoneally. The control group was left untreated until the average tumor volume reached ~1,000 mm3, then were treated with a single IT dose of conjugate 3A (2 or 20 μmol / kg, 100 μL injection). The tumor volume (caliper measurement, V= 0.5x(length x width2) and body weights were measured twice weekly. At all dose levels, IT 3A inhibited tumor growth > 8 weeks (Figures 4A-4B). The measurement of an IT injection of MSs can contribute upwards of ~100 mm3of the measured tumor volume. Therefore, approximately 50% of the record tumor volume can be attributed to the MS injection. The high dose SC injection of 3A (20 µmol / kg) also showed potent anti-tumor efficacy, although tumor regrowth was eventually observed after 6 weeks.ny-2904315102Docket No.: 67057-20030.40

[0388] Equimolar dosing of MS~SN-38 and PLX038A (7.5 µmol / kg) shows the advantage of MS~SN-38. After approximately 3 weeks, tumors begin to exponentially grow in animals treated with PLX038A, whereas no tumor growth was observed in the MS~SN-38 group until after 8 weeks (Figures 4A-4B).

[0389] IT doses of conjugate 3A (at 0.2 to 7.5 µmol / kg) were compared with SC doses (0.2 or 2.0 µmol / kg). The lowest IT dose of 0.2 µmol / kg resulted in a TGI ~55% and a 13-day extension in overall survival; the highest 7.5 µmol / kg dose resulted in ~90% TGI and 41-day extension in overall survival. In comparison, SC injection of conjugate 3A at 0.2 µmol / kg gave insignificant TGI, while the 2.0 µmol / kg gave 50% TGI and survival, comparable to 0.2 µmol / kg IT administered 3A. also It was also determined that IT 3A could control growth of very large tumors. When 22Rv1 ATM- / -tumors reached an average tumor volume ~1,000 mm3, 2- or 20 µmol / kg 3A was administered IT. Although 2 µmol / kg 3A resulted in a modest ~1 week delay in tumor growth and a 15 day increase in median survival, IT administration of 20 µmol / kg 3A resulted in >42 day delay in tumor growth and a median survival of 45 days (Figures 5A-5F). It was also discovered that 20 µmol / kg of conjugate 3A, delivered IT or SC, was tolerated with mice losing less than 10% body weight through the duration of the experiments (Fig. 22). Hence, conjugate 3A is about 10-fold more efficacious when administered intratumorally as compared to systemically, and can suppress growth of very large tumors.

[0390] Efficacy of MS~SN-38 in syngeneic model of colorectal cancer (CT26): CT26 tumors were established in the flank of female BALB / c mice. When tumor volumes reached ~125 mm3(approximately 10-14 days), mice were randomized into groups (n=5-8 / group). Group one mice were treated with a single IT dose of sham MSs containing 10 mg / mL PEG or left untreated. Groups 2-6 were treated with a single IT dose of MS~SN-38 (0.3 – 20 µmol / kg). Group 7 was treated with a single SC dose of MS~SN-38 (20 µmol / kg). The tumor volume and weight of all mice were measured 2 times a week for up to 60 days or until a humane endpoint was reached. CT26 tumors are homologous recombination proficient tumors and are not expected to be overly sensitive to DNA damaging agents and TOP1 inhibitions. As shown in Figures 6A-6B, a small but significant anti-tumor response was observed when 20 µmol / kg MS~SN-38 was delivered IT compared to SC MS~SN-38 and the vehicle control group. IT MS~SN-38 treated animals had a significant (*, p<0.033) decrease in tumor volume at day 15 and increase in overall median survival compared to the vehicle control and SC 20 µmol / kg MS~SN-38 (Figures 6A-6B). Example 8 Preparation of releasable hydrogel microsphere-talazoparib conjugatesny-2904315103Docket No.: 67057-20030.40

[0391] Preparation of releasable hydrogel microsphere-talazoparib conjugates of Formula (V), as disclosed herein, wherein: R1= SO2Me; R2= H; each R4= Me; n = 1; D = prodrug- talazoparib; Z* = 1,2,3-triazole; w = 0; y = 4; C* = carboxamide; R1a= SO2NMe2’ R2a= H; each R41= Me; q = 1; B* = 1,2,3-triazole; P1and P2= 10 kDa 4-armed PEG; r = 4.

[0392] Degradable hydrogel microspheres releasing the PARP inhibitor talazoparib were prepared by adaptation of previously disclosed methods. Bicyclononyne-hydrogel microspheres (BCN-microspheres) of formula (III) wherein Z comprises a bicyclononyne; w = 0; y = 4; C* = carboxamide; R1a= SO2NMe2’ R2a= H; each R41= Me; q = 1; B* = 1,2,3-triazole; P1and P2= 10kDa 4-armed PEG; and r = 4 were prepared as previously described (Schneider et al., 2016 Bioconj. Chem. 27: 1210-5) by reaction of the corresponding hydrogel microspheres wherein Z = NH2with (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN- OSu). The sterile amino microsphere slurry (4.3 g) stored in 100 mM NaOAc pH 4.0 was dispensed into a 15 mL conical tube and washed by suspension followed by centrifugation in 3x10 mL of sterile water, 3x10 mL of 50%v / v sterile water / acetonitrile, and finally 6x12mL of sterile acetonitrile (sterile filtered using a 0.2 µm PTFE PureFlo 50 mm Disc filter. The packed amino microsphere slurry in acetonitrile containing 11.2 μmol amine was combined with triethylamine (4 eq., 44.7 μmol) and BCN-HSC (1.5 eq., 16.8 μmol). The reaction was mixed end-over-end for 2.5 hr and a qualitative color test using trinitrobenzenesulfonate confirmed loading of the amines. Acetic anhydride was added (1 eq, 11.2 μmol) to cap any remaining free amines and after a 0.5 hr reaction the slurry was washed 5 times with 11 mL of sterile acetonitrile to provide the BCN-microspheres.

[0393] A 24 mM solution of the N3-linker-prodrug-talazoparib wherein Z = N3; R1= SO2Me; R2= H; each R4= Me; n = 1; and D-NH = N-[2-methoxyethyl]-N-[talazoparib-2-yl]methylamine (1.5 eq, 0.7 mL, 16.8 µmol) in sterile acetonitrile was added to the BCN-microspheres. Theny-2904315104Docket No.: 67057-20030.40 reaction solution was mixed end over end at ambient temperature for 4 hr. The loaded microspheres were washed 5x12 mL of acetonitrile to remove excess reagent before a 4 hr incubation with 1-azidoheptaethylene glycol to cap any unreacted BCN groups. Finally, the microspheres were washed 5x12 mL of sterile MeCN followed by 6x12 mL of 10 mM sodium acetate, 143 mM NaCl, 0.05% Tween 20, pH 5.0, 10 mM methionine. The resulting slurry of con jugate 6 was pelleted and stored at 4°C. The mass of the packed slurry was 1.22 g.

[0394] The concentration and loading efficiency in the preparation of conjugate 6 was determined by solubilizing 10 μL of the packed slurry (10 mg) in 9 volumes of 50 mM NaOH (90 μL) for 1 hour at room temperature, in triplicate. The talazoparib content (3.6 mM) was determined by absorbance at 310 nm (ε= 9,876 M-1cm-1). A total of 4.4 µmol talazoparib was loaded onto the microspheres. The PEG content (23 mg / mL) was determined using the previously described PEG assay. The percent loading of the microsphere slurry, determined from the ratio of talazoparib concentration to the calculated PEG reactive end-groups, was ~79% (157 nmol talazoparib / mg PEG). Unbound talazoparib in the preparation was determined by mixing 10 µL of the packed slurry in 4 volumes of MeCN (40 µL). After a thirty minute incubation at ambient temperature, the MSs were pelleted by centrifugation (10,000xg, 5 minutes) and 20 µL of the supernatant was analyzed by HPLC. Release of talazoparib and dissolution of the hydrogel was measured under accelerated conditions (pH 9.4, 37oC), and gave a release t1 / 2= 680 h and a degelation time of 2600 h extrapolated to pH 7.4, 37oC. Example 9 Pharmacokinetics of releasable hydrogel microsphere-antibody conjugates

[0395] Pharmacokinetics of the conjugates MS~IgG (“gdm1 MeSO2 linker” and “gdm1 ClPhSO2 linker”) from Example 3 and Example 12, administered S.C., were measured in immunodeficient, male NSG mice. Free drug (IgG) was used as control. Dosing solutions, 3.33 nmol / ml, were prepared by diluting MS~IgGs stocks or dissolving lyophilized IgG stock in Buffer Cit with 10 mM L-Methionine and 1.25 %w / v Hyaluronic Acid. Protein concentration in dosing solutions was assessed by A280(ε280= 210,000 M–1cm1). For MS~IgG, 10 µl of dosing solution samples were previously mixed with 40 µl 50 mM NaOH, incubated for 1 hour at ambient temperature and mixed with 10 µl Tris 1M, pH 7.5. Data are shown in Figure 8, which depicts mouse pharmacokinetics of IgG released from s.c. hydrogel-IgG conjugate, compared with bolus injection of IgG.

