Immunotherapy construct and method of use

Abstract

Disclosed herein are immunotherapy constructs comprising delivery particles, at least one adjuvant, and one or more therapeutic agents / compounds that induce antigen release and / or modulate the immunosuppressive tumor microenvironment. These immunotherapy constructs generate adaptive or anti-cancer immune responses, which can be used to prevent and treat a wide range of cancers. Further disclosed are uses of the immunotherapy constructs, including the prevention and treatment of cancers in humans and animals. TIFF2022540867000015.tif97158

Description

[Technical Field]

[0001] Description of research and development funded by the federal government. This invention was developed with government support under grants R44CA217534 and R43TR001906 awarded by the National Institutes of Health. The government has certain rights to this invention.

[0002] Field of Invention This disclosure relates to compositions and methods for treating and preventing cancer and other diseases and conditions. The compositions include immunotherapy constructs comprising a delivery system (such as particles) containing at least one therapeutic activator (which induces tumor antigen release and / or modulates the immunosuppressive tumor microenvironment) and at least one adjuvant (or immunostimulant), which induce adaptive immunity by utilizing the antigen within the target's own precancerous cells or cancer cells. [Background technology]

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

[0004] Classical cancer vaccines utilize immune stimulants (called adjuvants) and oncoproteins (called antigens). Ideally, neoantigens present only on cancer cells should be used. However, these neoantigens vary greatly across tumor types and patients, making the development of personalized vaccines difficult and costly. To circumvent the need to identify these antigens, radiation, chemotherapy, and engineered viruses, such as tarimozine-laharpalepbek (T-VEC), are used to kill tumors and release antigens, thereby inducing an adaptive immune response in situ. However, these techniques create an undesirable environment in the immunosuppressive tumor microenvironment, which reduces or inactivates anti-tumor T cell numbers (e.g., by increasing chemical stressors called oxidants, or by promoting immunosuppressive pathways). Furthermore, in-situ tumor vaccination strategies suffer from the tumor's immunosuppressive microenvironment and the inability to retain vaccine components and effectively deliver them to target cells (e.g., antigen-presenting cells). [Overview of the Initiative]

[0005] To overcome the aforementioned drawbacks, the inventors have developed a new class of immunotherapeutic agents that utilize an in-situ tumor vaccination strategy. In-situ tumor vaccination is a strategy in which a tumor is locally destroyed in the presence of immune stimulation, releasing tumor antigens, which together prime systemic adaptive immunity against the tumor. In certain cases, tumor antigens already present in the tumor microenvironment (TME) are utilized. This strategy is highly promising because it avoids the need to pre-identify tumor (neo) antigens, as is the case with conventional 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] Engineered particles for enhancing the CD8+ T cell repertoire and inducing systemic antitumor immunotherapy effects by co-delivering an adjuvant and a compound capable of inducing antigen release and / or modulating an immunosuppressive environment are described herein. This technique may be referred to as AIRISE, an abbreviation for Augmenting Immune Response and Inhibiting Suppressive Environment of Tumors.

[0007] A novel class of immunotherapeutic agents (generally known as immunotherapy constructs) based on engineered particles that enable co-delivery of an adjuvant and a therapeutic activator capable of inducing antigen release (e.g., by killing cancer cells) and / or modulating an immunosuppressive environment (e.g., tumor microenvironment, TME) is described herein. These immunotherapy constructs may also utilize antigens already present in the TME. The immunotherapy constructs enhance the CD8+ T cell repertoire and induce a systemic antitumor immunotherapy effect without needing to know which antigens are associated with the cancer being treated. While cellular immunity is described broadly herein, humoral immunity (antibody production) also plays a role and follows the same concepts.

[0008] Examples of therapeutic activators 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, polyI:C, etc.) prime and activate adaptive immune cells against the tumor antigens. Activated effector cells can recognize and attack tumors at any site in the body (including sites far from the local delivery of the immunotherapy construct), and can even reduce or prevent the spread or development of new tumors having one or more of the same tumor antigens as the treated tumor. This phenomenon is sometimes called the abscopal effect. The death of cancer cells further amplifies the adaptive immune loop with long-lasting effects. Memory adaptive immunity is also thought to be established for continuous anti-tumor immune surveillance.

[0009] Immunotherapy constructs can be administered locally, intratumorally, intranasally, intraperitoneally, intracerebrospinally, subcutaneously, intra-articularly, intra-synovially, intrathecally, orally, transdermally, intravenously, or by inhalation to easily accessible tumors, such as melanoma, head and neck cancer, breast cancer, colon cancer, ovarian cancer, bladder cancer, and lymphoma; or systemically to other cancers, such as lung cancer, liver cancer, pancreatic cancer, prostate cancer, brain cancer, kidney cancer, hematological cancer, and metastatic cancer.

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

[0011] Optionally, the immunotherapy constructs also contain one or more homing agents (such as 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 immunotherapy constructs provided herein can be used alone or in combination with standard therapies including, without limitation, 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 oncogenic proteins, or medical radioisotopes) can be directly loaded onto / within the immunotherapy constructs as therapeutic active agents.

