Combination cancer immunotherapy with hydrogels

Abstract

Compositions and methods for treatment of cancer are disclosed herein. In some embodiments, a composition for treating cancer includes a first dynamic hydrogel having a polymer non-covalently crosslinked with a plurality of nanoparticles and a first immunotherapeutic agent, and a second dynamic hydrogel having a polymer non-covalently crosslinked with a plurality of nanoparticles and a second immunotherapeutic agent, where the first dynamic hydrogel may be configured to be administered within a tumor in a subject to form an intratumoral depot and the second dynamic hydrogel may be configured to be administered to the subject external to the tumor to form a peritumoral depot.

Description

Atorney Docket No.: 079445-015310PC-1533440Stanford Ref. No. S24-412 FortemRef. No. APL.014WOCOMBINATION CANCER IMMUNOTHERAPY WITH HYDROGELSCROSS REFERENCE TO RELATED APPLICATION(S)

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 759,986, filed February 18, 2025, the disclosure of which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] The present technology generally relates to drug delivery, and in particular, to combination cancer immunotherapy with hydrogels.BACKGROUND

[0003] Despite the incredible progress made in recent decades in developing new cancer immunotherapies, cancer remains an immense healthcare burden; in 2024, cancer was the second leading cause of death in the United States, with projections of over two million new diagnoses and over 600,000 cancer fatalities. Even more, the incidence of six of the top ten cancers is increasing. These trends disproportionately affect countries with lower development indices, indicating the need for broadly translatable next generation immunotherapies.[0004 [ Advances in immunotherapy have revolutionized the current standard of care for cancer patients, but unfortunately, most approaches still fail to mount a robust anti-cancer effect. This is in part due to a highly immunosuppressive tumor microenvironment which has developed bio-orthogonal mechanisms of immune escape. To address this challenge, the field has turned to combination immunotherapies, but systemic administration of potent combination therapies has resulted in severe immune related adverse effects and toxicity. Combination immunotherapies may benefit from administration of immune agonists in a way that more closely resembles the endogenous cancer-immunity cycle, a tightly regulated sequence of cues in both space and time.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on illustrating clearly the principles of the present disclosure.

[0006] FIG. 1 illustrates polymer nanoparticle (PNP) hydrogels formed through the interactions of PEG-PLA nanoparticles (NPs) and dodecyl-modified hydroxypropyl methylcellulose polymers (HPMC-C12).

[0007] FIG. 2A is a schematic illustration of the endogenous cancer-immunity cycle.

[0008] FIG. 2B is a schematic illustration of a system including an intratumoral (IT) depot and a peritumoral (PT) depot to modulate the cancer-immunity cycle.

[0009] FIGS. 3A-3L illustrate data demonstrating that PNP hydrogels increased site specific cargo accumulation. Data are reported as mean + / - SEM. Statistics are ordinary oneway ANOVA run in GraphPad Prism with Tukey’s multiple comparisons test.

[0010] FIG. 3A is a schematic of an experimental hypothesis indicating that PNP hydrogels can be used to selectively distribute immunotherapies as a function of site of administration, comparing IT and PT injection.

[0011] FIG. 3B is a schematic of a PNP hydrogel with HPMC-C12 and PEG-PLA nanoparticles as functional building blocks.

[0012] FIG. 3C shows an experimental timeline, groups, and doses for tumor induction, treatment, and tumor or lymph node excision and imaging via an in vivo imaging system (N=4 per group per timepoint).

[0013] FIG. 3D is a series of representative images showing excised tumors on days 1, 3, and 5 from each treatment group (left) and quantification of total exposure via area under the curve of total flux in the tumor over 7 days (right).

[0014] FIG. 3E is a series of representative images of excised tumor draining lymph nodes (tdLNs) on days 1, 3, and 5 from each treatment group (left) and quantification of total exposure via area under the curve of total flux in the tumor over 5 days (right).

[0015] FIG. 3F is a graph showing the total flux in excised tumors for each time point.

[0016] FIG. 3G is a graph showing the total flux in excised tumor draining lymph nodes for each time point.

[0017] FIG. 3H is a photograph illustrating qualitative inspection of MC38 tumor burden upon excision on day 3 after treatment for fluorescent imaging.

[0018] FIG. 31 is a graph showing the quantification of average radiance in excised tumors for each time point.

[0019] FIG. 3J is a graph showing the ratio of average radiance of different routes of administration to systemic administration in excised tumors over time.

[0020] FIG. 3K is a graph showing the quantification of average radiance in excised tumor draining lymph nodes for each time point.

[0021] FIG. 3L is a graph showing the ratio of average radiance of different routes of administration to systemic administration in excised tumor draining lymph nodes over time.

[0022] FIGS. 4A-5C illustrate data demonstrating that IT cytokine immunotherapy extends survival over PT administration. Survival curves were compared by log-rank Mantel-Cox test.

[0023] FIG. 4A is a schematic showing an experimental overview of the tumor induction, treatment, and measurement timeline (N=10 for all groups).

[0024] FIG. 4B is a series of graphs showing tumor growth over 25 days post tumor inoculation for mice treated with empty gel, IT gel, PT gel, or IT soluble containing 5 pg of IL- 12, left to right, respectively.

[0025] FIG. 4C is a graph showing a survival curve comparing the therapeutic effect of IT gel to IT soluble administration of cytokine.

[0026] FIG. 4D is a graph showing a survival curve comparing the therapeutic effect of IT gel to PT gel delivery of cytokine.

[0027] FIG. 4E is a series of graphs illustrating animal weight monitoring post treatment for all studies as a proxy for toxicity. Weight loss is shown for animals in studies corresponding to (i) IL- 12 monotherapy study reported in FIG. 4 A, (ii) dose response study of IL- 12 at 20 pg (high) and 5 pg (low), (iii) IL-12 / IL-2 cytokine study reported in FIG. 5 A, (iv) OX40a monotherapy study reported in FIG. 13 A, (v) cytokine and antibody combination therapy study done in Bl 6F 10 reported in FIG. 21 A, (vi) cytokine and antibody combination study done in MC38 reported in FIG. 21 A, and (vii) single-site cytokine and antibody combination study reported in FIG. 22A.

[0028] FIG. 5A is a schematic showing an experimental overview of the tumor induction, treatment, and measurement timeline (N=7 for vehicle control, N=6 for untreated, N=10 for all others). Cytokine treatment consisted of 5 pg IL-2 and 20 pg IL- 12.

[0029] FIG. 5B is a series of graphs showing tumor growth curves through day 20 post inoculation of IT administered empty gel with immune checkpoint blockade (ICB), IT administered PNP hydrogel with cytokines in combination with ICB, untreated, and PT administered PNP hydrogel with cytokines in combination with ICB, from left to right, respectively.

[0030] FIG. 5C is a graph showing the probability of survival through 50 days post tumor inoculation.[0031| FIGS. 6A-8B illustrate data from intratumoral hydrogel administration of cytokines. N=6 for all groups. Data is reported as mean + / - SEM. Values are reported as percentages of CD45+cells (CD4+and CD8+T cells) or of parent T cell population. Statistics are determined by two-tailed Mann Whitney test in GraphPad Prism.

[0032] FIG. 6A is a plot showing a representative population of CD8+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0033] FIG. 6B is a series of graphs showing quantification of the percentage of CD8+T cells (upper left), quantification of the percentage of CD44+CD62L+central memory T cells (upper right), quantification of the percentage of CD44+CD62L effector memory T cells (lower left), and quantification of the percentage of CD44 CD62L T cells (lower right).

[0034] FIG. 6C is a plot showing representative populations of interferon-gamma (IFNy) producing CD8+T cells gated on CD8a and IFNy.

[0035] FIG. 6D is a graph showing quantification of the percentages of IFNy+CD8+T cells.

[0036] FIG. 7A is a plot showing a representative population of CD4+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0037] FIG. 7B is a series of graphs showing quantification of the percentages of CD4+T cells (upper left), quantification of the percentages of CD44+CD62L+central memory T cells (upper right), quantification of the percentages of CD44+CD62L effector memory T cells (lower left), quantification of the percentages of CD44 CD62L T cells (lower middle), and quantification of the percentages of IFNy CD4+T cells (lower right).

[0038] FIG. 8A is a plot showing a representative population of FOXP3+regulatory T cells gated on CD25 and FoxP3.

[0039] FIG. 8B is a graph showing quantification of percentages of regulatory T cells.

[0040] FIG. 9 is a series of graphs showing flow cytometry data of tumors on day 7 post cytokine therapies.

[0041] FIG. 10 is a series of graphs showing flow cytometry data of tdLNs on day 7 post cytokine therapies.

[0042] FIG. 11 is a series of graphs showing flow cytometry data of tumors on day 3 post cytokine therapies.

[0043] FIG. 12 is a series of graphs showing flow cytometry data of tdLNs on day 3 post cytokine therapies.

[0044] FIGS. 13A-13C illustrate data demonstrating that PT administration of OX40a controlled tumor burden and extended murine survival in a B16F10 tumor induction model.

[0045] FIG. 13 A is a schematic showing an experimental scheme (N=6 for IT OX40a, N=8 for all other groups; one animal from the PT OX40a group was censored on Day 31 due to the development of an ulcer).

[0046] FIG. 13B is a series of graphs showing tumor growth curves through day 20 post inoculation of IT administered OX40a with ICB, PT administered OX40a with ICB, ICB only, and untreated, left to right respectively.

[0047] FIG. 13C is a graph showing probability of survival through 50 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test.

[0048] FIGS. 14A-16B illustrate data demonstrating that PT administration of OX40a hydrogels augments anti-cancer T cell phenotypes. N=6 for all groups. Data is reported as mean + / - SEM. Values are reported as percentages of CD45+cells (CD4+and CD8+T cells) or of parent T cell population. Statistics are determined by two-tailed Mann Whitney test in GraphPad Prism.

[0049] FIG. 14A is a plot showing a representative population of CD8+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0050] FIG. 14B is a series of graphs showing quantification of the percentage of CD8+T cells (upper left), quantification of the percentage of CD44+CD62L+central memory T cells(upper right), quantification of the percentage of CD44+CD62L effector memory T cells (lower left), and quantification of the percentage of CD44 CD62L T cells (lower right).

[0051] FIG. 14C is a plot showing representative populations of IFNy producing CD8+T cells gated on CD8a and IFNy.

[0052] FIG. 14D is a graph showing quantification of the percentages of IFNy CD8+T cells.

[0053] FIG. 15A is a plot showing a representative population of CD4+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0054] FIG. 15B is a series of graphs showing quantification of the percentages of CD4+T cells (upper left), quantification of the percentages of CD44+CD62L+central memory T cells (upper right), quantification of percentages of CD44+CD62L effector memory T cells (lower left), quantification of the percentages of CD44-CD62L+T cells (lower middle), and quantification of the percentages of IFNy CD4+T cells (lower right).

[0055] FIG. 16A is a plot showing representative populations of FOXP3+regulatory T cells gated on CD25 and FoxP3.

[0056] FIG. 16B is a graph showing quantification of percentages of regulatory T cells.

[0057] FIG. 17 is a series of graphs showing flow cytometry data of tumors on day 7 post antibody therapies.

[0058] FIG. 18 is a series of graphs showing flow cytometry data of tdLNs on day 7 post antibody therapies.

[0059] FIG. 19 is a series of graphs showing flow cytometry data of tumors on day 14 post antibody therapies.

[0060] FIG. 20 is a series of graphs showing flow cytometry data of tdLNs on day 14 post antibody therapies.

[0061] FIGS. 21A-22C illustrate data demonstrating therapeutic efficacy of IT and PT administered combination immunotherapy. Survival curves were compared by log-rank Mantel-Cox test.

[0062] FIG. 21 A shows an experimental scheme. In the B16F10 study, N=6 for IT cytokine+PT OX40a, N=8 for all other groups. In the MC38 study, N=8 for all groups.

[0063] FIG. 2 IB is a series of graphs showing tumor growth curves for mice induced with B16F10 through day 20 post inoculation of IT administered IL-12 and IL-2 with ICB, IT administered IL-12 and IL-2 and PT administered OX40a with ICB, ICB only, and untreated clockwise from top left, respectively.

[0064] FIG. 21C is a graph showing the probability of survival for mice induced with B16F10 through 50 days post tumor inoculation.

[0065] FIG. 2 ID is a series of graphs showing tumor growth curves for mice induced with MC38 through day 20 post inoculation of IT administered IL-12 and IL-2 with ICB, IT administered IL-12 and IL-2 and PT administered OX40a with ICB, ICB only, and untreated clockwise from top left, respectively.

[0066] FIG. 2 IE is a graph showing the probability of survival for mice induced with MC38 through 50 days post tumor inoculation.

[0067] FIG. 22A shows an experimental scheme (N=8 for all groups).

[0068] FIG. 22B is a graph showing the probability of survival for mice induced with B16F10 through 50 days post tumor inoculation.

[0069] FIG. 22C is a series of graphs showing tumor growth curves for mice induced with B16F10 through day 20 post inoculation of IT delivered cytokine-antibody combination, PT administered cytokine-antibody combination, ICB only, and untreated, left to right respectively.|0070] FIGS. 23 A-25B illustrate results / effects of combination immunotherapies from tumors. N=6 for all groups. Data reported as mean + / - SEM. Statistics determined by Kruskal-Wallis test with Dunn’s multiple comparisons test in GraphPad Prism.

[0071] FIG. 23 A is a plot showing a representative population of CD8+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0072] FIG. 23B is a graph showing quantification of percentages of CD8+T cells of CD3+T cells.

[0073] FIG. 23C is a graph showing quantification of percentages of CD44+CD62L+central memory T cells of CD8+T cells.

[0074] FIG. 23D is a graph showing quantification of percentages of CD44+CD62L effector memory T cells of CD8+T cells.

[0075] FIG. 23E is a graph showing quantification of percentages of CD44 CD62L+naive and stem-like T cells of CD8+T cells.

[0076] FIG. 23F is a plot showing representative populations of CD8+T cells gated on CD8a and IFNy for quantification of IFNy expressing subsets from murine Bl 6F 10 tumors.

[0077] FIG. 23G is a graph showing quantification of percentages of IFNy expressing T cells among CD8+T cells.

[0078] FIG. 24A is a plot showing a representative population of CD4+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0079] FIG. 24B is a graph showing quantification of percentages of CD4+T cells of CD3+T cells.

[0080] FIG. 24C is a graph showing quantification of percentages of CD44+CD62L+central memory T cells of CD4+T cells.[0081 { FIG. 24D is a graph showing quantification of percentages of CD44+CD62L effector memory T cells of CD4+T cells.

[0082] FIG. 24E is a graph showing quantification of percentages of CD44 CD62L+naive and stem-like T cells of CD4+T cells.

[0083] FIG. 24F is a plot showing a representative population of CD4+T cells gated on CD4 and IFNy for quantification of IFNy expressing subsets from murine Bl 6F 10 tumors.[0084| FIG. 24G is a graph showing quantification of percentages of IFNy expressing T cells among CD4+T cells.

[0085] FIG. 25A is a plot showing a representative population of CD4+regulatory T cells gated on CD25 and FoxP3.

[0086] FIG. 25B is a graph showing quantification of percentages of CD25+FoxP3+regulatory T cells.[0087| FIG. 26 is a series of graphs showing flow cytometry data of tumors on day 7 post combination therapies.

[0088] FIGS. 27A-28E illustrate results / effects of combination immunotherapies on lymph nodes. N=6 for all groups. Data reported as mean + / - SEM. Statistics determined by Kruskal -Wallis test with Dunn’s multiple comparisons test in GraphPad Prism.

[0089] FIG. 27A is a plot showing a representative population of CD8+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0090] FIG. 27B is a graph illustrating quantification of percentages of cytotoxic T cells (CD8+) of CD3+T cells.

[0091] FIG. 27C is a graph illustrating quantification of the percentages of CD44+CD62L+central memory T cells of CD8+T cells.

[0092] FIG. 27D is a graph illustrating quantification of percentages of CD44+CD62L effector memory T cells of CD8+T cells.

[0093] FIG. 27E is a graph illustrating quantification of percentages of CD44 CD62L+naive and stem-like memory T cells of CD8+T cells.

[0094] FIG. 28A is a plot showing a representative population of CD4+T cells gated on CD62L and CD44 for subset phenotypes from murine B16F10 tumors.

[0095] FIG. 28B is a graph illustrating quantification of percentages of helper T cells (CD4+) of CD3+T cells.

[0096] FIG. 28C is a graph illustrating quantification of percentages of CD44+CD62L+central memory T cells of CD4+T cells.

[0097] FIG. 28D is a graph illustrating quantification of percentages of CD44+CD62L effector memory T cells of CD4+T cells.[0098| FIG. 28E is a graph illustrating quantification of percentages of CD44 CD62L+naive and stem-like memory T cells of CD4+T cells.

[0099] FIGS. 29A and 29B illustrate results showing that combination IT IL-12 therapy and systemic adoptive cells improved overall survival.

[0100] FIG. 29A illustrates an experimental scheme (N=8 for groups 1-3, N=10 for group 4). Mice received single injections of 20 pL 1:5 PNP hydrogels formulated with 15 pg IL- 12 intratum orally (groups 3 and 4) and were lymphodepleted intraperitoneally with 4 mg cyclophosphamide on day 7. On day 8, mice received 100 pL of 50 x 106pmel-1 adoptive cells 5 * 106cells total) (groups 2 and 4).

[0101] FIG. 29B is a graph illustrating the probability of survival of mice monitored every 2-3 days for a total of 50 days post tumor inoculation. The combination IL-12 ITtreatment and systemic adoptive cells improved overall survival over both single administered therapies. This combination therapy resulted in 70% long term survivors at day 50.10102] FIG. 30A illustrates an experimental scheme (N=8-12). Mice were induced with Bl 6F 10 cells on both left and right flanks at the same time. Right-flank tumors only were treated 7 days thereafter.[0103| FIG. 30B is a graph illustrating tumor growth curves through 20 days post inoculation; left untreated, right treated.

[0104] FIG. 30C is a graph illustrating the probability of survival for mice induced with two tumors through 60 days post tumor inoculation. Mice were considered to have met euthanasia criteria when either left or right tumor met or exceeded 150 mm2in area. Survival curves were compared by log-rank Mantel-Cox test.[0105| FIG. 31A illustrates an experimental scheme to evaluate differences in efficacy between antibody monotherapies and antibody combination therapy, (N=9-10 per group). All animals except those in the untreated group receive clinically relevant checkpoint blockade (aPDl) for 3x administrations of 250 pg each. Antibody doses included 100 pg 4-1BB and 10 pg of 0X40 per dose, or the combination thereof, where indicated, delivered in 100 pL peritumoral subcutaneous injections of 1:5 PNP hydrogels or systemically via intraperitoneal injections, as indicated.|0106] FIG. 3 IB is a graph illustrating tumor growth curves for mice induced with B16F10 through day 20 post inoculation.

[0107] FIG. 31C is a graph illustrating the probability of survival for mice induced with B16F10 through 50 days post tumor inoculation comparing antibody monotherapies to antibody combination therapy. Survival curves were compared by log-rank Mantel-Cox test.

[0108] FIG. 3 ID is a graph illustrating the probability of survival for mice induced with B16F10 through 50 days post tumor inoculation comparing antibody combination therapies delivered systemically or via hydrogel. Survival curves were compared by log-rank Mantel-Cox test.[0109| FIG. 32 A illustrates an experimental scheme to evaluate abscopal effects of antibody combination therapy in a dual-tumor induction model (N=8-9 per group). All animals except those in the untreated group receive clinically relevant checkpoint blockade (aPDl) for 3x administrations of 250 pg each and antibody doses.

[0110] FIG. 32B is a graph illustrating left and right tumor growth curves for mice induced with Bl 6F 10 through day 16 post inoculation.10111] FIG. 32C is a graph illustrating the probability of survival for mice induced with dual-flank B16F10 tumors through 60 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test.[0112| FIG. 33A illustrates an experimental scheme to investigate therapeutic effects of antibody combination therapies with adoptive cell therapy (PMEL CD8+ cells) in mice induced with B16F10 and lymphodepleted before treatment as indicated, (N=10 per group).10113] FIG. 33B is a graph illustrating the probability of survival for mice induced with B16F10 tumors through 50 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test.

[0114] FIG. 34A illustrates an experimental scheme to investigate therapeutic effects of cytokine combination therapies with adoptive cell therapy (PMEL CD8+ cells) in mice induced with B16F10 and with or without lymphodepletion. Cytokines were delivered as 20 pg IL12 and 5 pg IL2 in 20 pL intratumoral injections of 1:5 PNP hydrogels, as indicated (N=6-15 per group).

[0115] FIG. 34B is a graph illustrating the probability of survival for mice that were not lymphodepleted and induced with B 16F10 tumors through 70 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test.

[0116] FIG. 34C is a graph illustrating the probability of survival for mice that were lymphodepleted and induced with B16F10 tumors through 70 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test.DETAILED DESCRIPTION[0117| The present technology relates to compositions, systems, and methods for treatment of cancer. In some embodiments, for example, a system for treating cancer includes a first dynamic hydrogel having a polymer non-covalently crosslinked with a plurality of nanoparticles and a first immunotherapeutic agent, and a second dynamic hydrogel having a polymer non-covalently crosslinked with a plurality of nanoparticles and a second immunotherapeutic agent, where the first dynamic hydrogel is configured to be administered within a tumor in a subject to form an intratumoral (IT) depot and the second dynamic hydrogel is configured to be administered to the subject external to the tumor to form a peritumoral (PT)depot. The first dynamic hydrogel can be configured to modulate the tumor microenvironment, e.g., by converting the tumor microenvironment from an immune-suppressing state to an immune-activating state. The second dynamic hydrogel can be configured to enhance the activity of immune cells present in a draining lymph node associated with the tumor, e.g., such as by enhancing antigen uptake, antigen processing, antigen presentation, and / or T cell stimulation.(011.81 As another example, a system for treating cancer can include a first immunotherapeutic agent configured to be administered within a tumor in a subject and a second immunotherapeutic agent configured to be administered to the subject external to the tumor. In some embodiments, the first immunotherapeutic agent is administered as part of a first dynamic hydrogel including a polymer non-covalently crosslinked with a plurality of nanoparticles, where the first dynamic hydrogel forms a depot in vivo (e.g., an IT depot). The second immunotherapeutic agent may be administered without any hydrogel, e.g., via systemic injection.[0119 { The present technology can provide many advantages compared to conventional systems and methods for cancer immunotherapy. For instance, immune checkpoint blockade therapy has been adopted as a frontline cancer immunotherapy for improving progression-free survival in patients, but unfortunately, clinical trials report that up to 75% of patients still fail to respond to treatment. These results are likely due to a highly immunosuppressive tumor microenvironment (TME) that has evolved multiple, bio-orthogonal mechanisms of immunosuppression, indicating the need for combination immunotherapies that target both innate and adaptive axes of anti-cancer immunity.

[0120] For the former, immunostimulatory cytokines like IL-12 and IL-2 are promising agonists for their complementary activities inducing interferon-g mediated T-cell cytotoxicity and stimulating NK cells, repolarizing the tumor microenvironment from a “cold” immune excluded state to a “hot” anti-cancer state. Despite their potential, little progress has been made in broadly translating these therapies since the early approval of IL-2 for melanoma over 30 years ago. One challenge with many immune agonists is their exceptionally small therapeutic window; many cytokines, for example, are pleiotropic, resulting in global immune stimulation when delivered systemically. This on-target (correct receptor) / off-tumor (wrong tissue localization) may require high doses to achieve a therapeutic effect, inadvertently producing immune related adverse effects and dose-limiting toxicities.

[0121] These limitations are thus symptomatic of the way in which immune agonists are conventionally delivered — principally, systemically via intravenous infusion. To address this, the field has turned to IT administration of potent immune agonists, leveraging advancements in radiology, laparoscopy, and endoscopy that render many solid tumors accessible for minimally invasive injection. IT administration alone, however, is not without its limitations, such as lack of long-term retention of the cargo within the tumor. Conventional approaches for achieving sustained, localized intratumoral delivery of immune agonists typically involve modifying the biologic of interest, limiting the scope of cargo that could be administered, and repeat administrations, a process that reduces translatability. Further, many strategies have focused on stimulating cytotoxicity without intentionally trying to generate a long-term immunological memory, necessary to treat abscopal tumors, metastasis, or recurrences.

[0122] The present technology can address these and other challenges associated with conventional cancer immunotherapies by delivering immunotherapeutic agents to multiple locations within the body, thereby targeting multiple immunological processes of the cancerimmunity cycle to provide a more effective therapy. For instance, an IT depot may be used in conjunction with a PT to target both the tumor microenvironment and the immune cells within a tumor-draining lymph node (tdLN). In some embodiments, combination treatment systems including both an IT depot and a PT depot containing different immunotherapeutic agents can provide greater flexibility in targeting the unique immunological processes that occur at each location and thus may demonstrate superior efficacy, e.g., in comparison to systems including a single depot only (e.g., a single IT depot or a single PT depot).

[0123] In some embodiments of the present technology, the immunotherapeutic agents are customized to the particular depot location (e.g., an IT depot or a PT depot) to enhance the immune response at that location. For example, pro-inflammatory cytokines such as IL- 12 and IL-2 may exhibit complementary immune activities, such as inducing IFN-y mediated T cell cytotoxicity and stimulating natural killer (NK) cells. In some embodiments, intratumoral administration of such cytokines can cause the tumor microenvironment to become repolarized to an immunogenic state, resulting in more effective targeting of tumor cells by the immune system. As another example, targeting immune processes in the lymph nodes, such as differentiation and clonal expansion of T lymphocytes upon antigen stimulation and interaction with antigen presenting cells (APCs), may be beneficial in mounting a robust memory response.