[0396] Based on the observed plasma levels of the IgG antibody released from the hydrogel conjugate, the intratumoral concentrations of antibody after an intratumoral injection wereny-2904315105Docket No.: 67057-20030.40 modeled based on the two-compartment model of Chang et al (Pharmaceutics 2023, 15, 1132) (Figure 9). Dotted line is the predicted concentration for IgG in the plasma; solid line is the predicted concentration of IgG released into a 0.2 mL volume around the injection site, simulating injection into a 200 mm3tumor, that gives rise to the modeled plasma concentrations. Example 10 Preparation of releasable hydrogel microsphere-immunomodulator conjugate

[0397] Preparation of releasable hydrogel microsphere-immunomodulator conjugate of formula (IV), wherein: D = TLR7 / 8 agonist DV1001; R1= PhSO2Me; R2= H; each R4= Me; n = 1; D = 1-[[4-(aminomethyl)phenyl]methyl]-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (DV1001); Z* = 1,2,3-triazole; w = 0; y = 4; C* = carboxamide; R1a= SO2NMe2’ R2a= H; each R41= Me; q = 1; B* = 1,2,3-triazole; P1and P2= 10 kDa 4-armed PEG; r = 4.

[0398] Acylation of DV1001 for MS loading: In a 20 mL scintillation vial, 4-azido-3,3- dimethyl-1-(phenylsulfonyl)-2-butyl succinimidyl carbonate (60.4 mg, 155 µmol) dissolved in DMF (4.3 mL) and DV1001 (49.7 mg, 138 µmol) dissolved in DMF (5 mL) were combined and incubated at room temperature. After 15 minutes, HPLC analysis of the reaction mixture indicated the presence of unreacted DV1001. Additional N3-linker (9.9 mg, 25.3 µmol) was added and the reaction was incubated at room temperature for an additional 15 minutes. The reaction mixture was transferred to a 125 mL separatory funnel containing ~ 10 mL ethyl acetate. The reaction mixture was washed a total of 4 times with ddH2O (~25 mL). The mixture was washed with ~10 mL brine and after removal, transferred to a 125 mL Erlenmeyer flask containing MgSO4. The reaction mixture was filtered and a rotovap was used to dry the reaction to a yellow / brown oil. The crude reaction mixture oil was dissolved in ~2 mL DCM and purified by flash chromatography. Briefly, the reaction mixture was loaded to a Silicycle silica column (4g) pre-equilibrated with hexanes. A step gradient (50 mL / step), using mobile phases of 25%, 50%, 75% Acetone in Hexanes and 100% acetone, was used to purify and isolate N3-linker- DV1001 Mod: GDM1-PhSO2. Fractions containing N3-linker-DV1001 Mod: GDM1-PhSO2 were first tested by TLC and then by HPLC. Fractions were then combined and dried to a colorless oil using the rotovap. The oil was stored at -20C until use in subsequent experiments.ny-2904315106Docket No.: 67057-20030.40

[0399] Loading of N3-linker-DV1001 Mod: GDM1-PhSO2 to BCN~MSs: A 24.5 mM solution of N3-linker-DV1001 Mod: GDM1-MeSO2- (3.5 mL, 85 µmol) in acetonitrile was added to 18.8 g BCN loaded microspheres in acetonitrile (50 µmol, 2.65 μmol BCN / g slurry, 151JH01 lot, crosslinker = α-amine linked gem dimethyl linker, Mod=SO2NMe2). The reaction solution was mixed end over end for 16 hr at room temperature. Progress was monitored by following absorbance in the supernatant at 250 nm (ε250nm = 31,000 M-1cm-1). The loaded microspheres were washed 4 times with 40 mL of acetonitrile to. Unreacted BCN~MS were capped with PEG7-N3(50 µmol, 1 eq. 1.5 h). The MS slurry was then washed 6x 40 mL 10 mM NaAcOH 143 mM NaCl 0.05% Tween 20 pH 5.0 containing 10 mM methionine. The resulting slurry was pelleted, transferred to a new sterile 50 mL falcon tube and stored at 4°C. The mass of the packed slurry was 9.86 g.

[0400] The concentration and loading efficiency of the MSs was determined by solubilizing 20 μL of the packed slurry (20 mg) in 9 volumes of 50 mM NaOH (180 μL) for 1 hour at room temperature, in triplicate. The DV1001 content was determined after a 10 fold dilution in 250 mM NaOAc pH 5.0 by measuring the absorbance at 321 nm (ε321nm= 12,695 M-1cm-1). The PEG content in each solution was determined using the previously described PEG assay. The percent loading of the microsphere slurry was determined to be 96.5%, the ratio of DV1001 concentration to the calculated PEG reactive end-groups.

[0401] In vitro release kinetics and dissolution of MS~DV1001 (Mod: GDM1-PhSO2): Samples of MS~DV1001 (Mod: GDM-PhSO2) (70 μL) were placed in disk sample holders with permeable mesh sides. The disks were place in 5 mL tubes containing 3.75 mL of 100 mM Na borate buffer pH 9.4 pre-equilibrated to 37°C in a temperature-controlled heat block with stirring. A robot was programmed to pull 120 μL samples from the reaction buffer at t=0 and various timepoints over 20 hr and placed in sealed HPLC vials for subsequent analyses. The concentration of DV1001 in the reaction supernatant was determined by 5 fold dilution into 250 mM NaOAc pH 5.0 and recording the absorbance at 321 nm (ε321nm= 12,675 M-1cm-1) on an HP8450 UV-Vis. The plot of DV1001 concentration versus were fit to a single exponential using Prism software to determine the release rate. Similarly, the PEG content in each aliquot was determined using the previously described PEG assay to determine the dissolution time (tRG).

[0402] Characterization of MS~DV1001 Mod: GDM PhSO2: DV1001 release from the MSs was measured in triplicate under accelerated release conditions (pH 9.4). Using the ProLynx- made liquid handling robot, the reaction mixture was sampled at predetermined time intervals. The DV1001 and PEG content was assessed by UV-Vis and BaCl2 / I2 spectrometry, to measureny-2904315107Docket No.: 67057-20030.40 the t1 / 2 and tRG respectively. The in vitro t1 / 2 of release for DV1001 was 2.14 h at pH 9.4, extrapolated to an expected 214 h at pH 7.4 (Figure 10A). This rate is ~5.5x slower than the t1 / 2 observed from PEG~DV1001 Mod: GDM1-PhSO2. The purity of DV1001 on the MSs was determined to be >95% pure, as assessed by HPLC (Figure 10B).

[0403] Efficacy of MS~DV1001 in CT26 bearing mice: CT26 tumors were established in the flank female Balb / c mice, treated by IT injection of DV1001, resiquimod or the MS~DV1001 conjugate and tumor volume and survival were recorded over 60 days (Figures 11A-11B). The dose concentration of MS~DV1001 was selected based on the estimated amount of material released per day, calculated using the in vitro half-life (t1 / 2 pH 7.4= 210 h). Doses of 1.5, 5, and 20 µg MS~DV1001 are expected to release an average of 0.091, 0.30 and 1.2 µg / d respectively, for the first 7 d (0.63 µg, 2.12 µg and 8.5 µg total in 7 d). Similar tumor growth inhibition was observed with the low dose MS~DV1001 (1.5 µg) and free DV1001 (0.5 µg); one complete response was recorded in the group treated with free DV1001. However, mice in the mid- and high-dose groups of MS~DV1001 (5 and 20 µg) resulted in superior TGI compared to free DV1001 (Figures 11A-11B and 12A-12F). In addition, one complete response was recorded in the group treated with 20 µg MS~DV1001. Finally, mice treated with 20 µg MS~DV1001 had an improved overall survival compared to mice treated with free DV1001 or resiquimod. Overall, these data demonstrate a single IT administration of MS~DV1001 results in tumor growth inhibition and increase in the overall long-term survival of CT26 tumor bearing mice. Example 11 Preparation of a hydrogel-antibody conjugate of formula IV, wherein D = Antibody-Drug Conjugate (ENHERTU)

[0404] A suspension of the activated hydrogel of Formula (XXXV) from Example 1, but 1 pared using a solution of the Z-linker-X of formula (I) wherein Z = N3; R= PhS2preO2; R = H;4 each R = Me; n = 4; and X = N-succinimidyloxy; was washed twice with 9 volumes of phosphate buffered saline (PBS, 9.8 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.3). An aliquot (1 mL) was mixed with 1000 μl of 40.5 mg / ml ENHERTU (fam-trastuzumab deruxtecan- nxki) solution in PBS (270 nmol, 40.5 mg ENHERTU) and incubated for 18 hours at ambient temperature with mixing. Then, unreacted ENHERTU was removed by washing 3 times with 10 volumes of PBS and 3 times with 10 volumes of Histidine buffer (Buffer His, 25 mM L- Histidine, 0.05%w / v Polysorbate 20, pH 5.5) .

