Inter- and intratumoral delivery of cytokines using mesoporous silica rods as an immunomodulatory system

Abstract

Embodiments of the present invention include methods for treating conditions ameliorated by stimulating an immune response. In aspects, the methods include injecting mesoporous silica rods (MSRs) into or near the affected tissue. The MSRs can include cytokines (e.g., IL-2 or IL-12) and / or adjuvants. The MSRs can induce an innate immune response to treat cancer, infectious diseases, and other illnesses. The methods can include administering an additional pharmaceutical agent, such as an immune checkpoint inhibitor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 437,747, filed January 8, 2023, and U.S. Provisional Patent Application No. 63 / 599,507, filed May 15, 2023, the contents of which are incorporated herein by reference.

[0002] The present invention relates to cancer therapeutics, and more particularly to structures and methods for treating cancer using mesoporous silica materials to deliver cytokines. [Background technology]

[0003] An organism's immune system works in concert with a myriad of biological processes to protect itself from disease, injury, and illness. The immune system does these processes by detecting and responding to a wide variety of pathogens and foreign substances that are not native to the organism's body.

[0004] Dysfunction of the immune system can lead to several diseases, disorders and illnesses, including autoimmune diseases, inflammatory diseases and cancer. Another disease that affects the immune system is immunodeficiency. Immune deficiency occurs when the activity and response of the immune system typically falls below a healthy threshold, which can lead to recurrent and life-threatening infections. Immune deficiency can be manifested as a result of genetic diseases or acquired conditions such as HIV / AIDS, or by the use of immunosuppressive drugs.

[0005] Autoimmunity refers to a collection of autoimmune disorders in which the organism literally attacks itself. Autoimmune disorders result from an overactive immune system, which attacks normal tissues as if they were pathogens, physical or pathogenic insults, or other foreign organisms. Examples of autoimmune diseases include type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus.

[0006] In the human body, the innate immune system recognizes pathogenic insults and dead and / or defective cells within the body to mount a defensive response. This recognition occurs via a series of germline-encoded pattern recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).

[0007] Cytoplasmic PRRs include NOD-like receptors (NLRs) and retinoic acid-inducible gene I-like receptors (also known as RIG-I-like receptors). NLRs recognize ligands from various microbial pathogens, host cells, and environmental sources. Based on their domain structure, NLRs are subdivided into NLRPs and NLRCs. Among these, NLRP1 (murine NLRP1b), NLRP3, and NLR family inhibitor of apoptosis protein / NLCR4 are well-established NLRs for their ability to assemble inflammasomes.

[0008] Inflammasomes are multimeric cytosolic protein complexes that assemble in response to DAMPs and PAMPs, leading to the activation of inflammatory responses. Inflammasome assembly initiates an inflammatory form of cell death known as pyroptosis and the release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. The NLRP3 inflammasome responds to cellular perturbations and a wide variety of microorganisms.

[0009] The NLRP3 inflammasome, like other inflammasomes, is a multimeric cytosolic protein complex that assembles in response to cellular perturbations. This assembly leads to the activation of caspase-1, which promotes the maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and inflammatory cell death (i.e., pyroptosis). Inflammatory cytokines contribute to the development of systemic low-grade inflammation, and aberrant NLRP3 activation may promote a chronic inflammatory state in the body and regulate the pathogenesis of inflammation-related diseases. Therefore, targeting NLRP3 or other downstream signaling molecules, such as caspase-1, IL-1β, or IL-18, has the potential for significant therapeutic benefit. However, NLRP3 inflammasome-mediated inflammatory cytokines play a dual role in mediating human disease. They are detrimental in the pathogenesis of inflammatory and metabolic diseases but have beneficial roles in numerous infectious diseases and some cancers. Therefore, fine-tuning of NLRP3 inflammasome activity is essential for maintaining proper cellular homeostasis and health. The mechanisms of NLRP3 inflammasome activation play diverse roles in the pathogenesis of inflammation-related diseases such as cancer, atherosclerosis, diabetes, and obesity, and therefore offer therapeutic potential if targeted to modify the pathway.

[0010] Foam cells, also known as lipid-laden macrophages, are cholesterol-containing cells that can form plaques and cause inflammatory diseases such as atherosclerosis, which can lead to heart attacks and strokes. Foam cells are lipid-rich cells that typically have an M2 macrophage-like phenotype, but can also exhibit an M1 phenotype depending on the cues. However, certain foam cells can originate from smooth muscle tissue, and these specific foam cells exhibit a restricted macrophage-like phenotype. The presence of the NLRP3 inflammasome is known to promote foam cell formation, and this has been observed in some histological samples. However, many studies have also reported that NLRP3 inflammasome activation promotes macrophage foam cell formation via IL-1β.

[0011] Aluminum hydroxide, represented by the chemical formula Al(OH)3 and commonly known as "alum" in pharmaceutical and related fields, is composed of a bilayer of hydroxyl groups, with aluminum ions occupying two-thirds of the octahedral cavities between the bilayers. Among its several uses, aluminum hydroxide is used as a pharmaceutical adjuvant in some vaccines. Additionally, aluminum hydroxide is known to stimulate the immune system by inducing the release of uric acid, ultimately leading to the stimulation of T and B cells.

[0012] Recent studies suggest that the formation and growth of cancerous tumors are directly linked to inflammatory processes. It has long been known that cancer development and its response to treatment are regulated by inflammation, which can promote or suppress tumor progression. While chronic inflammation promotes tumor progression and resistance to treatment, the induction of acute inflammatory responses often stimulates dendritic cell (DC) maturation and antigen presentation, leading to antitumor responses.

[0013] Conventional cancer treatments are aimed at removing or "killing" cancerous tissue and preventing it from spreading. Such treatment options include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and palliative care. Treatments are typically pursued based on the type, location, and grade of the cancer, as well as the patient's health and preferences. These options have limitations; they can be ineffective, especially if the cancer has metastasized. Furthermore, chemotherapy and radiation therapy have various side effects related to cytotoxicity.

[0014] Cancer cells divide faster than most normal cells, so they can be sensitive to chemotherapy drugs. However, chemotherapy drugs also attack other cells in the body, particularly rapidly dividing cells such as blood cells and cells lining the mouth, stomach, and intestines, resulting in a narrow therapeutic window. The side effects of drugs often prevent their continued use, negatively affecting the quality of life of patients. Therefore, there is a need for improved medicines and cancer treatment methods that are more targeted and have reduced harmful side effects.

[0015] Recent efforts to develop new cancer treatments have focused on immunology. For example, checkpoint therapy can block inhibitory checkpoints and restore immune system function. However, the use of checkpoint inhibitors has limitations and often results in adverse immunological effects. Alterations in checkpoint inhibition can have diverse effects on most organ systems in the body. Colitis (i.e., inflammation of the colon) is a common side effect. Infusion of checkpoint inhibitors has also been associated with acute seronegative myasthenia gravis. Furthermore, clinical benefit is variable, with some patients becoming "hyperprogressors" with accelerated tumor growth rates and rapid deterioration.

[0016] Researchers have also developed cancer vaccines, which treat existing cancers or prevent the onset of cancer. Vaccines that treat existing cancers are known as therapeutic cancer vaccines or tumor antigen vaccines. Some vaccines are "autologous," meaning they are prepared from samples taken from the patient and are specific to that patient. However, clinical trials of cancer vaccines in humans have been somewhat disappointing. While general immune activation against the target antigens contained within cancer vaccines has been demonstrated in most cases, reductions in tumor burden have not been observed frequently. Tumor progression and metastasis usually occur, perhaps after only a short period of remission. The failure of cancer vaccines to fulfill their promise stems from the very relationship between the host and the tumor. Through natural selection processes, the host selectively enriches for clones of highly aggressive, neoplastically transformed cells that are apparently dedifferentiated and no longer express cancer cell-specific molecules. Specific activation of the immune system in such cases results only in the lysis of the remaining cells that express specific tumor-associated antigens (TAAs) in association with specific human leukocyte antigen (HLA) subclasses and required costimulatory molecules. However, the most dangerous clones of tumor cells lack these characteristics, making cancer vaccines rarely used.

[0017] Thus, conventional treatments for cancer have limitations and improved therapies are needed. Specifically, there is an unmet need for minimally invasive tumor control. This need is met by embodiments of the present invention, which are further described throughout the following disclosure. Summary of the Invention [Problem to be solved by the invention]

[0018] The invention described and claimed herein has many attributes and embodiments, including, but not limited to, those described or illustrated or referenced in this brief summary. The invention described and claimed herein is not limited to or by the features or embodiments identified in this summary, which are included for purposes of example only and not limitation. [Means for solving the problem]

[0019] The present invention relates to a method for treating a condition ameliorated by stimulating an immune response. In an embodiment, the method comprises injecting mesoporous silica rods (MSRs) into or near the affected tissue. In an embodiment, the MSRs induce an innate immune response. In an embodiment, the MSRs stimulate an inflammatory response.

[0020] In embodiments, the mesoporous silica rods carry a cytokine payload for injection into a tumor or infection. In aspects, the cytokine payload is time-released or gradually released (e.g., over 24 hours, 2 days, 5 days, etc.).

[0021] Thus, one embodiment is a mesoporous silica rod with a cytokine payload for injection into a tumor or infection. The cytokine payload can be interleukin-12 (IL-12) and / or interleukin-2 (IL-2). The mesoporous silica rod can also have an adjuvant (e.g., aluminum hydroxide, lipopolysaccharide, or a Toll-like receptor agonist) to stimulate the immune response. In an aspect, the mesoporous silica rod also has an immune checkpoint inhibitor (e.g., an anti-PD1 antibody) or is administered together with an immune checkpoint inhibitor.

[0022] Another embodiment is a method of treating a disease. The method can include (a) identifying diseased tissue and (b) inserting or injecting mesoporous silica rods into or near the diseased tissue. The mesoporous silica rods can carry a cytokine payload and / or an adjuvant. In an aspect, the diseased tissue is tumor tissue. In an aspect, the diseased tissue is infected tissue (e.g., bacterial, fungal, or viral infection).

[0023] Another embodiment is a method of enhancing an endogenous immune response in diseased tissue. The method can be used, for example, to convert a "cold" tumor into a "hot" tumor. The method can include (a) identifying diseased tissue (e.g., a cold tumor) and (b) inserting or injecting mesoporous silica rods into or near the diseased tissue. The mesoporous silica rods can carry a cytokine payload and / or a chemokine payload and / or an adjuvant.

[0024] Another embodiment is a method for the intertumoral delivery of cytokines to target tumor areas to induce an innate immune response via injection and implantation of mesoporous silica rods (MSR) or equivalent mesoporous silica structures or materials.

[0025] In embodiments, the methods described herein further comprise administration of an anti-PD-1 antibody (or portion of an antibody).

[0026] Another embodiment is a method for the intertumoral delivery of cytokines to targeted areas of inflammation to induce an innate immune response via injection and / or implantation of mesoporous silica rods (MSR) or equivalent mesoporous silica structures or materials.

[0027] Another embodiment is a mesoporous silica rod structure carrying a cytokine payload. The mesoporous silica rod can be cylindrical with multiple pores. Each pore can have a diameter of about 2 nanometers. In aspects, the pore diameter is about 5 nanometers, about 10 nanometers, about 15 nanometers, about 20 nanometers, about 25 nanometers, about 30 nanometers, about 35 nanometers, about 40 nanometers, or more. In aspects, the pore diameter varies (e.g., from about 2 nanometers to about 50 nanometers or more).

[0028] In embodiments, the cytokine is an interleukin (e.g., IL-2 or IL-12), or multiple interleukins. In embodiments, the mesoporous silica rods are cylindrical in shape.

[0029] In another embodiment, mesoporous silica rod structures are coated with a cytokine payload and an adjuvant (e.g., aluminum hydroxide or a TLR agonist) for injection and / or implantation into a tumor or other area of ​​inflammation within a subject.

[0030] In embodiments, the mesoporous silica rod structures deliver a physical insult to the tumor, creating a local innate immune response that leads to tumor regression.

[0031] In embodiments, the mesoporous silica rod structures deliver physical injury to the infected area (e.g., by bacteria, fungi, or viruses), creating a local innate immune response that helps fight the infection.

[0032] Other treatment areas targeted by the above embodiments include, for example, sebaceous cysts, acne vulgaris, lipomas, abscesses, or any set of aggregated cells that do not form healthy tissue structures within the human body, organism, or integumentary system of the human body or organism.

[0033] Another embodiment is a method of stimulating and inducing NETosis via injection and / or implantation of mesoporous silica rods (MSR) or equivalent mesoporous silica structures or materials.

[0034] In embodiments, MSR with IL-12 induces lymphoid aggregates and / or tertiary lymphoid structures.

[0035] In embodiments, the methods described herein result in tumor regression and / or an abscopal effect of the primary tumor. In embodiments, the methods increase survival time of a subject suffering from a disease (e.g., cancer).

[0036] Another embodiment is a method of stimulating and / or enhancing an endogenous immune response in diseased tissue, which can include (a) identifying the diseased tissue and (b) inserting a mesoporous silica rod into or near the diseased tissue.

[0037] Another embodiment is a method for converting a "cold" tumor into a "hot tumor." The method can include (a) identifying a cold tumor in tissue of a subject, and (b) inserting a mesoporous silica rod into or near the tissue.

[0038] Embodiments also include methods for producing and manufacturing mesoporous silica rod structures for modulating immune responses.

[0039] An embodiment includes a method of producing mesoporous silica rods, comprising: (a) adding poloxamer to water to form a solution; (b) mixing the solution; (c) adding an acid; (d) adding a silicon dioxide source; (e) incubating the solution; (f) sieving and vacuum filtering the solution; and (g) heating the solution to obtain mesoporous silica rods in the solution. The final solution can be sterilized. MSRs are generally stable in lyophilized form for storage / transport.

[0040] The method may also include mixing the mesoporous silica rods with granulocyte-macrophage colony-stimulating factor (GM-CSF). The method may also include mixing the mesoporous silica rods with an adjuvant. The method may also include mixing the mesoporous silica rods with cytosine guanosine dinucleotide (CpG) oligodinucleotide. The method may also include mixing the mesoporous silica rods with a cytokine. The method may also include freeze-drying the mesoporous silica rods.

[0041] Other features and advantages of embodiments of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of embodiments of the present invention. [Brief explanation of the drawings]

[0042] [Figure 1A] Figure 1 shows a melanoma mouse model, CT26 colorectal cancer model, for comparing survival times between control and treated mice.

[0043] [Figure 1B] FIG. 1 is a multivariate line graph showing the effect of interleukin-12 (IL-12) as it mediates antitumor activity in the B16F10 melanoma mouse model by stimulating T cells, natural killer (NK) cells, and NK T cells via its angiogenic effects.

[0044] [Figure 2A] 1 is a graph showing survival studies of mouse models exposed to a 3 μg bolus of IL-12, a 3 μg dose of MSR IL-12, and untreated controls.

[0045] [Figure 2B] 1 is a graph showing survival studies of mouse models exposed to a 6 μg bolus of IL-12, a 6 μg dose of MSR IL-12, and untreated controls.

[0046] [Figure 2C] 1 is a graph showing survival studies of mouse models exposed to 6 μg bolus IL-12, 6 μg dose MSR IL-12, MSR intratumoral treatment, and untreated controls.

[0047] [Figure 2D] 1 is a graph showing survival studies of mouse models exposed to 20 μg bolus IL-12, 20 μg dose MSR IL-12, MSR intratumoral treatment, and untreated controls.

[0048] [Figure 3A] FIG. 2C is a multivariate line graph showing tumor regression and abscopal effect of treated primary tumors in the combined survival study of FIGS. 2A and 2B.

[0049] [Figure 3B] FIG. 2C is a multivariate line graph showing tumor regression of untreated contralateral tumors and abscopal response in the combined survival study of FIGS. 2A and 2B.

[0050] [Figure 4A] 10 is an image of a histological sample of a 20 μg bolus injection of IL-12.

[0051] [Figure 4B] 10 shows images of histological samples of tumors treated with 20 μg of MSR IL-12.

[0052] [Figure 4C] 16 shows images of histological samples of untreated contralateral tumors in a mouse model.

[0053] [Figure 4D] Images of various histological samples obtained from a mouse model in which mesoporous silica rods (MSR) were injected into the tumor site.

[0054] [Figure 5A] 1 is a graph showing the results of a CT-26 colon cancer tumor study (mean tumor volume over time) comparing mice treated with MSR to control (PBS).

[0055] [Figure 5B] 1 is a graph showing the results (mean tumor volume over time) of a CT-26 colon cancer tumor study comparing mice treated with MSR to mice treated with IL-12 (6 μg) and MSR + IL-12 (6 μg).

[0056] [Figure 5C] 1 is a graph showing the results (mean tumor volume over time) of a CT-26 colon cancer tumor study comparing mice treated with MSR to mice treated with IL-12 (20 μg) and MSR + IL-12 (20 μg).

[0057] [Figure 6A] 1 is a graph showing the results of a CT-26 colon cancer tumor study (mean tumor volume over time) comparing mice treated with MSR to control (PBS).

[0058] [Figure 6B] 1 is a graph showing the results (mean tumor volume over time) of a CT-26 colon cancer tumor study comparing mice treated with MSR to mice treated with IL-12 (6 μg).

[0059] [Figure 6C]1 is a graph showing the results (mean tumor volume over time) of a CT-26 colon cancer tumor study comparing mice treated with MSR to mice treated with IL-12 (6 μg) and MSR + IL-12 (6 μg).

[0060] [Figure 6D] 1 is a graph showing the results (mean tumor volume over time) of a CT-26 colon cancer tumor study in which mice treated with MSR are compared to mice treated with IL-12 (20 μg).

[0061] [Figure 6E] 1 is a graph showing the results (mean tumor volume over time) of a CT-26 colon cancer tumor study comparing mice treated with MSR to mice treated with MSR + IL-12 (20 μg).

[0062] [Figure 7A] Graph showing mean tumor volume (primary) over time after treatment with (a) PBS, (b) ATT-02 it.pl, (c) ATT-02+it.pl, (d) ATT-02 pl2 flank, and (e) ATT-02+it.pl2 flank.

[0063] [Figure 7B] Graph showing mean tumor volume (secondary) over time following treatment with (a) PBS, (b) ATT-02 it.pl, (c) ATT-02+it.pl, (d) ATT-02 pl2 flank, and (e) ATT-02+it.pl2 flank.

[0064] [Figure 8A] Graph showing primary tumor growth (days post-inoculation) after treatment with PBS (control).

[0065] [Figure 8B] Graph showing primary tumor growth after treatment with ATT-02 it.pl.

[0066] [Figure 8C] Graph showing primary tumor growth after treatment with ATT-02+it.pl.

[0067] [Figure 8D] 1 is a graph showing primary tumor growth after treatment with ATT-02 pl flank.

[0068] [Figure 8E] Graph showing tumor growth after treatment with ATT-02+p12 flank.

[0069] [Figure 9A] Graph showing subsequent tumor growth (days post-inoculation) after treatment with PBS (control).

[0070] [Figure 9B] Graph showing secondary tumor growth after treatment with ATT-02 it.pl.

[0071] [Figure 9C] Graph showing secondary tumor growth after treatment with ATT-02+it.pl.

[0072] [Figure 9D] 1 is a graph showing secondary tumor growth after treatment with ATT-02 pl flank.

[0073] [Figure 9E] Graph showing secondary proliferation after treatment with ATT-02+pl2 flank.

[0074] [Figure 10A] 1 is a graph comparing IL-12 levels (pg / mL) over time (hours post treatment).

[0075] [Figure 10B] 1 is a graph comparing IFN gamma levels (pg / mL) over time (hours post treatment).

[0076] [Figure 11A] Quantification of T and B cells at day 6 post-vaccination is shown.

[0077] [Figure 11B] Quantification of T and B cells 12 days post-vaccination is shown.

[0078] [Figure 11C] Quantification of macrophages 6 days after vaccination is shown.

[0079] [Figure 11D] Quantification of monocytes and neutrophils 6 days after vaccination is shown.

[0080] [Figure 11E] Quantification of monocytes and neutrophils 12 days after vaccination is shown.

[0081] [Figure 12] IL-12 detection in mice administered a single subcutaneous injection of 1 mg MSR and 20 μg IL-12, 5 mg MSR and 20 μg IL-12 is shown.

[0082] [Figure 13A] White blood cell (WBC) counts are shown versus days post-immunization.

[0083] [Figure 13B] Lymphocyte (LYM) counts are shown versus days post-immunization.

[0084] [Figure 13C] Monocyte (MON) counts are shown versus days post-immunization.

[0085] [Figure 13D] Neutrophil (NEU) counts are shown versus days post-immunization.

[0086] [Figure 14A]The percentage of T cells in splenocytes (quantitation of T cells and B cells) 6 days after vaccination is shown.

[0087] [Figure 14B] The percentage of T cells in splenocytes (quantitation of T cells and B cells) 14 days after vaccination is shown.

[0088] [Figure 14C] The percentage of macrophages and neutrophils in splenocytes 6 days after vaccination is shown.

[0089] [Figure 14D] The percentage of macrophages and neutrophils in splenocytes 14 days after vaccination is shown.

[0090] [Figure 14E] The percentage of monocytes and neutrophils within splenocytes 6 days after vaccination is shown.

[0091] [Figure 14F] The percentage of monocytes and neutrophils within splenocytes 14 days after vaccination is shown.

[0092] [Figure 15A] Graph showing mean primary tumor growth versus time (days after tumor inoculation) in primary tumors, a single treatment of ATT-02 (1 mg MSR 20 μg IL-12) and ATT-02 cytosine guanosine dinucleotide (CpG) (1 mg MSR 20 μg IL-12).

[0093] [Figure 15B] Graph showing mean subsequent tumor growth versus time (days after tumor inoculation).

[0094] [Figure 15C] Graph showing primary tumor growth versus time (days after tumor inoculation).

[0095] [Figure 15D]Graph showing primary tumor growth versus time (days after tumor inoculation).

[0096] [Figure 15E] Graph showing subsequent tumor growth versus time (days after tumor inoculation).

[0097] [Figure 15F] Graph showing subsequent tumor growth versus time (days after tumor inoculation).

[0098] [Figure 16A] Graph showing total CD8 T cells after ATT-02 and ATT-02 CpG treatment.

[0099] [Figure 16B] Graph showing total CD8 T cells after ATT-02 and ATT-02 CpG treatment.

[0100] [Figure 16C] 1 is a graph showing effector memory T cells among CD8 T cells after ATT-02 and ATT-02 CpG treatment.

[0101] [Figure 16D] 1 shows the results of detecting IFN-γ-secreting splenocytes by ELISPOT assay.

[0102] [Figure 17A] Graph showing mean primary volume versus time (days post tumor inoculation) for control, ATT-02 it, ATT-02 pt, and ATT-02 pl treatments.

[0103] [Figure 17B] 1 is a graph showing the survival probability of mice in each group.

[0104] [Figure 17C] Graph showing tumor volume versus time (days post tumor inoculation) relative to controls.

[0105] [Figure 17D] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 it.

[0106] [Figure 17E] Graph tumor volume versus time (days post tumor inoculation) for ATT-02 pts.

[0107] [Figure 17F] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 pl.

[0108] [Figure 18A] Graph showing mean primary volume versus time (days post tumor inoculation) for control, ATT-02 it, ATT-02 pt, and ATT-02 pl treatments.

[0109] [Figure 18B] 1 is a graph showing the survival probability of mice in each group.

[0110] [Figure 18C] Graph showing tumor volume versus time (days post tumor inoculation) relative to controls.

[0111] [Figure 18D] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 it.

[0112] [Figure 18E] Graph tumor volume versus time (days post tumor inoculation) for ATT-02 pts.

[0113] [Figure 18F] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 pl.

[0114] [Figure 19A]FIG. 1 is a graph showing mean primary tumor volume versus time (days post tumor inoculation) for dual ATT-02 and ATT-02 CpG intratumoral (it) and perilymphatic (pl) treatments on both flanks of a B16F10 melanoma cancer model.

[0115] [Figure 19B] 1 is a graph showing the survival probability of mice in each group.

[0116] [Figure 19C] Graph showing tumor volume versus time (days post tumor inoculation) relative to controls.

[0117] [Figure 19D] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 itpl.

[0118] [Figure 19E] Graph tumor volume versus time (days after tumor inoculation) for ATT-02 Cpg.

[0119] [Figure 19F] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 pl (2 flanks).

[0120] [Figure 19G] Graph tumor volume versus time (days post tumor inoculation) for ATT-02 Cpg. (2 flanks).