[0124] In some embodiments, the present technology capitalizes on the progress made in the field of intratumoral retention while exploring synergistic combinations of immune agonists that drive both immediate cytotoxicity and immunological memory, the combination of which may be beneficial for eradicating large, established immunosuppressive tumors. One strategy for mounting a robust memory response is to target immunological processes that occur in the lymph nodes, such as the differentiation and clonal expansion of T lymphocytes upon antigen stimulation and interaction with antigen presenting cells (APCs). Among the T-cell co-stimulatory receptors belonging to the tumor necrosis factor receptor superfamily, 0X40 is implicated in critical immunoregulation, including formation of memory T-cell subsets. 0X40 agonism exhibits promising therapeutic effects both as a monotherapy and a combination therapy.10125] Moreover, the dynamic hydrogels described herein can provide many advantages for encapsulation and delivery of immunotherapeutic agents. Conventional hydrogel-based depot technologies typically exhibit several critical shortcomings, including complicated manufacturing, poor formulation stability, challenging administration, and burst release that can contribute to poor tolerability of the therapy. In contrast to conventional covalently crosslinked hydrogels, the dynamic hydrogels of the present technology are formed through strong yet dynamic physical interactions. As a result, these materials can address the shortcomings of other hydrogel-based depot technologies by exhibiting: (i) mild formulation requirements favorable for facile formulation with therapeutic cargo, and for maintaining stability of the therapeutic cargo during manufacturing and storage; (ii) shear-thinning properties allowing for straightforward injectability through standard syringes and needles; (iii) rapid self-healing of hydrogel structure and depot formation to avoid burst release of the therapeutic cargo; (iv) sufficiently high yield stress to form a robust depot that persists under the normal stresses of the subcutaneous space following administration; (v) controlled delivery of therapeutic cargo over clinically desirable timeframes; and / or (vi) biodegradability.

[0126] Embodiments of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The examples set forth herein are non-limiting examples and are merely examples among other possible examples.

[0127] The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed present technology. Embodiments under any one heading may be used in conjunction with embodiments under any other heading.I. Dynamic Hydrogels

[0128] The present technology utilizes dynamic hydrogels that can serve as a versatile platform for controlled release of therapeutic cargo, such as the therapeutics described in Section II below. In some embodiments, the dynamic hydrogels exhibit dynamic behavior, such as shear-thinning behavior, self-healing behavior, and / or highly tunable viscoelastic mechanical properties. The shear-thinning, self-healing, and / or viscoelastic properties of the dynamic hydrogels can result from non-covalent, supramolecular interactions between the components of the hydrogel (e.g., polymers and nanoparticles, as described further below). The non-covalent interactions can include physical crosslinking, which may encompass various types of crosslinking arising from weak physical interactions such as hydrogen bonding, hydrophobic interactions, ionic interactions, van der Waals interactions, host-guest interactions, crystal formation, physical entanglement, or combinations thereof. The non-covalent interactions can allow for the formation of dynamic, reversible crosslinks between components of the hydrogel that are capable of dissociating and reforming, e.g., spontaneously and / or in response to applied stress.10129] The dynamic hydrogels described herein can provide many advantages for therapeutic applications. For instance, the dynamic hydrogels described herein can exhibit high drug loading capacity, gentle conditions for encapsulation of biologic cargo, sustained delivery of cargo, and / or mechanical tunability. However, unlike traditional covalently crosslinked hydrogels, the dynamic hydrogels herein can be easily administered via techniques such as direct injection, catheter delivery, spreading, or spraying, due to their shear-thinning and / or self-healing properties. Additionally, the dynamic hydrogels herein can exhibit unique dynamic network rearrangements that provide highly tunable release characteristics for the therapeutic cargo. The dynamic hydrogels provided herein can also be synthesized in a straight-forward, cost-effective manner that is easily scalable.A. Polymer Nanoparticle Hydrogels

[0130] In some embodiments, the dynamic hydrogels described herein are polymer nanoparticle (PNP) hydrogels. PNP hydrogels are a type of supramolecular hydrogel formed from non-covalent interactions between polymers and nanoparticles. A PNP hydrogel can self-assemble rapidly upon mixing of a polymer solution with a nanoparticle solution. Selfassembly of the PNP hydrogel network can occur when polymers are linked together by adsorption of segments of the polymer chains onto the surfaces of the nanoparticles through multivalent, transient interactions. PNP hydrogel formation can be an entropy-driven process in which solvent molecules (e.g., water) solvating the polymer chains and nanoparticle surfaces are released into the bulk solution upon binding of the polymer chains to the nanoparticle surfaces, thus producing large gains in translational entropy. The interactions between the polymers and nanoparticle surfaces can be transient and reversible, thus allowing the PNP hydrogel to flow under applied shear stress, followed by rapid self-healing when the stress is relaxed.

[0131] The PNP hydrogels described herein can be composed of any suitable combination of polymers and nanoparticles that are capable of interacting non-covalently with each other to form crosslinks with the desired dynamic behavior. In some embodiments, the nanoparticle and polymer are selected to have a sufficiently strong affinity to produce efficient crosslinking. That is, the free energy gain (c) resulting from the adsorption of a polymer chain to the surface of a nanoparticle can be greater than or comparable to the thermal energy (knT). In addition, the average number of interactions per polymer chain and particle can be greater than 2 to achieve percolation of the hydrogel network. Moreover, to favor polymer bridging of multiple nanoparticles (as opposed to polymer wrapping around individual particles), the nanoparticle diameter can be comparable to or less than the persistence length of the polymer chains. When some or all of these criteria are met, the nanoparticles can serve as crosslinkers between the polymer chains, while the polymer chains can bridge many different particles, thus enabling hydrogel formation. In some embodiments, the modulus (G) of the PNP hydrogel is related to the number of dynamic hydrogel interactions per unit volume (n) and the energy associated with each interaction (aknT) according to the following relation: G ~ naknT. In some embodiments, the nanoparticle surfaces are hydrophobic, such that the adsorption of the polymer chains to the nanoparticle surfaces are at least partially influenced by the general level of hydrophobicity along the polymer chain (e.g., the size and / or number of hydrophobic groups attached to the polymer chain).

[0132] For example, as shown in FIG. 1, the PNP hydrogels described herein can be formed through dynamic interactions between hydrophobically-modified cellulose derivatives and nanoparticles, such as dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12) and biodegradable polymeric nanoparticles composed of poly(ethylene glycol)-block-poly(lacticacid) (PEG-PLA). The polymers can bridge between nanoparticles and dynamically interact with the nanoparticle surfaces. Additional examples of nanoparticles and polymers suitable for use in the PNP hydrogels herein are provided in Sections I.A.l and I.A.2 below, respectively.

[0133] The PNP hydrogels described herein can be differentiated from conventional drug delivery systems that include nanoparticles embedded in a covalently crosslinked hydrogel. Such conventional systems typically include gel-forming polymers that are covalently crosslinked with each other to form the gel network, while the nanoparticles serve as an optional additive that plays no role in gel formation, and thus can be freely substituted with other additives or omitted altogether. In contrast, the PNP hydrogels herein may be specifically formed through the interactions between the nanoparticles and polymers. In some embodiments, the polymers and nanoparticles used in the PNP hydrogels herein each independently do not form a gel alone, or are not used at concentrations where the polymer alone or the nanoparticle alone form a gel, such that gel formation occurs only when the polymer and nanoparticle are combined.[01341 In some embodiments, the PNP hydrogels herein include one or more polymers combined with one or more nanoparticles, such that the loss modulus of a solution of the one or more polymers and the loss modulus of a solution of the one or more particles are each greater than their respective storage moduli at an angular frequency within a range from 0.1 rad / s to 100 rad / s (e.g., 10 rad / s) as measured by oscillatory shear rheometry in the linear viscoelastic region. The storage modulus of the PNP hydrogel produced by combining the one or more polymers with the one or more particles may be greater than the loss modulus of the PNP hydrogel at an angular frequency within a range from 0.1 rad / s to 100 rad / s (e.g., 10 rad / s) as measured by oscillatory shear rheometry in the linear viscoelastic region. In some embodiments, the dynamic shear viscosity of the PNP hydrogel at a shear rate within a range from 0.1 s ' to 100 s ' (e.g., 10 s'1) is greater than the sum of the dynamic shear viscosity of the solution of the one or more polymers and dynamic shear viscosity of the solution of the one or more nanoparticles at the shear rate within the range from 0.1 s ' to 100 s '. For example, the dynamic shear viscosity of the PNP hydrogel can be greater than the sum of the dynamic shear viscosities of the polymer solution and the nanoparticle solution by a multiplicative factor within a range from 2 to 100,000, 2 to 1000, 2 to 100, or 2 to 10.

[0135] The PNP hydrogels described herein can include any concentration of polymers and nanoparticles suitable for providing desired hydrogel properties. For instance, higher polymer concentrations can produce PNP hydrogels with a higher stiffness and / or slowerdegradation rate. Higher nanoparticle concentrations can produce PNP hydrogels with a higher viscosity, stiffness, and yield stress, and / or slower degradation rate. The hydrogel properties may depend not only on the overall amount of solid content in the hydrogel, but also on the stoichiometry of polymer content to nanoparticle content. For example, increasing the nanoparticle concentration at a constant polymer concentration can produce a hydrogel having a more solid-like rheological response (e.g., lower tan delta), increased strain-to-yield, and increased yield stress. Increasing the polymer concentration at a constant nanoparticle concentration can produce a hydrogel having a more liquid-like rheological response (e.g., higher tan delta and greater frequency dependency of the storage modulus) and reduced strain-to-yield.

[0136] In some embodiments, the PNP hydrogels described herein include at least 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt % polymer; and / or at least 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 12 wt%, or 15 wt% nanoparticles. Alternatively or in combination, the concentration of polymer within the PNP hydrogel can be within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%; and / or the concentration of nanoparticles within the PNP hydrogel can be within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%. The nomenclature “X-Y hydrogel” or “X: Y hydrogel” is used herein to refer to a hydrogel having X wt% polymer and Y wt% nanoparticles.

[0137] In some embodiments, the PNP hydrogels herein are prepared by simple mixing of the polymers, nanoparticles, therapeutic cargo, and any optional additives. For example, the PNP hydrogel can be prepared by forming a polymer solution (e.g., by dissolving the polymer in an aqueous solvent such as water or a buffered solution such as phosphate-buffered saline (PBS)), forming a nanoparticle solution (e.g., by suspending the nanoparticles in an aqueous solvent), and forming a solution containing the therapeutic cargo (e.g., by dissolving or suspending the therapeutic cargo in an aqueous solvent). The solutions can then be combined, optionally with external agitation, to form the PNP hydrogel including the therapeutic cargo.1. Nanoparticles

[0138] The PNP hydrogels described herein can include a plurality of nanoparticles. The nanoparticles can be any suitable shape, such as spheres, cubes, rods, tubes, plates, fibers, etc. The nanoparticles can have a mean particle size (e.g., diameter) within a range from 1 nm to 1000 nm, 1 nm to 500 nm, 1 nm to 250 nm, 1 nm to 150 nm, 1 nm to 100 nm, 1 nm to 50 nm, 1 nm to 25 nm, 1 nm to 10 nm, 10 nm to 1000 nm, 10 nm to 500 nm, 10 nm to 250 nm, 10 nm to 150 nm, 10 nm to 100 nm, 10 nm to 50 nm, 10 nm to 25 nm, 25 nm to 1000 nm, 25 nm to 500 nm, 25 nm to 250 nm, 25 nm to 150 nm, 25 nm to 100 nm, 25 nm to 50 nm, 50 nm to 1000 nm, 50 nm to 500 nm, 50 nm to 250 nm, 50 nm to 150 nm, 50 nm to 100 nm, 100 nm to 1000 nm, 100 nm to 500 nm, 100 nm to 250 nm, 100 nm to 150 nm, 150 nm to 1000 nm, 150 nm to 500 nm, 150 nm to 250 nm, 250 nm to 1000 nm, 250 m to 500 nm, or 500 nm to 1000 nm. In some embodiments, the nanoparticles have a mean particle size less than or equal to 500 nm, 250 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. As described herein, to facilitate hydrogel formation, the mean particle size of the nanoparticles can be similar to or less than the persistence length of the polymer in the PNP hydrogel, such as less than or equal to 125%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the persistence length of the polymer. As used herein, “mean particle size” may refer to the statistical mean particle size (e.g., diameter) of the particles in the PNP hydrogel composition. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer to the hydrodynamic diameter or to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art, such as dynamic light scattering.[0139| The nanoparticles can be made out of a single material or can be made out of a combination of multiple different materials (e.g., two, three, four, five, or more different materials). The material(s) can be biodegradable and / or biocompatible. For example, in some embodiments, the nanoparticles are made partially or entirely out of one or more biodegradable and / or biocompatible polymers. Generally, biodegradable polymers can degrade by enzymatic hydrolysis, exposure to water in vivo, surface erosion, and / or bulk erosion. Biodegradable polymers can include synthetic polymers, naturally occurring polymers, or combinations thereof. Examples of synthetic biodegradable polymers include polyhydroxy acids (e.g., poly(lactic acid), poly(glycolic acid)), polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone),poly(hydroxybutyrate), poly(lactide-co-glycolide), poly(lactide-co-caprolactone), poly(ethylene-co-maleic anhydride), poly(ethylene maleic anhydride-co-L-dopamine), poly(ethylene maleic anhydride-co-phenylalanine), polyethylene maleic anhydride-co-tyrosine), poly(butadiene-co-maleic anhydride), poly(butadiene maleic anhydride-co-L-dopamine) (pBMAD), poly(butadiene maleic anhydride-co-phenylalanine), poly(butadiene maleic anhydride-co-tyrosine), and combinations (e.g., mixtures, copolymers) thereof. Examples of naturally occurring biodegradable polymers include polysaccharides (e.g., cellulose, alginate, collagen, chitosan, hyaluronic acid, starch, agarose, agar, xanthan gum), proteins (e.g., collagen, fibrin, albumin, zein, gelatin), and derivatives thereof (e.g., derivatives of cellulose such as cellulose nanocrystals, cellulose nanofibers), and combinations thereof.

[0140] Alternatively or in combination, the nanoparticles can be made partially or entirely out of one or more non-biodegradable polymers. Examples of non-biodegradable polymers include polystyrenes, polyalkylene glycols, poly(meth)acrylates, poly (meth)acrylamides, polyalkylenes (e.g., polyethylene, polyvinyls, poly(vinyl acetate), poly(ethylene terephthalate)), and combinations thereof.

[0141] The polymer(s) used to form the nanoparticles herein can have any suitable molecular weight, such as a molecular weight (e.g., number-average molecular weight (Mn)) within a range from 500 Da to 10,000 kDa, 1 kDa to 1000 kDa, or 10 kDa to 100 kDa. As used herein, “molecular weight” may refer to the relative average chain length of the bulk polymer, and can be estimated or characterized in various ways including gel permeation chromatography (GPC) and capillary viscometry. GPC molecular weights are reported as the number-average molecular weight (Mn) as opposed to the weight-average molecular weight (Mw). Capillary viscometry provides estimates of molecular weight (Mv) as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0142] In some embodiments, the nanoparticles are made partially or entirely out of one or more inorganic materials, such as clays (e.g., silicates) or other types of minerals (e.g., sulfides, oxides, halides, carbonates, sulfates, phosphates, apatites), or combinations thereof. Alternatively or in combination, the nanoparticles can be made partially or entirely out of one or more metals, such as gold, silver, copper, platinum, palladium, ruthenium, or combinations thereof. Optionally, the nanoparticles can be made partially or entirely out of carbon nanotubes (e.g., single-walled or multi -walled nanotubes), graphene, graphene oxide, or other ultrathin single crystals, including black phosphorous and boron based nanosheets.

[0143] In some embodiments, the nanoparticles are core-shell particles (also known as “core-corona particles”). A core-shell particle can have a core containing or formed from a first material, and a shell or corona containing or formed from a second, different material. For example, a core-shell particle can include at least two polymers, such that the core is made from a first polymer, and the shell or corona is made from a second, different polymer. As another example, the core-shell particle can include a single block copolymer, such that the core is made from a first block of the block copolymer, and the shell or corona can be made from a second block of the block copolymer. In some embodiments, one or both of the components of the core-shell particle is a non-polymeric material.

[0144] A core-shell particle can be composed of two compositionally disparate phases, of which one (either the core or shell / corona) is hydrophobic and the other (core or shell / corona) is hydrophilic. Suitable hydrophobic components include polyamides (e.g., poly(amino acids)), polyesters (e.g., poly(lactic acid), poly(caprolactone)), polypropylene oxides, polystyrenes, and combinations thereof. Suitable hydrophilic components include polysaccharides, proteins, polyamides (e.g., poly(amino acids)), naturally occurring polymers, synthetic polymers, and combinations thereof. Suitable block copolymers include combinations of polyethylene glycol and polyesters (e.g., PEG-PLA, poly(ethylene glycol)-block-poly(caprolactone) (PEG-PCL)) and combinations of polyethylene glycol and polypropylene glycol (e.g., poloxamers). In some embodiments, the core-shell particle is composed of an amphiphilic polymer including (1) one or more hydrophobic polymers selected from polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, and / or copolymers thereof, and (2) one or more hydrophilic polymers selected from polysaccharides, proteins, poly(amino acids), and / or polyalkylene oxides.

[0145] Alternatively, the nanoparticles can be homogenous nanoparticles. A homogenous nanoparticle can be uniformly formed from a single material, or can be formed from multiple materials that are not separated into disparate phases within the particle as in core-shell particles.

[0146] The nanoparticles can be prepared using techniques known in the art. The technique to be used can depend on a variety of factors, including the materials used to form the nanoparticles, the desired size range of the resulting nanoparticles, and suitability for the material to be encapsulated. Examples of suitable techniques include, but are not limited to, solvent evaporation, solvent removal, hot melt microencapsulation, spray drying, phaseinversion, polyelectrolyte condensation, single and double emulsion (e.g., probe sonication), nanoparticle molding, and electrostatic self-assembly.10147] The concentration of the nanoparticles in the PNP hydrogel can be varied to produce the desired hydrogel properties. In some embodiments, for example, the concentration of the nanoparticles in the PNP hydrogel is within a range from 1 wt% to 15 wt%, 2 wt% to 12 wt%, 3 wt% to 10 wt%, 5 wt% to 8 wt%, 5 wt% to 15 wt%, 5 wt% to 10 wt%, 10 wt% to 15 wt%, or 10 wt% to 12 wt%. The concentration of the nanoparticles in the PNP hydrogel can be about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, or about 15 wt%. In some embodiments, the concentration of the nanoparticles in the PNP hydrogel can be greater than or equal to 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, or 14 wt%. Alternatively or in combination, the concentration of the nanoparticles in the PNP hydrogel can be less than or equal to 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, or 15 wt%.2. Polymers

[0148] The PNP hydrogel can be formed when the nanoparticles are mixed with and interact with one or more polymers. The shear-thinning and / or self-healing properties of the PNP hydrogel can be derived from reversible, non-covalent interactions between the nanoparticles and the polymer chains, as described herein. The PNP hydrogel can include a single type of polymer or can include a combination of multiple different polymers (e.g., two, three, four, five, or more different polymers). The polymer(s) can be biodegradable and / or biocompatible. The polymer(s) can include naturally occurring polymers, synthetic polymers, or derivatives or combinations thereof. Examples of naturally occurring polymers include polysaccharides (e.g., cellulose, alginate, collagen, chitosan, hyaluronic acid, starch, agarose, agar, xanthan gum), proteins (e.g., collagen, fibrin, albumin, zein, gelatin), and combinations thereof. Examples of synthetic polymers include polyacrylamide, poly(lactic acid), polyethylene glycol, polyethylene glycol-co-propylene glycol (PEO-PPO), poly(acrylates) (e.g., poly(2-hydroxy ethyl methacrylate)), and combinations thereof. In some embodiments, the PNP hydrogel includes a derivative of a naturally occurring polymer, such as a cellulose derivative. Examples of cellulose derivatives include hydroxypropylmethylcellulose (HPMC), hydroxyethyl cellulose (HEC), hydroxypropylcellulose (HPC), ethylcellulose (EC),methylcellulose (MC), hydroxyethylmethylcellulose (HEMC), carboxymethylcellulose (CMC), carboxymethyl ethyl cellulose (CMEC), and combinations thereof.

[0149] In some embodiments, the PNP hydrogels herein include at least one polymer that is modified with a hydrophobic moiety. Hydrophobic modification of polymers may increase the energy associated with each polymer nanoparticle interaction (akBT), thereby increasing the modulus of the dynamic hydrogel given the same number of interactions per unit volume. Such modification may facilitate favorable interactions between the hydrophobic moiety on the polymer chain and the hydrophobic core of the nanoparticle, thereby enhancing the adsorption energy of the polymer to the nanoparticles. The hydrophobic moiety can include a plurality of carbon atoms (e.g., from 2 to 50 carbon atoms, 2 to 30 carbon atoms, or 2 to 18 carbon atoms), and can be a saturated molecule or an unsaturated molecule. Examples of hydrophobic moi eties that may be used include, but are not limited to, alkyl moi eties (e.g., C4 to C18 alkyls such as butyl (-C4), hexyl (-C6), octyl (-C8), decyl (-C10), dodecyl (-C12), tetradecyl (-C14), pentadecyl (-C15), hexadecyl (-C16), heptadecyl (-C17), octadecyl (-C18)), alkenyl moieties (e.g., oleyl, linoleyl), aryl moieties (e.g., phenyl, benzyl, pyryl, naphthyl, anthracene), and cycloalkyl moieties (e.g., adamantyl, cyclohexyl, cholesterol). In some embodiments, the degree of modification of the polymer (e.g., percentage of reactive groups on the polymer have been functionalized with the hydrophobic moiety) is within a range from 1% to 50%, 5% to 30%, 5% to 25%, or 10% to 15%. For example, the degree of modification can be about 5%, 10%, 15%, 20%, or 25%.

[0150] The concentration of the polymer(s) in the PNP hydrogel can be varied to produce the desired hydrogel properties (e.g., stiffness, storage modulus, degradation rate). In some embodiments, for example, the concentration of the polymer(s) in the PNP hydrogel is within a range from 0.25 wt% to 10 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 2 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%. The concentration of the polymer(s) in the PNP hydrogel can be about 0.1 wt%, 0.25 wt%, 0.5 wt% 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%. In some embodiments, the concentration of the polymer(s) in the PNP hydrogel can be greater than or equal to 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, or 4.5 wt%. Alternatively or in combination, the concentration of the polymer(s) in the PNP hydrogel can be less than or equal to 0.5 wt%, 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%.3. Additional Components|0151] The PNP hydrogels herein can optionally include one or more additional components to facilitate gel formation and / or modify the properties of the hydrogel. For example, the PNP hydrogels herein can include at least one enhancer compound that enhances the interactions between the polymers and nanoparticles, e.g., by providing bridging-type non-covalent interactions between the polymers and nanoparticles. In some embodiments, a portion of an enhancer compound interacts non-covalently with the polymer and a second portion of the enhancer compound interacts non-covalently with the nanoparticle. Non-limiting examples of such interactions include ionic interactions such as cationic / anionic interactions, electrostatic interactions, and hydrogen bonding interactions.

[0152] For example, in embodiments where the polymer is negatively charged at physiological pH (e.g., hyaluronic acid, carboxymethyl cellulose), a cationic surfactant can be used to enhance adsorption of the anionic polymer to the nanoparticles via electrostatic interactions. Examples of positively charged surfactants include cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium iodide, cetyltrimethylammonium fluoride, and cetyltrimethylammonium chloride. Conversely, in embodiments where the polymer is positively charged at physiological pH (e.g., chitosan, aminopolysaccharides, poly(lysine), cationic acrylate polymers, cationic vinyl polymers), an anionic surfactant can be used to enhance adsorption of the cationic polymer to the nanoparticles via electrostatic interactions. Examples of negatively charged surfactants include sodium dodecyl sulfate, sodium stearate, and charged fatty acid surfactants.10153] In some embodiments, molecular recognition between at least two compounds can provide the enhancement. For example, the adsorption of polymers, such as polysaccharides, to nanoparticles can be enhanced by an enhancer compound which includes a carbohydrate in one portion of the enhancer and a polymer tail that interacts with the nanoparticle.

[0154] The concentration of the enhancer compound can be varied to produce the desired effect on hydrogel formation. In some embodiments, for example, the concentration of the enhancer compound in the PNP hydrogel is within a range from 0.25 wt% to 10 wt%, 0.5 wt% to 5 wt%, 0.5 wt% to 2 wt%, 1 wt% to 5 wt%, or 1 wt% to 2 wt%. The concentration of the enhancer compound in the PNP hydrogel can be about 0.1 wt%, 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5wt%, 4 wt%, 4.5 wt%, or 5 wt%. In some embodiments, the concentration of the enhancer compound in the PNP hydrogel can be greater than or equal to 0.25 wt%, 0.5 wt%, 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, or 4.5 wt%. Alternatively or in combination, the concentration of the enhancer compound in the PNP hydrogel can be less than or equal to 0.5 wt%, 0.75 wt%, 1 wt%, 1.25 wt%, 1.5 wt%, 1.75 wt%, 2 wt%, 2.25 wt%, 2.5 wt%, 2.75 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%. Optionally, the PNP hydrogel may not include any enhancer compounds.B. Hydrogel Properties

[0155] The dynamic hydrogels described herein (e.g., the PNP hydrogels of Section I. A) can exhibit favorable physical and biological properties that contribute to their efficacy as drug delivery platforms. The properties of the dynamic hydrogels herein can be tuned in various ways, such as by modifying the types of components used to form the hydrogel (e.g., polymers, nanoparticles, and / or additional components as previously described in Section I. A; and / or the therapeutic cargo carried by the hydrogel as described below in Section II), the concentrations of the components, and / or the chemical functionalities of the components. Accordingly, the properties of the dynamic hydrogels herein can be adapted to the particular therapeutic application, such as forming a stable and / or persistent depot when delivered in vivo, providing a desired release profile for the therapeutic cargo (e.g., short-term release versus long-term release), providing a desired release mechanism for the therapeutic cargo (e.g., diffusion-based release versus erosion-based release), compatibility with a desired route of administration (e.g., injecting, infusing, spraying, spreading), biodegradability, biocompatibility, and / or allowing for cellular infiltration. Any reference herein to a property of a dynamic hydrogel may refer to the property of the dynamic hydrogel without any therapeutic cargo (e.g., a PNP hydrogel composed only of polymers and nanoparticles), the property of the dynamic hydrogel including the therapeutic cargo (e.g., a PNP hydrogel including polymers, nanoparticles, and the encapsulated therapeutic cargo), or both, unless otherwise stated or otherwise evident from the context.