[0405] A 10 μl sample of resulting MS~ENHERTU slurry was mixed with 50 μl of 50 mM NaOH and incubated 30 minutes at ambient temperature. After incubation, 10 μl of 1 M Tris, pHny-2904315108Docket No.: 67057-20030.40 7.5 were added to the mixture. ENHERTU concentration was obtained by UV spectrophotometry. PEG content was obtained by BaCl2 / I2spectrophotometry. MS~ENHERTU prepared slurry contained 2.2 mg / ml ENHERTU and 16.5 mg / ml PEG, indicating 135 μg ENHERTU per mg of PEG. Example 12 Preparation of a hydrogel-antibody conjugate of formula IV, wherein D = human IgG

[0406] Preparation of Lnk1-IgG. In 0.5 mL of PBS, reaction mixture contained 141 nmol (21.2 mg, 280 μM) IgG, 140 nmol (59 μg, 280 μM, 1 Eq) of linker reagent of formula (I) 1 =ClPhSO ; R2=H; each4wherein R2 R= Me; n = 1; Z = N3; and X = N-succinimidyloxy (“gdm1ClPhSO2OSu linker”). The reaction proceeded for 14 hours at ambient temperature in the dark. Excess reagents were removed using a 3.5 mL PD-10 column (Cytiva) previously equilibrated in Buffer Cit with 10 mM L-Methionine. Protein concentration was determined by –1-1 A280(ε280= 210,000 Mcm) using a NanoDrop spectrophotometer. Gel-shift analysis determined this Lnkx-IgG to comprise 64% unreacted IgG + 29% Lnk1-IgG + 7% Lnk2-IgG. 11wherein R= Me2Preparations of Lnk -IgG were similarly performed with linkersSO2; R = H;41each R = H; n = 4; Z = N3; and X = N-succinimidyloxy (“std MeSO2OSu linker”); wherein R 2 2= H; each4= MeSO ; RR= Me; n = 1; Z = N3; and X = N-succinimidyloxy (“gdm1MeSO21 ein R= Ph2 4OSu linker”) and wherSO2; R = H; each R= Me; n = 1; Z = N3; and X = N- succinimidyloxy (“gdm1PhSO2OSu linker”).

[0407] Lnk~IgG loading to MS~BCN: Preparation of MS~IgG was prepared following 11similar methods in Example 3. Briefly, Lnk -IgG (R = ClPhSO2; “gdm1ClPhSO2OSu linker”) (21.2 mg, 141 nmol IgG: 64% unreacted IgG + 29% Lnk1-IgG + 7% Lnk2-IgG) was sterilized by 0.2 μm pore filtration and mixed with MS~BCN (0.15 μmol BCN) and 100 μl Buffer Cit with 10 mM L-Methionine were added to sum 0.3 ml reaction volume. The SPAAC was allowed to progress for 150 hours before excess reagents were removed and unreacted MS~BCN was capped with PEG7-N3. The resulting MS~IgG was formulated and stored Buffer Cit with 10 mM L-Methionine. MS~IgG prepared slurry contained 34 mg / ml IgG and 10.1 mg / ml PEG, implying 22.5 nmol IgG per mg of PEG. Preparations of MS~IgG were similarly performed by conjugating Lnk1-IgG Mod prepared using the “std MeSO2OSu linker”. Example 13ny-2904315109Docket No.: 67057-20030.40 Pharmacokinetics of releasable hydrogel microsphere-ENHERTU conjugate after subcutaneous or intratumoral administration in NCI-N87 tumor bearing mice

[0408] Dosing solutions, 3.3 nmol / ml, were prepared by diluting MS~ENHERTU stock of Example 11 or dissolving lyophilized ENHERTU stock in Buffer His with 10 mM L-Methionine and 1.25 %w / v Hyaluronic Acid. Syringes were backfilled under sterile conditions to deliver of 50 µg of MS~ENHERTU or free ENHERTU. The contents of each syringe containing material from Example 11 were administered either SC or IT; syringes containing free ENHERTU were administered IV to male Balb / c nude mice bearing NCI-N87 tumors. Blood samples were drawn over 35 days, plasma was collected and samples frozen at -80ºC until analysis by ELISA.

[0409] Data are shown in Figure 13, which depicts mouse plasma concentrations of ENHERTU released from subcutaneous or intratumoral hydrogel-ENHERTU conjugate, or following IV injection of free ENHERTU. The release t1 / 2 of the hydrogel-ENHERTU conjugate were determined to be 4.2 days and 6.2 days, after subcutaneous or intratumoral administration, respectively. Example 14 Pharmacokinetics of releasable hydrogel microsphere-IgG conjugate after subcutaneous or intratumoral administration in DU-145 tumor bearing mice

[0410] Dosing solutions, 3.3 nmol / ml, were prepared by diluting MS~IgG from Example 12 in Buffer Cit with 10 mM L-Methionine and 1.25 %w / v Hyaluronic Acid. The protein concentration in dosing solutions was assessed by A280 (ε280= 210,000 M–1cm1) following release from the microspheres. Syringes were backfilled under sterile conditions to deliver of 50 µg of MS~IgG. The contents of each syringe containing material from Example 12 were administered either SC or IT to male NSG mice bearing DU-145 xenografts. Blood samples were drawn over 28 days, plasma was prepared and samples frozen at -80ºC until analysis by ELISA.

[0411] Data are shown in Figure 14, which depicts tumor bearing mouse pharmacokinetics of IgG released from subcutaneous or intratumoral hydrogel-IgG conjugate in male NSG mice bearing DU-145 xenografts. The release t1 / 2 of the hydrogel-IgG conjugate were determined to be 4.2 days and 10.4 days, after subcutaneous or intratumoral administration, respectively. Example 15 Combination of MS~SN-38 IT and oral TLZ in 22RV1 ATM- / -Xenograft

[0412] The 22Rv1 ATM- / -cell line was tested negative for Mycoplasma contamination prior to the initiation of in vivo studies. Tumor xenograft studies were conducted at Murigenics. Male,ny-2904315110Docket No.: 67057-20030.40 6-8 week old NSG mice were injected SC with 22Rv1 ATM- / -cells (1 x107in 100 µL of 1:1 PBS:Matrigel). When the average tumor volume reached ~125 mm3, mice were treated with IT MS~SN-38 (0.07 µmol / kg), SC MS~SN-38 (0.07 µmol / kg), PO TLZ (0.4 µmol / kg) QDx21 or a combination of IT or SC MS~SN-38 and PO TLZ. The tumor volume (caliper measurement, V= 0.5x(length x width2) and body weights were measured twice weekly.

[0413] Tumor inhibition by IT MS~SN-38 in combination with systemic talazoparib (TLZ). TOP1i and PARPi are highly synergistic towards BRCA and ATM deficient tumors, but have overlapping hematologic toxicities (A. Thomas, S. D. Fontaine, M. E. Diolaiti, P. Desai, R. Kumar, N. Takahashi, L. Sciuto, S. Nichols, A. Ashworth, F. Y. Feng, G. W. Ashley, M. Nguyen, Y. Pommier, D. V. Santi, PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors. Mol Cancer Ther 21, 1722-1728 (2022); M. A. Bjornsti, S. H. Kaufmann, Topoisomerases and cancer chemotherapy: recent advances and unanswered questions. F1000Res 8, (2019)). It was tested whether SN-38 that was localized in a tumor by IT MS~SN- 38 in combination with systemic administration of a PARPi would have synergistic anti-tumor effects. The effects of a combination of IT MS~SN-38 with the PARPi TLZ were measured on growth of TOP1i and PARPi sensitive 22Rv1 ATM- / -xenografts. Tumor growth (TG) and tumor growth inhibition (TGI) were calculated as described in D. Zhang, P. S. Dragovich, S. F. Yu, Y. Ma, T. H. Pillow, J. D. Sadowsky, D. Su, W. Wang, A. Polson, S. C. Khojasteh, C. Hop, Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues. Drug Metab Dispos 47, 1146-1155 (2019). The interaction of the drug combination was assessed using an additivity index determined as TGcalc / TGobsd where TGcalc is the product of TG values of the individual drugs and TGobsdis the observed tumor growth in the presence of both drugs. Here, an index <1 indicates infra-additive, an index of 1 indicates additivity and an index >1 indicates a supra-additive or synergistic interaction.

