Immunotherapy constructs and methods of use thereof

Abstract

To provide immunotherapeutic constructs for treating and preventing cancer and other diseases and conditions.SOLUTION: Disclosed herein are immunotherapeutic constructs comprising a delivery particle, at least one adjuvant, and one or more therapeutic agents / compounds that cause antigen release and / or modulate an immunosuppressive tumor microenvironment. These immunotherapeutic constructs generate adaptive immune or anti-cancer immune responses that can be used, for example, to prevent and treat a wide variety of cancers. Further disclosed is the use of the immunotherapeutic construct, including preventing and treating cancer in humans and animals.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants R44CA217534 and R43TR001906 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] FIELD OF THE INVENTION The present disclosure relates to compositions and methods for treating and preventing cancer and other diseases and conditions. The compositions include immunotherapeutic constructs that include a delivery system (such as a particle) containing at least one therapeutically active agent (which causes tumor antigen release and / or modulates the immunosuppressive tumor microenvironment) and at least one adjuvant (or immunostimulatory agent), and that generate adaptive immunity by utilizing antigens within the subject's own pre-cancerous or cancerous cells. [Background technology]

[0003] Background of the Invention Immune checkpoint inhibitors, such as those against PD-L1, PD-1, and CTLA-4, have shown promising results in the clinic and have received FDA fast-track approval for many cancer types. However, this treatment only works for a small proportion of cancer patients (approximately 10% to 40%). The lack of response is typically due to a lack of pre-existing anti-tumor immunity (e.g., CD8+ T cells), increasing the need for vaccines to boost the number of anti-tumor T cells in the body.

[0004] Classical cancer vaccines utilize immune stimulators (called adjuvants) and tumor proteins (called antigens). Ideally, neoantigens present only on cancer cells should be used. However, these neoantigens vary widely across tumor types and patients, making it difficult and expensive to develop personalized vaccines for each patient. To circumvent the need to identify these antigens, radiation, chemotherapy, and engineered viruses, such as talimogene-laherparepvec (T-VEC), have been used to kill tumors and release antigens to elicit adaptive immune responses in situ. However, these approaches create an unfavorable immunosuppressive tumor microenvironment (e.g., by increasing chemical stressors called oxidants or by promoting immunosuppressive pathways) that reduces the number of antitumor T cells or disables them. Furthermore, in situ tumor vaccination strategies suffer from the immunosuppressive tumor microenvironment and the inability to retain and effectively deliver vaccine components to target cells (e.g., antigen-presenting cells). Summary of the Invention

[0005] To overcome the aforementioned drawbacks, the present inventors have developed a new class of immunotherapeutic agents that utilizes an in situ tumor vaccination strategy. In situ tumor vaccination is a strategy in which tumors die locally in the presence of immune stimuli, releasing tumor antigens that together prime systemic adaptive immunity against the tumor. In some cases, tumor antigens already present in the tumor microenvironment (TME) are utilized. This strategy is highly promising because it avoids the need to identify tumor (neo)antigens in advance, as in traditional cancer vaccine development. It is also a personalized therapy because a unique set of tumor antigens is released, priming specific immunity for each patient.

[0006] This paper describes engineered particles that can simultaneously deliver adjuvants and compounds that can induce antigen release and / or regulate the immunosuppressive environment to enhance CD8+ T cell repertoire and induce systemic anti-tumor immunotherapy effects.This technology can be called AIRISE, which stands for Augmenting Immune Response and Inhibiting Suppressive Environment of Tumors.

[0007] Described herein is a new class of immunotherapeutic agents (generally, immunotherapeutic constructs) based on engineered particles that enable the simultaneous delivery of adjuvants and therapeutically active agents that can induce antigen release (e.g., by killing cancer cells) and / or modulate the immunosuppressive environment (tumor microenvironment, TME, etc.). These immunotherapeutic constructs can also use antigens already present in the TME. The immunotherapeutic constructs enhance the CD8+ T cell repertoire and induce systemic antitumor immunotherapeutic effects without the need to know which antigens are associated with the cancer being treated. While cellular immunity is broadly described herein, humoral immunity (antibody production) also plays a role and follows the same concept.

[0008] Examples of therapeutically active agents delivered to cancer cells by the provided engineered immunotherapy constructs (e.g., siRNA, miRNA, antisense oligonucleotides, mRNA, shRNA, DNA, other oligonucleotides and polynucleotides, small molecule inhibitors, chemotherapeutic drugs, antibodies, etc.) kill cancer cells, release tumor antigens, and / or manipulate the immunosuppressive tumor microenvironment, while co-delivered adjuvants (e.g., CpG, R848, poly I:C, etc.) prime and activate adaptive immune cells against tumor antigens. Activated effector cells can recognize and attack tumors at any site in the body (including sites distant from the local delivery of the immunotherapy construct) and can reduce or even prevent the spread or development of new tumors bearing one or more of the same tumor antigens as the treated tumor. This phenomenon is sometimes referred to as the abscopal effect. Cancer cell death further amplifies the adaptive immune loop with long-lasting effects. Memory adaptive immunity is also believed to be established for continuous antitumor immune surveillance.

[0009] Immunotherapeutic constructs can be administered locally, intratumorally, intranasally, intraperitoneally, intracerebrospinal, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, topically, transdermally, intravenously, or by inhalation, for example, for easily accessible tumors such as melanoma, head and neck cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, and lymphoma, or can be administered systemically for other cancers such as lung cancer, liver cancer, pancreatic cancer, prostate cancer, brain cancer, kidney cancer, blood cancer, and metastatic cancers.

[0010] Engineered immunotherapeutic constructs can have diameters in the nanometer or micrometer range and can be made from any material (e.g., lipids, inorganic materials, polymers, and combinations thereof) that can be loaded with therapeutic / adjuvant cargoes, deliver them to target sites (cancer cells, immune cells, extracellular matrix, etc.), and enable them to have the desired function.

[0011] Optionally, the immunotherapeutic constructs also contain one or more homing agents (antibodies, aptamers, ligands, peptides, etc.) that enable them to be preferentially delivered to and / or taken up by target cancer cells and / or various immune cell types (e.g., dendritic cells (DCs), macrophages, monocytes, T cells).

[0012] The immunotherapeutic constructs provided herein can be used alone or in combination with standard therapies, including, but not limited to, immune checkpoint inhibitors, chemotherapy, surgery, targeted therapy, and radiation therapy. Alternatively, checkpoint inhibitors (siRNAs, inhibitors, or antibodies against PD-L1 / PD-1, CTLA-4, etc.), other targeted therapeutic agents (e.g., small molecule inhibitors or antibodies targeting other oncoproteins, or medical radioisotopes) can be directly loaded onto / within the immunotherapeutic construct as therapeutically active agents.

[0013] The immunotherapeutic constructs can optionally be formulated into topical or microneedle formulations for localized delivery.

[0014] In certain embodiments, immunotherapeutic constructs are provided that include a delivery system containing at least one therapeutic agent that triggers tumor antigen release and / or modulates an immunosuppressive tumor microenvironment and at least one adjuvant. The immunotherapeutic construct may not include a tumor-specific antigen or ovalbumin. In another embodiment, the immunotherapeutic construct does not include any protein other than the one therapeutic agent or at least one adjuvant, if either is a protein. The therapeutic agent and adjuvant may be loaded into the delivery system, attached to the surface of the delivery system, bound to the delivery system, encapsulated within the delivery system, or contained within the delivery system. In certain embodiments of the provided immunotherapeutic constructs, the delivery system is a nanoparticle having a hydrodynamic size of 5 nm to 999 nm (e.g., about 80 nm to about 200 nm or about 90 nm to about 130 nm) when measured in an aqueous medium (such as PBS, Tris buffer, or water). In yet another example, the immunotherapeutic construct is a microparticle having a hydrodynamic size of 1 micron to 1,000 microns. In some embodiments, the delivery system has a size of about 5 nm to about 200 nm, about 5 nm to about 90 nm, about 5 nm to about 20 nm, about 30 nm to about 100 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm, about 40 nm to about 80 nm, about 70 nm to about 90 nm, or about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm.

[0015] In various embodiments of the immunotherapeutic construct, the therapeutic agent comprises an oligonucleotide (e.g., siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, shRNA or sgRNA (CRISPR-cas9 element)), a polynucleotide, a peptide, a protein, a chemotherapeutic drug, a toxin, an antioxidant, a small molecule inhibitor, an antibody, or a radiotherapeutic agent.

[0016] In the example of an immunotherapeutic construct, the adjuvant compound has immunostimulatory activity. For example, the adjuvant compound may be a TLR-binding DNA substituent, such as a CpG oligonucleotide (e.g., ISS 1018; Amplivax; CpG ODN 7909, CpG ODN 1826, CpG ODN D19, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN 2395, ODN M362, or SD-101); a DNA TLR agonist containing a CpG sequence (e.g., dSLIM); a non-CpG DNA TLR agonist (e.g., EnanDIM); an RNA TLR agonists (e.g., poly I:C or poly-ICLC); aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum chloride, or aluminum potassium sulfate); anti-CD40 antibodies (e.g., CP-870, 893); cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF); cationic peptide-conjugated CpG oligonucleotides (e.g., IC30, IC31); small molecule TLR agonists (e.g., imiquimod, resiquimod, gardikimod, or 3M-052); fusion proteins (e.g., ImuFact IMP321 and ONTAK); oil / surfactant-based adjuvants, such as MF59, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide ISA-51; QS21 stimulon derived from saponin (Aquila Biotech, Worcester, Mass., USA); mycobacterial extracts or synthetic bacterial cell wall mimics, such as lipopolysaccharides (e.g., monophosphoryl lipid A, OM-174, OM-197-MP-EC, or Pam3Cys); xanthenone derivatives (e.g., vadimezan); mixtures thereof (e.g., AS-15); or proprietary adjuvants, such as Ribi's Detox, Quil, or Superfos. Also provided are methods of using the immunotherapeutic constructs described herein, for example, in methods of treating or preventing cancer or another hyperproliferative disease.As one example, in the case of melanoma, immunotherapeutic constructs can be used as a prophylactic vaccine for patients with numerous atypical or other seemingly benign nevi (including, particularly, those with a genetic predisposition). As another example, immunotherapeutic constructs can be administered to accessible tumors / lesions via intratumoral / intralesional injection prior to surgical removal (i.e., in a neoadjuvant setting) to reduce the likelihood of recurrence and / or mobilize the immune system to kill any detectable or undetectable metastases. As another example, immunotherapeutic constructs can be administered to tumors by intratumoral injection, even if the tumor is unresectable. This activates and mobilizes the immune system to attack both treated and untreated tumors elsewhere in the body. As another example, immunotherapeutic constructs can be administered systemically to initiate an anti-tumor adaptive immune response. In certain embodiments, immunotherapeutic constructs can be administered to the area surrounding the tumor (peritumoral) or to the area remaining after tumor removal (adjuvant setting). As another example, immunotherapeutic constructs can be administered systemically, thereby developing adaptive immunity against cancer anywhere in the body. In certain embodiments, immunotherapeutic constructs may be administered directly to lymph nodes (whether or not there is a detectable tumor).

[0017] Another embodiment is a method of treating cells obtained from a subject exhibiting symptoms of cancer, comprising contacting the cells with a therapeutically effective amount of an immunotherapeutic construct of any one of the described embodiments or a composition comprising the immunotherapeutic construct. In an example of this embodiment, the cells obtained from the subject are cancer cells. In other embodiments, the cells are not cancer cells. For example, in some examples, the non-cancerous (e.g., normal) cells are immunological / immune cells. In an example method of treating cells, the method further comprises administering at least one treated cell back to the subject.

[0018] Method embodiments are also provided in which administration of the immunotherapeutic construct is combined with at least one other treatment, for example, treatment of cancer or another hyperproliferative disease or condition.

[0019] The step of administering the immunotherapeutic construct in any of the described method embodiments may include one or more of: direct injection into the tumor of the subject; systemic injection in the subject; local application to the subject; inhalation by the subject; hepatic artery injection into the subject; convection-enhanced delivery to the subject; or microneedle application to the subject.

[0020] In some embodiments of the methods provided, the subject (to be treated, to which the construct or composition is administered, or to which the cells are obtained) is a mammal. For example, in certain embodiments, the mammal is a human.

[0021] It is specifically contemplated that any of the embodiments of the immunotherapeutic constructs provided herein, and embodiments of the methods of using such constructs, include instances in which the immunotherapeutic construct does not comprise a tumor-specific antigen or ovalbumin. [The present invention 1001] A delivery system comprising at least one therapeutic agent that causes tumor antigen release and / or modulates an immunosuppressive tumor microenvironment and at least one adjuvant. 1. An immunotherapeutic construct comprising: The immunotherapeutic construct does not comprise a tumor-specific antigen or ovalbumin. [The present invention 1002] The immunotherapeutic construct of the present invention 1001, wherein the delivery system comprises a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, or a hybrid thereof. [The present invention 1003] The immunotherapeutic construct of the present invention 1002, wherein the delivery vehicle is a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, or an inorganic particle coated with a polymer or lipid. [The present invention 1004] The immunotherapy construct of the present invention 1003, wherein the delivery vehicle is an inorganic particle and comprises one or more of mesoporous silica, gold, aluminum, silver, iron oxide, calcium phosphate, or antioxidant particles. [The present invention 1005] 1004. The immunotherapeutic construct of claim 10, wherein the inorganic particles comprise antioxidant particles comprising cerium oxide. [The present invention 1006] The immunotherapeutic construct of the present invention 1004, wherein the delivery vehicle comprises mesoporous silica particles. [The present invention 1007] The delivery vehicle may be a fullerene, an endohedral metallofullerene, or 1001. The immunotherapeutic construct of the present invention, comprising one or more of: trimetal nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, silicon, silica microspheres and nanospheres, polymer microspheres and nanospheres, silica shells, biodegradable PLGA microspheres and nanospheres, gold particles, cerium oxide particles, zinc oxide particles, silver particles, aluminum particles, carbon particles, iron particles, iron oxide particles, adjuvant particles, and / or modified micelles. [The present invention 1008] The immunotherapeutic construct of any of claims 1001 to 1007, wherein the delivery vehicle is a polymer particle comprising one or more of PLGA, PLL, dextran, dendrimer, polyarginine, PEG, PEI, or chitosan. [The present invention 1009] 1001-1008. The immunotherapeutic construct of any of claims 1001-1008, having a hydrodynamic size of 5 nm to 999 nm. [The present invention 1010] 9. The immunotherapeutic construct of any of claims 1001 to 1008, having a hydrodynamic size of between 1 micron and 1000 microns. [The present invention 1011] 1006. The immunotherapeutic construct of claim 1006, wherein the delivery vehicle comprises mesoporous silica nanoparticles having a size of about 5 to about 200 nm. [The present invention 1012] 1011. An immunotherapeutic construct of the present invention, wherein mesoporous silica nanoparticles are coated with cross-linked polyethyleneimine and polyethylene glycol. [The present invention 1013] The immunotherapeutic construct of any of claims 1001 to 1012, wherein at least one therapeutic agent comprises an siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-cas9 element), oligonucleotide, polynucleotide, peptide, protein, chemotherapeutic drug, toxin, antioxidant, small molecule inhibitor, antibody, or radiotherapeutic agent. [The present invention 1014] The immunotherapeutic construct of the present invention 1013, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, or DNA. [The present invention 1015] The immunotherapeutic construct of the present invention 1014, wherein at least one therapeutic agent comprises an siRNA. [The present invention 1016] The immunotherapeutic construct of the present invention 1015, wherein at least one therapeutic agent comprises an siRNA that inhibits expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, BRAF, BRAF V600E, or MTDH. [The present invention 1017] The immunotherapeutic construct of any one of claims 1015 to 1016, wherein at least one therapeutic agent comprises an siRNA that inhibits the expression or activity of STAT3. [The present invention 1018] 10. The immunotherapeutic construct of any of claims 1015 to 1017, wherein at least one therapeutic agent comprises an siRNA that inhibits expression of HER2 activity. [The present invention 1019] The immunotherapeutic construct of any of claims 1001 to 1012, wherein at least one therapeutic agent inhibits expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, BRAF, BRAF V600E, or MTDH. [The present invention 1020] The immunotherapeutic construct of any of claims 1001 to 1019, wherein at least one therapeutic agent comprises one or more anti-cancer agents selected from an antibiotic, a plant alkaloid, a PLK1 inhibitor, a mitotic phase kinase inhibitor, an immune checkpoint inhibitor, a platinum-based chemotherapeutic agent, a HER2 small molecule inhibitor, an anti-EGFR antibody, and an anti-HER2 antibody. [The present invention 1021] The immunotherapeutic construct of the present invention 1020, wherein at least one therapeutic agent comprises an immune checkpoint inhibitor, and the immune checkpoint inhibitor is an antibody against PD-L1, PD1, or CTLA4. [The present invention 1022] The immunotherapeutic construct of the present invention 1021, wherein the immune checkpoint inhibitor is an antibody against PD-L1. [The present invention 1023] The immunotherapeutic construct of any of claims 1001 to 1022, wherein at least one therapeutic agent comprises a PLK1 inhibitor. [The present invention 1024] The immunotherapeutic construct of the present invention 1023, wherein the PLK1 inhibitor is volasertib. [The present invention 1025] The immunotherapeutic construct of any of claims 1001 to 1024, wherein at least one therapeutic agent comprises one or more of docetaxel, mitoxantrone, or cabazitaxel. [The present invention 1026] The immunotherapeutic construct of any of claims 1001 to 1025, wherein at least one therapeutic agent comprises an anti-EGFR antibody. [The present invention 1027] The immunotherapeutic construct of the present invention 1026, wherein the anti-EGFR antibody is cetuximab. [The present invention 1028] The immunotherapeutic antibody of any of claims 1001 to 1025, wherein at least one therapeutic agent comprises an anti-HER2 antibody. [The present invention 1029] The immunotherapeutic antibody of the present invention 1028, wherein the anti-HER2 antibody is trastuzumab. [The present invention 1030] The immunotherapeutic construct of any of claims 1001 to 1029, wherein the adjuvant has immunostimulatory activity and comprises one or more of a CpG oligonucleotide, a DNA TLR agonist containing a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, a lipopolysaccharide, a plant extract, or a derivative thereof. [The present invention 1031] The immunotherapeutic construct of any of claims 1001 to 1030, wherein the adjuvant comprises a CpG oligonucleotide, imiquimod, resiquimod, gardikimod, poly I:C, poly ICLC, dSLIM, or EnanDIM. [The present invention 1032] The immunotherapeutic construct of any of claims 1001 to 1031, wherein the adjuvant comprises a CpG oligonucleotide. [The present invention 1033] An immunotherapeutic construct according to any one of claims 1001 to 1032 of the present invention; and at least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof. A composition comprising: [The present invention 1034] A method for treating cancer, comprising the step of administering to a subject having cancer an effective amount of the immunotherapeutic construct of any of claims 1001 to 1032 or the composition of claim 1033. [This invention 1035] The method of claim 1034, wherein the subject is a mammal. [The present invention 1036] The method of claim 1035, wherein the mammal is a human. [This invention 1037] 1. A method of treating cells exhibiting a cancer symptom, comprising: contacting the cells with a therapeutically effective amount of any one of the immunotherapeutic constructs of the present inventions 1001 to 1032 or the composition of the present invention 1033; The method comprising: [The present invention 1038] 1. A method of treating cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, comprising: contacting the cells with a therapeutically effective amount of any one of the immunotherapeutic constructs of the present inventions 1001 to 1032 or the composition of the present invention 1033; The method comprising: [This invention 1039] 1. A method of treating cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, comprising: contacting the cells ex vivo with a therapeutically effective amount of any one of the immunotherapeutic constructs of the present inventions 1001 to 1032 or the composition of the present invention 1033; The method comprising: [The present invention 1040] The method of any one of claims 1038 to 1039, wherein the cell is a cancer cell. [This invention 1041] The method of any one of claims 1038 to 1039, wherein the cell is not a cancer cell. [The present invention 1042] The method of claim 1041, wherein the cell is an immune cell. [This invention 1043] 1039. The method of any one of claims 1038 to 1039, wherein the cells are immortalized. [This invention 1044] The method of any of claims 1037 to 1043, further comprising the step of administering at least one treated cell back to the subject. [This invention 1045] 1. A method of treating a subject diagnosed with or at increased risk for developing a hyperproliferative disease or condition, comprising: administering to the subject an effective amount of the composition of the present invention. The method comprising: [The present invention 1046] The method of claim 1045, wherein the subject is a mammal. [This invention 1047] The method of claim 1046, wherein the mammal is a human. [This invention 1048] The method of any of claims 1045 to 1047, wherein the hyperproliferative disease or condition comprises one or more of cancer, precancer, or cancer metastasis. [This invention 1049] The method of any of claims 1045-1048, wherein the hyperproliferative disease comprises one or more of melanoma, lung cancer, breast cancer, pancreatic cancer, brain cancer, prostate cancer, head and neck cancer, renal cancer, colorectal cancer, lymphoma, gastric cancer, colon cancer, liver cancer, or a rare cancer. [The present invention 1050] The administering step comprises: injection into or at the subject's tumor; local injection into or at the subject's tumor; Systemic injection in subjects, Systemic injection in a subject; or Local application to the subject Any of the methods of 1045 to 1049 of the present invention, comprising: [This invention 1051] The method of any of claims 1045 to 1050, wherein the administering step comprises applying a microneedle to the subject. [This invention 1052] 1. A method of enhancing the efficacy of anti-cancer therapy in a subject in need thereof, comprising: An effective amount of any one of the immunotherapeutic constructs of the present invention 1001 to 1032 or the composition of the present invention 1033; with at least one anticancer drug to a subject in need thereof. The method comprising: [This invention 1053] The method of claim 1052, wherein the anti-cancer agent is a chemotherapeutic agent or a targeted therapy agent. [This invention 1054] 1. A method of enhancing the efficacy of checkpoint blockade immunotherapy in a subject diagnosed with a neoplasm, comprising: An effective amount of any one of the immunotherapeutic constructs of the present invention 1001 to 1032 or the composition of the present invention 1033; At least one immune checkpoint inhibitor to a subject in need thereof. The method comprising: [This invention 1055] 1. A method of enhancing the effectiveness of radiation therapy in a subject diagnosed with a neoplasm, comprising: An effective amount of any one of the immunotherapeutic constructs of the present invention 1001 to 1032 or the composition of the present invention 1033; At least one radiation therapy to a subject in need thereof. The method comprising: [This invention 1056] The method of any of claims 1052 to 1055, wherein the immunotherapeutic construct or composition and the anti-cancer therapy are administered sequentially or simultaneously. [This invention 1057] The method of any one of claims 1052 to 1056, wherein the subject is a mammal. [This invention 1058] The method of claim 1057, wherein the mammal is a human. [This invention 1059] An immunotherapeutic construct according to any one of claims 1001 to 1032 of the present invention; with at least one anticancer drug Includes a kit. [The present invention 1060] The kit of the present invention 1059, wherein the anti-cancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor. [Brief explanation of the drawings]