[0156] The storage modulus (G1) of the dynamic hydrogel can correlate to the overall stiffness of the hydrogel, which in turn can dictate the time scale of degradation of the hydrogel (e.g., hydrogels having a higher storage modulus may be stiffer and degrade more slowly than hydrogels having a lower storage modulus). Accordingly, in embodiments where the therapeutic cargo of the dynamic hydrogel is released primarily or entirely via an erosion-basedmechanism, the release rate of the therapeutic cargo can be tuned by adjusting the storage modulus of the hydrogel (e.g., a higher storage modulus can produce a slower degradation rate and thus a slower release rate of the therapeutic cargo, while a lower storage modulus can produce a higher degradation rate and thus a faster release rate of the therapeutic cargo). For example, in embodiments where the dynamic hydrogel is a PNP hydrogel, the storage modulus of the PNP hydrogel can be increased or decreased by increasing or decreasing the polymer concentration, and / or by increasing or decreasing the nanoparticle concentration. In some embodiments, the dynamic hydrogels herein have a storage modulus within a range from 1 Pa to 10,000 Pa, 1 Pa to 5000 Pa, 1 Pa to 2500 Pa, 1 Pa to 1000 Pa, 1 Pa to 500 Pa, 1 Pa to 200 Pa, 1 Pa to 10 Pa, 10 Pa to 10,000 Pa, 10 Pa to 5000 Pa, 10 Pa to 2500 Pa, 10 Pa to 1000 Pa, 10 Pa to 500 Pa, 10 Pa to 200 Pa, 10 Pa to 100 Pa, 10 Pa to 50 Pa, 50 P to 10,000 Pa, 50 Pa to 5000 Pa, 50 Pa to 2500 Pa, 50 Pa to 1000 Pa, 50 Pa to 500 Pa, 50 Pa to 200 Pa, 50 Pa to 100 Pa, 100 Pa to 10,000 Pa, 100 Pa to 5000 Pa, 100 Pa to 2500 Pa, 100 Pa to 1000 Pa, 100 Pa to 500 Pa, 100 Pa to 200 Pa, 200 Pa to 10,000 Pa, 200 Pa to 5000 Pa, 200 Pa to 2500 Pa, 200 Pa to 1000 Pa, 200 Pa to 500 Pa, 500 Pa to 10,000 Pa, 500 Pa to 5000 Pa, 500 Pa to 2500 Pa, 500 Pa to 1000 Pa, 1000 Pa to 10,000 Pa, 1000 Pa to 5000 Pa, 1000 Pa to 2500 Pa, 2500 Pa to 10,000 Pa, 2500 Pa to 5000 Pa, or 5000 Pa to 10,000 Pa. The storage modulus can be measured, for example, using an oscillatory shear test in a parallel plate rheometer at an angular frequency of 10 rad / s, a strain within the linear viscoelastic region of the hydrogel (e.g., 1% strain), and a temperature of 25 °C.10157] The yield stress (ry) of the dynamic hydrogel can correlate to the ability of the hydrogel to form and maintain a cohesive depot in vivo (e.g., materials lacking a yield stress may flow rather than forming a cohesive depot). The dynamic hydrogels herein can exhibit little or no flow when subjected to stresses below the yield stress. When subjected to stresses above the yield stress, the dynamic hydrogels can flow, corresponding to a significant drop in observed viscosity (e.g., a decrease of at least one or two orders of magnitude). In embodiments where the dynamic hydrogel is a PNP hydrogel, the yield stress can be increased or decreased by increasing or decreasing the nanoparticle concentration, respectively. In some embodiments, the dynamic hydrogels herein have a yield stress within a range from 0.1 Pa to 1000 Pa, 0.1 Pa to 500 Pa, 0.1 Pa to 200 Pa, 0.1 Pa to 100 Pa, 0.1 Pa to 50 Pa, 0.1 Pa to 20 Pa, 0.1 Pa to 10 Pa, 0.1 Pa to 1 Pa, 1 Pa to 1000 Pa, 1 Pa to 500 Pa, 1 Pa to 200 Pa, 1 Pa to 100 Pa, 1 Pa to 50 Pa, 1 Pa to 10 Pa, 10 Pa to 1000 Pa, 10 Pa to 500 Pa, 10 Pa to 200 Pa, 10 Pa to 100 Pa, 10 Pa to 50 Pa, 10 Pa to 20 Pa, 20 Pa to 1000 Pa, 20 Pa to 500 Pa, 20 Pa to 200 Pa, 20 Pa to 100 Pa, 20 Pato 50 Pa, 50 Pa to 1000 Pa, 50 Pa to 500 Pa, 50 Pa to 200 Pa, 50 Pa to 100 Pa, 100 Pa to 500 Pa, 100 Pa to 200 Pa, 200 Pa to 1000 Pa, 200 Pa to 500 Pa, or 500 Pa to 1000 Pa. The yield stress can be measured, for example, using a stress ramp or stress sweep (e.g., from 1 Pa to 100 Pa, or from 1 Pa to 1000 Pa) in a parallel plate rheometer at a temperature of 25 °C to identify the stress at which the hydrogel exhibits a drop in viscosity.

[0158] The tan delta of the dynamic hydrogel (the ratio of the loss modulus (G") over the storage modulus (G1) (tan(6) = G" / G')) can describe the overall viscoelasticity of the hydrogel (e.g., lower tan delta values correspond to more solid-like behavior, higher tan delta values correspond to more liquid-like behavior), and can correlate to the degradation rate of the hydrogel. In some embodiments, the dynamic hydrogels herein have a tan delta less than or equal to 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. The tan delta can be within a range from 0.1 to 1, 0.1 to 0.5, 0.1 to 0.3, 0.2 to 1, 0.2 to 0.5, or 0.5 to 1. The tan delta can be measured, for example, using an oscillatory shear test in a parallel plate rheometer at an angular frequency of 10 rad / s, a strain within the linear viscoelastic region of the hydrogel (e.g., 1% strain), and a temperature of 25 °C.

[0159] In some embodiments, the dynamic hydrogels herein exhibit shear-thinning behavior, in that the viscosity of the dynamic hydrogel decreases with increasing shear rate and / or shear stress. Shear-thinning behavior can be advantageous, for example, to allow the dynamic hydrogel to be administered via injection. In some embodiments, the viscosity of the gel decreases with increasing shear rate at a shear rate within a range from 0.1 s'1to 1000 s’1, for example, as observed on an oscillatory rheometer (e.g., a parallel plate rheometer) at 25 °C. In some embodiments, the dynamic hydrogels herein have a viscosity within a range from 10 mPa-s to 2000 mPa-s, 10 mPa-s to 1000 mPa-s, 10 mPa-s to 500 mPa-s, 10 mPa-s to 200 mPa-s, 10 mPa-s to 100 mPa-s, 10 mPa-s to 50 mPa-s, 50 mPa-s to 2000 mPa-s, 50 mPa-s to 1000 mPa-s, 50 mPa-s to 500 mPa-s, 50 mPa-s to 200 mPa-s, 50 mPa-s to 100 mPa-s, 100 mPa-s to 2000 mPa-s, 100 mPa-s to 1000 mPa-s, 100 mPa-s to 500 mPa-s, 100 mPa-s to 200 mPa-s, 200 mPa-s to 2000 mPa-s, 200 mPa-s to 1000 mPa-s, 200 mPa-s to 500 mPa-s, 500 mPa-s to 2000 mPa-s, 500 mPa-s to 1000 mPa-s, or 1000 mPa-s to 2000 mPa-s at a shear rate of 1000 s’1. The viscosity can be less than 10,000 mPa-s, 1000 mPa-s, or 100 mPa-s at a shear rate of 1000 s’1. The viscosity can be measured, for example, using steady shear measurements in a parallel plate rheometer at a temperature of 25 °C.

[0160] In some embodiments, the dynamic hydrogels herein exhibit self-healing behavior. Self-healing may refer to a process in which a gel that exhibits reduced resistance toflow when subjected to an external stress regains some or all of its rigidity and / or strength after the external stress is removed. Self-healing behavior can be advantageous, for example, to allow the dynamic hydrogel to form a cohesive depot after administration via injection and / or to limit burst release. In some embodiments, the dynamic hydrogels herein stop flowing and recover their mechanical properties in no more than 5 seconds, 10 seconds, 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, or 10 minutes after the external stress is removed. Optionally, the modulus and / or viscosity of the dynamic hydrogel can recover to at least 90% of the initial value before application of the external stress within 5 minutes in a step-strain measurement (conducted with strains of 0.5% and 500%) or step-shear measurement (conducted with shear rates of 0.1 s'1and 100'1), respectively, on an oscillatory rheometer.

[0161] In some embodiments, the dynamic hydrogels herein exhibit viscoelastic behavior, in that the storage modulus (G1) of the hydrogel is dominant over the loss modulus (G") at some point, for example, as observed in an oscillatory frequency sweep measurement in a range from 0.1 rad / s to 100 rad / s on an oscillatory rheometer performed in the linear viscoelastic region, yet the hydrogel exhibits complete stress relaxation following application of a constant strain of 500% within 15 minutes.

[0162] In some embodiments, the dynamic hydrogels described herein are biocompatible. A biocompatible material can be a material that is, along with any metabolites or degradation products thereof, generally non-toxic to the subject, and do not cause any significant adverse effects to the subject, at concentrations resulting from the degradation of the administered materials. A biocompatible material can be a material that does not elicit a significant inflammatory or immune response when administered to a subject.

[0163] In some embodiments, the dynamic hydrogels described herein are biodegradable. A biodegradable material can be a material that will degrade or erode under physiological conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. For example, upon in vivo administration to a subject, the dynamic hydrogel can dissolve as the non-covalent bonds dissociate. The degradation rate of the dynamic hydrogel can be varied as desired, e.g., depending on the desired release profile for the therapeutic cargo. In some embodiments, following in vivo administration, the dynamic hydrogels are designed to persist at the administration site (e.g., remain as a cohesive depot) for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, or 12 months. Alternatively or incombination, the dynamic hydrogels herein can persist at the administration site for no more than 12 months, 9 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 28 days, 21 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day.II. Systems and Methods for Treatment of Cancer[0164| In some embodiments, the present technology provides systems and methods for treatment of cancer. Examples of cancers that may be prevented and / or treated by the compositions described herein include biliary tract cancer, bladder cancer, brain cancer (e.g., glioblastomas, medulloblastomas), breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia), liver cancer, lymphoma (e.g., Hodgkin's disease, non-Hodgkin lymphoma), lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer (e.g., renal cell adenocarcinoma, nephroblastoma), sarcoma (e.g., fibrosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma, melanoma), testicular cancer, and thyroid cancer.

[0165] FIG. 2A is a schematic illustration of an endogenous anti-tumor immune response, also referred to herein as the cancer-immunity cycle. Tumor antigens released from tumor cells can be taken up by APCs (e.g., dendritic cells (DCs)) that subsequently home to a tumor-draining lymph node (tdLN). The APCs can present the tumor antigens to T cells within the tdLN, resulting in the priming, activation, and proliferation of effector T cells (e.g., CD8+T cells). In some instances, T cell priming and proliferation may additionally or alternatively occur within the tumor itself. The effector T cells may then be trafficked back to the tumor to infiltrate the tumor and recognize and kill tumor cells. The killing of tumor cells may result in the additional release of tumor antigens, which may strengthen the anti -tumor immune response as the cancer-immunity cycle repeats.

[0166] In some embodiments, there are barriers to the effective functioning of this cancer-immunity cycle as the tumor develops. For example, activated T cells and innate immune cells (e.g. NK cells, macrophages) may require a pro-inflammatory, immune-activating environment to demonstrate effective anti-cancer activity. However, the tumor microenvironment has evolved ways of suppressing such anti-cancer activity, such as by expressing factors that promote angiogenesis, expressing factors that inhibit T cell responses,and blocking the migration of T cells via collagen-rich, fibrotic stroma. Moreover, negative regulators to T cell activation in the tdLN (e.g., checkpoints) may result in a suboptimal immune response and / or lack of immunological memory.

[0167] Accordingly, to overcome these and other challenges in cancer immunotherapy, it may be advantageous to modulate the microenvironments of both the tumor and the tdLN to stimulate a robust immune response and / or to repolarize the tumor microenvironment from a “cold” immune-suppressing state to a “hot” immune-activating state that is favorable for anticancer immune cell activity. In some embodiments, the present technology provides a combination depot system including multiple depots that are placed at different positions relative to the tumor to simultaneously or sequentially deliver immunotherapeutic agents to both the tumor and the tdLN.

[0168] FIG. 2B is a schematic illustration of a combination depot system according to some embodiments of the present technology. In some embodiments, the combination depot system includes an intratumoral (IT) depot configured to be implanted (e.g., injected) within a tumor and a peritumoral (PT) depot configured to be implanted (e.g., injected) external to the tumor. The IT depot can be configured to modulate the tumor microenvironment, such as by converting the tumor microenvironment from an immune-suppressing state to an immune-activating state. Without wishing to be bound by theory, it is hypothesized that immunotherapeutic agents that act to modulate the tumor microenvironment may be more efficacious when delivered intratumorally rather than peri turn orally. Accordingly, the IT depot may contain a first immunotherapeutic agent that effectuates such modulation, such as by recruiting immune cells into the tumor, activating immune cells within the tumor, enhancing recognition of cancer cells of the tumor by immune cells, enhancing killing of cancer cells of the tumor by immune cells, or a combination thereof. For instance, the first immunotherapeutic agent can be or include a pro-inflammatory cytokine (e.g., IL-2, IL-12, IL-15, IL-18, IL-21, IFN-a), a chemokine (e.g., GM-CSF, CCL21), an immune checkpoint blockade inhibitor (e.g., an anti-PD-Ll antibody, an anti-CTLA-4 antibody), and / or an adjuvant (e.g., CpG, an anthracy cline). The placement of the IT depot within the tumor may allow the first immunotherapeutic agent to act directly on cells within the tumor (e.g., tumor cells and / or immune cells), e.g., by localized release of the first immunotherapeutic agent within the tumor and / or infiltration of the IT depot by immune cells that are then exposed to the first immunotherapeutic agent while the immune cells are within the IT depot.

[0169] The PT depot can be implanted proximate to a tdLN associated with the tumor. The PT depot can be configured to release a second immunotherapeutic agent to the tdLN, e.g., to target and / or enhance the activity of immune cells present in the tdLN, such as APCs (e.g., DCs), T cells, B cells, etc. For instance, the second immunotherapeutic agent can enhance antigen uptake, antigen processing, antigen presentation, and / or co-stimulation of T cells interacting with APCs within the tdLN. In some embodiments, the second immunotherapeutic agent drives processes that result in robust immunological memory, which may be beneficial given that metastasis remains one of the leading complications resulting in patient death. For example, the second immunotherapeutic agent can be an antibody that binds to a receptor on an immune cell, (e.g., anti-CD40 antibody, an anti-CD205 antibody, an anti-CSFIR antibody, an anti-FLT3L antibody). Without wishing to be bound by theory, it is hypothesized that peritumoral delivery of immunotherapeutic agents that affect the development of immunological memory and / or other processes occurring in the lymph node may be more efficacious than intratumoral delivery.

[0170] The tdLN can be a lymph node selected from any of the following: a lymph node of the head (e.g., occipital lymph node, mastoid lymph node, parotid lymph node), a lymph node of the neck (e.g., cervical lymph node (e.g., submental lymph node, submandibular lymph node), deep cervical lymph node (e.g., deep anterior cervical lymph node, deep lateral cervical lymph node), inferior deep cervical lymph node (e.g., jugulo-omohyoid lymph node, jugulodigastric lymph node), supraclavicular lymph node (e.g., Virchow’s node)), a lymph node of the thorax (e.g., mediastinal lymph node, lung lymph node (e.g., subsegmental lymph node, segmental lymph node, lobar lymph node, interlobar lymph node, hilar lymph node)), a lymph node of the abdomen (e.g., periaortic lymph node, preaortic lymph node, celiac lymph node, hepatic lymph node, gastric lymph node, splenic lymph node, superior mesenteric lymph node, inferior mesenteric lymph node, retroaortic lymph node, common iliac lymph node, internal iliac lymph node, external iliac lymph node, sacral lymph node, retroperitoneal lymph node), a lymph node of the upper limbs (e.g., superficial lymph node of the arm (e.g., supratrochlear lymph node, deltoideopectoral lymph node), deep lymph node of the arm (e.g., lateral lymph node, anterior or pectoral lymph node, posterior or subscapular lymph node, central or intermediate lymph node, medial or subclavicular lymph node)), or a lymph node of the lower limbs (e.g., superficial inguinal lymph node, deep inguinal lymph node, popliteal lymph node).

[0171] The systems and depots described herein can be configured to encapsulate and release many different types of immunotherapeutic agents. Immunotherapeutic agents may include small molecule drugs, inorganic compounds, peptides, proteins (e.g., antibodies, antibody fragments), glycoproteins, polysaccharides, lipids, nucleic acids, cells, etc. Examples of immunotherapeutic agents include cytokines, chemokines, immune checkpoint inhibitors, adjuvants, and immune cells.

[0172] An immunotherapeutic agent can be configured to modulate activity of one or more immune cell types, such as APCs (such as DCs (e.g., cDCls, cDC2s, iDCs), monocytes, macrophages, B cells), T cells (e.g., cytotoxic T cells (CD8+T cells), helper T cells (CD4+T cells) (such as THI cells, TH2 cells, and TH17 cells), regulatory T cells, memory T cells, yb T cells), natural killer (NK) cells, neutrophils, basophils, eosinophils, and other myeloid and non-myeloid cells). Modulation of immune cell activity can include, for example, recruitment of an immune cell, activation of an immune cell, increasing antigen uptake by an immune cell, increasing antigen presentation by an immune cell, stimulation or co-stimulation of an immune cell, enhancing proliferation of an immune cell, enhancing recognition of tumor cells by an immune cell, enhancing tumor cell killing by an immune cell, reducing apoptosis of an immune cell, reducing exhaustion of an immune cell, triggering differentiation of an immune cell, improving immune cell priming, enhancing migration and / or lymphoid homing of an immune cell, etc.

[0173] Alternatively or in combination, an immunotherapeutic agent can be configured to promote conversion of the tumor microenvironment from an immune-suppressing state to an immune-activating state. This can be accomplished, for example, by recruiting immune cells into the tumor, activating immune cells within the tumor, enhancing recognition of cancer cells by immune cells, and / or enhancing killing of tumor cells by immune cells.

[0174] In some embodiments, the immunotherapeutic agent includes a cytokine, such as pro-inflammatory cytokine. Examples of cytokines that may be used include FLT3L, IFN-a, IFN-y, TNF-a, IL-1, IL-2, IL-6, IL-10, IL-7, IL-12, IL-15, IL-17, IL-18, and IL-21.

[0175] In some embodiments, the immunotherapeutic agent includes a chemokine, such as a chemokine that promotes recruitment and infiltration of immune cells (e.g., APCs, T cells, B cells). Examples of chemokines that may be used include GM-CSF, CCL1, CCL2, CCL3, CCL5, CCL7, CCL8, CCL13, CCL17, CCL21, CCL22, CXCL8, CXCL9, CXCL10, CXCL11, XCL1, and CX3CL1.

[0176] In some embodiments, the immunotherapeutic agent includes an antibody (which may be a whole antibody or an antibody fragment). The antibody may be an immune checkpoint inhibitor, such as an immune checkpoint blockade antibody (e.g., an antibody or antibody fragment that binds to an immune checkpoint protein). Examples of antibodies that may be used include anti-CTLA4 antibodies, anti-CD40 antibodies, anti-CD205 antibodies, anti-CSFIR antibodies, anti-FLT3L antibodies, anti-OX40 antibodies, anti-PD-Ll antibodies, anti-PDl antibodies, anti-4-lBB antibodies, anti-TGFp antibodies, and anti-CSFIR antibodies.

[0177] In some embodiments, the immunotherapeutic agent includes an adjuvant. Examples of adjuvants include a lipid-based adjuvant (e.g., a bacterial lipopolysaccharide-based adjuvant such as monophosphoryl lipid A (MPLA), an emulsion-based adjuvant such as MF59), a saponin-based adjuvant (e.g., QS-21), a polynucleotide adjuvant (e.g., cytosine phosphoguanine (CpG), immunomodulatory GpG, polyinosinic-polycytidylic acid (poly(EC))), a metal-based adjuvant (e.g., an aluminum-based adjuvant such as amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, or potassium aluminum sulfate (alum)), or a combination thereof. In some embodiments, the adjuvant is a toll-like receptor (TLR) agonist, such as a TLR7 / 8 agonist (e.g., an imidazoquinoline such as R848, 3M052), a TLR1 / 2 agonist (e.g., Pam3CSK4), or a TLR2 / 6 agonist (e.g., Pam2CSK4). In some embodiments, the adjuvant is a NODI agonist, a N0D2 agonist (e.g., L18-MDP), or a NOD 1 / 2 agonist (e.g., murabutide). The adjuvant can be a small molecule or peptide that has been functionalized with a lipid (e.g., 3M052, Pam3CSK4, Pam2CSK4, L18-MDP). In some embodiments, the adjuvant is a STING agonist (e.g., cGAMP).

[0178] In some embodiments, the immunotherapeutic agent includes an immune cell. Examples of immune cells include T cells, macrophages, NK cells, DCs, cells expressing chimeric antigen receptors (CARs), cells expressing transgenic T cell receptors (TCRs), tumorinfiltrating lymphocytes, etc. The immune cell may be an adoptive cell (e.g., an adoptive T cell). The immune cell may be engineered, e.g., to express non-native receptors such as CARs or transgenic TCRs.

[0179] An immunotherapeutic agent may be present in a depot at any suitable concentration. In some embodiments, the immunotherapeutic agent is present in the depot at a concentration of about 1 pg, about 5 pg, about 10 pg, about 20 pg, about 50 pg, about 100 pg, about 200 pg, about 250 pg, about 300 pg, about 400 pg, or about 500 pg. In some embodiments, the immunotherapeutic agent is present in the depot at a concentration of less than 1 pg, less than 5 pg, less than 10 pg, less than 20 pg, less than 50 pg, less than 100 pg,less than 200 jug, less than 250 jug, less than 300 jug, less than 400 jug, or less than 500 gg. In some embodiments, the immunotherapeutic agent is present in the depot at a concentration of greater than 1 pg, greater than 5 pg, greater than 10 pg, greater than 20 pg, greater than 50 pg, greater than 100 pg, greater than 200 pg, greater than 250 pg, greater than 300 pg, greater than 400 pg, or greater than 500 pg. In some embodiments, the immunotherapeutic agent is present in the depot at a concentration within a range from about 1 pg to about 5 pg, from about 5 pg to about 10 pg, from about 5 pg to about 20 pg, from about 10 pg to about 20 pg, from about 10 pg to about 50 pg, from about 20 pg to about 100 pg, from about 50 pg to about 200 pg, from about 100 pg to about 200 pg, from about 200 to about 250 pg, or from about 250 pg to about 500 pg.[01801 A depot (e.g., an IT depot, a PT depot) can include any suitable number of immunotherapeutic agents, such as one, two, three, four, five, or more immunotherapeutic agents. The immunotherapeutic agent(s) within an IT depot or a PT depot can be independently selected from any of the embodiments described herein. In some embodiments, the IT depot and PT depot include one or more different immunotherapeutic agents. While the advantages of intratumoral versus peritumoral delivery of certain immunotherapeutic agents are described herein, this is not intended to be limiting, and it will be appreciated that the IT depot and the PT depot may include one or more of the same immunotherapeutic agents. Many of the immunotherapeutic agents discussed herein have effects on multiple classes of cells and may activate complementary signaling pathways, (e.g., downstream biological cascades) that produce beneficial effects when administered either intratumorally or peri turn orally.10181] The depots of the present technology may be formed by administering one or more dynamic hydrogels into a subject, such as a first dynamic hydrogel that is administered into a tumor (to form an IT depot) and / or a second dynamic hydrogel that is administered external to the tumor (to form a PT depot). An immunotherapeutic agent can be encapsulated in the dynamic hydrogel via physical entrapment, interactions with hydrogel components (e.g., hydrophobic interactions), or suitable combinations thereof. For instance, immunotherapeutic agents including a lipophilic moiety (e.g., lipid-based and / or lipid-functionalized adjuvants) can adhere to hydrophobic components of the hydrogel (e.g., hydrophobic side chains of the polymer and / or hydrophobic surfaces of the nanoparticles). As another example, immunotherapeutic agents that are larger than the mesh size of the dynamic hydrogel can be encapsulated in the hydrogel via physical entrapment.