[0414] Fig. 15 shows the growth of 22Rv1 ATM- / -xenografts in mice (n=6) treated with 0.2 μmol / kg MS~SN-38 as a single IT dose, 0.4 μmol (0.15 mg) / kg of TLZ PO QD, and a combination of both. The TG was 0.82 for TLZ and 0.77 for IT MS~SN-38 giving a TGcalc for the combination of 0.63. The TGobsdof the combination was 0.17 so the additivity index was estimated as 3.7 (0.63 / 0.17), indicating very strong synergy of the two drugs. The TGobsdof the SC MS~SN-38 and TLZ combination was 0.99 indicating no observed synergy or additivity. The IT combination caused prolong tumor growth delay and improved overall survival, whereas the SC combination did not show improved antitumor activity or survival over single agent TLZny-2904315111Docket No.: 67057-20030.40 (Table 4). Thus, the IT delivered SN-38 is not diffusing from the tumor and causing a systemic effect. Table 4. Median survival of miceExample 16A Materials and Methods for Preparation and Characterization of MS~RLI

[0415] Materials: RLI (>95% pure) was produced at ATUM (Newark, CA) based on previously reported methods (Mortier, E., J Biol Chem. (2006) 281:1612–9). MS~IL-15 was prepared according to published procedures (Hangasky JA et al., J Immunother Cancer. (2022) 10:e004104-18). All other reagents were purchased from commercial vendors and used as received.

[0416] Synthesis of MS~RLI: Reductive alkylation using NaCNBH3 was used to conjugate N3-PEG4-linker(MeSO2)-CHO to RLI. The excess reagents were removed using prepacked PD- 10 columns (GE Healthcare). The azido-linker-RLI was attached to bicyclononyne(BCN)- derivatized MSs via strain promoted azide-alkyne cycloaddition (SPAAC). Then, N3-PEG7 (Sigma Aldrich) was used to cap the unreacted BCN groups. Finally, the MS~RLI conjugate was extensively washed and equilibrated in 25 mM Citrate, 500 mM NaCl, 30 mM methionine and 0.05% tween-20, pH 6.0 and stored at 4°C.

[0417] Cell-based assay: A U2OS cell-based assay kit for IL-2 / IL-15Rbg binding was performed according to the manufacturer’s (DiscoverX, Part #93-0998E3CP5).

[0418] In vivo PK and PD studies: For PK studies, MS~RLI was administered SC to either male C57BL / 6J mice or male NSG mice. Alternating groups of animals were bled and blood samples were collected in EDTA collection tubes containing HALT protease inhibitor. Plasma was prepared and stored at –80°C until analysis. Plasma samples were thawed on ice prior to analysis by either a hIL-15 / IL-15Ra complex specific ELISA (R&D Systems, hIL-15 / IL-15Rany-2904315112Docket No.: 67057-20030.40 complex DuoSet ELISA, Catalog #DY6924) or ELLA protein simple kit (Catalog SPCKB-PS- 000500). For PD studies, MS~RLI was administered SC to male C57BL / 6J mice. Mice were bled over a 28 day period. EDTA whole blood was collected and immunophenotyped by flow cytometry.

[0419] CT26 syngeneic model: CT26 tumors were established in the flank of female BALB / c mice by injection of 1 x105in 100 μL of serum-free medium. When the tumor volume reached ~100mm3mice were randomized into groups (n=7-8 / group) and mice were administered empty MSs IT, 10 μg MS~RLI IT, or empty MSs IT plus 10 μg MS~RLI SC. The experiment was performed two times. In the second experiment, mice (n=4 / group) were sacrificed on day 5 and EDTA whole blood, tumors and spleens were harvested for immunophenotyping. Kaplan- Meier mouse survival plots were generated based on the mouse survival, monitored based on humane end point criteria. The tumor volume was measured by calipers and calculated by the equation: V = 1 / 2(long dimension)(short dimension)2.

[0420] EO771 orthotopic model of Triple Negative Breast Cancer (TNBC): EO771 tumors were established by orthotopic injection of 3x105EO771 cells into the 4th mammary pad of C57BL / 6 mice. A single 50 μL IT dose of MS~IL-15 (1.2 mg or 6 mg) or MS~RLI (2 mg or 10 mg) was administered when the tumors reached ~40 mm3; empty MS (50 μL) were used as the negative control. Blood was collected 6 days before treatment, one day after MS administration and at the end of the experiment. Tumor volume was measured by calipers and calculated by the following equation: L*W*H*p / 6. All mice were sacrificed on day 15 of the treatment. Tumor- infiltrating immune cells and splenocytes were analyzed by flow cytometry. Lungs were embedded in paraffin and the metastatic lesions were evaluated by hematoxylin / eosin staining.

[0421] Flow cytometry: For PD studies in naïve mice, EDTA whole blood was incubated with a fixable viability dye, followed by incubation with CD16 / 32 antibody and subsequently surface stained with previously determined optimized Ab concentrations (Table A). Red blood cells (RBCs) were lysed and peripheral blood mononuclear cells (PBMCs) were fixed using a 1- Step Fix / Lyse solution (Invitrogen). A 1x permeabilization buffer (Invitrogen) was used to permeabilize PBMCs for intracellular staining. Table A. Antibodies used for immunophenotypingny-2904315113Docket No.: 67057-20030.40

[0422] For immunophenotyping of tumor bearing animals, EDTA whole blood was stained as described above, but RBCs were lysed with 1x RBC lysis buffer (Invitrogen) and for intracellular staining, cells were fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer Set (Invitrogen). Splenocytes were obtained from harvested spleens following mechanical disruption and filtering through a 40-μm cell strainer. RBCs were lysed using 1x RBC Lysis Buffer. Tumor infiltrating lymphocytes (TILs) were obtained from excised tumors by mechanical and enzymatic digestion and the lymphocytes were purified using Lympholyte- Mammal Cell Separation Media gradient (Cedarlane). Single cell suspensions (1x106cells) were stained with previously determined optimized Ab concentrations (Table A). Cells were fixed and stained intracellularly using the Foxp3 Transcription Factor Staining Buffer Set.

[0423] Stained single cell suspensions were read using a Attune NxT flow cytometer (BD Biosciences) and analyzed using FlowJo cytometry analysis software (TreeStar, Ashland, OR). A representative gating strategy is shown in Figure 43. CD8+and memory CD8+T cells were identified as CD3+CD8+and CD3+CD8+CD44hi, respectively; NK cells were identified as CD3- NK1.1+or CD3-CD49b+NKp46+. Treg cells were identified as CD3+CD4+CD25+Foxp3+. Cells with proliferative capacity were defined as Ki67+.

[0424] Statistics: One-way ANOVA followed by Tukey’s multiple comparison test were used for analysis of immunophenotyping data. Tumor volumes were plotted as mean ± standard error of the mean and compared using two-way ANOVA or mixed-effects model. Kaplan-Meier survival was analyzed by Mantel-Cox. All statistical testing was performed using GraphPad Prism v.9.5.1. A p value of less than or equal to 0.05 was considered statistically significant in all analyses.ny-2904315114Docket No.: 67057-20030.40 Analytical procedures for RLI HPLC analysis

[0425] HPLC analyses were performed on a Shimadzu LC-20AD HPLC system equipped with a Phenomenex Jupiter 5 µM C18 column (300 Å, 150 x 4.6 mm) heated to 40°C and a SPD- M20A photodiode-array detector. The elution program consisted of a 10-min linear gradient from 20- to 100% CH3CN containing 0.1% TFA with a flow rate of 1 mL / min. U2OS Dimerization Cell Based Assay

[0426] A U2OS cell-based assay kit for IL-2Rβγ binding was performed according to the manufacturer’s instructions (DiscoverX, Part #93-0998E3CP5). Briefly, cells were plated (100 µL, ~5,000 cells / well) in 96 well white-walled assay plates and incubated for 48 hours at 37ºC, 5% CO2. Cells were treated with varying concentrations of the RLI or RLIAPand incubated for an additional 6 hours at 37ºC, 5% CO2. Treated cells were then incubated with the PathHunter reagent substrate for 1 hour at ambient temperature protected from light. Plates were read for chemiluminescence signal detection using a Spectramax i3 plate reader using a 250 ms integration time. ELISA

[0427] The RLI concentrations in plasma were assessed using either a hIL-15 / IL-15Rα complex specific ELISA (R&D Systems, hIL-15 / IL-15Rα complex DuoSet ELISA, Catalog #DY6924) or ELLA protein simple kit (Catalog SPCKB-PS-000500) performed according to the manufacturer’s instructions. Plasma samples were thawed on ice prior to 4- to 10-fold dilution in the provided standard diluent. RLI concentrations were plot as a function of time and fit using GraphPad Prism software. Cell staining protocols

[0428] Pharmacodynamic studies in naïve mice. EDTA whole blood (25 µL) was transferred to a 96 deep well plate and incubated with a fixable viability dye to label dead cells. FcRγII / II receptors were blocked with CD16 / 32 (20 µL, 1.6 µg) before staining for cell-surface antigens (all reagents from Invitrogen). The whole blood samples were incubated at 4°C for 30 minutes with previously determined optimal antibodies concentrations for surface staining of peripheral blood mononuclear cells (PBMCs) (Table A). Red blood cells were lysed and PBMCs were fixed by incubation with 2 mL of 1-Step Fix / Lyse solution for 30 minutes at room temperature. Fixedny-2904315115Docket No.: 67057-20030.40 cells were washed once with 2 mL permeabilization buffer and then intracellularly stained (Ki- 67-APC) using a 30 minute incubation period. After the cells were stained, samples were washed 2X with 2 mL of FACS buffer. The cells were resuspended in 400 µL FACS buffer and stored at 4°C until analysis.