[0121] [Figure 20] 1 is a graph showing mean tumor volume versus time (days post tumor inoculation) for dual ATT-02 and ATT-02 CpG intratumoral (it) and perilymphatic (pl) treatments on both flanks of a B16F10 melanoma cancer model.

[0122] [Figure 21A] Graph showing tumor volume versus time (days post tumor inoculation) relative to controls.

[0123] [Figure 21B] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 itpl.

[0124] [Figure 21C] Graph tumor volume versus time (days after tumor inoculation) for ATT-02 Cpg.

[0125] [Figure 21D] Graph showing tumor volume versus time (days post tumor inoculation) for ATT-02 pl (2 flanks).

[0126] [Figure 21E] Graph tumor volume versus time (days post tumor inoculation) for ATT-02 Cpg. (2 flanks).

[0127] [Figure 22A] Quantification of total splenocytes (CD8+ T cells) after MSR, ATT-02 and ATT-02 CpG treatment is shown.

[0128] [Figure 22B] Quantification of total splenocytes (CD8+ T cells, as well as KLRG-1+ and CD127+ effector memory T cells) following MSR, ATT-02, and ATT-02 CpG treatment is shown.

[0129] [Figure 22C] Quantification of CD127+ effector memory T cells after MSR, ATT-02 and ATT-02 CpG treatment is shown.

[0130] [Figure 22D] Quantification of macrophages after MSR, ATT-02 and ATT-02 CpG treatment is shown.

[0131] [Figure 22E]Quantification of monocytes after MSR, ATT-02 and ATT-02 CpG treatment is shown.

[0132] [Figure 23A] The mean tumor volume (mm3) after treatment with PBS (control) versus time (days post-inoculation) is shown.

[0133] [Figure 23B] The mean tumor volume (mm3) after treatment with IL-12 versus time (days post-inoculation) is shown.

[0134] [Figure 23C] The mean tumor volume (mm3) after treatment with PD-1 versus time (days post-inoculation) is shown.

[0135] [Figure 23D] Tumor volumes (mm3) for treatment group ATT-02 are shown.

[0136] [Figure 23E] Individual tumor volumes (mm3) for treatment groups MSR+anti-PD-1 are shown.

[0137] [Figure 23F] Tumor volumes (mm3) for treatment groups ATT-02 + anti-PD-1 are shown.

[0138] [Figure 24A] Mean tumor volumes (mm3) for all treatment groups are shown. Mice were inoculated subcutaneously with 1M CT-26 cells in the right flank on day 0. On day 7, mice began treatment with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, or ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days.

[0139] [Figure 24B] The percent change in mean body weight for the treatment groups is shown.

[0140] [Figure 25A] The survival probability of mice in each group is shown.

[0141] [Figure 25B] The mean tumor volume (mm3) of naive, ATT-02 and ATT-02+PD1 treated mice is shown.

[0142] [Figure 26A] The mean tumor volume (mm3) after treatment with PBS (control) versus time (days post-inoculation) is shown.

[0143] [Figure 26B] The mean tumor volume (mm3) after treatment with IL-12 versus time (days post-inoculation) is shown.

[0144] [Figure 26C] The mean tumor volume (mm3) after treatment with PD-1 versus time (days post-inoculation) is shown.

[0145] [Figure 26D] Tumor volumes (mm3) for treatment group ATT-02 are shown.

[0146] [Figure 26E] Individual tumor volumes (mm3) for treatment groups MSR+anti-PD-1 are shown.

[0147] [Figure 26F] Tumor volumes (mm3) for treatment groups ATT-02 + anti-PD-1 are shown.

[0148] [Figure 27A] Shown are mean tumor volumes (mm3) in mice injected with 500K B16F10 cells. Treatment groups included PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days.

[0149] [Figure 27B] The percent change in mean body weight for the treatment groups is shown.

[0150] [Figure 28] The survival probability of mice in each group is shown.

[0151] [Figure 29A] Tumor volume (mm3) versus time (days post tumor inoculation) for treatment groups is shown.

[0152] [Figure 29B] Tumor volume (mm3) versus time (days post tumor inoculation) for treatment groups is shown.

[0153] [Figure 29C] Tumor volume (mm3) versus time (days post tumor inoculation) for treatment groups is shown.

[0154] [Figure 30A] The mean tumor volume (mm3) after treatment with PBS (control) versus time (days post-inoculation) is shown.

[0155] [Figure 30B] The mean tumor volume (mm3) after treatment with IL-12 versus time (days post-inoculation) is shown.

[0156] [Figure 30C] The mean tumor volume (mm3) after treatment with PD-1 versus time (days post-inoculation) is shown.

[0157] [Figure 30D] Tumor volumes (mm3) for treatment group ATT-02 are shown.

[0158] [Figure 30E] Individual tumor volumes (mm3) for treatment groups MSR+anti-PD-1 are shown.

[0159] [Figure 30F] Tumor volumes (mm3) for treatment groups ATT-02 + anti-PD-1 are shown.

[0160] [Figure 31A] The mean tumor volume (mm3) after treatment with PBS (control) versus time (days post-inoculation) is shown.

[0161] [Figure 31B] The mean tumor volume (mm3) after treatment with IL-12 versus time (days post-inoculation) is shown.

[0162] [Figure 31C] The mean tumor volume (mm3) after treatment with PD-1 versus time (days post-inoculation) is shown.

[0163] [Figure 31D] Tumor volumes (mm3) for treatment group ATT-02 are shown.

[0164] [Figure 31E] Individual tumor volumes (mm3) for treatment groups MSR+anti-PD-1 are shown.

[0165] [Figure 31F] Tumor volumes (mm3) for treatment groups ATT-02 + anti-PD-1 are shown.

[0166] [Figure 32A] Mean tumor volume (mm3) versus time (days post-inoculation) is shown. PBS-treated mice showing primary (p) versus secondary (c) tumor volume, assessing the abscopal effect in 2 of 5 mice.

[0167] [Figure 32B] Shown are mean tumor volumes (mm) versus time (days post-inoculation) after treatment with IL-12. MSR-treated mice showed primary (p) versus secondary (c) tumor volumes, assessing the abscopal effect in 3 out of 5 mice.

[0168] [Figure 32C]Shown are mean tumor volumes (mm3) versus time (days post-inoculation) after treatment with PD-1. IL-12 treated mice showing primary (p) versus secondary (c) tumor volumes assessing the abscopal effect in 2 of 5 mice.

[0169] [Figure 32D] Shown is the mean tumor volume (mm3) versus time (days post-inoculation) after treatment with PD-1. ATT-02-treated mice showed primary (p) versus secondary (c) tumor volumes, assessing the abscopal effect in 4 of 5 mice.

[0170] [Figure 33] The survival probability of mice in each group is shown.

[0171] [Figure 34A] Tumor volumes (mm3) of treatment groups compared to IL-12 it and PBS control are shown.

[0172] [Figure 34B] Tumor volume (mm3) of PBS control compared to ATT-02 treatment is shown.

[0173] [Figure 34C] Tumor volume (mm3) of treated ATT-02 compared to IL-12 is shown.

[0174] [Figure 34D] The percent change in mouse body weight over the course of the study is shown.

[0175] [Figure 35] The mean tumor volume (mm3) of the treatment groups versus time (days after tumor inoculation) is shown.

[0176] [Figure 36] The survival probability of mice in each group is shown.

[0177] [Figure 37A]The mean primary tumor burden volume (mm3) of treatment groups in single-dose mice is shown.

[0178] [Figure 37B] Primary right flank tumor volume (mm3) of PBS control is shown.

[0179] [Figure 37C] Primary right flank tumor volume (mm3) in ATT-02 low treatment is shown.

[0180] [Figure 37D] Primary right flank tumor volume (mm3) in ATT-02 high treatment group is shown.

[0181] [Figure 37E] The mean secondary tumor burden volume (mm3) of treatment groups in single-dose mice is shown.

[0182] [Figure 37F] Secondary left flank tumor volume (mm3) of PBS controls is shown.

[0183] [Figure 37G] Figure 1 shows the secondary left flank tumor volume (mm3) of ATT-02 low treatment.

[0184] [Figure 37H] Figure 1 shows secondary left flank tumor volume (mm3) of ATT-02 high treatment.

[0185] [Figure 38A] The mean primary tumor burden volume (mm3) of treatment groups in single-dose mice is shown.

[0186] [Figure 38B] Primary right flank tumor volume (mm3) of PBS control multiple doses is shown.

[0187] [Figure 38C] Primary right flank tumor volume (mm3) of ATT-02 low-treatment multiple-administration patients is shown.

[0188] [Figure 38D] Primary right flank tumor volume (mm3) of ATT-02 high-treatment multiple doses is shown.

[0189] [Figure 38E] The mean secondary tumor burden volume (mm3) of treatment groups in single-dose mice and multiple-dose mice is shown.

[0190] [Figure 38F] Figure 1 shows the subsequent left flank tumor volume (mm3) of ATT-02 high multiple dose treatment.

[0191] [Figure 39] The survival probability of mice in each group is shown.

[0192] [Figure 40A] AT055 Att-02 therapeutic efficacy after a single dose in the B16F10 model (day 7, total CD8+ T cells, KLRG1 and CD127+ cells).

[0193] [Figure 40B] AT055 Att-02 therapeutic efficacy after a single dose in the B16F10 model (day 7, total CD8+ T cells, KLRG1 and CD127+ cells).

[0194] [Figure 40C] AT055 Att-02 therapeutic efficacy after a single dose in the B16F10 model (day 14, total CD8+ T cells, KLRG1 and CD127+ cells).

[0195] [Figure 40D] AT055 Att-02 therapeutic efficacy after a single dose in the B16F10 model (day 14, total CD8+ T cells, KLRG1 and CD127+ cells).

[0196] [Figure 41]The mean primary tumor burden volume (mm3) of the treatment group (either perilymphatic (pl) or intratumoral (it) with PBS or ATT-02 + / - CpG) is shown.

[0197] [Figure 42A] The mean primary tumor burden volume (mm3) of the treatment groups and PBS control is shown.

[0198] [Figure 42B] Tumor volume (mm3) for MSR perilymphatic treatment is shown.

[0199] [Figure 42C] Tumor volume (mm3) of ATT-02 it treatment is shown.

[0200] [Figure 42D] Treatment group ATT-02 shows perilymphatic tumor volume (mm3)

[0201] [Figure 42E] Tumor volume (mm3) for treatment group ATT-02+CpG it is shown.

[0202] [Figure 42F] The tumor volume (mm3) around lymph nodes in the treatment group ATT-02+CpG is shown.

[0203] [Figure 43] The mean secondary tumor burden volume (mm3) of the treatment groups is shown.

[0204] [Figure 44A] The mean primary tumor burden volume (mm3) of the treatment groups and PBS control is shown.

[0205] [Figure 44B] Tumor volume (mm3) for MSR perilymphatic treatment is shown.

[0206] [Figure 44C] Tumor volume (mm3) of ATT-02 it treatment is shown.

[0207] [Figure 44D] Treatment group ATT-02 shows perilymphatic tumor volume (mm3)

[0208] [Figure 44E] Tumor volume (mm3) for treatment group ATT-02+CpG it is shown.

[0209] [Figure 44F] The tumor volume (mm3) around lymph nodes in the treatment group ATT-02+CpG is shown.

[0210] [Figure 45] The survival probability of mice in each group is shown.

[0211] [Figure 46A] Total CD8+ T cell population (AT063:CD8+ T cell population after treatment) is shown.

[0212] [Figure 46B] It is a CD8+TCF-1+ population.

[0213] [Figure 46C] It is the CD8+KLRG1+ population.

[0214] [Figure 46D] It is the CD8+CD127+ population.

[0215] [Figure 46E] It is the CD8+CD44+ population.

[0216] [Figure 47A] The total CD11b+GR-1+ population (AT063:CD11b+ population after treatment) is shown.

[0217] [Figure 47B] The CD11b+CD86+ population is shown.

[0218] [Figure 47C] The CD11b+MHCII+ population is shown.

[0219] [Figure 48A] The total CD11c+GR-1+ population is shown.

[0220] [Figure 48B] The CD11c+CD86+ population is shown.

[0221] [Figure 48C] The CD11c+MHCII+ population is shown.

[0222] [Figure 49] IFN-gamma expression by treatment group is shown (AT063 Att-02 therapeutic effect after a single dose in the B16F10 model).

[0223] [Figure 50] This paper outlines the fabrication of mesoporous silica rods. A symmetric triblock copolymer (Pluronic P123) composed of poly(ethylene oxide) and poly(propylene oxide) is used to create rod-shaped micelles in solution. When tetraethyl orthosilicate (TEO) is added to the solution, silica is deposited on the micelles, creating a hexagonal pore structure. The Pluronic P123 is then rinsed and calcined (high-temperature treatment) to remove the polymer, leaving behind a silica mesoporous structure.

[0224] [Figure 51] 1 is a process flow chart for synthesizing mesoporous silica rods (MSR).

[0225] [Figure 52A] Representative SEM sizing image of standard MSR width.

[0226] [Figure 52B] Representative SEM sizing images of standard MSR lengths.

[0227] [Figure 53] 1 is a process flow chart of a proposed manufacturing process for mesoporous silica rods (MSR).

[0228] [Figure 54A] Representative SEM sizing images of MSR length and MSR width are shown. [Figure 54B] Representative SEM sizing images of MSR length and MSR width are shown. [Figure 54C] Representative SEM sizing images of MSR length and MSR width are shown. [Figure 54D] Representative SEM sizing images of MSR length and width are shown. Modified MSR length (Figure 54A) and width (Figure 54B). Standard MSR length (Figure 54C) and width (Figure 54D). D[n,0.1] (μm), D[n,0.5] (μm), and D[n,0.9] (μm) = biodistribution of MSRs sized below 10%, below 50%, and below 90%, respectively, within the total number average.

[0229] [Figure 55] 1 shows the IL-12 in situ vaccine paradigm.

[0230] [Figure 56A] The mean primary tumor burden volume (mm3) versus time (days after tumor inoculation) for the different treatment groups is shown (P=0.01, IL-12 vs. ATT-02, 17 days).

[0231] [Figure 56B] The survival probability of mice in each group is shown (P=0.01, IL-12 vs. ATT-02, log-rank).

[0232] [Figure 56C] The mean primary tumor burden volume (mm3) versus time (days after tumor inoculation) for the different treatment groups is shown.

[0233] [Figure 56D] The survival probability of mice in each group is shown (P=0.02, IL-12 vs. ATT-02, log-rank).

[0234] [Figure 56E] The mean primary tumor burden volume (mm3) versus time (days after tumor inoculation) for the different treatment groups is shown (P=0.01, IL-12 vs. ATT-02, 20 days).

[0235] [Figure 56F] The survival probability of mice in each group is shown.

[0236] [Figure 57A] The mean primary tumor burden volume (mm3) versus time (days after tumor inoculation) for the different treatment groups is shown (P<0.01, PBS vs. ATT-02 low, 21 days; P<0.01, PBS vs. ATT-02 high, 21 days).

[0237] [Figure 57B] The mean secondary tumor burden volume (mm3) versus time (days after tumor inoculation) for the different treatment groups is shown (P<0.01, PBS vs. ATT-02 low, 21 days; P<0.01, PBS vs. ATT-02 high, 21 days).

[0238] [Figure 57C] The survival probability of mice in each group is shown (ATT-02 low dose 1x vs. ATT-02 3x, p=0.002, log-rank).

[0239] [Figure 57D] Fold change in gene expression compared to untreated controls is shown (primary / untreated).

[0240] [Figure 57E] Fold change in gene expression compared to untreated controls is shown (secondary / treated).

[0241] definition References herein to "one embodiment / aspect" or "embodiment / aspect" mean that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the present disclosure. Use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places herein does not necessarily all refer to the same embodiment / aspect, nor does it refer to separate or alternative embodiments / aspects that are mutually exclusive of other embodiments / aspects. Furthermore, various features are described that may be exhibited by some embodiments / aspects but not other embodiments / aspects. Similarly, various requirements are described that may be requirements of some embodiments / aspects but not other embodiments / aspects. Embodiments and aspects may be used interchangeably in some cases.

[0242] The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and within the specific context in which each term is used. Certain terms used to describe this disclosure are explained below or elsewhere herein to provide additional guidance to the practitioner regarding the description of this disclosure. It is understood that the same thing can be said in more than one way.

[0243] Thus, alternative language and synonyms may be used for any one or more of the terms described herein. Also, no particular significance is attached to whether a term is recited or explained herein. Synonyms for particular terms are provided. The recitation of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term described herein, is illustrative only and is not intended to further limit the scope and meaning of the disclosure or any exemplified term. Similarly, the disclosure is not limited to the various embodiments provided herein.

[0244] Without intending to further limit the scope of the present disclosure, examples of devices, apparatuses, methods and their related results according to embodiments of the present disclosure are provided below. Please note that in the examples, titles or subtitles may be used for the convenience of the reader and are not intended to limit the scope of the present disclosure in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification, including definitions, shall prevail.

[0245] Where applicable, the terms "about" or "generally" as used herein in the specification and appended claims mean a margin of + / - 20% unless otherwise specified. Also, where applicable, the term "substantially" as used herein in the specification and appended claims means a margin of + / - 10% unless otherwise specified. It is understood that not all uses of the above terms are quantifiable so that the referenced ranges can be applied.

[0246] The term "mesoporous" generally refers to materials having pores between 1 and 50 nanometers (nm) in size.

[0247] The term "mesoporous silica" refers to a form of silica characterized by its mesoporous structure (i.e., having pores with diameters ranging from 1 nm to 50 nm). Mesoporous silica is a relatively recent development in nanotechnology. The most common types of mesoporous nanoparticles are MCM-41 and SBA-15. Research continues for these particles, which have applications in catalysis, drug delivery, and imaging. Mesoporous ordered silica films with a variety of pore morphologies have also been obtained.

[0248] The term "mesoporous silica rod" or "MSR" refers to nanoparticles composed of mesoporous silica in a substantially rod-like shape (i.e., a linear, substantially cylindrical structure longer than it is wide). MSRs have also been used in sensing and interparticle communication protocols. Their large surface area and high loading capacity make them suitable for the delivery of various drugs, antibodies, genes, proteins, and peptides. MSRs can act as vaccine adjuvants; vaccine vehicles for the delivery of cancer antigens and pathogen-associated molecular patterns to APCs; tools for T cell priming, expansion, trafficking, infiltration, and recognition of cancer cells; agents that trigger the release of tumor antigens through photodynamic and photothermal therapy; chemotherapeutic agents that directly kill malignant cells or inhibit immune checkpoints; agents for starvation therapy, or a combination thereof.

[0249] The term "MCM-41" or "Mobil Composition of Matter No. 41" refers to a mesoporous material with a hierarchical structure from the family of silicate and alumosilicate solids. MCM-41 consists of a regular arrangement of cylindrical mesopores forming a one-dimensional pore system. MCM-41 is characterized by independently adjustable pore diameters, a sharp pore distribution, a large surface, and a large pore volume. The pores are larger than those in zeolites, and the pore distribution can be easily adjusted. The mesopores have diameters ranging from 2 nm to 6.5 nm.

[0250] The term "SBA-15" or "Santa Barbara Amorphous-15" refers to a stable mesoporous silica sieve with high hydrothermal and mechanical stability resulting from a uniform hexagonal pore framework characterized by a narrow pore size distribution and tunable pore diameter (i.e., 5 nm to 15 nm). Most importantly, it possesses relatively thick walls ranging from 3.1 nm to 6.4 nm. SBA mesoporous silica 15 has a high internal surface area, making it suitable for a variety of applications, including environmental adsorption and separation, advanced optics, and catalysis.

[0251] The term "active agent" or "active ingredient" refers to a substance, compound, or molecule that is biologically active or otherwise induces a biological or physiological effect in a subject to which it is administered. In other words, "active agent" or "active ingredient" refers to the component or components of a composition to which all or part of the composition's effect is attributed. An active agent can be a primary active agent, or in other words, the component of a composition to which all or part of the composition's effect is attributed. An active agent can be a secondary agent, or in other words, the component of a composition to which additional portions and / or other effects of the composition are attributed.

[0252] A "pharmaceutical composition" can comprise a combination of an active agent, such as a therapeutic peptide, with an inert or active carrier in a sterile composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0253] As used herein, the term "prevention" refers to any action whereby the onset of a disease is inhibited or delayed.

[0254] The term "treating" or "treatment" refers to one or more of: (1) inhibiting the disease (i.e., preventing further progression of pathology and / or symptomology), and (2) ameliorating the disease (i.e., reversing the pathology and / or symptomology), e.g., reducing the severity of the disease.

[0255] The term "administration" refers to the introduction of a certain amount of a given substance into a patient by a specific suitable method. The compositions disclosed herein can be administered via any common route, such as, but not limited to, inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration, as long as the desired tissue can be reached.

[0256] The term "inflammation" refers to part of the complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, or irritants. It is a defensive response involving immune cells, vascular, and molecular mediators. The function of inflammation is to eliminate the initial cause of cellular injury, remove necrotic cells and damaged tissue from the original injury and inflammatory process, and initiate tissue repair. Five major symptoms are fever, pain, redness, swelling, and loss of function. Because inflammation is a general response, it is considered a mechanism of innate immunity compared to adaptive immunity, which is specific to each pathogen. Too little inflammation can lead to progressive tissue destruction by harmful stimuli (e.g., bacteria) and impair the survival of the organism. In contrast, excessive inflammation in the form of chronic inflammation is associated with various diseases such as hay fever, periodontal disease, atherosclerosis, and osteoarthritis. Inflammation can be classified as either acute or chronic. Acute inflammation is the body's initial response to harmful stimuli and is achieved by increased migration of plasma and leukocytes (especially granulocytes) from the blood to damaged tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vasculature, the immune system, and various cells within the injured tissue. Persistent inflammation, known as chronic inflammation, results in the progressive migration of cell types present at the site of inflammation, e.g., mononuclear cells, and is characterized by the simultaneous destruction and healing of tissue from the inflammatory process.

[0257] The term "inflammatory disorder" or "inflammatory condition" refers to a condition in which the immune system mistakenly attacks the body's own cells or tissues. This causes abnormal inflammation, which can result in chronic pain, redness, swelling, stiffness, and damage to other healthy body tissues. Inflammatory conditions can affect the nervous system (e.g., encephalitis, myelitis, meningitis, arachnoiditis, and neuritis). Inflammatory conditions can affect the eyes (e.g., dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, and uveitis). Inflammatory conditions can affect the ear (e.g., otitis externa, otitis media, labyrinthitis, and mastoiditis). Inflammatory conditions can affect the cardiovascular system (e.g., endocarditis, myocarditis, pericarditis, arteritis, phlebitis, and capillaritis). Inflammatory conditions can affect the respiratory system (e.g., sinusitis, rhinopharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonitis, pleuritis, and mediastinitis). Inflammatory conditions can affect the mouth and digestive system (e.g., stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis / parotitis, cheilitis, pulpitis, mandibular inflammation, esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, Inflammatory conditions can affect the digestive appendages (e.g., hepatitis, ascending cholangitis, cholecystitis, pancreatitis, and peritonitis). Inflammatory conditions can affect the integumentary system (e.g., dermatitis, folliculitis, cellulitis, and hidradenitis). Inflammatory conditions can affect the musculoskeletal system (e.g., arthritis, dermatomyositis, myositis, synovitis / tendonitis, bursitis). Inflammatory conditions can affect the urinary system (e.g., nephritis, glomerulonephritis, pyelonephritis, ureteritis, cystitis, and urethritis). Inflammatory conditions can affect the female reproductive system (e.g., oophoritis, salpingitis, endometritis, parametritis, cervicitis, vaginitis, vulvitis, and mastitis). Inflammatory conditions can affect the male reproductive system (e.g., orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, preputitis, and balanoposthitis). Inflammatory conditions can affect the endocrine system (e.g., insulitis, hypophysitis, thyroiditis, parathyroiditis, and adrenal inflammation). Inflammatory conditions can also affect the lymphatic system (e.g., lymphangitis and lymphadenitis).

[0258] The term "autoimmune disease" or "autoimmune disorder" refers to a condition resulting from an abnormal immune response against a functioning body part. Common autoimmune diseases include Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, and pernicious anemia.

[0259] The term "immunotherapy" refers to the treatment of a subject afflicted with a disease or at risk of developing a disease or at risk of developing a recurrence of a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0260] The term "enhancing an endogenous immune response" refers to increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0261] The term "neoplasm" refers to a disease caused by or resulting in an inappropriately high level of cell division, an inappropriately low level of apoptosis, or both. For example, cancer is an example of a neoplasm. Examples of cancer include leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma). otheliosarcoma), synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Lymphoproliferative disorders are also considered to be proliferative diseases.

