Hydrogel-based delivery system and method for inducing three-level lymphatic structure in tumor and enhancing immune checkpoint blocking

Abstract

The present invention provides a system for drug delivery that utilizes a hydrogel to deliver a bioactive agent to stimulate three-level lymphatic structure formation within a mature tumor and enhance immune checkpoint blocking. Provided herein is an injectable hydrogel that can stimulate the formation of immune structures within a tumor, improve cancer prognosis and therapeutic response. The invention provides application of the system to treatment of cancer. Methods of making the drug delivery system are also provided.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 584,248, filed on September 21, 2023, which is incorporated by reference in its entirety. 1. Technical Field

[0003] The present invention discloses a hydrogel delivery system for use in tumors for immunotherapy to improve cancer prognosis and treatment response. 2. Background technology

[0004] The World Health Organization (WHO) estimates that there are approximately 2 to 3 million cases of skin cancer worldwide each year. Melanoma is the most dangerous type of skin cancer, accounting for 132,000 of these cases and is responsible for the majority of skin cancer deaths. 1 Due to the high probability of disease recurrence 2 , therapeutic intervention for advanced melanoma is challenging. In the past decade, various immunotherapies, including cytokines / chemokines, immune checkpoint blockade (ICB), and their combinations have been explored to address this challenge. 3 However, these immunotherapies have limitations, such as off-target toxicity and low patient response rates. 4 Limited drug response is associated with a suppressive tumor microenvironment that results in a paucity of tumor-infiltrating T cells 5-6 Therefore, therapeutic advances aimed at enhancing protective immunity against tumors are of clinical interest.

[0005] Tertiary lymphoid structures (TLS) are ectopic tissues of lymphoid immune cells that stimulate antigen-driven responses during chronic inflammatory states. 7-8 In disease-induced inflammation, TLS resembles secondary lymphoid organs that partially form within non-lymphoid tissues, such as the spleen. 9 TLS is an immune microenvironment composed of various immune cell types, including B cells, T cells, and antigen-presenting cells, which drives a protective response against tumor progression. 11 The favorable prognosis associated with TLS can be attributed to four major mechanisms: accelerated immune response, efficient interaction between antigens and lymphocytes, establishment of direct contact between lymphocytes and tumor cells, and lymphocyte homeostasis promoted by secreted survival factors. 12-14 .

[0006] Previous studies have investigated the basic cellular and molecular mechanisms of TLS formation, among which the B cell pathway involving the CXC chemokine ligand 13 / CXC chemokine receptor type 5 axis and the CC chemokine ligand (CCL) 19 / CCL21 / CC-chemokine receptor 7 axis play a key role in TLS generation. 16Furthermore, the interaction between LTα and LTi cells initiates the recruitment of cells to produce TLS via cytokine and chemokine signaling. 17 The key factors involved in this process include CXCL13, CCL21, IL-4, IL-7, IL-2 and LIGHT 14 The chemokine CXCL13 has been implicated as a TLS inducer in tumors, recruiting B cells to B cell-rich structures that promote TLS formation. 18 This chemokine is also used as a potential biomarker for TLS formation in the tumor microenvironment 19 Alternatively, the cytokine LIGHT is another bona fide TLS inducer 20 When combined with immunotherapy in mouse models, LIGHT has been successfully used in pilot studies to induce TLS and boost the generation of effector and memory T cells. 21-22 However, it is still unclear which factors are critical for upregulating TLS density and maturation in the tumor microenvironment. 23-24 .

[0007] Previously, TLS was induced by direct intratumoral injection of cytokines, chemokines, antibodies, or antigen-presenting cells. 25 Due to the high cost of injecting antigen-presenting cells, more research focuses on direct injection of cytokines / chemokines, including the above-mentioned CXCL13 and LIGHT 26 However, these approaches have limitations, such as the high dose-dependency of cytokines / chemokines for TLS induction, the generation of immature and imperfect TLS structures, and reduced interactions and activity between B cells and T cells. 27 Therefore, there is an increasing need to develop new methods for sustained delivery of cytokines / chemokines and control of immune cell infiltration and tissue localization. 28 .

[0008] Compared to other biomaterials, hydrogels are non-invasive, minimally invasive and low-technique sensitive. 29 Hydrogels are usually composed of a network of highly hydrophilic polymers through chemical or physical crosslinking. 30 In contrast to chemical crosslinks, which are stable, irreversible, and permanent, physical crosslinks are transient, reversible, and dynamic. Hydrogels formed by physical crosslinks exhibit injectability and self-healing properties due to their non-covalent bonding nature. 31 In addition, the release of drugs from hydrogels can be controlled by adjusting the mesh size, swelling ratio and degradation rate of the hydrogels. 32 .

[0009] The problem that this patent application attempts to solve is the different results of current cancer immunotherapies, such as cancer vaccines or immune checkpoint blockade ("ICB"), in patients. This inconsistency in therapeutic efficacy can be attributed to the fact that some tumors are "immunologically cold tumors" with low immune response. Therefore, it is necessary to change the immune tumor microenvironment to stimulate the immune system, thereby enhancing the effectiveness of immunotherapy. 3. Summary of the invention

[0010] 3.1 Overview

[0011] The disclosure of the present invention implements a groundbreaking drug delivery method that utilizes physically cross-linked hydrogels to deliver CXCL13 and LIGHT proteins, aiming to stimulate mature TLS formation and enhance ICB therapy in melanoma. Provided herein is a hydrogel-based delivery system composed of cytokines and chemokines for inducing intratumoral tertiary lymphoid structures ("TLS") and enhancing immune checkpoint blockade. In the past, cytokines / chemokines were injected indirectly to induce TLS, but the resulting TLS were immature and poorly structured. The method of the present invention uses hydrogels for locally controlled release of cytokines / chemokines to induce mature intratumoral TLS. The method of the present invention uses hydrogels to deliver cytokines / chemokines to induce mature TLS structures and improve ICB efficacy. This is the first time that hydrogels have been used to induce TLS formation.

[0012] This article provides an injectable hydrogel that can stimulate the formation of immune structures within tumors, improving cancer prognosis and treatment response. In this study, a single injection of a hydrogel containing specific immune molecules inhibited tumor growth in a mouse model and increased the density of these immune structures. The hydrogel also improved the effectiveness of a specific cancer treatment called immune checkpoint blockade, achieving better survival rates and tumor suppression. This article provides methods for using hydrogels as drug carriers to enhance the immune system's ability to fight cancer.

[0013] In one embodiment, the present disclosure utilizes the association of TLS with better prognosis in many cancers, and their ability to improve clinical outcomes of immunotherapy. The present disclosure utilizes hydrogels to deliver the cytokine LIGHT and the chemokine CXCL13, inducing intratumoral TLS to enhance the efficacy of immunotherapy.

[0014] Provided herein is a compound comprising hyaluronic acid and 4-(4-chlorophenyl)pyridine (HA-CPP) or a salt thereof.

[0015] In one embodiment, the compound comprises at least one moiety of formula (1) or a salt thereof:

[0016]

[0017] wherein n is an integer and X is a counterion.

[0018] Provided herein are compositions comprising in combination with one or more biologically active agents of compounds.

[0019] In certain embodiments, Binds to the bioactive agent via non-covalent binding to form a complex.

[0020] In certain embodiments, the bioactive agent is a chemokine, a cytokine, an immune checkpoint inhibitor, a cancer vaccine, a chimeric antigen receptor, or a combination thereof.

[0021] In certain embodiments, the bioactive agent is CXCL13, CCL21, IL-4, IL-7, IL-2, LIGHT, CCL19, CXCL12, CXCL13, lymphotoxin, TNF-α, or a combination thereof.

[0022] In certain embodiments, the chemokine is CXCL13 and the cytokine is LIGHT.

[0023] Provided herein are compositions comprising a disclosed compound and a pharmaceutically acceptable carrier.

[0024] In one embodiment, the pharmaceutically acceptable carrier is water.

[0025] In certain embodiments, the composition is an injectable composition and comprises about 1-5% water by weight.

[0026] In certain embodiments, the composition is administered by injection to form tertiary lymphoid structures and enhance immune checkpoint blockade.

[0027] The present invention provides use of the disclosed composition in the manufacture of a medicament for treating cancer in a subject.

[0028] In certain embodiments, the composition inhibits tumor growth, prolongs survival, increases TLS density, promotes TLS maturation, upregulates OVA antigen spread in secondary lymphoid organs, or a combination thereof.

[0029] In certain embodiments, the composition further comprises an anti-PD1 drug, an anti-CTLA-4 drug, anti-PD1, anti-PDL1, anti-CTLA-4, or a combination thereof.

[0030] In one embodiment, the composition is formulated for direct intratumoral injection.

[0031] In certain embodiments, the cancer is melanoma, colorectal cancer, lung cancer, pancreatic cancer, oral squamous cell carcinoma, and invasive breast cancer.

[0032] In certain embodiments, the subject is a human, a feline, a canine, a pig, a rabbit, or a rodent.

[0033] Provided herein is a method for preparing HA-CPP, comprising: (a) reacting 4-(4-chlorophenyl)pyridine with a compound of formula X-(CH2)3-NH2, wherein X is a leaving group; and (b) reacting the product of step (a) with hyaluronic acid (HA) having a molecular weight of about 6 kDa to 1000 kDa.

[0034] Provided herein is a method for preparing HA-CPP, comprising: (i) reacting 4-(4-chlorophenyl)pyridine with 3-halopropylamine to form pyridinium salt CPP-NH2, and (ii) performing an amidation reaction with at least one glucuronic acid group of hyaluronic acid (HA), wherein the molecular weight of the HA is about 6 kDa to 1000 kDa.

[0035] In certain embodiments, the method further comprises combining HA-CPP and cucurbit[8]uril (CB[8]) to form

[0036] In certain embodiments, the stoichiometric ratio between CPP and CB[8] is 2:1 or greater. 4. Description of the drawings

[0037] Figure 1 : Schematic diagram of mature TLS formation.