[0429] Tissue immunophenotyping in CT26 tumor bearing mice. Single cell suspensions of splenocytes were obtained from harvested spleens following mechanical disruption and filtering through a 40-µm cell strainer. RBCs were lysed using 1X RBC Lysis Buffer (Invitrogen). Tumor infiltrating lymphocytes (TILs) were obtained by excision and dissociation of tumor tissue. Tumors were minced into small pieces and treated with 3 mg / mL of Collagenase 4 (ThermoFisher) and 0.5 mg / mL of DNase I (Roche) for 30 min. at 37ºC. Then, the dissociated tissue was filtered through a 40-µm cell strainer and cell counts were determined using a Via1 Cassette (ChemoMetec). Single cell suspensions (<2x107cells / ml) were prepared and lymphocytes were purified by centrifugation on a Lympholyte-Mammal Cell Separation Media gradient (Cedarlane) for 20 min at 1250 g, 20ºC with slow acceleration and brakes turned off.

[0430] Aliquots of these cell suspensions (80 µL; ~1x106cells), as well as aliquots of blood (50 µL), were stained for FACS analysis. Briefly, a fixable viability dye was used to label dead cells (LIVE / DEAD Fixable Aqua Dead Cell Stain Kit, Invitrogen), followed by an incubation with CD16 / 32 antibody to block FcRγII / II. Then, samples were surface stained with previously determined optimized Ab concentrations for three different panels (all reagents from eBioscience, Table A). RBCs from blood samples, were lysed with 1x RBC lysis buffer (Invitrogen). Intracellular marker staining was performed using the Foxp3 Transcription Factor Staining Buffer Set (Invitrogen) and following supplier instructions.

[0431] Stained single cell suspensions were read using a Attune NxT flow cytometer (BD Biosciences) and analyzed using FlowJo cytometry analysis software (TreeStar, Ashland, OR). The absolute cell numbers of the samples were determined by direct cell analysis (volumetric counting by the Attune NxT). Positive populations were identified based on fluorescence minus one control. Expression and characterization of RLI

[0432] RLI (>95% pure) was produced at ATUM (Newark, CA) based on previously reported methods (E. Mortier, A. et a., J Biol Chem 281, 1612-1619 (2006); H. Perdreau et al., Eur Cytokine Netw 21, 297-307 (2010)). The purified protein was observed as distinct bands by SDS-PAGE and eluted as two peaks in HPLC chromatograms. When treated with PNGase Fny-2904315116Docket No.: 67057-20030.40 deglycosylase and evaluated by SDS-PAGE, conversion of the upper band into the lower band was observed indicating the higher molecular weight band is glycosylated RLI (Fig. 44). Preparation of MS~RLI conjugates

[0433] Optimization of azido-linker-RLI yield. Small scale reductive alkylation reactions of RLI varying the linker concentration were performed to determine optimal reaction conditions for stoichiometric linker addition to RLI. Reactions (50 µL) used 10 nmol (0.2 mg, 200 µM) RLI in 25 mM Citrate, pH 6.0, 500 mM NaCl and 0.05% tween-20 (Buffer A), 1.5- to 5 equivalents of N3-PEG4-L(MeSO2)-CHO (9 µg to 29 µg, 300 µM to 1 mM) and 750 nmol (47 µg, 10 mM) NaCNBH3. After 20 hours at room temperature, mixtures were treated with 0.5 mM DBCO- PEG5kDafor 4 hours, and analyzed by SDS-PAGE (Fig. 24) (E. L. Schneider et al., Bioconjug Chem 27, 2534-2539 (2016)).

[0434] Preparation of azido-linker-RLI. In 4 mL of Buffer A, reaction mixtures contained 0.8 µmol (18 mg, 200 µM) RLI, 1.6 µmol (0.9 mg, 400 µM) of N3-PEG4-L(MeSO2)-CHO and 40 µmol NaCNBH3 (10 mM). The reaction was allowed to proceed for 20 hours at ambient temperature protected from light. The excess reagents were removed using two 14.5 x 50 mm PD-10 columns (GE Healthcare) previously equilibrated in Buffer A. A small aliquot of the reaction mixture was reacted with DBCO-PEG5kDa for 4 hours and analyzed by SDS-PAGE. The gel-shift assay indicated the sample contained ~39% unreacted-, 51% monoalkylated- and 10% dialkylated-RLI. The reaction mixture was concentrated to ~0.8 mL using an Amicon Ultra 10,000 MW cut-off concentrator and the protein concentration (17 mg, 94% recovery) was determined by A280 (ε280= 22,960 M–1cm1) using a NanoDrop spectrophotometer.

[0435] Preparation of MS~RLI. A slurry of 0.51 mL of BCN-derivatized MSs containing 1.6 µmol BCN / mL in a 10 mL sterile syringe was washed with 5 x 7 mL of Buffer A. The azido- linker-RLI reaction mixture containing 0.74 µmol (17 mg) of ~50% mono-alkylated RLI in 1.5 mL Buffer A was added to the syringe through a 0.22 µm sterile filter. The mixture was rotated end-over end at ambient temperature for 48 hours, and washed with 5 x 6 mL of Buffer A to remove unbound RLI. The unreacted BCN groups were capped by treatment with 0.1 mL of 50 µmol / mL N3-PEG7(Sigma Aldrich) for 24 hours, then MSs with 6 x 7 mL of Buffer A containing 30 mM Met and stored at 4°C. Protein content of the microspheres was determined by A280in 10 mg aliquots of slurry after dissolution in 40 µL of 50 mM NaOH (ε280= 22,960 M–1cm1). The PEG content of the conjugate was determined following dissolution using BaCl2 / I2 spectrophotometry.ny-2904315117Docket No.: 67057-20030.40 Characterization of MS~RLI

[0436] In vitro release kinetics and reverse gelation time of MS~RLI. The release kinetics and reverse gelation time of MS~RLI were determined under accelerated release conditions. The MS~RLI slurry was washed twice using a 25-fold dilution in 125 mM borate buffer pH 9.4. After the second wash, MS~RLI was diluted 10-fold in 125 mM sodium borate pH 9.4, and incubated at 37°C. At predefined time intervals, the reaction mixture was centrifuged and the supernatant was sampled. A Nanodrop spectrophotometer was used to determine the A280of supernatant (ε280= 22,960 M–1cm1). Each sample was also analyzed by HPLC. The release rate was calculated by fitting the released A280vs time or RLI peak area vs time to a first-order rate equation in GraphPad Prism 8.0, and the t1 / 2 of RLI release at pH 7.4 was calculated using the equation: t1 / 2, pH 7.4= t1 / 2, pHx 10(pH-7.4)The in vitro solubilization of the MSs was monitored by quantitating the amount of PEG in the supernatant by BaCl2 / I2 spectrophotometry. Time matched samples (5 µL) of the reaction supernatant from the in vitro release assay were diluted 20-fold in water. The diluted sample (100 µL) was acidified with perchloric acid (0.5 M, 500 µL). In a 96 well plate, the acidified sample (200 µL) was treated with a solution (75 µL) of BaCl2 (3.3% w / v), I2 (0.06%) and KI (0.12%) and the absorbance was measured at 535 nm. The amount of PEG in the solution was calculated based on a linear standard curve generated by the same procedure using a solution of linear PEG8KDa (7.5 – 60 µg / mL).

[0437] Purity of RLI on MS~RLI. Purity of the RLI conjugated to the microsphere was determined by monitoring the released proteins at pH 9.4, 37ºC by HPLC. The MS~RLI slurry was washed two times and diluted in 125 mM sodium borate pH 9.4, and incubated at 37 ºC. At intervals spanning over 8 h , (~1 half-life of release) samples were centrifuged and 20 µL of the supernatants were analyzed by HPLC. The fraction of each released protein was determined as peak area / total peak areas, was plotted. The y-intercept of the plot reveals the fraction of each protein present on the MSs at t=0 (Figure 26).