[0262] The term "cancer" refers to human cancers and carcinomas, including solid tumors, renal cancer, breast cancer, lung cancer, kidney cancer, bladder cancer, urinary tract cancer, urethral cancer, penile cancer, vulvar cancer, vaginal cancer, cervical cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, and liver cancer, sarcomas, adenocarcinomas, etc. In any of the above embodiments, one or more cancer therapies, such as chemotherapy, radiation therapy, immunotherapy, surgery, or hormonal therapy, may be further co-administered with the methods described herein.

[0263] The term "abscopal effect" refers to the hypothesis in the treatment of metastatic cancer that shrinkage of untreated tumors occurs simultaneously with shrinkage of tumors within the area of ​​local treatment. In the abscopal effect, the immune system is thought to be stimulated to fight cancer throughout the body as a result of local therapy.

[0264] The term "infectious disease" refers to bacterial, protozoan, and viral pathogens that infect and cause disease in humans. Viral pathogens include, for example, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and herpes viruses. Bacterial and protozoan pathogens can include Escherichia coli (E. coli), Staphylococcus species, Streptococcus species, Mycobacterium tuberculosis, Giardia, Malaria, Leishmania, and Pseudomonas aeruginosa. Infectious pathogens can be capable of establishing chronic infections (e.g., persistent or persistent).

[0265] The term "skin and soft tissue infection" or "SSTI" encompasses any type of microorganism (i.e., bacteria, virus, or fungus) that can enter any break in the skin and invade the subcutaneous tissue (soft tissue under the skin), fascia (connective tissue), and muscle.

[0266] The term "cytokine" or "cytokines" generally refers to any of a variety of small regulatory proteins that regulate cells of the immune system. Examples include substances such as interferons, interleukins, and growth factors, which are secreted by certain cells of the immune system and affect other cells.

[0267] The term "chemokine" refers to any of a class of cytokines whose functions include attracting leukocytes to sites of infection. Chemokines are a family of small cytokines or signaling proteins secreted by cells that induce the directional movement of leukocytes and other cell types, including endothelial and epithelial cells. In addition to playing a key role in activating the host immune response, chemokines are important for biological processes including morphogenesis and wound healing, as well as in the pathogenesis of diseases such as cancer.

[0268] The term "intratumorally" generally refers to "within a tumor."

[0269] The term "intertumorally" generally refers to "between tumors."

[0270] The term "physical injury" generally refers to the effect or cause of any kind of injury, disturbance or destruction on the body of an organism, including, but not limited to, any effect or cause of any kind of injury, disturbance or destruction on the tissue of an organism.

[0271] The term "inflammasome" generally refers to a multiprotein complex that contributes to inflammatory rheumatic diseases through the activation of caspases.

[0272] The term "pyroptosis" generally refers to a form of programmed cell death associated with the antimicrobial response during inflammation.

[0273] The term "immunogenic cell death" generally refers to any type of cell death that elicits an immune response.

[0274] The term "neutrophil" refers to a type of white blood cell, also known as a neutrocyte or pseudoeosinophil, that forms an essential part of the innate immune system.

[0275] The term "macrophage" refers to a white blood cell that phagocytose necrotic cellular debris and foreign material, including viruses, bacteria, and tattoo ink.

[0276] The term "dendritic cell" refers to any cell that has branching processes and forms part of the mammalian immune system.

[0277] The term "interleukin / IL" refers generally to any of a group of cytokine proteins important in the regulation of lymphocyte function.

[0278] The term "interleukin-12 / IL-12" generally refers to a potent anti-tumor cytokine that belongs to a family of cytokine proteins.

[0279] The term "bolus injection" generally refers to a single dose of a drug or other pharmaceutical preparation that is administered all at once.

[0280] The term "adjuvant" generally refers to a substance that increases or modulates the immune response to a vaccine. Adjuvants can help generate a stronger immune response in people receiving the vaccine. Common adjuvants include aluminum, AS01B, AS04, CpG 1018, MatrixM™, and MF59.

[0281] The terms "adjuvant therapy" or "neoadjuvant therapy" refer to the use of an adjuvant in combination with a primary treatment (e.g., surgery or radiation) to reduce the chance of cancer recurrence. It is often used to make the primary treatment more effective.

[0282] The term "NLRP3 inflammasome" refers to a key component of the innate immune system that mediates caspase-1 activation and secretion of the pro-inflammatory cytokines IL-1β / IL-18 in response to microbial infection and cellular injury. However, aberrant activation of the NLRP3 inflammasome has been associated with several inflammatory disorders, including cryopyrin-associated periodic syndrome, Alzheimer's disease, diabetes, and atherosclerosis. The NLRP3 inflammasome is activated by diverse stimuli and multiple molecular and cellular events, including ion flux, mitochondrial dysfunction, and production of reactive oxygen species. Lysosomal injury has been shown to trigger its activation.

[0283] The terms "CCL2," "chemokine (C-C motif) ligand 2," "monocyte chemoattractant protein 1 (MCP1)," or "small inducible cytokine A2" refer to a small cytokine belonging to the C-C chemokine family. CCL2 recruits monocytes, memory T cells, and dendritic cells to sites of inflammation caused by either tissue injury or infection. CCL2 is involved in the pathogenesis of several diseases characterized by monocyte infiltration, such as psoriasis, rheumatoid arthritis, and atherosclerosis.

[0284] The terms "PD-1," "programmed cell death protein 1," or "CD279" refer to a cell surface protein that regulates the immune system's response to the body's cells by downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. Engagement of PD-1 by either of its ligands, PD-L1 or PD-L2, on neighboring cells inhibits TCR signaling and TCR-mediated proliferation, transcriptional activation, and cytokine production. This prevents autoimmune disease but may also prevent the immune system from killing cancer cells. Therapeutic antibodies designed to block the PD-1 / PD-L1 interaction have potential for the treatment of cancer. PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells to regulate activation or inhibition. Several inhibitors of programmed cell death-1 (PD-1) and programmed death-ligand-1 (PD-L1) have been approved as a form of immunotherapy for several cancers. Examples include pembrolizumab (Keytruda), nivolumab (Opdivo), and cemiplimab (Libtayo).

[0285] As used herein, the term "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen via one or more immunoglobulin variable regions. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody is most critical for binding specificity and affinity and is encoded by the variable domain. Antibodies can be whole antibodies, antigen-binding fragments, or single chains thereof.

[0286] The term "agonist antibody" refers to an antibody that stimulates or activates an organ. Antibodies act as receptor agonists, essentially replacing the activity of the normal ligand. Agonist activity can occur when an antibody binds to a receptor in a manner that mimics the binding of a physiological ligand, resulting in antibody-mediated agonism. For example, in Graves' disease, agonist antibodies against the thyrotropin receptor stimulate the thyroid gland to release thyroid hormone, causing hyperthyroidism. Agonist antibodies can also stimulate through the Fc portion of the antibody, engaging Fc receptors in trans or cis upon clustering, or through antigen-mediated clustering. The latter clustering mechanism requires antigen engagement by one half of the bispecific molecule and engagement of the stimulatory receptor by the second half of the bispecific molecule. Exemplary stimulatory receptors are CD3, CD28, and 4-1BB, which stimulate T cells.

[0287] The term "antibody fragment" or "antigen-binding fragment" is used in reference to a portion of an antibody, e.g., Fab', Fab, Fv, scFv, etc. An antibody fragment, regardless of structure, binds with the same antigen that is recognized by the intact antibody. The term "antibody fragment" also includes diabodies and any synthetic or genetically engineered protein containing an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex.

[0288] The term "immunogenicity" refers to the ability of a cell / tissue to elicit an immune response, which is generally considered to be an undesirable physiological response.

[0289] The term "immunogenic tumor" refers to a tumor with sufficient antigen and priming to induce a good T cell response in tumor-draining lymph nodes.In contrast, tumors with low immunogenicity cannot generate a T cell response.The ability of a tumor to respond to T cell control is not necessarily related to its ability to prime a T cell response.

[0290] The terms "immunogenic cell death," "ICD," or "immunogenic apoptosis" refer to a form of cell death that results in the regulated activation of the immune response. This cell death is characterized by an apoptotic form that maintains membrane integrity. Endoplasmic reticulum (ER) stress, which is often accompanied by the production of reactive oxygen species (ROS), is generally recognized as a causative agent of ICD. Two groups of ICD inducers have been recognized: type I inducers cause stress in the ER only as collateral damage, primarily targeting DNA or the chromatin maintenance apparatus or membrane components; type II inducers specifically target the ER. ICD can be induced by several cytostatic drugs, such as anthracyclines, oxaliplatin, and bortezomib, or by radiation therapy and photodynamic therapy (PDT). Several viruses can be listed among the biological causes of ICD. Just as immunogenic death of infected cells induces an immune response against infectious agents, immunogenic death of cancer cells can induce effective antitumor immune responses through the activation of dendritic cells (DCs) and the consequent activation of specific T cell responses. This effect can be used in antitumor therapy.

[0291] The term "immune checkpoint" or "checkpoint" refers to a regulator of the immune system. Immune checkpoints are important for self-tolerance, which prevents the immune system from indiscriminately attacking cells. However, some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint inhibitor therapy is a form of cancer immunotherapy. This therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can attenuate the immune response to immunological stimuli. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints and restore immune system function.

[0292] Tumor types can be categorized into distinct groups based on their response rates to immune checkpoint inhibitors (ICIs). In practice, tumor types are classified as either "hot" or "cold," a distinction that may be more clearly defined in the medical field. Hot tumors have a high mutational load and respond more quickly to ICIs. They accumulate many mutations, which causes tumor cells to produce specific molecules, or neoantigens, on their cell surface. These neoantigens make tumors more susceptible to recognition by the immune system, making them more likely to elicit a strong immune response. Malignant tumors considered "hot" include bladder, head and neck cancer, kidney cancer, liver cancer, melanoma, and non-small cell lung cancer, as well as various tumor types with high rates of microsatellite instability. Immune checkpoint inhibitors are effective against these tumor types. Cold tumors have low response rates and are often compared to impregnable fortresses surrounded by moats. Their "walls" contain few T cells, making it difficult for them to mobilize an immune response. Common cancers with "cold" tumors include glioblastoma, ovarian cancer, prostate cancer, and pancreatic cancer.

[0293] The term "administration" refers to the introduction of a certain amount of a predetermined substance into a patient by a specific suitable method. The compositions disclosed herein can be administered via any common route, such as, but not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration, as long as the desired tissue can be reached.

[0294] The term "subject" or "patient" refers to any single animal, more preferably a mammal (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human animals such as non-human primates), for which treatment is desired. Most preferably, the patient herein is a human.

[0295] Any numerical values, including ranges, such as pH, temperature, time, concentration and molecular weight, should be understood as approximate values ​​according to common practice in the art.As used herein, the term "about" can include a variation of (+) or (-) 1%, 5% or 10% of the described amount, depending on the context.Although not always explicitly stated, it should be understood that the reagents described herein are merely exemplary, and equivalents of such reagents are known in the art.

[0296] Many known useful compounds and the like can be found in Remington's Pharmaceutical Sciences (13th Ed), Mack Publishing Company, Easton, PA, a standard reference for various types of administration. As used herein, the term "formulation" means a combination of at least one active ingredient with one or more other ingredients, generally also called excipients, which may be independently active or inactive. The term "formulation" may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals, and may include compositions that are useful intermediates for storage or research purposes.

[0297] Other technical terms used herein have their ordinary meaning in the art in which they are used, as exemplified by various technical dictionaries. The specific values ​​and configurations described in these non-limiting examples may vary and are cited only to illustrate at least one embodiment and are not intended to limit its scope. DETAILED DESCRIPTION OF THE INVENTION

[0298] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the claimed subject technology. Additional features and advantages of the subject technology will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the description and claims.

[0299] Immunotherapy refers to a type of cancer treatment that uses substances made by the body to strengthen the immune system and help the body find and destroy cancer cells. Immunotherapy has emerged in recent years as a powerful and useful approach for cancer treatment. Some of its advantages over traditional cancer treatments (i.e., chemotherapy, radiation therapy, and surgery) are significant improvements in patients' quality of life and survival chances. Immunotherapy can be used to treat many different types of cancer and can be used in combination with chemotherapy and / or other cancer treatments.

[0300] Thus, the present invention is based on the surprising discovery that injection of mesoporous silica rods (MSRs) stimulates immune activity to high levels within target tissues. MSRs can trigger the activation of the cancer-immune cycle while avoiding harming healthy cells. Therefore, MSRs can be used as a scaffold for intratumoral delivery of cytokines. In embodiments, the methods described herein are suitable for treating conditions known or predicted to be improved by immune stimulation (e.g., cancer or infectious diseases).

[0301] Mesoporous Silica Rods (MSR) Mesoporous silica is a form of silica characterized by its mesoporous structure (i.e., having pores with diameters ranging from 1 nm to 50 nm). Mesoporousness is generally defined as between microporous (i.e., <2 nm) and macroporous (i.e., >50 nm). Mesoporous silica is a relatively recent development in nanotechnology. Ordered mesoporous silica films with a variety of pore morphologies have also been obtained.

[0302] Mesoporous silica rods (MSRs) can be injected into patients rather than surgically implanted like other scaffolds. MSRs can directly assemble into a 3D microenvironment for dendritic cells in the body. In some embodiments, long, rod-shaped microparticles are used that are several orders of magnitude larger than the size of a single immune cell. In embodiments, these microparticles are injected into tissues (e.g., using a standard 23-gauge syringe). Due to their size, the microparticles do not diffuse from the injection site.

[0303] The large surface area of ​​the pores allows the particles to be loaded with drugs or cytotoxins. Like Trojan horses, the particles can be detected via receptors or taken up by specific biological cells via endocytosis, depending on which chemicals are attached to the outside of the particle.

[0304] Mesoporous silica can also enhance the in vitro and in vivo dissolution of poorly water-soluble drugs. Many drug candidates derived from drug discovery have poor water solubility. Such drugs can be administered with MSRs, providing a promising drug delivery mechanism.

[0305] cytokines Cytokines are a broad and broad category of small proteins (e.g., 5–25 kDa) important in cell signaling. Cytokines are also peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators.

[0306] Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although there is some overlap in the terms). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by multiple cell types. Cytokines act through cell surface receptors and are particularly important in the immune system. Cytokines regulate the balance between humoral and cellular immune responses and modulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways. Cytokines are important in the host immune response in health and disease, particularly to infection, inflammation, trauma, sepsis, cancer, and reproduction.

[0307] Interleukin-12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigenic stimulation. IL-12 belongs to the interleukin-12 family. The IL-12 family is unique in that it contains only one heterodimeric cytokine: IL-12, IL-23, IL-27, and IL-35. IL-12 also possesses antiangiogenic activity (i.e., it can block the formation of new blood vessels) and can therefore be used as an anti-neoplastic agent. Despite sharing many structural features and molecular partners, IL-12 mediates a surprisingly diverse range of functional effects.

[0308] Interleukin-2 (IL-2) is an interleukin, a type of cytokine signaling molecule in the immune system. IL-2 regulates the activity of white blood cells (leukocytes, often lymphocytes) involved in immunity. IL-2 is part of the body's natural response to microbial infection and in distinguishing between foreign ("non-self") and "self." IL-2 mediates its effects by binding to the IL-2 receptor expressed by lymphocytes. The primary source of IL-2 is activated CD4+ and CD8+ T cells.

[0309] Applicants have discovered surprising results utilizing configured mesoporous silica rods (MSR) as scaffolds for intratumoral delivery of cytokines. The combination of MSR and cytokines modulates immune cell populations and activation states in a manner that can alter the tumor microenvironment, resulting in a more effective response by the immune system and improved survival in cancer studies.

[0310] Without being bound by theory, the applicants propose that MSRs may trigger multiple pathways in the immune system. MSRs can increase the probability of high endothelial venule (HEV) formation within the tumor microenvironment. HEV detection within tumors has been associated with better responses to immune checkpoint inhibitors in clinical trials. MSRs can also recruit neutrophils, lymphocytes, dendritic cells, and macrophages (including foam cells) to the injection site. The present invention helps to shift the therapeutic window of cytokines such as IL-2 and IL-12 by locally exposing tumors and inflamed areas to high concentrations in combination with inflammatory signals induced by implanted and / or injected silica materials, thereby reducing cytokine toxicity through controlled and localized cytokine release. In embodiments, the agent (e.g., cytokine) and MSR act synergistically with each other.

[0311] Treatment of a subject with mesoporous silica rods can trigger an enhanced immune response through physical injury. Through this mechanism, large populations of immune cells are recruited to the target area of ​​treatment, influencing and activating innate immune pathways. The controlled chemistry and composition of MSR and other suitable silica materials activate specific immune pathways in combination with cytokines, IL-12, IL-2, inflammasomes, and other regulatory proteins. Immune cells organize around the MSR.

[0312] The methods described herein offer several advantages over conventional treatments for cancer / inflammation. The MSR delivery system can be modified or configured as follows: a) release different cargoes (e.g., cytokines) at different rates; b) Utilizing the high surface area of ​​MSRs to effectively deliver large cargoes; c) increasing the half-life of the target cytokine; d) controlling the exposure and concentration of cytokine release; e) Retaining cargoes and releasing them safely over time (e.g., from days to months). One or more additional active substances can be included on the MSR. In embodiments, an adjuvant can be included on the MSR. In embodiments, the MSR described herein can target specific tissues, such as tumors. The MSR can also be used to treat diseases associated with infection and / or inflammation.

[0313] The treatments described herein allow for the creation of an alternative immunological environment within the tumor and / or inflammation site that creates a response that improves tumor and inflammation regression. The material composition of the mesoporous silica structure further enables immunogenic cell death (ICD), which creates an antigen source for further signaling immune cell proliferation, thus concentrating agents such as neutrophils, macrophages, and dendritic cells in the target treatment area for favorable outcomes such as tumor suppression, tumor regression, tumor size reduction, tumor elimination, inflammation reduction, inflammation suppression, inflammation regression, and elimination of inflammation within the target area.

[0314] Although the examples below describe the use of interleukin-12 (IL-12) and / or interleukin-2 (IL-2), the present invention can be used with other cytokines, including IL-1, IL-2, IL-3, GM-CSF, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-28, TNF-alpha, IL-23, IL-16, IL-17, TNF-α, TNF-β, interferons, IL-10, IL-19, IL-20, IL-22, and IL-24.

[0315] Adjuvants Adjuvants can be defined as vaccine components that enhance the magnitude, breadth, and / or durability of immune responses. Due to the various mechanisms and connections between innate and adaptive immune responses, adjuvants enhance innate immune responses, resulting in enhanced adaptive immune responses. Specifically, adjuvants can exert their immune-enhancing effects according to five immune functional activities:

[0316] First, adjuvants can aid in the translocation of antigens to lymph nodes where they can be recognized by T cells. This ultimately leads to greater T cell activity and enhanced pathogen clearance throughout the organism. Second, adjuvants can provide physical protection for antigens, thereby conferring long-term delivery. This means that the organism is exposed to antigens for a longer period of time, making the immune system more robust by utilizing additional time by upregulating the production of B and T cells necessary for greater immunological memory in the adaptive immune response. Third, adjuvants can increase the ability to generate a local response at the injection site (during vaccination) and help induce greater release of danger signals by chemokine-releasing cells such as helper T cells and mast cells. Fourth, adjuvants can induce the release of proinflammatory cytokines, which not only recruit B and T cells at the site of infection but also help increase transcriptional events that result in a net increase in immune cells overall. Finally, adjuvants are thought to increase the innate immune response to antigens by interacting with pattern recognition receptors (PRRs) on or within accessory cells.

[0317] In some embodiments, an adjuvant is used together with the MSR. The adjuvant can be co-administered with the MSR or carried by the MSR. In some embodiments, the adjuvant is a cytosine-guanosine oligonucleotide (CpG-ODN) sequence, granulocyte-macrophage colony-stimulating factor (GM-CSF), ovalbumin (OVA), monophosphoryl lipid A (MPL), poly(I:C), MF59, alum, aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, Quinoline. A, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), FIA, Montanide, adjuvant 65, lipovant, poly(DL-lactide-co-glycolide) microspheres, paraffin oil, squalene, virosomes, AS03, AS04, IL-1, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, IL-18, toll-like receptor ligand, CD40L, pathogen-associated molecular pattern (PA) MP), damage-associated molecular pattern molecules (DAMPs), Freund's complete adjuvant, Freund's incomplete adjuvant, antibodies against immunosuppressive molecules, lipopolysaccharide (LPS), Fas ligand, Trail, lymphotactin, mannan (M-FP), APG-2, Hsp70 and Hsp90. QS-21, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, (Oxpapc), and alpha-galactosylceramide. In embodiments, a combination (i.e., multiple) of adjuvants is used in the methods described herein.

[0318] How to use Thus, one aspect of the present invention is to alter the therapeutic window and utilization of cytokine proteins to achieve improved outcomes in tumor treatment through the suppression, reduction, and in certain cases, elimination of tumors by application of cytokine proteins to target areas.

[0319] Another aspect of the present invention is to change the therapeutic range and use of cytokine proteins to achieve favorable outcomes in inflammation treatment through the application of cytokine proteins to target areas to suppress, reduce, and in some cases eliminate the inflammatory area.Conditions that can be improved by immune stimulation include, for example, infectious diseases (e.g., bacterial, fungal, viral, and parasitic infectious diseases).Immunostimulation can also improve conditions associated with uncontrolled cell proliferation (e.g., cancer).

[0320] Delivery of the mesoporous silica rods can be accompanied by an adjuvant that acts as a secondary signal for inflammasome activation. Examples of adjuvants contemplated by the present invention include, but are not limited to, Toll-like receptor ("TLR") agonists (e.g., TLR9), lipopolysaccharide (LPS), and aluminum hydroxide (alum).

[0321] The internal geometry of the mesoporous silica rod structure and mesoporous silica material can vary based on the application. The internal structure can be labyrinthine or contain internal pores of various diameters to allow for the controlled inflow of inflammatory response agents produced by the organism while metering the excretion of any of the therapeutic payloads and / or adjuvants described above. The internal geometry can also include some dead ends, as the internal pores provide openings at the surface of the mesoporous silica rod or mesoporous silica structure, thereby not necessarily providing a time-release mechanism for the excretion of the therapeutic product.

[0322] The overall shape of the mesoporous silica material is also not limited to that of a cylinder, and other commonly equivalent shapes are contemplated, including, but not limited to, spheres, icosahedrons, bars, irregular rods, pyramids, cubes, rectangular prisms, prisms, octahedrons, dodecahedrons, triangular prisms, octagonal prisms, pentagonal prisms, ellipsoids, irregular ellipsoids, tetrahedrons, square pyramids, hexagonal pyramids, and any other geometric equivalents and alternatives known in the art.

[0323] Manufacturing method of mesoporous silica rods (MSR) In embodiments, the product comprises mesoporous silica rods (MSR) loaded (by adsorption) with the cytokine GM-CSF (Leukine®) and the adjuvant CpG 7909 (CpG). The dosage form may be a lyophilized powder that is reconstituted in water (WFI) prior to administration. As noted above, the formulation may be administered by subcutaneous injection.

[0324] MSR can be 3-6 μm wide and 70-100 μm long, and the unique structure of the material provides a high pore volume and large surface area, allowing for loading and controlled release of components (leukin and CpG). Synthetic amorphous silica is known to have a good biocompatibility toxicity profile and in situ dissolution / excretion properties.

[0325] Tetraethyl orthosilicate silica (TEOS) can be used as a raw material source of silicon dioxide in the manufacturing process. TEOS acts as a precursor to silicon dioxide. Therefore, TEOS material can be used in the manufacturing process to produce mesoporous silica. As shown below, TEOS is converted to silicon dioxide when water is added. Si(OC2H5)4+2 H2O→SiO2+4 C2H5OH

[0326] Mesoporous silica rod structures can be created using poloxamers (e.g., pluronic P123 symmetric triblock copolymers) as structural templates onto which silicon dioxide can form three-dimensional structures. After silica has formed around the polymer template, the polymer can be removed using high temperatures. Removal of the polymer leaves longitudinal pores.

[0327] Figure 50 shows an overview of the manufacturing process for producing mesoporous silica rods according to an embodiment of the present invention. A symmetric triblock copolymer (Pluronic P123) composed of poly(ethylene oxide) and poly(propylene oxide) is used to create rod-shaped micelles in solution. When tetraethyl orthosilicate (TEO) is added to the solution, silica deposits on the micelles, creating a hexagonal pore structure. The Pluronic P123 is rinsed and calcined (high-temperature treatment) to remove the polymer, leaving behind the silica mesoporous structure.