[0038] Figure 2: Scheme 1 (A) Synthesis route of HA-CPP. (B) Schematic diagram of the formation of supramolecular networks.

[0039] Figure 3: (A) CPP guest, (B) HA-CPP and (C) Chemical structure and 1H NMR spectrum of the complex.

[0040] Figure 4: Material characterization of the hydrogel. (A) The hydrogel was injected through a syringe needle. The “HKU” pattern formed by the hydrogel. (B) The hydrogel was injected into deionized water. (C) Shear viscosity of hydrogel. (D) Oscillatory rheological frequency sweep of hydrogels. (E) Two differently dyed Photo of the hydrogel self-healing into a single piece. (F) During continuous step strain measurement G' and G" of hydrogel.

[0041] Figure 5: Pass Hydrogels delivered CXCL13 and LIGHT to B16 tumor-bearing mice in vivo. (A) Hydrogels encapsulated drugs in the B16 mouse model. Treatment regimen for hydrogels. Mice were inoculated with B16-OVA melanoma cell line by intraperitoneal injection on day 0. On day 7, intraperitoneal injections of drug-encapsulated hydrogels (hydrogels + LIGHT + CXCL13), drugs alone (LIGHT + CXCL13), vehicle hydrogel controls, or PBS controls were given (n = 5 per group). At the end point (day 23), mice were sacrificed for further analysis. For survival experiments, mice were closely monitored until the end point of humane euthanasia (day 40). (B) Primary injection site tumor size of different experimental groups at the end point (day 23). (C) Quantification of primary injection site tumor size of different experimental groups at the end point (day 23). (D) Quantification of total tumor weight of different experimental groups at the end point (day 23). Unpaired t-test was used, and multiple t-test adjustments were made. Data are expressed as mean ± SEM. (E) Survival analysis of experimental groups (n = 5). Mantel-Cox test was used. *p < 0.05, ns means "not significant".

[0042] Figure 6: Encapsulated drug The hydrogel induced the formation and maturation of TLS and promoted interferon-γ T cell responses. Immunofluorescence staining and grading of TLS in hydrogel-treated B16 tumors (AC). (A) TLS formation in B16 tumors in different experimental groups: hydrogel + LIGHT + CXCL13 (scale bar = 200 μm), LIGHT + CXCL13 (scale bar = 200 μm), control hydrogel only (scale bar = 100 μm). (B) Quantification of the number of TLS per square millimeter in each experimental cohort. (C) TLS grading (mature, immature, no TLS) after immunostaining of treated B16 tumors. T cells (CD3 marker), B cells (B220 marker), and antigen presenting cells (CD11c) are shown in the tumor microenvironment of different experimental groups from Figure 5. The Kolmogorov Smirnoff test was used to evaluate statistical differences. (D) Quantification of the percentage of TLS grading in each experimental cohort (n = 8 per group). Chi-square contingency analysis was performed. Encapsulated drug Antigen-activated T cell response after hydrogel treatment (D, E). (D) Interferon-γ ELISPOT results. On day 23, ELISPOT analysis was performed using spleen cells from the experimental group of Figure 5. Interferon-γ release curves of OVA-induced spleen cells were analyzed. (E) Quantification of interferon-γ ELISPOT from different experimental groups (n=4 per group). Statistical analysis was performed using an unpaired t-test with Welch correction. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns means "not significant".

[0043] Figure 7: Through Hydrogels delivered CXCL13 and LIGHT in combination with high-dose αPD1 treatment into B16 tumor-bearing mice in vivo. (A) In the B16 mouse model, the encapsulated drug Treatment regimen of hydrogel combined with high-dose αPD1 treatment. On day 0, mice were inoculated with B16-OVA melanoma cell line by intraperitoneal injection. On day 7, intraperitoneal injection of drug-encapsulated hydrogel (hydrogel + LIGHT + CXCL13) was given (n = 5). On days 14 and 19, αPD1 treatment (0.6 mg per injection) was given alone or in combination after drug-encapsulated hydrogel treatment (n = 5 per group). Vehicle control mice were injected with blank hydrogel and PBS (n = 5). At the end point (day 23), mice were sacrificed for further analysis. For survival experiments, mice were closely monitored until the end point of humane euthanasia (day 40). (B) Primary tumor size at the injection site of different experimental groups at the end point (day 23). (C) Quantification of injection site tumor size of different experimental groups at the end point (day 23). (D) Quantification of total tumor weight of different experimental groups at the end point (day 23). Unpaired t-test was used, and multiple t-test adjustment was performed. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ns means "not significant". (E) Survival analysis of experimental groups (n = 5). Mantel-Cox test was used. *p < 0.05, ns means "not significant".

[0044] Figure 8: Analysis of TLS and T cell infiltration in combined treated B16 tumor-bearing mice. (A) TLS formation was observed after immunostaining of treated B16 tumors. T cells (CD3 marker), B cells (B220 marker) and antigen presenting cells (CD11c) were observed in the tumor microenvironment of the different experimental groups from Figure 7. Mature TLS were observed in the combined group (scale bar = 200 μm). Immature TLS or no TLS were observed in the αPD1 group (scale bar = 200 μm) and the vehicle control group (50 μm), respectively. (B) Quantification of the number of TLS per square millimeter in each experimental cohort. The Kolmogorov Smirnoff test was used for statistical analysis. (C) Quantification of the percentage of TLS grades in each experimental cohort (n = 5 per group), and chi-square contingency analysis was performed. (DE) Flow cytometric analysis of (D) CD3+CD8+T cell populations and (E) CD3+CD4+T cell populations in B16 tumors of each experimental cohort. (FG) Flow cytometric analysis of (F) CD3+CD8+ T cell populations and (G) CD3+CD4+ T cell populations from spleens of each experimental cohort. Experiments were performed in triplicate. Unpaired t-tests with Welch correction were performed. Data are expressed as mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001, ns means "not significant".

[0045] Figure 9: Hydrogel-based delivery of LIGHT+CXCL13 in B16 tumor-bearing mice. (A) Schematic diagram of hydrogel vaccine administration after intraperitoneal inoculation of B16 tumors. The hydrogel delivered 0.6 μg LIGHT and 0.6 μg CXCL13 to each animal via intraperitoneal injection. (B) Serum ELISA of mice vaccinated on day 14. ELISA plate with Hydrogels were pre-coated to detect specific antibody responses in mouse sera. ns means “not significant”.

[0046] Figure 10: Hydrogel-based delivery of LIGHT in B16 tumor-bearing mice. (A) Schematic diagram of hydrogel vaccine administration after intraperitoneal inoculation of B16 tumors. The hydrogel delivered 0.6 μg of LIGHT to each animal via intraperitoneal injection. (B) Primary injection site tumors were recorded using an electronic caliper. (C) The total tumor of each mouse was weighed and compared between different groups. ns means "not significant".

[0047] Fig.11 : Multicolor staining of B16 tumors treated with hydrogel + LIGHT. Multicolor staining of B16 tumors treated with hydrogel + LIGHT (CD3, B220, CD11c, DAPI). Scale bar: 200 μm.

[0048] Fig.12: Infiltration of spleen cells into hydrogels. Cells were seeded in cell plates and cultured with hydrogels. Cell infiltration into hydrogels was observed by optical microscopy after 1 and 2 days. Scale bars are 0.5 mm (left) and 1 mm (right). Quantification per unit area (mm) on day 2 2 ) of spleen cells. **p<0.01.

[0049] Fig.13A -K: pass Hydrogels delivered CXCL13 and LIGHT mRNA in vivo into B16.f10 tumor-bearing mice. (A) Hydrogels loaded with liposome-encapsulated CXCL13 and LIGHT mRNA in a B16.f10 mouse model. Treatment regimen for hydrogels. Mice were inoculated with B16.f10 melanoma cell line by subcutaneous injection on day 0. Intratumoral injections of drug-encapsulated hydrogels (hydrogel-LIGHT+CXCL13 mRNA), drug alone (LIGHT+CXCL13 mRNA), carrier hydrogel controls, or carrier liposome controls were given on days 5, 8, and 11 (n=5 per group). At the end point (day 23), mice were sacrificed for further analysis. For survival experiments, mice were closely monitored until the end point of humane euthanasia (day 40). (B) Individual tumor growth kinetics of different experimental groups. (C) Primary injection site tumor size of different experimental groups at the end point (day 23). (D) Quantification of primary injection site tumor size of different experimental groups at the end point (day 23). Unpaired t-test was used, and multiple t-test adjustments were performed. Data are expressed as mean ± SEM. (E) Survival analysis of experimental groups (n=5). Mantel-Cox test was used. *p<0.05, ns means "not significant". In encapsulated drug Immunofluorescence staining and grading of TLS and immune cell density in hydrogel-treated B16.f10 tumors (FK). (F) Formation of TLS in B16.f10 tumors in different experimental groups: hydrogel-LIGHT+CXCL13 mRNA (scale bar = 200 μm), LIGHT+CXCL13 mRNA (scale bar = 100 μm), control hydrogel only (scale bar = 100 μm). T cells (CD3 marker), B cells (B220 marker), and antigen presenting cells (CD11c) are shown in the tumor microenvironment of different experimental groups. (G) Quantification of the number of TLS per square millimeter in each experimental cohort. Kolmogorov Smirnoff test was used to evaluate statistical differences. (H) Formation of mature germinal center-like (Ki67+) TLS in the hydrogel-LIGHT+CXCL13 mRNA group (scale bar = 100 μm) and immature TLS in the LIGHT+CXCL13 mRNA group (scale bar = 100 μm). (I) TLS classification (mature, immature, no TLS) after immunostaining of treated B16.f10 tumors (n=5 per group). Chi-square contingency analysis was performed. (J) Quantification of B220+ B cells per square millimeter of tumors from each experimental group. (K) Quantification of CD3+ T cells per square millimeter of tumors from each experimental group. Statistical analysis was performed using an unpaired t-test with Welch correction. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns means "not significant".