[0022] [Figure 1]Cancer treatment. Targeted therapy has significantly improved cancer prognosis compared with nonspecifically toxic chemotherapy. However, this effect is not sustained. Immune checkpoint inhibitors (ICIs) unleash the body's own immune system to attack cancer, potentially resulting in a cure. However, only a small proportion of patients respond to this treatment. Our goal is to develop a novel immunotherapy construct (Augmenting Immune Response and Inhibiting Suppressive Environment of tumors - AIRISE) that manipulates the tumor microenvironment (TME) to enhance the anti-tumor T cell repertoire, thereby increasing the cure rate for cancer patients treated with ICIs. [Figure 2]In situ tumor vaccination mechanism of our novel immunotherapy construct. The immunotherapy construct AIRISE is injected intratumorally into only one tumor (e.g., melanoma or breast tumor). In one example, i.e., AIRISE-01 or CpG / DTX-NP, docetaxel (DTX), a chemotherapy drug that also has adjuvant properties, kills local cancer cells and releases tumor antigens, while CpG oligonucleotides (adjuvant) activate local antigen-presenting cells (APCs), mainly dendritic cells (DCs). In another example, i.e., AIRISE-02 or siSTAT3-CpG-NP, siSTAT3 can kill some cancer cells, and knocking down STAT3 reduces the immunosuppressive tumor microenvironment, which prevents the priming, activation, and function of anti-tumor adaptive immune responses. Tumor antigens already in the tumor microenvironment (TME, including cancer, immune cells, etc.) or released by our treatment are taken up by AIRISE-activated APCs within the tumor and tumor-draining lymph nodes. APCs then (cross-)present these antigens to prime tumor antigen-specific T cells. These activated cytotoxic (effector CD8+) T cells proliferate and enter the systemic circulation. They specifically home to tumors, wherever they are located in the body, that share some of the same antigens as the treated tumor (e.g., they home to both treated and untreated metastatic tumors). The more cancer cells killed by cytotoxic T cells, the more tumor antigens are released, amplifying the proliferation of effector (already primed) T cells in a positive feedback loop. Antitumor humoral immunity is also activated according to the same concept. In these examples, mesoporous silica nanoparticles also possess antioxidant properties (Morry, J. Biomaterials, 66:41-52, 2015), which can further modulate the immunosuppressive TME and inhibit tumor-promoting activity. This vaccination, induced locally at the tumor site, generates a systemic antitumor immune response throughout the body. [Figure 3]Superior activation of dendritic cells (MHCII+CD80+CD11c+ cells) after treatment with CpG-NP over CpG. Mice were administered CpG or CpG-NP by footpad injection. One day after treatment, draining lymph nodes (DLN) and non-draining lymph nodes (NDLN) were harvested and processed into single cells for flow cytometry analysis to identify the percentage of activated dendritic cells. *p<0.05, **p<0.01, ***p<0.0001. CpG ODN 1826 (SEQ ID NO:7) was used throughout the examples unless otherwise specified. [Figure 4A] The efficacy of CpG-NPs administered to a melanoma mouse model (as shown in Figure 4A, mice implanted with bilateral tumors) in inducing in situ tumor vaccination was demonstrated by the inhibition of tumor growth curves of the local treated tumor (Figure 4B) and the distant untreated tumor (Figure 4C) and the prolongation of mouse survival curves (Figure 4D). 250,000 and 100,000 B16F10 cells were implanted into each mouse (C57BL / 6) to establish local and distant tumors, respectively. Eight days after tumor implantation, the local tumor was intratumorally injected with CpG-NPs or saline every three days for a total of three times. The dose (per injection) was 20 μg CpG and 0.2 mg NP. Tumor volumes are plotted as mean and SEM. Statistical significance (*) between CpG-NPs and saline was assessed. *p<0.05, ***p<0.001, ****p<0.0001. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A]The efficacy of CpG- and / or docetaxel (DTX)-loaded NPs (AIRISE-01) administered to a melanoma mouse model (as in Figure 5A) in inducing in situ tumor vaccination was demonstrated by the inhibition of tumor growth curves of local treated tumors (Figure 5B) and distant untreated tumors (Figure 5C) and the prolongation of mouse survival curves (Figure 5D). Mice (the same model as in Figure 4) were treated with CpG-NPs, CpG-DTX-NPs, or saline. Dose (per injection): CpG 20 μg; DTX 2 μg; NP 0.2 mg. Tumor volumes are plotted as mean and SEM. * indicates a statistically significant difference between saline and CpG-NPs. $ indicates a statistically significant difference between saline and CpG-DTX-NPs. **p and $$p<0.01; ****p and $$$$p<0.0001. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6] Increased cytotoxic CD8+ T cells in local and distant tumors, DLN of local (treated) and distant tumors, and non-DLN induced by CpG-DTX-NPs (referred to as NPs in this figure). All conditions were the same as in Figure 5A. Seven days after the initial injection, tumors and lymph nodes (tumor-draining and non-draining) were harvested (see Figure 5A). Cells were stained with a panel of antibodies to assess lymphoid cell populations and activity. p-values: *p<0.05, **p<0.01, ***p<0.001. [Figure 7A]Figure 7B shows the effect of siSTAT3-CpG-NP (AIRISE-02) administered to a melanoma mouse model (Figure 7A) in inducing in situ tumor vaccination, as shown by the inhibition of tumor growth curves of locally treated tumors (Figure 7B) and distant untreated tumors (Figure 7C) and the prolongation of mouse survival curves (Figure 7D). Tumor volumes are plotted as mean and SEM. Dose (per injection): CpG 20 μg; siSTAT3 4 μg; NP 0.2 mg. Statistical significance (indicated p-values, *) was assessed between CpG-NP and siSTAT3-CpG-NP (the two best-responding groups). [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8A] CD8-dependent effect of siSTAT3-CpG-NP (AIRISE-02). C57 / BL6 mice bearing B16F10 tumors were established and treated as in Figure 8A. Starting 1 day before the first intratumoral treatment of AIRISE-02, groups of mice were administered a CD8-depleting antibody (Clone 2.43, BioXcell, 200 μg / mouse, twice weekly, i.p.) throughout the study. CD8 depletion was shown to reduce the efficacy of AIRISE-02 in inhibiting local tumor growth (Figure 8B), distant untreated tumor growth (Figure 8C), and prolonging mouse survival (Figure 8D), indicating that the effect of AIRISE-02 is immune-dependent rather than directly cytotoxic. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 9A]siSTAT3-CpG-NP (AIRISE-02) enhanced the effects of checkpoint inhibitors (PD1 antibody and CTLA4 antibody). C57 / BL6 mice bearing B16F10 tumors were established and treated as described in (Figure 9A). Two groups of mice were administered with checkpoint inhibitors (PD1 mAb 200 μg / mouse and CTLA4 mAb 100 μg / mouse, i.p.)—one group concomitantly with intratumoral AIRISE-02 and the other group alone (i.e., three doses every three days). AIRISE-02 significantly enhanced the effects of the checkpoint inhibitor cocktail. The combination controlled both local tumors (Figure 9B) and distant untreated tumors (Figure 9C) and prolonged mouse survival better than AIRISE-02 or the checkpoint inhibitor cocktail alone (Figure 9D). Five of eight mice treated with the combination were cured (tumor-free). [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10] siSTAT3-CpG-NP (AIRISE-02) extended the survival time of mice bearing experimental metastatic lung tumors. C57 / BL6 mice were injected (via the tail vein) with 200,000 Lewis lung carcinoma (LLC-JSP) cells to establish lung tumors in the mice. Treatment was administered intravenously as shown in (Figure 10A). Survival time was significantly extended by intravenous AIRISE-02 as shown in (Figure 10B). [Figure 11A] CpG and siSTAT3 delivered with cationic lipid particles (DharmaFECT) produce an in situ vaccination effect. C57 / BL6 mice bearing B16F10 tumors were established and treated as in Figure 11A. The therapeutic construct reduced the treated tumor (Figure 11B) and distant tumors (Figure 11C) and prolonged mouse survival (Figure 11D). Dose (per injection): CpG 20 μg; siSTAT3 4 μg. The therapeutic construct has an average size of 1068 nm (1.1 microns) as measured by DLS. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 12A] NP-mediated co-delivery of siRNA and CpG to cancer cells and immune cells. NPs carrying STAT3 siRNA or scrambled siRNA (siSCR) or CpG-loaded NPs (CpG-NPs) were used to treat B16F10 (Figure 12A) and J774 (Figure 12B) cells (both mouse cell lines) and bone marrow dendritic cells (BMDCs) (Figure 12C) from C3H / HEJ mice. The dose of each siRNA was 50 nM and 2.0% by weight of the NPs, and the dose of CpG was 2% by weight of the NPs for B16F10 and J774, and 4% by weight of the NPs for BMDCs. Forty-eight hours after treatment, mRNA was analyzed using qRT-PCR. The data demonstrated the effectiveness of nanoparticles for transfecting both cancer cells and immune cells with siRNA (e.g., siSTAT3), and this effect was not highly affected by the CpG loaded onto the NPs. Unless otherwise specified throughout the examples, "NP" refers to mesoporous silica nanoparticles coated with cross-linked PEI and PEG, as described in Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015 and U.S. Patent Application Publication No. 2017 / 0173169. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 13]HCC1954 cells (human HER2+ cancer cells) were treated with trastuzumab-conjugated NPs (T-NPs) carrying siRNA against HER2 or STAT3. The dose of each siRNA was 30 nM throughout and 2.0% by weight of the NPs. Protein analysis by Western blot 72 hours after treatment showed that 80% knockdown of STAT3 was achieved. The data demonstrate that nanoparticles can deliver at least two siRNA sequences (e.g., siHER2 and siSTAT3) without losing efficacy compared to a single siRNA. [Figure 14A] Preferential uptake of nanoparticles containing antibodies as homing targeting agents. One hour after exposure, EGFR antibody (cetuximab)-conjugated nanoparticles (C-NP) were preferentially taken up by EGFR-overexpressing lung cancer cells (A549 and H460) over normal lung cells (NL20), as shown in Figure 14A. Figure 14B shows the level of EGFR expression in these cell lines as measured by flow cytometry. Similarly, Figure 14C shows that HER2 antibody (trastuzumab)-conjugated nanoparticles (T-siSCR-NP) were also preferentially taken up by HER2-overexpressing breast cancer cells (BT474, SKBR3) over MCF7, which had low HER2 expression as shown by Western blot analysis (inset in Figure 14C). This preferential effect was not observed with rituximab (CD20 antibody)-conjugated nanoparticles (R-siSCR-T). siSCR indicates scrambled siRNA. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 15]Addition of CpG (SEQ ID NO:7) to nanoparticles containing a PLK1 inhibitor (p-iPLK1-NP) enhanced therapeutic benefit, as demonstrated by Kaplan-Meier survival curves. C57BL / 6 mice were injected with 100K LLC-JSP cells (lung cancer cells) in the right flank and 40K cells in the left flank. Twelve days after tumor inoculation, mice received intratumoral treatment of the right (local) tumor with saline, PD-L1 antibody-coated nanoparticles (p-NP), nanoparticles loaded with a PLK1 inhibitor (iPLK1-NP), p-NP loaded with a PLK1 inhibitor (p-iPLK1-NP), or p-NP loaded with a PLK1 inhibitor and CpG (p-iPLK1-NP-CpG). 0.5 mg of NP (iPLK1 2.5 μg, PD-L1 antibody 20 μg, CpG 20 μg) in 50 μl was administered every 3 days for a total of 3 doses. [Figure 16A] Effect of CpG and mitoxantrone (MTX)-loaded NPs administered to a melanoma mouse model as in (Figure 16A) in inducing in situ tumor vaccination, as shown by the inhibition of tumor growth curves of local treated tumors (Figure 16B) and distant untreated tumors (Figure 16C) and the prolongation of mouse survival curves (Figure 16D). Mice were treated with CpG-MTX-NPs or saline. Dose (per injection): CpG 20 μg; MTX 2 μg; NP 0.2 mg. Tumor volumes are plotted as mean and SEM. For tumor volume, **p<0.01 for CpG-MTX-NPs vs. saline. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 17A]AIRISE-02 enhanced CD8+ T cell proliferation within local (treated) and untreated tumors and their tumor-draining lymph nodes (DLNs). The model, treatment dose, and schedule were as shown in Figure 7. Seven days after the first treatment, cells harvested from tumors and DLNs from both local (treated) and distant (untreated) tumors were analyzed to determine the ratio of CD8+ T cells to CD4+FoxP3+ regulatory T cells in the live CD45+CD3+ T cell populations in tumors (A) and DLNs (B), along with the proliferation status (Ki-67) of effector (CD44+) CD8+ T cells within lymph nodes (C). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (n=3 / group) for AIRISE-02 vs. saline, unless otherwise indicated by brackets. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 18] NP cellular uptake within the TME. Mice (same model as in Figure 7, (B16F10 tumor size approximately 100 mm3 n=3 / group, plotted as mean and SD) were injected intratumorally with Alexa 488-siRNA-CpG-NPs. Two hours after injection, cells within the treated tumors were profiled and analyzed for the presence of siRNA-CpG-NPs (NP+) in each population. [Figure 19] siSTAT3-NP can knock down STAT3 in multiple cell types across multiple species. siSTAT3-NP (50 nM) was used to treat D-17 (canine osteosarcoma), BMDC (bone marrow-derived dendritic cells from mice), J774 (mouse macrophages), B16F10 (mouse melanoma), and HCC1954 (human breast cancer) cells for 48 hours. qRT-PCR analysis of STAT3 and HPRT mRNA was performed using primers specific to the corresponding species. A single siSTAT3 sequence was used throughout. siSCR = scrambled siRNA control. ***p<0.001; ****p<0.0001. [Figure 20]AIRISE-02 (siSTAT3-CpG-NP) + ICI induced complete responses in mice bearing bilateral CT26 tumors. 250K and 100K CT26 cells were implanted into both flanks of each Balb / c mouse. 15 days after tumor implantation, mice were treated as outlined. Tumor growth curves for locally treated and distant untreated tumors are plotted as spider plots (each line represents an individual mouse). Injection dose: CpG 16 μg; siSTAT3 5 μg; NP 0.25 mg. Two groups of mice received checkpoint inhibitors (PD1 mAb 200 μg / mouse and CTLA4 mAb 100 μg / mouse, i.p.), one concurrently with intratumoral AIRISE-02 and one alone. [Figure 21] AIRISE-02 (siSTAT3-CpG-NP) + ICI was also effective against mice bearing aggressive bilateral 4T1 breast tumors. 100K and 40K 4T1 cells were implanted into the bilateral mammary fat pads of each Balb / c mouse. 11 days after tumor implantation, mice were treated as outlined. Tumor growth curves of the local treated tumor and the distant untreated tumor are plotted as spider plots (each line represents an individual mouse). Doses are the same as in Figure 20. [Figure 22] Figure 21 Mouse survival curves. [Figure 23] Safety profile of siSTAT3-CpG-NP (AIRISE-02). Three female Balb / c mice were intramuscularly administered AIRISE-02. Mice were depilated and injected once into the caudal thigh muscle. Images of the injection site were taken pre-injection, post-injection, 24 hours post-injection, and 72 hours post-injection. Injection dose: CpG 16 μg; siSTAT3 5 μg; NP 0.25 mg. [Figure 24]Safety profile of AIRISE-02 in mice. Mice (Balb / c) bearing bilateral MM3MG-HER2d16 tumors implanted in the mammary fat pad (as described in Tsao et al., JCI Insight, 4(24):e131882, 2019) were treated with AIRISE-02 by five intratumoral injections into one of two tumors in each mouse over a two-week period. Body weight was monitored as shown in (A). Mice were euthanized when tumors exceeded 2 cm in diameter or when the mice showed signs of pain or distress (15–55 days after treatment). After euthanasia, blood was collected and processed for serum. Serum biomarkers were measured by Beckman AU680 (IDEXX BioAnalytics, West Sacramento, CA) and reported in (B). Doses were the same as in Figure 20. [Figure 25] Schematic illustrating the safety profile of AIRISE-02 in cynomolgus monkeys. Cynomolgus monkeys (approximately 2 years old, 3.1 ± 0.2 kg, n = 3) were subcutaneously injected with three increasing doses of AIRISE-02, as indicated in the table. CpG 7909 / 2006 (human sequence) was used (SEQ ID NO: 8). Body weight, food consumption, cage side and detailed observations, mortality, morbidity, injection site reactions, PK, clinical pathology, cytokine levels, complement split products, and anti-drug antibodies were monitored. [Figure 26] Hydrodynamic size of mesoporous silica nanoparticles coated with cross-linked PEI and PEG (NP) loaded with different amounts of siRNA and CpG, specified as a weight percent of the total construct. Average size (Z-average) and polydispersity index (PDI) are shown from triplicate measurements using a Malvern Zetasizer. [Figure 27]CpG-NPs can generate antigen-specific (adaptive) immune responses in the presence of antigen. The figure shows the percentage of IFNγ-activated CD8+ T cells after incubation with SF (SIINFEKL peptide). Cells were obtained from lymph nodes of untreated mice, mice treated with SF and CpG-loaded NPs (CpG-SF-NPs), mice treated with SF-loaded NPs (SF-NPs), mice treated with CpG-loaded NPs (CpG-NPs), and mice treated with SF formulated with incomplete Freund's adjuvant (IFA / SF). *p<0.05. Doses used: 16 μg CpG and 40 μg SF. The route of administration in mice was footpad injection. [Figure 28] Hydrodynamic size of nanoparticles (MSNP-PEI-PEG) loaded with approximately 2 wt % and approximately 9 wt % Poly I:C measured in PBS. [Figure 29A] AIRISE-02. (A) TEM image of mesoporous silica nanoparticle core. (B) Schematic of AIRISE-02, which contains PEI-coated mesoporous silica nanoparticles crosslinked as previously described (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015), conjugated with PEG, resulting in nanoparticle constructs (NPs). NPs were loaded with siSTAT3 and CpG via electrostatic interactions by mixing in PBS for 10–40 min. (C) Hydrodynamic size of AIRISE-02 ((2%) siSTAT3-(6%) CpG-NPs). [Figure 29B] See legend to Figure 29A. [Figure 29C] See legend to Figure 29A. [Figure 30]Topical siRNA-NPs in pig skin with and without microneedle roller pretreatment. Fluorescence images of pig skin treated with a single topical application of Dy677-siSCR-NPs in Aquaphor for 1 hour, with and without pretreatment of the skin with a microneedle roller. The siRNA signal is indicated by the arrow. Hoechst 33342 was also used to stain the nuclei of the tissue. [Figure 31] Topical siRNA-NP / Tween-Aquaphor in mice with and without microneedle roller pretreatment. Fluorescence images of mouse skin treated with a single topical application of Dy677-siSCR-NP in Tween / Aquaphor for 1.5 hours, with and without pretreatment of the skin with a microneedle roller. The siRNA signal is indicated by the arrow. Hoechst 33342 was also used to stain the nuclei of the tissue. [Figure 32] EGFR knockdown effect of topical siRNA-NPs using a microneedle roller versus injected siRNA-NPs. Mouse skin was harvested 3 days after a single topical treatment with siEGFR-NPs or siSCR-NPs in Tween / Aquaphor using a microneedle roller (A) or 3 days after a single injection of siEGFR-NPs or siSCR-NPs in saline (B). Skin tissue was fixed and stained with a fluorescently labeled EGFR antibody for EGFR signal quantification. Four to eight images (20x magnification) were taken per condition, and three animals were treated per group. [Figure 33] Dextran-based microneedles containing Dy677-siRNA-loaded NPs [Figure 34] Viability of mouse (A) bone marrow-derived dendritic cells and (B) J774 cells 2 days after treatment with AIRISE-02 containing different amounts of CpG and 2% by weight of siSTAT3. Dose: 50 nM siRNA. [Figure 35]Co-delivery of non-targeted scrambled siRNA (siSCR) and CpG using NPs or Dharmafect® to dendritic cells harvested from C3H / HEJ mice. The dose of each siRNA was 50 nM and 2.0% by weight of the NPs, and the dose of CpG was 4% by weight of the NPs. The siRNA-Dharmafect® formulation was prepared according to the manufacturer's protocol. mRNA was analyzed using qRT-PCR 48 hours after treatment. "NPs" refers to mesoporous silica nanoparticles coated with cross-linked PEI and PEG, as described in Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015, and U.S. Patent Application Publication No. 2017 / 0173169. [Figure 36] siSTAT3 / siCXCR4-CpG-NP + ICI is effective against mice bearing aggressive 4T1 breast tumors. 100K and 40K 4T1 cells were implanted into the bilateral mammary fat pads of each mouse (Balb / c). Eight days after tumor implantation, mice were treated with ICI (three doses, every three days) with or without siSTAT3 / siCXCR4-CpG-NP in a manner similar to Figures 20 and 21. Tumor growth curves of the locally treated tumor and the distant untreated tumor are plotted as spider plots (each line represents an individual mouse). Injection dose: CpG 16 μg; siSTAT3 5 μg; siCXCR4 5 μg; NP 0.25 mg. DETAILED DESCRIPTION OF THE INVENTION

[0023] Reference to sequence listing The nucleic acid sequences described herein are shown using standard abbreviations for nucleotide bases, as defined in 37 CFR § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in the embodiments, where appropriate. A computer-readable text file entitled "51127-004WO2_Sequence Listing_07.13.20_ST25.txt," created on or about July 13, 2020, with a file size of 2 KB, contains the sequence listing for this application and is incorporated herein by reference in its entirety.

[0024] SEQ ID NO:1 is a representative sense sequence of STAT3-specific siRNA (siSTAT3): TIFF2026035730000002.tif3128 (the last two positions are deoxy bases).

[0025] SEQ ID NO:2 is a representative antisense sequence of STAT3-specific siRNA (siSTAT3): TIFF2026035730000003.tif3128 (the last two positions are deoxy bases).

[0026] SEQ ID NO:3 is a representative sense sequence of a HER2-specific siRNA (siHER2): TIFF2026035730000004.tif3128

[0027] SEQ ID NO:4 is a representative antisense sequence of HER2-specific siRNA (siHER2): TIFF2026035730000005.tif3128

[0028] SEQ ID NO:5 is a representative sense sequence of an siRNA specific for SCR (siSCR): TIFF2026035730000006.tif3128

[0029] SEQ ID NO:6 is a representative antisense sequence of siRNA specific for SCR (siSCR): TIFF2026035730000007.tif3128

[0030] SEQ ID NO:7 is the sequence of CpG ODN 1826 (mouse system) used throughout the examples: TIFF2026035730000008.tif3128This ODN contains a complete phosphorothioate backbone and is nuclease resistant.

[0031] SEQ ID NO:8 is the sequence of CpG ODN 2006 / 7909 used in the examples using monkey and human systems: TIFF2026035730000009.tif3128This ODN contains a complete phosphorothioate backbone and is nuclease resistant.

[0032] Detailed Description Described herein is an immunotherapy approach for cancer treatment called AIRISE (Augmenting Immune Response and Inhibiting Suppressive Environment of Tumors) (Figures 1 and 2), which utilizes the patient's own tumor as a depot for a personalized set of tumor antigens (in situ tumor vaccination). The provided immunotherapy construct carries at least one adjuvant (e.g., CpG oligonucleotide) and one or more therapeutic agents / compounds (e.g., siRNA, antisense, oligonucleotide, drug, small molecule, antibody, etc.) that trigger antigen release and / or modulate the immunosuppressive tumor microenvironment. Specific examples of such therapeutic agents are docetaxel and siRNA against STAT3.

[0033] When the provided immunotherapeutic construct is administered to the tumor site (e.g., via intratumoral injection or via tumor homing by systemic delivery), tumor antigens are released in the presence of immune stimulation (provided by the supplied adjuvant). This antigen release and immune stimulation together initiate and support antigen-specific adaptive immunity. Tumor antigens can be taken up by existing antigen-presenting cells (APCs), which present the antigen to naive T cells. T cells (against those tumor antigens) are thereby primed and activated to become effector T cells (either in lymph nodes or within the tumor site) and proliferate throughout the body, ultimately resulting in an increased and improved immune response against the treated tumor and against other tumors (e.g., metastatic sites) distant from the initial administration site. The effect on tumors distant from the initial administration site is also known in the art as the abscopal effect.

[0034] These anti-tumor T cells, trained to recognize specific tumor antigens, control tumors both at the injection site and elsewhere in the body (see Figure 2). Cargo combinations can be applied to any type of micro / nanoparticle; i.e., aspects of the immunotherapeutic construct and its method of use are delivery vehicle independent. Specific exemplary delivery vehicles are described herein.

[0035] The present invention provides an in situ tumor vaccination effect using the immunotherapy described herein, exemplified by immunotherapy constructs loaded with CpG and siSTAT3 (siSTAT3-CpG-NP) or CpG and docetaxel (CpG / DTX-NP). Of these two exemplary therapeutic active agents, docetaxel (DTX) kills local cancer cells and releases tumor antigens, and CpG activates local antigen-presenting cells (APCs, mainly DCs), while siSTAT3 kills some cancer cells, but its main role is to reduce the immunosuppressive tumor microenvironment (TME), which prevents the priming and activation of anti-tumor adaptive immune responses. Note that siSTAT3-CpG-NP is designed to be taken up by both cancer and APCs. Although siSTAT3-CpG-NP may have some killing effect in some cancer cells, it activates APCs rather than killing them by knocking down STAT3. Tumor antigens (already present in the TME or released by the treatment) are taken up by CpG- or AIRISE-activated APCs in tumors and tumor-draining lymph nodes. APCs then (cross-)present these antigens to prime tumor antigen-specific T cells. These activated cytotoxic (effector) T cells proliferate and enter the systemic circulation. They specifically home to tumors, wherever they are located in the body, that share some of the same antigens as the treated tumor (e.g., home to both the treated tumor and distant (untreated) tumors). The more cancer cells killed by cytotoxic T cells, the more tumor antigens are released, amplifying the proliferation of effector (already primed) T cells in a positive feedback loop. In certain embodiments, antioxidant mesoporous silica nanoparticles (MSNPs) can further modulate the local immunosuppressive TME and inhibit tumor-promoting activity. This vaccination, delivered locally at the tumor site, generates systemic antitumor immunity throughout the body.

[0036] In certain embodiments, the delivery vehicle comprises a drug-loading MSNP core (e.g., approximately 50 nm) coated with a bioreducible cross-linked cationic polymer, such as polyethyleneimine (PEI), for oligo loading and endosomal escape, and a stabilizer, such as polyethylene glycol (PEG), which prevents nanoparticle aggregation, protects the oligocargo from degradation by blood enzymes (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015), shields the charge of PEI, and enhances safety. Oligos (siRNA and / or CpG) are finally loaded into the construct and mixed in PBS at room temperature for several minutes (e.g., 5 minutes). Oligonucleotides (siRNA and / or CpG) are electrostatically bound to PEI in an oligo-sequence-independent manner and protected from enzymatic degradation under a PEG layer (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). The resulting nanoparticles (NPs) were highly optimized for siRNA delivery efficacy in terms of MSNP size, PEI and PEG molecular weight and composition, PEI crosslinking conditions (to increase buffering capacity and reduce charge), and oligo and (optionally) antibody loading (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). This embodiment of siRNA-NP has a rigid MSNP core size of 50 nm (by TEM) and a hydrodynamic size of 100 nm (NPs with a polymer coating) with a narrow size distribution. This embodiment of the siRNA-NP contains 13.5 wt% PEI and 18.2 wt% PEG and can be loaded with 2-4 wt% siRNA or up to 10 wt% CpG oligos. Drugs (e.g., taxanes) can be loaded within the MSNP core or onto the polymer at 0.5-3 wt%. All values ​​in this paragraph are by weight of the nanoconstruct. See also U.S. Patent Application Publication No. 2017 / 0172923.

[0037] In a first specific aspect, an immunotherapeutic construct is provided that includes a delivery system, at least one therapeutic agent, e.g., loaded into, attached to the surface of, bound to, encapsulated in, or contained within the delivery system, that causes tumor antigen release and / or modulates an immunosuppressive tumor microenvironment, and at least one adjuvant compound, e.g., attached to the surface of, bound to, encapsulated in, or contained within the delivery system.

[0038] In examples of this embodiment, the delivery system comprises a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, a polymer- or lipid-coated inorganic particle, or a hybrid thereof (or in other embodiments, the delivery system is a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, a polymer- or lipid-coated inorganic particle, or a hybrid thereof).

[0039] In various examples of immunotherapy constructs, the delivery vehicle is an inorganic particle and includes one or more of mesoporous silica, gold, aluminum, calcium phosphate, iron oxide, or antioxidant particles (such as cerium oxide).

[0040] In even more examples of immunotherapeutic constructs, the delivery vehicle is a fullerene, endohedral metallofullerene, or metallofullerenes), trimetal nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, silicon, silica microspheres and nanospheres, polymer microspheres and nanospheres, silica shells, biodegradable PLGA microspheres and nanospheres, gold particles, cerium oxide particles, zinc oxide particles, silver particles, aluminum particles, carbon particles, iron particles, iron oxide particles, adjuvant particles (e.g., virosomes or other virus-like particles), and / or modified micelles. Optionally, the delivery vehicle comprises a polymer. In certain examples, the polymer particles include one or more of PLGA, PLL, polyarginine, PEG, PEI, or chitosan.

[0041] In any of the embodiments of the immunotherapeutic constructs provided, examples are contemplated to be nanoparticles having a hydrodynamic size of 5 nm to 999 nm (e.g., about 80 nm to about 200 nm or about 90 nm to about 150 nm) when measured in an aqueous medium (such as PBS, Tris buffer, or water). In some embodiments, the immunotherapeutic construct has a hydrodynamic size of less than 150 nm when measured in an aqueous medium (such as PBS, Tris buffer, or water). In yet other examples, the immunotherapeutic construct is a microparticle having a hydrodynamic size of 1 micron to 1000 microns (e.g., 1 micron to 50 microns) when measured in an aqueous medium (such as PBS, Tris buffer, or water).

[0042] In various embodiments of the described immunotherapeutic constructs, the therapeutic agent comprises an siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-cas9 element), other oligonucleotides, other polynucleotides, peptides, proteins, chemotherapeutic drugs, toxins, antioxidants, small molecule inhibitors, antibodies, or radiotherapeutic agents. In specific examples, the therapeutic agent inhibits the expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, or MTDH.

[0043] In still further embodiments of the immunotherapy construct, the therapeutic agent is an anti-cancer agent comprising one or more of an antibiotic (e.g., docetaxel, doxorubicin, or mitoxantrone), a plant alkaloid (e.g., cabazitaxel), a PLK1 inhibitor, a mitotic kinase inhibitor, an immune checkpoint inhibitor (such as an antibody against PD-L1, PD1, or CTLA4), a platinum-based chemotherapeutic agent, a small molecule HER2 inhibitor, or a HER2-specific antibody. Examples of mitotic kinase inhibitors include, but are not limited to, inhibitors of at least one of polo-like kinase (PLK), aurora kinase, cyclin-dependent kinase (CDK) 1, CDK2, HASPIN, monopolar spindle 1 kinase (Mps1), or NimA-related kinase (NEK). In some embodiments, the mitotic kinase inhibitor comprises one or more of GSK461364, BI2536, Tak960, NMS-P937, BI6727 (vorasertib), Chk 1 kinase inhibitor LY2603618, AU14022, YK-4-279, or PMN.

[0044] In any exemplary embodiment of the immunotherapy construct, the adjuvant compound has immunostimulatory activity. For example, the adjuvant compound may include one or more of CpG oligonucleotides, DNA TLR agonists containing CpG sequences, non-CpG DNA TLR agonists, RNA TLR agonists, aluminum salts, anti-CD40 antibodies, fusion proteins, cytokines, small molecule TLR agonists, oil-based or surfactant-based adjuvants, lipopolysaccharides, plant extracts, or derivatives thereof. In a specific example, the adjuvant compound includes CpG oligonucleotides, imiquimod, resiquimod, gardikimod, polyIC, polyICLC, dSLIM, or EnanDIM.

[0045] It is specifically contemplated that any of the embodiments of the immunotherapeutic constructs provided herein may not include a tumor-specific antigen or ovalbumin.

[0046] Yet another provided embodiment is a composition comprising at least one immunotherapeutic construct provided herein and at least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof.

[0047] Methods of using the immunotherapeutic constructs described herein, eg, methods of treating or preventing cancer or another hyperproliferative disease, are also provided.

[0048] One provided method of treating cancer includes administering to a subject having cancer an effective amount of the immunotherapeutic construct of any one of the described embodiments, or a composition comprising the immunotherapeutic construct, to reduce one or more symptoms of the cancer.

[0049] Another method embodiment provided is a method of treating a cell exhibiting symptoms of cancer, comprising contacting the cell with a therapeutically effective amount of the immunotherapeutic construct or composition comprising the immunotherapeutic construct of any one of the described embodiments.

[0050] Another method embodiment provided is a method of treating cells obtained from a subject exhibiting symptoms of cancer, comprising contacting the cells with a therapeutically effective amount of an immunotherapeutic construct of any one of the described embodiments or a composition comprising the immunotherapeutic construct. In an example of this embodiment, the cells obtained from the subject are cancer cells. In other embodiments, the cells are not cancer cells. For example, in some examples, the non-cancerous (e.g., normal) cells are immunological cells. In an example method of treating cells, the method further comprises administering at least one treated cell back to the subject.

[0051] Another provided embodiment is a method for treating a subject diagnosed with a hyperproliferative disease or hyperproliferative condition or diagnosed as being at high risk of developing such a disease or condition, comprising administering to the subject an effective amount of a composition comprising at least one immunotherapeutic construct as described herein.For example, in various embodiments, it is contemplated that the hyperproliferative disease or hyperproliferative condition comprises one or more of cancer, precancer, or cancer metastasis.For example, the hyperproliferative disease may comprise one or more of melanoma, lung cancer, breast cancer, pancreatic cancer, brain cancer, prostate cancer, head and neck cancer, kidney cancer, colorectal cancer, lymphoma, colon cancer, or liver cancer.