[0438] Bioactivity of RLIAP. In a 1.5 mL microcentrifuge tube, 50 µL of the MS~RLI conjugate (0.2 mg, 8.7 nmol) was washed 1x with PBS pH 7.4 and then diluted 10-fold in the same buffer. To obtained RLIAP, the washed microsphere conjugate was incubated in a 37ºC water bath for 10 days. The supernatant of the reaction mixture, containing RLIAP, was concentrated to 0.18 mg / mL (7.8 µM) using an Amicon Ultra 3,500 MW cut-off concentrator.ny-2904315118Docket No.: 67057-20030.40 The in vitro activity of RLIAP compared to RLI was assessed using the U2OS cell-based assay kit for IL-2Rβγ binding. In vivo studies

[0439] Preparation of dosing solutions. Dosing solutions were prepared by diluting the MS~RLI slurry in 25 mM Na citrate buffer pH 5.9 containing 500 mM NaCl, 0.05% tween-20 and 1.25% (w / v) hyaluronic acid. The RLI concentration was confirmed by diluting aliquots of the MS~RLI dosing solution (~20 µL) in 50 mM NaOH (80 µL) and incubated for 1 hour at room temperature. Following a 1 hour room temperature incubation, the reaction mixture (20 µL) was analyzed using standard HPLC methods. A standard curve was prepared using native RLI. RLI standards (0.047 – 0.856 mg / mL) were prepared from 2-fold serial dilutions following the same procedure. The concentration of RLI in each dosing solution was determined by peak area interpolation of the standard curve.

[0440] Pharmacokinetics of MS~RLI. Syringes with fixed needles (27 G) were backfilled with the MS~RLI conjugate (100 µL). The contents of the syringes were administered SC to either normal, male C57BL / 6J mice or male NSG mice 6-8 weeks old. For studies in C57BL / 6J mice, blood samples were collected in EDTA collection tubes at -48, 8, 24, 48, 96, 120, 168 and 240 hours from alternating groups of mice (n=3 / group). For studies in NSG mice, blood samples were collected in EDTA collection tubes at -48, 8, 24, 48, 72, 96, 120, 168, 240, 288, 336, 360, 408,456, 504, 552, 624 and 672 hours from alternating groups of mice (n=3 / group). HALT protease inhibitor cocktail was added to all samples and the plasma frozen at –80°C until analysis by ELISA.

[0441] Pharmacodynamic Studies. Naïve, male C57BL / 6J mice (n=4-5 / group) 6-8 weeks old were administered SC MS~RLI (1 – 20 µg) or MS~IL-1550µg in the mid-back or a single IP dose of free RLI (10 µg), or two IP doses of 2 µg RLI (D0 and D2). Mice that were administered sequential doses of MS~RLI or free RLI, were administered a cycle of treatment starting on Day 35. Blood samples were drawn on day -2, 2, 5, 7, 14, 21 and 28. For mice that received multiple doses, additional blood samples were collected 2, 5, 7, 14, and 21 days post second injection. PBMCs were prepared and immunophenotyped with an Attune NxT flow cytometer using an 8 color T cell panel to quantitate B cells, NK cells, CD3, CD4, CD8, and CD44 expressing cells. The total AUC for each cell phenotype was determined using Prism. Then, the baseline cell count x 28 was subtracted to yield the AUC28d.ny-2904315119Docket No.: 67057-20030.40

[0442] Skin toxicity of MS~RLI. On Day 0, C57BL / 6J or NSG mice were weighed, anesthetized with isoflurane, and their backs were shaved. MS~RLI (10 – 30 µg, 0.43 – 1.3 nmol) was injected SC into the flank of each mouse (n=6-15 / group); a control group (n=5) received a single SC injection of RLI (20 µg, 0.9) in the flank. The location of each injection was marked with a sharpie and reapplied as needed to monitor the site over 28 days. If an injection site lesion formed, the injection site was photographed and the mouse euthanized. All surviving mice were sacrificed 28 days after injection. Therapeutic Studies

[0443] CT26 model. CT26 tumors were established in the flank of female BALB / c mice by injection of 1 x105in 100 µL of serum-free medium. When the tumor volume reached ~100mm3mice were randomized into groups (n=8 / group). Mice were administered a 50 µL intra-tumoral injection of empty microspheres or 10 µg MS~RLI or a 50 µL subcutaneous injection of 10 µg MS~RLI. The tumor volume was measured by calipers and calculated by the equation: V = 1 / 2(long dimension)(short dimension)2. On day 5 post treatment, mice (n=4 / group) were sacrificed and EDTA whole blood, tumors and spleens were harvested for immunophenotyping. Kaplan-Meier mouse survival plots were generated based on the mouse survival, monitored based on humane end point criteria. Orthotopic EO771 tumor bearing mice. EO771 cells (3x105cells) were orthotopically inoculated into the 4th mammary pad of C57BL / 6 mice. When the tumors reached ~40 mm3in size, a single dose of MS~IL-15 (1.2 μg or 6 μg) or MS~RLI (2 μg or 10 μg) was administered intratumorally in a volume of 50 µL. Empty microspheres (50 µL) were used as the negative control and were administered intratumorally. Blood was collected at 3 timepoints as follows: 6 days before treatment, one day after the first injection of the treatment and at the end of experiment (Fig. 36). Tumor volume (mm3) was measured by calipers and calculated by the following equation: L*W*H*p / 6. All mice were sacrificed on day 15 of the treatment. Tumor- infiltrating immune cells and splenocytes were analyzed by flow cytometry. Lungs were embedded in paraffin and the metastatic lesions were evaluated by hematoxylin / eosin staining. Example 16B Preparation and in vitro characterization of MS~RLI

[0444] The preparation of a long-acting RLI was sought out by attaching the agonist to hydrogel microspheres via a β-eliminative releasable linker. To preclude spontaneousny-2904315120Docket No.: 67057-20030.40 deamidation at N77, the N77A substitution was introduced into the protein sequence (Nellis DF et al., Pharm Res. (2012) 29:722–38). Then, the MS~RLI prodrug was prepared following the strategy used for the analogous MS~IL-15 (Fig. 23) ( J. A. Hangasky et al., J Immunother Cancer 10 (2022)).

[0445] First, site-specific reductive alkylation of RLI using NaCNBH3 was used to attach an azido-linker carbamate of aminopropyl aldehyde to the N-terminus (O. Kinstler, et al., Adv Drug Deliv Rev 54, 477-485 (2002)). Small scale reactions showed that a linker / RLI of 2:1 was optimal to give the highest balanced yield of monoalkylated N3-linker-RLI (Fig. 24). Then, 18 mg of RLI was treated with 2 equivalents of N3-linker-aldehyde and 10 mM NaCNBH3at pH 6.0. A PEG-shift SDS-PAGE assay ( E. L. Schneider et al., Bioconjugate chemistry 27, 2534- 2539 (2016)) showed ~45% unreacted RLI, ~55% RLI having one PEG, and ≤5% with more than one PEG; thus, ≥90% of the alkylated product was the desired monoalkylated RLI. Without further purification, the N3-linker-RLI was coupled to BCN-derivatized tetra-PEG microspheres by SPAAC, and unreacted BCN groups were capped with N3-PEG7. The microspheres were extensively washed to remove unbound RLI and exchanged into pH 6.0 buffer containing 30 mM Met, and stored at 4°C. The packed slurry of 1 mL contained 4.2 mg / mL RLI (184 nmol) and had a PEG content of 3.5 mg / mL. Thus, the packed MS slurry contained 52 nmol RLI / mg PEG indicating the microspheres were loaded to 52%.