[0050] Fig.14A -K: pass Hydrogels delivered CXCL13 and LIGHT mRNA in vivo into CT26 tumor-bearing mice. (A) Hydrogels loaded with liposome-encapsulated CXCL13 and LIGHT mRNA in a CT26 mouse model. Treatment regimen of hydrogels. Mice were inoculated with CT26 colon cancer cell line by subcutaneous injection on day 0. Intratumoral injections of drug-encapsulated hydrogel (hydrogel-LIGHT+CXCL13 mRNA), drug alone (LIGHT+CXCL13 mRNA), carrier hydrogel control or carrier liposome control were given on days 5, 8 and 11 (n=5 per group). At the endpoint (day 23), mice were sacrificed for further analysis. For survival experiments, mice were closely monitored until the humane euthanasia endpoint (day 40). (B) Individual tumor growth kinetics of different experimental groups. (C) Primary injection site tumor size of different experimental groups at endpoint (day 23). (D) Quantification of primary injection site tumor size of different experimental groups at endpoint (day 23). Unpaired t-test was used, and multiple t-test adjustment was performed. Data are expressed as mean ± SEM. (E) Survival analysis of experimental groups (n=5). Mantel-Cox test was used. *p<0.05, ns means "not significant". In the case of encapsulated drug Immunofluorescence staining and grading of TLS and immune cell density in hydrogel-treated CT26 tumors (FK). (F) Formation of TLS in CT26 tumors in different experimental groups: hydrogel-LIGHT+CXCL13 mRNA (scale bar = 200 μm), LIGHT+CXCL13 mRNA (scale bar = 100 μm), control hydrogel only (scale bar = 100 μm). T cells (CD3 marker), B cells (B220 marker), and antigen presenting cells (CD11c) are shown in the tumor microenvironment of different experimental groups. (G) Quantification of the number of TLS per square millimeter in each experimental cohort. The Kolmogorov Smirnoff test was used to evaluate statistical differences. (H) Formation of mature germinal center-like (Ki67+) TLS in the hydrogel-LIGHT+CXCL13 mRNA group (scale bar = 100 μm) and immature TLS in the LIGHT+CXCL13 mRNA group (scale bar = 100 μm). (I) TLS classification (mature, immature, no TLS) after immunostaining of treated CT26 tumors (n=5 per group). Chi-square contingency analysis was performed. (J) Quantification of B220+B cells per square millimeter of tumors from each experimental group. (K) Quantification of CD3+T cells per square millimeter of tumors from each experimental group. Statistical analysis was performed using an unpaired t-test with Welch correction. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns means "not significant".

[0051] Fig.15A -I: pass Hydrogels delivered CXCL13 and LIGHT mRNA in combination with anti-PD1 therapy to 4T1 tumor-bearing mice in vivo. (A) Hydrogels loaded with liposome-encapsulated CXCL13 and LIGHT mRNA in 4T1 mouse model Treatment regimen for hydrogel combined with anti-PD1 therapy. Mice were inoculated with 4T1 breast cancer cell line by subcutaneous injection on day 0. Intratumoral injections of drug-encapsulated hydrogel (hydrogel-LIGHT+CXCL13mRNA), drug alone (LIGHT+CXCL13 mRNA), carrier hydrogel control or carrier liposome control were given on days 5, 8 and 11 (n=5 per group). Anti-PD1 treatment was given intravenously in combination or alone on days 15 and 17. At the end point (day 23), mice were sacrificed for further analysis. For survival experiments, mice were closely monitored until the end point of humane euthanasia (day 40). (B) Individual tumor growth kinetics of different experimental groups. (C) Primary injection site tumor size of different experimental groups at the end point (day 23). (D) Quantification of primary injection site tumor size of different experimental groups at the end point (day 23). Unpaired t-test was used, and multiple t-test adjustment was performed. Data are expressed as mean ± SEM. (E) Survival analysis of experimental groups (n=5). Mantel-Cox test was used. *p<0.05, ns means “not significant”. Immunofluorescence staining and grading of TLS and immune cell density in hydrogel-treated 4T1 tumors (FK). (F) Formation of TLS in 4T1 tumors in different experimental groups: hydrogel-LIGHT+CXCL13 mRNA+anti-PD1 (scale bar = 200 μm), hydrogel-LIGHT+CXCL13 mRNA (scale bar = 100 μm), control anti-PD1 only (scale bar = 100 μm). T cells (CD3 marker), B cells (B220 marker), and antigen presenting cells (CD11c) are shown in the tumor microenvironment of different experimental groups. (G) Quantification of the number of TLS per square millimeter in each experimental cohort. The Kolmogorov Smirnoff test was used to evaluate statistical differences. (H) Formation of mature germinal center-like (Ki67+) TLS in the hydrogel-LIGHT+CXCL13 mRNA group (scale bar = 100 μm) and the formation of immature TLS in the LIGHT+CXCL13 mRNA group (scale bar = 100 μm). (I) TLS grading (mature, immature, no TLS) after immunostaining in treated 4T1 tumors (n=5 per group). Chi-square contingency analysis was performed. 5. Specific implementation methods

[0052] 5.1 Definitions

[0053] As used herein, the term "treatment" means obtaining a desired pharmacological and / or physiological effect, such as inhibiting cancer growth or reducing ischemic damage to an organ (e.g., the heart). The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or side effects caused by the disease. As used herein, "treatment" includes preventative (e.g., prophylactic), curative, or palliative treatment of a disease in a mammal, particularly a human; and includes: (1) preventative (e.g., prophylactic), curative, or palliative treatment of a disease or condition (e.g., cancer or ischemic disease) in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease; (2) inhibiting the disease (e.g., by preventing its development); or (3) alleviating the disease (e.g., reducing the symptoms associated with the disease).

[0054] The terms "administered," "administering," or "administration" are used interchangeably herein and refer to modes of delivery including, but not limited to, oral, nasal, pulmonary, transdermal, such as passive or iontophoretic delivery, or parenteral, e.g., rectal, depot, subcutaneous, intravenous, intramuscular, intraperitoneal, intraarterial, intracerebellar, eye drops or ointments.

[0055] The term "effective amount" as used herein refers to an effective amount for the treatment of a disease, at a necessary dosage and time period to achieve the desired result. For example, in the treatment of cancer, an agent (i.e., a compound or composition) that reduces, prevents, delays, or inhibits or prevents any symptoms of cancer will be effective. An effective amount of an agent does not require a cure for a disease or condition, but will provide treatment for a disease or condition so that the onset of a disease or condition is delayed, hindered, or prevented, or the symptoms of a disease or condition are alleviated. An effective amount can be divided into one, two, or more doses in a suitable form for administration once, twice, or more throughout a specified time period.

[0056] The term "subject" or "patient" refers to an animal, including a human, that can be treated with the methods of the present invention. Thus, the term "subject" or "patient" includes any mammal that can benefit from the treatment methods of the present disclosure. The term "subject" includes, but is not limited to, mammals. Non-limiting examples include rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cattle and horses, and humans.

[0057] The term "cancer" as used herein refers to any cellular malignancy characterized by the loss of normal control, resulting in uncontrolled growth, lack of differentiation, and the ability to invade local tissues and metastasize. Cancer can form in any organ or tissue and can be breast cancer, cervical cancer, ovarian cancer, endometrial cancer, melanoma, uveal melanoma, brain tumor, lung cancer, liver cancer, lymphoma, neuroepithelioma, kidney cancer, bladder cancer, pancreatic cancer, prostate cancer, stomach cancer, colon cancer, uterine cancer, lymphoid hematopoietic tumors, myeloid leukemia, thyroid cancer, thyroid follicular carcinoma, myelodysplastic syndrome (MDS), mesenchymal tumors, teratoma, Neuroblastoma, glioma, glioblastoma, keratoacanthoma, anaplastic large cell lymphoma, esophageal squamous cell carcinoma, follicular dendritic cell carcinoma, intestinal cancer, muscle invasive carcinoma, seminal vesicle tumor, epidermal carcinoma, spleen cancer, head and neck cancer, stomach cancer, bone cancer, retinal cancer, bile duct cancer, small intestine cancer, salivary gland cancer, uterine sarcoma, testicular cancer, connective tissue cancer, prostate hypertrophy, myelodysplasia, Waldenstrom's macroglobulinemia The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like. The present invention relates to a tumor of the present invention and the like.

[0058] 5.2 Details

[0059] Tertiary lymphoid structures (TLS) within the tumor microenvironment are associated with good cancer prognosis and enhanced immune checkpoint blockade (ICB) response. The inventors designed an injectable hydrogel-based drug formulation to stimulate TLS formation in a B16 melanoma mouse model. The hydrogel is called , which is formed by supramolecular interactions between 4-(4-chlorophenyl)pyridine-modified hyaluronic acid (HA-CPP) and cucurbit[8]uril (CB[8]). Single injection of CXCL13 chemokine and LIGHT cytokine encapsulation The hydrogel effectively inhibited tumor growth, prolonged survival, increased TLS density, and promoted TLS maturation. Notably, hydrogel intervention also upregulated the spread of OVA antigens in secondary lymphoid organs. In addition, combining hydrogel-based drug formulations with anti-PD1 ICB therapy resulted in enhanced tumor suppression, improved survival, and increased TLS formation, contributing to the eradication of B16 melanoma. The present invention provides hydrogel-based drug carriers and methods for using hydrogel-based drug carriers as synthetic immune microenvironments for stimulating the formation of mature TLS within the B16 melanoma tumor microenvironment.

[0060] 5.2.1 Compounds

[0061] In some embodiments, the disclosure of the present invention provides a compound comprising hyaluronic acid and 4-(4-chlorophenyl)pyridine (HA-CPP) or a salt thereof. In some embodiments, the compound is a conjugate of hyaluronic acid (HA) and 4-(4-chlorophenyl)pyridine (CPP), wherein HA and CPP are covalently linked. In some embodiments, HA and CPP are directly linked. In some embodiments, HA and CPP are connected via a linker.