[0052] Method embodiments are also provided in which administration of an immunotherapeutic construct is combined with at least one other treatment, e.g., treatment of cancer or another hyperproliferative disease or condition. In a first example of such a combination method, a method comprising administering to a subject in need thereof an effective amount of an immunotherapeutic construct of any one of the described embodiments or a composition comprising the immunotherapeutic construct and at least one anti-cancer agent (e.g., a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor) enhances the effectiveness of anti-cancer therapy in a subject in need thereof. In another exemplary combination method, a method comprising administering to a subject in need thereof an effective amount of an immunotherapeutic construct of any one of the described embodiments or a composition comprising the immunotherapeutic construct and at least one immune checkpoint inhibitor enhances the effectiveness of checkpoint blockade immunotherapy in a subject diagnosed with a neoplasm. Yet another combination method is a method of enhancing the effectiveness of radiation therapy in a subject diagnosed with a neoplasm, comprising administering to a subject in need thereof an effective amount of an immunotherapeutic construct of any one of the described embodiments or a composition comprising the immunotherapeutic construct and at least one radiation therapy. In some embodiments of the combination method, examples are provided in which the immunotherapeutic construct or composition and a second agent (generally an anti-cancer therapeutic agent or treatment) are administered sequentially or simultaneously. As used herein, the term "enhance" with respect to the therapeutic effect of an anti-cancer therapy refers to an increase in the therapeutic effect of the anti-cancer therapy (e.g., treatment with an anti-cancer agent, radiation therapy, or checkpoint immunotherapy) over the therapeutic effect normally obtained when the anti-cancer therapy is administered without the immunotherapeutic construct of the present invention. An "increased therapeutic effect" occurs when there is an acceleration and / or an increase in the intensity and / or extent of the therapeutic effect obtained by the anti-cancer therapy. An "increased therapeutic effect" also includes an extension of the useful duration of the therapeutic benefit. An "increased therapeutic effect" may also occur when a relatively lower dose, frequency, or duration of treatment of the anti-cancer therapy is required when co-administered with an immunotherapeutic construct provided by the present invention to achieve the same benefit and / or effect as when a relatively higher dose, frequency, or duration of the anti-cancer therapy is administered alone.An enhancing effect preferably, but not necessarily, results in treatment of an acute condition for which anti-cancer therapy alone is ineffective or less therapeutically effective. Enhancement is achieved when the immunotherapeutic construct of the invention, when co-administered with an anti-cancer therapy, results in at least a 10% increase in therapeutic effect (e.g., at least 25%, at least 50%, at least 75%, or at least 100%) compared to administration of the anti-cancer therapy alone.

[0053] Administering the immunotherapeutic construct in any of the described method embodiments may include one or more of: direct injection into or around the subject's tumor, lesion, or resected tumor area; or systemic injection in the subject; or local application to the subject; or inhalation; or an implanted device; or microneedle application to the subject.

[0054] In some embodiments of the methods provided, the subject (to be treated, to which the construct or composition is administered, or to which the cells are obtained) is a mammal. For example, in certain embodiments, the mammal is a human.

[0055] Also provided herein are kits comprising the immunotherapeutic constructs described herein and at least one anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor.

[0056] Aspects of the present disclosure are described below with additional details and options to aid in the teaching of the present disclosure, as follows: (I) immunotherapeutic constructs; (II) therapeutic agents (which cause tumor antigen release and / or modulate the immunosuppressive tumor microenvironment); (III) adjuvant compounds; (IV) optional additional components; (V) delivery systems; (VI) pharmaceutical compositions and dosage formulations; (VII) exemplary methods of use; (VIII) kits; (IX) exemplary embodiments; and (X) examples.

[0057] (I) Immunotherapy construct Described herein is a new class of immunotherapeutic agents (generally, "immunotherapeutic constructs") comprising engineered particles that co-deliver adjuvants and therapeutically active agents to cancer cells. Embodiments provide therapeutically active agents that induce antigen release (specifically, tumor antigen release) and / or modulate the immunosuppressive environment (such as the tumor microenvironment). These immunotherapeutic constructs enhance the CD8+ T cell repertoire and induce systemic antitumor immunotherapeutic effects without the need to know or identify which antigens are associated with the cancer being treated.

[0058] This strategy has many important features: it is effective, personalized, safe due to local delivery, durable because it trains and harnesses the body's immune cells to attack cancer through a memory effect, inexpensive (e.g., low doses and frequency of administration are required), and applicable to many types of cancer.

[0059] It will be understood that the amount of each component (e.g., therapeutic agent, adjuvant, delivery vehicle, or any component of the delivery vehicle) in the immunotherapeutic construct can vary depending on the embodiment. By way of example, any individual component can constitute 0.001% to 80%, 0.01% to 75%, 0.5 to 50%, 0.5 to 10%, 0.5 to 5%, 1 to 10%, or 2 to 4% by weight of the immunotherapeutic construct. In some embodiments, the therapeutic agent comprises an oligonucleotide (e.g., an siRNA or any other oligonucleotide described herein), and the oligonucleotide constitutes 0.5 to 30% by weight of the immunotherapeutic construct, e.g., 0.5 to 10%, 1 to 5%, 5 to 15%, or 10 to 30%. In some embodiments, the therapeutic agent comprises an anti-cancer agent (e.g., a small molecule inhibitor, or any other anti-cancer agent described herein), and the anti-cancer agent constitutes 0.1-30% by weight, e.g., 0.5-10%, 1-5%, 5-15%, or 10-30% of the immunotherapeutic construct. In some embodiments, the therapeutic agent comprises an antibody, and the antibody constitutes 0.1-30% by weight, e.g., 0.5-10%, 1-5%, 5-15%, or 10-30% of the immunotherapeutic construct.

[0060] (II) Therapeutic Agent Examples of therapeutic active agents (e.g., therapeutic oligonucleotides including siRNA, miRNA, antisense oligonucleotides, sgRNA-Cas9, DNA, and mRNA, as well as small molecule inhibitors, chemotherapeutic drugs, antibodies, chemical agents, etc.) delivered to cancer cells and / or immune cells by the provided engineered immunotherapy constructs trigger tumor antigen release and / or modulate the immunosuppressive tumor microenvironment. In specific embodiments, the active agent kills cancer cells, thereby releasing tumor antigens, while the co-delivered adjuvant (e.g., CpG, R848, poly I:C, etc.) primes and activates adaptive immune cells against the released tumor antigens. The activated effector cells can recognize and attack tumors at any site in the body (including sites distant from the local delivery of the immunotherapy construct) and can reduce or even prevent the spread, recurrence, or development of new tumors bearing one or more of the same tumor antigens as the treated tumor. In certain embodiments, the dose of the therapeutic agent on the immunotherapy construct can be adjusted to reduce toxicity to beneficial immune cells. In certain embodiments, therapeutic agents that affect cancer viability without harming immune cells are utilized on the immunotherapy construct. In other examples, therapeutically active agents (e.g., siRNAs, inhibitors, or other drugs against STAT3, CD39, CD73, IDO-6, PD-L1, TGF-β, antioxidants, etc.) can be loaded onto / into the delivery vehicle (e.g., particle) to modulate the immunosuppressive tumor microenvironment, allowing for the priming and activation of immune cells to effectively attack cancer cells by utilizing antigens already within the tumor or whose release is triggered by the immunotherapy construct.Examples of therapeutic agents include STAT3, IDO-1, TGF-β, CD47, NOX1 to NOX5, HSP47, XBP1, BCL2, BCL-XL, AKT1, AKT2, AKT3, MYC, HER2, HER3, AR, survivin, GRB7, EPS8L1, RRM2, PKN3, EGFR, IRE1-α, VEGF-R1, RTP801, proNGF, keratin K6A, LMP2, LMP7, MECL1, HIF1α, furin, KSP, eiF-4E, p53, β-catenin, and A Therapeutic agents can target poB, PCSK9, SNALP, CD39, CD73, PD-L1, PD-1, CTLA-4, MIF, VEGF, PIGF, CXCR4, CCR2, PLK1, MTDH, Twist, Lcn2, IL-6, IL-10, SOCS1, TRAIL, p65, and mitotic kinases (e.g., PLK1, PLK2, PLK3, PLK4, CDK1, CDK2, CHK1, CHK2, BUB1, BUBR1, MPS1, NEK2, HASPIN, Aurora A). Therapeutic agents can also target other immunosuppressive genes known in the art (e.g., Liu et al., Database, bax094, 2017; Rabinovich et al., Annu Rev Immunol, 25:267, 2010). Therapeutic agents can also inhibit the activity of immune checkpoints known in the art. Immune checkpoints that, when inhibited, are beneficial for cancer therapy include, but are not limited to, PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, B7-H3, VISTA, A2AR, and IDO (Khair et al., Frontiers Immunology, 10:453, 2019). Collectively, immunotherapeutic constructs induce long-lasting immune-mediated anticancer effects. Memory adaptive immunity can also be established.

[0061] In certain embodiments, immunotherapeutic constructs are used to activate an immune response. Such embodiments are not limited to a particular manner of activating the immune response.

[0062] Therapeutic oligonucleotides. Various types of therapeutic oligonucleotides can be used, and these can include, but are not limited to, siRNA, miRNA, antisense oligonucleotides, ribozymes, aptamers, DNA, mRNA, sgRNA (for CRISPR) and CRISPR-cas9 elements. In other words, any chain of nucleotides can be used in this technical field as long as it can specifically regulate (interfere with or enhance) the action or synthesis of specific genes and proteins. Each specific oligonucleotide can have single or multiple targets. Examples of gene / protein targets of interest for the present invention include immune checkpoints, transcription factors, phosphatases, kinases, etc. Specific targets include STAT3, IDO-1, TGF-β, CD47, NOX1-5, HSP47, XBP1, BCL2, BCL-XL, AKT1, AKT2, AKT3, MYC, HER2, HER3, AR, survivin, GRB7, EPS8L1, RRM2, PKN3, EGFR, IRE1-α, VEGF-R1, RTP801, proNGF, keratin K6A, LMP2, LMP7, MECL1, HIF1α, furin, KSP, eiF-4E, p53, and β-catenin. , ApoB, PCSK9, SNALP, CD39, CD73, PD-L1, PD-1, CTLA-4, MIF, VEGF, PIGF, CXCR4, CCR2, PLK1, MTDH, Twist, Lcn2, IL-6, IL-10, SOCS1, TRAIL, p65, and mitotic kinases (e.g., PLK1, PLK2, PLK3, PLK4, CDK1, CDK2, CHK1, CHK2, BUB1, BUBR1, MPS1, NEK2, HASPIN, Aurora A). Therapeutic oligonucleotides can also target other immune suppressive genes known in the art (e.g., Liu et al., Database, bax094, 2017; Rabinovich et al., Annu Rev Immunol, 25:267, 2010). Therapeutic oligonucleotides can inhibit the expression and activity of immune checkpoints known in the art.Immune checkpoints that, when inhibited, are beneficial for cancer therapy include, but are not limited to, PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, B7-H3, VISTA, A2AR, and IDO (Khair et al., Frontiers Immunology, 10:453, 2019). Therapeutic oligonucleotides can also contain two strands targeting two genes (e.g., siRNAs for BLC2 and AKT1, siRNAs for AR and MYC). They can also contain immunostimulatory sequences / elements that can simultaneously enhance the immune response and regulate the expression of target genes. They can also be designed to target the aforementioned genes that have mutations.

[0063] In certain embodiments, the immunotherapeutic construct contains an oligonucleotide that mediates RNA interference as an active agent. RNA interference is a highly conserved mechanism triggered by double-stranded RNA (dsRNA) and can downregulate the transcripts of genes homologous to the dsRNA. dsRNA is first processed by Dicer into short, 21-23 nucleotide double strands called small interfering RNAs (siRNAs). Once incorporated into the RNA-induced silencing complex (RISC), dsRNA can mediate gene silencing through cleavage of target mRNA. "siRNA" or "small interfering ribonucleic acid" refers to two strands of ribonucleotides that hybridize along complementary regions under physiological conditions. An siRNA molecule contains a double-stranded region that is substantially identical to a region of the mRNA of a target gene. A region that has 100% identity to the corresponding sequence of the target gene is suitable. This state is referred to as "fully complementary." However, this region may also contain one, two or three mismatches compared with the corresponding region of target gene depending on the length of the region of targeted mRNA, and therefore may not be completely complementary.The method of analyzing and identifying siRNA that has sufficient sequence identity to effectively inhibit the expression of specific target sequence is known in the art.Preferred mRNA target region is considered to be coding region.Non-translated region, such as 5'-UTR, 3'-UTR and splice junction, is also suitable, as long as these regions are unique to mRNA target.

[0064] In some embodiments, siRNA encapsulated in or bound to an immunotherapeutic construct is utilized in methods and systems involving RNA interference. Such embodiments are not limited to a particular size or type of siRNA molecule. The length of the region of the siRNA complementary to the target can be, for example, 15-100 nucleotides, 18-25 nucleotides, 20-23 nucleotides, or more than 15, 16, 17, or 18 nucleotides. If there is a mismatch in the corresponding target region, the length of the complementary region generally needs to be somewhat longer.

[0065] In certain embodiments, it is contemplated that siRNA delivery techniques using the immunotherapeutic constructs disclosed herein (e.g., by loading siRNA onto the immunotherapeutic construct) can be used to inhibit the development of any gene of interest. Specific targets include STAT3, IDO-1, TGF-β, CD47, NOX1-5, HSP47, XBP1, BCL2, BCL-XL, AKT1, AKT2, AKT3, MYC, HER2, HER3, AR, survivin, GRB7, EPS8L1, RRM2, PKN3, EGFR, IRE1-α, VEGF-R1, RTP801, proNGF, keratin K6A, LMP2, LMP7, MECL1, HIF1α, furin, KSP, e, among other genes known to be drivers in cancer and other diseases. These include iF-4E, p53, β-catenin, ApoB, PCSK9, SNALP, CD39, CD73, PD-L1, PD-1, CTLA-4, MIF, VEGF, PIGF, CXCR4, CCR2, PLK1, MTDH, Twist, Lcn2, IL-6, IL-10, SOCS1, TRAIL, p65, and mitotic kinases (e.g., PLK1, PLK2, PLK3, PLK4, CDK1, CDK2, CHK1, CHK2, BUB1, BUBR1, MPS1, NEK2, HASPIN, Aurora A). Other known potential immunosuppressive genes are described in Liu et al., Database, bax094, 2017, and Rabinovich et al., Annu Rev Immunol, 25:267, 2010. Furthermore, it is specifically contemplated that the siRNA may be directed to a mutant or mutated gene rather than the wild-type gene.

[0066] Those skilled in the art will know how to utilize representative sequences for these targets, which are readily available in public sequence databases. The following table provides sample sequence information: TIFF2026035730000010.tif125162TIFF2026035730000011.tif249162TIFF2026035730 000012.tif253162TIFF2026035730000013.tif249162TIFF2026035730000014.tif54162

[0067] Such embodiments are not limited to the specific manner of evaluating the delivery profile of siRNA in vitro and / or in vivo.In some embodiments, siRNA molecules are labeled with imaging agents (for example, fluorescent dyes FITC, RITC, Cy™ dye, Dylight™ dye or Alexa Fluor™ dye) or radioactive tracers, thereby making it possible to visualize the biodistribution and intracellular delivery profile of siRNA molecules at organ level.In some embodiments, RT-PCR, FISH, IHC, flow cytometry and Western blotting are used to analyze target protein at mRNA level and protein level, respectively.

[0068] In some embodiments, the present disclosure provides a method for inhibiting target gene in cells, comprising introducing siRNA into cells, which can inhibit target gene by RNA interference, wherein the siRNA comprises two complementary RNA strands, and the siRNA is loaded onto immunotherapy construct.In some embodiments, the siRNA is modified with cholesterol at 3' sense strand.In some embodiments, the cell is in human or animal subject (for example, horse, dog, cat, or other domestic animal, livestock, or other animal with cancer).

[0069] MicroRNAs (miRNAs) or miRNA mimics are short non-coding RNAs that can target and substantially silence protein-coding genes via 3'-UTR elements. While the critical role of miRNAs in numerous biological processes has been established, comprehensive analysis of miRNA function in complex diseases is lacking. miRNAs are initially transcribed as primary miRNAs (pri-miRNAs), which are then cleaved by the nuclear RNAses Drosha and Pasha to yield precursor-miRNAs (pre-miRNAs). These precursors are further processed by cytoplasmic RNAse III Dicer to form short, double-stranded miR-miR* duplexes, one strand of which (miR) is then incorporated into the RNA-induced silencing complex (RISC), which contains the enzymes Dicer and Argonaute (Ago). The mature miRNA (approximately 17 to 24 nt) guides RISC to specific target sites located within the 3' UTR of target genes. Upon binding to their target sites, miRNAs repress translation through mRNA degradation, translational inhibition, and / or sequestration into processing bodies (P-bodies) (Eulalio et al., Cell, 132:9-14, 2008; Behm-Ansmant et al., Cold Spring Harb. Symp. Quant. Biol., 71:523-530, 2006; Chu and Rana, Plos. Biology., 4:e210, 2006). Recent estimates suggest that over 60% of protein-coding genes contain 3'-UTR miRNA target sites (Friedman et al., Genome Res., 19:92-105, 2009).In this regard, miRNAs act as key regulators of processes as diverse as early development (Reinhart et al., Nature, 403:901-906, 2000), cell proliferation and cell death (Brennecke et al., Cell, 113(1):25-36, 2003), apoptosis and fat metabolism (Xu et al., Curr. Biol., 13(9):790-795, 2003), and cell differentiation (Chen et al., Mol. Microbiol., 53843-856, 2004; Dostie et al., RNA-A Publication of the RNA Society, 9:180-186, 2003). Furthermore, examination of miRNA expression in chronic lymphocytic leukemia (Calin et al., Proc. Natl. Acad. Sci. USA, 105:5166-5171, 2008), colon adenocarcinoma (Michael et al., Mol. Cancer Res., 1:882-891, 2003), Burkitt lymphoma (Metzler et al., Genes Chromosomes Cancer, 39:167-169, 2004), heart disease (Zhao et al., Cell, 129:303-317, 2007), and viral infection (Pfeffer et al., Science, 304:734-736, 2004) suggests important associations between miRNAs and numerous diseases.

[0070] Previously observed miRNAs are typically 21-22 nucleotides long and arise from longer precursors transcribed from non-protein-coding genes. (Reviewed in Carrington and Ambros (Science, 301(5631):336-338, 2003)) The precursors form structures that fold back on themselves within self-complementary regions. They are then processed by the nuclease Dicer in animals (or DCL1 in plants). miRNA molecules interrupt translation through precise or imprecise base pairing with their targets. In some embodiments, miRNAs can be used as components of immunotherapeutic constructs that are therapeutically provided or administered to subjects, e.g., human patients, to treat diseases such as cancer. Alternatively, in some embodiments, a nucleic acid complementary to a miRNA can be therapeutically administered to a subject in vivo or used in vitro to produce a desired therapeutic result (e.g., miRNA-142-3p, miRNA-142-3p, miRNA-124, or miRNA-138). In this manner, the complementary nucleic acid can be used as a template to generate a desired therapeutic miRNA (e.g., miRNA-142-3p, miRNA-142-3p, miRNA-124, or miRNA-138).

[0071] Embodiments in which the therapeutic oligonucleotide is directed to or specific for STAT3 are particularly contemplated. STAT3 refers to the protein "Signal Transducer and Activator of Transcription 3" and its homologs. The term includes human proteins, regardless of whether the protein is wild-type or mutant. In embodiments, "STAT3" refers to a protein related to Entrez Gene 6774, OMIM 102582, UniProt P40763, and / or RefSeq(protein)NP 003141 (which refers to proteins and related nucleic acids known as of the filing date of this application). "Phosphorylated STAT3" refers to a STAT3 protein that is phosphorylated and activated by phosphorylation. In embodiments, phosphorylated STAT3 is phosphorylated on tyrosine 705 or on a residue corresponding to tyrosine 705 in a homolog. In embodiments, activation of STAT3 means that STAT3 can activate the transcription of other genes. In embodiments, activated STAT3 is phosphorylated on tyrosine 705 or on a residue corresponding to tyrosine 705, forms a dimer (e.g., a homodimer or a heterodimer), translocates to the nucleus, and / or activates transcription. In embodiments, activated STAT3 forms a homodimer. Examples of proteins that phosphorylate and thereby activate STAT3 include JAK2, EGFR, c-MET, and PDGF-R.

[0072] Anti-cancer agent. The phrase anti-cancer agent is used according to its simple ordinary meaning and refers to a composition (e.g., a compound, drug, antagonist, inhibitor, modulator) that has anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is a targeted therapy agent. In some embodiments, the anti-cancer agent is an immune checkpoint inhibitor. In some embodiments, the anti-cancer agent is an agent identified herein that has utility in methods for treating cancer. In some embodiments, the anti-cancer agent is an agent approved by the FDA or similar regulatory agency in a country other than the United States to treat cancer.

[0073] Examples of anti-cancer agents include, but are not limited to, MEK (e.g., MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g., XL518, CI-1040, PD035901, selumetinib / AZD6244, GSK1120212 / trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766, PD184352, SB239063, BAY 43-9006); alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, uramustine, chlorambucil, melphalan, ifosfamide), ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), alkylsulfonates (e.g., busulfan and hepsulfam), nitrosoureas (e.g., carmustine, lomusitne, semustine, and streptozocin) and triazenes (e.g., decarbazine); antimetabolites such as folic acid analogs (e.g., methotrexate, leucovorin, raltitrexed, and pemetrexed), pyrimidines analogs (e.g., fluorouracil, floxouridine, cytarabine, capecitabine, and gemcitabine) and purine analogs (e.g., mercaptopurine, thioguanine, pentostatin, fludarabine, and 5-azathioprine); plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, cabazitaxel, and homoharringtonine); topoisomerase inhibitors, such as camptothecin derivatives (e.g., irinotecan and topotecan), amsacrine, and epipodophyllotoxins (e.g., etoposide (VP16), etoposide phosphate, and teniposide);Antibiotics, such as anthracenediones (e.g., mitoxantrone), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and fluorodaunorunicin hydrochloride) hydrochloride), Streptomyces-derived antibiotics or derivatives thereof (e.g., dactinomycin, bleomycin, mitomycin, geldanamycin, plicamycin, and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin), clofazimine, and beta-lactam derivatives; platinum-based chemotherapeutic agents (e.g., cisplatin, oxaliplatin, carboplatin); substituted ureas (e.g., hydroxyurea); methylhydrazine derivatives (e.g., procarbazine), adrenocortical suppressants (e.g., mitotane and aminoglutethimide); angiogenesis inhibitors (e.g., L-asparaginase and arginine deiminase); PI3K inhibitors (e.g., wortmannin and LY294002); mTOR inhibitors (e.g., sertraline); DNA methyltransferase inhibitors (e.g., 5-aza-2'-deoxycytidine); antisense oligonucleotides; apoptosis gene modulators; Cis-regulatory factors (e.g., deoxyadenosine and triptolide); BCR / ABL antagonists; bFGF inhibitors; casein kinase inhibitors (ICOS); gallium nitrate; gelatinase inhibitors; glutathione inhibitors (e.g., etanidazole); immunostimulatory peptides; insulin-like growth factor 1 receptor inhibitors; leukemia inhibitory factor; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIF inhibitors; mismatched double-stranded RNA; mycobacterial cell wall extracts; nitric oxide modulators; phosphatase inhibitors; plasminogen activator inhibitors; proteasome inhibitors (e.g., bortezomib); protein A system immunomodulators; protein kinase C modulators; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; ribozymes; signal transduction inhibitors / modulators (e.g., itraconazole); single-chain antigen-binding proteins;stem cell inhibitors; stromelysin inhibitors; synthetic glycosaminoglycans; telomerase inhibitors; thyroid-stimulating hormones; translation inhibitors; urokinase receptor antagonists; gonadotropin-releasing hormone agonists (GnRH), such as goserelin and leuprolide (leuprorelin); steroids, such as corticosteroids (e.g., prednisone and dexamethasone); progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate); antiprogestrogens (e.g., mifepristone); estrogens (e.g., diethylstilbestrol and ethinyl estradiol); antiestrogens, such as aromatase inhibitors (e.g., exemestane, fadrozole, letrozole, pentrozole, and anastrozole), selective estrogen receptor modulators (e.g., tamoxifen analogs, tamoxifen androgens (e.g., testosterone propionate and fluoxymesterone); antiandrogens (e.g., flutamide, finasteride, and bicalutamide); immunostimulants, levamisole, interleukins (e.g., interleukin-2), and interferon / interferon agonists (e.g., α-interferon); monoclonal antibodies, e.g., anti-CD20 monoclonal antibodies monoclonal antibodies (e.g., rituximab), anti-HER2 monoclonal antibodies (e.g., trastuzumab), anti-CD52 monoclonal antibodies, anti-CD25 monoclonal antibodies (e.g., daclizumab), anti-HLA-DR monoclonal antibodies, and anti-VEGF monoclonal antibodies; immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas aeruginosa exotoxin conjugate, etc.); radioimmunotherapeutic agents (e.g., 111 In, 90 Y or 131 anti-CD20 monoclonal antibodies conjugated to IFN-I; statins (e.g., cerivastatin and pitavastatin); 5-T1 Breceptor agonists (e.g., 5-nonyloxytryptamine); BRAF kinase inhibitors (e.g., vemurafenib and dabrafenib); tyrosine kinase inhibitors, such as inhibitors of one or more of EGFR, HER2, KDR, FLT4, EphB4, and Src (e.g., gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY3 34543, AG-1478, dacomitinib / PF299804, OSI-420 / desmethylerlotinib, AZD8931, ARRY-380, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626, sorafenib, imatinib ( Gleevec®), sunitinib and dasatinib; immune checkpoint inhibitors (e.g., anti-CTLA4 antibodies, anti-PD1 / L1 antibodies); PLK1 inhibitors (GSK461364, BI2536, Tak960, NMS-P937, volasertib), etc., or mixtures thereof (e.g., leuprolide + estrogen + progesterone).

[0074] Furthermore, the immunotherapeutic constructs described herein can be used in combination with immunostimulants (e.g., Bacille Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), therapeutic monoclonal antibodies (e.g., anti-CD20 monoclonal antibodies, anti-HER2 monoclonal antibodies, anti-CD52 monoclonal antibodies, anti-HLA-DR monoclonal antibodies, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugates, anti-CD22 monoclonal antibody-Pseudomonas aeruginosa exotoxin conjugates, etc.), immune checkpoint inhibitors (e.g., anti-CTLA4 antibodies, anti-PD1 antibodies, anti-PD-L1 antibodies), and radioimmunotherapy (e.g., 111 In, 90 Y or 131 These immunotherapeutic agents may also be co-administered with conventional immunotherapeutic agents, including anti-CD20 monoclonal antibodies conjugated to I. These immunotherapeutic agents may also be directly loaded onto the immunotherapy construct to enhance their therapeutic efficacy, reduce toxicity, and shorten administration times.

[0075] In a further aspect, the immunotherapeutic constructs described herein include, but are not limited to: 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, 111 Ag, 111 In, 117 mSn, 149 Pm, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At and 212 It can be co-administered with conventional radiotherapeutic agents, including radionuclides such as Bi. These radiotherapeutic agents can also be directly loaded onto the immunotherapy construct to enhance therapeutic efficacy, reduce toxicity, and shorten administration times.

[0076] Instead of oligonucleotides, antibodies and small molecule inhibitors that interfere with or enhance the activity of target genes and proteins can be used in a similar manner. For example, instead of siRNA against PD-1, PD-1 antibodies can be loaded onto nanoparticles as therapeutic components in the immunotherapeutic constructs provided herein.

[0077] (III) Adjuvant The immunotherapeutic constructs provided herein include at least one adjuvant component, for example, contained within a delivery vehicle or otherwise associated with the delivery vehicle. The immunotherapeutic construct embodiments are not limited to a particular type of adjuvant, although specific examples are provided herein. The adjuvant may also be part of or conjugated to a therapeutic agent. For example, an siRNA that knocks down a target gene may be designed to contain an immunostimulatory sequence. In some embodiments, the at least one adjuvant constitutes 0.5-20% by weight of the immunotherapeutic construct.