[0182] The dynamic hydrogel used to form an IT depot and / or PT depot may be any of the embodiments described in Section I above. For example, the dynamic hydrogel may be shear-thinning, self-healing, and / or include highly tunable viscoelastic mechanical properties. In some embodiments, the dynamic hydrogel is a PNP hydrogel including a polymer that is non-covalently crosslinked with a plurality of nanoparticles. The polymer of the PNP hydrogel may be a hydrophobically modified polysaccharide, such as a hydrophobically modified cellulose derivative (e.g., HPMC-C12). The nanoparticles of the PNP hydrogel may be a polymeric and / or amphiphilic nanoparticle, such as a PEG-PLA nanoparticle.

[0183] The dynamic hydrogels used to form the IT depots and PT depots described herein may be configured with varying compositions and / or properties as discussed in Section I above. In some embodiments, the IT depot has the same composition (e.g., polymer type, polymer concentration, nanoparticle type, nanoparticle concentration), physical properties (e.g., stiffness, storage modulus, loss modulus, tan delta, yield stress, viscosity), release profile, and / or degradation rate as the PT depot. Alternatively, the IT depot may differ from the PT depot with respect to composition, physical properties, release profile, and / or degradation rate. This may be advantageous, for example, for modulating the release of immunotherapeutic agents at different locations to coordinate with different processes involved in the cancerimmunity cycle that may occur according to different time frames (e.g., certain receptors are not constitutively expressed and are induced only after cell activation via an immunostimulatory molecule).

[0184] In some embodiments, the IT depot is formed from a first dynamic hydrogel including a polymer and a plurality of nanoparticles, and the PT depot is formed from a second dynamic hydrogel including a polymer and a plurality of nanoparticles. The first dynamic hydrogel and the second dynamic hydrogel can be formed from the same polymer or can be formed from different polymers. The concentration of the polymer in the first dynamic hydrogel can be the same as the concentration of the polymer in the second dynamic hydrogel, or can be different from the concentration of the polymer in the second dynamic hydrogel. In some embodiments, the concentration of the polymer in the first dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%. In some embodiments, the concentration of the polymer in the second dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

[0185] The first dynamic hydrogel and the second dynamic hydrogel can be formed from the same nanoparticles or can be formed from different nanoparticles. The concentration of the nanoparticles in the first dynamic hydrogel can be the same as the concentration of the nanoparticles in the second dynamic hydrogel, or can be different from the concentration of the nanoparticles in the second dynamic hydrogel. In some embodiments, the concentration of the concentration of the nanoparticles in the first dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt% to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%. In some embodiments, the concentration of the concentration of the nanoparticles in the second dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.10186] The first dynamic hydrogel can have the same storage modulus as the second dynamic hydrogel, or can have a different storage modulus than the second dynamic hydrogel. In some embodiments, the first dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa, e.g., when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the first dynamic hydrogel. In some embodiments, the second dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa, e.g., when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the second dynamic hydrogel.

[0187] The first dynamic hydrogel can have the same yield stress as the second dynamic hydrogel, or can have a different yield stress than the second dynamic hydrogel. In some embodiments, the first dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa, e.g., when measured at 25 °C. In some embodiments, the second dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa, e.g., when measured at 25 °C.

[0188] The first dynamic hydrogel can have the same viscosity as the second dynamic hydrogel, or can have a different viscosity than the second dynamic hydrogel. In some embodiments, the first dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s, e.g., when measured at 25 °C at a shear rate of 1000 s'1. In some embodiments, the second dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s, e.g., when measured at 25 °C at a shear rate of 1000 s'1.

[0189] In some embodiments, the first dynamic hydrogel is configured to release a first immunotherapeutic agent according to a first release profile, and the second dynamic hydrogel is configured to release a second immunotherapeutic agent according to a second release profile. The first release profile and second release profile may be different (e.g., different release rates and / or release durations) or may be substantially the same (e.g., substantially the same release rate and / or release duration). The release profile may be controlled by the degradation rate of the dynamic hydrogel, which in turn may depend on the physical properties and composition of the dynamic hydrogel. For instance, as discussed in Section I B. above, hydrogels having a higher storage modulus may be stiffer and degrade more slowly than hydrogels having a lower storage modulus, and thus may produce a slower release rate. In embodiments where the dynamic hydrogel is a PNP hydrogel, the storage modulus of the PNP hydrogel can be increased or decreased by increasing or decreasing the polymer concentration, and / or by increasing or decreasing the nanoparticle concentration.

[0190] In some embodiments, the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year. In some embodiments, the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0191] In some embodiments, the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year. In some embodiments, the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0192] In some embodiments, rapid release of immunotherapeutic agents within the tumor may be desirable (e.g., to quickly activate immune cells, initiate cytotoxic tumor cell killing, repolarize the tumor microenvironment, and / or induce certain ligands), whereas immunotherapeutic agents that are delivered peritumorally may benefit from slower and / or sustained release over longer time frames (e.g., to induce immunological memory and / or to promote a longer-acting immune response). Accordingly, the first dynamic hydrogel (IT depot) may be configured to produce a rapid release profile while the second dynamic hydrogel (PT depot) may be configured to produce a sustained release profile. In other embodiments, however, the second dynamic hydrogel can be configured to produce a rapid release profile and the first dynamic hydrogel can be configured to produce a sustained release profile), or both dynamic hydrogels can produce substantially the same release profile (e.g., a rapid release profile or a sustained release profile).

[0193] In some embodiments, the first dynamic hydrogel that degrades at a first degradation rate, and the second dynamic hydrogel that degrades at a second degradation rate. The first degradation rate and second degradation rate may be different or may be substantially the same. The degradation rate of the dynamic hydrogel may determine how long the dynamic hydrogel persists in vivo (e.g., remains as a cohesive depot at the implantation site). The degradation rate of the dynamic hydrogel may depend on the stiffness of the dynamic hydrogel, as discussed elsewhere herein.

[0194] In some embodiments, the first dynamic hydrogel is configured to persist in vivo or a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year. In some embodiments, the first dynamic hydrogel is configured to persist in vivo for a duration of nomore than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.[0195| In some embodiments, the second dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year. In some embodiments, the second dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.[0196| The dynamic hydrogels described herein can be administered to the subject via any suitable route, such as a parenteral route. For example, in some embodiments, the dynamic hydrogel is administered to the subject via injection (e.g., subcutaneous injection or intramuscular injection for forming a PT depot, intratumoral injection for forming an IT depot). The shear-thinning properties of the dynamic hydrogel can allow for facile delivery of the hydrogel and the encapsulated therapeutic cargo via injection. Injection of the dynamic hydrogel can be performed using any suitable tubular device having a lumen configured for delivery of a hydrogel, such as needles (e.g., hypodermic needles, surgical needles, infusion needles), injector pens, catheters, trocars, cannulas, tubing, etc. The dynamic hydrogel can be formulated to have a volume that is sufficiently small for injection, such as a volume less than or equal to 2 mL, 1.75 mL, 1.5 mL, 1.25 mL, 1 mL, 0.75 mL, 0.5 mL, or 0.25 mL.

[0017] In some embodiments, immune cells at the injection site may infiltrate into the depot formed by the dynamic hydrogel, while nonimmune cells at the injection site (e.g., muscle cells, skin cells, adipocytes, fibroblasts, tumor cells) may be mostly or entirely excluded from the hydrogel. The immune cells can include APCs, such as dendritic cells (e.g., cDCls, cDC2s, iDCs), monocytes, macrophages, and B cells. The depot can optionally serve as a local immunological niche by recruiting and / or activating other types of immune cells, such as T cells (e.g., cytotoxic T cells (CD8+T cells), helper T cells (CD4+T cells) (such as THI cells,TH2 cells, and TH17 cells), regulatory T cells, memory T cells, 76 T cells), NK cells, neutrophils, basophils, eosinophils, and other myeloid and non-myeloid cells).10198] The dynamic hydrogel can be administered to the subject according to any suitable timing. For instance, the dynamic hydrogel can be administered on a periodic basis, such as once per week, once per 2 weeks, once per 4 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once 9 months, once per year, once per 2 years, once per 5 years, or once per 10 years. In some embodiments, the first dynamic hydrogel for forming an IT depot is administered before, concurrently with, or after the second dynamic hydrogel for forming a PT depot. The first dynamic hydrogel for forming an IT depot may be administered to the subject at the same or a different frequency than the second dynamic hydrogel for forming a PT depot.

[0019] In some embodiments, the present technology provides methods for treating a subject by administering a system including one or more dynamic hydrogels to the subject as described herein. The system can prevent and / or treat a disease or condition by producing a desired therapeutic effect in the subject, such as alleviation of symptoms, a reduction in the severity of the disease or condition, inhibiting an underlying cause of the disease or condition, steadying the disease or condition in a non-advanced state, delaying the progress of a disease or condition, improving or alleviating the disease or condition, and / or preventing the subject from contracting a disease or condition.]0200| For example, the disease or condition can be a cancer, such as biliary tract cancer, bladder cancer, brain cancer (e.g., glioblastomas, medulloblastomas), breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia), liver cancer, lymphoma (e.g., Hodgkin's disease, non-Hodgkin lymphoma), lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer (e.g., renal cell adenocarcinoma, nephroblastoma), sarcoma (e.g., fibrosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma, melanoma), testicular cancer, or thyroid cancer.[0201 [ In some embodiments, a method of preventing and / or treating cancer includes administering one or more dynamic hydrogels (e.g., PNP hydrogels) of the present technology to a subject in need thereof. The dynamic hydrogel(s) can be administered to the subject viainjection (e.g., a subcutaneous, intramuscular, or intratumoral injection). The dynamic hydrogel(s) can include a therapeutically effective amount of one or more immunotherapeutic agents for preventing and / or treating the cancer. In some embodiments, the method includes administering a first dynamic hydrogel including a first immunotherapeutic agent and a polymer non-covalently crosslinked with a plurality of nanoparticles into a tumor in a subject to form an IT depot, and administering a second dynamic hydrogel including a second immunotherapeutic agent and a polymer non-covalently crosslinked with a plurality of nanoparticles to the subject at a location external to the tumor.

[0202] In some embodiments, the present technology provides methods for preparing one or more dynamic hydrogels for treating a subject as described herein. The method can include combining the components of a dynamic hydrogel (e.g., polymer and nanoparticles) with an immunotherapeutic agent, thus forming a dynamic hydrogel encapsulating the immunotherapeutic agent. The combining of the hydrogel components and immunotherapeutic agent can be performed using simple mixing under gentle conditions, such as physiological pH (e.g., pH 7.0 to 7.4) at room temperature (e.g., 25 °C) or physiological temperature (e.g., 37 °C).10203] In some embodiments, the dynamic hydrogel is prepared no more than 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes, or 1 minute before administering the dynamic hydrogel to the subject. Alternatively or in combination, the dynamic hydrogel can be prepared at least 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 1 hour before administering the dynamic hydrogel to the subject. The dynamic hydrogel can be mostly or fully formed before the dynamic hydrogel is administered to the subject. For example, the dynamic hydrogel can be sufficiently crosslinked (e.g., non-covalently crosslinked) to exhibit the shear-thinning, self-healing, and / or viscoelastic properties described herein before the dynamic hydrogel is administered to the subject.

[0204] In some embodiments, the present technology provides kits for preparing a dynamic hydrogel as described herein. The kit can include a solution containing an immunotherapeutic agent and one or more solutions containing the components of a dynamic hydrogel (e.g., a solution containing a polymer and a solution containing nanoparticles, or a single solution containing a polymer and nanoparticles). The solutions can be provided in tubes, bottles, ampoules, syringes, or any other suitable storage container. In some embodiments, the solutions each independently include a suitable pharmaceutically acceptable diluent. The pharmaceutically acceptable diluent can be any diluent that does not substantially produceadverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject. Examples of pharmaceutically acceptable diluents include, but are not limited to, saline, Ringer’s solution, dextrose solution, phosphate buffered saline, water, or a combination thereof. The pharmaceutically acceptable diluent can include an isotonicity imparting agent, such as sodium chloride, potassium chloride, or monosodium phosphate. The pharmaceutically acceptable diluent can include a buffer, such as bicarbonate, TRIS, HEPES, MOPS, CHES, CHAPS, or phosphate buffered saline. The pharmaceutically acceptable diluent can include stabilizers and / or preservatives, as appropriate. Additional examples and details of pharmaceutically acceptable diluents can be found in Martin, Remington’s Pharmaceutical Sciences, 21st Ed., Mack Publ. Co., Easton, Pa. (2005), which is incorporated herein by reference in its entirety.

[0205] Although certain embodiments of the present technology are described in terms of administering a first dynamic hydrogel to form an IT depot and administering a second dynamic hydrogel to form a PT depot, the systems and methods herein may involve administration of more than two dynamic hydrogels to form additional depots. For instance, a system can include a third dynamic hydrogel that is administered intratumorally or peri turn orally, a fourth dynamic hydrogel that is administered intratumorally or peri turn orally, and so on. In embodiments where multiple IT depots are formed, some or all of the IT depots may be formed in the same tumor, or some or all of the IT depots may be formed in different tumors (e.g., if the tumor has metastasized). Similarly, in embodiments where multiple PT depots are formed, some or all of the PT depots may be formed proximate to the same tumor and / or tdLN, or some or all of the PT depots may be formed proximate to different tumors and / or tdLNs. Any suitable number and combination of IT and PT depots may be used, e.g., a system can include a single IT depot and multiple PT depots, a single PT depot and multiple IT depots, multiple IT depots and multiple PT depots, etc.A. Antibody Combination Therapy

[0206] In some embodiments, the systems and methods herein may involve administration of multiple antibodies via a dynamic hydrogel to form a depot in vivo. For example, a depot (e.g., an IT depot, a PT depot) can include any suitable number of immunotherapeutic agents, such as one, two, three, four, five, or more immunotherapeutic agents. The immunotherapeutic agent(s) within an IT or PT depot can be independently selected from any of the embodiments described herein. In some embodiments, the IT depotand PT depot may include one or more different immunotherapeutic agents. Many of the immunotherapeutic agents discussed herein have effects on multiple classes of cells and may activate complementary signaling pathways (e.g., downstream biological cascades) that produce beneficial effects when administered either intratumorally or peri turn orally.[0207| In some embodiments, the one or more immunotherapeutic agents may include an antibody (which may be a whole antibody or an antibody fragment). The antibody may be an immune checkpoint inhibitor, such as an immune checkpoint blockade antibody (e.g., an antibody or antibody fragment that binds to an immune checkpoint protein). Examples of antibodies that may be used include anti-CTLA4 antibodies, anti-CD40 antibodies, anti-CD205 antibodies, anti-CSFIR antibodies, anti-FLT3L antibodies, anti-OX40 antibodies, anti-PD-Ll antibodies, anti-PDl antibodies, anti-4-lBB antibodies, anti-TGFp antibodies, and anti-CSFIR antibodies.

[0208] In some examples, a depot can include a first immunotherapeutic agent comprising a first antibody (e.g., an anti-OX40 antibody) and a second immunotherapeutic agent comprising a second antibody (e.g., an anti-4-lBB antibody). In such embodiments, the one or more immunotherapeutic agents may be selected such that they activate complementary signaling pathways. For example, in an embodiment of a depot containing two antibodies, an anti-OX40 antibody may be selected to bind and activate the 0X40 receptor which is expressed on CD4 T cells and provide a costimulatory signal for T cell activation, and an anti-4-lBB antibody may be selected to stimulate the response of CD8 T cells. The depot can be administered intratumorally or peritumorally, which as previously discussed can reduce the toxicity concerns associated with systemic administration. For example, without wishing to be bound by theory, it is hypothesized that the use of an IT or PT depot for the targeted delivery of multiple immunotherapeutic agents can limit the toxicity and patient burden of treatment by reducing the required dose size (as compared to systemic delivery) and by localizing the treatment to the desired region.B. Additional Combination Therapies

[0209] Moreover, in some embodiments, the present technology provides combination therapies that involve administering a first immunotherapeutic agent via a dynamic hydrogel to form a depot in vivo (e.g., an IT depot or a PT depot), and administering a second immunotherapeutic agent without any hydrogel. For instance, the second immunotherapeutic agent may be administered as a part of a pharmaceutical composition (e.g., including apharmaceutically acceptable diluent), where the composition does not form a depot in vivo. The second immunotherapeutic agent may be administered via any suitable route, such as through the space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally), etc. In some embodiments, the first immunotherapeutic agent is delivered locally (e.g., due to being encapsulated in the dynamic hydrogel), while the second immunotherapeutic agent is delivered systemically (e.g., via systemic injection). The first and second immunotherapeutic agents may be independently selected from any of the embodiments described herein. The first and / or second immunotherapeutic agent can include one or more antibodies, one or more cytokines, and / or one or more immune cells (e.g., adoptive immune cells).

[0210] In some embodiments, the first immunotherapeutic agent comprises one or more antibodies and the second immunotherapeutic agent comprises immune cells (e.g., adoptive immune cells). In some embodiments, the first immunotherapeutic agent is an antigen-agnostic cargo, an antibody-drug conjugate, an antigen-specific antibody, or a bispecific antibody. Examples of antibodies that may be used include anti-CTLA4 antibodies, anti-CD40 antibodies, anti-CD205 antibodies, anti-CSFIR antibodies, anti-FLT3L antibodies, anti-OX40 antibodies, anti-PD-Ll antibodies, anti-PDl antibodies, anti-4-lBB antibodies, anti-TGFp antibodies, anti-CSFIR antibodies, anti -LAG-3 antibodies, anti-CD20 antibodies, anti -HER antibodies, anti-EGFR, anti-VEGF antibodies, bispecific T-cell engagers (CD19xCD3, BCMAxCD3, CD20xCD3, DLL3xCD3), and antibody drug conjugates (ADCs). According to some methods, a treatment course of a solid tumor may comprise lymphodepletion of the patient, followed by administration of adoptive immune cells (e.g., without administration of a hydrogel) after a first period of time, followed by administration of one or more antibodies (e.g., via an IT depot, PT depot, or both) after a second period of time. The first period of time can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days, and the second time period can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days. The first period of time can be greater than the second period of time, less than the second period of time, or substantially the same as the second period of time. In someembodiments, the duration of the second period of time is selected based on the time required for host immune cell regeneration following lymphodepletion. In some embodiments, the second period of time may be a duration of time that allows the adoptive immune cells sufficient time to engraft before the administration of the antibodies.[02111 In some embodiments, the first immunotherapeutic agent is a combination of anti-OX40 and anti-4-lBB antibodies, and the second immunotherapeutic agent comprises immune cells (e.g., adoptive immune cells). In such embodiments, methods of treatment include lymphodepletion of the patient, administration of adoptive immune cells (e.g., without administration of a hydrogel) after a first period of time, and administration of a combination of anti-OX40 and anti-41BB antibodies (e.g., via an IT depot, PT depot, or both) after a second period of time. The inventors have discovered that in treatment regimens including lymphodepletion and systemic adoptive cell therapy, the administration of the combination anti-OX40 and anti -4- IBB antibody treatment via a hydrogel (e.g., IT, PT, or both) confers greater long-term survival than the administration of the combination antibody treatment delivered systemically. Additional details can be found at Example 11 below.

[0212] Without wishing to be bound by theory, it is believed that the effects of systemic administration of adoptive cells can be augmented by an additional immunotherapeutic agent (e.g., antibodies) delivered via a hydrogel depot. In some embodiments, the combination therapy can act synergistically, limit toxicity of the immunotherapeutic agents delivered via the hydrogel, and enable more potential treatment options. As described herein, hydrogel depots can be used to administer immunotherapeutic agents with a diverse array of targets including adoptive cells or endogenous patient cells, or that act to create an advantageous local inflammatory environment. Hydrogel depots may also be advantageous because they enable regional specificity of treatments. Additionally, the combination therapy can be incorporated into the current clinical standard of care of systemic infusion of adoptive cells, facilitating a clear path for clinical translation.

[0213] In some embodiments, the first immunotherapeutic agent is one or more cytokines, and the second immunotherapeutic agent comprises immune cells (e.g., adoptive immune cells). Methods of treatment include lymphodepletion of the patient, administration of one or more cytokines (e.g., via an IT depot, PT depot, or both), and administration of adoptive immune cells. In some embodiments, the one or more cytokines may include one or more cytokines. Examples of cytokines that may be used include FLT3L, IFN-a, IFN-y, TNF-a, IL-1, IL-2, IL-6, IL-10, IL-7, IL-12, IL-15, IL-17, IL-18, and IL-21. In some embodiments, thefirst immunotherapeutic agent is a combination of chemokines and adjuvants. Examples of chemokines and adjuvants that may be used include chemoattractants such as CCL4, CCL5, CXCL9, CXCL10, CXCL11, and CXCL16 and adjuvants or immunostimulators such as TLR agonists (poly IC, CpG ODN, MPLA, resiquimod, GM-CSF). The one or more cytokines may be administered at the same time as the lymphodepleting therapy, or may be administered before or after the lymphodepleting therapy. For example, in some embodiments the cytokine may be administered at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days before the lymphodepletion. In some examples, the cytokine may be administered at least 6 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after the lymphodepletion. Likewise, the adoptive immune cells may be administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after administration of the cytokine.[0214| In some embodiments, the first immunotherapeutic agent is IL-12, and the second immunotherapeutic agent comprises immune cells (e.g., adoptive immune cells). In several of such examples, the first immunotherapeutic agent does not include IL-2 and / or another cytokine. In such embodiments, methods of treatment include lymphodepletion of the patient, administration of IL-12 (e.g., via an IT depot, PT depot, or both), and administration of adoptive immune cells (e.g., without administration of a hydrogel). The inventors have discovered that in treatment regimens including lymphodepletion and systemic adoptive cell therapy, the administration of IL-12 (without IL-2) via a hydrogel (e.g., IT, PT, or both) confers greater long-term survival than the administration of the combination of IL-12 and IL-2 (via a hydrogel). Additional details can be found at Example 12 below.

[0215] IL-2 acts as both an anti-tumor and a pro-tumor cytokine and is critical for regulatory T cell survival and function. As regulatory T cells are immune suppressing and work to down regulate the anti-cancer response, it is important to closely control the IL-2 concentration within the tumor. Specifically, low level persistent IL-2 signaling can lead to cell dysfunction, including increased T cell exhaustion, despite causing initial increases in proliferation. Without wishing to be bound by theory, it is believed that in treatment regimens without lymphodepletion, the endogenous immune cells compete with the adoptive immune cells for the available cytokines. However, as lymphodepletion depletes the number of endogenous immune cells, there is reduced competition for both the endogenous andexogenous cytokines available to the adoptive immune cells in such treatments. In treatment regimens with lymphodepletion, it is believed that in mice administered IL-12 only, the combination of the exogenous IL- 12 and the endogenous IL-2 facilitates increased adoptive cell proliferation and expansion, promoting long-term survival. However, for mice treated with a combination of IL-12 and IL-2, it is believed that IL-2 concentrations within the tumor microenvironment may become too high (from a combination of the exogenous IL-2 and the endogenous IL-2). As a result, the number of activated regulatory T cells may increase, leading to an anti -turn or response, and eventually to a decrease in overall survival.

[0216] Without wishing to be bound by theory, it is believed that the combination of systemically delivered adoptive immune cells and hydrogel-depot delivered cytokines may be advantageous as it allows for treatments designed with regional specificity and directed towards a range of potential targets. Additionally, the combination therapy can be incorporated into the current clinical standard of care, thus facilitating clinical translation.

[0217] In some examples of combination adoptive cell therapy based on the present technology, a treatment course of a solid tumor may not require lymphodepletion. For instance, methods of treatment can include administration of one or more cytokines via a dynamic hydrogel to form a depot (e.g., an IT depot, a PT depot, or both) followed by administration of adoptive immune cells, without any lymphodepleting therapy. The adoptive immune cells may be administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days after administration of the cytokine. Without wishing to be bound by theory, it is hypothesized that the administration of various immunotherapeutic agents via IT or PT hydrogel depots may cause biochemical remodeling of the tumor in a way that promotes adoptive cell engraftment in a manner that mirrors the effects of lymphodepletion. These effects are enhanced by the hydrogel depots as compared to systemic administration of immunotherapeutic agents because the depots facilitate long-term retention of the cargo within the tumor and help to avoid the previously described problems related to systemic administration.

[0218] For example, in some embodiments, the treatment course of a solid tumor without lymphodepletion may comprise the administration of one or more first immunotherapeutic agents via a dynamic hydrogel to form a depot (e.g., an IT depot), followed by administration of a second immunotherapeutic agent (e.g., adoptive immune cells), after afirst waiting period. In some embodiments, the treatment course may include one or more doses of a clinically relevant checkpoint blockade (e.g., aPDl).

[0219] In some embodiments, the first immunotherapeutic agent is a combination of IL-12 and IL-2, and the second immunotherapeutic agent comprises immune cells (e.g., adoptive immune cells). In such embodiments, methods of treatment include administration of IL-12 and IL-2 (e.g., via an IT depot, PT depot, or both), and administration of adoptive immune cells (e.g., without administration of a hydrogel). The inventors have discovered that in treatment regimens including systemic adoptive cell therapy without lymphodepletion, the administration of a combination of IL-12 and IL-2 via a hydrogel (e.g., IT, PT, or both) confers greater long-term survival than the administration of either IL-12 or IL-2 only (via a hydrogel). Additional details can be found at Example 12 below.

[0220] Without wishing to be bound by theory, it is believed that the increased survival seen in treatment regimens without lymphodepletion and receiving a combination of IL- 12 and IL-2 can be attributed to the concentration of available cytokine within the tumor. In treatment regimens without lymphodepletion, the number of endogenous immune cells is not reduced. As such, it is believed that the endogenous IL-2 in the tumor is being consumed by the endogenous immune cells instead of by the adoptive immune cells. Thus, there is not an excess of endogenous IL-2 available to increase the regulatory T cell survival and to confer negative survival benefits as seen in the lymphodepleted systems. As a result, the exogenous IL-2 is necessary for adoptive cell function and the administration of both IL-2 and IL-12 may improve adoptive cell survival and efficacy as compared to the administration of IL-12 only.