[0062] Hyaluronic acid (HA) is a linear polysaccharide with a high molecular weight, which consists of a repeating disaccharide of D-glucuronic acid and N-acetyl-D-glucosamine. These disaccharides are linked by glucuronic acid β (1→3) bonds. The compounds of the disclosure of the present invention include at least one 4-(4-chlorophenyl)pyridine (CPP) linked to HA.

[0063] In some embodiments, a linker is used to connect the CPP to HA. The linker can be any moiety with a functional group that can form a bond with one or more functional groups on HA. In some embodiments, the linker is an alkylenamine. The CPP can react with an alkylamine derivative containing a leaving group to form an amine derivative of the CPP, such as CPP-(CH2) m -NH2, wherein m is an integer, such as 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The amine group can then be linked to HA, for example by forming a bond with one or more carboxylic acid groups of the D-glucuronic acid subunit. In some embodiments, the linker is -(CH2)3-NH-.

[0064] The degree of substitution of CPP on the HA backbone (i.e., how many disaccharide subunits are replaced by CPP) can vary. In some embodiments, about 10-30% of the hyaluronic acid disaccharide subunits are replaced by CPP (i.e., the degree of substitution is about 0.1-0.3). In some embodiments, about 15% of the hyaluronic acid disaccharide subunits are replaced by CPP (i.e., the degree of substitution is about 0.15). In some embodiments, the degree of substitution of CPP on the HA backbone is about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3 or higher.

[0065] In some embodiments, the HA-CPP comprises at least one moiety of formula (1) or a salt thereof:

[0066]

[0067] wherein n is an integer and X is a counterion.

[0068] The integer can be any number between 1 and 1000.

[0069] In some embodiments, the compound comprises repeating units of the compound of formula (1) and unsubstituted HA

[0070]

[0071] wherein each of n and m is independently an integer from 1 to 1000 at each occurrence.

[0072] In some embodiments, the total ratio of n to m is about 0.1. In some embodiments, the total ratio of n to m is about 0.15. In some embodiments, the total ratio of n to m is about 0.2. In some embodiments, the total ratio of n to m is about 0.25. In some embodiments, the total ratio of n to m is about 0.3.

[0073] In some embodiments, the HA-CPP comprises at least one moiety of formula (1a):

[0074]

[0075] In some embodiments, the HA-CPP comprises about 5% to about 50% of the moiety of formula (1a). In some embodiments, the HA-CPP comprises about 10% to about 30% of the moiety of formula (1a). In some embodiments, the HA-CPP comprises about 15% of the moiety of formula (1a). In some embodiments, the HA-CPP comprises about 20% of the moiety of formula (1a). In some embodiments, the HA-CPP comprises about 25% of the moiety of formula (1a). In some embodiments, the HA-CPP comprises about 30% of the moiety of formula (1a).

[0076] In some embodiments, the present disclosure provides a composition comprising (a) a compound comprising hyaluronic acid and 4-(4-chlorophenyl)pyridine (HA-CPP) or a salt thereof and cucurbitac[n]uril (CB[n]), wherein n is 5, 6, 7 or 8.

[0077] In some embodiments, n is 5 In some embodiments, n is 6 In some embodiments, n is 7 In some embodiments, n is 8

[0078] In some embodiments, the composition is in the form of a hydrogel.

[0079] In some embodiments, the composition is an injectable composition and comprises about 1-5% water by weight.

[0080] In some embodiments, the HA-CPP and CB[n] are linked by non-covalent binding.

[0081] In some embodiments, the compositions further comprise one or more bioactive agents. Can be bound to a bioactive agent by non-covalent binding to form a complex. In some embodiments, Can bind to biologically active agents via non-covalent binding to form a complex.

[0082] Any bioactive agent can be used in the composition of the disclosure of the present invention. In some embodiments, the bioactive agent is a chemokine, a cytokine, an immune checkpoint inhibitor, a cancer vaccine, a chimeric antigen receptor, or a combination thereof. In some embodiments, the bioactive agent is CXCL13, CCL21, IL-4, IL-7, IL-2, LIGHT, CCL19, CXCL12, CXCL13, lymphotoxin, TNF-α, or a combination thereof. In some embodiments, the chemokine is CXCL13 and the cytokine is LIGHT.

[0083] 5.2.2 Preparation method

[0084] In some embodiments, the disclosure of the present invention provides a method for preparing HA-CPP, comprising: (a) combining 4-(4-chlorophenyl)pyridine with a compound of formula X-(CH2)3-NH2, wherein X is a leaving group; and (b) reacting the product of step (a) with hyaluronic acid (HA). In some embodiments, the molecular weight of HA is about 6 kDa to 1000 kDa. In some embodiments, the leaving group is a halogen (e.g., F, Cl, Br, I). In some embodiments, the leaving group is a sulfonate (e.g., an alkyl sulfonate or an aryl sulfonate).

[0085]

[0086] In some embodiments, the present disclosure provides a method for preparing HA-CPP, comprising: (i) combining 4-(4-chlorophenyl)pyridine with 3-halopropylamine to form pyridinium salt CPP-NH2, and (ii) performing an amidation reaction with at least one glucuronic acid subunit of hyaluronic acid (HA). In some embodiments, the molecular weight of HA is about 6 kDa to 1000 kDa.

[0087]

[0088] In some embodiments, the method further comprises combining HA-CPP and cucurbit[n]uril (CB[n]) to form wherein n is 5, 6, 7 or 8. In some embodiments, n is 8, and the method comprises combining HA-CPP and cucurbit[8]uril (CB[8]) to form

[0089] In some embodiments, the stoichiometric ratio between CPP and CB[8] is 2: 1. In some embodiments, the stoichiometric ratio between CPP and CB[8] is greater than 2:1.

[0090] 5.2.3 Preparation

[0091] The compounds of the disclosure of the present invention can be formulated into liquid pharmaceutical compositions, which are sterile solutions or suspensions that can be administered by, for example, intravenous, intramuscular, subcutaneous or intraperitoneal injection; and the compositions can be in any dosage form, including, but not limited to, solid or liquid dosage forms for oral, parenteral, nasal or sublingual administration. Suitable diluents or solvents for making sterile injectable solutions or suspensions of the mixtures of the disclosure of the present invention include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution and isotonic sodium chloride solution. Fatty acids, such as oleic acid and its glyceride derivatives can also be used to prepare injections, as can natural pharmaceutically acceptable oils, such as olive oil or castor oil. These oil solutions or suspensions may also contain alcohol diluents or carboxymethylcellulose or similar dispersants. Other commonly used surfactants, such as Tween or Spans or other similar emulsifiers or bioavailability enhancers, which are commonly used to prepare pharmaceutically acceptable dosage forms, may also be used for formulation purposes. Oral administration may be in the form of liquid or solid compositions.

[0092] The composition is administered to the subject to elicit a desired response, factors such as the severity of the disease state or condition to be alleviated, the age, sex, weight of the patient, the subject's existing state and the severity of the pathological condition to be treated, the subject's subsequent concurrent medication or special diet, and other factors that will be recognized by those skilled in the art, with the appropriate dosage ultimately being determined by the attending physician. The dosage regimen can be adjusted to provide the desired response. The composition of the disclosure of the present invention is administered in an amount and for a period of time such that at least one dose of the compound (e.g., 2, 3, 4 or even more doses) is administered to the subject to achieve an improved therapeutic response.

[0093] 5.3 Examples

[0094] 5.3.1 Based on Development and characterization of hydrogel-based drug delivery systems

[0095] We developed an injectable hyaluronic acid (HA) hydrogel-based drug delivery system for CXCL13 and LIGHT proteins. To synthesize HA polymers modified with 4-(4-chlorophenyl)pyridine (HA-CPP), we followed a well-defined synthetic route ( Figure 2A ). Initially, we combined CPP and 3-chloropropylamine, resulting in the formation of a pyridinium salt called CPP-NH2. We then performed an amination reaction with HA (molecular weight (MW) 60K) and successfully generated the desired HA-CPP ( Figure 2B ).

[0096] To determine the average degree of CPP substitution, we integrated the peaks in the NMR spectrum and calculated a value of 0.15. We then mixed HA-CPP with CB[8] at a stoichiometric ratio of 2:1 CPP:CB[8] to form a complex, i.e. (Scheme 1B) The complexation was confirmed by 1H-NMR, which showed a clear shift in the spectrum of the aromatic protons of the CPP, indicating that the side-chain CPP guest was successfully inserted into the CB[8] cavity (Figure 3).

[0097] We also prepared the lyophilized complex by dissolving it in deionized (DI) water at 2% by weight. Hydrogel. 2% The hydrogel exhibits ideal injectability because it passes smoothly through a syringe needle via shear thinning and retains its shape after the shear force disappears ( Figure 4A and B). The hydrogel also exhibited shear-thinning properties in the flow ramp experiment, which further demonstrated its injectability ( Figure 4C ).

[0098] We performed rheological tests to check the Dynamic viscoelastic behavior of the network generated by supramolecular crosslinking. The sample was first subjected to amplitude sweep to determine the linear viscoelastic range, and then tested by oscillating frequency sweep within this range. The critical frequency (ω_c) when G'=G" is close to the lowest angular frequency tested (0.1rad / s), indicating that a hydrogel with G'>G" is formed in the entire tested frequency range ( Figure 4D ). In addition, due to the reversible host-guest complexation between polymer side chains, The hydrogels exhibit self-healing properties. We demonstrated this by connecting two differently dyed hydrogels and observing the self-healing effect optically ( Figure 4E We also conducted a series of experiments under small (1%) and large (100%) strains. The hydrogel was subjected to cyclic step strain measurements to verify the self-healing ability of the supramolecular hydrogel. The hydrogel formed a self-supporting structure at 1% strain, where G' exceeded G", but transformed into a sol state at 100% strain, where the G' and G" values ​​switched ( Figure 4F ). This pattern is repeated in subsequent step strain cycles. The dynamic, injectable, and self-healing properties of the hydrogel ensure that the injected hydrogel is applied near the tumor and maintains its shape at that location.