[0078] Generally, an adjuvant is any substance that, when mixed into a vaccine composition, increases or otherwise modifies the immune response to a (cancer) antigen. Adjuvants with immunostimulatory activity are particularly contemplated. Adjuvants induce nonspecific activation of the immune system unless associated with an antigen (e.g., adjuvants in vaccines). The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies elicited against the antigen, and an increase in T-cell activity is typically manifested by increased antigen-specific T-cell proliferation, target cell death, or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a predominantly humoral or Th2 response to a predominantly cellular or Th1 response.

[0079] Suitable adjuvants include TLR-binding DNA substituents, such as CpG oligonucleotides (e.g., ISS1018; Amplivax; CpG ODN 7909, CpG ODN 1826, CpG ODN D19, CpG ODN 1585, CpG ODN 2216, CpG ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN 2395, ODN M362, and SD-101), DNA TLR agonists containing CpG sequences (e.g., dSLIM), non-CpG DNA TLR agonists (e.g., EnanDIM), and cationic peptide-conjugated CpG oligonucleotides (e.g., IC30, IC31); RNA TLR agonists (e.g., poly I:C and poly-ICLC); aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum chloride, and aluminum potassium sulfate); anti-CD40 antibodies (e.g., CP-870, 893); cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF); small molecule TLR agonists (e.g., imiquimod, resiquimod, gardikimod, and 3M-052); fusion proteins (e.g., ImuFact IMP321, CyaA, and ONTAK); oil-based or surfactant-based adjuvants, such as MF59, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide ISA-51; plant extracts, such as QS21 stimulon derived from saponin (Aquila Biotech, Worcester, Mass., USA); mycobacterial extracts and synthetic bacterial cell wall mimics, such as lipopolysaccharides (e.g., monophosphoryl lipid A, OM-174, OM-197-MP-EC and Pam3Cys); xanthenone derivatives (e.g., vadimezan); mixtures thereof (e.g., AS-15); and other proprietary adjuvants, such as Ribi's Detox, Quil or Superfos.Several immunological adjuvants (e.g., dendritic cell-specific MF59) and their preparations have been previously described (Dupuis et al., Cell Immunol. 186(1):18-27, 1998; Allison, Dev Biol Stand.; 92:3-11, 1998).

[0080] Cytokines may also be used. Some cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), promoting dendritic cell maturation into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589), and functioning as immune adjuvants (e.g., IL-12) (Gabrilovich et al., J Immunother Emphasis Tumor Immunol. (6):414-418, 1996). Toll-like receptors (TLRs), or agents that activate TLRs, may also be used as adjuvants. Toll-like receptors (TLRs) are important members of a family of pattern recognition receptors (PRRs) that recognize conserved motifs shared by many microorganisms, called "pathogen-associated molecular patterns" (PAMPS).

[0081] In some embodiments, the adjuvant comprises a CpG oligonucleotide. CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Without being bound by any specific mechanistic theory, CpG oligonucleotides function at least in part by activating the innate (non-adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates, in both prophylactic and therapeutic vaccines. More importantly, CpG-induced TLR9 activation promotes the maturation and differentiation of dendritic cells, even in the absence of CD4 T cell help, leading to the activation of T cells. H TLR9 stimulates T cells and enhances their activation, generating potent cytotoxic T lymphocytes (CTLs). H 1 bias is usually T HThis effect is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which promote a 2-bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or coadministered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce strong responses when the antigen is relatively weak. They also accelerate immune responses, allowing in some experiments to reduce antigen doses by two orders of magnitude while obtaining antibody responses comparable to full-dose vaccines without CpG (Krieg, Nature Reviews, Drug Discovery, 5:471-484, 2006). U.S. Patent No. 6,406,705 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 agonist is dSLIM (double stem loop immunomodulator) manufactured by Mologen (Berlin, Germany). Other TLR binding molecules may also be used, for example, RNA-binding TLR 7, RNA-binding TLR 8 and / or RNA-binding TLR 9.

[0082] Xanthenone derivatives, such as vadimezan or AsA404 (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)), can also be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives can also be administered in parallel with the vaccines of the present invention, e.g., via systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, such xanthenone derivatives are thought to function by stimulating interferon (IFN) production through stimulators of the IFN gene (IFN-ISTING) receptor (see, e.g., Conlon et al., J Immunology, 190:5216-5225, 2013; and Kim et al., ACS Chem Biol, 8:1396-1401, 2013). Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex™, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may function therapeutically and / or as adjuvants. In the context of the present invention, the amounts and concentrations of useful adjuvants and additives can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0083] Poly-ICLC is a synthetically prepared double-stranded RNA containing poly(I) and poly(C) strands with an average length of 5000 nucleotides. It is stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. This compound activates the RNA helicase domains of TLR3 and MDA5, both members of the PAMP family, leading to the activation of DCs and natural killer (NK) cells and the production of a "natural mixture" of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct, broad-spectrum host-targeted anti-infective and possibly anti-tumor effects mediated by two IFN-inducible nuclear enzyme systems: 2'5'-OAS and Pl / eIF2a kinase, also known as PKR(4-6), as well as RIG-I helicase and MDA5.

[0084] Examples of immunological adjuvants that can be combined with the immunotherapeutic construct include TLR ligands, C-type lectin receptor ligands, NOD-like receptor ligands, RLR ligands, and RAGE ligands. TLR ligands can include lipopolysaccharide (LPS) and its derivatives, as well as lipid A and its derivatives, including monophosphoryl lipid A (MPL), glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4'-monophosphoryl lipid A. In a specific embodiment, the immunological adjuvant is MPL. In another embodiment, the immunological adjuvant is LPS. TLR ligands can also include TLR3 ligands (e.g., polyinosinic-polycytidylic acid (poly(I:C))), TLR7 ligands (e.g., imiquimod and resiquimod), and TLR9 ligands.

[0085] As used herein, the term "TLR-binding DNA substituent" refers to a substituent or moiety capable of binding to a toll-like receptor ("TLR"), comprising at least one deoxyribonucleic acid. In embodiments, the TLR-binding DNA substituent is a nucleic acid. In embodiments, the TLR-binding DNA substituent comprises at least one nucleic acid analog. In embodiments, the TLR-binding DNA substituent comprises at least one nucleic acid analog with an alternative backbone (e.g., a phosphodiester derivative (e.g., phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boronphosphonate, or O-methylphosphoramidite), a peptide nucleic acid backbone, LNA, or linkage). In embodiments, the TLR-binding DNA substituent comprises DNA. In embodiments, all nucleotide sugars in the TLR-binding DNA substituent are deoxyribose (e.g., all nucleotides are DNA). In embodiments, the TLR-binding DNA substituent comprises or is DNA having internucleotide linkages selected from phosphodiesters and phosphodiester derivatives (e.g., phosphoramidates, phosphorodiamidates, phosphorothioates, phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methylphosphonates, boron phosphonates, O-methyl phosphoramidites, or combinations thereof). In embodiments, the TLR-binding DNA substituent comprises DNA having internucleotide linkages selected from phosphodiesters and phosphorothioates. In embodiments, the TLR-binding DNA substituent comprises or is DNA having backbone linkages selected from phosphodiesters and phosphorodithioates. In embodiments, the TLR-binding DNA substituent comprises or is DNA having phosphodiester backbone linkages. In embodiments, the TLR-binding DNA substituent comprises or is DNA having phosphorothioate backbone linkages. In embodiments, the TLR-binding DNA substituent comprises or is DNA having phosphorodithioate backbone linkages. In embodiments, the TLR-binding DNA substituent preferentially binds to TLR9 over other TLRs.In embodiments, the TLR-binding DNA substituent specifically binds to TLR9. In embodiments, the TLR-binding DNA substituent specifically binds to TLR3. In embodiments, the TLR-binding DNA substituent specifically binds to TLR7. In embodiments, the TLR-binding DNA substituent specifically binds to TLR8. In embodiments, the TLR-binding DNA substituent specifically binds to a cell subcompartment (e.g., endosome)-associated TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In embodiments, the TLR-binding DNA substituent comprises or is a G-rich oligonucleotide. In embodiments, the TLR-binding DNA substituent comprises a CpG motif, where C and G are nucleotides and p is a phosphate linking C and G. In embodiments, the CpG motif is unmethylated. In embodiments, the TLR-binding DNA substituent is a class A CpG oligodeoxynucleotide (ODN). In embodiments, the TLR-binding DNA substituent is a class B CpG oligodeoxynucleotide (ODN). In embodiments, the TLR-binding DNA substituent is a class C CpG oligodeoxynucleotide (ODN). In embodiments, the TLR-binding DNA substituent (e.g., a TLR9-binding DNA substituent) comprises a deoxyribonucleic acid having an A, G, C, or T base and a phosphodiester and / or phosphodiester derivative linkage (e.g., a phosphorothioate linkage).

[0086] The phrase "CpG motif" refers to a 5' C nucleotide linked to a 3' G nucleotide via a phosphodiester or phosphodiester derivative internucleotide linkage. In embodiments, a CpG motif comprises a phosphodiester internucleotide linkage. In embodiments, a CpG motif comprises a phosphodiester derivative internucleotide linkage.

[0087] As used herein, the term "class A CpG ODN" or "A-class CpG ODN" or "D-type CpG ODN" or "class A CpG DNA sequence" is used according to its general meaning in biological science and chemical science, and refers to a CpG motif comprising a poly-G sequence at 5', 3' or both ends; an internal palindromic sequence containing a CpG motif; or an oligodeoxynucleotide comprising one or more of the following: one or more phosphodiester derivatives that bind deoxynucleotides.In an embodiment, class A CpG ODN comprises a poly-G sequence at 5', 3' or both ends, an internal palindromic sequence that contains a CpG motif, and one or more phosphodiester derivatives that bind deoxynucleotides.In an embodiment, the phosphodiester derivative is phosphorothioate.Examples of class A CpG ODN include ODN D19, ODN 1585, ODN 2216 and ODN 2336.

[0088] The terms "class B CpG ODN" or "B class CpG ODN" or "K-type CpG ODN" or "class B CpG DNA sequence" are used according to their common meaning in biological and chemical sciences and refer to a hexameric motif containing a CpG motif; a CpG motif containing an oligodeoxynucleotide containing one or more phosphodiester derivatives that bind every deoxynucleotide. In embodiments, a class B CpG ODN contains a hexameric motif containing a CpG motif and one or more copies of a phosphodiester derivative that binds every deoxynucleotide. In embodiments, the phosphodiester derivative is phosphorothioate. In embodiments, a class B CpG ODN contains one hexameric motif containing a CpG motif. In embodiments, a class B CpG ODN contains two copies of a hexameric motif containing a CpG motif. In embodiments, a class B CpG ODN contains three copies of a hexameric motif containing a CpG motif. In some embodiments, the class B CpG ODN contains four copies of a hexameric motif containing a CpG motif. Examples of class B CpG ODNs include ODN 1668, ODN 1826, ODN 2006, and ODN 2007.

[0089] The term "class C CpG ODN" or "C-class CpG ODN" or "C-type CpG DNA sequence" is used according to its general meaning in biological and chemical sciences, and refers to an oligodeoxynucleotide that contains a palindromic sequence containing a CpG motif and a phosphodiester derivative (phosphorothioate) that binds every deoxynucleotide. Examples of class C CpG ODN include ODN 2395 and ODN M362.

[0090] (IV) Optional Additional Ingredients targeting part One or more targeting moieties (also known as targeting molecules) may be included, e.g., loaded into the delivery vehicle, attached to the surface of the delivery vehicle, and / or encapsulated within the delivery vehicle. In embodiments, the targeting moiety is displayed on the exterior surface of the delivery vehicle. In certain embodiments, such targeting moieties enable specific cell targeting (e.g., preventing delivery of toxic therapeutic agents to immune cells, enriching / targeting delivery of therapeutic agents with cell-specific functions). In certain embodiments, the targeting moiety may also have therapeutic activity and thus function as a therapeutic agent (e.g., a PD-L1 antibody) of the immunotherapeutic constructs provided herein. Such targeting moieties may be particularly beneficial for systemic delivery.

[0091] Exemplary targeting molecules include proteins, peptides, ligands, nucleic acids, lipids, sugars, antibodies, aptamers, affibody molecules, ligands, small molecules, or polysaccharides that bind to one or more targets associated with organs, tissues, cells, or extracellular matrices, or specific types of tumors or infected cells. The degree of specificity with which a delivery vehicle is targeted can be controlled by selecting a targeting molecule with appropriate affinity and specificity. For example, antibodies are highly specific. They can be polyclonal, monoclonal, fragments, recombinant, or single-chain, many of which are commercially available or easily obtained using standard techniques. T cell-specific molecules, antigens, and tumor-targeting molecules can be attached to the surface of immunotherapy constructs. The targeting molecule can be conjugated to the terminus of one or more PEG chains present on the surface of the particle.

[0092] In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a single-chain variable fragment) that specifically recognizes a cell or tumor marker that is present exclusively or in high abundance on target cells, such as malignant cells (e.g., a tumor antigen). Suitable targeting molecules that can be used to guide immunotherapeutic constructs to cells and tissues of interest, as well as methods for conjugating targeting molecules to nanoparticles, are known in the art. See, for example, Ruoslahti et al. (Nat. Rev. Cancer, 2:83-90, 2002). Exemplary tumor antigens that can be targeted using antigen-binding molecules such as antibodies are described above with respect to vaccine antigens. In certain cases, therapeutic agents may be toxic to both cancer cells and immune cells, resulting in suboptimal effects. Therefore, in certain embodiments, immunotherapeutic constructs can be conjugated to a targeting moiety to enrich the delivery of therapeutic agents and adjuvants only to cancer cells. Examples include antibodies against HER2, EGFR, PSMA, PD-L1, etc., which are expressed or optionally overexpressed on cancer cells. In some embodiments, immunotherapeutic constructs can be conjugated to targeting moieties to enrich delivery of therapeutic agents and adjuvants exclusively to immune cells.

[0093] Targeting molecules may also include neuropilin and endothelial targeting molecules, integrins, selectins, adhesion molecules, bone targeting molecules such as zoledronic acid and alendronic acid (e.g., for targeting cancer that has metastasized to bone), stromal, and fibroblast targeting molecules.

[0094] In some embodiments, the targeting moiety targets the immunotherapeutic construct to antigen-presenting cells (APCs), particularly to a subclass of APCs known as dendritic cells. Dendritic cells express several cell surface receptors that can mediate endocytosis. In some embodiments, the immunotherapeutic construct enhances the activity of DCs to process tumor antigens, thereby better directing in situ tumor vaccination. Targeted delivery to DCs may also be achieved. Targeting exogenous antigens to internalized surface molecules on antigen-presenting cells distributed throughout the body can promote particle uptake and overcome a major rate-limiting step in therapy.

[0095] Dendritic cell targeting molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the surface of dendritic cells. Dendritic cell targeting molecules also include ligands that bind to cell surface receptors on dendritic cells. One such receptor, the lectin DEC-205, has been used in vitro and in mice to enhance both humoral (antibody-based) and cellular (CD8 T cell) responses by two to four orders of magnitude (Hawiger et al., J. Exp. Med., 194(6):769-79, 2001; Bonifaz et al., J. Exp. Med., 196(12):1627-38, 2002; Bonifaz et al., J. Exp. Med., 199(6):815-24, 2004). In these reports, antigens were fused to anti-DEC205 heavy chains, and recombinant antibody molecules were used for immunization.

[0096] Various other endocytic receptors, including mannose-specific lectins (mannose receptors) and IgG Fc receptors, have also been targeted in this way, similarly improving antigen presentation efficiency.Other suitable receptors that can be targeted include DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptors, and scavenger receptors.Targeting moieties for these receptors can be attached to immunotherapy constructs for preferential uptake into immune cells that express these receptors.An example is mannose attached to immunotherapy constructs for targeted delivery to macrophages and DCs, which have high levels of mannose receptors.

[0097] Other receptors that can be targeted include toll-like receptors (TLRs). TLRs recognize and bind to pathogen-associated molecular patterns (PAMPs). PAMPs target TLRs on the surface of dendritic cells and signal internally, potentially increasing DC antigen uptake, maturation, and T cell stimulatory capacity. PAMPs that can be conjugated or co-encapsulated on particle surfaces include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial), zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly(inosinic-cytidylic) acid (bacterial), lipoteichoic acid (bacterial), or imidazoquinoline (synthetic).

[0098] Targeting molecules can be covalently attached to delivery vehicles using a variety of methods known in the art. In preferred embodiments, the targeting moiety is attached to the delivery vehicle by PEGylation or biotin-avidin bridging.

[0099] CD40 agonist. In certain embodiments, the targeting moiety targets CD40. The moiety can be a CD40 agonist. The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is widely expressed by immune cells, hematopoietic cells, vascular cells, epithelial cells, and other cells, including a wide range of tumor cells. As a potential target for cancer therapy, CD40 may mediate tumor regression through both the indirect effect of immune activation and the direct cytotoxic effect on tumors, resulting in a "two-to-one" mechanism of action of CD40 agonists. CD40 agonists are known in the art and are reviewed in Vonderheide (Clin Cancer Res, 13(4):1083-1088, 2007). Exemplary agonists include recombinant CD40L (recombinant human trimer), CD-870, 893 (fully human IgG2 mAb), SGN-40 (humanized IgG1), and HCD 122 (fully human IgG1 mAb). Soluble agonist CD40 antibodies have been shown to replace T cell help provided by CD4+ lymphocytes in a mouse model of T cell-mediated immunity (Khalil et al., Update Cancer Ther., 2:61-65, 2007).

[0100] Integrin ligand. In another embodiment, the targeting moiety is a ligand for integrin. Studies have shown that integrins are overexpressed on the surface of tumor cells and can serve as markers to distinguish tumor cells from normal cells. Certain integrins also activate TGF-β via an extracellular pathway. After being released from tumor cells, latent TGF-β binds to integrins on the surface of tumor cells, resulting in the activation of latent TGF-β. Increased TGF-β concentration in the tumor microenvironment supports immunosuppression and recruits regulatory T cells to the tumor environment.

[0101] RGD peptides can perform dual functions. RGD peptides are not only typical integrin targeting ligands (Ruoslahti et al., Annu. Rev. Cell Dev. Biol., 12:697-715, 1996), but also function as immune danger signals that activate APCs (Altincicek et al., Biol Chem., 390, 1303-11, 2009). Therefore, in preferred embodiments, RGD peptides are loaded into delivery vehicles, attached to the surface of delivery vehicles, and / or encapsulated within delivery vehicles.

[0102] T cell receptors that recognize the p53 antigen. In certain embodiments, the targeting moiety is a T cell receptor (TCR) that recognizes the p53 antigen in the context of human MHC. T cell receptor recombinant proteins derived from bacterial, eukaryotic, or yeast cells, including T cell receptors composed of alpha, beta, or gamma / delta chains (α / β TCR or γ / Δ TCR).

[0103] IL-15 / IL-15Rα. In another embodiment, the targeting moiety is an IL-15 / IL-15Rα complex. Interleukin-15 (IL-15) is a cytokine that shares specific receptor subunits with IL-2 and therefore has some overlapping mechanisms of action. IL-15 is expressed by dendritic cells and provides an important signal for the proliferation and priming of natural killer (NK) cells. Therefore, the IL-15 / IL-15Rα complex can be used to target nanoparticulate compositions to, for example, natural killer (NK) cells.

[0104] (V) Delivery System Embodiments of the immunotherapeutic constructs provided herein are independent with respect to the delivery system used for delivery of the therapeutic agent and adjuvant. Thus, in various embodiments, the delivery system may use or be based on any type of known or to be developed particulate delivery vehicle. These include nanoparticles, fullerenes, endohedral metallofullerenes, trimetal nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, lipid-based nanoparticles, lipoplexes, polymeric nanoparticles, calcium phosphate particles, aluminum salt particles, polyplexes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass microspheres and nanospheres, polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, adjuvant particles (e.g., virosomes or other virus-like particles), silver nanoparticles, carbon nanoparticles, iron nanoparticles, porous and non-porous silica nanoparticles, and modified micelles. Hybrid particles containing several classes of materials can also be used. Nanometer- and micron-sized particles can be used. Particles can be of any shape, structure, and porosity. Therapeutic agents, adjuvants, and any additional compounds can be included with the delivery agent by any suitable means, for example, loaded into the delivery system, attached to the surface of the delivery system, bound to the delivery system, encapsulated within the delivery system, or contained within the delivery system. Such agents can be encapsulated, covalently bonded, or non-covalently bonded (e.g., by electrostatic interactions, hydrophobic interactions, van der Waals interactions, or compound-specific interactions such as nucleic acid base pairing, ligand-receptor, antibody-antigen, biotin-avidin, etc.).

[0105] In some embodiments, the delivery system comprises mesoporous silica nanoparticles (MSNPs), such as those described in U.S. Patent Application Publication No. US2017 / 0173169 or U.S. Patent Application Publication No. US2017 / 0172923, the MSNPs described therein being incorporated herein by reference.

[0106] In some embodiments, the average particle size of the mesoporous nanoparticles (or different nanoparticles) is about 5 nm to about 200 nm, about 5 nm to about 90 nm, about 5 nm to about 20 nm, about 30 nm to about 100 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm, about 40 nm to about 80 nm, about 70 nm to about 90 nm, or about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In some embodiments, the mesoporous silica nanoparticles are coated with a cationic polymer or other compound. The cationic polymer can be attached to the surface of the nanoparticles using any suitable means. In some embodiments, the cationic polymer is attached to the nanoparticles via electrostatic interactions. The cationic polymer can be any polymer having a positive charge, such as, but not limited to, PEI, polyamidoamine, poly(allylamine), poly(diallyldimethylammonium chloride), chitosan, poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid), poly(L-lysine), diethylaminoethyl-dextran, poly(N-ethyl-vinylpyridinium bromide), poly(dimethylamino)ethyl methacrylate), or poly(ethylene glycol)-co-poly(trimethylaminoethyl methacrylate chloride). Other cationic polymers will be apparent to those skilled in the art and can be found, for example, in Polymer Handbook, 4th Edition, Edited by: Brandrup, E.H. Immergut, and E.A. Grukle; John Wiley & Sons, 2003.

[0107] The cationic polymer can be linear or branched. In some embodiments, the cationic polymer can range in size from about 500 Da to about 25 kDa and can be branched or linear. For example, branched PEI with an average size of 1.8 kDa to 10 kDa can be loaded onto the nanoparticle core. The ratio of cationic polymer to nanoparticles can vary depending on the desired results. The cationic polymer can be present at 1-50% by weight of the nanoconstruct, e.g., 5-40%, 10-30%, 20-30%, 5-15%, 5-20%, 5-25%, 5-30%, 10-20%, 10-25%, or 25-40% by weight, e.g., about 5, about 10, about 15, about 20, about 25, about 30, or about 35% by weight. In some embodiments, the cationic polymer is present at 10-20% by weight.

[0108] In some embodiments, the cationic polymer is crosslinked, e.g., by a cleavable disulfide bond, before or after coating onto the nanoparticles. In some embodiments, the attached cationic polymer is crosslinked after binding to the nanoparticles, e.g., MSNP, using, e.g., DSP (dithiobis[succinimidylpropionate]), DTSSP (3,3'-dithiobis(sulfosuccinimidylpropionate) and DTBP (dimethyl 3,3'-dithiobispropionimidate). Crosslinking can occur in the absence or presence of free cationic polymer in solution. In other embodiments, the cationic polymer is not crosslinked.

[0109] The stabilizer can be conjugated to the MSNP (or a different nanoparticle) and / or the cationic polymer, for example, by any suitable means. In some embodiments, the stabilizer is conjugated to an amine group or other reactive group of a cross-linked cationic polymer coated on the nanoparticle (e.g., MSNP). Exemplary stabilizers include, but are not limited to, PEG, dextran, polysialic acid, hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, and polyacrylamide, or a combination thereof.

[0110] The stabilizer may have multiple chemically reactive groups for attachment to, for example, nanoparticles, cationic polymers, and / or other components. For example, a reactive stabilizer, such as a PEG derivative, may have two electrophilic moieties, such as maleimide-PEG-N-hydroxysuccinimidyl ester (Mal-PEG-NHS), which contains both a Michael acceptor and an activated ester. The stabilizers used in conjunction with the compositions and methods of the present invention, such as PEG, generally have molecular weights ranging from 500 Da to 40 kDa, e.g., 2 to 10 kDa. The stabilizer may be present in an amount of 1 to 50% by weight of the nanoconstruct, e.g., 5 to 30%, 10 to 20%, 10 to 25%, 5 to 15%, 5 to 20%, 5 to 25%, or 1 to 10% by weight, e.g., about 5, about 10, about 15, about 20, about 25, about 35, about 40, or about 45% by weight.

[0111] As used herein, "average particle size" generally refers to the statistical average particle size (diameter) of particles in a particle population.The diameter of essentially spherical particles can refer to physical diameter or hydrodynamic diameter.The diameter of non-spherical particles can preferentially refer to hydrodynamic diameter.As used herein, the diameter of non-spherical particles can refer to the maximum linear distance between two points on the surface of the particle.The average hydrodynamic particle size can be measured using methods known in the art, for example, dynamic light scattering.

[0112] "Monodisperse" and "uniform size distribution" are used interchangeably herein to describe a population of nanoparticles or microparticles in which all particles are the same or approximately the same size. As used herein, a monodisperse distribution refers to a particle distribution in which 90% of the distribution is within 15% of the median particle size, more preferably within 10% of the median particle size, and most preferably within 5% of the median particle size.

[0113] As used herein, "nanoparticle" generally refers to a particle having a diameter of 5 nm to less than 1 micron, preferably 20 nm to 1 micron. Particles can have any shape. Nanoparticles having a spherical shape are generally referred to as "nanospheres." The present disclosure is not limited to a particular type or variety of nanoparticles for complexing with adjuvants and therapeutic agents configured to treat or prevent cancer and related hyperproliferative disorders.

[0114] Examples of nanoparticles include fullerenes (also known as C 60 , C 70 , C 76 , C 80 , C 84 ), endohedral metallofullerenes (EMIs) containing additional atoms, ions, or clusters inside their fullerene cages, trimetallic nitride-templated endohedral metallofullerenes (TNT EMEs, which are highly symmetric four-atom molecular cluster endohedrals formed within a trimetallic nitride template within a carbon cage), single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods (nanotubes with internal metallofullerene and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, lipid particles (liposomes, lipoplexes, polymer nanoparticles, polyplexes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, adjuvant particles (e.g., virosomes or other virus-like particles), and cellulose nanoparticles. Other exemplary nanoparticles include glass microspheres and nanospheres, polymer microspheres and nanospheres, biodegradable PLGA microspheres and nanospheres, gold nanoparticles, silver nanoparticles, platinum nanoparticles, carbon nanoparticles, and iron nanoparticles.

[0115] In some embodiments, the nanoparticles are modified micelles. In these embodiments, the modified micelles comprise a polyol polymer modified to include a hydrophobic polymer block. As used in this disclosure, the term "hydrophobic polymer block" refers to a segment of a polymer that is itself hydrophobic. As used herein, the term "micelle" refers to an aggregate of molecules dispersed in a liquid. Typical micelles in aqueous solution form aggregates with hydrophilic "head" regions in contact with the surrounding solvent, isolating a single hydrophobic tail region in the center of the micelle. In some embodiments, the head region can be, for example, a surface region of the polyol polymer, and the tail region can be, for example, a hydrophobic polymer block region of the polyol polymer.

[0116] The present invention further encompasses the use of micrometer-scale particles in addition to nanometer-scale particles. When microparticles are used, they are preferably relatively small, on the order of 1 to 50 micrometers. For ease of explanation, the use of "nanoparticles" herein encompasses true nanoparticles (1 nm to 1000 nm in size), microparticles (e.g., 1 micrometer to 50 micrometers), or both.

[0117] Examples of nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers, dendrimers with covalently bound metal chelates, nanofibers, nanohorns, nanoonions, nanorods, nanoropes, adjuvant particles (e.g., virosomes or other virus-like particles), and quantum dots. In some embodiments, the nanoparticles are metal nanoparticles (e.g., nanoparticles of gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or alloys of two or more thereof). The nanoparticles can include a core, or a core and a shell, such as core-shell nanoparticles. Hybrid particles containing several classes of materials can also be used.