[0221] Although certain embodiments of the present technology are described in terms of administering a first dynamic hydrogel to form a depot and administering a second therapeutic agent without a hydrogel, the systems and methods herein may involve administration of more than one dynamic hydrogel to form additional depots. For example, in some embodiments, the present technology provides combination therapies that may involve administering a first immunotherapeutic agent via a dynamic hydrogel to form an IT depot in vivo, administering a second immunotherapeutic agent via a dynamic hydrogel to form a PT depot in vivo, and administering a third immunotherapeutic agent without any hydrogel.Examples[022 1 The present technology is further illustrated by the following non-limiting examples.Example 1: Preparation of Hydrogels and Immunotherapeutic Formulations

[0223] This example describes the preparation of hydrogels and immunotherapeutic formulations for treatment of cancer. These materials were prepared for subsequent studies (Examples 2-12) exploring the ability of an injectable hydrogel depot to enable the rational localization of potent immunotherapeutic cytokines (IL- 12, IL-2) and antibodies (OX40a, 4-1BB). Specifically, these studies investigated whether sustained locoregional delivery of distinct immune agonists could be used to intentionally skew biodistribution either to the tumor microenvironment or to the tumor draining lymph node. It was hypothesized that PNP hydrogels could provide highly localized and sustained release of cytokines (IL-12 and IL-2) and agonistic (anti-OX40 or anti-4-lBB antibodies. These immune agonists were evaluated as monotherapies and combination therapies, delivered either intratumorally or peritumorally, in the poorly immunogenic Bl 6F 10 melanoma model, and the lead combination was validated in the MC38 adenocarcinoma model. These studies examined how site of administration of these therapies skewed biodistribution via in vivo imaging of excised organs and explored how this altered biodistribution could be leveraged to enable synergistic combination immunotherapies driving tumor repolarization and effective immunological memory. These responses were characterized via flow cytometry, demonstrating that site of administration is involved in mounting distinct immunophenotypes in the lymph nodes and tumors in treated mice. Notably, this platform approach was demonstrated to be both cargo and tumor agnostic, improving efficacy in multiple tumor models without requiring modification of the cargo, suggesting a promising path for future exploration of novel combination immunotherapies.

[0224] HPMC-C12 Synthesis. Hydroxypropylmethylcellulose (HPMC) (1.0 g) was dissolved in anhydrous NMP (40 mL) with stirring at 80 °C for 1 hour. Once the polymer had completely dissolved and the solution cooled to room temperature, a solution of 1-dodecylisocyanate (0.5 mmol) in NMP (5 mL) was added dropwise, followed by DIPEA (catalyst, 125 |iL). The reaction was maintained at 50 °C for 30 minutes, then the heat was shut off, and the mixture was left stirring overnight at room temp. The solution was then precipitated from acetone and HPMC-C12 was purified by dialysis against MilliQ water for 3-4 days (MWCO 3.5 kDa) and lyophilized, yielding HPMC-C12 as a white amorphous powder. The polymer was dissolved at 60 mg / mL in sterile PBS, pH 7.4, prior to use in hydrogels.

[0225] Synthesis of PEG-b-PLA. Prior to use, recrystallized lactide (10 g) was fully dissolved in cryo-distilled dichloromethane (DCM) (45 mL) under N2 (g) with mild heating. Methoxy poly(ethylene glycol) (PEG) (5 kDa; 2.5 g) was heated to 100 °C under vacuum for1-2 h, allowed to cool under N2, and then dissolved in cryodistilled DCM (5 mL). Once dissolved, the full PEG solution was added to the lactide solution under N2 and mixed with hand swirling. A solution of DBU (150 pL cryodistilled DBU per 1 mL cryodistilled DCM) was prepared and 500 pL added to the lactide-PEG solution under N2. The reaction was swirled by hand and allowed to react for 8 min before quenching with acetic acid (~2 drops in 500 pL acetone). The PEG-PLA copolymer was precipitated from excess 50:50 mixture ethyl ether and hexanes, collected, and dried under vacuum to yield a white amorphous powder.

[0226] PEG-b-PLA Nanoparticle Formation. Briefly, a solution (1 mL) of PEG-PLA in 25:75 DMSO: acetonitrile (50 mg / mL) was added dropwise to water (10 mL) at a stir rate of 600 rpm. NPs were purified by ultracentrifugation over a filter (MWCO 10 kDa; Millipore Amicon Ultra-15) followed by resuspension in PBS to a final concentration of 200 mg / mL. NP size and dispersity were characterized by DLS (Wyatt DynaPro PlateReader-II; average diameter = 34.1 nm, PDI <= 0.05).

[0227] PNP Hydrogel Formulation. To prepare PNP hydrogels, HPMC-C12 was dissolved at 6 wt% in PBS and loaded into a 1 mL luer-lock syringe. A 20 wt% solution of PEG-A-PLA nanoparticles (“PEG-PLA NPs”) in PBS was added to a solution of PBS with adjuvant, cytokine, and / or antibodies, depending on formulation, and loaded into a second 1 mL syringe. The two syringes were connected with a female-female luer lock elbow, with care to avoid air at the interface of the HPMC-C12 and nanoparticle solution, and gently mixed until a homogenous PNP hydrogel was formed. Hydrogels were formulated with final concentrations of 1 wt% HPMC-C12 and 5 wt% NPs (“1:5”). Each dose included 20 pL hydrogel injection either intratumorally (IT) or peritumorally (PT) through a 27-gauge needle, as indicated. IT soluble / bolus controls were administered as a 20 pL injection in PBS through a 27-gauge needle, while systemic treatments of anti -PDI were delivered as 100 pL soluble intraperitoneal injections.

[0228] Immunotherapy Formulations. Immunotherapy inj ections were formulated with varying concentrations of cytokines, antibodies, and adjuvants in either soluble form (in PBS) or in PNP hydrogels. IL-12 was dosed at 5 or 20 pg per injection, as indicated (Sino Biological CT022-M08H). IL-2 was dosed at 5 pg per injection (Sino Biological, 51061-MNAE). Anti-0X40 antibody was dosed at 10 pg per injection (BioX Cell, BE0031). Anti-41BB was dosed at 100 pg per injection (BioX Cell, BE0296). ODN 2395 (CpG) was dosed at 20 pg per injection (Invivogen, tlrl-2395). Anti-PDl antibody was dosed at 200 or 250 pg per injection, as indicated (BioX Cell, Clone RMP1-14, BE0146).Example 2: Site of Administration Alters Biodistribution[022*>] This example describes an in vivo study investigating the impact of the administration site (IT vs. PT) on the distribution of immunotherapeutic agents.

[0230] AlexaFluor 647 Conjugation of Anti-OX40 Antibody. Anti-OX40 antibody (BioX Cell, BE0031) was conjugated with AlexaFluor 647 NHS-ester (ThermoFisher, A20006) according to the manufacturer’s specifications. AlexaFluor 647 NHS-ester was prepared at 10 mg / mL in anhydrous DMSO and combined with the antibody in 8 molar excess. The reaction was left to stir and incubate for 1 hour at room temperature before purification of the conjugated antibody via repeated washes through Amicon ultra centrifugal filters, 10 kDa MWCO (Millipore, UFC501008).]0231| Mammalian Cell Culture. Murine B16F10 melanoma was purchased from ATCC (CRL-6475). Aliquots of MC38 murine colon adenocarcinoma were provided as a gift from the Davis Lab at Stanford and subsequently purchased from Sigma Aldrich (SCC172). Cells were maintained in RPMI media (Cytiva, SH30027.FS) , supplemented with 10% fetal bovine serum (R&D Systems, SI 1550) and 1% penicillin-streptomycin (Thermo Fisher, 15140122) prior to tumor inoculation.

[0232] Murine Tumor Induction and Monitoring.

[0233] All animal studies were performed in accordance with the National Institutes of Health (NIH) guidelines, with the approval of the Stanford Administrative Panel on Laboratory Animal Care. Seven- to 8-week-old female C57BL / 6 were purchased from Charles River and housed in the animal facility at Stanford University. Mice were allowed to acclimate in the Stanford facilities for 1 week prior to beginning experimental procedures. Right-side flanks of mice were shaven prior to injections.| 234] MC38 flank xenografts were generated on 9-week-old female C57BL / 6 mice by injecting a 50 pL of MC38 cells (10 x io6cells / mL) encapsulated in a 1 wt% alginate hydrogel subcutaneously above the right hind leg. Cell loaded alginate hydrogels were prepared using a dual syringe mixing technique as described previously. A stock solution of sterile alginate (Pronova UP LVG) was dissolved at 5 wt% by adding saline to the polymers and allowing them to dissolve over 1 day at 4 °C. A stock solution of calcium sulfate (100 mM) was prepared in water in a large container in the form of a slurry, mixed vigorously. Calcium sulfate and cells were combined at the volume needed to reach the desired final concentration and loaded into a 1 mL syringe, while alginate (at the volume needed to reach the desired finalconcentration) was loaded into a separate 1 mL syringe. The two syringes were connected with a female-female luer lock elbow, with care to avoid air at the polymer-solution interface and mixed gently until a homogenous alginate hydrogel (1 wt% alginate, 10 mM calcium sulfate) was formed.[0235| B16F 10 melanoma models were generated similarly; 9-week-old female C57BL / 6 mice were injected with a 50 pL alginate hydrogel loaded with B16F10 cells (6 x 106cells / mL) subcutaneously above the right hind leg.

[0236] Following tumor inoculation, mice were monitored for palpable tumors. Treatments began on day 7 after tumor inoculation, at which point tumors grew to approximately 5 mm in diameter. Treatment groups were formed to maintain consistent tumor burden at the time of treatment, resulting in groups of N=6-12, depending on the study design. Mice were cage blocked or randomized depending on the study. Tumor burden was measured on day -1 before treatment to ensure fair distribution of tumors across treatment groups. Mouse weight was tracked to assess acute toxicity, with euthanasia criteria for sustained losses of more than 20% of body mass relative to the start of treatment. Tumor burden was tracked three times a week using caliper (Mitutoyo) measurements to calculate total tumor area (L * ) mice were euthanized once total tumor burden grew to 150 mm2or larger.

[0237] In Vivo Biodistribution of Anti-OX40 Antibody. PNP hydrogels and immunotherapy formulations were prepared as described in Example 1 above. C57BL / 6 mice were injected either peritumorally or intratumorally with 1:5 PNP hydrogels loaded with 10 pg of AF647 labeled anti-OX40 antibody. At the indicated time points, mice were euthanized with CO2 and either the lymph node or tumors were excised and imaged using an in vivo imaging system (IVIS Lago). Excised tissues of interest were imaged at 640 / 670 excitation / emission with medium binning, fstop = 8 for 2 second exposure.

[0238] Statistical analysis. For imaging analysis, N=4-10 samples were imaged per timepoint per group, and data are presented as mean + / - standard error of mean (SEM) as specified in the corresponding figure captions. Statistics are ordinary one-way ANOVA run in GraphPad Prism with Tukey’s multiple comparisons test. For survival studies and flow cytometry, a sample size of N=6-12 was used and data are presented as mean + / - standard error of mean (SEM) as specified in the corresponding figure captions. Comparisons between two treatment groups (e.g., intratumoral to peritumoral) were determined by two-tailed Mann Whitney test in GraphPad prism. Survival curves were compared by log-rank Mantel-Cox testin GraphPad prism. Comparisons between multiple groups were determined by Kruskal-Wallis test with Dunn’s multiple comparisons test in GraphPad Prism, p values less than 0.2 are shown.

[0239] FIG. 3A is a schematic of an experimental hypothesis indicating that PNP hydrogels can be used to selectively distribute immunotherapies as a function of site of administration, comparing intratumoral and peritumoral injection. PNP hydrogels have been used to substantially alter the pharmacokinetics of an anti-CD40 antibody (CD40a) to favor drug exposure in target tissues, resulting in retention at the injection site and accumulation in the tdLNs. It was hypothesized that this feature of sustained locoregional delivery via PNP hydrogels could be exploited to selectively retain cargo in the tumor via IT administration or in the tdLNs via PT.

[0240] FIG. 3B is a schematic illustrating the components of the PNP hydrogels. Anti-0X40 agonist antibodies (OX40a) were fluoro-labeled with AlexaFluor 647 NHS-ester, according to the manufacturer’s recommendations, and OX40a loaded PNP hydrogels were prepared by physically mixing aqueous solutions of dodecyl-modified hydroxypropyl methylcellulose (HPMC-C12), poly(ethylene glycol)-block-poly(lactic acid) (PEG-b-PLA) nanoparticles, and labeled OX40a.

[0241] As shown in FIG. 3C, to assess retention in the tumor from IT injections, C57BL / 6 mice were inoculated with 5 * 105MC38 cells or 3 x 105B16F10 cells and treated 7 days thereafter with 1 : 5 PNP hydrogels (where x:y refers to the wt% of polymer to nanoparticle, respectively, in the final formulation). Tumors were excised at days 1, 3, and 5 post treatment and imaged on an in vivo imaging system.

[0242] FIGS. 3D and 3E show representative images of excised tumors (FIG. 3D) and tdLNs (FIG. 3E) on days 1, 3, and 5 from each treatment group. As an indicator of total bioaccumulation, area under the curve of total flux was quantified over the time series of imaging in both tumors and lymph nodes. It was found that IT administration resulted in significantly greater retention and accumulation in the tumor compared to either PT or systemic administration of the labeled antibody (FIG. 3D). Contrarily, PT administration resulted in an increase in antibody exposure to the lymph nodes when compared to IT or systemic administration (FIG. 3E). These trends in the AUC are commensurate with site specific exposure across the time points at which images were taken.

[0243] As shown in FIGS. 3F and 3G, IT administration maintained meaningfully increased tumor localization of cargo through day 7 post administration while PT administration increased lymph node exposure through day 5 post administration compared to alternate sites of administration.

[0244] As shown in FIG. 3H, differences in tumor burden across site of administration were observed, with those mice receiving PT OX40a having the smallest tumors.

[0245] FIG. 31 shows the quantification of average radiance in excised tumors for each time point. At each point across a five-day time series, it was found that IT administration resulted in significantly greater retention and accumulation in the tumor compared to PT administration of the labeled antibody. FIG. 3K shows the quantification of average radiance in excised tumor draining lymph nodes for each time point. In reversal of the trend seen in FIG.31, PT administration resulted in a meaningful increase in antibody exposure to the tdLNs when compared to IT or systemic administration (FIG. 3K); this result was most notable at the critically relevant timepoint of D3, at which point peak 0X40 receptor upregulation post activation was expected (FIG. 3K, p = 0.0009 /

[0246] FIG. 3 J shows the ratio of average radiance of different routes of administration to systemic administration in excised tumors over time. FIG. 3L shows the ratio of average radiance of different routes of administration to systemic administration in excised tumor draining lymph nodes over time. FIGS. 3J and 3L quantify the impact of locoregional, sitespecific administration relative to current clinical approaches for protein therapeutics, namely intravenous infusion. In reporting the ratios IT: Systemic and PT: Systemic of average radiance, it was demonstrated that IT delivery via PNP hydrogels increases tumor exposure by up to 21.8x relative to systemic administration, and that PT delivery via PNP hydrogels increases lymph node exposure by up to 3.4x relative to systemic administration (FIGS. 3 J and 3L). Interestingly, when quantifying signal in the tdLNs, PT administration resulted in over 2x greater exposure relative to systemic administration for all four time points evaluated whereas IT administration only crossed this threshold at 24 hours, demonstrating that overall, PT administration enables more targeted locoregional delivery to tdLNs compared to IT or systemic routes of administration. Data are reported as mean + / - SEM. Statistics are two-tailed unpaired Welch’s t test, run in GraphPad Prism.

[0247] Collectively, these data demonstrate that PNP hydrogels can be leveraged to selectively skew cargo accumulation as a function of site of administration.Example 3: IT Hydrogel Administration of Cytokines Confers Survival Benefit over PT Administration

[0248] This example describes an in vivo study investigating the survival benefit conferred by cytokines administered via IT or PT hydrogels. Having demonstrated unique pharmacokinetics and biodistribution from IT, PT, and soluble systemic administration (Example 2), studies were performed to examine whether these differences resulted in changes in therapeutic efficacy when delivering potent proinflammatory cytokines. These studies were performed according to the protocols of Examples 1 and 2 above.

[0249] FIG. 4A shows a treatment schedule for a monotherapy study using IL-12. The effect and tolerability of a single administration of low dose IL-12 was evaluated in C57B / 6 mice induced with 3 x 105B16F10 cells and treated 4 days thereafter.

[0250] As shown in FIG. 4B, it was first observed that IT administration of IL-12 via 1:5 PNP hydrogels better controlled tumor growth through 25 days post tumor induction compared to soluble and PT administration of the same therapy. As shown in FIG. 4C, when comparing long-term survival benefit, IT PNP administration resulted in significantly longer survival of mice compared to a soluble IT administration of the same treatment, with median survival extending to 32 days compared to 29 days, respectively (p = 0.045). Further, mice receiving IT hydrogel administration had survivors through day 49 post tumor induction compared to day 39 for mice receiving intratumoral soluble cytokine. The survival benefit between mice treated with PT hydrogel and IT hydrogel was also compared. As shown in FIG.4D, there was a notable difference in extending the median survival from 26 days for mice treated with peritumoral hydrogel to 32 days for mice treated with intratumoral cytokine (p = 0.0642).

[0251] Having demonstrated the potential for IT administration of cytokines to elicit a meaningful anti-cancer response, it was next investigated if a more robust response could be achieved and tolerated with higher doses and combinations of cytokines. Based on doses reported in studies using other tumor-anchoring systems, a dose response study was performed. As shown in FIG. 4E, either 5 pg (low dose) or 20 qg (high dose) of IL-12 was delivered on day 7 after inducing B16F10 tumors (N=8 for all groups), and weight loss was monitored for two weeks post treatment as an indicator of treatment induced toxicity. Mice did not lose more than 5% of their body weight even when treated with high dose hydrogel, and any weight loss was quickly recovered within one week post treatment. Moreover, it was qualitatively observedthat mice treated with high dose gels had more controlled tumor burden over the period observed than those mice treated with low dose gels.

[0252] Therefore, subsequent studies were performed to examine the therapeutic efficacy of an IT injected hydrogel loaded with both 20 pg of IL- 12 and 5 pg of IL-2, complementary cytokines that activate both the JAK and STAT pathways. As shown in FIG.5A, all mice, except for those in the untreated group, also received immune checkpoint blockade (ICB) as 200 pg doses of aPDl delivered intraperitoneally every three days for five total administrations. As shown in FIG. 5B, mice receiving IT hydrogels containing cytokine, in combination with ICB, had well controlled tumor burden through day 20 post tumor inoculation compared to those receiving other treatments; mice receiving empty hydrogel and those that were untreated had tumors that started growing exponentially around 10 days after tumor inoculation, while 3 of 10 mice receiving the PT treatment had tumors that started growing exponentially by day 20. As shown in FIG. 5C, only the mice receiving IT therapy had long term survivors, extending median survival from 31.5 to 37 days compared to those receiving PT administration of the same treatment (p = 0.0018).Example 4: Intratumoral Hydrogel Administration of Cytokines Polarizes the Tumor Microenvironment

[0253] This example describes in vivo studies investigating the frequency of various T cell subtypes following IT or PT administration of hydrogels loaded with cytokines. To gain insight into the mechanism of action driving the observed survival benefit, the frequency of T cell subsets in the tumor seven days following IT or PT administration of cytokine-loaded hydrogels was assessed.[02541 Sample Preparation for Flow Cytometry of Lymph Nodes and Tumors. Immunotherapy formulations were prepared and tumor induction was performed according to the protocols of Examples 1 and 2 above. Tumor draining inguinal lymph nodes were harvested from mice at days 3, 7, and 14 post intratumoral or peritumoral immunotherapy administration, as indicated. LNs were mechanically disrupted into single cell suspensions using Corning single-frosted micro slides (2948-75X25), passed through 70 micron filters (Celltreat, 229484), spun at 500 ref for 5 minutes, resuspended in PBS, and counted using acridine orange / propidium iodide cell viability stain (Vitascientific, LGBD10012) and a Luna-FL dual fluorescence cell counter (Logos Biosystems). 1 million live cells per sample were transferred to a 96-well conical bottom plate (Thermo Scientific, 249570) and stained. Tumors weresimilarly excised, weighed and mechanically disrupted via surgical scissors. Bulk homogenate was enzymatically digested with an enzyme mixture consisting of collagenase / hyaluronidase (STEMCELL Technologies, # 07912) and DNAse 1 (STEMCELL Technologies, #100-0762) at 0.1x:0.15x ratios, respectively, in RPMI media (Cytiva, SH30027.FS). Samples and enzymatic mixture were transferred to 5.0 mL protein lobind tubes (Eppendorf, #0030108302) and left to digest at 37C while shaking at 300 rpm for 25 minutes. Digestion was stopped by adding complete RPMI media, previously described, supplemented with 1% 0.5 M EDTA (BioVision, 2103-100). Samples were spun at 300 ref for 10 min with brake on low, resuspended in PBS, counted and plated as described for LNs.[0255 | Staining Protocol and Flow Cytometry. Lymph node and tumor samples were first stained with 200uL Ghost Dye Violet 510 live / dead (Tonbo Biosciences, 13-0870-T100) for 5 minutes on ice, quenched with 100 pL FACS buffer (PBS, 3% heat inactivated FBS, 1 mM EDTA), and spun at 935xg for 2 minutes (with brake on low for tumor samples). Samples were then incubated with 50 pL anti-mouse CD16 / CD32 (1:50 dilution, BD, 553142) for 5 minutes on ice before staining with 50 pL surface antibody stain for 30 min on ice. Samples were spun as before. For intracellular cytokine staining, samples were incubated in 200 pL lx IC Fixation Buffer for 30 minutes at room temperature in the dark. Cells were spun at 500xg for 5 minutes, washed in 200 pL of lx Permeabilization Buffer, and spun as before. Samples were then incubated in lOOuL of full intracellular antibody stain diluted in lx Permeabilization buffer for 30 minutes at room temperature in the dark. Cells were quenched in 200 pL of lx Permeabilization buffer, centrifuged at 500xg for 5 minutes, then washed and spun once more. Samples were resuspended in 200uL FACS buffer and run on the Agilent NovoCyte Penteon Flow Cytometer in the Stanford Shares FACS Facility. Data was analyzed in FlowJo. FoxP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, 00-5523-00) were used for fixation and permeabilization according to the manufacturer’s instructions.|0256] LN full antibody stain included anti-mouse CD44 (1:80 dilution; BV750; BioLegend, 103079), anti-mouse / rat XCR1 (1:160 dilution; AF647; BioLegend, 148213), antimouse CDllc (1:200 dilution; PE; BioLegend, 117308), anti-mouse CD19 (1:200 dilution; PE-Cy7; BioLegend, 115520), anti-mouse CD161 (NK1.1) (1:100 dilution; BV605; BioLegend, 108740), anti-mouse I-A / I-E (MHCII) (1:400 dilution; FITC; BioLegend, 107606), anti-mouse CD45 (1:400 dilution; AF700; BioLegend, 103128), anti-mouse CD8a (1:200 dilution; BV785; BioLegend, 100750), anti-mouse CD3 (1:200 dilution; PerCP / eFluor710; Invitrogen, 46003283), anti-mouse CDllb (1:100 dilution; BUV395; BD,563553), anti-mouse F4 / 80 (1:125 dilution; BV421; BioLegend, 123132), anti-mouse CD4 (1:200 dilution; BUV805; BD, 612900), and anti-mouse CD62L (1:80 dilution; BUV737; BD, 612833).

[0257] Tumor surface antibody stain included anti-mouse CD25 (1:50 dilution; FITC; BioLegend, 101908), anti-mouse / rat XCR1 (1:100 dilution; AF647; BioLegend, 148213), antimouse CD206 (1:50 dilution; BV650; BioLegend, 141723), anti-mouse Ly-6C (1 : 100 dilution; BV570; BioLegend, 128030), anti-mouse Ly-6G (1:100 dilution; BV711; BioLegend, 127643), anti-mouse CDllc (1:100 dilution; PE; BioLegend, 117308), anti-mouse CD19 (1:100 dilution; PE-Cy7; BioLegend, 115520), anti-mouse CD161 (NK1.1) (1:50 dilution; BV605; BioLegend, 108740), anti-mouse I-A / I-E (MHCII) (1:200 dilution; FITC; BioLegend, 107606), anti-mouse CD45 (1:200 dilution; AF700; BioLegend, 103128), anti-mouse CD8a (1:100 dilution; BV785; BioLegend, 100750), anti-mouse CD3 (1:100 dilution; PerCP / eFluor710; Invitrogen, 46003283), anti-mouse CDllb (1:100 dilution; BUV395; BD, 563553), anti-mouse F4 / 80 (1:80 dilution; BV421; BioLegend, 123132), and anti-mouse CD4 (1:100 dilution; BUV805; BD, 612900). Tumor intracellular antibody stain included antimouse IFN-y (1:20 dilution; APC; BioLegend, 505810) and anti-mouse FoxP3 (1:20 dilution; PE; BioLegend, 126403).