[0328] The mesoporous silica rods are combined with the sterile-filtered components (GM-CSF, CpG) and thoroughly mixed to allow adsorption. The solution is then placed in a vial, frozen, lyophilized, and stored at -20°C. The proposed configuration of this system consists of a sterile vial containing the three components (MSR, GM-CSF, and CpG) as lyophilized powders to be reconstituted with water for injection.

[0329] The processed solution is an aqueous mixture that is subsequently freeze-dried. Figure 51 shows a detailed flow chart of the manufacturing process. j refers to the jacket temperature, and T r refers to the internal temperature.

[0330] Using an EasyMax 402® system, heat Pluronic P123 (P123) to 40°C. Add water for injection (WFI) and mix the solution (450-600 rpm). Add acid (37% hydrochloric acid) along with tetraethyl orthosilicate (TEOS). Silica rods form in the solution. Allow the solution to stand (i.e., age) at 100°C for approximately 48 hours.

[0331] On the third day, water (WFI) is added and the solution is sieved (180 μm) and then vacuum filtered (30 μm). The product is then dried (calcined at 550° C. for 5 hours). The final product can then be sterilized. As above, the product can be stored and transported in lyophilized form. The product remains stable at -20° C.

[0332] Porosity / Volume Determination The mesoporous silica rod material provides a large surface area for adsorbing the components of the system, which can facilitate controlled delivery when injected into the subcutaneous space.

[0333] Pore ​​size distribution analysis is performed using a static pressure (volumetric) analyzer using a gas adsorption technique. This technique measures the amount of inert gas adsorbed on the surface of the sample at various relative pressures. As the pressure is gradually reduced (desorption), the condensed gas evaporates from the pores. From the resulting isotherms, the Barrett, Joyner, and Halenda (BJH) theory is used to determine the equivalent cylindrical pore volume and pore area from the amount of adsorbed and desorbed gas. Surface area can also be determined using the Brunauer-Emmett-Teller (BET) theory.

[0334] Silica purity (thermogravimetric analysis) Thermogravimetric analysis (TGA) measures the thermal mass loss profile of a sample. This instrument utilizes a microbalance housed within a furnace that utilizes a sample carrier and thermocouple combination to accurately record the change in sample mass over time as the temperature is increased. Because the sample can be held constant at a desired temperature, mass change over time can also be observed. Because mesoporous silica consists of a high percentage of SiO2, TGA provides the percentage of the sample that is silica.

[0335] Impurities (inductively coupled plasma mass spectrometry) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is an analytical technique for determining trace multi-element and isotopic concentrations in liquid, solid, or gas samples. ICP-MS combines an ion-generating argon plasma source with the high sensitivity detection limits of mass spectrometry detection. This technique is utilized to identify the absence of residual metal and carbon-based impurities in mesoporous silica.

[0336] Silica size (Malvern® Morphologi G3S image analyzer) MSR particle size and shape analysis is performed using a Malvern® Morphologi G3S or Malvern® Morphologi 4 image analyzer. This instrument is an automated microscope that uses a series of objectives with varying magnifications, a motorized stage, and a digital camera to capture images of particles and determine their size and shape. The analytical range of this technique is approximately 0.50 μm to 1,000 μm, but measurements up to 10,000 μm can be achieved in certain applications.

[0337] The instrument captures images of particles that fall within a selected field of view of interest (see Figures 52A and 52B). The instrument determines the size of individual pixels at the selected magnification and creates a projected two-dimensional image. The instrument then converts the pixels in the two-dimensional image into a circle with the same pixel area as the two-dimensional image and reports the circular equivalent (CE) diameter.

[0338] Figures 52A and 52B are scanning electron microscope (SEM) sizing images of typical MSR lengths and widths as analyzed by a Malvern® Morphologi G3S instrument. Typical MSR lengths (left) and widths (right). D[n,0.1] (μm), D[n,0.5] (μm), and D[n,0.9] (μm) = biodistribution of MSRs sized below 10%, 50%, and 90%, respectively, within the total number average.

[0339] The key functional properties of mesoporous silica rods relate to their physical characteristics, including dimensions, surface area, pore size, and volume, as determined by emission standards. Additionally, the identity and purity of the material (SiO2) are determined by inductively coupled plasma mass spectrometry (ICP-MS), which identifies the elemental content of the substrate. By increasing the temperature and comparing the combustion thermal transitions with pure components, its chemical composition can be revealed. TGA is used to determine the purity of synthesized nanomaterials by comparing them to standards.

[0340] In an embodiment, the following routine test is applied to the mesoporous silica rods: [Table 1]

[0341] Administration Mesoporous silica structures may be administered alone to create physical injury to tumors or areas of inflammation, thus prompting a natural immune response from the human body or organism, which in effect leads to tumor regression through natural cellular processes such as phagocytosis.

[0342] Therapeutic drug molecules of various sizes are also contemplated by the present invention and can be used within the mesoporous silica rod structures or mesoporous silica materials. Molecules of various sizes can be applied as a coating to the exterior surface of the mesoporous silica rod structures or mesoporous silica materials. Furthermore, the various molecules can be combined with any number of other therapeutic agents, adjuvants, prodrugs, buffers, agents, etc.

[0343] The cytokine payloads of the present invention may be injected directly into and / or adjacent to the target tumor area.

[0344] The mesoporous silica rod structure or mesoporous silica material can have at least six pores designated for excretion of a therapeutic payload, or the mesoporous silica rod structure or silica material can have at least one pore, no more than two pores, no more than three pores, no more than four pores, no more than five pores, no more than six pores, no more than seven pores, no more than eight pores, no more than nine pores, no more than ten pores, no more than eleven pores, no more than twelve pores, no more than thirteen pores, no more than fourteen pores, no more than fifteen pores, no more than twenty pores, no more than twenty-five pores, no more than thirty pores, no more than forty pores, no more than fifty pores, no more than sixty pores, no more than seventy-five pores, no more than sixty-five pores, no more than seventy-five pores, no more than sixty-five pores, no more than seventy-five pores, no more than sixty-five pores, no more than seventy-five pores, no more than seventy-five pores, no more than one hundred pores, no more than one hundred twenty pores, no more than one hundred fifty ... The porous membrane may have up to 200 pores, up to 250 pores, up to 300 pores, up to 350 pores, up to 400 pores, up to 500 pores, up to 600 pores, up to 750 pores, up to 1,000 pores, up to 1,200 pores, up to 1,500 pores, up to 2,000 pores, up to 3,000 pores, up to 5,000 pores, up to 10,000 pores, up to 15,000 pores, up to 20,000 pores, up to 30,000 pores, up to 50,000 pores, up to 100,000 pores, or up to 1,000,000 pores.

[0345] The mesoporous silica rod structure or mesoporous silica material can have at least seven pores designated for excretion of a therapeutic payload. Alternatively, the mesoporous silica rod structure or silica material can have at least eight pores, at least nine pores, at least ten pores, at least eleven pores, at least twelve pores, at least thirteen pores, at least fourteen pores, at least fifteen pores, at least twenty pores, at least twenty-five pores, at least thirty pores, at least forty pores, at least fifty pores, at least sixty pores, at least seventy-five pores, at least one hundred pores, at least one twenty pores, at least one fifty pores, at least one seventy-five pores, at least one hundred pores, at least one hundred twenty pores, at least one hundred fifty pores, at least one hundred seventy-five ... The porous membrane may have at least 300 pores, at least 350 pores, at least 400 pores, at least 500 pores, at least 600 pores, at least 750 pores, at least 1,000 pores, at least 1,200 pores, at least 1,500 pores, at least 2,000 pores, at least 3,000 pores, at least 5,000 pores, at least 10,000 pores, at least 15,000 pores, at least 20,000 pores, at least 30,000 pores, at least 50,000 pores, at least 100,000 pores, or at least 1,000,000 pores.

[0346] The pores of the mesoporous silica rod structure or mesoporous silica material can have a uniform diameter, or in other embodiments, have a variety of diameters. In still other embodiments, the pores of the mesoporous silica rod structure may have an array of diameters arranged in a pattern of alternating diameters and dimensions. In still other embodiments, the variety of pore diameters of the mesoporous silica rod structure may be randomly created or determined by human input, algorithms, recursive algorithms, computer programs, nanofabrication, nanoengineering, 3-D printing, 3-D printing programs, chemical processes, and any other process and method of design suitable for the purposes of the present invention and known and recognized in the art.

[0347] Therapeutic payloads include all of the payloads listed above and any other art-recognized equivalents or any therapeutic agent known in the art.

[0348] The mesoporous silica rod structures or mesoporous silica materials may also dissolve within the target area and / or remain localized at the treatment site.

[0349] The mesoporous silica rod structure or mesoporous silica material can carry or be coated with a therapeutic payload, which can be any of the substances, drugs, adjuvants, and proteins described herein, as well as any equivalents known in the art and combinations of art-recognized therapeutic equivalents. In some embodiments, the MSR has one or more surface modifications (e.g., treated with substances such as glycolic acid or lactic acid, conjugated to amine, thiol, chloro, or phosphonate groups, or compounds such as PEI added to the MPS rod). In embodiments, the surface-modified MSR is an MSR to which free PEI has been added.

[0350] In certain embodiments, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are about 2 nm (nanometers), about 3 nm to 50 nm, about 36 nm to 50 nm, about 37 nm to 50 nm, about 38 nm to 50 nm, about 39 nm to 50 nm, about 40 nm to 50 nm, about 41 nm to 50 nm, about 42 nm to 50 nm, about 43 nm to 50 nm, about 44 nm to 50 nm, about 45 nm to 50 nm, about 46 nm to 50 nm, about 47 nm to 50 nm, about 48 nm to 50 nm, or about 49 nm to 50 nm.

[0351] In certain embodiments of the present invention, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are 2 nm to 3 nm, 2 nm to 4 nm, 2 nm to 5 nm, 2 nm to 10 nm, 2 nm to 15 nm, 2 nm to 20 nm, 2 nm to 25 nm, 2 nm to 30 nm, 2 nm to 35 nm, 2 nm to 40 nm, 2 nm to 45 nm, 5 nm to 10 nm, 5 nm to 15 nm, 5 nm to 20 nm, 5 nm to 25 nm, 5 nm to 30 nm, 5 nm to 35 nm, 5 nm to 40 nm, 5 nm to 45 nm, 5 nm to 50 nm, 10 nm to 15 nm, 10 nm to 20 nm, 10 nm to 25 nm, 10 nm to 25 nm, 10 nm to 35 nm, 10 nm to 40 nm, 10 nm to 45 nm, 10 nm to 50 nm, 10 nm to 15 nm, 10 nm to 20 ... The ranges may be nm to 30nm, 10nm to 35nm, 10nm to 40nm, 10nm to 45nm, 10nm to 50nm, 15nm to 20nm, 15nm to 25nm, 15nm to 30nm, 15nm to 35nm, 15nm to 40nm, 15nm to 45nm, 20nm to 25nm, 20nm to 30nm, 20nm to 35nm, 20nm to 40nm, 20nm to 45nm, 25nm to 30nm, 25nm to 35nm, 25nm to 40nm, 25nm to 45nm, 30nm to 35nm, 30nm to 40nm, 30nm to 45nm, 35nm to 40nm, 35nm to 45nm, 40nm to 45nm.

[0352] In certain embodiments of the present invention, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, about 75 nm, about 76 nm, about 77 nm, about 78 nm, about 79 nm, about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 120 nm, about It can be 5 nm, about 50 nm, about 55 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 175 nm, about 190 nm, about 200 nm, about 215 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm or more.

[0353] In certain aspects of the invention, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are 2 nm or less, 3 nm or less, 4 nm or less, 5 nm or less, 6 nm or less, 7 nm or less, 8 nm or less, 9 nm or less, 10 nm or less, 11 nm or less, 12 nm or less, 13 nm or less, 14 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 35 nm or less, 40 nm or less, 45 nm or less, 50 nm or less, The thickness may be 55 nm or less, 60 nm or less, 70 nm or less, 80 nm or less, 90 nm or less, 100 nm or less, 110 nm or less, 120 nm or less, 120 nm or less, 130 nm or less, 140 nm or less, 150 nm or less, 160 nm or less, 175 nm or less, 190 nm or less, 200 nm or less, 215 nm or less, 225 nm or less, 250 nm or less, 275 nm or less, 300 nm or less, 325 nm or less, 350 nm or less, or 500 nm or less.

[0354] In certain aspects of the invention, the pore size of the mesoporous silica materials and mesoporous silica rods disclosed herein is at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least At least 55 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 120 nm, at least 130 nm, at least 140 nm, at least 150 nm, at least 160 nm, at least 175 nm, at least 190 nm, at least 200 nm, at least 215 nm, at least 225 nm, at least 250 nm, at least 275 nm, at least 300 nm, at least 325 nm, at least 350 nm, or at least 500 nm.

[0355] In certain embodiments of the present invention, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are in the range of 2 nm to 55 nm, 2 nm to 60 nm, 2 nm to 65 nm, 2 nm to 70 nm, 2 nm to 75 nm, 2 nm to 80 nm, 2 nm to 85 nm, 2 nm to 90 nm, 2 nm to 95 nm, 2 nm to 100 nm, 2 nm to 105 nm, 2 nm to 110 nm, 2 nm to 115 nm, 2 nm to 120 nm, 5 nm to 55 nm, 5 nm to 60 nm, 5 nm to 65 nm, 5 nm to 70 nm, 5 nm to 75 nm, 5 nm to 80 nm, 5 nm to 85 nm , 5nm~90nm, 5nm~95nm, 5nm~100nm, 5nm~105nm, 5nm~110nm, 5nm~115nm, 5nm ~120nm, 10nm~55nm, 10nm~60nm, 10nm~65nm, 10nm~70nm, 10nm~75nm, 10nm~8 0nm, 10nm~85nm, 10nm~90nm, 10nm~95nm, 10nm~100nm, 10nm~105nm, 10nm~1 10nm, 10nm~115nm, 10nm~120nm, 20nm~55nm, 20nm~60nm, 20nm~65nm, 20nm~7 0nm, 20nm~75nm, 20nm~80nm, 20nm~85nm, 20nm~90nm, 20nm~95nm, 20nm~100 nm, 20nm~105nm, 20nm~110nm, 20nm~115nm, 20nm~120nm, 30nm~55nm, 30nm~6 0nm, 30nm~65nm, 30nm~70nm, 30nm~75nm, 30nm~80nm, 30nm~85nm, 30nm~90n m, 30nm~95nm, 30nm~100nm, 30nm~105nm, 30nm~110nm, 30nm~115nm, 30nm~12 0nm, 40nm~55nm, 40nm~60nm, 40nm~65nm, 40nm~70nm, 40nm~75nm, 40nm~80n m, 40nm~85nm, 40nm~90nm, 40nm~95nm, 40nm~100nm, 40nm~105nm, 40nm~110n m, 40nm~115nm, 40nm~120nm, 50nm~55nm, 50nm~60nm, 50nm~65nm, 50nm~70n m, 50nm~75nm, 50nm~80nm, 50nm~85nm, 50nm~90nm, 50nm~95nm, 50nm~100nm,50nm~105nm, 50nm~110nm, 50nm~115nm, 50nm~120nm, 60nm~65nm, 60nm~70nm , 60nm~75nm, 60nm~80nm, 60nm~85nm, 60nm~90nm, 60nm~95nm, 60nm~100nm, 6 0nm~105nm, 60nm~110nm, 60nm~115nm, 60nm~120nm, 70nm~75nm, 70nm~80nm, 70nm~85nm, 70nm~90nm, 70nm~95nm, 70nm~100nm, 70nm~105nm, 70nm~110nm, It can be in the range of 70nm to 115nm, 70nm to 120nm, 80nm to 85nm, 80nm to 90nm, 80nm to 95nm, 80nm to 100nm, 80nm to 105nm, 80nm to 110nm, 80nm to 115nm, 80nm to 120nm, 90nm to 95nm, 90nm to 100nm, 90nm to 105nm, 90nm to 110nm, 90nm to 115nm, 90nm to 120nm, 100nm to 105nm, 100nm to 110nm, 100nm to 115nm, 100nm to 120nm, 110nm to 115nm, 110nm to 120nm.

[0356] In certain embodiments of the present invention, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are from about 2 nm to about 50 nm, from about 2 nm to about 5 nm, from about 2 nm to about 10 nm, from about 2 nm to about 15 nm, from about 2 nm to about 20 nm, from about 2 nm to about 30 nm, from about 2 nm to about 40 nm, from about 5 nm to about 10 nm, from about 5 nm to about 15 nm, from about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 10 nm to about 15 nm, from about 10 nm to about 20 nm, from about 10 nm to about 25 nm, from about 10 nm to about 30 nm, from about 10 nm to about 40 nm, from about 10 nm to about 50 nm, from about 15 nm to about 20 nm, from about 15 nm to about 25 nm, from about 15 nm to about 30 nm, from about 15nm to about 40nm, about 15nm to about 50nm, about 20nm to about 25nm, about 20nm to about 30nm, about 20nm to about 35nm, about 20nm to about 40nm, about 20nm ~about 50nm, about 25nm to about 30nm, about 25nm to about 35nm, about 25nm to about 40nm, about 25nm to about 50nm, about 30nm to about 35nm, about 30nm to about 40nm m, about 30 nm to about 45 nm, about 30 nm to about 50 nm, about 35 nm to about 40 nm, about 35 nm to about 45 nm, about 35 nm to about 50 nm, about 40 nm to about 45 nm, about 40 nm to about 50 nm, about 45 nm to about 50 nm, about 46 nm to about 50 nm, about 47 nm to about 50 nm, about 48 nm to about 50 nm, or about 49 nm to about 50 nm.

[0357] In certain embodiments of the present invention, the pore sizes of the mesoporous silica materials and mesoporous silica rods disclosed herein are from about 2 nm to about 50 nm, from about 2 nm to about 55 nm, from about 2 nm to about 60 nm, from about 2 nm to about 65 nm, from about 2 nm to about 65 nm, from about 2 nm to about 80 nm, from about 2 nm to about 100 nm, from about 2 nm to about 120 nm, from about 2 nm to about 150 nm, from about 2 nm to about 200 nm, from about 5 nm to about 55 nm, from about 5 nm to about 60 nm, from about 5 nm to about 65 nm, from about 5 nm to about 80 nm, from about 5 nm to about 100 nm, from about 5 nm to about 120 nm, from about 5 nm to about 150 nm. m, about 10nm to about 55nm, about 10nm to about 60nm, about 10nm to about 65nm, about 10nm to about 80nm, about 10nm to about 100nm , about 10nm to about 120nm, about 10nm to about 150nm, about 15nm to about 55nm, about 15nm to about 60nm, about 15nm to about 80nm , about 15nm to about 100nm, about 15nm to about 120nm, about 15nm to about 150nm, about 20nm to about 55nm, about 20nm to about 60nm m, about 20nm to about 65nm, about 20nm to about 80nm, about 20nm to about 100nm, about 20nm to about 100nm, about 20nm to about 120 nm, about 20nm to about 150nm, about 30nm to about 55nm, about 30nm to about 80nm, about 30nm to about 100nm, about 30nm to about 12 0nm, about 30nm to about 150nm, about 40nm to about 55nm, about 40nm to about 80nm, about 40nm to about 100nm, about 40nm to about 1 20nm, about 40nm to about 150nm, about 50nm to about 60nm, about 50nm to about 65nm, about 50nm to about 80nm, about 50nm to about 1 00nm, about 50nm to about 120nm, about 50nm to about 150nm, about 60nm to about 65nm, about 60nm to about 70nm, about 60nm to about 80nm, approximately 60nm ~ approximately 100nm, approximately 60nm ~ approximately 120nm, approximately 60nm ~ approximately 150nm, approximately 70nm ~ approximately 75nm, approximately 70nm ~ Approximately 80nm, approximately 70nm to approximately 100nm, approximately 70nm to approximately 110nm, approximately 70nm to approximately 120nm, approximately 70nm to approximately 130nm, approximately 70n m ~ about 140nm, about 70nm - about 150nm, about 80nm - about 85nm, about 80nm - about 90nm, about 80nm - about 100nm, about 80 nm~about 120nm, about 80nm~about 150nm, about 90nm~about 100nm, about 90nm~about 110nm, about 90nm~about 120nm,It may be in the range of about 90 nm to about 150 nm, about 100 nm to about 110 nm, about 100 nm to about 120 nm, about 100 nm to about 130 nm, about 100 nm to about 140 nm, about 100 nm to about 150 nm, about 110 nm to about 120 nm, about 110 nm to about 140 nm, about 110 nm to about 150 nm, about 120 nm to about 135 nm, about 120 nm to about 150 nm, about 130 nm to about 140 nm, about 130 nm to about 150 nm, about 140 nm to about 145 nm, or about 140 nm to about 150 nm.

[0358] Dosages of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs, and other equivalents of the present invention are from 1 ng (nanogram) to about 100 ng, 1 ng to about 500 ng, 1 ng to about 1 μg (microgram), 1 ng to about 2 μg, about 1 ng to about 3 μg, about 1 ng to about 4 μg, about 1 ng to about 5 μg, about 1 ng to about 6 μg, about 1 ng to about 7 μg, about The range may be from 1 ng to about 8 μg, from about 1 ng to about 9 μg, from about 1 ng to about 10 μg, from about 1 ng to about 12 μg, from about 1 ng to about 15 μg, from about 1 ng to about 17 μg, from about 1 ng to about 20 μg, from about 1 ng to about 25 μg, from about 1 ng to about 30 μg, from about 1 ng to about 35 μg, from about 1 ng to about 40 μg, from about 1 ng to about 50 μg, from about 1 ng to about 60 μg, from about 1 ng to about 75 μg, from about 1 ng to about 100 μg, from about 1 ng to about 1 mg, or from about 1 ng to about 1 g.

[0359] In other embodiments, the dosage of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs, and other equivalents of the present invention is from about 1 μg to about 2 μg, from about 1 μg to about 3 μg, from about 1 μg to about 4 μg, from about 1 μg to about 5 μg, from about 1 μg to about 6 μg, from about 1 μg to about 8 μg, from about 1 μg to about 10 μg, from about 1 μg to about 15 μg, from about 1 μg to about 2 μg, The range may be 0 μg, about 1 μg to about 30 μg, about 1 μg to about 40 μg, about 1 μg to about 50 μg, about 1 μg to about 100 μg, about 1 μg to about 500 μg, about 1 μg to about 1 mg, about 1 μg to about 5 mg, about 1 μg to about 10 mg, about 1 μg to about 20 mg, about 1 μg to about 50 mg, about 1 μg to about 100 mg, about 1 μg to about 200 mg, about 1 μg to about 500 mg, about 1 μg to about 750 mg, or about 1 μg to about 1 g.

[0360] Dosages of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs and other equivalents of the present invention are about 1 ng, about 2 ng, about 3 ng, about 4 ng, about 5 ng, about 6 ng, about 7 ng, about 8 ng, about 9 ng, about 10 ng, about 11 ng, about 12 ng, about 13 ng, about 14 ng, about 15 ng, about 16 ng, about 17 ng, about 19 ng, about 20 ng, about 25 ng, about 30 ng, about 35 ng, about 40 ng, about 45 ng, about 50 ng, about 55 ng, about 60 ng, about 65 ng, about 70 ng, about 75 ng, about 80 ng, about 85 ng, about 90 ng, about 95 ng , about 100 ng, about 110 ng, about 120 ng, about 130 ng, about 140 ng, about 150 ng, about 160 ng, about 175 ng, about 190 ng, about 200 ng, about 225 ng, about 250 ng, about 275 ng, about 300 ng, about 325 ng, about 350 ng, about 375 ng, about 400 ng, about 425 ng, about 450 ng, about 475 ng, about 500 ng, about 550 ng, about 600 ng, about 650 ng, about 700 ng, about 750 ng, about 800 ng, about 850 ng, about 900 ng, about 950 ng, about 1,000 ng, about 1,200 ng, about 1,500 ng, about 2,000 ng, about 3,500 ng, or about 5,000 ng.