[0118] Immunotherapeutic construct-containing compositions are disclosed, each comprising one or more active agents and one or more adjuvant compounds loaded into a delivery vehicle, attached to the surface of the delivery vehicle, and / or encapsulated within the delivery vehicle. Nanoparticulate compositions offer several advantages over delivering one or more active agents to target cells in solution. For example, nanoparticulate compositions present a local concentration of one or more active agents on or within the nanoparticles, increasing the avidity of the nanoparticles when they encounter target cells. Nanoparticulate compositions can also function as depots for active agents, with tunable release kinetics that can extend over several days to extend the effective systemic half-life and efficacy of the agent(s).

[0119] Typically, two or more active agents (including one therapeutic agent and one adjuvant) are loaded into the delivery vehicle, attached to the surface of the delivery vehicle, and / or encapsulated in the delivery vehicle. The relative concentration of each of the two or more active agents and their location on or within the delivery vehicle can be manipulated during the preparation of the composition to match the desired dosage and presentation received by target cells. By loading two or more active agents into the same delivery vehicle or on the same delivery vehicle, it is possible to present two or more active agents to target cells or the same tumor microenvironment simultaneously, or to present them to target cells in other predetermined sequences.

[0120] The delivery vehicle can be, for example, a nanolipogel, a polymer particle, a silica particle, a liposome, or a multilamellar vesicle. In certain embodiments, the particulate delivery vehicle is a nanoscale composition, e.g., from 10 nm to less than 1 micron. However, it will be understood that in some embodiments and applications, the particles can be smaller or larger (e.g., microparticles, etc.). While the exemplary immunotherapeutic constructs disclosed herein may be referred to as nanoparticulate compositions, it will be understood that in some embodiments and applications, the particulate composition can be somewhat larger than nanoparticles. For example, the particulate composition can also be from 1 micron to 1000 microns. Such compositions can be referred to as fine particulate compositions.

[0121] In embodiments for treating cancer, it is desirable that particles have a size suitable for reaching tumor microenvironment.In certain embodiments, particles have a size suitable for reaching tumor microenvironment and / or tumor cells by the enhanced permeability and retention (EPR) effect.EPR refers to the property that molecules of a certain size (for example, the microparticle composition described herein) tend to accumulate in tumor tissue much more than in normal tissue.Therefore, in compositions for treating cancer, delivery vehicles are preferably within the range of 25 nm to 500 nm, and more preferably within the range of 30 nm to 300 nm.

[0122] Nanolipogels. Nanolipogels are core-shell nanoparticles that combine the advantages of both liposomes and polymer-based particles for sustained delivery of active agents. In some of these embodiments and applications, nanolipogels can exhibit increased loading efficiency, enhanced sustained release, and improved therapeutic efficacy for macromolecule-molecule combinations compared to conventional nanoparticle compositions.

[0123] Typically, the outer shell of a nanolipogel protects the cargo, provides biocompatibility, and a surface for functionalization with targeting molecules. The outer shell encapsulates components so that they are not exposed until desired, for example, in response to environmental conditions or stimuli, creates a monodisperse and reproducible particle population, and mediates internalization into desired cell types. The inner core can be a dendrimer or other polymer and has a distinct and additional function relative to the outer shell. For example, the inner shell can allow secondary deposition of drugs, vaccines, or imaging agents; increase the loading of components with different physicochemical properties into the particle; enable tunable release of contents from the particle; and increase the cytosolic availability of DNA / RNA, drugs, and / or proteins by disrupting endosomes, all of which can enhance drug efficacy, antigen presentation, and transfection / silencing.

[0124] Nanolipogels have a polymer matrix core containing one or more host molecules. The polymer matrix is ​​preferably a hydrogel, e.g., a crosslinked block copolymer containing one or more poly(alkylene oxide) segments, such as polyethylene glycol, and one or more aliphatic polyester segments, such as polylactic acid. One or more cargo molecules are dispersed within or covalently attached to the polymer matrix. The hydrogel core is surrounded by a liposome shell.

[0125] Nanolipogels can be constructed to incorporate various active agents that can then be released in a controlled manner. The active agents can be dispersed within the hydrogel matrix, dispersed within the liposome shell, covalently attached to the liposome shell, or combinations thereof. Active agents can be selectively incorporated into each of these locations within the nanolipogel. Furthermore, the release rate of the active agent from each of these locations can be independently tailored. Because each of these locations has different properties, including size and hydrophobicity / hydrophilicity, the chemical components independently incorporated into each of these locations can vary dramatically in size and composition. For example, nanolipogels can be loaded with one or more compounds dispersed within the polymer matrix, a conjugated small molecule hydrophobic drug, and an adjuvant. Nanolipogels can provide simultaneous sustained release of agents with widely differing chemical compositions and molecular weights.

[0126] Nanolipogels are typically spherical, with an average particle size ranging from 50 nm to 1000 nm, more preferably from 75 nm to 300 nm, and most preferably from 90 nm to 200 nm. In certain embodiments, the nanolipogels have an average particle size of 100 nm to 140 nm. The particles may be non-spherical.

[0127] Depending on the nature of the lipids present within the liposomal shell of the nanolipogel, nanolipogels may be prepared with a positive, negative, or near-neutral surface charge. In certain embodiments, the nanolipogels have a near-neutral surface charge. In certain embodiments, the nanolipogels have a zeta potential of 10 mV to -10 mV, more preferably 5 mV to -5 mV, even more preferably 3 mV to -3 mV, and most preferably 2 mV to -2 mV.

[0128] Hydrophobic active agents, such as proteins, may be covalently bound to the surface of the nanolipogel, while hydrophilic active agents may be covalently bound to the surface of the nanolipogel or dispersed within the liposome shell. In certain embodiments, the liposome shell comprises one or more PEGylated lipids. In these cases, one or more active agents may be conjugated to the terminus of one or more PEG chains present on the surface of the liposome shell.

[0129] In another embodiment, the lipid is modified to include an avidin moiety, allowing for the attachment thereto of a biotinylated targeting moiety, detectable label, or other active agent, if desired.

[0130] In certain embodiments, one or more active agents are covalently attached to the surface of the nanolipogel via linking groups that are cleaved in response to an external chemical or physical stimulus, such as a change in ambient pH, to trigger release of the active agent(s) at the desired physiological location.

[0131] Core. The nanolipogel core is formed from a polymer matrix. The matrix can include one or more host molecules, as described in more detail below. The nanolipogel core can further include one or more active agents. The active agents can be complexed to the host molecule, dispersed in the polymer matrix, or a combination thereof.

[0132] The polymer matrix of a nanolipogel can be formed from one or more polymers or copolymers. By varying the composition and morphology of the polymer matrix, various controlled-release characteristics can be achieved, allowing for the delivery of moderately constant doses of one or more active agents over extended periods of time.

[0133] The polymer matrix can be formed from a non-biodegradable or biodegradable polymer, although preferably the polymer matrix is ​​biodegradable. The polymer matrix can be selected to degrade over a period ranging from 1 day to 1 year, more preferably 7 days to 26 weeks, even more preferably 7 days to 20 weeks, and most preferably 7 days to 16 weeks. Biodegradable crosslinkers may be used to increase the molecular weight of the polymer, allowing it to be removed from the body as small fragments after degradation of the crosslinker.

[0134] Generally, synthetic polymers are preferred, although natural polymers may also be used. Representative polymers include poly(hydroxy acids), such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), polyhydroxyalkanoates, such as poly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactone; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); poly(glycolide-co-caprolactone); polycarbonates, such as tyrosine polycarbonate; polyamides (synthetic polyamides and Poly(amino acids), including natural polyamides; Polypeptides and poly(amino acids); Polyesteramides; Other biocompatible polyesters; Poly(dioxanone); Poly(alkylene alkylates); Hydrophilic polyethers; Polyurethanes; Polyetheresters; Polyacetals; Polycyanoacrylates; Polysiloxanes; Poly(oxyethylene) / poly(oxypropylene) copolymers; Polyketals; Polyphosphates; Polyhydroxyvalerates; Polyalkylene oxalates; Polyalkylene succinates; Poly(maleic acid, hydroxypropyl methyl acrylates); poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone; poly(alkylene oxides), such as polyethylene glycol (PEG); derivatized celluloses, such as alkyl celluloses (e.g., methyl cellulose), hydroxyalkyl celluloses (e.g., hydroxypropyl cellulose), cellulose ethers, cellulose esters, nitrocellulose, polymers of acrylic acid, methacrylic acid, or copolymers or derivatives thereof including esters, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (collectively referred to herein as "polyacrylic acid"), and derivatives, copolymers, and blends thereof.

[0135] As used herein, "derivative" includes polymers having substitutions, addition of chemical groups, and other modifications to the polymer backbone as commonly performed by those skilled in the art.Natural polymers, including proteins such as albumin, collagen, gelatin, prolamins such as zein, and polysaccharides such as alginate and pectin, may be incorporated into the polymer matrix.Various polymers may be used to form the polymer matrix, but generally, the resulting polymer matrix is ​​a hydrogel.In some cases, when the polymer matrix contains a natural polymer, the natural polymer is a biopolymer that degrades by hydrolysis, such as polyhydroxyalkanoate.

[0136] The polymer matrix may optionally contain one or more cross-linkable polymers. Preferably, the cross-linkable polymer contains one or more photopolymerizable groups, which allows cross-linking of the polymer matrix after nanolipogel formation. Examples of suitable photopolymerizable groups include vinyl groups, acrylate groups, methacrylate groups, and acrylamide groups. When present, the photopolymerizable groups may be incorporated into the backbone of the cross-linkable polymer, into one or more side chains of the cross-linkable polymer, into one or more terminal ends of the cross-linkable polymer, or a combination thereof.

[0137] The polymer matrix can be formed from polymers of various molecular weights to form nanolipogels with properties, including drug release rates, that are optimal for a particular application. Generally, the polymers that make up the polymer matrix have an average molecular weight ranging from 500 Da to 50 kDa. When the polymer matrix is ​​formed from a non-crosslinked polymer, the polymer typically has an average molecular weight ranging from 1 kDa to 50 kDa, more preferably from 1 kDa to 70 kDa, and most preferably from 5 kDa to 50 kDa. When the polymer matrix is ​​formed from a crosslinked polymer, the polymer typically has a lower average molecular weight ranging from 500 Da to 25 kDa, more preferably from 1 kDa to 10 kDa, and most preferably from 3 kDa to 6 kDa. In certain embodiments, the polymer matrix is ​​formed from a crosslinked polymer having an average molecular weight of 5 kDa.

[0138] In some embodiments, the polymer matrix is ​​formed from a poly(alkylene oxide) polymer or a block copolymer comprising one or more poly(alkylene oxide) segments. The poly(alkylene oxide) polymer or poly(alkylene oxide) polymer segment may contain 8 to 500 repeating units, more preferably 40 to 300 repeating units, and most preferably 50 to 150 repeating units. Suitable poly(alkylene oxides) include polyethylene glycol (also known as polyethylene oxide or PEG), polypropylene 1,2-glycol, poly(propylene oxide), polypropylene 1,3-glycol, and copolymers thereof.

[0139] In some embodiments, the polymer matrix is ​​formed from an aliphatic polyester or a block copolymer comprising one or more aliphatic polyester segments. Preferably, the polyester or polyester segment is poly(lactic acid) (PLA), poly(glycolic acid) PGA, or poly(lactide-co-glycolide) (PLGA).

[0140] In some embodiments, the polymer matrix is ​​formed from a block copolymer comprising one or more poly(alkylene oxide) segments, one or more aliphatic polyester segments, and, optionally, one or more photopolymerizable groups. In these cases, the one or more poly(alkylene oxide) segments impart the necessary hydrophilicity to the polymer so that the resulting polymer matrix forms a suitable hydrogel, while the polyester segments provide the polymer matrix with tunable hydrophobic / hydrophilic properties and / or desired in vivo degradation characteristics.

[0141] The degradation rate of the polyester segment, and often the corresponding drug release rate, can be varied from a few days (for pure PGA) to several months (for pure PLA) and can be easily manipulated by changing the ratio of PLA to PGA within the polyester segment. Furthermore, poly(alkylene oxides), such as PEG, and aliphatic polyesters, such as PGA, PLA, and PLGA, have been established as safe for human use. These materials have been used in human clinical applications, including drug delivery systems, for over 30 years.

[0142] In certain embodiments, the polymer matrix is ​​formed from a triblock copolymer comprising a central poly(alkylene oxide) segment, adjacent aliphatic polyester segments attached to either end of the central poly(alkylene oxide) segment, and one or more photopolymerizable groups. Preferably, the central poly(alkylene oxide) segment is PEG and the aliphatic polyester segments are PGA, PLA, or PLGA.

[0143] Generally, the average molecular weight of the central poly(alkylene oxide) segment is greater than the average molecular weight of the adjacent polyester segments. In certain embodiments, the average molecular weight of the central poly(alkylene oxide) segment is at least three times the average molecular weight of one of the adjacent polyester segments, more preferably at least five times the average molecular weight of one of the adjacent polyester segments, and most preferably at least ten times the average molecular weight of one of the adjacent polyester segments.

[0144] In some cases, the central poly(alkylene oxide) segment has an average molecular weight ranging from 500 Da to 10,000 Da, more preferably from 1,000 Da to 7,000 Da, and most preferably from 2,500 Da to 5,000 Da. In certain embodiments, the average molecular weight of the central poly(alkylene oxide) segment is 4,000 Da. Typically, each adjacent polyester segment has an average molecular weight ranging from 100 Da to 3,500 Da, more preferably from 100 Da to 1,000 Da, and most preferably from 100 Da to 500 Da.

[0145] Examples of natural polymers include proteins such as albumin, collagen, gelatin, and prolamins such as zein, as well as polysaccharides such as alginate, cellulose derivatives, and polyhydroxyalkanoates such as polyhydroxybutyrate. The in vivo stability of microparticles can be adjusted during production by using polymers such as poly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG). If PEG is exposed on the exterior, the hydrophilicity of PEG may increase the circulation time of these materials.

[0146] Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers, and mixtures thereof.

[0147] The matrix can also be made from gel-type polymers, such as alginate, produced by conventional ionic gelation techniques. The polymer is first dissolved in an aqueous solution, mixed with barium sulfate or some bioactive agent, and then extruded through a microdroplet-forming device, which optionally uses a stream of nitrogen gas to disrupt the droplets. A slowly agitated (e.g., 100-170 RPM) ionic hardening bath is placed below the extrusion device to capture the microdroplets as they form. The microparticles are incubated in the bath for 20-30 minutes to allow sufficient time for gelation to occur. Microparticle size is controlled by using various sizes of extruders or by varying the flow rate of nitrogen gas or the polymer solution. Chitosan microparticles can be prepared by dissolving the polymer in an acidic solution and crosslinking it with tripolyphosphate. Carboxymethylcellulose (CMC) microparticles can be prepared by dissolving the polymer in an acidic solution and precipitating the microparticles with lead ions. In the case of negatively charged polymers (eg, alginate, CMC), positively charged ligands of different molecular weights (eg, polylysine, polyethyleneimine) can be ionically bound.

[0148] Perhaps the most widely used are aliphatic polyesters, particularly hydrophobic poly(lactic acid) (PLA), relatively hydrophilic poly(glycolic acid) PGA, and their copolymer, poly(lactide-co-glycolide) (PLGA). The degradation rates of these polymers, and often the corresponding drug release rates, can vary from days (PGA) to months (PLA) and are easily manipulated by changing the ratio of PLA to PGA. Second, the physiological compatibility of PLGA and its homopolymers PGA and PLA has been established for safe use in humans. These materials have a history of over 30 years in various human clinical applications, including drug delivery systems. PLGA nanoparticles can be formulated in a variety of ways to improve pharmacokinetics and biodistribution to target tissues through either passive or active targeting. Microparticles are designed to release encapsulated or attached molecules over days to weeks. Factors affecting release duration include the pH of the surrounding medium (relatively fast release rates at pH 5 or below due to acid-catalyzed hydrolysis of PLGA) and polymer composition. Aliphatic polyesters vary in hydrophobicity, which affects their degradation rate. Specifically, hydrophobic poly(lactic acid) (PLA), relatively hydrophilic poly(glycolic acid) PGA, and their copolymer, poly(lactide-co-glycolide) (PLGA), have different release rates. The degradation rates of these polymers, and often the corresponding drug release rates, can vary from days (PGA) to months (PLA) and are easily manipulated by changing the ratio of PLA to PGA.

[0149] Shell Component. Nanolipogels contain a liposomal shell composed of one or more concentric lipid monolayers or lipid bilayers. The shell can further contain one or more active agents, targeting molecules, or a combination thereof.

[0150] Nanolipogels contain a liposomal shell composed of one or more concentric lipid monolayers or bilayers. The composition of the liposomal shell may be varied to affect the release rate of one or more active agents in vivo. Lipids may also be covalently crosslinked, if desired, to alter in vivo drug release.

[0151] The lipid shell can be formed from a single lipid bilayer (monolayer) or several concentric lipid bilayers (multilayer). The lipid shell may be formed from a single lipid. However, in preferred embodiments, the lipid shell is formed from a combination of multiple lipids. The lipids can be neutral, anionic, or cationic at physiological pH.

[0152] Suitable neutral lipids and anionic lipids include sterols and lipids, such as cholesterol, phospholipids, lysolipids, lysophospholipids and sphingolipids.Neutral lipids and anionic lipids include phosphatidylcholine (PC) (egg PC, soybean PC, etc.) containing 1,2-diacyl-glycero-3-phosphocholine; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids, such as sphingomyelin; sphingoglycolipids (also known as 1-ceramidylglucosides), such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols containing carboxylic acid groups, such as cholesterol; or derivatives thereof; and 1,2-diacyl-sn-glycero-3-phosphoethanolamines, including 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-dioleoylglycerylphosphatidylethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). Natural derivatives of these lipids (e.g., tissue-derived L-α-phosphatidylcholines: egg yolk, heart, brain, liver, soybean) and / or synthetic derivatives (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholines, 1-acyl-2-acyl-sn-glycero-3-phosphocholines, 1,2-diheptanoyl-sn-glycero-3-phosphocholines) are also suitable.

[0153] Suitable cationic lipids include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts, also known as TAP lipids, such as methyl sulfate.Suitable TAP lipids include DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-) and DSTAP (distearoyl-).Other suitable cationic lipids include dimethyldioctadecylammonium bromide (DDAB), 1,2-diacyloxy-3-trimethylammonium propane, N-[1-(2,3-dioleoyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dialkyloxy-3 -dimethylammonium propane, dioctadecylamidoglycylspermine (DOGS), 3-[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanam-inium trifluoroacetate (DOSPA), β-alanylcholesterol, cetyltrimethylammonium bromide (CTAB), diC 14-amidine, N-tert-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(α-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N,N,N',N'-tetramethyl-,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy 1,4-butanediammonium iodide, 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives, such as 1-[2-(9(Z)-octadecenoyloxy)ethyl]2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)-imidazolinium chloride (DOTIM) and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM), and 2,3-dialkyloxypropyl quaternary ammonium derivatives containing a hydroxyalkyl moiety on the quaternary amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropylammonium bromide (DORIE-HP ... These include 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DPRIE) and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).

[0154] Other suitable lipids include PEGylated derivatives of the neutral, anionic, and cationic lipids listed above. By incorporating one or more PEGylated lipid derivatives into the lipid shell, nanolipogels can be obtained that display polyethylene glycol chains on their surface. The resulting nanolipogels may exhibit increased in vivo stability and circulation time compared to nanolipogels lacking PEG chains on their surface. Examples of suitable PEGylated lipids include distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), including DSPE-PEG (2000 MW) and DSPE-PEG (5000 MW), dipalmitoyl-glycero-succinate polyethylene glycol (DPGS-PEG), stearyl-polyethylene glycol, and cholesteryl-polyethylene glycol.

[0155] In certain embodiments, the lipid shell is formed from a combination of multiple lipids. In certain embodiments, the lipid shell is formed from a mixture of at least three lipids. In particular embodiments, the lipid shell is formed from a mixture of phosphatidylcholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) and cholesterol.

[0156] In some embodiments, the lipid shell is formed from a mixture of one or more PEGylated phospholipids and one or more additional lipids or sterols. In certain cases, the molar ratio of the one or more PEGylated lipids to the one or more additional lipids or sterols ranges from 1:1 to 1:6, more preferably from 1:2 to 1:6, and most preferably from 1:3 to 1:5. In certain embodiments, the molar ratio of the one or more PEGylated lipids to the one or more additional lipids or sterols is 1:4.

[0157] In some embodiments, the lipid shell is formed from a mixture of one or more phospholipids and one or more additional lipids or sterols. In certain cases, the molar ratio of the one or more phospholipids to the one or more additional lipids or sterols ranges from 1:1 to 6:1, more preferably from 2:1 to 6:1, and most preferably from 3:1 to 5:1. In certain embodiments, the molar ratio of the one or more phospholipids to the one or more additional lipids or sterols is 4:1.

[0158] In a preferred embodiment, the lipid shell is formed from a mixture of a phospholipid, such as phosphatidylcholine (PC), a PEGylated phospholipid, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), and cholesterol. In a particular embodiment, the lipid shell is formed from a mixture of phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG), and cholesterol in a 3:1:1 molar ratio.

[0159] Polymer particles. The delivery vehicle can also be a polymer particle, such as a microparticle or nanoparticle. The particle can be biodegradable or non-biodegradable. Exemplary polymers that can be used to manufacture the polymer particle are described above with respect to the polymer matrix component of the nanolipogel.

[0160] Examples of preferred biodegradable polymers include polymers of hydroxy acids, such as lactic acid and glycolic acid, and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), blends and copolymers thereof. In a preferred embodiment, the particles are composed of one or more polyesters.

[0161] For example, the particles may contain one or more of the following polyesters: homopolymers containing glycolic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA," and caprolactone units, such as poly(ε-caprolactone), collectively referred to herein as "PCL"; and copolymers containing lactic acid units and glycolic acid units, characterized by a lactic acid:glycolic acid ratio, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and polyacrylates, and their derivatives. Exemplary polymers also include copolymers of polyethylene glycol (PEG) with the aforementioned polyesters, such as various forms of PLGA-PEG copolymers or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers." In certain embodiments, a PEG region can be covalently attached to a polymer via a cleavable linker to yield a "PEGylated polymer." Alginate polymers can also be used.

[0162] In some embodiments, the particles are composed of PLGA. PLGA is a safe, FDA-approved polymer. PLGA particles are advantageous because they can protect active agents (i.e., encapsulate), promote long-term release, and are suitable for incorporating targeting moieties.

[0163] The particle can contain one or more polymer conjugates, including end-to-end linkages between the polymer and targeting moiety, detectable label or other active agent.For example, the modified polymer can be PLGA-PEG-phosphonate.In another example, the particle can be modified to include an avidin moiety, and biotinylated targeting moiety, detectable label or other active agent can be attached thereto.

[0164] Examples of preferred natural polymers include proteins such as albumin, collagen, gelatin, and prolamins such as zein, as well as polysaccharides such as alginate, cellulose derivatives, and polyhydroxyalkanoates such as polyhydroxybutyrate.The in vivo stability of particles can be adjusted during production by using polymers such as poly(lactide-co-glycolide) copolymerized with polyethylene glycol (PEG).If PEG is exposed on the outer surface, the hydrophilicity of PEG may increase the circulation time of these materials.

[0165] Examples of non-biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers, and mixtures thereof.

[0166] Nanolipogels. Nanolipogels are nanoparticles that combine the advantages of both liposomes and polymer-based particles for sustained delivery of nucleic acids, proteins, and / or small molecules. Nanolipogels can be in the form of spheres, disks, rods, or other shapes with different aspect ratios. Nanospheres can be relatively large, i.e., microparticles. Nanolipogels are typically formed from synthetic or natural polymers that can encapsulate drugs via distal loading and whose properties can be tailored to promote different release rates. The release rate can be adjusted by varying the polymer-to-lipid ratio from 0.05 to 5.0, more preferably from 0.5 to 1.5.

[0167] Nanolipogels are designed to be loaded with drugs either before, during, or after formation and then function as controlled-release vehicles for the drugs. Nanolipogels can be loaded with multiple drugs so that controlled release of multiple drugs is then achieved.

[0168] The nanolipogels are loaded with one or more therapeutic agents and / or adjuvants during and / or after formation by the process of rehydrating the nanolipogel in the presence of the agents. For example, the nanolipogels are loaded with molecules that function as adjuvants, and the nanolipogels then incorporate one or more anti-cancer agents after formation to deliver and release the adjuvants along with the anti-cancer agents.

[0169] polymer nanoparticles Emulsion Method. In some embodiments, polymeric nanoparticles are prepared using an emulsion solvent evaporation method. For example, a polymeric material is dissolved in a water-immiscible organic solvent and mixed with a drug solution or a combination of drug solutions. The water-immiscible organic solvent can be one or more of chloroform, dichloromethane, and acyl acetate. The drug can be dissolved in one or more of acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethyl sulfoxide (DMSO). An aqueous solution is then added to the resulting mixture to obtain an emulsion solution by emulsification. The emulsification technique can be probe sonication or homogenization with a homogenizer. The peptide, fluorophore, or drug can be bound to the surface of the polymer matrix of the particle, encapsulated within the polymer matrix of the particle, surrounded by the polymer matrix of the particle, and / or distributed throughout the polymer matrix of the particle.

[0170] Nanoprecipitation method. In another embodiment, polymeric nanoparticles are prepared using nanoprecipitation method or microfluidic device. A polymeric material is mixed with a drug or a combination of drugs in a water-miscible organic solvent. The resulting mixture is then added to an aqueous solution to obtain a nanoparticle solution.

[0171] Exemplary Preparation Methods: The particles can be manufactured from a variety of polymers using a variety of methods that can be selected based on criteria including the polymer composition of the particles, and the drug is loaded into or associated with the particles according to methods known in the art. Exemplary methods are provided below.

[0172] Solvent evaporation. In this method, the polymer is dissolved in a volatile organic solvent such as methylene chloride. The drug (soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution containing a surfactant such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporates, leaving behind solid particles. The resulting particles are washed with water and dried overnight in a freeze dryer. This method can yield particles with different sizes (0.5 to 1000 microns) and morphologies. This method is useful for relatively stable polymers such as polyester and polystyrene.

[0173] However, unstable polymers such as polyanhydrides may decompose during the manufacturing process due to the presence of water. For these polymers, the following two methods, which are carried out in completely anhydrous organic solvents, are more useful.

[0174] Hot-melt microencapsulation. In this method, the polymer is first melted and then mixed with solid particles. The mixture is suspended in an immiscible solvent (such as silicone oil) and heated to 5°C above the melting point of the polymer with continuous stirring. Once the emulsion is stabilized, it is cooled until the polymer particles solidify. The resulting particles are washed by decantation with petroleum ether to obtain a free-flowing powder. This method yields particles with sizes ranging from 0.5 to 1,000 microns. The outer surfaces of spheres prepared by this technique are typically smooth and dense. This procedure is used to prepare particles made from polyesters and polyanhydrides. However, this method is limited to polymers with molecular weights between 1,000 and 50,000.

[0175] Solvent Removal. This technique is primarily designed for polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent, such as methylene chloride. This mixture is then suspended in an organic oil (e.g., silicone oil) by stirring to form an emulsion. Unlike solvent evaporation, this method can be used to create particles from polymers with high melting points and various molecular weights. This procedure can yield particles ranging from 1 to 300 microns. The external morphology of the spheres produced by this technique depends largely on the type of polymer used.

[0176] Spray drying. In this method, the polymer is dissolved in an organic solvent. A known amount of active drug is suspended (insoluble drug) or co-dissolved (soluble drug) in the polymer solution. The solution or dispersion is then spray-dried. Typical process parameters for a mini spray dryer (Buchi) are as follows: polymer concentration = 0.04 g / mL, inlet temperature = -24 °C, outlet temperature = 13-15 °C, aspirator setting = 15, pump setting = 10 mL / min, spray flow rate = 600 Nl / h, and nozzle diameter = 0.5 mm. Microparticles in the 1-10 micron range are obtained, with a morphology that depends on the type of polymer used.

[0177] Hydrogel Particles. Particles made from gel-type polymers such as alginate are produced by conventional ionic gelation techniques. The polymer is first dissolved in an aqueous solution, mixed with barium sulfate or some bioactive agent, and then extruded through a microdroplet-forming device, which optionally uses a stream of nitrogen gas to disrupt the droplets. A slowly agitated (e.g., 100-170 RPM) ionic hardening bath is placed below the extrusion device to capture the microdroplets as they form. The particles are incubated in the bath for 20-30 minutes to allow sufficient time for gelation to occur. Particle size is controlled by using various sizes of extruders or by varying the flow rate of nitrogen gas or the polymer solution. Chitosan particles can be prepared by dissolving the polymer in an acidic solution and crosslinking with tripolyphosphate. Carboxymethylcellulose (CMC) particles can be prepared by dissolving the polymer in an acidic solution and precipitating the particles with lead ions. For negatively charged polymers (e.g., alginate, CMC), positively charged ligands of different molecular weights (e.g., polylysine, polyethyleneimine) can be ionically attached.