[0258] FIGS. 6A and 7A show flow cytometry data of the frequencies of CD8+and CD4+T cells from murine Bl 6F 10 tumors, respectively. While the overall frequencies of CD8+and CD4+T cells among CD45+cells were not different between the mice receiving IT vs PT injections, the composition of T cell subsets were meaningfully skewed toward an anti -tumor phenotype in mice receiving IT therapy compared to those receiving PT therapy. As shown in FIGS. 6A-6D, with respect to CD8+T cell subsets, mice receiving IT cytokine therapy had a greater percentage of central memory, IFNy-expressing, and naive and stem-like memory cells. FIGS. 7A and 7B show that similar trends were observed among the CD4+T cells. It was shown that IT therapy drives a significant increase in the fraction of CD4+central memory T cells when compared to those generated by PT therapy and, as shown in FIGS. 8 A and 8B, a decrease in CD25+FOXP3+regulatory T cells.

[0259] Assessing important myeloid populations, as shown in FIG. 9, it was also observed that IT administration augmented the fraction of MHCII+CDllc+DCs, and it particularly increased the percent of inflammatory DCs when compared to PT administrations; to the contrary, PT administration of cytokine drove higher fractions of cDC2s and cDCls inthe tumor. Other populations, including B cells and NK cells, were similar across treatment groups in the tumors.

[0260] Important lymphoid and myeloid populations in the tdLN were concomitantly examined on day 7 post treatment. As shown in FIG. 10, differences in T cell subsets were marginal, with a modest increase in the fraction of cytotoxic CD8+T cells, NK cells, and MHCII+CDllc+DCs when treated with IT therapy compared to PT therapy. Of note, IT cytokine administration reduced the fraction of cDC2s compared to PT administration.

[0261] Recognizing that the cytokine cargo has pleiotropic effects that may occur across multiple timescales, studies were performed to capture the immune landscape of the tumor microenvironment and the tdLNs more quickly after treatment. A similar analysis was conducted to assess the frequency of T cell subsets on day three. As shown in FIG. 11, at this early time point, fewer differences in cell phenotype were observed between the two treatment groups, though IT therapy drove an increase in the percent of cDC2s and cDCls in the tumor while PT administration increased the representation of iDCs and NK cells. As summarized in FIG. 12, frequencies of important lymphoid and myeloid cells were then quantified in the tdLNs on day three and it was found that IT administration of cytokines resulted in a slightly increased frequency of effector memory CD8+T cells, a decrease in naive and stem-like memory CD8+T cells, an increase in cDCls and a decrease in cDC2s.Example 5: PT Hydrogel Administration of QX40a Confers Survival Benefit over IT Administration

[0262] This example describes in vivo studies investigating the therapeutic potential of OX40a. Having examined the effect of administration site on anti-cancer immunity in the context of cytokine immunotherapies, it was hypothesized that complementary trends would be observed when delivering certain antibodies, such as agonistic anti-OX40, which helps sustain T cell activity and drives a long-term response through the clonal expansion of certain T cell memory subsets. Since much of this expansion happens in the tdLNs, where tumor antigens, T-cells, and APCs interact, it was speculated that PT administration of OX40a would be superior to intratumoral administration.

[0263] To assess the therapeutic potential of OX40a, B16F10 melanoma tumors were induced in C57BL / 6 mice and treated with 1:5 PNP hydrogels loaded with 10 pg of OX40a and 20 pg of CpG per dose on day 7, in accordance with the protocols of Examples 1 and 2 above. FIG. 13 A shows the experimental scheme. All mice, excluding those that wereuntreated, were also given 200 pg doses of aPDl via intraperitoneal injection every three days for five administrations. As shown in FIG. 13B, it was observed that tumors remained well controlled in both groups receiving hydrogel-delivered therapies, while mice receiving only aPDl or those that were untreated experienced rapid tumor growth, by two weeks post administration. However, as shown in FIG. 13C, only PT administration of the OX40a therapy, but not IT administration, resulted in long-term survivors, extending median survival from 26 days to 31 days.Example 6: PT Administration of QX40a Alters Tumor and Lymph Node Immunophenotype

[0264] This example describes in vivo studies characterizing how IT vs. PT administration of immune-agonizing cargo via hydrogels altered the immunophenotypes of the tumor and tdLN microenvironments. Studies were performed according to the protocols in Examples 1, 2, and 4 above.

[0265] First, the frequency of T cell subsets in the tumor seven days following IT or PT administration of OX40a-loaded hydrogels was assessed. As shown in FIGS. 14A-14D, the frequency of CD8+T cells and CD8+central memory T cells were similar across both treatment groups, but it was found that CD8+T cells were enriched for effector memory subsets when treated via PT administration rather than IT administration. It was also observed that PT OX40a administration noticeably drove the frequency of naive and stem-like memory CD8+T cells down compared to when treated via IT administration while both treatment groups resulted in comparable frequencies of IFNy+cytotoxic CD8+T cells.

[0266] As shown in FIGS. 15A and 15B, PT administration of OX40a-loaded hydrogels dramatically increased the proportion of CD4+T cells and, among them, significantly boosted the fraction with an effector memory phenotype (with a commensurate reduction in the proportion with central- or naive and stem-like memory phenotypes) and those expressing IFNy. Additionally, PT administration reduced the fraction of regulatory T cells when compared to IT administration of the same therapy.

[0267] Complementing these data, myeloid populations in the tumor were assessed on day 7 post treatment. As shown in FIG. 17, there were modest increases in frequencies of MHCII+CD1 lc+DCs, cDC2s, cDCls, B cells, NEC cells, and CD4+T cells in the tumors from mice treated with PT OX40a hydrogels while IT therapy led to an increase in cell frequency for only iDCs and CD8+T cells.

[0268] Concurrently, similar populations in the tdLNs on day 7 post treatment were examined. As shown in FIG. 18, similar trends were observed: PT administration of OX40a, but not IT administration, augmented the frequency of central memory CD8+T cells and effector memory CD8+T cells while decreasing the corresponding frequency of naive and stem-like memory CD8+T cells. A marginal increase was found in the frequency of helper CD4+T cells, but similar frequencies of CD4+T cell subsets between treatment groups in the lymph nodes and similar percentages of NK cells and MHCII+CD1 lc+DCs. PT administration of the OX40a therapy resulted in decreased fractions of cDCls and cDC2s in the tdLNs compared to IT administration.[0269| Recognizing that one parameter of anti-cancer immunity is the generation of memory T cell subsets and that the context in which T cells recognize antigen contributes to T cell expansion, contraction and memory phases, studies were performed to characterize the tumor microenvironment and lymph nodes at a later stage post treatment. The same flow cytometric analysis was performed on tumors and tdLNs from mice treated with OX40a-loaded hydrogels 14 days after treatment (day 21 post tumor induction). FIG. 19 shows a series of graphs summarizing the frequencies of various immunophenotypes present within the tumor, and FIG. 20 shows the frequencies of various immunophenotypes present within the tdLN. In the tumors, it was noted that PT administered OX40a increased the contribution of MHClL CD1 lc+DCs and cDC2s while decreasing the proportion of iDCs and cDCls when compared to IT administration. Similar fractions of B cells and a small decrease in the proportion of NK cells from PT administration were observed compared to IT administration. A meaningful decrease in the percentage of CD8+T cells and an increase in the frequency of CD4+T cells were observed when treating with PT OX40a-loaded hydrogels. As shown in FIG. 20, meaningful differences in frequency of lymphoid and myeloid cells persisted in the tdLNs through day 14, evidence of a sustained response.Example 7: Combination of IT Administered Cytokines and PT Delivered QX40a Mounts Robust Anti-Cancer Response

[0270] This example describes in vivo studies investigating the synergy between cytokines delivered via IT administration and OX40a delivered via PT administration. Noting the individual efficacies of these therapeutic approaches, it was hypothesized that the combination of IT delivered cytokines and PT administered OX40a would capitalize on bothtumor polarization and mount a robust memory response to confer superior anti-cancer efficacy. Studies were performed according to the protocols in Examples 1, 2, and 4 above.

[0271] FIG. 21A shows the experimental scheme. To assess the potency of this combination, B16F10 melanoma tumors were induced in C57BL / 6 mice and treated via IT administration with 1:5 PNP hydrogels loaded with cytokines as before (IL-12 and IL-2) with and without concomitant PT administration of 1:5 PNP hydrogels loaded with OX40a and CpG; all groups except for untreated received 250 pg of aPDl delivered every five days for three total administrations. As shown in FIG. 2 IB, reporting tumor burden through day 20 post tumor induction, it was found that mice receiving hydrogel treatments had better controlled tumor growth than those that did not, consistent with previous findings. Additionally, tumor growth was better controlled in the group receiving the IT-PT therapy combination than in the group receiving IT cytokine alone. As shown in FIG. 21C, this trend persisted through the duration of the study, where it was found that 50% of mice treated with the IT-PT combination survived past day 50 post tumor inoculation compared to no mice surviving past day 41 when treated with IT cytokine and ICB alone. The combination therapy extended median survival to 46 days compared to 32.5 days for mice receiving only IT cytokine and ICB.

[0272] To verify that this was a tumor agnostic effect, the study was repeated in C57BL / 6 mice inoculated with 5 x 105MC38 murine adenocarcinoma cells and treated with the same regime shown in FIG. 21 A. FIGS. 2 ID and 2 IE are graphs showing the resulting tumor burdens and survival probabilities, respectively. Tumor burden remained well controlled over the duration of the study in both groups receiving hydrogel delivered immunotherapy, with 100% of animals being tumor free by day 50 post tumor induction in those two cohorts.

[0273] To validate that the observed synergy is a consequence of rational localization of the respective cargo, the efficacy of the combination immunotherapy was investigated when co-delivered at a single site. FIG. 22A shows the experimental scheme. Here, mice were induced with B16F10 melanoma tumors and treated via either IT or PT administration with single 1:5 PNP hydrogels loaded with the entire immunotherapy combination including IL-12, IL-2, OX40a and CpG. As shown in FIG. 22B, mice receiving the IT therapy had a median overall survival of 36 days compared to those receiving PT therapy, which had a median overall survival of 30.5 days. As shown in FIG. 22C, mice receiving the IT combination therapy had well controlled tumor burden through day 20 post tumor induction whereas 25% of those receiving PT therapy had tumors that reached exponential growth by day 23. Despite the difference in immediate tumor growth, which resulted in earlier mouse euthanasia, the long-term survival of both cohorts converged by day 40 with 25% long term survivors, significantly fewer than when the therapies were delivered separately.

[0274] As before, studies were performed to characterize this response at the cellular level. B16F10 tumors were induced, treated, and excised tumors and lymph nodes were analyzed via flow cytometry on day 7 post treatment. T cell populations were then assessed. As shown in FIGS. 23 A and 23B, it was observed that the spatially distinct combination therapy resulted in the greatest frequency of CD8+T cells compared to either of the monotherapies or the IT combination. The IT combination therapy resulted in a lower frequency of CD8+T cells than any other treatment, indicating a deleterious effect of having all cargo IT. In examining CD8+T cell subsets, it was observed that IT cytokine therapy drives an expansion of central memory cells relative to the other groups, and especially compared to PT antibody therapy. As shown in FIG. 23C, this is nevertheless recovered when the therapies are delivered together, either in a co-localized or spatially distinct fashion. As shown in FIG. 23D, the combination of IT and PT therapy increased the frequency of CD8+effector memory cells relative to IT cytokine alone, an improvement that is not realized when the therapies are co-localized. As shown in FIG. 23E, the expansion of these critical populations is reinforced by the dramatic reduction in naive and stem-like central memory CD8+T cells in tumors from mice receiving IT and PT combination therapy, especially compared to IT cytokine monotherapy. As seen in FIGS. 23F and 23G, it was also observed that the IT and PT combination therapy results in the greatest frequency of IFNy producing CD8+T cells compared to all other groups, salvaging the otherwise low frequency of this population when receiving PT OX40a alone.

[0275] These quantifications were repeated for CD4+T cell subsets. As shown in FIGS.24A and 24B, was observed that PT OX40a drives an expansion of CD4+T cells relative to other treatment groups (which is mostly preserved in the combination therapy regimes and still a noticeable increase from IT cytokine therapy alone). As shown in FIGS. 24C and 24D, while the combination therapies decreased CD4+central memory populations, this is complemented by a subtle increase in CD4+effector memory fractions. As shown in FIG. 24E, once again, these differences parallel decreases in the naive and stem-like central memory CD4+T cells. However, as shown in FIGS. 24F, 24G, 25 A, and 25B, the IT and PT combination immunotherapy had a synergistic effect on IFNy+CD4+T cells and regulatory T cells. The IT and PT combination therapy redeemed the IFNy CD4+T cell population relative to the PT mAb therapy (and still meaningfully drives the population up from the IT cytokine therapy) while significantly decreasing the frequency of Tregs, a property not directly observed foreither of the monotherapies alone. This feature is observed in the IT and PT combination therapy but not the IT combination therapy.

[0276] The characterization of the tumor was continued by examining the frequencies of various myeloid populations. As shown in FIG. 26, the IT and PT combination therapy resulted in a moderate decrease in the proportion of MHCII+CDllc+DCs relative to the monotherapies, a moderate decrease in the percentage of cDC2cs relative to PT mAb, an increase in iDCs compared to PT mAb, and a decrease in cDCls compared to both monotherapies.

[0277] Next, T cell populations and their subsets were characterized in the tdLNs on day 7 post treatment, as before. As shown in FIGS. 27A and 27B, it was found that the IT and PT combination therapy group significantly increased the percent of CD8+T cells compared to PT mAb alone while the IT combination did not. As shown in FIGS. 27A, 27C, and 27D, the IT and PT combination therapy resulted in a decrease in the frequency of central memory CD8+T cells but dramatically increased the frequency of effector memory CD8+T cells, a population which was not particularly augmented by any therapy alone. As shown in FIG. 27E, both combination groups resulted in a consistent decrease in the naive and stem-like memory CD8+T cells, though the IT and PT combination therapy group did so more significantly when compared to IT cytokine alone. As shown in FIGS. 28A-28E, in the case of the CD4+T cell population and its subsets, a skew was observed in the effector memory CD4+population, where the IT and PT combination therapy increased frequency compared to the monotherapies.

[0278] The discovery and adoption of cancer immunotherapies, notably immune checkpoint inhibitors, has revolutionized the standard of care in cancer treatment and significantly improved some patient outcomes; these successes notwithstanding, many approaches still fail to induce favorable response rates in a majority of patients and are associated with undesirable immune related adverse events. Advancements in characterizing the cancer immunity cycle have, in turn, demonstrated the need for next generation cancer immunotherapy to leverage combination approaches to uniquely overcome multiple, bio-orthogonal methods of immune evasion. Successful combination approaches generally engage both innate and adaptive immunity to eradicate fully established tumors and mount a robust memory anti-cancer response. Here, the capacity of a PNP hydrogel system to successfully deliver distinct immunomodulating cargo to a tumor and tdLN to promote anti-cancer immunity has been demonstrated. Building on reports demonstrating that these hydrogels render CD40a combination immunotherapytractable, significantly reducing toxicides while improving efficacy by changing the biodistribution of the cargo when compared to soluble, systemic administration, it has been demonstrated that site of administration can optimize the therapeutic efficacy of different immune agonists based on their mechanism of action.[0279| IT anchoring of cytokines can improve efficacy while reducing toxicity, e.g., the combination of engineered IL-2 and IL- 12 that binds to collagen can potentiate both checkpoint blockade and tumor specific antibodies to prolong survival and clear tumors in multiple syngeneic murine tumor models. Consistent with the findings disclosed herein, IT administration of combination cytokine immunotherapy polarized T cell phenotypes, critically increasing IFNY+CD8+T cell populations, which underlie cytotoxic cancer clearance, in the tumor microenvironment. This effector response may be driven specifically by the combination of IL-2 and IL-12, whereby IL-2 upregulates the IL-12 receptor and poses effector cells for enhanced Thl signaling. This cascade in turn enhances production of anti -turn oral cytokines like ITNy and the proliferation of effector cells. IT administration of these cytokines resulted in significant expansion of central memory subsets of both CD4+and CD8+T cells compared to PT administration of the same therapy. Central memory T cells have enhanced survival and proliferative potential, providing a reservoir of antigen-specific T cells ready to expand in response to challenges. While the exact role of different T cell memory subsets remains an area of active investigation, some studies suggest that central memory T cells confer superior antitumor immunity compared with effector memory T cells. Collectively, central memory T cells are well positioned to respond to secondary antigen exposure and survive in lymphoid structures, suggesting that the enhanced composition of T cells in the IT cytokine group that may confer an advantage in protecting against metastasis or relapse.[0280| Opposite trends have been observed when treating with PT OX40a. 0X40 agonism has been shown to critically promote T effector cell activation and inhibit T regulatory cell function, encouraging especially the expansion and survival of CD4+T cells; these properties have been readily exploited to eradicate local, distal and spontaneous malignancies. These reports are consistent with the data disclosed herein, where PT administration of an 0X40 agonist augmented T effector memory cell subsets (both CD8+and CD4+), CD4+T cells (as a fraction of all CD45+cells), and downregulated immunosuppressive regulatory T cells relative to IT administration. While CD8+T cells have canonically been the focus for cancer immunotherapy due to their cytotoxic effector functions, recent evidence has shown that CD4+T cells play a pivotal role in directing the anti-tumor immune response.

[0281] These results underscore the advantages of targeting therapy in a rational, spatiotemporal fashion. Localization to the tumor microenvironment may be insufficient to fully capitalize on a therapy’s potential depending on its mechanism of action. Central memory T cell populations traffic to the lymph node where they interact with tumor antigen presenting DCs, expand, and acquire effector function. The presence of the correct co-stimulatory cues can be deterministic to the ultimate composition of the expanded T cell population and subsequent anti-cancer immune response. It is hypothesized that cytokine driven expansion of central memory T cells is complimentary to 0X40 agonized lymph node interactions that drive a more robust effector memory T cell population. As suggested by the results of adoptive immunotherapy studies, a heterogeneous population of central memory and effector memory T cells may potentiate the best clinical outcomes. In fact, it has been proposed that central and effector memory phenotypes do not represent distinct subsets but rather a continuum in a differentiation pathway that may be herein exploited for successful immunotherapy.

[0282] Indeed, combining IT cytokine-loaded hydrogels with PT OX40a-loaded hydrogels resulted in synergistic effects that altered the immune landscape of the tumor microenvironment and tdLNs in distinct ways not captured by either monotherapy alone and which were not recapitulated by co-delivery of the immune agonists at a single site. The combination therapy resulted in a tumor immunophenotype consisting of both expanded central memory and effector memory T cell subsets, increased IFNy producing CD4+and CD8+T cells, and nearly complete regression of immunosuppressive regulatory T cells, results that realized superior efficacy compared to either of the leading monotherapies alone.

[0283] Together, this data demonstrates a single administration technique to bridge innate and adaptive immune pathways toward an endogenous, antigen-agnostic anti-cancer response. The strategy of combining IT and PT injection depends largely on the mechanism of action of the cargoes selected, not the expression of tumor or ECM specific motifs onto which the cargo will bind. Therefore, it represents a facile and translatable way to explore many promising combination immunotherapies. The data disclosed herein characterizes one such set of combination immunotherapy; however, other combination immunotherapy formulations are possible. For example, the mechanism and safety profile of the present technology could be generalized to other antibodies, cytokines, or molecules. As immuno-oncology continues to advance, additional targets are likely to be identified that the skilled artisan would readily adapt for use in this system. Nevertheless, the present disclosure demonstrates that PNP hydrogels are an emerging immunoengineering system that can potentiate powerful combinationimmunotherapies and address unique immunological questions regarding the precise spatiotemporal delivery of potent immune agonists.Example 8: Combination Immunotherapy with IT Depot and Systemic Adoptive Cell Therapy

[0284] This example describes an in vivo study investigating a combination immunotherapy involving delivery of cytokines from an IT depot and systemic delivery of adoptive T cells.

[0285] Preparation of PNP hydrogels and tumor induction in mice was performed according to the protocols in Example 1 and 2 above.

[0286] Pmel-1 adoptive T cell culture, activation and transfer. On day 0, splenocytes were isolated from spleens and peripheral lymph nodes were harvested from female pmel-1 mice (Jackson Laboratory) and dissociated into a single cell suspension. Red blood cells were lysed using ACK lysis buffer (Thermo Fisher, A1049201). Cells were resuspended at 2 x 106cells / mL and plated into 96-well plates in culture media of complete RPMI 1640 with lx non-essential amino acids (Thermo Scientific, 11140050), 1 mM sodium pyruvate (Thermo Scientific, 11350070), 0.4x vitamin solution (Sigma-Aldrich, M6895), 92 pM 2-mercaptoethanol (Gibco, 21985023), 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) supplemented with 60 lU / mL hIL-2 (PeproTech, 200-02) and 10 pg / mL human gplOO (Anaspec, AS-62589). After two days, cells were split and fed with 50 pL culture media supplemented with 50 lU / mL hIL-2. On day 4, cells were fed with 50 pL culture media supplemented with 50 lU / mL hIL-2. Additionally, anti-CD3 96 well plates were made by coating 96-well plates with 50 pL of 5 pg / mL solution of anti-CD3 (clone 145-2C11, BioX cell) and incubated at 4 °C for 24 hours. On day 5, anti-CD3 plates were washed with sterile PBS to remove excess anti-CD3 and cells were harvested and resuspended at 1 x 106cells / mL in culture media supplemented with 60 lU / mL hIL-2 and 2 pg / mL anti-CD28 (clone 27.51 BioX cell). On day 7, cells were supplemented with 50 pL culture media supplemented with 120 lU / mL hIL-2. On day 8, cells were harvested, resuspended at 1 x 106cells / mL in culture media supplemented with 50 lU / mL IL-2 and moved to clean 96-well plates. On day 10, cells were ready for adoptive transfer. On day 10, activated pmel-1 T cells were harvested and resuspended at 50 x 106cells / mL in sterile PBS. Prior to administration, cells were passed through a 70 pm cell strainer (Celltreat, 229484). 100 pL of cells were adoptively transferred retro orbitally into female C57BL / 6 mice using U-100 insulin syringes (BD, 329461).

[0287] Lymphodepletion of mice. Mice were lymphodepleted 1 day prior to adoptive pmel-1 transfer with 4 mg of cyclophosphamide (Sigma-Aldrich C0768) intraperitoneally.

[0288] FIG. 29A illustrates the experimental scheme for the study. Mice received single IT injections of 20 pL 1:5 PNP hydrogels formulated with 15 pg IL-12 (“IT Gel IL-12,” groups 3 and 4). Mice were lymphodepleted intraperitoneally with 4 mg cyclophosphamide on day 7. On day 8, mice received a 100 pL dose (5 x io6pmel-1 adoptive cells) from a 50 x io6cell / mL suspension (“ACT,” groups 2 and 4).

[0289] FIG. 29B illustrates the probability of survival of mice monitored every 2-3 days for a total of 50 days post tumor inoculation. The combination IL-12 IT treatment and systemic adoptive cells improved overall survival over both single administered therapies. This combination therapy resulted in 70% long term survivors at day 50.Example 9: Abscopal Effects of Site-Specific Combination Immunotherapy of IT Administered Cytokines and PT Administered Antibody

[0290] This example describes in vivo studies investigating the abscopal effects of sitespecific combination immunotherapy. Given the distinct skew of effector, memory, and regulatory T-cell subsets in tumors and tumor draining lymph nodes, it was investigated whether the lead combination therapy could drive systemic anti-tumor immunity.

[0291] Preparation of PNP hydrogels and tumor induction in mice was performed according to the protocols in Example 1 and 2 above.

[0292] FIG. 30A illustrates the experimental scheme for the study. B16F10 melanoma tumors were induced on both left and right flanks of C57BL / 6 mice at the same time to model aggressive metastases. Right flank tumors only were treated 7 days thereafter via IT administration with 1:5 PNP hydrogels loaded with cytokines as before (5 pg IL-2 and 20 pg IL-12) with and without concomitant PT administration of 1:5 PNP hydrogels loaded with OX40a (10 pg OX40a); all groups except for untreated received 250 pg of aPDl delivered every five days for three total administrations.

[0293] FIG. 30B illustrates the tumor growth curves through 20 days post inoculation with the untreated tumors on the left and the treated tumors on the right. As before, tumor growth was monitored via caliper measurements, where both treated and untreated tumors in animals receiving only aPDl and in untreated animals grew rapidly in the first two weeks post inoculation while those receiving cytokine or cytokine + antibody combination were wellcontrolled. Mice receiving combination IT cytokine and PT OX40a therapy demonstrated a greater reduction in tumor size as compared to mice treated with IT cytokine only. Additionally, a comparable reduction in untreated (left) and treated (right) tumor size was observed for the mice treated with the combination IT cytokine and PT OX40a therapy which is indicative of an abscopal effect from the site-specific immunotherapy.

[0294] FIG. 30C illustrates the survival for mice induced with two tumors through 60 days post tumor inoculation. Mice were considered to have met euthanasia criteria when either left or right tumor met or exceeded 150 mm2in area. Survival curves were compared by logrank Mantel-Cox test. Mice treated with combination IT cytokine and PT OX40a therapy demonstrated improved overall survival over all other groups. Survival was remarkably similar to that seen for the single-tumor efficacy study. It was found that only the combination of intratumoral cytokines and peritumoral antibodies resulted in long-term, progression free survivors (33%), and that the site-specific combination immunotherapy resulted in significantly better overall survival compared to IT cytokines (p = 0.0207). Collectively, these results demonstrate the capacity for site-specific, locoregional delivery of immune agonists to impart robust, systemic anti-cancer immunity by clearing abscopal tumors.Example 10: Comparison of PT Delivered Antibody Monotherapy with PT Delivered Antibody Combination Therapy

[0295] This example illustrates in vivo studies investigating the therapeutic efficacy of antibody monotherapy compared to combination therapy.

[0296] Preparation of PNP hydrogels and tumor induction in mice was performed according to the protocols in Example 1 and 2 above.