[0446] The MS~RLI were characterized in vitro to ensure the release rate was suitable and that the released RLIAPwas biologically active. At pH 9.4, the base-catalyzed β-elimination of the MS~RLI resulted in complete release of RLIAP, with a t1 / 2,pH9.4of 7 h corresponding to 700 h at pH 7.4 (Fig. 25). The MS gel contained GDM DMS modulators in the crosslinks, and the in vitro time to reverse gelation (tRG) at pH 9.4 was 28 h, corresponding to 2,800 h at pH 7.4 (Fig. 25). The purity of RLI on the MS~RLI assessed by HPLC quantitation of released protein vs time at pH 9.4 was ≥93% ( Hangasky JA et al., Immunother Cancer. (2022) 10:e004104-18.) (Fig. 26). Finally, using an engineered U2OS cell line expressing the human IL-2 / IL-15Rβ / γc the RLIAPreleased from the MS at pH 7.4 retained an EC50equipotent to native RLI, demonstrating that coupling to and the release of RLI from microspheres does not affect receptor engagement (Fig. 27). These results showed that MS~RLI slowly releases RLIAP with a long in vitro t1 / 2, that the released RLIAPretained full bioactivity, and that the MS~RLI was suitable for in vivo studies. Example 16 MS~RLI increases the t1 / 2 and exposure of RLIAPny-2904315121Docket No.: 67057-20030.40

[0447] To evaluate the PK properties of MS~RLI in mice, plasma concentrations of RLIAP were determined after a single SC dose (Fig. 28). The C vs t plot of MS~RLI10µg in C57BL / 6J immunocompetent mice showed a terminal t1 / 2of ~30 h for the released RLIAP. This is a 10-fold increase in t1 / 2 over free RLI (3 h), but much lower than expected from the 700 h t1 / 2 for in vitro cleavage. It has been observed that MS~drug conjugates typically show in vivo half-lives of release that are ~2- to 3-fold lower than the in vitro release ( D. V. Santi et al., Proc Natl Acad Sci U S A 109, 6211-6216 (2012)). Moreover, although the MS~RLI conjugate uses the same linker (Mod = MeSO2-) as MS~IL-15, it shows a much faster rate of release (t1 / 230 vs 168 h). Without wishing to be bound by theory, the higher than expected clearance of RLI could be due to the rapid formation of an immune cell cytokine sink ( J. A. Hangasky et al., Front Immunol 11, 1813 (2020)). Therefore, the PK of MS~RLI10µg was determined in NSG mice lacking IL- 2 / IL-15Rγc and deficient in NK, B and T cells. The C vs t plot of the RLIAP released from MS~RLI10µgin NSG mice showed sustained plasma concentrations for at least one month with a t1 / 2 of ~700 h (Fig. 28).This >20-fold increase of t1 / 2 in immunodeficient mice is consistent with the presence of an immune-cell cytokine sink at the onset, or induced very rapidly by the RLIAP in immunocompetent mice. The above results show that a) the in vivo t1 / 2of RLIAPwas extended 10-fold compared to RLI, b) the observed t1 / 2 was shorter than expected from the same linker used with MS~IL-15, and c) without wishing to be bound by theory, the short t1 / 2 of released RLIAPis likely due to an immune cell cytokine sink.

[0448] The construction of a pharmacokinetic model that supports the pharmacokinetic data was next sought out. The standard equation describing the plasma concentration of a drug released from a depot in a first-order process with rate constant k1 and subsequently eliminated from the plasma in a first-order process with rate constant k2(Eqn 1) was modified to include a time-dependent elimination rate constant (Eqn 2) wherein a basal non-target mediated elimination rate, kbasal, (renal filtration, for example) is augmented by an immune cell-mediated elimination rate, kIC, that is a function of the number of immune cells present. Without wishing to be bound by theory, it is assumed that upon stimulation by the released cytokine, the immune cells initially present (N0) begin expanding with a doubling time Td, such that the rate of elimination of the cytokine from the plasma increases with time. C(t) = Dose•F / Vd• k1 / (k2– k1) • (e-k1t– e-k2t) (Eqn 1) k2(t) = kbasal + N0 • kIC • Ʃ(2t / Td) (Eqn 2)

[0449] For mixed immune cell populations having different doubling times, for example NK cells and T cells, the total cytokine sink is taken as the sum of the different cell populations eachny-2904315122Docket No.: 67057-20030.40 with its own Td. The simple form of the model assumes that each immune cell type is equivalent in its ability to clear cytokine.

[0450] As NSG mice are NK and T cell-deficient (N0= 0), the value of k2that best fits the data from NSG mice is taken as kbasal. This then allows a determination of the cytokine release rate (t1 / 2 = 700 h) and basal plasma clearance (0.75 mL / h) from fitting the data to a dose of 10 µg (0.44 nmol). Basal plasma clearance was parsed into Vd / F = 21.6 mL and t1 / 2(basal)= 20 h based on the observed Tmax = 72 h. This Vd / F is in agreement with the value of 26 mL reported for i.p. administration of RLI in C57BL / 6J mice (A. Bessard, et al., Mol Cancer Ther 8, 2736-2745 (2009)). Thus, the high release rate of MS~RLI in immune-competent mice could be closely simulated by a target-mediated drug disposition model (TMDD), where the amount of target increases exponentially over time due to RLI-stimulated NK and T cell expansion – a “cytokine sink”. Example 17 MS~RLI induces high and prolonged CD8+and NK cell expansion in naïve mice

[0451] The effects of MS~RLI on the target immune cells – NK, CD8+and CD44hiCD8+T cells – were then determined and compared with those of MS~IL-1550µg. The effects of 1- to 10 µg MS~RLI were studied on the expansion of immune cells and compared to the optimal 50 µg dose of MS~IL-1550µg (Fig. 29). The effects at the lower doses, 1-to 2.5 µg, were near- indistinguishable from control. The highest 10 µg dose (~8.8-fold lower equivalents than MS~IL-1550µg) more effectively expanded NK cells, but showed comparable expansion of CD44hiCD8+T cells. Although a saturating target immune cell response was not observed and 10 μg MS~RLI was well tolerated (Figure 31), higher doses resulted in injection site toxicities preventing longitudinal PD measurements. Thus, MS~RLI10µgwas selected as a standard dose for subsequent studies to avoid toxicities and synchronize effects with MS~IL-1550µg on CD44hiCD8+T cells.

[0452] Next, the effects of MS~RLI10µgwere quantified on the target immune cells and compared to those of MS~IL-1550µgand a bolus injection of free RLI. Figure 30A and Figure 30B show the longitudinal immune cell expansion, and Table 4 quantitates the expansion over time by a previously reported AUC method ( J. A. Hangasky et al., J Immunother Cancer (2022) 10:e004104-18)). First, MS~RLI10µgwas compared to 10 µg free RLI. MS~RLI10µgcaused the same or slightly longer duration of expansion of target immune cells, a ~2-fold increase in the CD44hiCD8+T cell AUC28d, a high 10-fold expansion in the NK cell AUC28d, and a ~12-foldny-2904315123Docket No.: 67057-20030.40 increase in the Ki67+NK cell AUC28d. Next, MS~RLI10µg was compared to the long-acting MS~IL-1550µg. MS~RLI10µg had a similar expansion of CD44hiCD8+T cells compared to MS~IL-1550µg, but a 2-fold higher NK cell AUC28d, and a 3-fold higher AUC28dfor Ki67+NK cells. Finally, a second dose of MS~RLI at d 35 failed to induce proliferation of both NK and CD44hiCD8+T cells; other agonists predominantly cause only NK hypo-responsiveness (Fig. 32) (Frutoso M. et al., J Immunol. (2018) 201:493–506)). Therefore, long-acting MS~IL-15 and MS~RLI both exhibit potent and prolonged immunostimulatory effects on NK cells and CD44hiCD8+ T cells. However, achieving these PD responses requires lower doses of MS~RLI. Table 4. Duration of immune cell expansion and AUC28d in PBMCs.Example 18 Local toxicity of MS~RLI

[0453] The injection site toxicity of MS~RLI was assessed in both immunocompetent and immunodeficient mice. When dosed SC in immunocompetent C57BL / 6J mice, at ≥ 20 µg MS~RLI resulted in ≥90% (26 / 30) of mice forming observable injection site lesions lasting 7- to 14 days post dose administration. Without wishing to be bound by theory, it was suspected that high local concentrations of RLI was inducing a strong local immune cell activation at the injection site. Indeed, the 10 µg dose used here for PD studies showed only 2.5% (1 / 40) of mice showing injection site toxicity; no lesions (0 / 10) were observed with an equimolar bolus dose of RLI which does not remain at the injection site. H&E staining of the injection site in mice receiving ≥20 µg MS~RLI revealed a range of histologic findings including mononuclear inflammation, epidermal hyperplasia and transmural coagulative necrosis of the dermis, subcutis and brown adipose tissue. Without wishing to be bound by theory, it was suspected that high local concentration of RLI was inducing a strong immune cell activation at the injection site.ny-2904315124Docket No.: 67057-20030.40 Accordingly, there were no observable injection site lesions in immunodeficient NSG mice (n=0 / 10) at 30 µg MS~RLI, indicating that the observed lesions result from the high, prolong exposure from MS~RLI. Thus, while 10 μg MS~RLI appears safe and effective, ≥20 µg MSI~RLI causes injection site reactions consequent to immune cell activation by the slow- releasing depot – a therapeutic index of only ~2. Example 19 Intra-tumoral administration of MS~RLI and MS~IL-15 have anti-metastatic activity

[0454] In view of the skin necrosis and consequent low therapeutic index for SC MS~RLI, the effects of intra-tumoral (IT) administration were investigated. Initially, the effects of IT MS~RLI on both CT-26 colon carcinoma and EO771 TNBC were examined to assess which was most appropriate for IT studies; the effects of systemic RLI on CT-26 ( M. Desbois et al., IJ Immunol 197, 168-178 (2016)); and IT hetIL-5 on EO771 ( D. Stellas et al., Cell Rep 42, 112501 (2023)) have been reported. CT26 tumor bearing mice well tolerated both SC and IT 10 μg MS~RLI with no signs of body weight loss (Figure 34). However, SC MS~RLI had no effect on tumor growth, whereas IT administration resulted in 40% tumor (Figure 33A and Figure 33B). It was also observed that both SC and IT MS~RLI caused a ~2-fold increase in the total number of NK cells and high levels of proliferating NK cells in the tumor, blood and spleen (Figure 35A, Figure 35B, and Figure 35C). Without wishing to be bound by theory, it is understood that the systemic effect of the high 10 μg IT MS~RLI is likely a manifestation of exposure after released RLIAPexited the tumor. In early studies of EO771 tumors, the same 10 μg dose of IT MS~RLI caused a similar TGI as in CT-26 tumors. Because both tumors had similar responses to IT MS~RLI, EO771 was selected for further focus to benefit from the information reported from studies of IT administration of the IL-15 agonist hetIL-15 (D. Stellas et al., Cell Rep 42, 112501 (2023)).