[0099] 5.3.2 In vivo based Hydrogel delivery of CXCL13 and LIGHT exhibits antitumor effects in the intraperitoneal B16-OVA mouse model

[0100] We first injected blank hydrogels without encapsulated drugs (control hydrogels) intraperitoneally into female C57 mice to evaluate potential in vivo toxicity. Mice injected with control hydrogels showed no signs of discomfort or weight loss after injection. Serum ELISA results obtained on day 14 showed no Hydrogel-related immunogenic responses (Figure 9).

[0101] Our preliminary study first immunized B16 tumor-bearing mice with LIGHT-encapsulated hydrogel formulations by intraperitoneal challenge (Figure 10). Enhanced T cell cluster recruitment was uniquely observed in the LIGHT-encapsulated hydrogel group ( Fig.11 ). However, there were no observable changes in primary tumor size or total tumor weight at the endpoint (Figure 10). This result suggests that recruiting T cells by LIGHT alone may not be sufficient for anti-tumor activity. Therefore, we combined LIGHT (T cell recruitment factor) and CXCL13 (B cell recruitment factor) for the final drug-encapsulated hydrogel formulation 12.

[0102] We found that under in vitro culture conditions, cells loaded with CXCL13 and LIGHT proteins The hydrogels recruited significantly more immune cells than the control hydrogel alone ( Fig.12 ).

[0103] In our subsequent experiments, we injected randomized female C57 mice intraperitoneally with B16-OVA melanoma cells (3 × 10 5 cells / animal), followed by injection of drug-encapsulated hydrogel formulations (0.6 μg CXCL13+0.6 μg LIGHT delivered by hydrogel), drug alone (0.6 μg CXCL13+0.6 μg LIGHT in PBS), vehicle-hydrogel control, or PBS control ( Figure 5A We observed no signs of discomfort in the mice in the different experimental groups. At the end point (day 23), all mice were sacrificed and their tumors and spleens were surgically removed for further immunological analysis. Interestingly, both the drug-encapsulated hydrogel formulation and the drug alone group showed partial primary tumor inhibition ( Figure 5B and 5C When injection site tumor size (primary intraperitoneal tumor) was compared to vehicle-hydrogel control and PBS control tumors ( Figure 5B ), both the drug-encapsulated hydrogel formulation and the drug-alone group formed significantly smaller B16-OVA tumors (mean tumor size was 47.2 mm 2 and 45.7mm 2In contrast, the average tumor size in the vehicle-hydrogel control group was 78.8 mm 2 )( Figure 5C ).

[0104] We also quantified the total tumor weight (primary and metastatic intraperitoneal tumors) from all groups. Our results revealed that only the drug encapsulated hydrogel formulation group resulted in a significant tumor weight reduction (mean tumor weight of 0.83 g) when compared to the vehicle control (mean tumor weight of 1.25 g) ( Figure 5D This result can be attributed to the enhanced recruitment of cancer-suppressing immune cell infiltration generated by the hydrogel matrix. 35 It is worth noting that compared with the control group, the drug-encapsulated hydrogel formulation group also significantly prolonged the survival of mice (at the end of the 40th day of the experiment, 1 / 5 of the mice had no death) ( Figure 5E This result can also be attributed to the enhanced recruitment of cancer-suppressing immune cell infiltration generated by the hydrogel matrix. 35 Therefore, we performed further studies to analyze the immune microenvironment within the tumor microenvironment and secondary lymphoid organs in different experimental groups.

[0105] 5.3.3 In vivo based Hydrogel delivery of CXCL13 and LIGHT generates mature TLS within the B16 tumor microenvironment

[0106] Using frozen tumor samples (harvested at the end point of day 23) from the aforementioned experimental groups (Figure 5), we performed immunohistochemical staining to reveal the presence of T cell (CD3 surface marker), B cell (B220 surface marker) and antigen presenting cell (CD11c surface marker) infiltration within the tumor microenvironment.

[0107] We characterized mature TLS as densely packed germinal center-like B cell clusters surrounded by T cell clusters and adjacent antigen-presenting dendritic cells. Interestingly, we observed these mature structures primarily in the tumor microenvironment of the drug-encapsulated hydrogel formulation group ( Fig. 6A and 6C ).

[0108] Although we observed TLS-like structures in other experimental groups, most were immature TLS, which are characterized by dispersed distribution of aggregated B cells and T cell populations ( Fig. 6A ). Notably, the drug-encapsulated hydrogel formulation group produced significantly greater TLS numbers than the other groups (mean TLS number was 0.45 per square millimeter) ( Figure 6B A significantly greater proportion of TLS-positive samples from the drug-encapsulated hydrogel formulation group showed mature TLS structures when compared to the PBS control group ( Figure 6C). In conclusion, these immunohistochemical staining results confirm the efficacy of the drug-encapsulated hydrogel formulation.

[0109] 5.3.4 In vivo based Hydrogel delivery of CXCL13 and LIGHT generates potent OVA-antigen T cell responses in secondary lymphoid organs

[0110] To determine whether intraperitoneal hydrogel-based delivery of CXCL13 and LIGHT could stimulate antigen spreading responses in secondary lymphoid organs, we harvested spleens adjacent to the injection site for interferon-γ ELISPOT assays. Our results revealed that OVA antigen-activated splenocytes from both the drug-encapsulated hydrogel formulation group and the drug-alone group stimulated a potent interferon-γ T cell response, which plays a key role in tumor elimination ( Fig.6D and E) 36. Notably, OVA-activated splenocytes from the drug-encapsulated hydrogel formulation group (0.6 μg CXCL13 + 0.6 μg LIGHT delivered by hydrogel) produced significantly stronger interferon-γ T cell responses compared to the drug-alone group and the PBS control group ( Fig. 6E This finding confirms the antitumor efficacy observed earlier ( Figure 5D ).

[0111] 5.3.5 In vivo based Hydrogel delivery of CXCL13 and LIGHT in combination with anti-PD1 ICBs leads to potent B16-OVA melanoma inhibition

[0112] It has been previously reported that increased TLS formation is positively correlated with a good clinical response to ICB therapy and improved cancer survival 12,15 .

[0113] Therefore, our next experiment aimed to improve B16 tumor eradication by combining high-dose anti-PD1 (0.6 mg αPD1) drug with a hydrogel formulation of drug encapsulation (0.6 μg CXCL13 + 0.6 μg LIGHT delivered by hydrogel) in the intraperitoneal B16 model ( Fig. 7A As expected, the combination of the drug-encapsulated hydrogel formulation with αPD1 resulted in the most significant tumor inhibition at the end point of day 23 ( Figure 7B -D). Compared with the vehicle control group (mean tumor size 89.2 mm 2 , mean tumor weight 1.24 g), both injection site tumor and total tumor weight were significantly lower in the combination group (mean tumor size 30 mm 2 , average tumor weight 0.61g), and the combined group was superior to the αPD1 group alone ( Figure 7C and 7DFinally, compared with other experimental groups, the combined treatment significantly prolonged the survival of B16 tumor-bearing mice (by the end of the 40th day, 2 / 5 mice had no death), confirming the therapeutic efficacy of the drug-encapsulated hydrogel formulation combined with anti-PD1 ( Fig. 7E ).

[0114] 5.3.6 In vivo based Hydrogel delivery of CXCL13 and LIGHT in combination with anti-PD1 ICBs generates mature TLS within the tumor microenvironment and enhances T cell responses in secondary lymphoid organs

[0115] We used frozen tumor samples from the previous experimental groups (collected at the end point of day 23) to detect the presence of TLS in the B16 tumor microenvironment and grade the maturity of TLS (Figure 8). The combination group produced significantly higher numbers of TLS in the tumor than the αPD1 and vector control groups (the average number of TLS was 0.524 per square millimeter) ( Figure 8B ). Notably, mature TLS were mainly observed in the combination group, while the αPD1 group and the vehicle control group mainly exhibited immature TLS or no TLS, respectively ( Figure 8C ).

[0116] We also performed flow cytometry analysis to explore the potential role of T cell subsets within the tumor microenvironment in different experimental groups. We observed a higher CD8-positive T cell population in the combination group compared with the vehicle control group, confirming the cytotoxic role of T cells in eliminating cancer cells within the tumor microenvironment, although there was no significant difference in the CD4-positive population ( Fig.8D and 8E In the spleen, the combined treatment induced a larger population of CD8-positive and CD4-positive T cells compared with the vehicle control ( Figure 8F and 8G ). Taken together, these results provide insight into the tumor- and spleen-based T cell-tumoricidal activity generated by combined treatment.

[0117] 5.3.7 Hydrogel-based mRNA therapy for enhancing tertiary lymphoid structure formation and eradication of solid cancers

[0118] In this work, we developed a unique hydrogel formulation to enhance the expression and drug activity of messenger ribonucleic acid (mRNA). We have conducted several experiments to study the therapeutic efficacy of our novel hydrogel-based mRNA therapy in various preclinical cancer models (B16.f10 skin cancer, CT26 colon cancer, and 4T1 breast cancer). These experiments highlight the prospects of our hydrogel platform in delivering potent mRNA drugs that effectively treat multiple types of solid cancers. In summary, our results confirm that the anti-tumor efficacy of our hydrogel-based mRNA therapy is enhanced compared to mRNA therapy alone and vehicle control. Importantly, we demonstrated that our hydrogel-based mRNA therapy can outperform pre-existing anti-PD1 immune checkpoint blockade therapy in inhibiting breast cancer, or be given as a combination therapy against cancer.