[0297] FIG. 31A illustrates the experimental scheme used to evaluate differences in efficacy between antibody monotherapies and antibody combination therapy. B16F10 melanoma tumors were induced in C57BL / 6 mice. Antibody doses included 100 pg 4-1BB and 10 ug of 0X40 per dose, or the combination thereof, where indicated, delivered in 100 pL peritumoral subcutaneous injections of 1:5 PNP hydrogels or systemically via intraperitoneal injections, as indicated. All animals except those in the untreated group receive clinically relevant checkpoint blockade (aPDl) for 3x administrations of 250 pg each, delivered 7 days after tumor induction and every 5 days thereafter.

[0298] FIG. 3 IB illustrates the tumor growth curves for mice induced with B16F10 through 20 days post inoculation. Mice receiving antibody combination therapy demonstratedgreater tumor size control than mice receiving antibody monotherapy, suggesting a synergistic effect between the two antibodies.

[0299] FIG. 31C illustrates the probability of survival for mice induced with B16F10 through 50 days post tumor inoculation comparing antibody monotherapies to antibody combination therapy when delivered via a PT hydrogel. Survival curves were compared by logrank Mantel-Cox test. Administration of the antibody combination therapy, but not antibody monotherapy, resulted in long-term survivors. The mice receiving the combination therapy demonstrated about 40% survival after 50 days.

[0300] FIG. 3 ID illustrates the probability of survival for mice induced with B16F10 through 50 days post tumor inoculation comparing antibody combination therapies delivered systemically or via hydrogel. Survival curves were compared by log-rank Mantel-Cox test. Administration of antibody combination therapies delivered both systemically and via hydrogel resulted in long-term survivors without a significant difference in survival between the systemically treated and PT hydrogel treated groups. Without wishing to be bound by theory, it is hypothesized that a difference in survival between systemically treated and PT hydrogel treated groups may be only observed at higher dose concentrations than tested in this study. As previously discussed, the systemic administration of antibodies at high doses can be toxic to patients such that local administration through a PT hydrogel at higher doses may help to limit toxicity.]0301| FIGS. 32A-32C illustrate in vivo studies demonstrating that antibody combination therapy drives a systemic anti-cancer immune response. FIG. 32A illustrates the experimental scheme used to evaluate the abscopal effects of antibody combination therapy in a dual -turn or induction model. B16F10 melanoma tumors were induced in the left and right flanks of C57BL / 6 mice. All animals except those in the untreated group received clinically relevant checkpoint blockade (aPDl) for 3x administrations of 250 ug, delivered 7 days after tumor induction and every 5 days thereafter. Mice were treated on day 7 via PT gel administration or systemically with antibody combination therapy. The antibody combination therapy comprised 100 pg 4- IBB and 10 ug 0X40 per dose, delivered in 100 pL peritumoral subcutaneous injections of 1:5 PNP hydrogels or systemically via intraperitoneal injections, as indicated.

[0302] FIG. 32B illustrates the left and right tumor growth curves for mice induced with B16F10 through day 16 post inoculation. Administration of the antibody combination therapy resulted in reduced tumor area for mice treated both systemically and via PT depots.

[0303] FIG. 32C illustrates the probability of survival for mice induced with dual-flank B16F10 tumors through 60 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test. Mice receiving both systemic and PT administered antibody combination therapy demonstrated increased long-term survival as compared to the other treatment groups. Mice treated via PT depots showed about 40% survival as compared to mice treated systemically which showed about 20% survival after 60 days, although there was no statistically significant difference between the PT depot and systemically treated groups (p = 0.63). These results indicate that the combination antibody therapy is tolerable at the tested dosage and increases the probably of survival.Example 11: PT Administered Antibody Combination Therapy Synergizes with Adoptive Cell Therapy

[0304] This example illustrates in vivo studies demonstrating that antibody combination therapy synergizes with adoptive cell therapy.

[0305] Preparation of PNP hydrogels and tumor induction in mice was performed according to the protocols in Example 1, 2, and 8.

[0306] FIG. 33A illustrates the experimental scheme used to investigate therapeutic effects of antibody combination therapy with adoptive cell therapy. B16F10 melanoma tumors were induced in C57BL / 6 mice. On day 7, mice were lymphodepleted intraperitoneally with 4 mg cyclophosphamide as described in Example 8. On day 8, all except those in the untreated group received a 100 pL dose (5 * 106PMEL CD8+ cells) from a 50 * 106cell / mL suspension (“ACT”). On day 11, mice were treated with antibody combination therapy via PT gel administration or systemically. The antibody combination therapy comprised 100 pg 4 IBB and 10 ug 0X40 per dose, delivered in 100 pL peritumoral subcutaneous injections of 1:5 PNP hydrogels or systemically via intraperitoneal injections, as indicated.

[0307] FIG. 33B illustrates the probability of survival for mice induced with B16F10 tumors through 50 days post tumor inoculation. Survival curves were compared by log-rank Mantel-Cox test. Mice receiving the PT combination antibody therapy in addition to the ACT demonstrated significantly greater long-term survival than all other treatment groups (p = 0.0337 as compared to systemic antibody combination therapy). At day 50, only the groupreceiving PT combination antibody therapy paired with ACT demonstrated modest long-term survival.Example 12: IT Administered Cytokine Therapy Synergizes with Adoptive Cell Therapy

[0308] This example illustrates in vivo studies demonstrating that cytokine combination therapy synergizes with adoptive cell therapy.

[0309] Preparation of PNP hydrogels and tumor induction in mice was performed according to the protocols in Example 1, 2, and 8.

[0310] FIG. 34A illustrates the experimental scheme used to investigate therapeutic effects of cytokine combination therapies with adoptive cell therapy in mice with or without lymphodepletion (“LD”). B16F10 melanoma tumors were induced in C57BL / 6 mice. On day 7, some groups of mice were lymphodepleted intraperitoneally with 4 mg cyclophosphamide as described in Example 8. Cytokines were delivered as 20 pg IL- 12, 5 pg IL-2 in 20 pL, or as a combination of IL-12 / IL-2 through intratumoral injections of 1:5 PNP hydrogels, concurrently with the lymphodepletion. On day 8, all except those in the untreated group received a 100 pL dose (5 x io6PMEL CD8+ cells) from a 50 x io6cell / mL suspension (“ACT”).[0311 [ FIGS. 34B and 34C illustrate the probability of survival for mice induced with B16F10 tumors through 70 days post tumor inoculation without (FIG. 34B) and with lymphodepletion (LD) (FIG. 34C). Survival curves were compared by log-rank Mantel-Cox test. For mice without lymphodepletion, mice receiving the IL-12 / IL-2 and ACT combination therapy demonstrated the highest rate of long-term survival at about 80% after 70 days. For mice receiving lymphodepletion, mice treated with the IL- 12 and ACT combination therapy demonstrated the highest long-term survival at about 75% after 70 days.Additional Examples

[0312] Additional examples of aspects of the present technology are described below as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and do not limit the subject technology.

[0313] Clause 1. A system for the treatment of cancer, comprising:a first dynamic hydrogel configured to be administered within a tumor in a subject to form an intratumoral depot, wherein the first dynamic hydrogel comprises: a polymer non-covalently crosslinked with a plurality of nanoparticles, anda first immunotherapeutic agent; anda second dynamic hydrogel configured to be administered to the subject external to the tumor to form a peritumoral depot, wherein the second dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a second immunotherapeutic agent.|03l4] Clause 2. The system of Clause 1, wherein the first dynamic hydrogel is configured to modulate a tumor microenvironment of the tumor.

[0315] Clause 3. The system of Clause 2, wherein the modulation comprises converting the tumor microenvironment from an immune-suppressing state to an immune-activating state.

[0316] Clause 4. The system of any one of Clauses 1-3, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent into the tumor.

[0317] Clause 5. The system of any one of Clauses 1-4, wherein the first dynamic hydrogel is configured to be infiltrated by immune cells within the tumor, such that the immune cells are exposed to the first immunotherapeutic agent while within the first dynamic hydrogel.

[0318] Clause 6. The system of any one of Clauses 1-5, wherein the first immunotherapeutic agent is configured to recruit immune cells into the tumor, activate immune cells within the tumor, enhance recognition of cancer cells of the tumor by immune cells, enhance killing of cancer cells of the tumor by immune cells, or a combination thereof.

[0319] Clause 7. The system of any one of Clauses 1-6, wherein the first immunotherapeutic agent comprises a pro-inflammatory cytokine, a chemokine, an immune checkpoint inhibitor, an adjuvant, or a combination thereof.

[0320] Clause 8. The system of Clause 7, wherein the first immunotherapeutic agent comprises the pro-inflammatory cytokine, and wherein the pro-inflammatory cytokine comprises IL-2, IL-12, IL-15, IL-18, IL-21, or IFN-a.

[0321] Clause 9. The system of Clause 7, wherein the first immunotherapeutic agent comprises the chemokine, and wherein the chemokine comprises GM-CSF or CCL21.

[0322] Clause 10. The system of Clause 7, wherein the first immunotherapeutic agent comprises the immune checkpoint inhibitor, and wherein the immune checkpoint inhibitor comprises an anti-PD-Ll antibody or an anti-CTLA-4 antibody.

[0323] Clause 11. The system of Clause 7, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises a small molecule or a nucleic acid.

[0324] Clause 12. The system of Clause 7, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises CpG or an anthracycline.

[0325] Clause 13. The system of any one of Clauses 1-12, wherein the second dynamic hydrogel is configured to be administered proximate to a draining lymph node associated with the tumor.

[0326] Clause 14. The system of Clause 13, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent to the draining lymph node associated with the tumor.

[0327] Clause 15. The system of Clause 13 or 14, wherein the second immunotherapeutic agent is configured to enhance activity of immune cells present in the draining lymph node associated with the tumor.

[0328] Clause 16. The system of Clause 15, wherein the immune cells comprise B cells, antigen-presenting cells, T cells, or a combination thereof.[0329| Clause 17. The system of Clause 15 or 16, wherein the activity comprises antigen uptake, antigen processing, antigen presentation, T cell costimulation, or a combination thereof.|0330] Clause 18. The system of any one of Clauses 1-17, wherein the second immunotherapeutic agent comprises an antibody that binds to a receptor on an immune cell.

[0331] Clause 19. The system of Clause 18, wherein the antibody comprises an anti-CD40 antibody, an anti-CD205 antibody, an anti-CSFIR antibody, or an anti-FLT3L antibody.

[0332] Clause 20. The system of any one of Clauses 1-19, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are the same.

[0333] Clause 21. The system of any one of Clauses 1-19, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are different.

[0334] Clause 22. The system of any one of Clauses 1-21, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to afirst release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is different from the first release profile.

[0335] Clause 23. The system of Clause 22, wherein the first release profile differs from the second release profile with respect to one or more of release duration or release rate.[0336[ Clause 24. The system of any one of Clauses 1-21, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to a first release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is substantially the same as the first release profile.[0337[ Clause 25. The system of any one of Clauses 1-24, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0338] Clause 26. The system of any one of Clauses 1-25, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0339] Clause 27. The system of any one of Clauses 1-26, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0340] Clause 28. The system of any one of Clauses 1-27, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0341] Clause 29. The system of any one of Clauses 1-28, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0342] Clause 30. The system of any one of Clauses 1-29, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0343] Clause 31. The system of any one of Clauses 1-30, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.]0344| Clause 32. The system of any one of Clauses 1-31, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0345] Clause 33. The system of any one of Clauses 1-32, wherein at least one of the polymer of the first dynamic hydrogel or the polymer of the second dynamic hydrogel comprises a hydrophobically modified polysaccharide.

[0346] Clause 34. The system of Clause 33, wherein the hydrophobically modified polysaccharide comprises a hydrophobically modified cellulose derivative.

[0347] Clause 35. The system of Clause 34, wherein the hydrophobically modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).|0348] Clause 36. The system of any one of Clauses 1-35, wherein at least one of the plurality of nanoparticles of the first dynamic hydrogel or the plurality of nanoparticles of the second dynamic hydrogel comprises a plurality of polymeric nanoparticles.[0349 | Clause 37. The system of Clause 36, wherein the plurality of polymeric nanoparticles is amphiphilic.

[0350] Clause 38. The system of Clause 37, wherein the plurality of polymeric nanoparticles comprises polyethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

[0351] Clause 39. The system of any one of Clauses 1-38, wherein a concentration of the polymer in the first dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

[0352] Clause 40. The system of any one of Clauses 1-39, wherein a concentration of the polymer in the second dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

[0353] Clause 41. The system of any one of Clauses 1-40, wherein a concentration of the polymer in the first dynamic hydrogel is the same as a concentration of the polymer in the second dynamic hydrogel.

[0354] Clause 42. The system of any one of Clauses 1-40, wherein a concentration of the polymer in the first dynamic hydrogel is different from a concentration of the polymer in the second dynamic hydrogel.

[0355] Clause 43. The system of any one of Clauses 1-42, wherein a concentration of the nanoparticles in the first dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

[0356] Clause 44. The system of any one of Clauses 1-43, wherein a concentration of the nanoparticles in the second dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

[0357] Clause 45. The system of any one of Clauses 1-44, wherein a concentration of the nanoparticles in the first dynamic hydrogel is the same as a concentration of the nanoparticles in the second dynamic hydrogel.

[0358] Clause 46. The system of any one of Clauses 1-44, wherein a concentration of the nanoparticles in the first dynamic hydrogel is different from a concentration of the nanoparticles in the second dynamic hydrogel.

[0359] Clause 47. The system of any one of Clauses 1-46, wherein the first dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the first dynamic hydrogel.

[0360] Clause 48. The system of any one of Clauses 1-47, wherein the second dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the second dynamic hydrogel.

[0361] Clause 49. The system of any one of Clauses 1-48, wherein a storage modulus of the first dynamic hydrogel is the same as a storage modulus of the second dynamic hydrogel.

[0362] Clause 50. The system of any one of Clauses 1-48, wherein a storage modulus of the first dynamic hydrogel is different from a storage modulus of the second dynamic hydrogel.

[0363] Clause 51. The system of any one of Clauses 1-50, wherein the first dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

[0364] Clause 52. The system of any one of Clauses 1-51, wherein the second dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

[0365] Clause 53. The system of any one of Clauses 1-52, wherein a yield stress of the first dynamic hydrogel is the same as a yield stress of the second dynamic hydrogel.

[0366] Clause 54. The system of any one of Clauses 1-52, wherein a yield stress of the first dynamic hydrogel is different from a yield stress of the second dynamic hydrogel.

[0367] Clause 55. The system of any one of Clauses 1-54, wherein the first dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

[0368] Clause 56. The system of any one of Clauses 1-55, wherein the second dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

[0369] Clause 57. The system of any one of Clauses 1-56, wherein a viscosity of the first dynamic hydrogel is the same as a viscosity of the second dynamic hydrogel.

[0370] Clause 58. The system of any one of Clauses 1-56, wherein a viscosity of the first dynamic hydrogel is different from a viscosity of the second dynamic hydrogel.

[0371] Clause 59. The system of any one of Clauses 1-58, wherein the first dynamic hydrogel is shear-thinning and self-healing.

[0372] Clause 60. The system of any one of Clauses 1-59, wherein the second dynamic hydrogel is shear-thinning and self-healing.

[0373] Clause 61. The system of any one of Clauses 1-60, wherein at least one of the first dynamic hydrogel or second dynamic hydrogel is configured to be administered to a patient via injection.

[0374] Clause 62. The system of any one of Clauses 1-61, wherein the cancer is biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, liver cancer, lymphoma, lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, skin cancer, testicular cancer, or thyroid cancer.

[0375] Clause 63. A method for treating cancer, comprising:administering a first dynamic hydrogel into a tumor in a subject to form an intratumoral depot, wherein the first dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, anda first immunotherapeutic agent; andadministering a second dynamic hydrogel to the subject external to the tumor to form a peri turn oral depot, wherein the second dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a second immunotherapeutic agent.

[0376] Clause 64. The method of Clause 63, wherein the first dynamic hydrogel is configured to modulate a tumor microenvironment of the tumor.

[0377] Clause 65. The method of Clause 64, wherein the modulation comprises converting the tumor microenvironment from an immune-suppressing state to an immune-activating state.]0378| Clause 66. The method of any one of Clauses 63-65, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent into the tumor.

[0379] Clause 67. The method of any one of Clauses 63-66, wherein the first dynamic hydrogel is configured to be infiltrated by immune cells within the tumor, such that the immune cells are exposed to the first immunotherapeutic agent while within the first dynamic hydrogel.

[0380] Clause 68. The method of any one of Clauses 63-67, wherein the first immunotherapeutic agent is configured to recruit immune cells into the tumor, activate immune cells within the tumor, enhance recognition of cancer cells of the tumor by immune cells, enhance killing of cancer cells of the tumor by immune cells, or a combination thereof.[0381 J Clause 69. The method of any one of Clauses 63-68, wherein the first immunotherapeutic agent comprises a pro-inflammatory cytokine, a chemokine, an antibody, an adjuvant, or a combination thereof.

[0382] Clause 70. The method of Clause 69, wherein the first immunotherapeutic agent comprises the pro-inflammatory cytokine, and wherein the pro-inflammatory cytokine comprises IL-2, IL-12, IL-15, IL-18, IL-21, or IFN-a.

[0383] Clause 71. The method of Clause 69, wherein the first immunotherapeutic agent comprises the chemokine, and wherein the chemokine comprises GM-CSF or CCL21.

[0384] Clause 72. The method of Clause 69, wherein the first immunotherapeutic agent comprises the antibody, and wherein the antibody comprises an anti-PD-Ll antibody or an anti-CTLA-4 antibody.

[0385] Clause 73. The method of Clause 69, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises a small molecule or a nucleic acid.

[0386] Clause 74. The method of Clause 69, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises CpG or an anthracycline.

[0387] Clause 75. The method of any one of Clauses 63-74, wherein the second dynamic hydrogel is administered proximate to a draining lymph node associated with the tumor.

[0388] Clause 76. The method of Clause 75, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent to the draining lymph node associated with the tumor.

[0389] Clause 77. The method of Clause 75 or 76, wherein the second immunotherapeutic agent is configured to enhance activity of immune cells present in the draining lymph node associated with the tumor.

[0390] Clause 78. The method of Clause 77, wherein the immune cells comprise B cells, antigen-presenting cells, T cells, or a combination thereof.[0391| Clause 79. The method of Clause 77 or 78, wherein the activity comprises antigen uptake, antigen processing, antigen presentation, T cell costimulation, or a combination thereof.|0392] Clause 80. The method of any one of Clauses 63-79, wherein the second immunotherapeutic agent comprises an antibody that binds to a receptor on an immune cell.

[0393] Clause 81. The method of Clause 80, wherein the antibody comprises an anti-CD40 antibody, an anti-CD205 antibody, an anti-CSFIR antibody, or an anti-FLT3L antibody.

[0394] Clause 82. The method of any one of Clauses 63-81, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are the same.

[0395] Clause 83. The method of any one of Clauses 63-81, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are different.

[0396] Clause 84. The method of any one of Clauses 63-83, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to afirst release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is different from the first release profile.

[0397] Clause 85. The method of Clause 84, wherein the first release profile differs from the second release profile with respect to one or more of release duration or release rate.[0398| Clause 86. The method of any one of Clauses 63-83, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to a first release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is substantially the same as the first release profile.[039 [ Clause 87. The method of any one of Clauses 63-86, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0400] Clause 88. The method of any one of Clauses 63-87, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.|'0401] Clause 89. The method of any one of Clauses 63-88, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0402] Clause 90. The method of any one of Clauses 63-89, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0403] Clause 91. The method of any one of Clauses 63-90, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0404] Clause 92. The method of any one of Clauses 63-91, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0405] Clause 93. The method of any one of Clauses 63-92, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0406] Clause 94. The method of any one of Clauses 63-93, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

[0407] Clause 95. The method of any one of Clauses 63-94, wherein at least one of the polymer of the first dynamic hydrogel or the polymer of the second dynamic hydrogel comprises a hydrophobically modified polysaccharide.

[0408] Clause 96. The method of Clause 95, wherein the hydrophobically modified polysaccharide comprises a hydrophobically modified cellulose derivative.

[0409] Clause 97. The method of Clause 96, wherein the hydrophobically modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).|0410] Clause 98. The method of any one of Clauses 63-97, wherein at least one of the plurality of nanoparticles of the first dynamic hydrogel or the plurality of nanoparticles of the second dynamic hydrogel comprises a plurality of polymeric nanoparticles.[0411 | Clause 99. The method of Clause 98, wherein the plurality of polymeric nanoparticles is amphiphilic.

[0412] Clause 100. The method of Clause 99, wherein the plurality of polymeric nanoparticles comprises polyethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

[0413] Clause 101. The method of any one of Clauses 63-100, wherein a concentration of the polymer in the first dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

[0414] Clause 102. The method of any one of Clauses 63-101, wherein a concentration of the polymer in the second dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

[0415] Clause 103. The method of any one of Clauses 63-102, wherein a concentration of the polymer in the first dynamic hydrogel is the same as a concentration of the polymer in the second dynamic hydrogel.

[0416] Clause 104. The method of any one of Clauses 63-102, wherein a concentration of the polymer in the first dynamic hydrogel is different from a concentration of the polymer in the second dynamic hydrogel.

[0417] Clause 105. The method of any one of Clauses 63-104, wherein a concentration of the nanoparticles in the first dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

[0418] Clause 106. The method of any one of Clauses 63-105, wherein a concentration of the nanoparticles in the second dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

[0419] Clause 107. The method of any one of Clauses 63-106, wherein a concentration of the nanoparticles in the first dynamic hydrogel is the same as a concentration of the nanoparticles in the second dynamic hydrogel.

[0420] Clause 108. The method of any one of Clauses 63-106, wherein a concentration of the nanoparticles in the first dynamic hydrogel is different from a concentration of the nanoparticles in the second dynamic hydrogel.

[0421] Clause 109. The method of any one of Clauses 63-108, wherein the first dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the first dynamic hydrogel.

[0422] Clause 110. The method of any one of Clauses 63-109, wherein the second dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the second dynamic hydrogel.

[0423] Clause 111. The method of any one of Clauses 63-110, wherein a storage modulus of the first dynamic hydrogel is the same as a storage modulus of the second dynamic hydrogel.

[0424] Clause 112. The method of any one of Clauses 63-110, wherein a storage modulus of the first dynamic hydrogel is different from a storage modulus of the second dynamic hydrogel.

[0425] Clause 113. The method of any one of Clauses 63-112, wherein the first dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

[0426] Clause 114. The method of any one of Clauses 63-113, wherein the second dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

[0427] Clause 115. The method of any one of Clauses 63-114, wherein a yield stress of the first dynamic hydrogel is the same as a yield stress of the second dynamic hydrogel.

[0428] Clause 116. The method of any one of Clauses 63-114, wherein a yield stress of the first dynamic hydrogel is different from a yield stress of the second dynamic hydrogel.

[0420] Clause 117. The method of any one of Clauses 63-116, wherein the first dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

[0430] Clause 118. The method of any one of Clauses 63-117, wherein the second dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

[0431] Clause 119. The method of any one of Clauses 63-118, wherein a viscosity of the first dynamic hydrogel is the same as a viscosity of the second dynamic hydrogel.

[0432] Clause 120. The method of any one of Clauses 63-118, wherein a viscosity of the first dynamic hydrogel is different from a viscosity of the second dynamic hydrogel.

[0433] Clause 121. The method of any one of Clauses 63-120, wherein the first dynamic hydrogel is shear-thinning and self-healing.

[0434] Clause 122. The method of any one of Clauses 63-121, wherein the second dynamic hydrogel is shear-thinning and self-healing.

[0435] Clause 123. The method of any one of Clauses 63-122, wherein at least one of the first dynamic hydrogel or second dynamic hydrogel is configured to be administered to a patient via injection.

[0436] Clause 124. The method of any one of Clauses 63-123, wherein the cancer is biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, liver cancer, lymphoma, lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, skin cancer, testicular cancer, or thyroid cancer.

[0437] Clause 125. A system for the treatment of cancer, comprising:a dynamic hydrogel configured to be administered in a subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, anda first immunotherapeutic agent; anda second immunotherapeutic agent configured to be administered to the subject.

[0438] Clause 126. The system of Clause 125, wherein the dynamic hydrogel is configured to be administered into a tumor in the subject to form an intratumoral depot.

[0439] Clause 127. The system of Clause 125, wherein the dynamic hydrogel is configured to be administered external to a tumor in the subject to form a peritumoral depot.

[0440] Clause 128. The system of any one of Clauses 125-127, wherein the first immunotherapeutic agent comprises a cytokine.

[0441] Clause 129. The system of any one of Clauses 125-127, wherein the first immunotherapeutic agent comprises an antibody.

[0442] Clause 130. The system of any one of Clauses 125-127, wherein the first immunotherapeutic agent comprises at least two different antibodies.

[0443] Clause 131. The system of any one of Clauses 125-130, wherein the second immunotherapeutic agent is configured to be administered to the subject via systemic injection.

[0444] Clause 132. The system of any one of Clauses 125-131, wherein the second immunotherapeutic agent comprises an immune cell.

[0445] Clause 133. A method for treating cancer, comprising:administering a dynamic hydrogel to a subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second immunotherapeutic agent to the subject.

[0446] Clause 134. The method of Clause 133, wherein the dynamic hydrogel is administered into the tumor in the subject to form an intratumoral depot.

[0447] Clause 135. The method of Clause 133, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

[0448] Clause 136. The method of any one of Clauses 133-135, wherein the first immunotherapeutic agent comprises a cytokine.[0449J Clause 137. The method of any one of Clauses 133-135, wherein the first immunotherapeutic agent comprises an antibody.

[0450] Clause 138. The method of any one of Clauses 133-137, wherein the second immunotherapeutic agent is administered to the subject via systemic injection.