[0361] Dosages of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs and other equivalents of the present invention are about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13 μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg, about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about 24 μg, about 25 μg, about 26 μg, about 27 μg, about 28 μg, about 29 μg, about 30 μg, about 31 μg, about 32 μg, about 33 μg, about 34 μg, about 35 μg, about 36 μg, about 37 μg, about 38 μg, about 39 μg, about 40 μg, about 41 μg, about 42 μg, about 43 μg, about 44 μg, about 45 μg, about 46 μg, about 47 μg, about 48 μg, about 49 μg, about 50 μg, about 51 μg, about 52 μg, about 53 μg, about 54 μg, about 55 μg, about 56 μg, about 57 μg, about 58 μg, about 59 μg, about 60 μg, about 61 μg, about 62 μg, about 63 μg, about 64 μg, about 65 μg, about 66 μg, about 67 μg, g, about 17μg, about 18μg, about 19μg, about 20μg, about 21μg, about 22μg, about 23μg, about 24μg, about 25μg, about 30μg, about 32μg, about 35μg, about 37μg, about 40μg g, about 42μg, about 45μg, about 48μg, about 50μg, about 55μg, about 60μg, about 65μg, about 70μg, about 75μg, about 80μg, about 85μg, about 90μg, about 95μg, about 100μg g, about 110μg, about 120μg, about 130μg, about 140μg, about 150μg, about 160μg, about 170μg, about 180μg, about 190μg, about 200μg, about 215μg, about 230μg g, about 250μg, about 265μg, about 275μg, about 300μg, about 325μg, about 350μg, about 375μg, about 400μg, about 425μg, about 450μg, about 475μg, about 500μ g, about 550μg, about 600μg, about 650μg, about 700μg, about 750μg, about 800μg, about 850μg, about 900μg, about 950μg, about 1mg, about 1.5mg, about 2mg, about 2 .5mg, about 3mg, about 3.5mg, about 4mg, about 4.5mg, about 5mg, about 5.5mg, about 6mg, about 6.5mg, about 7mg, about 7.5mg, about 8mg, about 8.5mg, about 9mg, about 9.5mg, approximately 10mg, approximately 11mg, approximately 12mg, approximately 13mg, approximately 14mg, approximately 15mg, approximately 16mg, approximately 17mg, approximately 18mg, approximately 19mg, approximately 20mg, approximately 22mg, approximately 24mg, approximately 25mg, approximately 27mg, approximately 29mg, approximately 30mg, approximately 33mg, approximately 35mg, approximately 37mg, approximately 40mg, approximately 43mg, approximately 45mg, approximately 48mg, approximately 50mg, approximately 55mg, approximately 60mg, approximately 65mg, approximately 70mg Approximately 75mg, approximately 80mg, approximately 85mg, approximately 90mg, approximately 95mg, approximately 100mg, approximately 110mg, approximately 120mg, approximately 130mg, approximately 140mg, approximately 150mg, approximately 160mg, approximately 170mg, approximately 180mg, approximately 190mg, approximately 200mg, approximately 220mg, approximately 240mg, approximately 250mg, approximately 275mg, approximately 300mg, approximately 325mg, approximately 350mg, approximately 375mg, approximately 400mg, approximately 425mg. Approximately 450mg, approximately 475mg, approximately 500mg, approximately 550mg, approximately 600mg, approximately 650mg, approximately 700mg, approximately 750mg, approximately 800mg, approximately 850mg, approximately 900mg, approximately 950mg, approximately 1g, approximately 1.5g, approximately 2g, approximately 2.5g, approximately 3g, approximately 3.5g, approximately 4g, approximately 4.5g, approximately 5g, approximately 5.5g, approximately 6g, approximately 6.5g, approximately 7g, approximately 7.5g, approximately 8g, approximately 8.5g, approximately 9g, approximately 9.5g, approximately 10g Approximately 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 22g, 24g, 25g, 27g, 28g, 30g, 33g, 35g, 38g, 40g, 43g, 45g, 48g, 50g, 55g, 60g, 65g, 70g, 75g, 80g, 85g, 90g, 95g, and 100g.

[0362] Doses of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs and other equivalents of the present invention may be at least 1 ng, at least 2 ng, at least 3 ng, at least 4 ng, at least 5 ng, at least 6 ng, at least 7 ng, at least 8 ng, at least 9 ng, at least 10 ng, at least 11 ng, at least 12 ng, at least 13 ng, at least 14 ng, at least 15 ng, at least 16 ng, at least 17 ng, at least 19 ng, at least 20 ng, at least 25 ng, at least 30 ng, at least 35 ng, at least 40 ng, at least 45 ng, at least 50 ng, at least 55 ng, at least 60 ng, at least 65 ng, at least 70 ng, at least 75 ng, at least 80 ng, at least 85 ng, at least 90 ng, at least 95 ng, at least It can be 100 ng, at least 110 ng, at least 120 ng, at least 130 ng, at least 140 ng, at least 150 ng, at least 160 ng, at least 175 ng, at least 190 ng, at least 200 ng, at least 225 ng, at least 250 ng, at least 275 ng, at least 300 ng, at least 325 ng, at least 350 ng, at least 375 ng, at least 400 ng, at least 425 ng, at least 450 ng, at least 475 ng, at least 500 ng, at least 550 ng, at least 600 ng, at least 650 ng, at least 700 ng, at least 750 ng, at least 800 ng, at least 850 ng, at least 900 ng, at least 950 ng, at least 1,000 ng, at least 1,200 ng, at least 1,500 ng, at least 2,000 ng, at least 3,500 ng, or at least 5,000 ng.

[0363] Doses of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs and other equivalents of the present invention may be at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 6 μg, at least 7 μg, at least 8 μg, at least 9 μg, at least 10 μg, at least 11 μg, at least 12 μg, at least 13 μg, at least 14 μg, at least 15 μg, at least 16 μg, at least 17 μg, at least at least 18 μg, at least 19 μg, at least 20 μg, at least 21 μg, at least 22 μg, at least 23 μg, at least 24 μg, at least 25 μg, at least 30 μg, at least 32 μg, at least 35 μg, at least 37 μg, at least 40 μg, at least 42 μg, at least 45 μg, at least 48 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg, at least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, At least 95 μg, at least 100 μg, at least 110 μg, at least 120 μg, at least 130 μg, at least 140 μg, at least 150 μg, at least 160 μg, at least 170 μg, at least 180 μg, at least 190 μg, at least 200 μg, at least 215 μg, at least 230 μg, at least 250 μg, at least 265 μg, at least 275 μg, at least 300 μg, at least 325 μg, at least 350 μg, at least 375 μg, at least 400 μg, at least 425 μg , at least 450μg, at least 475μg, at least 500μg, at least 550μg, at least 600μg, at least 650μg, at least 700μg, at least 750μg, at least 800μg, at least 850μg, at least 900μg, at least 950μg, at least 1mg, at least 1.5mg, at least 2mg, at least 2.5mg, at least 3mg, at least 3.5mg, at least 4mg, at least 4.5mg, at least 5mg, at least 5.5mg, at least 6mg, at least 6.5mg, at least 7mg, at least 7.5mg, at least 8mg, at least 8.5mg, at least 9mg, at least 9.5mg, at least 10mg, at least 11mg, at least 12mg, at least 13mg, at least 14mg, at least 15mg, at least 16mg, at least 17mg, at least 18mg, at least 19mg, at least 20mg, at least 22mg, at least 24mg, at least 25mg, at least 27mg, at least 29mg, at least 30mg, at least 33mg, at least 35mg, at least 37mg, at least 40mg, at least 43mg, at least 45mg, at least 48mg, at least 50mg, at least 55mg, at least 60mg, at least 65mg, at least 70mg, at least 75mg, at least 80mg, at least 85mg, at least 90mg, at least 95mg, at least 100mg, at least 110mg, at least 120mg, at least 130mg, at least 140mg, at least at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, at least 200 mg, at least 220 mg, at least 240 mg, at least 250 mg, at least 275 mg, at least 300 mg, at least 325 mg, at least 350 mg, at least 375 mg, at least 400 mg, at least 425 mg, at least 450 mg, at least 475 mg, at least 500 mg, at least 550 mg, at least 600 mg, at least Also 650mg, at least 700mg, at least 750mg, at least 800mg, at least 850mg, at least 900mg, at least 950mg, at least 1g, at least 1.5g, at least 2g, at least 2.5g, at least 3g, at least 3.5g, at least 4g, at least 4.5g, at least 5g, at least 5.5g, at least 6g, at least 6.5g, at least 7g, at least 7.5g, at least 8g, at least 8.5g, at least 9g, at least 9.It can be 5g, at least 10g, at least 11g, at least 12g, at least 13g, at least 14g, at least 15g, at least 16g, at least 17g, at least 18g, at least 19g, at least 20g, at least 22g, at least 24g, at least 25g, at least 27g, at least 28g, at least 30g, at least 33g, at least 35g, at least 38g, at least 40g, at least 43g, at least 45g, at least 48g, at least 50g, at least 55g, at least 60g, at least 65g, at least 70g, at least 75g, at least 80g, at least 85g, at least 90g, at least 95g, or at least 100g.

[0364] Dosages of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs and other equivalents of the present invention may be 1 ng or less, 2 ng or less, 3 ng or less, 4 ng or less, 5 ng or less, 6 ng or less, 7 ng or less, 8 ng or less, 9 ng or less, 10 ng or less, 11 ng or less, 12 ng or less, 13 ng or less, 14 ng or less, 15 ng or less, 16 ng or less, 17 ng or less, 19 ng or less, 20 ng or less, 25 ng or less, 30 ng or less, 35 ng or less, 40 ng or less, 45 ng or less, 50 ng or less, 55 ng or less, 60 ng or less, 65 ng or less, 70 ng or less, 75 ng or less, 80 ng or less, 85 ng or less, 90 ng or less, 95 ng or less, It can be 100ng or less, 110ng or less, 120ng or less, 130ng or less, 140ng or less, 150ng or less, 160ng or less, 175ng or less, 190ng or less, 200ng or less, 225ng or less, 250ng or less, 275ng or less, 300ng or less, 325ng or less, 350ng or less, 375ng or less, 400ng or less, 425ng or less, 450ng or less, 475ng or less, 500ng or less, 550ng or less, 600ng or less, 650ng or less, 700ng or less, 750ng or less, 800ng or less, 850ng or less, 900ng or less, 950ng or less, 1,000ng or less, 1,200ng or less, 1,500ng or less, 2,000ng or less, 3,500ng or less, 5,000ng or less.

[0365] Dosages of the payloads, silica materials, cytokine proteins, inflammasomes, interleukins, adjuvants, pharmaceutical compositions, therapeutic agents, prodrugs and other equivalents of the present invention may be 1 μg or less, 2 μg or less, 3 μg or less, 4 μg or less, 5 μg or less, 6 μg or less, 7 μg or less, 8 μg or less, 9 μg or less, 10 μg or less, 11 μg or less, 12 μg or less, 13 μg or less, 14 μg or less, 15 μg or less, 16 μg or less, 17 μg or less Bottom, 18μg or less, 19μg or less, 20μg or less, 21μg or less, 22μg or less, 23μg or less, 24μg or less, 25μg or less, 30μg or less, 32μg or less, 35μg or less, 37μg or less, 40μg or less, 42μg g or less, 45 μg or less, 48 ​​μg or less, 50 μg or less, 55 μg or less, 60 μg or less, 65 μg or less, 70 μg or less, 75 μg or less, 80 μg or less, 85 μg or less, 90 μg or less, 95 μg or less, 100 μg or less, 110μg or less, 120μg or less, 130μg or less, 140μg or less, 150μg or less, 160μg or less, 170μg or less, 180μg or less, 190μg or less, 200μg or less, 215μg or less, 230μg or less, 250μg or less, 265μg or less, 275μg or less, 300μg or less, 325μg or less, 350μg or less, 375μg or less, 400μg or less, 425μg or less, 450μg or less, 475μg or less, 500μg or less, 550μg or less, 600μg or less, 650μg or less, 700μg or less, 750μg or less, 800μg or less, 850μg or less, 900μg or less, 950μg or less, 1mg or less, 1.5mg or less, 2mg or less, 2.5m g or less, 3 mg or less, 3.5 mg or less, 4 mg or less, 4.5 mg or less, 5 mg or less, 5.5 mg or less, 6 mg or less, 6.5 mg or less, 7 mg or less, 7.5 mg or less, 8 mg or less, 8.5 mg or less, 9 mg or less, 9.Less than 5mg, less than 10mg, less than 11mg, less than 12mg, less than 13mg, less than 14mg, less than 15mg, less than 16mg, less than 17mg, less than 18mg, less than 19mg, less than 20mg, less than 22mg, less than 24mg, less than 25mg, less than 27mg, less than 29mg, less than 30mg, less than 33mg, less than 35mg, less than 37mg, less than 40mg, less than 43mg, less than 45mg, less than 48mg, less than 50mg, less than 55mg, less than 60mg, less than 65mg, less than 70mg Below 75mg, below 80mg, below 85mg, below 90mg, below 95mg, below 100mg, below 110mg, below 120mg, below 130mg, below 140mg, below 150mg, below 160mg, below 170mg, below 180mg, below 190mg, below 200mg, below 220mg, below 240mg, below 250mg, below 275mg, below 300mg, below 325mg, below 350mg, below 375mg, below 400mg, below 425mg, below 450mg Less than 1g, less than 475mg, less than 500mg, less than 550mg, less than 600mg, less than 650mg, less than 700mg, less than 750mg, less than 800mg, less than 850mg, less than 900mg, less than 950mg, less than 1g, less than 1.5g, less than 2g, less than 2.5g, less than 3g, less than 3.5g, less than 4g, less than 4.5g, less than 5g, less than 5.5g, less than 6g, less than 6.5g, less than 7g, less than 7.5g, less than 8g, less than 8.5g, less than 9g, less than 9.5g, less than 10g, 11 Below 100g: 12g or less, 13g or less, 14g or less, 15g or less, 16g or less, 17g or less, 18g or less, 19g or less, 20g or less, 22g or less, 24g or less, 25g or less, 27g or less, 28g or less, 30g or less, 33g or less, 35g or less, 38g or less, 40g or less, 43g or less, 45g or less, 48g or less, 50g or less, 55g or less, 60g or less, 65g or less, 70g or less, 75g or less, 80g or less, 85g or less, 90g or less, 95g or less, 100g or less.

[0366] In one embodiment, treatment with a compound disclosed herein reduces inflammation (e.g., signs and symptoms) by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0367] In one embodiment, treatment with a compound disclosed herein reduces inflammation (e.g., signs and symptoms) by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0368] In one embodiment, treatment with a compound disclosed herein reduces inflammation (e.g., signs and symptoms), e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0369] In one embodiment, treatment with a compound disclosed herein reduces tumor growth (e.g., size and / or number of tumors), e.g., by about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0370] In one embodiment, treatment with a compound disclosed herein reduces tumor growth (e.g., tumor size and / or number) by, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%.

[0371] In one embodiment, treatment with a compound disclosed herein reduces tumor growth (e.g., size and / or number of tumors), e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0372] In one embodiment, treatment with a compound disclosed herein reduces tumor perimeter by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0373] In one embodiment, treatment with a compound disclosed herein reduces tumor circumference by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0374] In one embodiment, treatment with a compound disclosed herein reduces tumor perimeter by, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0375] In one embodiment, treatment with a compound disclosed herein reduces tumor diameter by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0376] In one embodiment, treatment with a compound disclosed herein reduces tumor diameter by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0377] In one embodiment, treatment with a compound disclosed herein reduces tumor diameter by, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0378] In one embodiment, treatment with a compound disclosed herein reduces tumor volume by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0379] In one embodiment, treatment with a compound disclosed herein reduces tumor volume by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0380] In one embodiment, treatment with a compound disclosed herein reduces tumor volume by, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0381] In one embodiment, treatment with a compound disclosed herein reduces tumor burden by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0382] In one embodiment, treatment with a compound disclosed herein reduces tumor burden by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0383] In one embodiment, treatment with a compound disclosed herein reduces tumor burden by, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0384] In one embodiment, treatment with a compound disclosed herein reduces lesion growth (e.g., size and / or number) by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0385] In one embodiment, treatment with a compound disclosed herein reduces lesion growth (e.g., size and / or number) by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%.

[0386] In one embodiment, treatment with a compound disclosed herein reduces lesion growth (e.g., size and / or number) by, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0387] In one embodiment, treatment with a compound disclosed herein reduces lesion diameter by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0388] In one embodiment, treatment with a compound disclosed herein reduces lesion diameter by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0389] In one embodiment, treatment with a compound disclosed herein reduces lesion diameter by, e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0390] In one embodiment, treatment with a compound disclosed herein reduces lesion size by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0391] In one embodiment, treatment with a compound disclosed herein reduces lesion size by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0392] In one embodiment, treatment with a compound disclosed herein reduces lesion size, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0393] In one embodiment, treatment with a compound disclosed herein reduces lesion volume by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0394] In one embodiment, treatment with a compound disclosed herein reduces lesion volume by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0395] In one embodiment, treatment with a compound disclosed herein reduces lesion burden, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.

[0396] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst perimeter by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0397] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst perimeter by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0398] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst perimeter by, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0399] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst diameter by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0400] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst diameter by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0401] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst diameter, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0402] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst volume by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0403] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst volume by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0404] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst volume by, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0405] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst volume by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0406] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst volume by, for example, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0407] In one embodiment, treatment with a compound disclosed herein reduces sebaceous cyst volume, e.g., by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0408] The present specification also provides pharmaceutical compositions for administering to subjects.The pharmaceutical compositions disclosed herein can further comprise pharmaceutically acceptable carriers, excipients or diluents.As used herein, the term " pharmaceutically acceptable " means that the composition is sufficient to achieve therapeutic effect without harmful side effects, and can be easily determined according to the type of disease, patient's age, weight, health condition, sex and drug sensitivity, administration route, administration mode, administration frequency, treatment period, the drugs used in combination with or simultaneously with the compositions disclosed herein and other factors known in medicine.

[0409] The composition can be used by blending with various pharmaceutically acceptable carriers such as physiological saline or organic solvents. To enhance stability or absorption, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers may be used.

[0410] The dosage and frequency of administration of the pharmaceutical compositions disclosed herein will be determined by the type of active ingredient, as well as by various factors such as the disease being treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

[0411] The total effective dose of the compositions disclosed herein may be administered to a patient in a single dose, or may be administered over a long period of time in multiple doses according to a divided treatment protocol. In the pharmaceutical compositions disclosed herein, the content of the active ingredient may vary depending on the severity of the disease. However, the effective dose of the disclosed compositions is determined by taking into account various factors, including the patient's age, weight, health condition, sex, disease severity, diet, and secretion rate, as well as the administration route and treatment frequency of the pharmaceutical composition. Taking this into consideration, those skilled in the art can easily determine the effective dose suitable for a specific use of the pharmaceutical compositions disclosed herein. The pharmaceutical compositions disclosed herein are not particularly limited in terms of formulation, administration route, and administration mode, as long as they show favorable effects.

[0412] In various embodiments, the formulation may include one or more preservatives and / or additives known in the art. Similarly, the formulation may be further formulated into any of a variety of known delivery formulations, including, but not limited to, surfactants, adjuvants, biodegradable polymers, hydrogels, and the like, any such components, their chemical and functional characteristics known in the art. Similarly, formulations that promote rapid, sustained, or delayed release of bioactive agents after administration are known in the art. The described formulations may be produced to include these or other formulation components known in the art.

[0413] Thus, the composition can be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routine work performed by those skilled in the art. The appropriate dosage can be confirmed by using appropriate dose-response data. In various embodiments, the bioactive agent in the formulations described herein can be administered to patients over an extended period of time, such as, but not limited to, chronic administration for chronic conditions.

[0414] Packaging and equipment for administration may be determined by various considerations, including, but not limited to, the volume of material to be administered, conditions for storage, whether administration is by skilled medical personnel or patient self-compliance, the dosing regimen, the geopolitical environment (e.g., exposure to extreme temperature conditions in developing countries), and other practical considerations.

[0415] Injection devices include pen-type syringes, auto-injectors, safety syringes, injection pumps, infusion pumps, glass pre-filled syringes, plastic pre-filled syringes, and needleless syringes, which may be pre-filled with liquid or may be dual-chambered for use with, for example, freeze-dried materials.An example of a syringe for such use is Lyo-Ject™, a dual-chamber pre-filled freeze-dried syringe available from Vetter GmbH, Ravensburg, Germany.Another example is LyoTip, a pre-filled syringe designed to easily deliver freeze-dried formulations, available from LyoTip, Inc., Camarillo, California, USA.Administration by injection can be, but is not limited to, intravenous, intramuscular, intraperitoneal, or subcutaneous, as needed.Administration by non-injection route can be, but is not limited to, nasal, oral, ocular, cochlear, cutaneous, or pulmonary, as needed. The above-described injection devices may be used in combination with catheters, cannulas, ports, shunts, and the like.

[0416] In certain embodiments, the kit can include one or more single-chamber or multi-chamber syringes (e.g., liquid syringes and lyophilized syringes) for administering one or more formulations described herein. In various embodiments, the kit can include formulation components for parenteral, subcutaneous, intramuscular, or IV administration, sealed in a vial under partial vacuum, ready to be filled into a syringe and administered to a subject. In this regard, the composition can be placed therein under partial vacuum. In all of these embodiments and others, the kit can contain one or more vials according to any of the above, each vial containing a single unit dose for administration to a subject.

[0417] The kits can include a lyophilate disposed as described herein that, when reconstituted, provides the composition accordingly. In various embodiments, the kits can contain a lyophilate and a sterile diluent for reconstituting the lyophilate.

[0418] Also described herein are methods for treating a subject in need thereof, comprising administering to the subject an effective amount of a formulation described herein. The therapeutically effective amount or therapeutically effective dose of the formulation depends on the disease or condition of the subject and the actual clinical situation.

[0419] In one embodiment, the formulations described herein can be administered by any suitable route, particularly parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration. It is also understood that the preferred route varies depending on the condition and age of the recipient and the disease being treated. Methods for determining the most effective administration means and dosage are known to those skilled in the art and vary depending on, but not limited to, the composition used for treatment, the purpose of treatment, and the subject being treated. Single or multiple administrations can be performed, and, but not limited to, the dose level and dose pattern are selected by the treating physician. Suitable dosage formulations and methods for administering active substances are known in the art.

[0420] The formulations described herein can be used in the manufacture of medicinal products and for the treatment of humans and other animals by administration in accordance with conventional procedures.

[0421] Compositions according to embodiments described herein have desirable properties, such as desirable solubility, viscosity, syringeability, and stability. Lyophilizates according to embodiments described herein also have desirable properties, such as desirable recovery, stability, and reconstitution.

[0422] In one embodiment, the pH of the pharmaceutical formulation is at least about 3.5, at least about 3.75, at least about 4, at least about 4.25, at least about 4.5, at least about 4.75, at least about 5, at least about 5.25, at least about 5.5, at least about 5.75, at least about 6, at least about 6.25, at least about 6.5, at least about 6.75, at least about 7, at least about 7.25, at least about 7.5, at least about 7.75, at least about 8, at least about 8.25, at least about 8.5, at least about 8 0.75, at least about 9, at least about 9.25, at least about 9.5, at least about 9.75, at least about 10, at least about 10.25, at least about 10.5, at least about 10.75, at least about 11, at least about 11.25, at least about 11.5, at least about 11.75, at least about 12, at least about 12.25, at least about 12.5, at least about 12.75, at least about 13, at least about 13.25, at least about 13.5, at least about 13.75 or at least about 14.

[0423] In one embodiment, the pH of the pharmaceutical formulation is about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 8, about 6 to about 7, about 6 to about 9, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 11, about 8 to about 9 about 9, about 8 to about 10, about 8 to about 11, about 8 to about 12, about 8 to about 13, about 8 to about 14, about 3 to about 10, about 3 to about 11, about 3 to about 12, about 3 to about 13, about 3 to about 14, about 5 to about 10, about 5 to about 12, about 5 to about 13, about 5 to about 14, about 6 to about 10, about 6 to about 11, about 6 to about 12, about 6 to about 13, about 6 to about 14, about 7 to about 12, about 7 to about 13, about 7 to about 14. [Example]

[0424] The compositions and methods described herein will be further understood by reference to the following examples, which are intended to be purely illustrative. The compositions and methods described herein are not limited in scope by the exemplified embodiments, which are intended only as illustrations of single aspects. Any functionally equivalent method is within the scope of the invention. In addition to those explicitly described herein, various modifications of the compositions and methods described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are within the scope of the invention. [Example]

[0425] Synthesis of mesoporous silica rods Methods for producing mesoporous silica rods (MSRs) are known in the art. The size, morphology, pore size, and pore structure of MSRs can be rationally designed, and the synthesis process can be freely controlled. Particle size and shape can also affect their blood circulation, cellular uptake, and tumor penetration, which are determinant parameters for achieving therapeutic efficacy. Regarding particle size, a diameter range of 50–300 nm may be advantageous for optimal cellular uptake, long circulation time, high drug loading, and high accumulation within tumors.