[0119] 5.3.7.1 Skin cancer model

[0120] For the skin cancer model, we inoculated B16.f10 melanoma (2 × 10 5 cells / animal), followed by injections of co-delivered hydrogel mRNA formulations (CXCL13 mRNA + LIGHT mRNA in cationic liposomes), mRNA drugs alone (CXCL13 mRNA + LIGHT mRNA in cationic liposomes), hydrogel controls, or carrier liposome controls ( Fig.13A We observed no signs of discomfort in the mice in the different experimental groups. At the end point (day 23), all mice were euthanized and their tumors were extracted for further analysis. Interestingly, both co-delivery of the hydrogel mRNA formulation and co-delivery of the mRNA drug alone showed potent primary tumor inhibition ( Fig. 13B and C). When primary tumor size was compared to hydrogel control and PBS control ( Fig. 13B and C), both co-delivery of the hydrogel mRNA formulation and co-delivery of the mRNA drug alone resulted in significantly smaller B16.f10 tumors (mean tumor size was 218.8 mm, respectively). 3 and 446mm 3 In contrast, the average tumor size in the hydrogel control group was 1032.9 mm 3 )( Fig.13D ). The results also demonstrated that co-delivery of hydrogel mRNA formulations can better inhibit tumor growth than co-delivery of individual mRNA drugs. It is worth noting that the co-delivery of hydrogel mRNA formulations also significantly prolonged mouse survival (by the end of the experiment on day 40, 3 / 5 mice survived) compared to the control group (all 5 mice died) ( Fig.13EThis result can be attributed to the enhanced recruitment of cancer-suppressing immune cell infiltration produced by the sustained release of cytokines from the hydrogel. 52 Increased presence of TLS in tumors provides prediction of strong clinical response to cancer immunotherapy 53,54,55,56 Therefore, we conducted further studies to analyze the immune microenvironment within the tumors of different experimental groups. Using frozen tumor samples from the experimental groups, we performed immunohistochemical staining and confocal quantification to reveal the presence of TLS through the infiltration of T cells (CD3 surface marker), B cells (B220 surface marker), and antigen-presenting cells (CD11c surface marker) into the tumors ( Fig.13F We also characterized mature TLS as densely packed germinal center-like Ki67-positive cells surrounded by clusters of CD3-positive T cells. Interestingly, we observed these mature structures primarily in tumors from the co-delivered hydrogel mRNA formulation group ( Fig.13H Although we observed TLS-like structures in other experimental groups, most of them were immature TLS, which were characterized by clustered B cells, Ki67-positive cells, and dispersed distribution of T cell populations ( Fig.13H and I). Notably, the co-delivery hydrogel mRNA formulation group produced significantly greater TLS numbers (average TLS number of 0.47 per square millimeter) compared to the other groups ( Figure 13G A significantly greater proportion of TLS-positive samples from the co-delivered hydrogel mRNA formulation group showed mature TLS structures when compared to the PBS control group ( Figure 13G ), and increased density of tumor-infiltrating B and T cells ( Fig.13J and K). In conclusion, the immunohistochemical staining results confirmed the efficacy of CXCL13 mRNA and LIGHT mRNA co-delivery using the hydrogel formulation.

[0121] 5.3.7.2 Colon cancer model

[0122] For the colon cancer model, we inoculated CT26 cells (2 × 10 5 cells / animal), followed by injections of co-delivered hydrogel mRNA formulations (CXCL13 mRNA + LIGHT mRNA in cationic liposomes), mRNA drugs alone (CXCL13 mRNA + LIGHT mRNA in cationic liposomes), hydrogel controls, or carrier liposome controls ( Fig.14AWe observed no signs of discomfort in the mice in the different experimental groups. At the end point (day 23), all mice were euthanized and their tumors were extracted for further analysis. Interestingly, both co-delivery of the hydrogel mRNA formulation and co-delivery of the mRNA drug alone showed potent primary tumor inhibition ( Fig. 14B and C). When primary tumor size was compared to hydrogel control and PBS control ( Fig. 14B Both co-delivery of the hydrogel mRNA formulation and co-delivery of the mRNA drug alone resulted in significantly smaller CT26 tumors (mean tumor size was 135 mm, respectively). 3 and 381mm 3 In contrast, the average tumor size in the hydrogel control group was 1120.4 mm 3 )( Fig.14D ). The results also demonstrated that co-delivery of hydrogel mRNA formulations can better inhibit tumor growth than co-delivery of individual mRNA drugs. It is worth noting that the co-delivery of hydrogel mRNA formulations also significantly prolonged mouse survival (by the end of the 40th day of the experiment, 3 / 5 mice survived) compared to the control group (all 5 mice died) ( Fig.14E We conducted further studies to analyze the immune microenvironment within the tumors of different experimental groups. Interestingly, we observed these mature structures mainly in the tumors of the co-delivered hydrogel mRNA formulation group ( Fig.14F ). Notably, the co-delivery hydrogel mRNA formulation group produced significantly greater TLS numbers (average TLS number was 0.43 per square millimeter) compared to the other groups ( Figure 14G A significantly greater proportion of TLS-positive samples from the co-delivered hydrogel mRNA formulation group showed mature TLS structures when compared to the PBS control group ( Fig.14H and I), and increased tumor-infiltrating B and T cell densities ( Fig.14J and K). In conclusion, the results of immunohistochemical staining of CT26 showed a trend similar to the aforementioned results in the B16.f10 model.

[0123] 5.3.7.3 Breast Cancer Model

[0124] For the breast cancer model, we inoculated 4T1 cells (1 × 10 5Cells / animal) were then injected into the same tumor site with co-delivered hydrogel mRNA formulations (CXCL13 mRNA + LIGHT mRNA in cationic liposomes), mRNA drugs alone (CXCL13 mRNA + LIGHT mRNA in cationic liposomes), or carrier liposome controls on days 5, 8, and 11. On days 15 and 17, intravenous injections of anti-PD1 treatments were given alone or as a combination treatment ( Fig.15A ). We observed no signs of discomfort in the mice in the different experimental groups. At the end point (day 23), all mice were euthanized and their tumors were extracted for further analysis. Interestingly, both co-delivery of the hydrogel mRNA formulation plus anti-PD1 and co-delivery of the hydrogel mRNA formulation alone showed potent primary tumor inhibition ( Fig. 15B and C). When primary tumor size was compared to vehicle control ( Fig. 15B and C), co-delivery of the hydrogel mRNA formulation plus anti-PD1 and co-delivery of the hydrogel mRNA formulation alone both resulted in significantly smaller 4T1 tumors (mean tumor size was 21.6 mm, respectively). 3 and 245.2mm 3 In contrast, the average tumor size in the hydrogel control group was 1665 mm 3 )( Fig.15D ). Both the hydrogel-based combination drug group and the hydrogel-based mRNA formulation group outperformed their non-hydrogel counterparts. Notably, the co-delivery of the hydrogel mRNA formulation plus anti-PD1 group most significantly prolonged mouse survival (1 / 5 mice survived at the end of the experiment on day 40) compared to the control group (all 5 mice died) ( Fig.15E We conducted further studies to analyze the immune microenvironment within the tumors of different experimental groups. Interestingly, we mainly observed these mature structures in tumors of the co-delivery hydrogel mRNA formulation plus anti-PD1 group and the co-delivery hydrogel mRNA formulation group ( Fig.15F ). Notably, the group co-delivering the hydrogel mRNA formulation plus anti-PD1 produced the greatest number of TLS (mean TLS number was 0.83 per square millimeter) ( Figure 15G A significantly greater proportion of TLS-positive samples from the co-delivered hydrogel mRNA formulation plus anti-PD1 group showed mature TLS structures when compared to the other groups ( Fig.15H and I). In conclusion, the results demonstrate the development of a novel injectable hydrogel-based formulation for enhancing the drug activity of mRNA therapeutics against cancer. Our study is unique in that we discovered co-delivered mRNA cytokine and chemokine druggable targets that can stimulate the formation of mature TLS in cancer, which effectively eradicates various cancers and can enhance anti-PD1 therapeutic response.

[0125] 5.3.7.4 Discussion and Conclusion

[0126] In this study, we developed an injectable hydrogel-based formulation for the delivery of CXCL13 chemokine and LIGHT cytokine. The favorable properties of hydrogels, including controlled drug release, biocompatibility, and porosity for cell infiltration, have selected them as drug delivery vehicles. 37 . Specifically, The hydrogels were used to encapsulate biomolecules (CXCL13, LIGHT) and serve as suitable delivery vehicles for intraperitoneal drug administration. This drug delivery approach can overcome the limitations of reduced drug immunogenicity and rapid drug degradation, which is particularly relevant in the context of cancer, where the immunosuppressive environment can hinder drug efficacy. 38 CXCL13 and LIGHT are essential proteins involved in TLS production in non-lymphoid tissues. TLS induction within the tumor microenvironment promotes anticancer immunity to resist cancer progression by maintaining and recruiting immunostimulatory lymphocytes and upregulating central memory T and B cells. 39 Therefore, the combination of a novel hydrogel drug delivery platform with key TLS inducing factors can enhance tumor control by generating a synthetic anticancer immune microenvironment within the tumor microenvironment.

[0127] Using the B16 melanoma mouse model, we demonstrated that a single injection of the hydrogel-based drug (CXCL13, LIGHT) formulation resulted in significant primary tumor site and total tumor inhibition compared to PBS and vehicle controls (Figure 5). Although the individual drug groups also showed significant tumor inhibition effects at the primary tumor site ( Figure 5C ), but it did not induce significant overall tumor suppression ( Figure 5D ). It is worth noting that the drug-encapsulated hydrogel formulation group prolonged the survival of mice compared with the control group ( Figure 5E To identify the immunological features that determine the therapeutic efficacy of hydrogel-based drug formulations, we further analyzed TLS organization in B16 tumor samples. Increased TLS presence in tumors can be used as a predictive biomarker for strong clinical response to cancer immunotherapy 40 Previous studies have also demonstrated the presence of TLS-like structures within tumor tissue derived from intraperitoneal cancer-bearing mouse models. 41-42 In this study, given that CXCL13 and LIGHT are known to recruit B cells and T cells, respectively, to produce TLS 43 We uniquely observed an increase in TLS number and maturity within the tumor microenvironment after hydrogel-based drug (CXCL13, LIGHT) treatment ( Figure 6B and 6C).