[0178] Other Delivery Vehicles In some embodiments, the delivery vehicle is a liposome or lipid nanoparticle. Liposomes are typically spherical vesicles composed of lamellar lipid bilayers. Liposomes can be, for example, multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), or cochleate vesicles. Liposomes, micelles, and other lipid-based delivery vehicles useful for preparing the disclosed nanoparticulate compositions are known in the art. See, for example, Torchilin et al. (Adv Drug Delivery Rev, 58(14):1532-55, 2006). It is anticipated that a wide variety of liposomes and exosomes can be used with the present invention. Liposomes can include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) or Lipofectamine™. In some embodiments, a delivery system comprising chitosan can be used, for example, as described in Lu et al. (Cancer Cell, 18:185-197, 2010). In some embodiments, nanovectors can be used to deliver miRNA to a subject. Nanovectors are described, for example, in Pramanik et al. (Mol Cancer Ther, 10:1470-1480, 2011).

[0179] The delivery vehicle can be silica particles. Suitable silica particles useful for preparing the disclosed nanoparticulate compositions are also known in the art. See, for example, Barbe et al. (Adv Materials, 16(21):1959-1966, 2004), Ngamcherdtrakul et al. (Adv Func Materials, 25:2646-2659, 2015) and Argyo et al. (Chem. Mater., 26(1):435-451, 2014). For example, in some embodiments, silicone nanoparticles (e.g., as described in Bharali et al. PNAS, 102(32):11539-11544, 2005) can be used to deliver adjuvants and other therapeutically active agents to cells. The solubility of silica or silicon in the body provides sustained release of the drug carried by the particle. Additionally, biodegradable polymers or bioreducible crosslinkers can be used to modify the silica or silicon particles to provide sustained release capabilities.

[0180] (VI) Pharmaceutical Compositions and Dosage Formulations Provided herein is a composition for use in the treatment of cancer, pre-cancer and other proliferative diseases.The composition comprises at least two active ingredients / drugs, one of which is a therapeutic active agent that (1) causes tumor antigen release and / or (2) regulates immunosuppressive tumor microenvironment, and the other is an adjuvant.As described herein, the active agent is combined with delivery / delivery vehicle in delivery vehicle such as liposome, organic (nano- or micro) particle or inorganic (nano- or micro) particle.

[0181] The composition can be provided to cells directly, for example, by contacting with cells, or indirectly, for example, by the action of any biological process.For example, the composition can be formulated in a physiologically acceptable carrier or vehicle and injected into the tissue or fluid surrounding cells.The composition can pass through the cell membrane by simple diffusion, endocytosis, or any active or passive transport mechanism.

[0182] When therapeutic compound (such as delivery system combined with at least one therapeutic agent and at least one adjuvant) is formulated into pharmaceutical composition, it can be mixed with pharmaceutically acceptable carrier or excipient.As used herein, the phrase " pharmaceutically acceptable " refers to molecular entities and compositions that are generally considered physiologically tolerable and do not typically cause allergic reactions or similar adverse reactions, such as stomach upset, dizziness, etc., when administered to human or veterinary subjects.

[0183] As used herein, the term "pharmaceutically acceptable derivative" refers to any pharmaceutically acceptable salt, solvate, or prodrug, e.g., ester, of a desired active agent, which, upon administration to a recipient, can provide (directly or indirectly) the desired active agent or its active metabolite or residue. Such derivatives are recognizable to those skilled in the art without undue experimentation. Nevertheless, see the teachings of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol. 1: Principles and Practice. Pharmaceutically acceptable derivatives include salts, solvates, esters, carbamates, and phosphate esters.

[0184] While the compositions for treatment can be used as is, it may be preferable to administer them in a pharmaceutical formulation, e.g., by mixing them with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Thus, in one aspect, a pharmaceutical composition or formulation comprises at least one active composition or a pharmaceutically acceptable derivative thereof in combination with a pharmaceutically acceptable excipient, diluent, and / or carrier. The excipient, diluent, and / or carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not significantly deleterious to the recipient thereof.

[0185] Any composition formulation disclosed herein, whether for research, prophylactic and / or therapeutic treatment, can advantageously contain any other pharmaceutically acceptable carrier, including those that do not cause significant adverse allergic reactions or other untoward reactions that outweigh the benefits of administration.Exemplary pharmaceutically acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (AR, Gennaro edited. 2005) and Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.In addition, formulations can be prepared to meet the sterility, pyrogenicity, general safety, and purity standards required by the US FDA Office of Biological Standards and / or other relevant foreign regulatory authorities.Pharmaceutical excipients, pharmaceutical diluents, and pharmaceutical carriers can be selected based on the intended route of administration and standard pharmaceutical practice.

[0186] Such pharmaceutical preparations can be prepared for use in a conventional manner using one or more suitable excipients, diluents, and carriers. Pharmaceutically acceptable excipients assist or enable the formation of a dosage form for a bioactive material and include diluents, binders, lubricants, glidants, disintegrants, colorants, and other ingredients. Preservatives, stabilizers, dyes, and even flavoring agents can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, ascorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used. An excipient is pharmaceutically acceptable if, in addition to performing its desired function, it is non-toxic, well tolerated upon ingestion, and does not interfere with the absorption of the bioactive material.

[0187] Exemplary commonly used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonicity agents, absorption delaying agents, salts, stabilizers, buffers, chelating agents (e.g., EDTA), gels, binders, disintegrants and / or lubricants.

[0188] Exemplary buffers include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and / or trimethylamine salts.

[0189] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halide, hexamethonium chloride, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0190] Exemplary isotonicity agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0191] Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol; sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.

[0192] A "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a compound that, when administered to a subject for treating a condition, disorder, or pathology, is sufficient to achieve such a condition, disorder, or pathology. The "therapeutically effective amount" varies depending on the compound, the disease and its severity, and the age, weight, physical condition, and responsiveness of the mammal being treated. The exact dosage and formulation will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science, and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003); and Pickar, Dosage Calculations (1999)). In certain cases, the term "therapeutically effective amount" is used to refer to an amount or dose sufficient to modulate, e.g., increase or decrease, the desired activity, for example, by 10%, 50%, or 90%. Generally, a therapeutically effective amount is sufficient to cause a clinically significant improvement in the host's condition after a treatment regimen containing one or more therapeutic agents. The concentration or amount of the active ingredient depends on the desired dosage and administration regimen, as described herein.

[0193] The actual dose to be administered to a particular subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as physical, physiological, and psychological factors, including the target, body weight, stage of cancer, type of cancer, previous or concurrent therapeutic interventions, idiopathic disease of the subject, and route of administration.

[0194] The amount effective for this use depends on the severity and location of the disease, particularly if metastatic sites are involved, as well as the weight and general condition of the patient being treated. Generally, dosages range from 0.01 mg / kg to 100 mg / kg of host body weight per day of the immunotherapeutic construct, with doses of 0.1 mg / kg to 10 mg / kg per day, e.g., 3-7 mg / kg, being more commonly used. Long-term maintenance doses may be adjusted as needed. However, dosages may vary depending on the patient's requirements, the severity of the condition being treated, and the compound being used. For example, dosages may be empirically determined for a particular patient, taking into account the type and stage of cancer diagnosed. In the context of the present invention, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose also depends on the presence, nature, and extent of any adverse side effects associated with the administration of a particular vector or transduced cell type to a particular patient. Determining the appropriate dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with relatively low dosages that are less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

[0195] The selected dosage can be influenced by the desired therapeutic effect, the route of administration, the desired duration of treatment, and the specific immunotherapeutic conjugate used. Generally, immunotherapeutic conjugates can be administered in the range of 0.001 mg / kg to 100 mg / kg per administration (e.g., daily; or 2, 3, 4, 5, or more times per week; or 2, 3, 4, 5, or more times per month, etc., as described in further detail below). The route of administration can also be considered when determining the dosage. For example, in certain embodiments, immunotherapeutic constructs are administered by intravenous or intraperitoneal routes in the range of 0.01 mg / kg to 100 mg / kg (e.g., daily; or 2, 3, 4, 5 or more times per week; or 2, 3, 4, 5 or more times per month, etc.), or by subcutaneous route (e.g., local injection into or adjacent to the tumor or TME) in the range of 0.0001 mg / kg to 1 mg / kg (e.g., daily; or 2, 3, 4, 5 or more times per week; or 2, 3, 4, 5 or more times per month, etc.). Further exemplary dosages are described below.

[0196] Suitable dosages can range from 0.01 mg / kg to 100 mg / kg of body weight per day, week, or month. Exemplary doses can include 0.05 mg / kg to 10.0 mg / kg of an active compound (immunotherapeutic construct) disclosed herein. The total daily dose can be 0.05 mg / kg to 30.0 mg / kg of drug administered to a subject one to three times per day, including administration of a total daily dose of 0.05-3.0 mg / kg, 0.1-3.0 mg / kg, 0.5-3.0 mg / kg, 1.0-3.0 mg / kg, 1.5-3.0 mg / kg, 2.0-3.0 mg / kg, 2.5-3.0 mg / kg, or 0.5-3.0 mg / kg / day using a 60-minute oral, intravenous, or other administration regimen. In one particular example, the dose may be administered QD or BID to a subject, for example, at a total daily dose of 1.5 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, or 7.5 mg / kg of a composition comprising up to 92-98% wt / v of a compound disclosed herein.

[0197] Additional useful doses can often be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In other examples, doses can include 1 μg / kg, 10 μg / kg, 20 μg / kg, 40 μg / kg, 80 μg / kg, 200 μg / kg, 0.1-5 mg / kg, or 0.5-1 mg / kg. In other examples, doses can include 1 mg / kg, 10 mg / kg, 20 mg / kg, 40 mg / kg, 80 mg / kg, 200 mg / kg, 400 mg / kg, 450 mg / kg, or more.

[0198] The treatment materials of the present disclosure may be used in serious disease states, i.e., life-threatening or potentially life-threatening situations, in which case substantial over-administration of these compositions is possible and may be felt to be desirable by the treating physician.

[0199] As understood by those skilled in the art, specific dosage is affected by the pharmacokinetics of active compound.For administration, therapeutically effective amount (also referred to herein as dosage) can be initially estimated based on the results obtained from in vitro assay and / or animal model test.This information can be used to more accurately determine the dosage that is useful for the target of interest.Useful preclinical tests include pharmacodynamic analysis, toxicity analysis, etc.

[0200] A therapeutically effective amount can be achieved by administering a single dose or multiple doses over the course of a treatment regimen (e.g., every hour, every 2 hours, every 3 hours, every 4 hours, every 6 hours, every 9 hours, every 12 hours, every 18 hours, daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, or monthly).

[0201] The effective amount of a compound containing an active agent includes a dose that partially or completely achieves the desired therapeutic, preventive and / or biological effect.The actual amount that is effective for a specific application depends on the pathology to be treated and the route of administration.The effective amount for human use can be determined from animal models.For example, the dose for human can be formulated to achieve a local (e.g., intratumoral) level or circulating level that has been found to be effective in animals.

[0202] The compositions may be administered with one or more anesthetic agents including ethanol, bupivacaine, chloroprocaine, levobupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane, isoflurane, ketamine, propofol, sevoflurane, codeine, fentanyl, hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone, remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine, dibucaine, ethyl chloride, xylocaine, and / or phenazopyridine.

[0203] In certain embodiments involving treating or preventing cancer (including, e.g., cancer metastasis), the compositions disclosed herein can be used in combination with other cancer therapies, such as chemotherapeutic agents, radiation therapy, and / or immunotherapy. The compositions described herein can be administered simultaneously or sequentially with another treatment within a selected time frame, e.g., within a 10-minute, 1-hour, 3-hour, 10-hour, 15-hour, 24-hour, or 48-hour time frame, or when the complementary treatment falls within a clinically relevant therapeutic window.

[0204] The pharmaceutical compositions may be for parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous) administration, by infusion or in a depot, formulated in a dosage form appropriate for each administration route.

[0205] In some embodiments, the compositions are administered systemically, for example, intravenously or intraperitoneally, in an amount effective to deliver the composition to target cells. Other routes include infusion or mucosally.

[0206] In certain embodiments, the composition is administered locally, for example, by direct injection into the site to be treated.In some embodiments, the composition is directly injected or administered into one or more tumors or diseased tissues.Typically, local injection causes an increase in the local concentration of the composition, which is higher than the concentration that can be achieved by systemic administration.In some embodiments, the composition is delivered locally to appropriate cells by using a catheter or syringe.Other means of locally delivering such compositions to cells include using an infusion pump or incorporating the composition into a polymer implant, which can cause sustained release of the composition in the immediate vicinity of the implant.

[0207] By way of example, in certain embodiments, immunotherapeutic constructs are administered locally, e.g., to easily accessible tumors, e.g., melanoma, head and neck cancer, breast cancer, and lymphoma, or systemically for other cancers, e.g., lung cancer, liver cancer, pancreatic cancer, prostate cancer, and metastatic cancer.

[0208] Thus, the therapeutic compositions described herein can be administered (alone or as part of a combination therapy) by a variety of routes, including any convenient method for use in human or veterinary medicine. A therapeutically effective amount of the desired active agent can be formulated into a pharmaceutical composition that is parenterally, transmucosally (e.g., orally, nasally, or rectally), or transdermally administered. In some embodiments, administration is parenteral, for example, via intravenous injection, or intraarteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Administration can be as a bolus, by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In certain embodiments, the immunotherapy constructs provided herein can also be delivered using implantable systems that allow for local delivery to non-cutaneous tumors, e.g., hepatic arterial infusion pumps, convection-enhanced delivery. In certain embodiments, for example, those involving the treatment of inflammatory conditions affecting joints, the pharmaceutical composition may be administered directly to the synovium, synovial fluid, or joint capsule, preferably by injection with a syringe. Administration may be local or systemic. The choice may be influenced by the condition being treated and the active agent and composition being administered.

[0209] For injection, the composition can be prepared as an aqueous solution, for example, in a buffer solution such as Hanks' solution, Ringer's solution, or physiological saline. The solution can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the composition can be in lyophilized and / or powder form, for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0210] Compositions containing immunotherapeutic constructs can be administered in aqueous solution via parenteral injection. Injectable preparations can be in the form of suspensions or emulsions, and optionally contain pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such injectable compositions can contain diluents such as sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline solutions of various pH and ionic strengths, and optionally additives such as detergents and solubilizers (e.g., TWEEN™ 20, TWEEN™ 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., thimerosal, benzyl alcohol). Examples of non-aqueous solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The injectable formulations may be lyophilized and resuspended, for example, immediately prior to use. The injectable formulations may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the composition, by irradiating the composition, or by heating the composition.

[0211] In other embodiments, the immunotherapeutic construct-containing composition is applied topically or by infusion. Topical administration can include application to the lung, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. These administration methods can be achieved by formulating the shell or coating of the delivery vehicle with a mucosal transport element. When delivered as either an aerosol or spray-dried particles with an aerodynamic diameter of less than 5 microns, the composition can be delivered to the lungs, crossing the lung epithelial lining and traveling to the bloodstream during inhalation.

[0212] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including, but not limited to, nebulizers, metered dose inhalers, and dry powder inhalers, all of which are well known to those skilled in the art.

[0213] Formulations for administration to mucosal membranes are typically spray-dried drug particles that can be incorporated into tablets, gels, capsules, suspensions, or emulsions. Standard pharmaceutical excipients are available from any formulator.

[0214] Transdermal formulations can be prepared.They are typically ointments, lotions, sprays or patches, and any of them can be prepared using standard techniques.Transdermal formulations can contain penetration enhancers.Combined with this method, chemical enhancers and physical methods including electroporation and microneedles can work.

[0215] Microneedles (MNs) are micron-sized needles, measuring 10-2000 μm in height and 10-50 μm in width, that can penetrate directly through the epidermal layer to the dermal tissue with minimal to no pain (Hao et al., J Biomed Nanotechnol, 13(12):1581-1597, 2017). Several types of microneedles can be used. In some embodiments, metallic or plastic microneedle rollers can be used to physically disrupt the skin surface to facilitate penetration of the applied topical agent (in this case, an immunotherapeutic construct). In some embodiments, degradable and dissolving microneedles can contain the immunotherapeutic construct. Upon administration to the skin, the microneedles can dissolve and release the construct deep within the skin layers. In some embodiments, non-degradable microneedles can be coated with the immunotherapeutic construct to deliver the coated construct deep within the skin layers. Microneedles can be fabricated from many classes of materials, including but not limited to polymers, sugars, polysaccharides, peptides, proteins, metals, inorganic compounds, etc. (Ye et al., Adv Drug Deliv Rev, 127:106-118, 2018). Any material and manufacturing method known in the art for microneedle technology can be applied to enhance delivery of this immunotherapeutic construct.

[0216] Any device that facilitates systemic or local delivery of therapeutic agents can also be applied to the immunotherapy constructs of the present inventors. For example, to deliver the immunotherapy constructs described herein, hepatic arterial infusion (HAI) pump (Cohen et al., The Oncologist, 8(6):553-566, 2003), an implantable chemotherapy device that delivers high concentrations of cytotoxic agents directly to liver metastases while minimizing systemic toxicity, can also be used.

[0217] (VII) Exemplary Uses: Provided herein are immunotherapeutic constructs comprising at least one adjuvant and at least one therapeutically active agent capable of inducing antigen release and / or modulating the immunosuppressive environment (such as the tumor microenvironment), thereby enabling methods for treating and / or preventing hyperproliferative diseases, disorders, or conditions, including cancer, cancer symptoms, cancer progression (including precancer through cancer), and cancer metastasis. Specific examples of hyperproliferative diseases, disorders, or conditions include cancer. In some embodiments, cancer can suppress the immune system of a subject or individual with cancer. In some embodiments, the immunotherapeutic constructs provided herein can suppress or reverse cancer-mediated immunosuppression, allowing immune recognition and elimination of malignant tumors.

[0218] As used herein, the term "treatment" or "treating" refers to any improvement in cancer that occurs in a treated subject compared to an untreated subject. Such improvement can be prevention of cancer worsening or progression (e.g., improved progression-free survival). Furthermore, such improvement can also be a reduction or cure of cancer or its associated symptoms (e.g., a reduction in tumor volume, partial remission, complete remission (e.g., over 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years or more), prevention of cancer recurrence or relapse, a reduction in metastasis, or a reduction in the number of tumors or lesions). It will be understood that a treatment may not be successful in 100% of treated subjects, but may be successful in some individuals compared to other treatments the individual has received, as determined by a skilled artisan (e.g., a physician). As used herein, the term "preventing" refers to avoiding the onset of cancer or its associated syndromes as used herein. It will be understood that prevention refers to avoiding the onset of cancer within a certain time frame in the future. The time frame preferably begins when the compound of the present invention is administered, and continues for at least 1 month, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, or even the remaining physiological lifespan of the subject.It will be understood that prevention may not be successful in 100% of the subjects treated, but may be successful in some individuals compared to other treatments that the individual has received, as determined by those skilled in the art (e.g., physicians).Prevention may also be related to the recurrence of cancer after remission, for example, as measured by a reduced probability of recurrence within a population.Prevention also refers to the elimination of cancer cells at any site in the body, which would otherwise re-grow and later show positive prognosis and disease symptoms.

[0219] The disclosed compositions can be used to treat benign or malignant cancers and tumors thereof. Treatment can directly target and kill cancer cells, indirectly target cancer cells by increasing an immune response against the cancer cells, or a combination thereof.

[0220] In mature animals, a balance is normally maintained between cell renewal and cell death within most organs and tissues. Various types of mature cells in the body have a finite lifespan. As these cells die, new cells are generated through the proliferation and differentiation of various types of stem cells. Under normal circumstances, the generation of new cells is regulated so that the number of any particular type of cell remains constant. However, occasionally, cells arise that no longer respond to normal growth control mechanisms. These cells grow to significant sizes and give rise to clones of cells that can give rise to tumors or neoplasms. Tumors that are unable to grow indefinitely and do not extensively invade healthy surrounding tissue are benign. Tumors that continue to grow and become increasingly invasive are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells shed from the tumor, invade blood or lymphatic vessels, and travel to other tissues, where they continue to grow. In this way, a primary tumor at one site can give rise to secondary tumors at other sites.

[0221] The disclosed compositions can slow or inhibit tumor growth in a subject, reduce tumor growth or size, or completely eliminate a tumor, inhibit or reduce tumor metastasis, and / or inhibit or reduce symptoms associated with tumor development or growth. For example, in some embodiments, the compositions reduce tumor burden in a subject or slow or prevent tumor growth over time.

[0222] Malignant tumors can be classified according to the embryonic origin of the tissue from which they originate. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the skin, or the epithelial lining of internal organs and glands. Sarcomas, which occur less frequently, originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of the hematopoietic cells of the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can appear in multiple organs or tissues of the body, establishing cancer.

[0223] Types of cancer that can be treated using the provided compositions and methods include, but are not limited to, vascular cancers such as multiple myeloma, and solid cancers, including adenocarcinomas and sarcomas of the bone, bladder, brain, breast, cervix, colon, rectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus. In some embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors at multiple locations.

[0224] Administration is not limited to the treatment of existing tumors, but can also be used to prevent or reduce the risk of developing such diseases in individuals, i.e., for prophylactic use, and to reduce the spread of cancer, for example, by metastasis. Potential candidates for prophylactic vaccination include individuals who are at high risk of developing cancer, i.e., who have a personal or family history of a particular type of cancer.

[0225] In one embodiment, an immunotherapy construct containing the adjuvant CpG, siRNA against BRAFV600E to kill melanoma and precursor cells (melanocytes in the nevi) and cause antigen release, and siRNA against STAT3 to alleviate the immunosuppressive environment can be used to generate adaptive immunity in subjects at high risk of developing melanoma and in melanoma patients. The immunotherapy construct not only prevents and treats melanoma, but also protects late-stage melanoma patients from future recurrence or relapse after surgery. Along with the targeting agent, siRNA against other genes may be incorporated onto or into the nanoparticle / construct.

[0226] Various treatment modalities, including systemic immunotherapy, chemotherapy, and biochemotherapy, have been tested in adjuvant settings, but they also result in systemic toxicity and side effects that can be overcome by local treatment with the immunotherapeutic constructs described herein. In certain embodiments, immunotherapeutic constructs (e.g., containing CpG or another adjuvant along with chemotherapy drugs, targeted therapy, and / or siRNA against STAT3) can be used to treat breast cancer in an adjuvant setting by intratumoral injection prior to surgical removal of the primary breast tumor, thereby preventing cancer recurrence and metastasis without the toxicity of systemic drugs.

[0227] As used herein, the term "cancer" refers to any type of cancer, neoplasm, or malignant tumor found in mammals, including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated using the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g., triple negative, ER positive, ER negative, chemotherapy resistant, Herceptin® resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary , metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck or esophageal), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma or multiple myeloma. Additional examples include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus, or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, cancer), malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid carcinoma, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma of the breast, carcinoma of pancreatic stellate cells, carcinoma of hepatic stellate cells, or prostate cancer. As used herein, the term "precancer" refers to a condition or growth that precedes or progresses to cancer.As used herein, the term "cancer metastasis" refers to the spread of cancer cells or tumors from one organ or part of the body to another organ or part of the body.

[0228] The term "leukemia" refers broadly to progressive malignant diseases of the blood-forming organs, generally characterized by a distortion of the proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and character of the disease: acute or chronic; (2) the type of cell involved: myeloid (myeloid), lymphocytic (lymphotropic), or monocytic; and (3) the increased or absent number of abnormal cells in the blood: leukemic or non-leukemic (subleukemic). Exemplary leukemias that can be treated using a compound, pharmaceutical composition, or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocythemic leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonic cell leukemia, eosinophilic leukemia, Gross' leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, These include lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, small myeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, or anaplastic cell leukemia.

[0229] The term "sarcoma" generally refers to a tumor composed of a substance like embryonic connective tissue and generally made up of closely packed cells embedded in a fibrous or homogeneous substance. Sarcomas that may be treated using the compounds, pharmaceutical compositions, or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abenethy's sarcoma, liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid rhabdomyosarcoma, chlorosarcoma, and leiomyosarcoma. sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, B-cell immunoblastic sarcoma, lymphoma, T-cell immunoblastic sarcoma, Jensen's sarcoma, Kaposi's sarcoma, Kupffer's astrocytic sarcoma, angiosarcoma, leukemia sarcoma, malignant mesenchymal sarcoma, parosteal osteosarcoma, reticulocytic sarcoma, Rous sarcoma, serosarcoma cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

[0230] The term "melanoma" is intended to mean a tumor arising from the melanocyte system of the skin and other organs. Melanomas that can be treated using the compounds, pharmaceutical compositions, or methods provided herein include, for example, acral lentiginous melanoma, amelanotic malignant melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

[0231] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated using a compound, pharmaceutical composition, or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, lobular carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatous, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchoalveolar carcinoma, bronchiolocarcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, cylindrical carcinoma, carcinoma, cylindrical cell carcinoma, ductal carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, medullary carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ulcerative carcinoma, fibrous carcinoma, gelatinous carcinoma, colloid adenocarcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, adrenal-like carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepithelial carcinoma, intraepithelial carcinoma carcinoma, Krompecher's carcinoma, Kurticayskii cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinomacarcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma, melanoma, moll carcinoma, mucinous carcinoma, mucinous secretory carcinoma, carcinoma mucocellulare, mucoepidermoid carcinoma, mucinous carcinoma, mucinous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcomatoid carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tubular carcinoma, tuberous carcinoma, verrucous carcinoma or choriocarcinoma.

[0232] (VIII) Kit In particular, active ingredients including at least one of the therapeutic constructs described (including a delivery vehicle containing or associated with at least one therapeutic agent and at least one adjuvant) can be provided as a kit. The kit can optionally include one or more containers containing (containing) one or more compounds or complexes described herein (e.g., anti-cancer agents), along with one or more additional agents for use in treatment. For example, some kits include a certain amount of at least one additional anti-cancer composition, or a certain amount of at least one additional anti-inflammatory agent, or both.

[0233] Any active ingredient in the kit may be provided in pre-measured doses, although this is not required. It is anticipated that certain kits will contain multiple doses.

[0234] The kit may also include a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting approval by the agency for manufacture, use, or sale for human administration. The notice may state that the provided active ingredient can be administered to a subject. The kit may include additional instructions for using the kit, such as instructions regarding administration, proper disposal of associated waste, etc. The instructions may be in the form of printed instructions provided within the kit, or the instructions may be printed on a part of the kit itself. The instructions may be in the form of a sheet, pamphlet, booklet, CD-ROM, or computer-readable device, or the instructions may be provided remotely, such as a website. In certain embodiments, the kit may also include some or all of the necessary medical supplies needed to effectively use the kit, such as applicators, ampoules, sponges, sterile adhesive strips, Chloraprep, gloves, etc. The contents of any of the kits described herein may be varied. The kit instructions direct the use of the active ingredients included in the kit to achieve the clinical and / or therapeutic uses described herein.

[0235] Suitable methods, materials, and examples to be used in the practice and / or testing of embodiments of the disclosed invention are described herein. Such methods and materials are illustrative only and are not intended to be limiting. Other methods, materials, and examples similar or equivalent to those described herein can be used.