[0426] In embodiments, five components are used in a simple manufacturing process with low cost and minimal complex chemistry. 1) Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), Pluronic® (P123) 2) Water for injection (WFI) 3) Hydrochloric acid 37% acid - fuming 4) Tetraethyl orthosilicate (TEOS) 5) Pure 70% ethanol

[0427] Mesoporous materials can vary in their structural arrangement and pore size. For example, MCM-48 has a cubic arrangement, whereas MCM-50 has a lamellar arrangement. Nonionic triblock copolymers, such as alkyl poly(ethylene oxide) (PEO) oligomeric surfactants and poly(alkylene oxide) block copolymers, have also been used in the synthesis of SBAs. The ratio of ethylene oxide to propylene oxide can be varied to achieve the desired symmetry of the mesoporous material: (SBA-11 (cubic), SBA-12 (3-d hexagonal), SBA-15 (hexagonal), and SBA-16 (cubic cage structure). It differs from MCM in that it has even larger pores of 4.6-30 nm and even thicker silica walls. FSM-16, i.e., folded sheets of mesoporous material, is another type of mesoporous material and can be synthesized using a quaternary ammonium surfactant as a template and the layered polysilicate kanemite.

[0428] Synthesis / modification of mesoporous silica rods Varying the process for synthesizing mesoporous silica can result in different shapes, sizes, pore volumes, and surface areas. Applicants have optimized a reproducible, controlled process for creating "standard" macroporous mesoporous silica rods (MSRs). Once a standard MSR process and characterization are established, the process can be tailored to achieve the ideal MSR for a specific application. To demonstrate this principle, longer and wider MSRs were synthesized with the intention of extending their lifespan and degrading more slowly in vivo.

[0429] To synthesize longer and wider mesoporous silica rods, Applicants adjusted parameters in the optimized standard MSR process. These parameters included slowing the stirring speed and reducing the addition of hydrochloric acid. First, P123 was weighed and melted in a Mettler Toledo Reactor Vessel*. Water for injection was then added and mixed at a constant speed of 400 rpm until the P123 was dissolved. Next, 11 ml of hydrochloric acid was added, followed by tetraethyl orthosilicate. The MSR formed over a 20-hour period. Mixing was then stopped, the temperature was increased, and the MSR was aged for 24 hours. After solidification, the solution was washed through a sieve with ethanol, and the MSR was collected by vacuum filtration and dried. The MSR was scraped into a porcelain dish and calcined at 550°C for 5 hours. After cooling, the MSR was pure and sterile, depyrogenated, and collected in a sterile container for use.

[0430] A Mettler Toledo EasyMax 402 System was used to precisely control the jacket temperature, internal solution temperature, and agitation speed. The instrument is integrated with iControl software, which monitors technical data in real time and provides batch reports.

[0431] The synthesis of mesoporous silica rods utilized five components: P123, EasyMax 402, WFI, HCl, and TEOS to simplify the manufacturing process, reduce costs, and reduce complex chemistry.

[0432] By decreasing the stirring speed and amount of hydrochloric acid added during synthesis, MSRs that were longer and wider than standard MSRs were produced (Figures 54A-54D). The results also demonstrated no adverse effects on other desired parameters, including thermogravimetric analysis (TGA), pore volume, and pore size (see below). The BET surface area increased as predicted by the modified MSRs. In conclusion, this method demonstrated the control of specific parameters in process development to produce desired modified MSR morphologies. [Table 2] [Table 3]

[0433] Vaccine Manufacturing Methods for producing mesoporous silica rod vaccines for humoral responses are known in the art. In this example, lyophilized MSR was suspended in PBS containing IL-12 (1-20 μg) and gently mixed. IL-12 was adsorbed onto the surface of the MSR at 37°C for 1-3 hours. The contents were then drawn into a syringe and injected directly into the tumor. IL-12 content and release profile can be confirmed using in vitro methods. [Example]

[0434] IL-12 release content IL-12 was combined with MSR as described above. An ELISA test was used to detect the presence of IL-12.

[0435] The MSR was left in PBS solution for 7 days. The amount of IL-12 released was measured over the course of this 7-day period. Only small amounts of IL-12 were detectably released (i.e., less than 3%). Furthermore, no cross-reactivity was observed with polyethyleneimine (PEI) L25K alone (diluted 1:10 in PBS). [Example]

[0436] IL-12 potency In vitro released IL-12 was collected from the vaccine and analyzed for potency. The collection protocol was as follows: 6 mL of phosphate-buffered saline (PBS) was introduced, resuspended, and mixed to a 6× vaccine dose. This solution was aliquoted into 6×1 mL tubes for a 1× vaccine dose. The 1× vaccine dose was centrifuged at 1000 rpm. 700 μl of the resultant was collected and stored at −20°C, then replaced with 700 μl of PBS and incubated at 37°C. After 7 days, 75 μl of mesoporous silica rods and supernatant (SUP) were tested for potency.

[0437] The efficacy results showed that quantitatively potent IL-12 was only found in the 7-day rods and in the T-0 (1 μg IL-12 dose, no PEI) rods. It was also discovered that quantitatively potent IL-12 was found in the 7-day rods containing PEI alone. However, PEI did not significantly affect the IL-12 release and efficacy observed in vitro. Furthermore, 25kJ PEI did not activate HEK cells, and the 5 μg dose was toxic at tested concentrations of 12.5 to 100 μg / ml. [Example]

[0438] Intertumoral IL-12 delivery One of the most effective antitumor cytokines is interleukin-12 (IL-12), which mediates its antitumor activity through the stimulation of T cells, natural killer (NK) cells, and NK T cells through its angiogenic effects. However, systemic IL-12 expression can cause toxicity due to the induction of high levels of interferon (IFN)-γ. Strategies have been developed that allow local delivery of IL-12 within tumors, resulting in high antitumor efficacy with reduced toxicity.

[0439] Due to the toxicity of the material and its strong clinical history of antitumor efficacy, we conceived the use of the MSR biomaterial as an intertumor delivery vehicle, which predicted long-term delivery of cytokines and potentially enhanced immune responses generated by the biomaterial's chemistry and composition.

[0440] The formulations were tested in the B16F10 melanoma mouse model and the CT26 colorectal cancer model. Figure 1A shows the B16F10 melanoma mouse model to compare survival times between control and treated mice. The details are summarized as follows: Mouse: Female C57 / BL6, 8 weeks old Primary tumor (+ / -50mm 3 Intratumoral vaccination with a 23G needle using vaccines containing 6 μg and 20 μg of IL-12 for Subcutaneous (sc) B16F10 mice injected with a 29G needle into two flanks (10^6 / mouse) The tumor is approximately 50 mm 3 Randomization when reached Tumor volume: Tumor measurement with calipers 3 times a week ·Weight record 3 times a week Sacrifice n=3 mice on day 7 after immunization TIL analysis of spleen and blood by flow cytometry on day 7 Collect tumors, implantation sites, and LNs at endpoint for histological analysis

[0441] Pilot Test The combined treatment (MSR+IL-12) group showed better tumor regression and survival rates (6 μg). [Example]

[0442] IL-12 MSR therapy in the abscopal B16F10 melanoma mouse model Female mice (C57 / BL6 - 8 weeks old) were inoculated with primary tumors (+ / - 50 mm) via a 23-gauge needle containing 6 μg and 20 μg of IL-12. 3 Intratumoral vaccine injections were administered to the tumor size.

[0443] Subcutaneous intraflank injections of B16F10 were given via a 29-gauge needle into two flanks.

[0444] Tumor volumes were measured by calipers three times a week.

[0445] Mouse body weights were recorded three times a week.

[0446] On day 7 of treatment, tumor infiltrating lymphocyte (TIL) analysis was performed on spleen and blood by flow cytometry.

[0447] Tumors, implantation sites, and LNs were collected at endpoint for histological analysis. [Example]

[0448] Cytokine MSR therapy in a B16F10 solid tumor mouse model Female mice (C57 / BL6-8 weeks old) received intratumoral vaccine injections via a 21-gauge needle containing 3 μg and 6 μg of cytokines.

[0449] B16F10 was injected subcutaneously into the flank via a 29-gauge needle.

[0450] Tumor volumes were measured with calipers three times a week, and body weights were recorded three times a week.

[0451] Tumor infiltrating lymphocyte (TIL) analysis was performed on spleen, lymph nodes, and blood by flow cytometry, and tumors were collected at endpoint for histological analysis. [Example]

[0452] CT-26 Colon Cancer Tumor Trial In this study, various doses of IL-12-coated MSR vaccine were administered intratumorally to mouse CT-26 colon cancer tumors to evaluate its therapeutic effect. The MSR vaccine was compared with an MSR-only group and a bolus control group. The results of this study are summarized in Table 1 below.

[0453] On day 0, mice were injected with 0.5 x 10 6 CT-26 tumor cells were inoculated sc. 3 (+ / -10mm 3 When the tumor volume reached 1000 μL, the mice were randomly divided into two groups receiving IL-12 MSR vaccine at two different doses (6 μg, 20 μg) by intratumoral injection through a 23-gauge needle (50 μL), IL-12, and MSR control. 2 Tumor dimensions were measured two days a week using a 2000 cc / 2000 scintigraphy machine. Mice were weighed three times a week during the study period, prior to challenge and treatment.

[0454] Tumor volume is 1500mm 3 Mice were euthanized if their weight loss was greater than 20% or if their body weight loss was greater than 20%. Other causes of euthanasia included tumor ulceration and bleeding, lethargy, and cachexia. Blood was collected for serum collection on days 6 and 21 post-immunization for quantification of IFNg and IL-12 by ELISA. On day 7, N3 mice were sacrificed and tumor explants were analyzed. At endpoint, inguinal lymph nodes and tumors were collected and analyzed for tumor-infiltrating lymphocyte (TIL) populations by histology. Spleens and blood were collected for CTL responses by flow cytometry. Survival rates were recorded. [Table 4] JPEG2026501821000006.jpg45168

[0455] result The mean tumor growth after 3 μg treatment with cytokines was reduced by more than six-fold when compared to non-vaccinated mice. Relative to the control (IT cytokine), treatment with 3 μg cytokines showed an approximately two-fold improvement in reduced mean tumor growth.

[0456] Figure 1B is a multivariate line graph showing the effect of interleukin-12 (IL-12) in mediating antitumor activity in a B16F10 melanoma mouse model by stimulating T cells, natural killer (NK) cells, and NK T cells through angiogenic effects. Group 1 mice (control) were unvaccinated (circles). Group 2 received a single dose (3 μg) of IL-12. Group 3 was treated with mesoporous silica rods containing IL-12 (3 μg). Group 4 received a single dose (6 μg) of IL-12. Mice treated with mesoporous silica rods containing IL-12 (6 μg) showed the smallest tumor size (hexagons).

[0457] Mice treated with mesoporous silica rods containing 6 μg of IL-12 also showed the greatest survival probability. Figures 2A-2D show the results of a mouse model survival study. Figure 2A shows the results for mice exposed to a 3 μg bolus of IL-12, a 3 μg dose of MSR IL-12, and an untreated control. Figure 2B shows the results for mice exposed to a 6 μg bolus of IL-12, a 6 μg dose of MSR IL-12, and an untreated control. As with other studies, the most significant results were observed in mice treated with mesoporous silica rods containing 6 μg of IL-12.

[0458] The mean tumor growth after 6 μg treatment with cytokines was reduced by more than 7-fold when compared to non-vaccinated mice. Relative to the control (IT cytokines), treatment with 6 μg cytokines showed an approximately 3-fold improvement in reduced mean tumor growth.

[0459] Figures 3A and 3B show the results of tumor regression and abscopal effect. As noted above, the greatest response (i.e., smallest tumor size) was observed in mice treated with mesoporous silica rods containing IL-12 (6 μg). Similar results were observed in the contralateral tumors.

[0460] Histology indicates that IL-12 / MSR treatment significantly affected tumor vasculature compared to other treatment groups. Additionally, melanophages (macrophages bearing melanocyte-derived melanin) were present in large numbers. Figures 4A-4D show histological images taken from treated and control mice. Figure 4A shows a histological sample from a 20 μg bolus injection of IL-12. Figure 4B shows a histological sample from a tumor treated with 20 μg of MSR IL-12. Figure 4C shows a histological sample from an untreated contralateral tumor. Figure 4D shows various histological samples from tumors injected with mesoporous silica rods (MSR). Histology indicates that IL-12 / MSR treatment significantly affected tumor vasculature compared to other treatment groups. Additionally, melanophages (macrophages bearing melanocyte-derived melanin) were present in large numbers.

[0461] Figures 5A-5C show the results (mean tumor volume over time) of a CT-26 colon cancer tumor study. Figure 5A shows a comparison of mice treated with MSR compared to control (PBS). Figure 5B shows the results of mice treated with MSR compared to mice treated with IL-12 (6 μg) and MSR + IL-12 (6 μg). Figure 5C shows the results of mice treated with MSR compared to mice treated with IL-12 (20 μg) and MSR + IL-12 (20 μg).

[0462] Similarly, Figures 6A-6E show the results of mean tumor volume over time. Figure 6A shows mice treated with MSR compared to control (PBS). Figure 6B shows mice treated with MSR compared to mice treated with IL-12 (6 μg). Figure 6C shows mice treated with MSR compared to mice treated with IL-12 (6 μg) and MSR + IL-12 (6 μg). Figure 6D shows mice treated with MSR compared to mice treated with IL-12 (20 μg). Figure 6E shows mice treated with MSR compared to mice treated with MSR + IL-12 (20 μg). Complete regression was observed in the following categories: a) IL-12 (6 μg) it; N1 < 0 mm out of 9 3 b) MSR + IL-12 (6 μg) it; N1 < 0 mm out of 9 3 c) IL-12 (20 μg) it; N2 < 0 mm out of 9 3 d) MSR + IL-12 (20 μg) it; N4 < 0 mm out of 9 3 [Example]

[0463] ATT-02 and ATT-02+ intratumoral and perilymphatic administration In this study, mice were inoculated subcutaneously (sc) with 1 million B16F10 tumor cells in the left flank (primary) and 250,000 in the right flank (secondary). 3 (right) and 25-50 mm 3 Mice bearing tumors ranging in size from 100 to 1200 mm (left) were randomized into treatment groups receiving 50 μl of Att-02 (IL-12 and MSR) and Att-02 + MSR (containing cytosine guanosine dinucleotide, "CpG"). Mice received either an intratumoral (it) dose and a perilymphatic dose (pl) on the ipsilateral side (larger tumor) or a pl dose on each flank (2 flanks). All treatments were administered 10 days after tumor inoculation.

[0464] Figures 7A and 7B show the results of this study (mean tumor volume over time). Figure 7A shows the mean primary tumor volume in mice treated with PBS (control) compared to: ATT-02 it.pl (intratumoral and perilymphatic IL-12 and MSR) ATT-02+it.pl (CpG-containing, intratumoral and perilymphatic IL-12 and MSR) ATT-02 pl2 flank (perilymphoid flank IL-12 and MSR) ATT-02+it.pl2 flank (containing CpG, perilymphatic flank IL-12 and MSR) Mice receiving intratumoral (it) + perilymphatic (pl) injections had the least primary tumor growth. Secondary tumor growth was minimal in both treatment variations. Overall, tumor growth was substantially less than in controls.

[0465] Primary tumor studies Similarly, Figures 8A-8E show the results of mice treated with each of the above variations. Figure 8A shows that tumors grew rapidly with PBS (control). Figure 8B shows primary tumor growth (days post-inoculation) after treatment with ATT-02 it.pl. Figure 8C shows primary tumor growth (days post-inoculation) after treatment with ATT-02 + it.pl. Figure 8D shows primary tumor growth (days post-inoculation) after treatment with ATT-02 pl flank. Figure 8E shows primary tumor growth (days post-inoculation) after treatment with ATT-02 pl2 flank. Figure 8F shows primary tumor growth (days post-inoculation) after treatment with ATT-02 + pl2 flank.

[0466] Secondary Tumor Trials Similarly, Figures 9A-9E show the results of mice treated with each of the above variations. Figure 9A shows that tumors grew rapidly with PBS (control). Figure 9B shows primary tumor growth (days post-inoculation) after treatment with ATT-02 it.pl. Figure 9C shows primary tumor growth (days post-inoculation) after treatment with ATT-02 + it.pl. Figure 9D shows primary tumor growth (days post-inoculation) after treatment with ATT-02 pl flank. Figure 9E shows primary tumor growth (days post-inoculation) after treatment with ATT-02 pl2 flank. Figure 9F shows primary tumor growth (days post-inoculation) after treatment with ATT-02 + pl2 flank. [Example]

[0467] IL-12 study in mice treated with MSR In this study, the levels of IL-12 levels were measured in mice treated with MSR and in controls.

[0468] Figure 10A is a graph comparing IL-12 levels (pg / mL) over time (hours post-treatment). Similarly, Figure 10B is a graph comparing IFN-gamma levels (pg / mL) over time (hours post-treatment).

[0469] In summary, IL-12 was detected in mice (n=6) that received two subcutaneous injections of 5 mg MSR and 20 μg IL-12. The control group received two injections of 20 μg ss IL-12. Serum samples were collected and analyzed 1 hour, 6 days, and 12 days after treatment. IL-12 (a) and IFN-gamma (b) levels were measured using ELISA kits (Life Technologies). Both cytokines were detected at baseline levels in the MSR IL-12 group.

[0470] Comparison of immune cell levels in mice treated with MSR Immune cell activity in splenocytes from mice (n = 6) treated with two subcutaneous injections of 5 mg MSR and 20 μg IL-12 compared to a control group receiving two injections of 20 μg IL-12. Splenocytes were isolated and analyzed by flow cytometry 6 and 12 days after treatment. Figures 11A-11E show quantification of T cells, B cells (11A, 11B); macrophages (11C, 11D), monocytes, and neutrophils (11E, 11F).

[0471] Comparison of IL-12 levels IL-12 detection in mice (n=5) administered a single subcutaneous injection of 1 mg MSR and 20 μg IL-12, or 5 mg MSR and 20 μg IL-12. The control group received a single injection of 20 μg IL-12. Serum samples were collected and analyzed 2 hours, 3 days, 7 days, and 14 days after treatment. IL-12(a) levels were measured using an ELISA kit (Life Technologies). Figure 12 shows IL-12 (pg / mL) versus time after treatment.

[0472] blood analysis In the next study, blood was analyzed. Results of a complete blood count from blood samples stored in EDTA tubes 14 days after treatment. Leukocytes (a), lymphocytes (b), monocytes (c), and neutrophils (d) were recorded.

[0473] Figure 13A shows white blood cell (WBC) counts versus days post-immunization. Figure 13B shows lymphocyte (LYM) counts. Figure 13C shows monocyte (MON) counts. Figure 13D shows neutrophil (NEU) counts. The results suggest a dose-response for MSR of immune cells.

[0474] Next, immune cell activity in splenocytes from mice (n=3) treated with a single subcutaneous injection of 1 mg MSR and 20 μg IL-12, or 5 mg MSR and 20 μg IL-12, compared with a control group that received a single injection of 20 μg IL-12. Splenocytes were isolated and analyzed by flow cytometry 6 and 14 days after treatment. Quantification of immune cells is shown for T cells, B cells (Figures 14A and 14B); macrophages (Figures 14C and 14D); monocytes, and neutrophils (Figures 14E and 14F).

[0475] Primary / Secondary Tumor Trials The next study involved single-dose treatment of primary tumors with ATT-02 and ATT-02 CpG. C57 / B16 mice (n=10 mice / group, n=10 mice / group with untreated ATT-02 intratumor, and n=10 mice / group with ATT-02 CpG intratumor) were inoculated sc with 10^6 and 0.25x10^6 B16F10 melanoma tumor cells in the left and right flanks, respectively. On day 10, left flank tumors were treated intratumorally with ATT-02 (1 mg MSR, 20 μg IL-12) and ATT-02 CpG (1 mg MSR, 20 μg IL-12, and 100 μg CpG). Graphs show mean tumor (Figure 15A, Figure 15D) and individual tumor growth curves (Figure 15B, Figure 15C, Figure 15E, Figure 15F) for treated and untreated tumors. Arrows indicate timing of treatment.

[0476] Figures 16A-16D show quantification of total splenocytes (a), CD8+ T cells (a, b represent zooms in the CD8 population), and KLRG-1+ and CD127+ effector memory T cells (b, c represent zooms in the KLRG-1 and CD127 populations) (mean ± SD) after ATT-02 and ATT-02 CpG treatment. Spleens were harvested from immunized mice 7 days after treatment. Detection of IFN-γ-secreting splenocytes was performed by ELISPOT assay. Splenocytes from ATT-02 and ATT-02 CpG-treated mice (n = 6) were stimulated with 30 μg / ml of neoantigen peptides (30 μg each (B16-M27, B16-M40, B16-M27, B16-M47, B16-M48) or 100 μg / ml of B16F10 cell lysate. Data shown represent the mean ± SD for each group. Unstimulated cells were used as a control. Spleens were harvested from immunized mice 7 days after treatment.

[0477] The following figures show the therapeutic effect of a single intratumoral (it), peritumoral (pt), or perilymphatic (pl) treatment with ATT-02 in a B16F10 melanoma model. C57 / Bl6 mice (n = 7 mice / group: untreated, n = 7 mice / group: ATT-02 it, n = 7 mice / group: ATT-02 pt, n = 7 mice / group: ATT-02 pl) were inoculated sc with 10^6 B16F10 melanoma tumor cells in the left flank. On day 10, left flank tumors were treated it, pt, and pl with ATT-02 (1 mg MSR, 20 μg IL-12); PBS was used as a control. Figures 17A, 17C-17F show spider plots of mean tumor and individual tumor growth curves for treated and untreated tumors, as well as overall survival (Figure 17B). Arrows indicate the timing of treatment.

[0478] Similarly, the following figure shows the therapeutic effect of dual intratumoral (it) and perilymphatic (pl) treatment of both flanks in a B16F10 melanoma cancer model. C57 Bl6 mice (naive n = 7 mice / group, ATT-02 it pl n = 7 mice / group, ATT-02 CpG it pl n = 7 mice / group, ATT-02 CpG pl n = 7 mice / group) were inoculated sc with 10^6 B16F10 melanoma tumor cells in the left flank. On day 10, the left flank tumor was treated intratumorally and perilymphatic with ATT-02 (1 mg MSR, 28 μg IL-12) and ATT-02 CpG (1 mg MSR, 28 μg IL-12, and 100 μg CpG). Figures 18A, 18C-18F show the mean tumor and individual tumor growth curves for treated tumors, and overall survival (Figure 18B). Arrows indicate timing of it and pl treatments. Arrows indicate timing of treatments.

[0479] The following figure shows the therapeutic effect of dual intratumoral (it) and perilymphatic (pl) treatment of both flanks in a B16F10 melanoma cancer model. C57 Bl6 mice (naive n = 7 mice / group, ATT-02 it pl n = 7 mice / group, ATT-02 CpG it pl n = 7 mice / group, ATT-02 CpG pl n = 7 mice / group) were inoculated sc with 10^6 B16F10 melanoma tumor cells in the left flank. On day 10, the left flank tumor was treated intratumorally and perilymphatic with ATT-02 (1 mg MSR, 28 μg IL-12) and ATT-02 CpG (1 mg MSR, 28 μg IL-12, and 100 μg CpG). Figures 19A, 19C-19G show the mean tumor and individual tumor growth curves for treated tumors, and overall survival (19B). Arrows indicate the timing of it and pl treatment.

[0480] The following figure shows the therapeutic effect of dual intratumoral (it) and perilymphatic (pl) treatment of both flanks in a B16F10 melanoma cancer model. C57 Bl6 mice (naive n = 7 mice / group, ATT-02 it pl n = 7 mice / group, ATT-02 CpG it pl n = 7 mice / group, ATT-02 CpG pl n = 7 mice / group) were inoculated sc with 10^6 B16F10 melanoma tumor cells in the left flank. On day 10, the left flank tumor was treated intratumorally and perilymphatic with ATT-02 (1 mg MSR, 28 μg IL-12) and ATT-02 CpG (1 mg MSR, 28 μg IL-12, and 100 μg CpG). Figures 20 and 21A-21E show individual tumor growth curves for the mean tumor and for untreated tumors. Arrows indicate the timing of treatment.

[0481] In the next study, mice were inoculated sc with 1 million B16F10 tumor cells in the left flank (primary) and 250,000 in the right flank (secondary). Figures 22A-22E show quantification of total splenocytes, CD8+ T cells (a, b represent zooms in the CD8 population), KLRG-1+ and CD127+ effector memory T cells (b, c represent zooms in the KLRG-1 and CD127 populations), macrophages, monocytes, and neutrophils (d) immune cells after MSR, ATT-02, and ATT-02 CpG treatment. Spleens were harvested from immunized mice 7 days after treatment. Data shown represent the mean ± SD for each group. [Example]

[0482] Solid tumor studies in mice treated with MSR In the following study, solid tumor levels were examined in mice treated with MSR and in controls.