[0128] Furthermore, we found that hydrogel-based drug formulations (CXCL13, LIGHT) treatment enhanced antigen diffusion ( Fig.6D and Fig. 6E ). Interferon-γ ELISPOT assay revealed that T cells in the spleen within the peritoneal cavity led to enhanced OVA antigen recognition and subsequent interferon-γ release. Elevated tumor antigen dissemination can lead to increased recognition of cancer by antigen-presenting cells and adaptive immune cells, which can enhance therapeutic response 44 Importantly, further studies showed that combining anti-PD1 drugs with a single injection of a hydrogel-based drug formulation improved melanoma treatment response and survival (Figure 7). Compared with tumors in the vehicle control group, tumors in the combined group showed increased TLS presence and maturity, which was positively correlated with treatment efficacy ( Fig. 8A Interestingly, anti-PD1 ICBs were previously reported to increase the number of TLS via circulating T follicular helper cells in mice bearing non-small cell lung cancer (NSCLC). 45 This may explain why a higher proportion of anti-PD1-treated mice showed mature and immature TLS compared with vehicle control mice. In addition, we observed that CD8 + A higher percentage of T cells and CD8 + and CD4 + A higher percentage of T cells ( Fig.8D -F). This result indicates that T cells are key mediators of tumor eradication in the combination group, confirming the well-established role of T cell-mediated anti-cancer immunity 46 Previous studies by Brenna et al. also demonstrated a similar role for TLS generation and enhanced T cell infiltration in promoting cancer elimination in the context of ovarian cancer. 47 .

[0129] This study has several limitations that we plan to address in future studies. First, the hydrogel-based drug delivery platform can be validated in combination with other ICB therapies (such as anti-CTLA-4 or anti-PDL1) across a range of cancer types. 48 Second, gaining a deeper understanding of the underlying mechanisms will involve classifying memory immune cell subtypes and examining the correlation between TLS duration and favorable prognosis in various tumor mouse models and humanized mouse models. 49 Third, current studies are limited in their ability to characterize the role of germinal center-like TLS, indicative of mature and functional TLS, in different cancer types and stages of disease progression. Gaining such insight is critical to determining whether TLS-inducing drugs can be universally exploited at any stage of disease.50 To address this issue, high-sensitivity characterization of mature TLS at different time points could be investigated using imaging mass cytometry and multiplex immunohistochemistry. However, more cost-effective and time-efficient analytical methods have not yet been developed. 51 .

[0130] In summary, we have reported the development of a novel hydrogel-based drug (CXCL13, LIGHT) formulation that effectively inhibits B16 melanoma tumor growth and improves the survival of tumor-bearing mice. In addition, the treatment stimulated the increase in the formation of mature TLS, providing valuable insights into the combined production of mature TLS by LIGHT cytokines and CXCL13 chemokines. The combination of hydrogel-based drugs and ICBs also enhances the therapeutic response. We expect that hydrogel-based drug (CXCL13, LIGHT) formulations will have the potential to be used as therapeutic agents for clinical melanoma in the future.

[0131] 5.3.8 Materials and methods

[0132] HA-CPP synthesis

[0133] In a 20 ml sealed tube, 4-(4-chlorophenyl)pyridine (CPP) (378 mg, 2 mmol) and 3-chloropropylamine (260 mg, 2 mmol) were added together with 5 ml of isopropanol as solvent. The solution was purged with nitrogen for 15 minutes and the tube was tightly sealed. The reaction mixture was heated to 90° C. in an oil bath and allowed to react for 12 hours.

[0134] After completion, the reaction mixture was cooled to room temperature and the precipitated solid was filtered. The obtained solid was further recrystallized using a mixture of dichloromethane (DCM) and hexane, resulting in the formation of the final product CPP-NH2.

[0135] Then, CPP-NH2 (53 mg, 0.15 eq) and hyaluronic acid (HA, molecular weight 60k) (0.5 g, 1 eq) were dissolved in 50 ml of MES buffer (pH 5). Then 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM) (0.343 g, 1 eq) was added to the solution. The mixture was stirred vigorously for 48 hours. After the reaction was completed, it was dialyzed with saline and then deionized water for a total of 3 days. Finally, the remaining solution was lyophilized to obtain a solid final product, HA-CPP.

[0136] Hydrogel formation

[0137] The lyophilized HA-CPP was dissolved in deionized water and the pH of the dissolved solution was balanced to neutral by adding a small amount of 0.1 M NaOH or HCl if necessary. These solutions were then mixed with CB[8] in a stoichiometric ratio, resulting in the formation of a hydrogel through non-covalent binding.

[0138] Dynamic Oscillatory Rheology

[0139] A modular compact rheometer (Anton Parr) was used to study the Rheological properties of the hydrogels were measured using a rheometer with 25 mm parallel plates and a gap set to 0.5 μm. An initial amplitude sweep was performed using parameters of 10 rad / s and 0.01-200% strain to ensure that the subsequent frequency sweep (using 1% strain and a range of 0.1-200 rad / s) occurred within the linear viscoelastic region. Shear viscosity measurements were performed on the hydrogels with a shear rate ramp from 0.1 to 100 s -1 In addition, step strain tests were performed using cyclic strain fluctuating between 1 and 100% at 10 rad / s.

[0140] Animal experiments

[0141] Four-week-old female C57BL / 6J mice were provided and maintained by the Centre of Comparative Medicine, Laboratory Building, University of Hong Kong. The murine B16-OVA cell line was cultured for three generations in Dulbecco's modified Eagle's medium (DMEM) containing 100 μg / mL penicillin / streptomycin in 10% fetal bovine serum (FBS) at a maximum confluence of 85% and tested negative for mycoplasma. The cells were suspended in filtered PBS. A total of 3 × 10 cells / mL of 100 μL were added using a Hamilton syringe and a 25S gauge bevel-tip needle. 5 10 cells were injected into the peritoneal cavity of female C57BL / 6 mice. Using a Hamilton syringe and a 22S gauge bevel tip needle, 2 wt % PBS was diluted in Hydrogels, hydrogel-encapsulated protein drugs (CXCL13, ACROBiosystems and recombinant LIGHT, Rndsystems) and anti-PD1 (Invivomab) treatments were injected into the peritoneal cavity of female C57BL / 6 mice. Animals were euthanized according to strict humane endpoint guidelines and based on veterinary observations of signs and symptoms of pain and distress to minimize harm to tumor-bearing animals. All animal experiments in this study were approved by the Committee on the Use of Live Animals in Teaching & Research, the University of Hong Kong (#CULATR 23-034).

[0142] At the tumor endpoint, mice were sacrificed and primary and metastatic tumors were surgically harvested for tumor size analysis using electronic calipers and tumor weight analysis using mass balances. Spleens were also surgically harvested and processed for further analysis by resuspending strained splenocytes in RPMI medium containing FBS, adding red blood cell lysis buffer, and washing in filtered PBS. The harvested tumors were treated overnight in 4% paraformaldehyde and then resuspended in 30% sucrose for immunohistochemistry. The harvested tumors were also processed into single cell suspensions for flow cytometry according to the manufacturer's protocol by a tumor dissociation kit (Miltenyi Biotec, 130-096-730). For survival studies, tumor-bearing mice were closely monitored until the 40th day endpoint and euthanized based on the advice of a veterinarian, considering the observation of signs and symptoms of pain and distress to minimize damage.

[0143] Immunohistochemical staining

[0144] Five micron frozen sections were prepared from OCT preserved tissues and fixed on slides. Immunohistochemistry was performed using a multi-antibody staining protocol according to the manufacturer's instructions. In brief, slides were washed in filtered PBS and blocked with PBS containing 5% FBS for 1 hour. The following first monoclonal antibodies were used: rabbit anti-mouse CD3 (Invitrogen, 1:25), rat anti-mouse B220 (Invitrogen, 1:25) and hamster anti-mouse APC-11c (Biolegend, 1:25). The first antibody was incubated overnight at 4 ° C in the dark and washed several times with PBS-Tween. The following second monoclonal antibodies were used: donkey anti-rat 647 antibody (Invitrogen, 1:50), goat anti-rabbit FITC antibody (Invitrogen, 1:50). The second antibody was incubated in the dark at room temperature for 1 hour and washed several times with PBS-Tween. According to the manufacturer's protocol, slides were encapsulated using a DAPI-injected mounting medium (Abcam, ab104139). Images were taken on a confocal microscope LSM900 and processed using ImageJ (v1.53k). For TLS quantification, sections were scanned and analyzed using Zen, ImageJ software or QuPath.

[0145] TLS defines standards

[0146] Murine TLS are identified by immunostaining of sections to determine the coexistence of CD3+, B220+, and CD11+ cells in a tight organization. Mature TLS are characterized by a clear separation of CD3+ zones from adjacent CD11c cells that surround B220+ zones in a germinal center-like tight organization. Immature TLS are described as the coexistence of CD3+ zones adjacent to tight clusters of B220+ zones. The absence of TLS is defined as the absence of CD3+, B220+, or CD11+ cells that aggregate in a tight organization.

[0147] Flow cytometric analysis

[0148] Mouse tumors and spleens were processed into single-cell suspensions after passing through a 70-μm cell strainer (Thermo Fisher Scientific). 0.5–2 × 10 6The cells were incubated with Block buffer (1×PBS, 2% FBS, 0.5% BSA) for 15 minutes, and then 50 μl Master Mix surface marker staining (FITC anti-mouse CD3, PE / Cy7 anti-mouse CD4, BV-421 anti-mouse CD8a, 1:50, Biolegend) was added for 30 minutes. The cells were then washed in filtered PBS and centrifuged at 2000RPM and then resuspended in 200 μl FAC buffer (1×PBS, 2% FBS, 0.002% sodium azide). For fluorescence-activated cell sorting, samples were analyzed using a NovoCyte Quanteon flow cytometer.