[0455] The effects of single IT doses of MS~RLI and MS~IL-15 were studied on the hetIL- 15-sensitive EO771 (D. Stellas et al., Cell Rep 42, 112501 (2023)). Mice bearing ~40 mm3EO771 orthotopic tumors were treated with single IT doses of 5 or 10 µg MS~RLI or equimolar doses of 1.2 or 6 µg MS~IL-15 (Figure 36). IT MS~IL-15 and MS~RLI were well tolerated with no observed body weight loss (Figure 37). Treatment with 10 µg IT MS~RLI resulted in a moderate ~42% TGI and a 76% reduction in terminal tumor weight, whereas equimolar MS~IL- 15 gave only ~20% TGI and insignificant ~30% tumor weight reduction (Figure 38A and Figure 39). Despite the fact that only the high dose MS~RLI showed significant TGI, histological analysis of lung tissues showed a significant reduction in the number of metastatic lesions inny-2904315125Docket No.: 67057-20030.40 mice treated with 1.2 μg MS~RLI IT or 2 μg MS~IL-15 IT (Figure 38B). Complete blood count of tumor-bearing animals on days -6, 2, and 14, showed in general no significant changes in circulating white blood cell populations. A ~2-fold increase in the %CD8+T cells in splenocytes was observed after IT MS~RLI and IT MS~IL-15 treatment (Figure 40 and Figure 41). Taken together, these results show that IT administration of the long-acting MS~RLI / IL-15 conjugates caused moderate tumor reduction and high anti-metastatic effects. Example 20 Summary of Experiments with MS~RLI and MS~IL-15

[0456] SC administration of a long-acting MS conjugate of the IL-15 superagonist RLI releases RLI with a half-life of 30 h – long compared to most IL-15 agonists (J. A. Hangasky et al., Front Immunol 11, 1813 (2020)), but short compared to MS~IL-15 because of a hyper-active cytokine sink. The released cytokine shows potent stimulation of NK and CD44hiCD8+cells, but causes skin lesions at doses ~2-fold higher than its most effective dose. Loco-regional IT injection of MS~RLI causes expected local effects of IL-15RAs and a highly effective anti- metastatic effect. A combinations of IL-15 with systemic treatment by other agents could serve the dual purpose of antitumor and anti-metastatic activities without toxic effects of systemic IL- 15 agonists. The anti-metastatic effects of IT-administered long-acting IL-15 agonists counters two of the major criticisms of loco-regional therapy: the need for frequent injections, and the enigma of managing metastasis (A. Marabelle et al., Ann Oncol 29, 2163-2174 (2018)); Ann Oncol 29, 2163-2174 (2018) et al., Ann Oncol 29, 2163-2174 (2018)).

[0457] All publication, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entireties, to the same extent as if each were incorporated by reference individually.

[0458] It is to be understood that, while the disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.ny-2904315126

Claims

Docket No.: 67057-20030.40 CLAIMS What is claimed is:

1. A method of treating cancer in a subject in need thereof, comprising intratumorally and / or peritumorally administering to the subject a pharmaceutically acceptable amount of a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI).

2. The method of claim 1, wherein the drug (D) component of the hydrogel is a small molecule, peptide, peptide-drug conjugate, protein, protein-drug conjugate, antibody, bispecific or trispecific antibody, PROTAC, or antibody-drug conjugate.

3. The method of claim 1, wherein D is a toll-like receptor agonist, exatecan or a prodrug having the formula HN(R7)CH2Q wherein R7is H, optionally substituted (C1-C6) alkyl, optionally substituted aryl, or optionally substituted heteroaryl; and Q is.

4. The method of claim 1, wherein D is SN38.

5. The method of claim 1, wherein D is an antibody or antibody-drug conjugate.

6. The method of claim 5, wherein D is an anti-PD1 antibody.

7. The method of claim 5, wherein D is an anti-PDL1 antibody.

8. The method of claim 5, wherein D is an anti-CTLA4 antibody.

9. The method of claim 1, wherein D is a cytokine.

10. The method of claim 9, wherein D is IL-15.

11. The method of claim 9, wherein D is receptor-linked IL-15 (RLI).ny-2904315127Docket No.: 67057-20030.40 12. The method of any one of claims 1-11, wherein the hydrogel is administered once weekly.

13. The method of any one of claims 1-11, wherein the hydrogel is administered biweekly.

14. The method of any one of claims 1-11, wherein the hydrogel is administered once every month.

15. The method of any one of claims 1-11, wherein the hydrogel is administered once every two months.

16. The method of any one of claims 1-11, wherein the hydrogel is administered once every three months.

17. The method of any one of claims 1-11, wherein the hydrogel is administered once every four months.

18. A method of treating cancer in a subject in need thereof, comprising intratumorally and / or peritumorally administering to the subject a pharmaceutically acceptable amount of a hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI) in combination with a second agent, wherein the second agent is administered systemically or subcutaneously.

19. The method of claim 18, wherein the second agent is a small molecule, peptide, protein, antibody, or antibody-drug conjugate.

20. The method of claim 18, wherein the drug (D) of the hydrogel comprises a conjugated topoisomerase I inhibitor and the second agent is a PARP inhibitor or an antibody.

21. The method of claim 20, wherein the topoisomerase I inhibitor is SN38.

22. The method of claim 20 or 21, wherein the PARP inhibitor is talazoparib, rucaparib, nirabarib, or olaparib.

23. The method of claim 18, wherein both the drug (D) of the hydrogel and the second agent are antibodies.ny-2904315128Docket No.: 67057-20030.40 24. The method of claim 23, wherein the drug (D) is an anti-CTLA4 antibody and the second agent is an anti-PD1 antibody.

25. The method of claim 23, wherein the drug (D) is an anti-CTLA4 antibody and the second agent is an anti-PDL1 antibody.

26. The method of claim 23, wherein the drug (D) is an anti-PD1 antibody and the second agent is an anti-CTLA4 antibody.

27. The method of claim 23, wherein the drug (D) is an anti-PDL1 antibody and the second agent is an anti-CTLA4 antibody.

28. The method of any one of claims 18-27, wherein the hydrogel is administered once weekly.

29. The method of any one of claims 18-27, wherein the hydrogel is administered biweekly 30. The method of any one of claims 18-27, wherein the hydrogel is administered once every month.

31. The method of any one of claims 18-27, wherein the hydrogel is administered once every two months.

32. The method of any one of claims 18-27, wherein the hydrogel is administered once every three months.

33. The method of any one of claims 18-27, wherein the hydrogel is administered once every four months.

34. The method of any one of claims 28-33, wherein the second agent is administered more frequently than the hydrogel.

35. The method of claim 34, wherein the second agent is administered once daily.

36. The method of any one of claims 1-35, wherein the electron-withdrawing groups of the hydrogel of formula (IV), (VII), (XXII), (XXIII), (XXIV), (XXV), (XXVI), (XXII), (XXIII), (XXIX), (XXX), or (XXXI) are selected from the group consisting of CN and SO2R3, wherein R3is optionally substituted C1-C6alkyl or cycloalkyl, optionally substituted aryl, optionallyny-2904315129Docket No.: 67057-20030.40 substituted heteroaryl, or N(R5)2 wherein each R5is independently H or optionally substituted C1-C6 alkyl or cycloalkyl.

37. The method of any one of claims 1-36, wherein the hydrogel is administered intratumorally.

38. The method of any one of claims 1-36, wherein the hydrogel is administered peritumorally.ny-2904315130

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