[0483] On day 0, mice were inoculated subcutaneously with 1M CT-26 cells in the right flank. On day 7, mice were treated with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, or ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of 5 doses of PD-1 at 200 μg / mouse.

[0484] Figure 23A shows the individual tumor volumes (mm ) of the treatment groups PBS control and MSR it. 3 ) Figure 23B shows the individual tumor volumes (mm ) of IL-12 treatment. 3 ) Figure 23C shows the individual tumor volumes (mm 3 ) Figure 23D shows the individual tumor volumes (mm) of treatment group ATT-02. 3 ) Figure 23E shows the individual tumor volumes (mm ) for the treatment groups MSR + anti-PD-1. 3 ) Figure 23F shows the individual tumor volumes (mm) of the treatment groups ATT-02 + anti-PD-1. 3 ) is shown.

[0485] Mice were then inoculated subcutaneously with 1M CT-26 cells in the right flank on day 0. Treatment of mice began on day 7 with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, or ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of 5 doses of PD-1 at 200 μg / mouse. Figure 24A shows the mean tumor volume (mm) across all treatment groups. 3 ) is shown. Figure 24B shows the percent change in mean body weight for the treatment groups.

[0486] On day 0, mice were subcutaneously inoculated with 1M CT-26 cells in the right flank. On day 7, mice were treated with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of 5 doses of PD-1 at 200 μg / mouse. Figure 25A shows the survival rate of mice by group that were terminated on day 65. Figure 25B shows that surviving cohorts of ATT-02 and ATT-02 + PD-1 mice were rechallenged with 3M CT-26 cells on day 65, and tumor volumes were measured up to day 25 until the end of the study.

[0487] In the next study, mice were inoculated subcutaneously with 500K B16F10 cells in the right flank on day 0. On day 6, treatment of mice began with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of 5 doses of PD-1 at 200 μg / mouse. On day 27, cohorts of surviving mice from the ATT-02 and ATT-02 + PD-1 treatment groups received a second dose of ATT-02. Figure 26A shows the individual tumor volumes (mm ) of the PBS control and MSR it treatment groups. 3 ) Figure 26B shows the individual tumor volumes (mm ) of IL-12 treatment. 3 ) Figure 26C shows the individual tumor volumes (mm ) of patients treated with PD-1. 3 ) is shown.

[0488] Figure 26D shows individual tumor volumes (mm) for treatment group ATT-02. 3 ) Figure 26E shows the individual tumor volumes (mm ) for the treatment groups MSR + anti-PD-1. 3 ) Figure 26F shows individual tumor volumes (mm ) for the treatment groups ATT-02 + anti-PD-1. 3 ) is shown.

[0489] Mice were then inoculated subcutaneously with 500K B16F10 cells in the right flank on day 0. On day 6, treatment of mice began with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of 5 doses of PD-1 at 200 μg / mouse. On day 27, cohorts of surviving mice from treatment groups ATT-02 and ATT-02 + PD-1 received a second dose of ATT-02. Figure 27A shows the mean tumor volume (mm ) of the treatment groups. 3 ) and Figure 27B shows the percent change in mean weight for the treatment groups.

[0490] On day 0, mice were inoculated subcutaneously with 500K B16F10 cells in the right flank. On day 6, mice were treated with PBS, IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) and PD-1 (ip) every 3 days, or ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of five doses of PD-1 at 200 μg / mouse. On day 27, cohorts of surviving mice from the ATT-02 and ATT-02 + PD-1 treatment groups received a second dose of ATT-02. Figure 28 shows the percentage of surviving mice by group, which terminated the study on day 64.

[0491] On day 0, mice were subcutaneously inoculated with CT-26 cells, 3M cells, and 1M cells in the right flank (primary) and left flank (secondary) respectively. On day 8, mice received PBS, MSR (it), IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) + PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of 5 doses of PD-1 at 200 μg / mouse. Figure 29A shows the mean total tumor burden volume (mm) of the treatment groups.3 ) and Figure 29B shows the mean primary right flank tumor volume (mm 3 ) Figure 29C shows the mean secondary left flank tumor volume (mm 3 ) is shown.

[0492] Mice were then subcutaneously inoculated with CT-26 cells, 3M cells, and 1M cells in the right flank (primary) and left flank (secondary) on day 0. On day 8, treatment of mice began with PBS, MSR (it), IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) + PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of five doses of PD-1 at 200 μg / mouse. Figure 30A shows the primary tumor volume (mm ) of the treatment groups, PBS control and MSR it. 3 ) Figure 30B shows the primary tumor volume (mm ) of IL-12 treatment. 3 ) Figure 30C shows the primary tumor volume (mm ) of PD-1 treatment. 3 ) in treatment group ATT-02. 3 ) Figure 30E shows the primary tumor volume (mm ) of the treatment group MSR + anti-PD-1. 3 ). Figure 30F shows the primary tumor volume (mm ) of the treatment groups ATT-02 + anti-PD-1. 3 ) is shown.

[0493] On day 0, mice were subcutaneously inoculated with CT-26 cells, 3M cells, and 1M cells in the right flank (primary) and left flank (secondary). On day 8, mice began treatment with PBS, MSR (it), IL-12 (20 μg, it), PD-1 ip every 3 days, ATT-02 (it, 20 μg), MSR (it) + PD-1 (ip) every 3 days, and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of five doses of PD-1 at 200 μg / mouse. Figure 31A shows the secondary tumor volume (mm ) of the treatment groups: PBS control and MSR it. 3) Figure 31B shows the secondary tumor volume (mm ) of IL-12 treatment. 3 ) Figure 31C shows the secondary tumor volume (mm 3 ) Figure 31D shows the secondary tumor volume (mm ) of treatment group ATT-02. 3 ) Figure 31E shows the secondary tumor volume (mm ) of the treatment group MSR + anti-PD-1. 3 ) Figure 31F shows the secondary tumor volume (mm ) of the treatment groups ATT-02 + anti-PD-1. 3 ) is shown.

[0494] On day 0, mice were subcutaneously inoculated with CT-26 cells, 3M cells, and 1M cells in the right flank (primary) and left flank (secondary) flanks. On day 8, mice received PBS, MSR (it), IL-12 (20 μg, it), and PD-1 ip every 3 days; ATT-02 (it, 20 μg), MSR (it) + PD-1 (ip) every 3 days; and ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of five doses of PD-1 at 200 μg / mouse. Figure 32A shows primary versus secondary tumor volumes in PBS-treated mice, assessing the abscopal effect in 2 of 5 mice. Figure 32B shows primary versus secondary tumor volumes in MSR-treated mice, assessing the abscopal effect in 3 of 5 mice. Figure 32C shows IL-12-treated mice showing primary (p) vs. secondary (c) tumor volume, assessing the abscopal effect in 2 of 5 mice, and Figure 32D shows ATT-02-treated mice showing primary (p) vs. secondary (c) tumor volume, assessing the abscopal effect in 4 of 5 mice.

[0495] On day 0, mice were subcutaneously inoculated with CT-26 cells, 3M cells, and 1M cells in the right flank (primary) and left flank (secondary) flanks. On day 8, mice received PBS, MSR (it), IL-12 (20 μg, it), or PD-1 ip every 3 days; ATT-02 (it, 20 μg), MSR (it) + PD-1 (ip) every 3 days; or ATT-02 (it, 20 μg) + PD-1 (ip) every 3 days. Mice receiving PD-1 received a total of five doses of PD-1 at 200 μg per mouse. Figure 33 shows the survival rate for each treatment group. The study was terminated on day 122.

[0496] On day 0, mice were inoculated subcutaneously with 1M EMT-6 cells in the right flank. On day 7, mice were administered PBS, IL-12 (20 μg, it), or ATT-02 (it, 20 μg). Figure 34A shows the tumor volume (mm ) of the treatment groups PBS control compared to IL-12 it. 3 ) Figure 34B shows tumor volume (mm3) of the PBS control compared to ATT-02 treatment. Figure 34C shows tumor volume (mm3) of treated ATT-02 compared to IL-12. Figure 34D shows the percent change in mouse body weight over the course of the study.

[0497] On day 0, mice were inoculated subcutaneously with 1M EMT-6 cells in the right flank. On day 7, mice were administered PBS, IL-12 (20 μg, it), or ATT-02 (it, 20 μg). Figure 35 shows the mean tumor volume (mm) of the treatment groups. 3 ) is shown.

[0498] Mice were then inoculated subcutaneously with 1M EMT-6 cells in the right flank on day 0. On day 7, mice were administered PBS, IL-12 (20 μg, it), or ATT-02 (it, 20 μg). Figure 36 shows survival analysis by treatment from a study terminated on day 80.

[0499] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the right flank (primary) and left flank (secondary) respectively. On day 8, mice were administered PBS or ATT-02 it at two dose levels: low dose (28 μg) or high dose (56 μg). Figure 37A shows the mean primary tumor burden volume (mm) of the treatment groups in single-dose mice. 3 ) Figure 37B shows the primary right flank tumor volume (mm ) of the PBS control. 3 ) Figure 37C shows the primary right flank tumor volume (mm ) in the ATT-02 low-treated group. 3 ) and Figure 37D shows the primary right flank tumor volume (mm ) of ATT-02 high treatment. 3 ) in single-dose mice. Figure 37E shows the mean secondary tumor burden volume (mm 3 ) Figure 37F shows the secondary left flank tumor volume (mm) of the PBS control. 3 ) Figure 37G shows the secondary left flank tumor volume (mm 3 ) Figure 37H shows the secondary left flank tumor volume (mm ) of ATT-02 high treatment. 3 ) is shown.

[0500] Mice were then inoculated subcutaneously with B16F10 cells, 1M cells in the right flank (primary), and 0.25M cells in the left flank (secondary) on day 0. On days 10, 17, and 24, mice were administered it with PBS or ATT-02 at two dose levels: low dose (28 μg) or high dose (56 μg). Figure 38A shows the mean primary tumor burden volume (mm) of the treatment groups in multiple-dosed mice. 3 ) and Figure 38B shows the primary right flank tumor volume (mm ) of the PBS control multiple doses. 3 ) and Figure 38C shows the primary right flank tumor volume (mm ) of ATT-02 low treatment multiple doses. 3 ) Figure 38D shows the mean secondary tumor burden volume (mm 3 ) Figure 38E shows the sequential left flank tumor volume (mm ) of ATT-02 low multiple dose treatment. 3 ) Figure 38F shows the sequential left flank tumor volume (mm ) of ATT-02 high multiple dose treatment. 3) is shown. Figure 39 shows the survival probability.

[0501] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the left flank (secondary). On day 8, the single-dose cohort (1x) received PBS or ATT-02 at two dose levels: low dose (28 μg) or high dose (56 μg). Splenocytes were collected on days 7 and 14 after the single dose. Figures 40A and 40B show total CD8+ T cells, KLRG1, and CD127+ cells on day 7. Figures 40C and 40D show total CD8+ T cells, KLRG1, and CD127+ cells on day 14. The following is a summary: 1M splenocytes collected for flow Gating on live populations · SCC-A vs. CD8+ T cells KLRG-1 and CD127 counts on CD8+ cells back-counted against total cell counts

[0502] On day 0, mice were inoculated subcutaneously with B16F10 cells, 1M cells in the right flank (primary), and 0.25M cells in the left flank (secondary). On day 10, mice were administered perilymphatic (pl) or intratumoral (it) with PBS or ATT-02 + / - CpG. Figure 41 shows the mean primary tumor burden volume (mm) of the treatment groups. 3 ) is shown.

[0503] Mice were then inoculated subcutaneously with B16F10 cells, 1M cells in the right flank (primary), and 0.25M cells in the left flank (secondary) on day 0. On day 10, mice were administered perilymphatic (pl) or intratumoral (it) with PBS or ATT-02 + / - CpG. Figure 42A shows the tumor volume (mm ) of the treated groups compared with the PBS control. 3 ) Figure 42B shows the tumor volume (mm ) of the MSR perilymphatic treatment. 3 ) Figure 42C shows the tumor volume (mm ) of ATT-02 it treatment. 3 ) Figure 42D shows the perilymphatic tumor volume (mm) of treatment group ATT-02. 3) in the treatment group ATT-02+CpG it. 3 ) Figure 42F shows the tumor volume (mm ) surrounding lymph nodes in the treatment group ATT-02+CpG. 3 ) is shown.

[0504] On day 0, mice were inoculated subcutaneously with B16F10 cells, 1M cells in the right flank (primary), and 0.25M cells in the left flank (secondary). On day 10, mice were administered perilymphatic (pl) or intratumoral (it) with PBS or ATT-02 + / - CpG. Figure 43 shows the mean secondary tumor burden volume (mm) of the treatment groups. 3 ) is shown.

[0505] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the right flank (primary) and left flank (secondary) respectively. On day 10, mice were administered perilymphatic (pl) or intratumoral (it) with PBS or ATT-02 + / - CpG. Figure 44A shows the secondary tumor volume (mm ) of the treated groups compared with the PBS control. 3 ) Figure 44B shows the secondary tumor volume (mm 3 ) Figure 44C shows the secondary tumor volume (mm 3 ). Figure 44D shows the secondary tumor volume (mm 2) of the treatment group ATT-02 perilymphatic 3 ) Figure 44E shows the secondary tumor volume (mm ) of the treatment group ATT-02+CpG it. 3 ). Figure 44F shows the secondary tumor volume (mm ) surrounding lymph nodes in the treatment group ATT-02+CpG. 3 ) is shown.

[0506] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells in the right flank (primary), and 0.25M cells in the left flank (secondary). Figure 45 shows survival analysis between treatment groups. The study was terminated on day 34.

[0507] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the left flank (secondary). On day 10, mice received PBS or ATT-02+ / -CpG perilymphatic (pl) or intratumoral (it). Splenocytes were collected 7 days after administration for FLOW analysis. 1M splenocytes were collected for FLOW analysis with gating on the raw population SCC-A versus CD8+ for T cells TCF-1+, KLRG1+, CD44+, and CD127 in the CD8+ population for regulatory T cells and activated T cells, back-counted against total cell counts. Figure 46A shows the total CD8+ T cell population. Figure 46B shows the CD8+TCF-1+ population. Figure 46C shows the CD8+KLRG1+ population. Figure 46D shows the CD8+CD127+ population. Additionally, Figure 46E shows the CD8+CD44+ population.

[0508] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the left flank (secondary). On day 10, mice received PBS or ATT-02+ / -CpG perilymphatic (pl) or intratumoral (it). Splenocytes were collected 7 days after administration for FLOW analysis. 1M splenocytes were collected for FLOW analysis with gating on the raw populations CD11b+GR-1+; CD11b+CD86+; CD11b+MHC-II+ for macrophages and monocytes, back-counted against total cell counts. Figure 47A shows the total CD11b+GR-1+ population. Figure 47B shows the CD11b+CD86+ population. Figure 47C shows the CD11b+MHCII+ population.

[0509] As described above, on day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the left flank (secondary) as described above. On day 10, mice received PBS or ATT-02+ / -CpG either perilymphatic (pl) or intratumoral (it). Splenocytes were collected 7 days after administration for FLOW analysis. 1M splenocytes were collected for FLOW analysis with gating on the raw populations CD11c+GR-1+; CD11c+CD86+; CD11c+MHC-II+ for macrophages and monocytes, back-counted against total cell counts. Figure 48A shows the total CD11c+GR-1+ population. Figure 48B shows the CD11c+CD86+ population. Figure 48C shows the CD11c+MHCII+ population.

[0510] On day 0, mice were subcutaneously inoculated with B16F10 cells, 1M cells, and 0.25M cells in the left flank (secondary). On day 10, mice received PBS or ATT-02 + / - CpG either perilymphatic (pl) or intratumoral (it). Splenocytes were collected 7 days after administration for interferon gamma (IFNg) analysis. 1M splenocytes were co-cultured with either 30 μg each of B16 peptides (B16-M27, B16-M40, B16-M27, B16-M47, B16-M48) or B16F10 cell lysate. Unstimulated cells were used as a control. Figure 49 shows IFNgamma expression by treatment group. [Example]

[0511] Further studies on mice treated with MSR Despite the remarkable clinical success of immune checkpoint inhibitor (ICI) therapy in advanced cutaneous melanoma (SKCM), a high unmet medical need remains, as only a subpopulation of these patients respond, and many of these patients subsequently progress. In patients with advanced SKCM treated with pembrolizumab monotherapy, for example, approximately 60% do not respond, with a median progression-free survival (PFS) of approximately 5.6 months. Myeloid cell populations execute a characteristic immunosuppressive program in the tumor microenvironment. Indeed, the presence of immunosuppressive tumor-associated macrophages (TAMs) and other anti-inflammatory myeloid cells strongly correlates with poor prognosis across multiple tumor types and treatments, including resistance to anti-PD(L)-1 therapeutics. While overly simplistic, the M1 / M2 paradigm captures the functional antipodes of macrophage states, where M1 macrophages are potently pro-inflammatory / immunogenic and M2 macrophages are potently immunosuppressive. TAMs are known to be phenotypically plastic, occupying metastable functional states rather than a locked-in phenotype, and remain responsive to local signals that influence their functional state, opening the possibility of therapeutically converting M2 immunosuppressive TAMs into immunogenic M1 populations.

[0512] Interleukin-12 (IL-12) is a potent pro-inflammatory cytokine expressed primarily by activated innate immune cells, including M1 macrophages, neutrophils, and dendritic cells, providing a crucial link between upstream “danger sensing” and downstream adaptive immune responses [16–19]. Unfortunately, attempts to deliver IL-12 systemically have been hampered by the toxicity associated with systemic exposure.

[0513] However, because its key site of action is within the tumor microenvironment (TME), intralesional IL-12 therapy is being actively pursued as an intratumoral therapy. Importantly, IL-12 activates NK cells and antigen-experienced T cells, leading to the secretion of IFNγ, which further upregulates IL-12 expression, creating a crucial feed-forward activation loop. Once established within the TME, this "IL-12 / IFNγ cycle" can convert immunosuppressive TAMs into immunogenic "M1" macrophages. In a landmark 2011 paper, Restifo et al. demonstrated that IL-12 upregulates key components of the antigen processing and presentation machinery in TAMs, enabling them to effectively cross-present tumor antigens from dying tumor cells. Furthermore, these converted inflammatory TAMs express important costimulatory molecules, pro-inflammatory cytokines, chemokines, and other factors necessary to drive effective type I antitumor immune responses. Thus, intralesional IL-12 is sufficient to drive effective "in situ vaccination." A clear advantage of the in situ vaccine approach over antigen-specific vaccines is that dead tumor cells serve as the antigen source, allowing the patient's own immune system to "personalize" the selection of the optimal immunogenic tumor antigen.

[0514] One of the leading IL-12-based in situ vaccine approaches involves intralesional IL-12 gene delivery using DNA-encoded IL-12 (tavokinogene telseplasmid; Tavo) and in vivo electroporation (Tavo-EP). Although clear immunological activity and sporadic clinical benefit have been observed in preclinical models and in patients with melanoma, Merkel cell carcinoma, and TNBC, this approach failed to meet a pre-specified ORR in combination with pembrolizumab in a recent phase 2 trial in patients with advanced cutaneous melanoma who were refractory or had progressed on prior anti-PD(L)-1 therapy. Applicants hypothesize that the clinical failure of Tavo-EP was the result of a difficult-to-control drug delivery system exacerbated by the inherent heterogeneity of the tumor microenvironment, which likely varied in terms of the density of both IL-12-responsive / IFNγ-secreting cells (NK cells and T cells) and IFNγ-responsive tumor amniotic membrane proteins (TAMs).

[0515] Importantly, analyses of both clinical trial samples and experimental models highlight the importance of activating the IFNγ / CXCL9 / CXCR3 cytokine / chemokine cascade to enable successful in situ vaccination and responses to anti-PD(L)-1 agents. Indeed, analysis of melanoma tumors from a phase 2 clinical trial of Tavo-EP plus pembrolizumab in patients with low TILs, who typically do not respond to pembrolizumab monotherapy, demonstrated that induction of intratumoral CXCR3 mRNA within the TME strongly correlated with clinical response, an effect presumably attributable to increased infiltration of CXCR3+ T cells and NK cells into the tumor. Furthermore, using an experimental murine TNBC model and patient samples from a phase 2 "window-of-opportunity" trial, Telli et al. identified that response to intralesional Tavo-EP required activation of the IL-12 / IFNγ cycle, accompanied by downstream induction of the IFNγ / CXCL9 / CXCR3 recruitment pathway. Collectively, these multiple preclinical and clinical studies demonstrate that activation of the IL-12 / IFNγ loop can successfully convert the TME / tumor immune cycle from a tolerogenic state to an inflammatory, immunogenic state. Importantly, however, the feedforward amplification required to achieve this TME "tipping point" depends on the presence of IL-12-responsive / IFNγ-secreting cells (e.g., T cells, NK cells) and IFNγ-responsive cells (e.g., TAMs, DCs) that secrete IL-12- and IFNγ-dependent chemokines, such as CXCL9 and CXCL10. Secretion of these T cell and NK cell chemokines can further amplify this cycle by recruiting IL-12-responsive CXCR3+ T cells and NK cells to the TME. Figure 55 outlines these key aspects of the IL-12 / IFNγ cycle. The purpose of the planned grant is to test a series of known immunomodulatory molecules for their ability to enhance the ability of IL-12 to trigger the IL-12 / INF gamma cycle within the TME and promote sustained antitumor T cell activity and tumor regression.

[0516] Figure 55 shows the IL-12 in situ vaccine paradigm. IL-12 in situ vaccine efficacy depends on the activation of the IL-12 / IFNγ feed-forward loop within the tumor microenvironment (TME). IL-12 activates intratumoral antigen-experienced T cells and NK cells, leading to the secretion of IFNγ. IFNγ then activates tumor-associated macrophages (TAMs). This converts immunosuppressive "M2-like" TAMs into inflammatory cross-presenting "M1-like" macrophages. IFNγ-stimulated TAMs then secrete chemokines (e.g., CXCL9 and CXCL10), which recruit new CXCR3+ antigen-experienced T cells and NK cells to the TME. When exposed to IL-12, these newly recruited inflammatory cells acquire enhanced cytotoxic activity and secrete IFNγ, further amplifying the cycle.

[0517] The MSR-based drug delivery system has several novel features that address the limitations of previous IL-12-based in situ vaccines. MSRs are silica rods approximately 100 microns long and 5 microns in diameter, providing a massive surface area (approximately 900 m / mg MSR) that facilitates the adsorption of immunomodulatory proteins (e.g., IL-12). They contain abundant small pores, averaging 5-10 nm in diameter. Additionally, MSRs exhibit inherent pro-inflammatory effects that have been demonstrated to act via NLR3-dependent inflammasome activation. In the context of in situ vaccines, the pro-inflammatory effects of mesoporous silica may add significant adjuvant effects. Thus, ATT-02 already represents a combinatorial immunomodulatory modality. Another key innovation inherent to this platform is its modularity. Individual batches of MSRs can be loaded with a single immune stimulatory protein, and combination products can then be easily assembled by simply mixing pre-loaded MSRs. This modularity facilitates the formulation and testing of candidate IL-12 combinations; testing multiple combinations, including dual, triple, and other combinations, is technically impractical in other systems. Finally, unlike many biomaterial drug delivery strategies, MSRs can be easily manufactured at a scale suitable for clinical trial use and commercialization.

[0518] In vitro testing of ATT-02 (IL-12 monotherapy) versus ATT-02+ (IL-12 in combination with an additional well-characterized immunostimulatory agent) to optimize the "conversion" of immunosuppressed immature bone marrow APCs into activated immunogenic APCs with enhanced capacity for cross-presentation (referred to as "Aim 1").

[0519] Because the goal of Aim 1 is to screen for potential immune activators that synergize with IL-12, we developed an in vitro system that allows for efficient screening by focusing on their ability to drive effective cross-presentation by macrophages using ovalbumin-specific CD8 T cells from OT-1 transgenic mice as a readout. Briefly, transwell inserts with 8-micron pore size are coated with Matrigel to facilitate injection of MSR. M2 macrophages are generated from bone marrow harvested from C57BL / 6 H2-Kb+ mice according to standard protocols. After polarization, M2 macrophages are harvested, counted, and 0.5 × 106 cells are plated onto Matrigel. To track proliferation by flow cytometry, 1 × 106 OT-1 CD8 T cells isolated from the spleen of OT-1 transgenic mice are labeled with the fluorescent dye CFSE. While the total amount of MSR is kept constant, a total of 1 mg of MSR is injected into the Matrigel layer of each insert. The MSR does not contain a payload, IL-12 alone or various combinations of immune stimulatory molecules as outlined below.