[0149] IFN-γ ELISPOT assay

[0150] Mice were killed, spleen cells were collected, and IFN-γELISPOT analysis was performed according to the manufacturer's protocol (Sigma-Aldrich). In brief, 100 μl of spleen cells were incubated on IFN-γELISPOT plates, which were pre-activated for 30 minutes using 200 μl of DMEM medium without FBS. Subsequently, 5 μg of OVA peptide (OVA257-264 and positive inducer) were added, and the cells were incubated at 37 ° C in 5% CO2 for 20 hours. After the perturbation culture medium, the cells were washed in washing buffer and incubated with the second biotinylated antibody at room temperature for 1 hour. Finally, the cells were incubated with biotin substrate, then washed and dried to produce visible spots in the positive wells.

[0151] Statistical analysis

[0152] All results were plotted in Prism 7 (GraphPad Software, Inc.). Statistical comparisons between groups were determined by unpaired t-test using Prism 7. Chi-square contingency test was used for TLS grading. Mantel-Cox test was used for survival analysis. For all tests, p<0.05 was considered statistically significant.

[0153] Exemplary products, systems, and methods are listed in the following items:

[0154] 1. A compound comprising hyaluronic acid and 4-(4-chlorophenyl)pyridine (HA-CPP) or a salt thereof.

[0155] 2. The compound described in item 1, wherein HA and CPP are directly linked.

[0156] 3. The compound described in item 1 or 2, wherein HA and CPP are connected via a linker.

[0157] 4. The compound described in item 3, wherein the linker is an alkyleneamine.

[0158] 5. The compound described in item 3 or 4, wherein the linker is -(CH2)3-NH-.

[0159] 6. The compound of any one of the preceding items, wherein about 10-30% of the hyaluronan disaccharide subunits are substituted with CPP.

[0160] 7. The compound of item 6, wherein about 15% of the hyaluronan disaccharide subunits are substituted with CPP.

[0161] 8. The compound according to any one of the preceding items, comprising at least one moiety of formula (1) or a salt thereof:

[0162]

[0163] wherein n is an integer and X is a counterion.

[0164] 9. The compound of item 8, comprising about 10-30% of the moiety of formula (1) wherein n is 1.

[0165] 10. The compound of item 8 or 9, comprising about 15% of the moiety of formula (1) wherein n is 1.

[0166] 11. A composition comprising the compound according to any one of items 1 to 10 above and cucurbituril.

[0167] 12. The composition of claim 11, wherein the cucurbituril is cucurbit[n]uril (CB[n]), wherein n is 5, 6, 7 or 8.

[0168] 13. The composition of item 11-12, wherein n is 8

[0169] 14. The composition of any one of items 11-13, which is in the form of a hydrogel.

[0170] 15. The composition of any one of items 11-14, wherein the composition is an injectable composition.

[0171] 16. The composition of any one of items 11-15, comprising about 1-5% by weight of water.

[0172] 17. The composition of any one of items 11-16, wherein HA-CPP and CB[n] are linked by non-covalent binding.

[0173] 18. The composition of any one of items 11-17, further comprising one or more biologically active agents.

[0174] 19. The composition of any one of items 11-18, wherein Binds to the bioactive agent via non-covalent binding to form a complex.

[0175] 20. The composition according to any one of items 11-19, comprising and one or more biologically active agents.

[0176] 21. The composition of any one of items 11-20, wherein Binds to the bioactive agent via non-covalent binding to form a complex.

[0177] 22. The composition of any one of items 11-21, wherein the bioactive agent is a chemokine, a cytokine, an immune checkpoint inhibitor, a cancer vaccine, a chimeric antigen receptor, or a combination thereof.

[0178] 23. The composition of any one of items 11-22, wherein the bioactive agent is CXCL13, CCL21, IL-4, IL-7, IL-2, LIGHT, CCL19, CXCL12, CXCL13, lymphotoxin, TNF-α or a combination thereof.

[0179] 24. The composition of any one of items 11-23, wherein the chemokine is CXCL13 and the cytokine is LIGHT.

[0180] 25. A method for treating cancer, comprising administering the composition of any one of items 11-24 to a subject in need thereof.

[0181] 26. The method of claim 25, wherein the composition is administered by injection to form tertiary lymphoid structures and enhance immune checkpoint blockade.

[0182] 27. The method of claim 25 or 26, wherein the composition inhibits tumor growth, prolongs survival, increases TLS density, promotes TLS maturation, upregulates OVA antigen spread in secondary lymphoid organs, or a combination thereof.

[0183] 28. The method described in item 25, 26 or 27, which also includes administering anti-PD1 drugs, anti-CTLA-4 drugs, anti-PD1, anti-PDL1, anti-CTLA-4 drugs or a combination thereof.

[0184] 29. The method of claim 25, 26 or 27, wherein administration comprises direct intratumoral injection.

[0185] 30. The method of item 25, 26 or 27, wherein the cancer is melanoma, colorectal cancer, lung cancer, pancreatic cancer, oral squamous cell carcinoma or invasive breast cancer.

[0186] 31. The method of item 25, 26 or 27, wherein the subject is a human, a feline, a canine, a pig, a rabbit or a rodent.

[0187] 32. A method for preparing HA-CPP, comprising: (a) reacting 4-(4-chlorophenyl)pyridine with a compound of formula X-(CH2)3-NH2, wherein X is a leaving group; and (b) reacting the product of step (a) with hyaluronic acid (HA) having a molecular weight of about 6 kDa to 1000 kDa.

[0188] 33. A method for preparing HA-CPP, comprising: (i) reacting 4-(4-chlorophenyl)pyridine with 3-halopropylamine to form pyridinium salt CPP-NH2, and (ii) performing an amidation reaction with at least one glucuronic acid group of hyaluronic acid (HA), wherein the molecular weight of the HA is about 6 kDa to 1000 kDa.

[0189] 34. The method of claim 33, wherein the halogen is chlorine.

[0190] 35. The method of item 32, 33 or 34, further comprising combining HA-CPP and cucurbit[n]uril (CB[n]) to form Where n is 5, 6, 7 or 8.

[0191] 36. The method of item 32, 33, 34 or 35, further comprising combining HA-CPP and cucurbit[8]uril (CB[8]) to form

[0192] 37. The method of any one of items 32-36, wherein the stoichiometric ratio between CPP and CB[8] is 2:1 or greater than 2:1.

[0193] 38. The method of any one of items 32-37, wherein the degree of substitution of the CPP is from about 10 to about 30%.

[0194] 39. The method of any one of items 32-38, wherein the degree of substitution of the CPP is about 15%.

[0195] The foregoing description of specific embodiments will reveal the general nature of the disclosure so fully that others can easily modify and / or adjust such specific embodiments for various applications without departing from the overall concept of the disclosure of the present invention by applying the knowledge within the technology of (multiple) related fields (including the cited documents and the contents incorporated herein by reference) without excessive experimentation. Therefore, based on the teachings and guidance set forth herein, such adjustments and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for descriptive purposes rather than for limiting purposes, so that the terms or wording of this specification are interpreted by those skilled in the art based on the teachings and guidance set forth herein, combined with the knowledge of the technicians in (multiple) related fields.

[0196] Although various embodiments of the disclosure of the present invention have been described above, it should be understood that they have been presented by way of example and not limitation. It will be apparent to those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the disclosure. Therefore, the disclosure of the present invention should not be limited by any of the above exemplary embodiments, but should only be limited according to the appended claims and their equivalents.

[0197] All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

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Claims

1. A compound comprising hyaluronic acid and 4-(4-chlorophenyl)pyridine (HA-CPP) or a salt thereof.

2. The compound of claim 1, wherein HA and CPP are connected via a linker, wherein the linker is an alkylene amine.

3. The compound of claim 2, wherein the linker is -(CH2)3-NH-.

4. The compound of claim 1, wherein about 10-30% of the hyaluronan disaccharide subunits are substituted with CPP.

5. The compound of claim 1, comprising at least one moiety of formula (1) or a salt thereof: wherein n is an integer and X is a counterion.

6. A composition comprising the compound of claim 1 and cucurbitac[n]uril (CB[n]), wherein n is 5, 6, 7 or 8 ( ).

7. The composition of claim 6, wherein n is 8 ( ).

8. The composition of claim 6, which is in the form of a hydrogel.

9. The composition of claim 6, wherein the composition is an injectable composition and comprises about 1-5% water by weight.

10. The composition of claim 6, wherein HA-CPP and CB[n] are linked by non-covalent binding.

11. The composition of claim 6, further comprising one or more bioactive agents.

12. The composition of claim 11, wherein Binds to the bioactive agent via non-covalent binding to form a complex.

13. The composition of claim 6, comprising and one or more biologically active agents.

14. The composition of claim 13, wherein Binds to the bioactive agent via non-covalent binding to form a complex.

15. The composition of claim 11, wherein the bioactive agent is a chemokine, a cytokine, an immune checkpoint inhibitor, a cancer vaccine, a chimeric antigen receptor, or a combination thereof.

16. The composition of claim 11, wherein the bioactive agent is CXCL13, CCL21, IL-4, IL-7, IL-2, LIGHT, CCL19, CXCL12, CXCL13, lymphotoxin, TNF-α, or a combination thereof.

17. The composition of claim 16, wherein the chemokine is CXCL13 and the cytokine is LIGHT.

18. Use of the composition of claim 10 in the manufacture of a medicament for treating cancer.

19. The use of claim 18, wherein the composition is an injectable composition, and forms tertiary lymphoid structures and enhances immune checkpoint blockade after injection.

20. The use of claim 18, wherein the composition inhibits tumor growth, prolongs survival, increases TLS density, promotes TLS maturation, upregulates OVA antigen spread in secondary lymphoid organs, or a combination thereof.

21. The use of claim 18, wherein the composition further comprises an anti-PD1 drug, an anti-CTLA-4 drug, anti-PD1, anti-PDL1, anti-CTLA-4 or a combination thereof.

22. The use of claim 18, wherein the medicament is formulated for direct intratumoral injection.

23. The use of claim 18, wherein the cancer is melanoma, colorectal cancer, lung cancer, pancreatic cancer, oral squamous cell carcinoma, or invasive breast cancer.

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