[0236] The following illustrative embodiments and examples are included to demonstrate particular aspects of the present disclosure. Those of skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0237] (IX) Exemplary Embodiments 1. a delivery system and at least one therapeutic agent, e.g., loaded into, attached to the surface of, bound to, encapsulated in, or contained within the delivery system, that causes tumor antigen release and / or modulates an immunosuppressive tumor microenvironment; For example, at least one adjuvant compound attached to the surface of the delivery system, bound to the delivery system, encapsulated within the delivery system, or contained within the delivery system. 1. An immunotherapeutic construct comprising: The immunotherapeutic construct does not comprise a tumor-specific antigen or ovalbumin. 2. The immunotherapeutic construct of embodiment 1, wherein the delivery system comprises a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, an inorganic particle coated with a polymer or lipid, or a hybrid thereof. 3. The immunotherapeutic construct of embodiment 2, wherein the delivery vehicle is a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, or an inorganic particle coated with a polymer or lipid. 4. The immunotherapeutic construct of embodiment 3, wherein the delivery vehicle is an inorganic particle and comprises one or more of mesoporous silica, gold, aluminum, iron oxide, calcium phosphate, or antioxidant particles. 5. The immunotherapeutic construct of embodiment 4, wherein the inorganic particles comprise antioxidant particles comprising cerium oxide. 6. The immunotherapeutic construct of embodiment 4, wherein the delivery vehicle comprises mesoporous silica particles. 7. The delivery vehicle is a fullerene, endohedral metallofullerene, 10. The immunotherapeutic construct of embodiment 1, comprising one or more of: trimetal nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, silicon, silica microspheres and nanospheres, polymer microspheres and nanospheres, silica shells, biodegradable PLGA microspheres and nanospheres, gold particles, cerium oxide particles, zinc oxide particles, silver particles, aluminum particles, carbon particles, iron particles, iron oxide particles, calcium phosphate, adjuvant particles, and / or modified micelles. 8. The immunotherapy construct of any one of embodiments 1-7, wherein the delivery vehicle is a polymer particle comprising one or more of PLGA, PLL, polyarginine, PEG, PEI, or chitosan. 9. The immunotherapeutic construct of any one of aspects 1 to 8, which is a nanoparticle having a hydrodynamic size of between 5 nm and 999 nm. 10. The immunotherapeutic construct of any one of aspects 1-8, which is a nanoparticle having a hydrodynamic size of between 1 micron and 1000 microns. 11. The immunotherapeutic construct of embodiment 6, wherein the delivery vehicle comprises mesoporous silica nanoparticles having a size of about 5 to about 200 nm. 12. The immunotherapeutic construct of embodiment 11, wherein the mesoporous silica nanoparticles are coated with cross-linked polyethyleneimine and polyethylene glycol. 13. The immunotherapeutic construct of any one of embodiments 1-12, wherein at least one therapeutic agent comprises an siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-cas9 element), oligonucleotide, polynucleotide, peptide, protein, chemotherapeutic drug, toxin, antioxidant, small molecule inhibitor, antibody, or radiotherapeutic agent. 14. The immunotherapeutic construct of embodiment 13, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, or DNA. 15. The immunotherapeutic construct of embodiment 14, wherein at least one therapeutic agent comprises an siRNA. 16. The immunotherapeutic construct of embodiment 15, wherein at least one therapeutic agent comprises an siRNA that inhibits expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, BRAF, BRAF V600E, or MTDH. 17. The immunotherapeutic construct of embodiment 15 or 16, wherein at least one therapeutic agent comprises an siRNA that inhibits STAT3 expression or activity. 18. The immunotherapeutic construct of any one of embodiments 15-17, wherein at least one therapeutic agent comprises an siRNA that inhibits expression of HER2 activity. 19. The immunotherapeutic construct of any one of embodiments 1-12, wherein at least one therapeutic agent inhibits expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, or MTDH. 20. The immunotherapeutic construct of any one of embodiments 1-19, wherein the therapeutic agent is an anti-cancer agent comprising one or more of an antibiotic, a plant alkaloid, a PLK1 inhibitor, a mitotic phase kinase inhibitor, an immune checkpoint inhibitor, a platinum-based chemotherapeutic agent, a HER2 small molecule inhibitor, or a HER2-specific antibody. 21. The immunotherapeutic construct of embodiment 20, wherein the therapeutic agent is a checkpoint inhibitor, and the checkpoint inhibitor is an antibody against PD-L1, PD1, or CTLA4. 22. The immunotherapeutic construct of embodiment 21, wherein the checkpoint inhibitor is an antibody against PD-L1. 23. The immunotherapeutic construct of any one of aspects 1-22, wherein at least one therapeutic agent comprises a PLK1 inhibitor. 24. The immunotherapeutic construct of embodiment 23, wherein the PLK1 inhibitor is volasertib. 25. The immunotherapeutic construct of any one of aspects 1-24, wherein at least one therapeutic agent comprises one or more of docetaxel, mitoxantrone, or cabazitaxel. 26. The immunotherapeutic construct of any one of aspects 1-25, wherein at least one therapeutic agent comprises an anti-EGFR antibody. 27. The immunotherapeutic construct of embodiment 26, wherein the anti-EGFR antibody is cetuximab. 28. The immunotherapeutic construct of any one of aspects 1-25, wherein at least one therapeutic agent comprises an anti-HER2 antibody. 29. The immunotherapeutic construct of embodiment 28, wherein the anti-HER2 antibody is trastuzumab. 30. The immunotherapeutic construct of any one of embodiments 1-29, wherein the adjuvant has immunostimulatory activity and comprises one or more of a CpG oligonucleotide, a DNA TLR agonist comprising a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, lipopolysaccharide, a plant extract, or a derivative thereof. 31. The immunotherapeutic construct of any one of aspects 1-30, wherein the adjuvant compound comprises a CpG oligonucleotide, imiquimod, resiquimod, gardikimod, poly I:C, poly ICLC, dSLIM, or EnanDIM. 32. The immunotherapeutic construct of any one of aspects 1-31, wherein the adjuvant compound comprises a CpG oligonucleotide. 33. An immunotherapeutic construct according to any one of embodiments 1 to 32; and and at least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof. A composition comprising: 34. A method of treating cancer, comprising administering to a subject having cancer an effective amount of the immunotherapeutic construct of any one of embodiments 1 to 32 or the composition of embodiment 33, to reduce one or more symptoms of the cancer. 35. The method of embodiment 34, wherein the subject is a mammal. 36. The method of embodiment 35, wherein the mammal is a human. 37. A method of treating cells exhibiting a cancer symptom, comprising: contacting the cells with a therapeutically effective amount of an immunotherapeutic construct of any one of embodiments 1 to 32 or a composition of embodiment 33. The method comprising: 38. A method of treating cells obtained from a subject exhibiting symptoms of cancer, comprising: contacting the cells with a therapeutically effective amount of an immunotherapeutic construct of any one of embodiments 1 to 32 or a composition of embodiment 33. The method comprising: 39. Contacting cells ex vivo with a therapeutically effective amount of the immunotherapeutic construct of any one of embodiments 1 to 32 or the composition of embodiment 33. A method comprising: 40. The method of embodiment 38 or 39, wherein the cell is a cancer cell. 41. The method of embodiment 38 or 39, wherein the cell is not a cancer cell. 42. The method of embodiment 41, wherein the cell is an immune cell. 43. The method of embodiment 38 or 39, wherein the cells are immortalized. 44. The method of any one of embodiments 37-43, further comprising administering at least one treated cell back to the subject. 45. A method of treating a subject diagnosed with or at increased risk for developing a hyperproliferative disease or condition, comprising: administering to said subject an effective amount of the composition of embodiment 33. The method comprising: 46. ​​The method of embodiment 45, wherein the subject is a mammal. 47. The method of embodiment 46, wherein the mammal is a human. 48. The method of any one of embodiments 45-47, wherein the hyperproliferative disease or condition comprises one or more of cancer, precancer, or cancer metastasis. 49. The method of embodiments 45-48, wherein the hyperproliferative disease comprises one or more of melanoma, lung cancer, breast cancer, pancreatic cancer, brain cancer, prostate cancer, head and neck cancer, renal cancer, colorectal cancer, lymphoma, colon cancer, or liver cancer. 50. The administering step comprises: direct injection into a tumor in a subject; Systemic injection in a subject; or Local application to the subject 50. The method of any one of embodiments 45-49, comprising one or more of: 51. The method of any one of aspects 45-50, wherein the administering step comprises applying a microneedle to the subject. 52. A method of enhancing the efficacy of anti-cancer therapy in a subject in need thereof, comprising: an effective amount of the immunotherapeutic construct of any one of embodiments 1 to 32 or the composition of embodiment 33; with at least one anticancer drug to a subject in need thereof. The method comprising: 53. The method of embodiment 52, wherein the anti-cancer agent is a chemotherapeutic agent or a targeted therapy agent. 54. A method of enhancing the efficacy of checkpoint blockade immunotherapy in a subject diagnosed with a neoplasm, comprising: an effective amount of the immunotherapeutic construct of any one of embodiments 1 to 32 or the composition of embodiment 33; At least one immune checkpoint inhibitor to a subject in need thereof. The method comprising: 55. A method of enhancing the effectiveness of radiation therapy in a subject diagnosed with a neoplasm, comprising: an effective amount of the immunotherapeutic construct of any one of embodiments 1 to 32 or the composition of embodiment 33; At least one radiation therapy to a subject in need thereof. The method comprising: 56. The method of any one of embodiments 52-55, wherein the immunotherapeutic construct or composition and the anti-cancer therapy are administered sequentially or simultaneously. 57. The method of any one of embodiments 52-56, wherein the subject is a mammal. 58. The method of embodiment 57, wherein the mammal is a human. 59. An immunotherapeutic agent according to any one of embodiments 1 to 32; Anticancer drugs and Includes a kit. 60. The kit of embodiment 59, wherein the anti-cancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor. [Example]

[0238] (X) Example Example 1: Using nanotechnology to engineer tumors to become depots for cancer vaccination to prime systemic anti-tumor immunity Immune checkpoint inhibitors (ICIs), such as those targeting PD-L1 / PD-1 and CTLA-4, have shown excellent results in the clinic (Sharon et al., Chin J Cancer, 33(9):434-44, 2014; Buchbinder & Desai, Am J Clin Oncol, 39(1):98-106, 2016). Immune checkpoint inhibitors release the brakes on the patient's own immune system to fight cancer, provide immunological memory, and result in long-lasting immune responses even after treatment has stopped. Therefore, ICIs can produce more durable responses than chemotherapy and targeted therapy in advanced cancers, as shown in Figure 1. However, this treatment only works for a small proportion of patients (approximately 10–40%) (Ribas, Update Cancer Therapeutics, 2(3):133–139, 2007; Topalian et al., N Engl J Med, 366(26):2443–54, 2012). The lack of response is typically due to a lack of pre-existing antitumor immunity (e.g., tumor-directed effector (CD8+) T cells) (Santarpia & Karachaliou, Cancer Biology & Medicine, 12(2):74–78, 2015; Tumeh et al., Nature, 515(7528):568–571, ​​2014). Therefore, the ability to prime the antitumor CD8+ T cell repertoire is essential for immunotherapy.

[0239] In situ tumor vaccination is a strategy in which tumors die locally in the presence of immune stimuli, releasing tumor antigens that together prime systemic adaptive immunity against the tumor (Pierce et al., Hum Vaccin Immunother, 11(8):1901-9, 2015). This strategy is highly promising because it circumvents the need for prior identification of tumor antigens in traditional cancer vaccine development. It is also personalized therapy because a unique set of tumor antigens is released, priming specific immunity for each patient.

[0240] The cancer immunotherapy approach provided herein utilizes the patient's own tumor as a depot (in situ tumor vaccination) for a personalized set of tumor antigens. An example of the system described herein is called AIRISE (Augmenting Immune Response and Inhibiting Suppressive Environment of Tumors). AIRISE aims to improve patient survival outcomes when used alone or together with checkpoint inhibitors (Figure 1). The provided particles (immunotherapy constructs) carry an adjuvant (e.g., CpG) and one or more compounds (siRNA, drugs, small molecules, etc.) that trigger antigen release and / or modulate the immunosuppressive tumor microenvironment (e.g., docetaxel, siRNA against STAT3). When the tumor site is treated with the provided immunotherapy constructs (e.g., via local intratumoral injection or via tumor homing with systemic delivery), tumor antigens are released in the presence of immune stimulation (provided by the adjuvant), initiating adaptive immunity. At the same time, compounds that modulate the immunosuppressive tumor microenvironment can be co-delivered on the same nanoparticles to maximize the in situ tumor vaccination effect. Tumor antigens can be taken up by AIRISE-activated antigen-presenting cells (APCs), which present the antigens to naive T cells. T cells (against these tumor antigens) are primed and activated to become effector T cells (either in lymph nodes or at the tumor site), which then proliferate throughout the body. Effector T cells specifically home to tumors that share the same tumor antigens, regardless of their location in the body (e.g., both locally treated tumors and untreated metastatic tumors elsewhere in the body). Cancer cell death by cytotoxic T cells releases even more tumor antigens, amplifying the process of antitumor T cell generation in a positive feedback loop. Notably, even if treatment is local (e.g., intratumoral injection), the vaccination effect induced locally at the tumor site generates a systemic, long-lasting antitumor immune response (Figure 2).For example, AIRISE can be injected directly into a melanoma lesion, and treatment can affect both the injected tumor and untreated metastatic melanoma tumors in the lung or liver.

[0241] These anti-tumor T cells, trained to recognize specific tumor antigens, control tumors both at the injection / treatment site and elsewhere in the body (Figure 2). Cargo combinations can be applied to any type of micro / nanoparticle to create AIRISE. The following example utilizes established mesoporous silica nanoparticles (U.S. Patent Application Publication No. 2017 / 0172923) as proof of concept. Another example, shown in Figure 11, utilizes cationic lipid particles with a hydrodynamic size of 1.1 microns for in situ vaccination with CpG and siSTAT3, yielding results similar to those in Figure 7, suggesting that various types and sizes of particles can be used.

[0242] Methods and Materials Nanoparticle synthesis and characterization: Mesoporous silica-based nanoparticles were synthesized as previously described (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015; Ngamcherdtrakul et al., International J Nanomed, 13:4015-4027, 2018). Briefly, mesoporous silica nanoparticles (MSNPs) were synthesized by sol-gel synthesis. The MSNP core was coated layer-by-layer with polyethyleneimine (PEI) and polyethylene glycol (PEG). Additionally, PEI on the MSNPs was crosslinked to enhance the efficacy and safety of oligonucleotide delivery, as previously described (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). The crosslinked PEI and PEG-coated MSNPs are hereafter referred to as "NPs" in this example.

[0243] Cargo loading onto NPs: siRNA and CpG (ODN 1826; SEQ ID NO:7) were electrostatically loaded onto nanoparticles (NPs) by mixing for 10 minutes, although shorter times (2–5 minutes) were also effective. Mitoxantrone was also loaded onto NPs by mixing in aqueous solution (e.g., PBS) at room temperature for 4 hours. Loading was achieved in a fully conjugated manner, as confirmed by the absence of free cargo molecules in the supernatant upon separation of the cargo-loaded NPs by centrifugation. Cargo content was measured spectrophotometrically. siRNA was conjugated with Dy677 dye (Dharmacon) and quantified by the fluorescent signal. CpG was measured using Nanodrop spectrophotometry. Unbound siRNA and CpG could also be measured by gel electrophoresis. Mitoxantrone (MTX) was quantified by measuring absorbance at 658 nm. The final NPs containing CpG and siRNA were characterized for hydrodynamic size in PBS by Zetasizer (Figure 26 and Figure 29).

[0244] Docetaxel was overloaded onto the nanoparticles before PEI conjugation. Briefly, MSNPs were mixed with an ethanol solution of docetaxel overnight before PEI conjugation. Unbound docetaxel and PEI were washed away in PBS. PEI-NPs (DTX) were then conjugated with PEG according to previous methods (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015; Ngamcherdtrakul et al., International J Nanomed, 13:4015-4027, 2018). The final products contained 0.5-1.7 wt% DTX with a starting DTX-to-MSNP mass ratio of 0.2-0.8. They had a DLS size of 100 nm.

[0245] B16F10 Bilateral Orthotopic Murine Melanoma Tumor Model: Six-week-old female C57BL / 6 mice were obtained from the Charles River NCI colony (Wilmington, MA). Each mouse was implanted intradermally with B16F10 cells in the left shoulder (local, 250,000 cells) and right shoulder (distal, 100,000 cells). Eight days after implantation, test compounds / constructs were injected intratumorally into the left (local) tumor only, while the right (distal) tumor was left untreated. Unless otherwise stated, test compounds / constructs were administered three times every three days. Both local and distal tumor burdens in mice were measured every 1-2 days using a Vernier caliper, V = 0.5 x length x width. 2 Tumor volume was calculated by the following formula: Survival was also monitored. 3 When the stool size exceeded 100 mg / kg, the mice were sacrificed.

[0246] For studies combining NP treatment with immune checkpoint inhibitors, a cocktail of PD-1 Ab (200 μg / mouse) and CTLA-4 Ab (100 μg / mouse) was administered intraperitoneally (three doses every three days) on the same day as intratumoral treatment of the NP compounds.

[0247] To confirm that the therapeutic effect was immune-mediated, mice treated with the test compounds / constructs were injected intraperitoneally with CD8 antibody (200 μg / mouse) starting one day before the first intratumoral treatment and continuing throughout the study.

[0248] For immune profiling, local and distant tumors and their respective draining lymph nodes were harvested and processed into single cells according to established protocols. The harvested cells were then stained for various surface proteins (e.g., CD45, CD8, CD4, CD44, TIM3, PD-1, CD39, LAG3, CD3, CD19, CD11b, CD11c, MHCII, CD80, Ly6C, Ly6G, F4 / 80, CD206) using a set of fluorescently labeled antibodies that together identify various immune cell populations and their status. Specific intracellular proteins (e.g., Ki67, FoxP3, STAT3) can also be stained according to the manufacturer's protocol (BD Biosciences). Flow cytometry was typically performed on a BD Fortessa (four lasers, up to 18 parameters) against two separate antibody panels (lymphoid and myeloid). To ensure the robustness of multicolor flow cytometry analysis, fluorescence compensation was performed according to established protocols known in the art. To test AIRISE uptake within the tumor microenvironment (Figure 18), mice bearing bilateral B16F10 tumors were intratumorally injected with AIRISE-02 (loaded with Alexa488 dye-conjugated siSCR instead of siSTAT3) as described above. Two hours after treatment, tumors (treated and untreated) were harvested, processed into single cells, and surface stained with the panel of antibodies described herein. The presence of AIRISE-02 within various cell populations within the tumor was analyzed by flow cytometry.

[0249] LLC-JSP metastatic mouse lung tumor model: LLC-JSP (200,000 cells) was injected intravenously (tail vein) into 6-week-old C57BL / 6 mice. Three days after cancer cell injection, mice were randomized and treated with the test compound / construct every three days for a total of four treatments. In this model, AIRISE-02 was injected intravenously via the tail vein rather than into the tumor.

[0250] CT26 bilateral ectopic tumor model. 250K and 100K CT26 (murine colorectal carcinoma) cells were implanted into both flanks of each mouse (Balb / c). 15 days after tumor implantation, the mice were treated with the test compound / construct.

[0251] 4T1 bilateral orthotopic tumor model. 100K and 40K 4T1 cells were implanted into the bilateral mammary fat pads of each mouse (Balb / c). 8 or 11 days after tumor implantation (as indicated), mice were treated with test compounds / constructs.

[0252] Results and Discussion CpG-loaded nanoparticles exhibit better adjuvant properties than free CpG C57BL / 6 mice (n = 3 / group) were injected with 4 μg of free CpG or 4 μg of CpG on NPs into one footpad. Twenty-four hours after injection, local draining lymph nodes (DLN) and non-draining lymph nodes (NDLN) were harvested and analyzed for CD11c, MHCII, and CD80 expression by flow cytometry. CpG-loaded nanoparticles activated dendritic cells in the local DLN significantly better than free CpG (Figure 3). CpG nanoparticles also enhance the possibility of simultaneously delivering several therapeutic cargoes to the same site. Furthermore, exemplary nanoparticles were highly optimized for the delivery of siRNA, which can modulate tumor immunosuppressive properties at the mRNA level. Co-delivery of CpG (or another adjuvant) with siRNA or other targeting molecules that address some features of tumor immunosuppression has been proposed to beneficially prime immunotherapeutic effects.

[0253] Adjuvant-loaded nanoparticles induce in situ tumor vaccination To evaluate the ability of CpG-NPs to induce in situ tumor vaccination, mice bearing two (bilateral) melanoma tumors were used. Only one of the two tumors was intratumorally injected with treatment, while the other tumor was left untreated. The growth of both tumors was monitored. Intratumoral treatment with CpG-NPs successfully primed a systemic immune response and induced a strong abscopal effect (inhibition of both the locally treated tumor and the distant untreated tumor) (Figure 4). Consequently, survival time was also extended. The immunotherapy construct technology described herein induces in situ vaccination, so there is no need to load tumor antigens onto or into either of the complexes.

[0254] Co-delivery of drug and CpG on the same NPs can induce effective in situ tumor vaccination Intratumoral injection of chemotherapy drugs to induce in situ tumor vaccination has not been widely explored. In fact, intratumoral injection of nanoparticles containing chemotherapy drugs and adjuvants has not been performed to date. This is due to the potential toxicity of chemotherapy drugs to immune cells, which may negate activated immunotherapy responses.

[0255] Surprisingly, simultaneous delivery of the chemotherapy drug docetaxel (DTX) and CpG (CpG-DTX-NPs) on the same nanoparticles did not worsen the in situ vaccination efficacy of CpG-NPs, despite the potential toxicity of chemotherapy to immune cells (Figure 5). In fact, CpG-DTX-NPs can control local tumors better than CpG-NPs. At the same time, CpG-DTX-NPs still induce slightly better in situ vaccination efficacy than CpG-NPs, as demonstrated by the control of distant tumors and long-term survival of mice. Furthermore, DTX-NPs do not show significant activity.

[0256] Co-delivery of another chemotherapeutic drug, mitoxantrone (MTX), and CpG on the same nanoparticles also induced an in situ vaccination effect of CpG-NPs (Figure 16).

[0257] Immune cell analysis shows a significant increase in activated CD8+ T cells within tumors and lymph nodes After treatment (Figure 5A), we characterized the T cell status within local and distant tumors 7 days after the first administration. Compared with saline, treatment with CpG-DTX-NP (abbreviated as "NP" in Figure 6) resulted in desirable characteristics of cytotoxic CD8+ T cells: relatively low expression of PD1 within treated and distant tumors (Figure 6A), relatively high proliferation of CD8+ T cells within treated tumors (Figure 6B), relatively low depletion of CD8+ T cells within treated tumors (Figure 6C), and a higher ratio of CD8+ T cells to regulatory T cells (Figure 6D). Similarly, more CD8+ T cells were found within the draining lymph nodes (DLN) of treated tumors (Figure 6E), and these CD8+ T cells were more activated (Figure 6F). Within the DLN of both tumors, activated CD8+ T cells were more proliferative (as measured by the Ki67 marker) (Figure 6G). Relatively high proliferation and activity of CD8+ T cells was observed within non-DLNs (Figure 6H), suggesting that T cells were transported outside of the local lymph nodes (e.g., in the blood) and contributed to the abscopal effect of the treatment. These T cell characteristics indicate that the nanoparticles described herein can increase the beneficial antitumor T cell repertoire (non-exhausted state) and generate systemic immunity. DTX on our NPs killed tumor cells and released tumor antigens, but did not harm CD8+ T cells and instead increased their proliferation.

[0258] Co-delivery of siRNA and CpG on the same NPs can induce effective in situ tumor vaccination This is the first time that a single NP has been used to co-deliver an adjuvant and siRNA via intratumoral injection. Although nanoparticles have previously been used to deliver immunogenic chemotherapeutic drugs, tumor antigens, or adjuvants, siRNA has never been co-delivered with an adjuvant on a nanoparticle.

[0259] Intratumoral co-delivery of CpG oligos and siSTAT3 on NPs induced systemic antitumor immune responses in a melanoma mouse model siSTAT3 (2 wt%) was loaded within the mesoporous silica core of the NPs, and CpG oligonucleotides (10 wt%) were loaded on the outer surface (bound to a cationic polymer layer but protected from enzymatic degradation by PEG). It has been proposed that tumor antigens (already present in the tumor or released by cancer death during treatment) prime antitumor immunity in the presence of CpG immunostimulation. siSTAT3 modulates the immunosuppressive tumor environment and amplifies immunotherapeutic responses. To demonstrate this, siSTAT3-CpG-NPs (AIRISE-02) were evaluated in a bilateral B16F10 melanoma tumor model in mice (Figure 7A). Eight days after tumor implantation, AIRISE-02 was injected into one of the tumors (locally) every three days for a total of three injections. AIRISE-02 significantly improved mouse survival (Figure 7D) and reduced both local (treated, Figure 7B) and distant (untreated, Figure 7C) tumors, suggesting successful in situ tumor vaccination and an abscopal effect of the treatment. Figures 7B-C also show that siSTAT3-CpG-NPs outperform CpG-NPs and siSTAT3-NPs.

[0260] In another experiment, mice were treated with AIRISE-02 (siSTAT3-CpG-NP) in the same manner as in Figure 7. One day after the third dose (or seven days after the first dose), mice were sacrificed. Tumors and associated draining lymph nodes (DLNs) were harvested and subjected to immune profiling by multicolor flow cytometry. Figure 17 shows that AIRISE-02 resulted in significantly higher CD8 / Treg ratios for both local tumors (treated) and distant tumors (untreated) as well as associated DLNs (p<0.05 for AIRISE-02 vs. saline), confirming successful in situ tumor vaccination. Within patient tumors, regulatory T cells (Tregs) are typically elevated and suppress antitumor immune responses, including CD8+ T cell activity. Therefore, a higher intratumoral CD8 / Treg ratio is desirable and is an indicator of long-term survival in cancer patients. CpG-NPs did not significantly increase CD8 / Tregs in either the tumor or lymph nodes at this time point, consistent with its lower effect than AIRISE-02 in Figure 7. Furthermore, in AIRISE-02-treated mice, effector CD8+ T cells in the lymph nodes were more proliferative (Ki-67) than in other control groups (Figure 17C).

[0261] In another experiment, mice were treated by intratumoral injection of siRNA-CpG-NPs (AIRISE-02) (a model similar to that shown in Figure 7). The siRNA was tagged with Alexa-488. Figure 18 shows that 2 hours after intratumoral injection, siRNA-CpG-NPs were taken up by 15% of cells in the TME, whereas a previous study reported that CpG-siSTAT3 conjugates were taken up by only 2% of cells in the TME 1 and 3 hours after intratumoral injection (Kortylewski et al., Nature Biotechnology, 27(10):925-932, 2009). In line with our findings that the TME consists of 80-90% cancer cells, among the cells that took up siRNA-CpG-NPs, 80% were cancer cells (CD45-) and 20% were immune cells (CD45+). In contrast, the previously described CpG-siSTAT3 conjugate relies on CpG for processing and is primarily taken up by TLR9+ cells, not TLR9- cancer cells. Delivery of siSTAT3 and CpG to both cancer cells and immune cells is desirable. Among immune cells, myeloid cells (CD45+CD3-CD19-) took up siSTAT3-CpG-NPs to the greatest extent. These included macrophages (F4 / 80+) and DCs (CD11c+MHCII+). NPs were not detected in distant untreated tumors (not shown), indicating no extravasation of NPs into untreated tumors.

[0262] We also confirmed that the therapeutic effect of AIRISE-02 was dependent on the immune response, rather than on the direct cytotoxic effect of the therapeutic agent. When CD8 was depleted from mice using an anti-CD8 antibody (Figure 8A), the therapeutic response of siSTAT3-CpG-NPs was significantly reduced (Figures 8B-8D). This indicates that the therapeutic effect was immune-mediated.

[0263] Furthermore, because NP treatment generates a relatively large CD8+ T cell repertoire, it has been proposed that this treatment could be beneficially combined with checkpoint inhibitor treatment to enhance anticancer efficacy. This was tested by using siSTAT3-CpG-NP (AIRISE-02, Figure 9A) with two checkpoint inhibitors currently used in the clinic (anti-PD-1 antibody and anti-CTLA4 antibody). NP treatment (intratumoral administration into only one of the two tumors) has a similar effect on survival as the checkpoint inhibitor cocktail (intraperitoneal administration) (Figure 9D). Combining AIRISE-02 with the checkpoint inhibitor cocktail substantially improved efficacy in terms of control of local tumors (Figure 9B) and distant tumors (Figure 9C) as well as mouse survival (Figure 9D). Notably, complete cure was achieved in five of eight mice (which remain tumor-free for 10 months to date), whereas AIRISE-02 or ICI alone did not cure any mice (Figure 10C). The curative effect in the B16F10 model is considered superior because this model is known in the literature to be aggressive and previous CpG-based vaccines plus ICIs have not shown curative effects when treatment is initiated one week after tumor implantation (though curative effects have sometimes been reported in preventive settings). In another group of cured mice, tumor rechallenge was performed by implanting B16F10 cells three months after the last treatment. The cured mice rejected tumor growth, suggesting the successful long-lasting antitumor effect (memory effect) of AIRISE plus ICI.