[0451] Clause 139. The method of any one of Clauses 133-138, wherein the second immunotherapeutic agent comprises an immune cell.

[0452] Clause 140. A method for treating cancer, comprising:lymphodepleting a subject;after lymphodepleting the subject, administering a first immunotherapeutic agent to the subject; andafter administering the first immunotherapeutic agent, administering a dynamic hydrogel to the subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a second immunotherapeutic agent.10453] Clause 141. The method of Clause 140, wherein the dynamic hydrogel is administered into the tumor in the subject to form an intratumoral depot.

[0454] Clause 142. The method of Clause 140, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

[0455] Clause 143. The method of any one of Clauses 140-142, wherein the first immunotherapeutic agent comprises an immune cell.

[0456] Clause 144. The method of any one of Clauses 140-143, wherein the second immunotherapeutic agent comprises one or more antibodies.[0457| Clause 145. The method of any one of Clauses 140-144, wherein the first immunotherapeutic agent is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after lymphodepleting the subject.

[0458] Clause 146. The method of any one of Clauses 140-145, wherein the dynamic hydrogel is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after administering the first immunotherapeutic agent.

[0459] Clause 147. A method for treating cancer, comprising:lymphodepleting a subject;administering a dynamic hydrogel to the subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second immunotherapeutic agent to the subject.

[0460] Clause 148. The method of Clause 147, wherein the dynamic hydrogel is administered into the tumor in the subject to form an intratumoral depot.

[0461] Clause 149. The method of Clause 147, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

[0462] Clause 150. The method of any one of Clauses 147-149, wherein the first immunotherapeutic agent comprises one or more cytokines.

[0463] Clause 151. The method of any one of Clauses 147-150, wherein the second immunotherapeutic agent comprises an immune cell.| 464] Clause 152. The method of any one of Clauses 147-151, wherein administering the dynamic hydrogel occurs during the lymphodepletion.

[0465] Clause 153. The method of any one of Clauses 147-151, wherein administering the dynamic hydrogel occurs before the lymphodepletion.[0466| Clause 154. The method of Clause 153, wherein administering the dynamic hydrogel occurs at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days before lymphodepleting the subject.

[0467] Clause 155. The method of any one of Clauses 147-151, wherein administering the dynamic hydrogel occurs after the lymphodepletion.

[0468] Clause 156. The method of Clause 155, wherein administering the dynamic hydrogel occurs at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after lymphodepleting the subject.|0469] Clause 157. The method of any one of Clauses 147-156, wherein administering the second immunotherapeutic agent occurs after administering the dynamic hydrogel.

[0470] Clause 158. The method of Clause 157, wherein administering the second immunotherapeutic agent occurs at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after administering the dynamic hydrogel.

[0471] Clause 159. A method for treating cancer, comprising:administering a dynamic hydrogel to a subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second immunotherapeutic agent to the subject,wherein administering the dynamic hydrogel and administering the second immunotherapeutic agent occur without a previous or concurrent lymphodepletion therapy.

[0472] Clause 160. The method of Clause 159, wherein the dynamic hydrogel is administered into the tumor in the subject to form an intratumoral depot.

[0473] Clause 161. The method of Clause 159, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.Conclusion

[0474] Although many of the embodiments are described above with respect to systems and methods related to the treatment of cancer, the technology is applicable to other applications and / or other approaches, such as the treatment of other disease or conditions. Moreover, other embodiments in addition to those described herein are within the scope of the technology. Additionally, several other embodiments of the technology can have different configurations, components, or procedures than those described herein. A person of ordinary skill in the art, therefore, will accordingly understand that the technology can have other embodiments with additional elements, or the technology can have other embodiments without several of the features shown and described above with reference to FIGS. 1-34C.

[0475] The descriptions of embodiments of the technology are not intended to be exhaustive or to limit the technology to the precise form disclosed above. Where the context permits, singular or plural terms may also include the plural or singular term, respectively. Although specific embodiments of, and examples for, the technology are described above for illustrative purposes, various equivalent modifications are possible within the scope of the technology, as those skilled in the relevant art will recognize. For example, while steps arepresented in a given order, alternative embodiments may perform steps in a different order. The various embodiments described herein may also be combined to provide further embodiments.

[0476] As used herein, the terms “generally,” “substantially,” “about,” and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent variations in measured or calculated values that would be recognized by those of ordinary skill in the art. The terms “about” and “approximately,” in reference to a number, are used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0477] Moreover, unless the word “or” is expressly limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of “or” in such a list is to be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list. As used herein, the phrase “and / or” as in “A and / or B” refers to A alone, B alone, and A and B. Additionally, the term “comprising” is used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and / or additional types of other features are not precluded.

[0478] As used herein, the term “subject” may refer to any animal, including but not limited to, humans and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0479] To the extent any materials incorporated herein by reference conflict with the present disclosure, the present disclosure controls.

[0480] It will also be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the technology. Further, while advantages associated with certain embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the disclosure and associated technology can encompass other embodiments not expressly shown or described herein.

Claims

CLAIMSWhat is claimed is:

1. A system for the treatment of cancer, comprising:a first dynamic hydrogel configured to be administered within a tumor in a subject to form an intratumoral depot, wherein the first dynamic hydrogel comprises: a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; anda second dynamic hydrogel configured to be administered to the subject external to the tumor to form a peritumoral depot, wherein the second dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a second immunotherapeutic agent.

2. The system of claim 1, wherein the first dynamic hydrogel is configured to modulate a tumor microenvironment of the tumor.

3. The system of claim 2, wherein the modulation comprises converting the tumor microenvironment from an immune-suppressing state to an immune-activating state.

4. The system of any one of claims 1-3, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent into the tumor.

5. The system of any one of claims 1-4, wherein the first dynamic hydrogel is configured to be infiltrated by immune cells within the tumor, such that the immune cells are exposed to the first immunotherapeutic agent while within the first dynamic hydrogel.

6. The system of any one of claims 1-5, wherein the first immunotherapeutic agent is configured to recruit immune cells into the tumor, activate immune cells within the tumor, enhance recognition of cancer cells of the tumor by immune cells, enhance killing of cancer cells of the tumor by immune cells, or a combination thereof.

7. The system of any one of claims 1-6, wherein the first immunotherapeutic agent comprises a pro-inflammatory cytokine, a chemokine, an immune checkpoint inhibitor, an adjuvant, or a combination thereof.

8. The system of claim 7, wherein the first immunotherapeutic agent comprises the pro-inflammatory cytokine, and wherein the pro-inflammatory cytokine comprises IL-2, IL-12, IL-15, IL-18, IL-21, or IFN-a.

9. The system of claim 7, wherein the first immunotherapeutic agent comprises the chemokine, and wherein the chemokine comprises GM-CSF or CCL21.

10. The system of claim 7, wherein the first immunotherapeutic agent comprises the immune checkpoint inhibitor, and wherein the immune checkpoint inhibitor comprises an anti-PD-Ll antibody or an anti-CTLA-4 antibody.

11. The system of claim 7, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises a small molecule or a nucleic acid.

12. The system of claim 7, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises CpG or an anthracycline.

13. The system of any one of claims 1-12, wherein the second dynamic hydrogel is configured to be administered proximate to a draining lymph node associated with the tumor.

14. The system of claim 13, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent to the draining lymph node associated with the tumor.

15. The system of claim 13 or 14, wherein the second immunotherapeutic agent is configured to enhance activity of immune cells present in the draining lymph node associated with the tumor.

16. The system of claim 15, wherein the immune cells comprise B cells, antigenpresenting cells, T cells, or a combination thereof.

17. The system of claim 15 or 16, wherein the activity comprises antigen uptake, antigen processing, antigen presentation, T cell costimulation, or a combination thereof.

18. The system of any one of claims 1-17, wherein the second immunotherapeutic agent comprises an antibody that binds to a receptor on an immune cell.

19. The system of claim 18, wherein the antibody comprises an anti-CD40 antibody, an anti-CD205 antibody, an anti-CSFIR antibody, or an anti-FLT3L antibody.

20. The system of any one of claims 1-19, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are the same.

21. The system of any one of claims 1-19, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are different.

22. The system of any one of claims 1-21, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to a first release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is different from the first release profile.

23. The system of claim 22, wherein the first release profile differs from the second release profile with respect to one or more of release duration or release rate.

24. The system of any one of claims 1-21, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to a first release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is substantially the same as the first release profile.

25. The system of any one of claims 1-24, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

26. The system of any one of claims 1-25, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

27. The system of any one of claims 1-26, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

28. The system of any one of claims 1-27, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

29. The system of any one of claims 1-28, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

30. The system of any one of claims 1-29, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

31. The system of any one of claims 1-30, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

32. The system of any one of claims 1-31, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

33. The system of any one of claims 1-32, wherein at least one of the polymer of the first dynamic hydrogel or the polymer of the second dynamic hydrogel comprises a hydrophobically modified polysaccharide.

34. The system of claim 33, wherein the hydrophobically modified polysaccharide comprises a hydrophobically modified cellulose derivative.

35. The system of claim 34, wherein the hydrophobically modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

36. The system of any one of claims 1-35, wherein at least one of the plurality of nanoparticles of the first dynamic hydrogel or the plurality of nanoparticles of the second dynamic hydrogel comprises a plurality of polymeric nanoparticles.

37. The system of claim 36, wherein the plurality of polymeric nanoparticles is amphiphilic.

38. The system of claim 37, wherein the plurality of polymeric nanoparticles comprises poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

39. The system of any one of claims 1-38, wherein a concentration of the polymer in the first dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

40. The system of any one of claims 1-39, wherein a concentration of the polymer in the second dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

41. The system of any one of claims 1-40, wherein a concentration of the polymer in the first dynamic hydrogel is the same as a concentration of the polymer in the second dynamic hydrogel.

42. The system of any one of claims 1-40, wherein a concentration of the polymer in the first dynamic hydrogel is different from a concentration of the polymer in the second dynamic hydrogel.

43. The system of any one of claims 1-42, wherein a concentration of the nanoparticles in the first dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

44. The system of any one of claims 1-43, wherein a concentration of the nanoparticles in the second dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

45. The system of any one of claims 1-44, wherein a concentration of the nanoparticles in the first dynamic hydrogel is the same as a concentration of the nanoparticles in the second dynamic hydrogel.

46. The system of any one of claims 1-44, wherein a concentration of the nanoparticles in the first dynamic hydrogel is different from a concentration of the nanoparticles in the second dynamic hydrogel.

47. The system of any one of claims 1-46, wherein the first dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the first dynamic hydrogel.

48. The system of any one of claims 1-47, wherein the second dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the second dynamic hydrogel.

49. The system of any one of claims 1-48, wherein a storage modulus of the first dynamic hydrogel is the same as a storage modulus of the second dynamic hydrogel.

50. The system of any one of claims 1-48, wherein a storage modulus of the first dynamic hydrogel is different from a storage modulus of the second dynamic hydrogel.

51. The system of any one of claims 1-50, wherein the first dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

52. The system of any one of claims 1-51, wherein the second dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

53. The system of any one of claims 1-52, wherein a yield stress of the first dynamic hydrogel is the same as a yield stress of the second dynamic hydrogel.

54. The system of any one of claims 1-52, wherein a yield stress of the first dynamic hydrogel is different from a yield stress of the second dynamic hydrogel.

55. The system of any one of claims 1-54, wherein the first dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

56. The system of any one of claims 1-55, wherein the second dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

57. The system of any one of claims 1-56, wherein a viscosity of the first dynamic hydrogel is the same as a viscosity of the second dynamic hydrogel.

58. The system of any one of claims 1-56, wherein a viscosity of the first dynamic hydrogel is different from a viscosity of the second dynamic hydrogel.

59. The system of any one of claims 1-58, wherein the first dynamic hydrogel is shear-thinning and self-healing.

60. The system of any one of claims 1-59, wherein the second dynamic hydrogel is shear-thinning and self-healing.

61. The system of any one of claims 1-60, wherein at least one of the first dynamic hydrogel or second dynamic hydrogel is configured to be administered to a patient via injection.

62. The system of any one of claims 1-61, wherein the cancer is biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, liver cancer, lymphoma, lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, skin cancer, testicular cancer, or thyroid cancer.

63. A method for treating cancer, comprising:administering a first dynamic hydrogel into a tumor in a subject to form an intratumoral depot, wherein the first dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second dynamic hydrogel to the subject external to the tumor to form a peritumoral depot, wherein the second dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a second immunotherapeutic agent.

64. The method of claim 63, wherein the first dynamic hydrogel is configured to modulate a tumor microenvironment of the tumor.

65. The method of claim 64, wherein the modulation comprises converting the tumor microenvironment from an immune-suppressing state to an immune-activating state.

66. The method of any one of claims 63-65, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent into the tumor.

67. The method of any one of claims 63-66, wherein the first dynamic hydrogel is configured to be infiltrated by immune cells within the tumor, such that the immune cells are exposed to the first immunotherapeutic agent while within the first dynamic hydrogel.

68. The method of any one of claims 63-67, wherein the first immunotherapeutic agent is configured to recruit immune cells into the tumor, activate immune cells within the tumor, enhance recognition of cancer cells of the tumor by immune cells, enhance killing of cancer cells of the tumor by immune cells, or a combination thereof.

69. The method of any one of claims 63-68, wherein the first immunotherapeutic agent comprises a pro-inflammatory cytokine, a chemokine, an antibody, an adjuvant, or a combination thereof.

70. The method of claim 69, wherein the first immunotherapeutic agent comprises the pro-inflammatory cytokine, and wherein the pro-inflammatory cytokine comprises IL-2, IL-12, IL-15, IL-18, IL-21, or IFN-a.

71. The method of claim 69, wherein the first immunotherapeutic agent comprises the chemokine, and wherein the chemokine comprises GM-CSF or CCL21.

72. The method of claim 69, wherein the first immunotherapeutic agent comprises the antibody, and wherein the antibody comprises an anti-PD-Ll antibody or an anti-CTLA-4 antibody.

73. The method of claim 69, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises a small molecule or a nucleic acid.

74. The method of claim 69, wherein the first immunotherapeutic agent comprises the adjuvant, and wherein the adjuvant comprises CpG or an anthracycline.

75. The method of any one of claims 63-74, wherein the second dynamic hydrogel is administered proximate to a draining lymph node associated with the tumor.

76. The method of claim 75, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent to the draining lymph node associated with the tumor.

77. The method of claim 75 or 76, wherein the second immunotherapeutic agent is configured to enhance activity of immune cells present in the draining lymph node associated with the tumor.

78. The method of claim 77, wherein the immune cells comprise B cells, antigenpresenting cells, T cells, or a combination thereof.

79. The method of claim 77 or 78, wherein the activity comprises antigen uptake, antigen processing, antigen presentation, T cell costimulation, or a combination thereof.

80. The method of any one of claims 63-79, wherein the second immunotherapeutic agent comprises an antibody that binds to a receptor on an immune cell.

81. The method of claim 80, wherein the antibody comprises an anti-CD40 antibody, an anti-CD205 antibody, an anti-CSFIR antibody, or an anti-FLT3L antibody.

82. The method of any one of claims 63-81, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are the same.

83. The method of any one of claims 63-81, wherein the first immunotherapeutic agent and the second immunotherapeutic agent are different.

84. The method of any one of claims 63-83, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to a first release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is different from the first release profile.

85. The method of claim 84, wherein the first release profile differs from the second release profile with respect to one or more of release duration or release rate.

86. The method of any one of claims 63-83, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent according to a first release profile, and wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent according to a second release profile that is substantially the same as the first release profile.

87. The method of any one of claims 63-86, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

88. The method of any one of claims 63-87, wherein the first dynamic hydrogel is configured to release the first immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

89. The method of any one of claims 63-88, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

90. The method of any one of claims 63-89, wherein the second dynamic hydrogel is configured to release the second immunotherapeutic agent over a release duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

91. The method of any one of claims 63-90, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

92. The method of any one of claims 63-91, wherein the first dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

93. The method of any one of claims 63-92, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

94. The method of any one of claims 63-93, wherein the second dynamic hydrogel is configured to persist in vivo for a duration of no more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 28 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 150 days, 180 days, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 1 year.

95. The method of any one of claims 63-94, wherein at least one of the polymer of the first dynamic hydrogel or the polymer of the second dynamic hydrogel comprises a hydrophobically modified polysaccharide.

96. The method of claim 95, wherein the hydrophobically modified polysaccharide comprises a hydrophobically modified cellulose derivative.

97. The method of claim 96, wherein the hydrophobically modified cellulose derivative is dodecyl-modified hydroxypropylmethylcellulose (HPMC-C12).

98. The method of any one of claims 63-97, wherein at least one of the plurality of nanoparticles of the first dynamic hydrogel or the plurality of nanoparticles of the second dynamic hydrogel comprises a plurality of polymeric nanoparticles.

99. The method of claim 98, wherein the plurality of polymeric nanoparticles is amphiphilic.

100. The method of claim 99, wherein the plurality of polymeric nanoparticles comprises poly(ethylene glycol)-block-poly(lactic acid) (PEG-PLA) nanoparticles.

101. The method of any one of claims 63-100, wherein a concentration of the polymer in the first dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

102. The method of any one of claims 63-101, wherein a concentration of the polymer in the second dynamic hydrogel is within a range from 0.5 wt% to 5 wt%, 0.5 wt% to 4 wt%, 0.5 wt% to 3 wt%, 0.5 wt% to 2 wt%, 0.5 wt% to 1 wt%, 1 wt% to 5 wt%, 1 wt% to 4 wt%, 1 wt% to 3 wt%, 1 wt% to 2 wt%, 2 wt% to 5 wt%, 2 wt% to 4 wt%, 2 wt% to 3 wt%, 3 wt% to 5 wt%, 3 wt% to 4 wt%, or 4 wt% to 5 wt%.

103. The method of any one of claims 63-102, wherein a concentration of the polymer in the first dynamic hydrogel is the same as a concentration of the polymer in the second dynamic hydrogel.

104. The method of any one of claims 63-102, wherein a concentration of the polymer in the first dynamic hydrogel is different from a concentration of the polymer in the second dynamic hydrogel.

105. The method of any one of claims 63-104, wherein a concentration of the nanoparticles in the first dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

106. The method of any one of claims 63-105, wherein a concentration of the nanoparticles in the second dynamic hydrogel is within a range from 1 wt% to 12 wt%, 1 wt% to 10 wt%, 1 wt% to 8 wt%, 1 wt% to 5 wt%, 1 wt% to 3 wt%, 3 wt% to 12 wt%, 3 wt% to 10 wt%, 3 wt% to 8 wt%, 3 wt% to 5 wt%, 5 wt % to 12 wt%, 5 wt% to 10 wt%, 5 wt% to 8 wt%, 8 wt% to 12 wt%, 8 wt% to 10 wt%, or 10 wt% to 12 wt%.

107. The method of any one of claims 63-106, wherein a concentration of the nanoparticles in the first dynamic hydrogel is the same as a concentration of the nanoparticles in the second dynamic hydrogel.

108. The method of any one of claims 63-106, wherein a concentration of the nanoparticles in the first dynamic hydrogel is different from a concentration of the nanoparticles in the second dynamic hydrogel.

109. The method of any one of claims 63-108, wherein the first dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the first dynamic hydrogel.

110. The method of any one of claims 63-109, wherein the second dynamic hydrogel has a storage modulus within a range from 10 Pa to 1000 Pa, or 50 Pa to 500 Pa when measured at 25 °C over an angular frequency of 0.1 rad / s to 100 rad / s within a linear viscoelastic region of the second dynamic hydrogel.

111. The method of any one of claims 63-110, wherein a storage modulus of the first dynamic hydrogel is the same as a storage modulus of the second dynamic hydrogel.

112. The method of any one of claims 63-110, wherein a storage modulus of the first dynamic hydrogel is different from a storage modulus of the second dynamic hydrogel.

113. The method of any one of claims 63-112, wherein the first dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25114. The method of any one of claims 63-113, wherein the second dynamic hydrogel has a yield stress within a range from 1 Pa to 500 Pa, or 20 Pa to 200 Pa when measured at 25 °C.

115. The method of any one of claims 63-114, wherein a yield stress of the first dynamic hydrogel is the same as a yield stress of the second dynamic hydrogel.

116. The method of any one of claims 63-114, wherein a yield stress of the first dynamic hydrogel is different from a yield stress of the second dynamic hydrogel.

117. The method of any one of claims 63-116, wherein the first dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

118. The method of any one of claims 63-117, wherein the second dynamic hydrogel has a viscosity within a range from 100 mPa-s to 1000 mPa-s when measured at 25 °C at a shear rate of 1000 s'1.

119. The method of any one of claims 63-118, wherein a viscosity of the first dynamic hydrogel is the same as a viscosity of the second dynamic hydrogel.

120. The method of any one of claims 63-118, wherein a viscosity of the first dynamic hydrogel is different from a viscosity of the second dynamic hydrogel.

121. The method of any one of claims 63-120, wherein the first dynamic hydrogel is shear-thinning and self-healing.

122. The method of any one of claims 63-121, wherein the second dynamic hydrogel is shear-thinning and self-healing.

123. The method of any one of claims 63-122, wherein at least one of the first dynamic hydrogel or second dynamic hydrogel is configured to be administered to a patient via injection.

124. The method of any one of claims 63-123, wherein the cancer is biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia, liver cancer, lymphoma, lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, skin cancer, testicular cancer, or thyroid cancer.

125. A system for the treatment of cancer, comprising:a dynamic hydrogel configured to be administered in a subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; anda second immunotherapeutic agent configured to be administered to the subject.

126. The system of claim 125, wherein the dynamic hydrogel is configured to be administered into a tumor in the subject to form an intratumoral depot.

127. The system of claim 125, wherein the dynamic hydrogel is configured to be administered external to a tumor in the subject to form a peritumoral depot.

128. The system of any one of claims 125-127, wherein the first immunotherapeutic agent comprises a cytokine.

129. The system of any one of claims 125-127, wherein the first immunotherapeutic agent comprises an antibody.

130. The system of any one of claims 125-127, wherein the first immunotherapeutic agent comprises at least two different antibodies.

131. The system of any one of claims 125-130, wherein the second immunotherapeutic agent is configured to be administered to the subject via systemic injection.

132. The system of any one of claims 125-131, wherein the second immunotherapeutic agent comprises an immune cell.

133. A method for treating cancer, comprising:administering a dynamic hydrogel to a subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second immunotherapeutic agent to the subject.

134. The method of claim 133, wherein the dynamic hydrogel is administered into a tumor in the subject to form an intratumoral depot.

135. The method of claim 133, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

136. The method of any one of claims 133-135, wherein the first immunotherapeutic agent comprises a cytokine.

137. The method of any one of claims 133-135, wherein the first immunotherapeutic agent comprises an antibody.

138. The method of any one of claims 133-137, wherein the second immunotherapeutic agent is administered to the subject via systemic injection.

139. The method of any one of claims 133-138, wherein the second immunotherapeutic agent comprises an immune cell.

140. A method for treating cancer, comprising:lymphodepleting a subject;after lymphodepleting the subject, administering a first immunotherapeutic agent to the subject; andafter administering the first immunotherapeutic agent, administering a dynamic hydrogel to the subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a second immunotherapeutic agent.

141. The method of claim 140, wherein the dynamic hydrogel is administered into a tumor in the subject to form an intratumoral depot.

142. The method of claim 140, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

143. The method of any one of claims 140-142, wherein the first immunotherapeutic agent comprises an immune cell.

144. The method of any one of claims 140-143, wherein the second immunotherapeutic agent comprises one or more antibodies.

145. The method of any one of claims 140-144, wherein the first immunotherapeutic agent is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after lymphodepleting the subject.

146. The method of any one of claims 140-145, wherein the dynamic hydrogel is administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after administering the first immunotherapeutic agent.

147. A method for treating cancer, comprising:lymphodepleting a subject;administering a dynamic hydrogel to the subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second immunotherapeutic agent to the subject.

148. The method of claim 147, wherein the dynamic hydrogel is administered into a tumor in the subject to form an intratumoral depot.

149. The method of claim 147, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

150. The method of any one of claims 147-149, wherein the first immunotherapeutic agent comprises one or more cytokines.

151. The method of any one of claims 147-150, wherein the second immunotherapeutic agent comprises an immune cell.

152. The method of any one of claims 147-151, wherein administering the dynamic hydrogel occurs during the lymphodepletion.

153. The method of any one of claims 147-151, wherein administering the dynamic hydrogel occurs before the lymphodepletion.

154. The method of claim 153, wherein administering the dynamic hydrogel occurs at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days before lymphodepleting the subject.

155. The method of any one of claims 147-151, wherein administering the dynamic hydrogel occurs after the lymphodepletion.

156. The method of claim 155, wherein administering the dynamic hydrogel occurs at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after lymphodepleting the subject.

157. The method of any one of claims 147-156, wherein administering the second immunotherapeutic agent occurs after administering the dynamic hydrogel.Ill158. The method of claim 157, wherein administering the second immunotherapeutic agent occurs at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days after administering the dynamic hydrogel.

159. A method for treating cancer, comprising:administering a dynamic hydrogel to a subject to form a depot, wherein the dynamic hydrogel comprises:a polymer non-covalently crosslinked with a plurality of nanoparticles, and a first immunotherapeutic agent; andadministering a second immunotherapeutic agent to the subject,wherein administering the dynamic hydrogel and administering the second immunotherapeutic agent occur without a previous or concurrent lymphodepletion therapy.

160. The method of claim 159, wherein the dynamic hydrogel is administered into a tumor in the subject to form an intratumoral depot.

161. The method of claim 159, wherein the dynamic hydrogel is administered external to a tumor in the subject to form a peritumoral depot.

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