[0520] (Table 3). In previous studies, Applicants established that successful IL-12-mediated in situ vaccination is characterized by the generation of a unique KLRG1hiPD-1loCTLA-4lo phenotype within the CD8 population, similar to what has been identified in viral immunology as short-lived effector cells (also known as SLECs). After 72 hours of incubation, cells are harvested and analyzed by flow cytometry to assess the degree of OT-1 proliferation and the proportion of CD8 T cells displaying this unique "SLEC-like" effector T cell phenotype. The flow panel includes: live / dead dye, CSFE, CD8, KLRG1, CD127, PD-1, CTLA-4, CD107a, CD44, and CD69. Positive controls include stimulation of proliferation with CD3 / 28-activating beads.

[0521] Expected Results / Pitfalls / Discussion: While this technology can accommodate combination approaches with numerous components, ATT-02+ may preferably be limited to a total of three payloads to limit the cost and complexity of product manufacturing. Proposed molecules combined with IL-12 have published data supporting either enhanced M1 macrophage polarization, cross-presentation, or in situ vaccine efficacy (references included in Table 3). Applicants anticipate the identification of several IL-12+ "X" doublets and IL-12+ "X" + "Y" triplet combinations that result in significant increases in (1) OT-1 proliferation and (2) the percentage of CD8+ cells with the KLRG1+ phenotype. Because all endpoints are quantitative with continuous variable readouts, ranking combinations is not anticipated to be problematic. Each of the 45 conditions (no treatment / MSR alone / IL-12 alone / IL-12 + "X" (n=6) / IL-12 + "X" + "Y" (n=36)) was repeated in quadruplicate to assess variance and achieve confidence in signal consistency.

[0522] A second experiment was then performed comparing "IL-12 alone" with the top five ranked combinations, using a 1-way ANOVA with Bonferroni correction for multiple pairwise comparisons. If ranking by OT-1 proliferation and percentage of KLRG1+OT-1 cells fails to distinguish these combinations, Applicants will consider increasing the stringency of the assay by enhancing the immunosuppressive phenotype of macrophages with hydrocortisone and IL-10. Goal of Aim 1: Compare the best-performing IL-12 combination to IL-12 alone (aka ATT-02) in vivo in Aim 2. If no combination increases OT-1 proliferation or the frequency of KLRG1+OT-1 cells by >50%, then ATT-02 alone will be further tested in Aim 2 and Aim 3. [Table 5]

[0523] ATT-02 (IL-12) monotherapy versus ATT-02+ will be tested in the two-tumor "abscopal" B16-OVA model. The primary endpoint is tumor growth inhibition of untreated tumors. Other endpoints include (1) tumor growth inhibition and total tumor burden in treated tumors; (2) survival rate; (3) assessment of antigen-specific T cell responses (i.e., SIINFEKL / Kb tetramer + CD8); (5) the percentage of TAMs presenting the SIINFEKL peptide within the H2-Kb groove (stained with clone 25-D1.16); and (6) flow-based immunophenotyping of T and NK cells. Attivare has conducted experiments testing ATT-02 (IL12+MSR) as an intralesional treatment in several syngeneic tumor models, including CT26, EMT-6, and the B16F10 model, and in each case, ATT-02 demonstrated significant tumor growth inhibition of treated tumors compared to vehicle (PBS) and recombinant IL-12.

[0524] Based on these data, Applicants tested ATT-02 in the difficult-to-treat B16F10 2-tumor "abscopal" model, treating only a single tumor and leaving the contralateral tumor untreated. In this study, the primary tumor was expanded to approximately 100 mm before treatment with ATT-02 was initiated. 3 ATT-02 demonstrated significant growth inhibition (the "abscopal effect") of both treated and untreated tumors, indicating that ATT-02 can generate a systemic antitumor immune response (Figure 57) and provide a significant survival benefit to ATT-02-treated mice. In this study, treated and untreated tumors from mice treated with low (28 mg / mg MSR) or high (56 mg / mg MSR) doses of ATT-02 were harvested for gene expression analysis 7 days after treatment. Both high and low doses of ATT-02 resulted in a significant induction of key INFγ-inducible genes in treated tumors (Figure 56D). These genes were also increased, albeit to a lesser extent, in distant untreated tumors at this early time point (Figure 56E).

[0525] Figure 56 shows that incorporation of IL-12 into mesoporous silica rods (MSR) significantly improves antitumor activity and survival compared to recombinant IL-12 alone. Three different mouse syngeneic tumor models (CT26 colorectal carcinoma, EMT-6 breast carcinoma, and B16F10 melanoma) were used to investigate the antitumor effect of intralesional injection of ATT-02. Generally, approximately 1 × 10 5 ~1×10 6 Tumor cells were implanted into a single site in the flank of female mice of the appropriate syngeneic strain, measuring approximately 50 mm in volume before treatment initiation. 3 Tumor volumes were measured using digital calipers. Total tumor volume was 1000-2000 mm. 3 Mice were sacrificed when tumor growth reached 100%. Significant tumor growth inhibition was observed in all three experimental systems. A clear survival benefit was observed in all three models, with complete responses observed in both the CT26 and EMT-6 experiments. (Figures 56A and 56B) On day 0, mice were inoculated subcutaneously with 1M CT-26 cells in the right flank. On day 7, mice were treated with PBS, mIL-12 (20 μg, it), or MSR (it). (Figures 56C and 56D) On day 0, mice were inoculated subcutaneously with 1M EMT-6 cells in the right flank. On day 7, mice were administered PBS, mIL-12 (20 μg, it), or ATT-02 (it, 20 μg IL-12, MSR). (Figures 56E and 56F) On day 0, mice were inoculated subcutaneously with 500K B16F10 cells in the right flank. On day 6, mice were treated with PBS, mIL-12 (20 μg, it), ATT-02 (it, 20 μg IL-12, MSR), or MSR (it). In the CT-26 experiment, 4 / 7 ATT-02-treated CT26 mice achieved a CR compared with 0 / 7 IL-12-treated mice. In the EMT-6 experiment, 7 / 9 mice treated with ATT-02 achieved a CR compared with 3 / 9 mice treated with IL-12.

[0526] In Aim 2, Applicants continued their studies with the two-tumor B16F10 model by using the related B16-OVA model to facilitate comparison of ATT-02 (IL-12+MSR) versus ATT-02+ (the best-performing combination from Aim 1) based on quantification of antigen-specific CD8 T cell responses (SIINFEKL-tetramer+CD8 T cells by flow cytometry) and tumor growth inhibition and survival. Briefly, 6-8 week-old female C57BL / 6 mice were injected with 1.0 x 10 6 tumor cells and, at the same time, a second inoculation (0.25 x 10 6 The larger tumor (right) was 100 mm 3 When tumor volume reached 1000-2000 mm, mice were randomized into three treatment groups (12 mice / group): PBS control, ATT-02, and ATT-02+. 3Tumor growth was measured 2–3 times weekly using digital calipers on the primary treated tumor (right) and the contralateral untreated tumor (left) until tumor growth approached 40% or until no progression was identified on day 45. Health checks and body weights were recorded at each tumor measurement. The same experiment was repeated three times to ensure the robustness of tumor growth inhibition. After establishing the reproducibility of growth inhibition in this model, additional experiments were performed to harvest tumors (treated and untreated) for gene expression (Nanostring) on ​​days 3, 7, and 10 to quantify the induction of IFNγ-dependent genes and flow cytometry immunophenotyping of lymphocyte and NK cell populations. Because these tumors express the SIINFEKL epitope of ovalbumin, a highly immunogenic antigen presented by the H2-Kb MHC class allele that presents SIINFEKL, Applicants were able to quantify and phenotype tumor antigen-specific CD8 T cell responses systemically (e.g., in the spleen) and in both treated and untreated tumor TILs. Furthermore, macrophages isolated from dissociated tumors were analyzed for the expression of SIINFEKL / Kb complexes and costimulatory molecules (e.g., CD80 / CD86), which are indicative of cross-presenting APCs. Our analytical approach in Aim 2 largely followed previously published work. Terminal bleeding was used for cytokine measurements (e.g., IL-12, INFγ, CXCL9, CXCL10) and serum markers of liver injury (AST / ALT).

[0527] ATT-02 was predicted to demonstrate significant tumor growth inhibition, including an increase in complete responses and a significant improvement in survival, with ATT-02+ significantly superior to ATT-02 in terms of anti-tumor efficacy (tumor growth inhibition of treated and untreated tumors).

[0528] Furthermore, tumor transcriptional analysis predicted a transformation from M2 to M1-dominant TME, including upregulation of APM (antigen processing and presentation machinery) and the influx of KLRG1+SIINFEKL / Kb tetramer+ CD8 T cells. Kaplan-Meier survival curves were generated using Prism software and compared using the log-rank test. Other continuously variable endpoints were compared and assessed for significance using ANOVA. Sample size calculations were performed using the following assumptions: a type 1 error rate (a) of 5%; power = 80%; two treatment groups (ATT-02, ATT-02+) and a PBS control; 1000 mm3 (mean < H0), and a very conservative estimate of SD (1000) from our previous experiments. Based on this analysis, a recommended group sample size of >11 was used.

[0529] Figures 57A-57E show that intralesional ATT-02 promotes significant primary and abscopal tumor growth inhibition and increased survival, correlating with the induction of an interferon-γ-dependent gene signature in a difficult-to-treat B16F10 syngeneic tumor model. On day 0, mice were inoculated subcutaneously with B16F10 cells, 1M cells, and 0.25M cells in the right flank (primary) and left flank (secondary). On days 10, 17, and 24, mice received intratumoral administration of PBS or ATT-02 at two dose levels: low (28 μg) or high (56 μg) doses into the primary tumor. Nanostring analysis was completed on samples 7 days after treatment.

[0530] Aim 3: (1) Optimize ATT-02+ dose and schedule in an autologous MMTV-PyMT mouse breast tumor model through analysis of dose / tumor burden / activity relationships, including the immunological and therapeutic impact of multiple simultaneous and repeated administration scenarios. A significant obstacle to the clinical development of Tavo-EP has been the lack of robust preclinical data addressing dose-activity relationships, particularly whether the ratio of treated tumor burden to overall tumor burden influences treatment efficacy. In this Aim, Applicants utilized the MMTV-PyMT breast cancer mouse model, which is characterized by high tumor formation at a young age (approximately 100% of hemizygous females within 60 days). Because these tumors are multifocal and grow slower in this autologous model than in typical syngeneic cell transplant models, (1) we can better test the hypothesis that multiple simultaneous and / or repeated treatments increase antitumor T cell activity and significantly delay tumor progression. Cohorts of hemizygous female mice will be monitored twice weekly for tumor growth by palpation and health checks. Tumor growth is monitored weekly using digital caliper measurements of any palpable tumors. Furthermore, 50 μl of blood sampled weekly from the submandibular vein for quantification of tumor circulating cell-free DNA (cfDNA) by quantitative PCR of the PyMT transgene in mouse models has proven useful as a measure of tumor burden and response to treatment. Tumor antigen-specific T cell responses to the PyMT oncogene are quantified by flow cytometry using the H-2Dq MT241-250 (LPSLLSNPTY) tetramer, as previously reported in conjunction with the KLRG1 / "SLEC" flow panel described in Aim2.

[0531] SubAim 3.1: To test whether the antitumor efficacy of ATT-02+ depends on the proportion of tumor burden treated, a cohort of 64 hemizygous female mice was monitored for tumor development and randomized into four groups of 16 mice each receiving a single treatment consisting of no treatment, injection of ATT-02+ into one tumor, injection of three tumors, or injection of every palpable tumor. Eight mice per group were followed for tumor growth rate and survival. Four mice per group were euthanized after 7 and 14 days for histopathological diagnosis, multiplex IHC (mIHC), and analysis of the tumor microenvironment for Nanostring-based gene expression, as described in Aim 2. The frequency of tumor antigen (e.g., PyMT 241-250)-specific CD8 (i.e., H-2Dq MT241-250 tetramer+) and KLRG1+ CD8 (SLECS) was quantified by flow cytometry. Tumor burden was assessed using digital caliper measurements and quantitative PCR for PyMT cfDNA as described above.

[0532] SubAim 3.2: This experiment will be analyzed with a similar design to SubAim 3.1, except that ATT-02+ will only be injected into a single tumor per treatment. Cohorts of 12 mice will be randomly divided into four groups: (1) no treatment; (2) a single dose; (3) two doses spaced one week apart; and (4) three doses spaced one week apart. Expected Results / Pitfalls / Discussions: We predict that the immunological activity, tumor growth inhibition, and survival benefit of ATT-02+ treatment will be significantly enhanced if a larger proportion of the tumor burden is treated and if tumors are repeatedly dosed. These studies will determine the dose-response relationship and, importantly, whether multiple simultaneous or repeated treatments result in a plateau effect. One obvious caveat to Aim 3 is that MMTV-PyMT is a breast cancer model, not a melanoma model. We argue that, at least in the development of immuno-oncology, the mechanisms of immune dysregulation and the corresponding TME phenotype are more intimate than the histogenesis of malignant cells. For example, when developing pembrolizumab, our preclinical IND-enabling model was MC38, a carcinogen-induced colon cancer cell line that produced an inflammatory TME enriched in PD-1+ exhausted CD8 cells, similar to PD-1-responsive melanoma.

[0533] Taken together, the multifocality, slow tumor growth rate, high M2 TAM content, and availability of a tumor antigen-specific tetramer assay for immune monitoring make this a useful model for addressing Aim 3. Objective: Completion of the study will help rationalize the dose and schedule strategies implemented in our upcoming Phase 1 clinical trials of ATT-02+.

[0534] Successful completion of these studies will define the composition of our IL-12 MSR-based drug candidate (ATT-02 or ATT-02+), provide preclinical efficacy, preliminary non-GLP safety data, and help inform the rationale for first-in-human dosing to support investigational new drug (IND) applications for the treatment of patients with advanced cutaneous melanoma refractory to anti-PD1 blockade. * * *

[0535] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect such variations to be utilized by those skilled in the art, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of all possible variations of the above-described embodiments is encompassed by this invention unless otherwise specified herein or clearly contradicted by context.

[0536] The grouping of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with the other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to include the modified group and thus fulfill the written description of all Markush groups used in the appended claims.

[0537] Unless otherwise specified, all numbers expressing features, items, quantities, parameters, characteristics, terms, etc. used in the specification and claims should be understood to be modified in all instances by the term "about." As used herein, the term "about" means that the so-modified feature, item, quantity, parameter, characteristic, or term encompasses a range of 10% above and below the value of the stated feature, item, quantity, parameter, characteristic, or term. Thus, unless specified to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical designation should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate numerical value falling within that range. Unless otherwise stated herein, each individual value of a numerical range is incorporated herein as if it were individually recited herein.

[0538] The terms "a," "an," "the," and similar referents as used in the context of describing the present invention (particularly in the context of the claims that follow) should be construed to encompass both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further clarify the invention and does not limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0539] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in a claim, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim, whether as filed or added by amendment. The transitional term "consisting essentially of" limits the scope of the claim to specific materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are essentially or explicitly described and enabled herein.

[0540] The grouping of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with the other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to include the modified group and thus fulfill the written description of all Markush groups used in the appended claims.

[0541] All patents, patent publications, and other publications referenced and identified herein are expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methods described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date of these documents or representations as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0542] Finally, while aspects of the present specification have been emphasized by reference to particular embodiments, it should be understood that those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to the particular methods, protocols, and / or reagents, etc., described herein. Accordingly, various modifications or variations to, or alternative configurations of, the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the specification. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Therefore, the present invention is not limited to that precisely as shown and described.

Claims

1. Mesoporous silica rods composed of cytokine payloads for injection into tumors or infections.

2. 2. The mesoporous silica rod of claim 1, wherein the cytokine payload is interleukin-12 (IL-12) and / or interleukin-2 (IL-2).

3. 10. The mesoporous silica rod of claim 1 further comprising an adjuvant.

4. 4. The mesoporous silica rod of claim 3, wherein the adjuvant is one or more of aluminum hydroxide (alum), lipopolysaccharide (LPS), and a toll-like receptor agonist (TLR agonist).

5. 10. The mesoporous silica rod of claim 1, which is substantially cylindrical.

6. 10. The mesoporous silica rod of claim 1, which is composed of an internal labyrinth structure.

7. A method of treating a disease, comprising: a) identifying said disease in affected tissue; and b) inserting the mesoporous silica rod of claim 1 into or near the affected tissue; A method comprising:

8. 8. The method of claim 7, further comprising administering a therapeutic amount of an anti-PD-1 antibody.

9. 8. The method of claim 7, wherein the disease is a skin and / or soft tissue infection.

10. 10. The method of claim 9, wherein the infection is a bacterial infection, a fungal infection, or a viral infection.

11. The method of claim 7, wherein the disease is cancer.

12. 12. The method of claim 11, wherein the cancer is at least one of breast, lung, kidney, bladder, urinary tract, urethra, penis, vulva, vagina, cervix, colon, ovary, prostate, pancreas, stomach, brain, head and neck, skin, uterus, testicle, esophagus, head or neck cancer, brain cancer (e.g., glioma or glioblastoma), and liver cancer.

13. 1. A method of enhancing or stimulating an endogenous immune response in an affected tissue, comprising: a) identifying the diseased tissue; and b) inserting a mesoporous silica rod into or near the affected tissue; A method comprising:

14. The method of claim 13 , wherein the mesoporous silica rods comprise a cytokine payload.

15. 15. The method of claim 14, wherein the cytokine payload is interleukin-12 (IL-12) and / or interleukin-2 (IL-2).

16. The method of claim 13 , wherein the mesoporous silica rods further comprise an adjuvant.

17. 17. The method of claim 16, wherein the adjuvant is one or more of aluminum hydroxide (alum), lipopolysaccharide (LPS), and a toll-like receptor agonist (TLR agonist).

18. The method of claim 13, wherein the mesoporous silica rods are substantially cylindrical.

19. The method of claim 13 , wherein the mesoporous silica rods comprise an internal labyrinth structure.

20. 14. The method of claim 13, wherein the diseased tissue is tumor tissue.

21. 21. The method of claim 20, wherein the tumor tissue is pre-malignant or malignant.

22. 14. The method of claim 13, wherein the diseased tissue is an infected tissue.

23. 23. The method of claim 22, wherein the infected tissue is infected with a bacteria, a fungus, or a virus.

24. The method of claim 13 , wherein the immune response is innate and / or adaptive.

25. 14. The method of claim 13, further comprising administering a therapeutic amount of an anti-PD-1 antibody.

26. 1. A method for converting a cold tumor into a hot tumor, comprising: a) identifying a cold tumor within a tissue of interest; and b) inserting a mesoporous silica rod into, at, or near the tumor; A method comprising:

27. 27. The method of claim 26, wherein the mesoporous silica rods comprise a cytokine payload.

28. 28. The method of claim 27, wherein the cytokine payload is interleukin-12 (IL-12) and / or interleukin-2 (IL-2).

29. 27. The method of claim 26, wherein the mesoporous silica rods further comprise an adjuvant.

30. 30. The method of claim 29, wherein the adjuvant is one or more of aluminum hydroxide (alum), lipopolysaccharide (LPS), and a toll-like receptor agonist (TLR agonist).

31. 27. The method of claim 26, wherein the mesoporous silica rods are substantially cylindrical.

32. 27. The method of claim 26, wherein the mesoporous silica rod comprises an internal labyrinth structure.

33. 27. The method of claim 26, wherein the cold tumor tissue is pre-malignant or malignant.

34. The method of claim 26, wherein the immune response is innate and / or adaptive.

35. 27. The method of claim 26, further comprising administration of one or more immune checkpoint inhibitors.

36. 1. A method of stimulating an immune response in a target tissue of a subject, comprising: a) identifying the target tissue; and b) inserting a mesoporous silica rod into or near said target tissue; Including, The method, wherein the mesoporous silica rods comprise a cytokine payload.

37. 37. The method of claim 36, wherein the cytokine payload is an interleukin.

38. 38. The method of claim 37, wherein the interleukin is interleukin-12 (IL-12) and / or interleukin-2 (IL-2).

39. 37. The method of claim 36, wherein the mesoporous silica rods further comprise an adjuvant.

40. 40. The method of claim 39, wherein the adjuvant is one or more of aluminum hydroxide (alum), lipopolysaccharide (LPS), and a toll-like receptor agonist (TLR agonist).

41. 37. The method of claim 36, wherein the mesoporous silica rods are substantially cylindrical.

42. 37. The method of claim 36, wherein the mesoporous silica rod comprises an internal labyrinth structure.

43. 37. The method of claim 36, wherein the target tissue is a tumor tissue.

44. 44. The method of claim 43, wherein the tumor tissue is pre-malignant or malignant.

45. 37. The method of claim 36, wherein the target tissue is an infected tissue.

46. 46. ​​The method of claim 45, wherein the infected tissue is infected with bacteria, fungi and / or viruses.

47. 37. The method of claim 36, wherein the immune response is innate and / or adaptive.

48. 1. A plurality of mesoporous silica rods for delivering a physical injury to a tumor or target area of ​​a subject, comprising: the mesoporous silica rods comprise a cytokine payload and an adjuvant; A plurality of mesoporous silica rods, wherein said cytokine payload and / or adjuvant are configured for sustained or delayed release into said tumor or inflamed area.

49. 49. The mesoporous silica rod of claim 48, wherein the cytokine payload is selected from interleukin-12 (IL-12) and interleukin-2 (IL-2).

50. 49. The mesoporous silica rod of claim 48, wherein the adjuvant is selected from aluminum hydroxide (alum), lipopolysaccharide (LPS) and a toll-like receptor agonist (TLR agonist).

51. 47. The mesoporous silica rod of claim 46, which is substantially cylindrical.

52. 49. A mesoporous silica rod according to claim 48, comprising an internal labyrinth structure.

53. 49. The mesoporous silica rods of claim 48, wherein each of said silica rods comprises about 3 μg of cytokine payload.

54. 49. The mesoporous silica rods of claim 48, wherein each of said silica rods comprises about 6 μg of cytokine payload.

55. 49. The mesoporous silica rods of claim 48, wherein each of said silica rods comprises about 20 μg of cytokine payload.

56. 49. A method of stimulating an immune response in a subject, comprising injecting a plurality of mesoporous silica rods according to claim 48 into the subject.

57. 57. The method of claim 56, wherein the subject is in need of treatment for tumor tissue or infected tissue.

58. 1. A mesoporous silica structure for delivering a physical injury to a tumor or diseased area, comprising: The mesoporous silica structure dissolves within the tumor or the affected area and induces an innate immune response.

59. 59. The mesoporous silica structure of claim 58, which dissolves over a period of one week.

60. 59. The mesoporous silica structure of claim 58, which dissolves over a period of about one month.

61. 59. The mesoporous silica structure of claim 58, comprising a cytokine payload.

62. 59. The mesoporous silica structure of claim 58, comprising an adjuvant.

63. 59. The mesoporous silica structure of claim 58, comprising a cytokine payload and an adjuvant.

64. 1. A method for producing mesoporous silica rods, comprising: a) adding poloxamer to water to form a solution; b) mixing the solutions; c) adding an acid; d) adding a silicon dioxide source; e) incubating the solution; f) sieving and vacuum filtering the solution; g) heating the solution to obtain the mesoporous silica rods; A method comprising:

65. 65. The method of claim 64, wherein the poloxamer is a polyoxypropylene having a molecular mass of about 10,000 g / mol and containing about 30% polyoxyethylene.

66. 65. The method of claim 64, wherein the silicon dioxide source is tetraethyl orthosilicate silica (TEOS).

67. 65. The method of claim 64, wherein the acid is hydrochloric acid.

68. 68. The method of claim 67, wherein the hydrochloric acid is about 37% hydrochloric acid.

69. 65. The method of claim 64, wherein said step of incubating said solution comprises incubating at about 100°C for about 48 hours.

70. h) mixing the mesoporous silica rods with granulocyte-macrophage colony-stimulating factor (GM-CSF) 65. The method of claim 64, further comprising:

71. h) mixing the mesoporous silica rods with an adjuvant 65. The method of claim 64, further comprising:

72. 72. The method of claim 71, wherein the adjuvant is one or more of aluminum hydroxide (alum), lipopolysaccharide (LPS), cytosine guanosine dinucleotide (CpG), and a toll-like receptor agonist (TLR agonist).

73. h) mixing the mesoporous silica rods with CpG oligodinucleotides 65. The method of claim 64, further comprising:

74. h) mixing the mesoporous silica rods with cytokines 65. The method of claim 64, further comprising:

75. 75. The method of claim 74, wherein the cytokine is interleukin-12 (IL-12), interleukin-2 (IL-2), or granulocyte-macrophage colony-stimulating factor (GM-CSF).

76. h) freeze-drying the mesoporous silica rods 68. The method of claim 67, further comprising:

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