[0264] Systemic co-delivery of CpG oligos and siSTAT3 on NPs extended survival in mice, suggesting potential immune priming and activation. In addition to intratumoral administration, AIRISE-02 can be administered systemically to treat cancers that are not easily accessible for intratumoral injection, such as lung cancer. Figure 10A shows the treatment schedule for AIRISE-02 via the tail vein of mice bearing Lewis Lung Carcinoma (LLC-JSP) tumors. Long-term survival was observed (Figure 10B), suggesting successful anti-cancer immunotherapy.

[0265] Intratumoral co-delivery of CpG oligos and siSTAT3 by cationic lipids also induces in situ tumor vaccination siSTAT3 and CpG were mixed with cationic lipids (Dharmafect, Dharmacon) to form lipid nanoparticles, which were administered to mice in the same manner as in Figure 7. Figures 11A-11C show that a very similar response was achieved with the cationic lipids as observed with mesoporous silica nanoparticles (Figure 7). This demonstrates that for intratumoral injection, various types of nanoparticles can be used to create therapeutics using the cargo combinations described herein.

[0266] The NPs can deliver siRNA together with CpG to both cancer cells and immune cells, resulting in target gene knockdown As shown in Figure 12A for B16F10 cancer cells, Figure 12B for J774 macrophages, and Figure 12C for mouse primary DCs, NPs can deliver siRNA to both cancer cells and immune cells, knocking down the STAT3 gene (as an example). Interestingly, siSCR-NPs were also found to reduce STAT3 levels in DCs (see Figure 12C vs. untreated). This was not caused by nanoparticle toxicity, as cell viability was unchanged compared to untreated controls (Figure 34), and STAT3 mRNA was normalized by housekeeping mRNA. Without being bound by any explanation, it is proposed that this may be due to the antioxidant properties of mesoporous silica nanoparticles, as antioxidants have previously been reported to counteract immunosuppressive pathways, including STAT3 activation (Yoon et al., Autophagy, 6(8):1125-1138, 2010). On the other hand, Dharmafect (a commercially available transfection agent based on cationic lipids (non-antioxidant) manufactured by Horizon Discovery) was found to increase STAT3 expression in DCs (Figure 35), potentially resulting in an undesirable immunosuppressive TME. This suggests that the use of the antioxidant mesoporous silica nanoparticle platform described in Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015, may be more advantageous than lipid nanoparticles for controlling STAT3-mediated pathways. Figure 19 also demonstrates that STAT3 is sufficiently conserved that the same siSTAT3 sequence can knockdown STAT3 in canine, murine, and human cells, facilitating direct translation of murine studies to canine and human studies. Therefore, the same siSTAT3 sequence was used across species throughout this application.

[0267] The ability to transfect cancer cells, DCs, and macrophages and reduce specific genes such as STAT3 suggests that the immunotherapy constructs described herein can be used for ex vivo manipulation of immune cells. Such ex vivo manipulated immune cells can be administered back to patients for therapeutic effects and immune responses (e.g., killing cancer cells). Cells can be derived from the patient being treated or from different healthy donors (e.g., stem cells and their derivatives (Senju et al., Int J Hematol, 91(3):392-400, 2010)). Cancer cells can also be treated ex vivo (with or without additional drugs) with our immunotherapy constructs to generate whole-cell cancer vaccines (Keenan et al., Int J Hematol, 91(3):392-400, 2010; Goldstein et al., Int J Hematol, 117:118-127, 2011).

[0268] AIRISE-02 (siSTAT3-CpG-NP) was also found to be effective in other tumor models, including colon cancer (Figure 20) and breast cancer (Figures 21 and 22). In particular, we show that in these two models, combining ICI (administered systemically via IP) with AIRISE administered locally to one of two tumors in each mouse results in better efficacy than either AIRISE or ICI alone.

[0269] Safety Profile of AIRISE-02. The safety of AIRISE was evaluated in both mice and monkeys. Intramuscular injection of AIRISE-02 into mice was found not to cause toxicity to the mouse skin (no edema or erythema was observed, Figure 23). There were no changes in body weight or serum biomarkers of renal and liver function when mice were treated with AIRISE-02 compared to saline (Figure 24). AIRISE-02 was also tested in monkeys and found to be safe. Specifically, AIRISE-02 was administered subcutaneously to cynomolgus monkeys in a weekly dose-escalation fashion (1, 2.8, and 9.5 mg / kg, n=3 / group, Figure 25). All three animals survived to the scheduled end of the study. There were no test article-related effects on clinical findings. Regarding skin findings, there were no drug-related skin findings at the low dose. At the mid-dose, test article-related edema (grade 2-3) was observed at 48 and / or 72 hours post-dose but resolved after 7 days. At the high dose, test article-related edema (grade 1-3) and erythema (grade 1) were observed in all animals beginning as early as 24 hours post-dose and continuing for 7 days (last observation), but were not of concern. There were no test article-related effects on body weight. There were no test article-related effects on hematology parameters. There were no test article-related effects on coagulation parameters. There were no test article-related effects on clinical chemistry parameters.

[0270] Example 2: Immunotherapeutic constructs (such as NPs) can also be loaded with two types of siRNA simultaneously without losing efficacy The immunotherapy constructs described herein can also simultaneously deliver multiple siRNAs, in addition to simultaneously delivering siRNA and CpG. For example, Figure 13 shows that when NPs (prepared essentially as described in Example 1) are loaded with two individual siRNAs against HER2 or STAT3, specific knockdown of each protein is achieved. Figure 13 also shows that when both siRNAs are loaded into the same NP vial (see T-siHER2 / siSTAT3-NP), similar knockdown of the two proteins is achieved as when they are loaded into two separate NP vials (see T-siHER2-NP+T-siSTAT3-NP). As another example, Figure 36 shows the effect of NPs loaded with siCXCR4, siSTAT3, and CpG (siSTAT3 / siCXCR4-CpG-NP) in enhancing the effect of immune checkpoint inhibitors on a melanoma model.

[0271] This demonstrates the versatility of the immunotherapy construct particles described herein in loading multiple types of oligonucleotides without losing efficacy.The nanoparticle delivery described herein is also beneficial because it allows loading multiple siRNAs, each of which can kill cancer cells and / or regulate multiple aspects of immunosuppression.

[0272] The data shown in Figure 13 clearly demonstrate that NPs can deliver siRNA, or a cocktail of two or more different siRNAs, which may have a cytotoxic effect on cancer cells, may override the immunosuppressive tumor microenvironment, may contain immunostimulatory sequences, or may have any combination of these characteristics.

[0273] Example 3: Cell-type specific / targeted immunotherapy constructs siRNA has great potential because it can precisely and effectively regulate any gene. Immunotherapy constructs (AIRISE) can address different immunosuppressive pathways or different immune cell populations by utilizing antibodies (or other targeting agents) on our nanoparticles for specific delivery. The immunotherapy constructs comprising the NPs provided herein can be conjugated with targeting agents for targeted delivery to specific cell populations, such as specific cells within tumors. Some antibodies, such as anti-PD-L1 antibodies, can function as both targeting agents and modulators of immunosuppressive pathways.

[0274] Figures 14A-14C provide examples of such targeting of NPs. NPs conjugated with cetuximab (an anti-EGFR antibody) show preferential uptake in EGFR+ cells over EGFR-low cells (Figures 14A-14B). Similarly, NPs conjugated with trastuzumab (an anti-HER2 antibody) show preferential uptake in HER2+ cells over HER2-low cells (Figure 14C) (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015).

[0275] This type of targeting is believed to work equally well with NPs loaded with adjuvants and active agents, as described herein.

[0276] Example 4: Treatment with multi-drug nanoparticles In this example, a PD-L1 antibody (mouse PD-L1 from BioXcell) is conjugated onto mesoporous silica nanoparticles loaded with a PLK1 inhibitor (volasertib) and CpG, prepared essentially as in Example 1. The PD-L1 antibody functions as both an ICI and a tumor-homing (targeting) agent.

[0277] method Volasertib (a PLK1 inhibitor; iPLK1) was overloaded onto the nanoparticles before PEI conjugation. Briefly, MSNPs were mixed with an ethanol / DMSO solution of volasertib overnight before PEI conjugation. Unbound docetaxel or volasertib and PEI were washed away in PBS. PEI-NPs (iPLK1) were then conjugated with PEG according to previously published methods (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015; Ngamcherdtrakul et al., International J Nanomed, 13:4015-4027, 2018). The final products contain 0.5-2 wt% iPLK1. They have a DLS size of 100 nm. For constructs containing PD-L1 antibody (p-NP), the PD-L1 antibody was thiolated and conjugated to the terminus of the PEG layer on the nanoparticles according to our previously published methods (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015; Ngamcherdtrakul et al., International J Nanomed, 13:4015-4027, 2018).

[0278] LLC-JSP Bilateral Mouse Lung Tumor Model: Six-week-old female C57BL / 6 mice were obtained from the Charles River NCI colony (Wilmington, MA). Each mouse was subcutaneously injected with LLC-JSP cells in the left flank (local, 100,000 cells) and right flank (distal, 40,000 cells). Twelve days after implantation, test compounds / constructs were injected intratumorally into the left (local) tumor only, while the right (distal) tumor was left untreated. Unless otherwise stated, test compounds / constructs were administered three times every three days. Both local and distal tumor burdens in mice were measured every 1-2 days using a Vernier caliper, with V = 0.5 x length x width. 2 Tumor volume was calculated by the following formula: Survival was also monitored. 3When the stool size exceeded 100 mg / kg, the mice were sacrificed.

[0279] result On day 12 after tumor inoculation, mice received intratumoral treatment of the left (local) tumor with saline, PD-L1 antibody-coated nanoparticles (p-NPs), PLK1 inhibitor-loaded nanoparticles (iPLK1-NPs), PLK1 inhibitor-loaded p-NPs (p-iPLK1-NPs), or PLK1 inhibitor and CpG-loaded p-NPs (p-iPLK1-NPs-CpG) (while the distal tumor remained untreated). 0.5 mg of NPs (2.5 μg iPLK1, 20 μg PD-L1 antibody, 20 μg CpG) in 50 μl were administered every 3 days for a total of three doses. The immunotherapy constructs extended mouse survival beyond the same immunotherapy constructs without CpG (Figure 15). The immunotherapy constructs were also significantly more effective than free PD-L1 antibody and volasertib, each administered at a concentration five times higher than that on the nanoparticles. Incorporating an adjuvant (CpG) into the same NPs further improved survival rates. For example, we found that incorporating CpG into p-iPLK1-NPs (referred to as p-iPLK1-NP-CpG) significantly improved survival rates in 2 of 7 mice, with 1 mouse remaining completely tumor-free.

[0280] Example 5: Topical formulation and application of AIRISE The immunotherapeutic constructs disclosed herein can be formulated into topical preparations. Several vehicles known in the art, such as Aquaphor (ointment-based) and Carbopol (gel-based), can be mixed with the constructs. Heat or surfactants (e.g., polysorbate 80 (Tween 80) as an emulsifier) ​​can be used to further enhance mixing of the vehicle with the aqueous suspension of AIRISE. As an example, we confirmed that 10% by weight of Tween-80 did not cause premature leakage of siRNA from the nanoparticles. Furthermore, we showed that 2.5% by weight of Tween-80 was sufficient to enhance mixing of siRNA-NPs with Aquaphor when the mixture was heated to 55°C.

[0281] Methods that simultaneously enhance penetration, such as ultrasound and a microneedle roller (e.g., a Dermaroller® with needle heights ranging from 0.5 mm to 1.5 mm), can be used. When tested on pig skin (Figure 30) and mice (Figure 31), application of microneedles with needle heights as short as 0.5 mm can enhance penetration of topical siRNA nanoparticle formulations.

[0282] Figure 30 shows that the microneedle roller enhances the penetration of siRNA nanoparticle constructs when tested on pig skin, which has a thickness similar to that of human skin. Pig skin was incubated with the formulation (Dy677-siSCR-NP in Aquaphor) for 1.5 hours (37°C; 5% CO2). After 1.5 hours, skin punches were taken from the treated area and processed for fluorescence imaging using standard techniques. Significant enhancement of skin penetration with the microneedle roller was observed. When the siRNA-NP in Aquaphor solution was administered without the roller, the siRNA signal (arrow) was limited to the outer surface of the pig skin, whereas we observed siRNA signal (arrow) extending beyond the epidermis into the dermis layer before application of the microneedles (Figure 30).

[0283] Figure 31 shows that the microneedle roller enhances localized delivery of siRNA-NPs. First, mice were shaved one day before treatment. Dy677-siSCR-NPs (0.72 nmol siRNA) were mixed with 100 μL of 2.5% Tween-Aquaphor (per application). Immediately before treatment, the dermal microroller was applied consistently in four directions to only one side of the back, while the other side was not pretreated. For comparison, the mixture was applied to a shaved area (approximately 2 cm ) with or without microneedle pretreatment. 2 After 1.5 hours of treatment time, treated skin samples were harvested and processed for imaging.

[0284] Figure 32 shows the gene knockdown obtained 3 days after application of the microroller + topical siRNA nanoconstruct. Compared to the saline-treated group, 55% EGFR knockdown (*p<0.05) was observed in the siEGFR-NP group (Figure 32A). In comparison, a single intradermal injection of siEGFR-NP (same siEGFR dose of 0.72 nmol) resulted in 40% EGFR knockdown compared to the saline-treated group (Figure 32B).

[0285] Microneedle morphology of AIRISE-02. As shown in Figure 33, for microneedle fabrication, we have considered the use of dissolving microneedles based on dextran, amylopectin, PVP, PEG, methylcellulose, chitosan, or other polymers or compounds known in the art. The use of dissolving microneedles allows for painless at-home treatment and allows for high needle density (100 needles / cm). 2 This method is highly effective in delivering AIRISE-02 due to its high affinity for siRNA. As an example (Figure 33), a dextran solution (300 mg / ml aqueous solution) containing NPs loaded with Dy677-conjugated siRNA was cast onto a microneedle mold. The solution was centrifuged or vacuumed to tightly pack the mold. The microneedles were dried in air, a desiccator, a vacuum oven, a refrigerator, or a combination thereof and then removed from the mold. The needle height varied from 300 to 800 microns depending on the template and optimization. siRNA-NPs were successfully loaded into these needle arrays (approximately 0.5 nmol siRNA / array), and the needles completely dissolved within 5 minutes after application to pig skin. Different dissolution times can be engineered by varying the components of the microneedles. Different templates can also be used to fabricate microneedle patches with different shapes and morphologies.

[0286] Example 6: Different nanoparticle materials can be used to deliver the disclosed cargo combinations and produce similar immunotherapeutic effects As an example, siSTAT3 and CpG were loaded onto cationic lipid particles (Dharmafect; commercially available) and administered to mice (AIRISE-02, based on mesoporous silica) in the same manner as in Example 1. CpG and siSTAT3 delivered with cationic lipid particles were also found to produce in situ tumor vaccination / immunostimulatory effects (Figure 11). Similar results were obtained using jetPEI (a commercially available PEI-based transfection agent that has reached clinical stage) as a delivery system. This demonstrates the versatility of the disclosed cargo combination and its delivery platform-independent nature. It is worth noting, however, that while lipid platforms are generally effective in delivering siRNA to cancer cells, layer-by-layer functionalized mesoporous silica nanoparticles (described herein) exhibit better siRNA knockdown activity in immune cells (e.g., primary dendritic cells) than their lipid counterparts (Figure 12).

[0287] As will be understood by those skilled in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its specified recited elements, steps, ingredients, or components. Accordingly, the terms "include" or "including" should be interpreted to recite "comprise," "consist of," or "consist essentially of." The transitional terms "comprise" or "comprises" mean including and allowing for the inclusion of even large amounts of unspecified elements, steps, ingredients, or components. The transitional phrase "consisting of" excludes any elements, steps, ingredients, or components not specified. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients, or components and those that do not substantially affect the embodiment. A significant effect, in this context, is a measurable reduction in the biological effect (such as an anti-cancer effect) of the immunotherapeutic construct.

[0288] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like used in the specification and claims are to be understood as being modified in each instance by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. 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 parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where further clarification is needed, the term "about," when used in conjunction with a stated numerical value or range, has the meaning reasonably given by one of ordinary skill in the art, i.e., to indicate something greater than or somewhat less than the stated value or range, within ±20% of the stated value, ±19% of the stated value, ±18% of the stated value, ±17% of the stated value, ±16% of the stated value, ±15% of the stated value, ±14% of the stated value, ±13% of the stated value, ±12% of the stated value, ±11% of the stated value, ±10% of the stated value, ±9% of the stated value, ±8% of the stated value, ±7% of the stated value, ±6% of the stated value, ±5% of the stated value, ±4% of the stated value, ±3% of the stated value, ±2% of the stated value, or ±1% of the stated value.

[0289] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported to the extent practicable, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0290] The terms "a," "an," "the," and similar referents as used in the context of describing the present invention (particularly in the context of the appended claims) should be construed to encompass both the singular and the plural unless otherwise specified herein or clearly contradicted by context. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise specified herein, each separate value is incorporated herein as if set forth individually herein. 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 better describe the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0291] Groupings of alternative elements or aspects of the invention disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be included in or deleted from a group. When any such inclusion or deletion occurs, the specification includes the modified group and is therefore deemed to satisfy the written description of all Markush groups used in the appended claims.

[0292] Certain aspects of this invention have been described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described aspects will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art as appropriate, and the inventors intend that the invention may 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. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0293] Additionally, throughout this specification, numerous references are made to patents, printed publications, journal articles and other writings (materials referenced herein), each of which is individually incorporated herein by reference in its entirety for the teachings referenced.

[0294] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Accordingly, the invention is not limited to the exact same that has been shown and described.

[0295] The details shown in this specification are by way of example and for the purpose of illustrative description of preferred embodiments of the present invention only, and are presented to provide what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt has been made to show the structural details of the present invention in more detail than necessary for a fundamental understanding of the invention, and the description using figures and / or examples will make clear to those skilled in the art how several forms of the present invention may be embodied in practice.

[0296] The definitions and explanations used in this disclosure are meant and intended to control any future constructions unless clearly and unambiguously changed in the examples or unless the application of meaning makes any construction meaningless or essentially meaningless. Definitions should be interpreted from Webster's Dictionary, 3rd Edition, or dictionaries known to those skilled in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004), when the construction of a term makes it meaningless or essentially meaningless.

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Claims

1. A delivery system comprising at least one therapeutic agent that causes tumor antigen release and / or modulates an immunosuppressive tumor microenvironment and at least one adjuvant.

1. An immunotherapeutic construct comprising: The immunotherapeutic construct does not comprise a tumor-specific antigen or ovalbumin.

2. 10. The immunotherapeutic construct of claim 1, wherein the delivery system comprises a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, or a hybrid thereof.

3. 3. The immunotherapeutic construct of claim 2, wherein the delivery vehicle is a liposome, a lipid-based particle, a polymeric particle, an inorganic particle, or an inorganic particle coated with a polymer or lipid.

4. 4. The immunotherapeutic construct of claim 3, wherein the delivery vehicle is an inorganic particle and comprises one or more of mesoporous silica, gold, aluminum, silver, iron oxide, calcium phosphate, or antioxidant particles.

5. 5. The immunotherapeutic construct of claim 4, wherein the inorganic particles comprise antioxidant particles comprising cerium oxide.

6. 5. The immunotherapeutic construct of claim 4, wherein the delivery vehicle comprises mesoporous silica particles.

7. 2. The immunotherapeutic construct of claim 1, wherein the delivery vehicle comprises one or more of fullerenes, endohedral metallofullerenes, trimetal nitride-templated endohedral metallofullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, silicon, silica microspheres and nanospheres, polymer microspheres and nanospheres, silica shells, biodegradable PLGA microspheres and nanospheres, gold particles, cerium oxide particles, zinc oxide particles, silver particles, aluminum particles, carbon particles, iron particles, iron oxide particles, adjuvant particles, and / or modified micelles.

8. The immunotherapeutic construct of any one of claims 1 to 7, wherein the delivery vehicle is a polymer particle comprising one or more of PLGA, PLL, dextran, dendrimer, polyarginine, PEG, PEI, or chitosan.

9. The immunotherapeutic construct of any one of claims 1 to 8, having a hydrodynamic size of 5 nm to 999 nm.

10. 9. The immunotherapeutic construct of any one of claims 1 to 8, having a hydrodynamic size of 1 micron to 1000 microns.

11. 7. The immunotherapeutic construct of claim 6, wherein the delivery vehicle comprises mesoporous silica nanoparticles having a size of about 5 to about 200 nm.

12. 12. The immunotherapeutic construct of claim 11, wherein the mesoporous silica nanoparticles are coated with cross-linked polyethyleneimine and polyethylene glycol.

13. 13. The immunotherapy construct of any one of claims 1 to 12, wherein at least one therapeutic agent comprises an siRNA, miRNA, an antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-cas9 element), an oligonucleotide, a polynucleotide, a peptide, a protein, a chemotherapeutic drug, a toxin, an antioxidant, a small molecule inhibitor, an antibody, or a radiotherapeutic agent.

14. 14. The immunotherapeutic construct of claim 13, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, or DNA.

15. 15. The immunotherapeutic construct of claim 14, wherein at least one therapeutic agent comprises an siRNA.

16. 16. The immunotherapeutic construct of claim 15, wherein at least one therapeutic agent comprises an siRNA that inhibits expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, BRAF, BRAF V600E, or MTDH.

17. 17. The immunotherapeutic construct of claim 15 or 16, wherein at least one therapeutic agent comprises an siRNA that inhibits STAT3 expression or activity.

18. The immunotherapeutic construct of any one of claims 15 to 17, wherein at least one therapeutic agent comprises an siRNA that inhibits expression of HER2 activity.

19. 13. The immunotherapeutic construct of any one of claims 1-12, wherein at least one therapeutic agent inhibits expression or activity of STAT3, CD39, CD73, TGF-β, PD-L1, PD1, CTLA4, MIF, PLK1, HIF, NOX1-4, HER2, EGFR, BCL2, AKT1, HIF1-α, NOX1-4, AR, MYC, BRAF, BRAF V600E, or MTDH.

20. 20. The immunotherapy construct of any one of claims 1-19, wherein the at least one therapeutic agent comprises one or more anti-cancer agents selected from an antibiotic, a plant alkaloid, a PLK1 inhibitor, a mitotic phase kinase inhibitor, an immune checkpoint inhibitor, a platinum-based chemotherapeutic agent, a HER2 small molecule inhibitor, an anti-EGFR antibody, and an anti-HER2 antibody.

21. 21. The immunotherapy construct of claim 20, wherein the at least one therapeutic agent comprises an immune checkpoint inhibitor, and the immune checkpoint inhibitor is an antibody against PD-L1, PD1, or CTLA4.

22. 22. The immunotherapeutic construct of claim 21, wherein the immune checkpoint inhibitor is an antibody against PD-L1.

23. The immunotherapeutic construct of any one of claims 1 to 22, wherein at least one therapeutic agent comprises a PLK1 inhibitor.

24. 24. The immunotherapeutic construct of claim 23, wherein the PLK1 inhibitor is volasertib.

25. 25. The immunotherapeutic construct of any one of claims 1 to 24, wherein the at least one therapeutic agent comprises one or more of docetaxel, mitoxantrone, or cabazitaxel.

26. The immunotherapeutic construct of any one of claims 1 to 25, wherein at least one therapeutic agent comprises an anti-EGFR antibody.

27. 27. The immunotherapeutic construct of claim 26, wherein the anti-EGFR antibody is cetuximab.

28. The immunotherapeutic antibody of any one of claims 1 to 25, wherein at least one therapeutic agent comprises an anti-HER2 antibody.

29. 29. The immunotherapeutic antibody of claim 28, wherein the anti-HER2 antibody is trastuzumab.

30. 30. The immunotherapy construct of any one of claims 1-29, wherein the adjuvant has immunostimulatory activity and comprises one or more of a CpG oligonucleotide, a DNA TLR agonist comprising a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil-based or surfactant-based adjuvant, lipopolysaccharide, a plant extract, or a derivative thereof.

31. 31. The immunotherapeutic construct of any one of claims 1 to 30, wherein the adjuvant comprises a CpG oligonucleotide, imiquimod, resiquimod, gardikimod, poly I:C, poly ICLC, dSLIM, or EnanDIM.

32. The immunotherapeutic construct of any one of claims 1 to 31, wherein the adjuvant comprises a CpG oligonucleotide.

33. An immunotherapeutic construct according to any one of claims 1 to 32; and at least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof. A composition comprising:

34. 34. A method of treating cancer, comprising administering to a subject having cancer an effective amount of the immunotherapeutic construct of any one of claims 1 to 32 or the composition of claim 33.

35. 35. The method of claim 34, wherein the subject is a mammal.

36. 36. The method of claim 35, wherein the mammal is a human.

37. 1. A method of treating cells exhibiting a cancer symptom, comprising: contacting said cells with a therapeutically effective amount of an immunotherapeutic construct according to any one of claims 1 to 32 or a composition according to claim 33. The method comprising:

38. 1. A method of treating cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, comprising: contacting said cells with a therapeutically effective amount of an immunotherapeutic construct according to any one of claims 1 to 32 or a composition according to claim 33. The method comprising:

39. 1. A method of treating cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, comprising: contacting the cells ex vivo with a therapeutically effective amount of the immunotherapeutic construct of any one of claims 1 to 32 or the composition of claim 33. The method comprising:

40. 40. The method of claim 38 or 39, wherein the cell is a cancer cell.

41. 40. The method of claim 38 or 39, wherein the cell is not a cancer cell.

42. 42. The method of claim 41, wherein the cell is an immune cell.

43. 40. The method of claim 38 or 39, wherein the cells are immortalized.

44. 44. The method of any one of claims 37-43, further comprising administering at least one treated cell back to the subject.

45. 1. A method of treating a subject diagnosed with or at increased risk for developing a hyperproliferative disease or condition, comprising: administering to said subject an effective amount of the composition of claim 33. The method comprising:

46. 46. ​​The method of claim 45, wherein the subject is a mammal.

47. 47. The method of claim 46, wherein the mammal is a human.

48. 48. The method of any one of claims 45-47, wherein the hyperproliferative disease or condition comprises one or more of cancer, precancer, or cancer metastasis.

49. 49. The method of any one of claims 45-48, wherein the hyperproliferative disease comprises one or more of melanoma, lung cancer, breast cancer, pancreatic cancer, brain cancer, prostate cancer, head and neck cancer, renal cancer, colorectal cancer, lymphoma, gastric cancer, colon cancer, liver cancer, or a rare cancer.

50. The administering step comprises: injection into or at the subject's tumor; local injection into or at the subject's tumor; Systemic injection in subjects, Systemic injection in a subject; or Local application to the subject 50. The method of any one of claims 45 to 49, comprising:

51. 51. The method of any one of claims 45 to 50, wherein the administering step comprises microneedling to the subject.

52. 1. A method of enhancing the efficacy of anti-cancer therapy in a subject in need thereof, comprising: an effective amount of an immunotherapeutic construct according to any one of claims 1 to 32 or a composition according to claim 33; with at least one anticancer drug to a subject in need thereof. The method comprising:

53. 53. The method of claim 52, wherein the anti-cancer agent is a chemotherapeutic agent or a targeted therapy agent.

54. 1. A method of enhancing the efficacy of checkpoint blockade immunotherapy in a subject diagnosed with a neoplasm, comprising: an effective amount of an immunotherapeutic construct according to any one of claims 1 to 32 or a composition according to claim 33; At least one immune checkpoint inhibitor to a subject in need thereof. The method comprising:

55. 1. A method of enhancing the effectiveness of radiation therapy in a subject diagnosed with a neoplasm, comprising: an effective amount of an immunotherapeutic construct according to any one of claims 1 to 32 or a composition according to claim 33; At least one radiation therapy to a subject in need thereof. The method comprising:

56. 56. The method of any one of claims 52 to 55, wherein the immunotherapeutic construct or composition and the anti-cancer therapy are administered sequentially or simultaneously.

57. The method of any one of claims 52 to 56, wherein the subject is a mammal.

58. 58. The method of claim 57, wherein the mammal is a human.

59. An immunotherapeutic construct according to any one of claims 1 to 32; with at least one anticancer drug Includes a kit.

60. 60. The kit of claim 59, wherein the anti-cancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor.

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