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

[0014] In certain embodiments, an immunotherapy construct is provided that includes 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. The immunotherapy construct may not contain tumor-specific antigens or ovalbumin. In another embodiment, the immunotherapy construct does not contain proteins other than one therapeutic agent or at least one adjuvant, if either is a protein. The therapeutic agent and adjuvant can be loaded within 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 immunotherapy construct, 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 still other examples, the immunotherapy construct is a microparticle having a hydrodynamic size of 1 micron to 1000 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 immunotherapy construct, the therapeutic agent includes oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, mRNA, DNA, shRNA, or sgRNA (CRISPR-cas9 elements)), polynucleotides, peptides, proteins, chemotherapeutic agents, toxins, antioxidants, small molecule inhibitors, antibodies, or radiotherapeutic agents.

[0016] In examples of immunotherapy constructs, adjuvant compounds possess immunostimulatory activity. Examples of adjuvant compounds include TLR-binding DNA substituents, e.g., CpG oligonucleotides (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); DNA TLR agonists containing CpG sequences (e.g., dSLIM); non-CpG DNA TLR agonists (e.g., EnanDIM); RNA TLR agonists (e.g., poly-I:C or poly-ICLC); aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum chloride, or potassium aluminum sulfate); anti-CD40 antibodies (e.g., CP-870, 893); cytokines (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF)); cationic peptide conjugate CpG oligonucleotides (e.g., IC30, IC31); small molecule TLR agonists (e.g., imiquimod, reximod, gardikimod, or 3M-052); fusion proteins (e.g., ImuFact IMP321 and ONTAK); oil / surfactant adjuvants (e.g., MF59, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide ISA-51); saponin-derived QS21 stimulon (Aquila Biotech, Worcester, Mass., USA); mycobacterial extracts or synthetic bacterial cell wall mimics, e.g., lipopolysaccharides (e.g., monophosphoryl lipid A, OM-174, OM-197-MP-EC, or Pam3Cys); xanthenon derivatives (e.g., badi mezan); mixtures thereof (e.g., AS-15); or proprietary adjuvants, e.g., one or more of Ribi's Detox, Quil, or Superfos. Methods of using the immunotherapy constructs described herein in a manner for treating or preventing cancer or other hyperproliferative diseases are also provided.As an example, in the case of melanoma, the immunotherapy construct may be used as a prophylactic vaccine for patients with multiple atypical nevi or other seemingly benign nevi (especially those with a genetic predisposition). As another example, the immunotherapy construct may be administered to an accessible tumor / lesion via intratumor / intrafocal injection prior to surgical removal (i.e., neoadjuvant setting) to reduce the likelihood of recurrence and / or mobilize the immune system to kill any detectable or undetectable metastases. As yet another example, the immunotherapy construct may be administered to a tumor by intratumor injection even if the tumor is unresectable. This activates and mobilizes the immune system to attack both the treated and untreated tumors elsewhere in the body. As yet another example, the immunotherapy construct may be administered systemically to initiate an adaptive anti-tumor immune response. In certain embodiments, the immunotherapy construct may be administered to the area surrounding the tumor (peritumor) or to the area remaining after tumor removal (adjuvant setting). As yet another example, the immunotherapy construct may be administered systemically, thereby developing adaptive immunity against cancer at any site in the body. In certain embodiments, immunotherapy constructs may be administered directly to lymph nodes (whether or not they contain detectable tumors).

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

[0018] Methods and embodiments of combining the administration of an immunotherapy construct with at least one other treatment, such as treatment for cancer or another hyperproliferative disease or condition, are also provided.

[0019] In any of the described methods and embodiments, the step of administering the immunotherapy construct may include one or more of the following: direct injection into the tumor of interest; systemic injection into the interest; topical application to the interest; inhalation by the interest; hepatic artery infusion into the interest; convection-enhanced delivery to the interest; or microneedle application to the interest.

[0020] In any example of the method embodiments provided, the subject (being treated, administered a construct or composition, or from which cells are obtained) is a mammal. For example, in certain embodiments, the mammal is a human.

[0021] In this specification, any embodiment of the immunotherapy construct provided, and any embodiment of the method of using such construct, is particularly intended to include examples in which the immunotherapy construct does not contain tumor-specific antigens or ovalbumin. [Invention 1001] A delivery system comprising at least one therapeutic agent that induces tumor antigen release and / or modulates the immunosuppressive tumor microenvironment, and at least one adjuvant. An immunotherapy construct including, The immunotherapy construct, which does not contain tumor-specific antigens or ovalbumin. [Invention 1002] An immunotherapy construct of the present invention 1001, wherein the delivery system comprises liposomes, lipid particles, polymer particles, inorganic particles, or hybrids thereof. [Invention 1003] An immunotherapy construct of the present invention 1002, wherein the delivery vehicle is a liposome, lipid-based particles, polymer particles, inorganic particles, or inorganic particles coated with a polymer or lipid. [Invention 1004] An immunotherapy construct of the present invention 1003, wherein the delivery vehicle is an inorganic particle comprising one or more of mesoporous silica, gold, aluminum, silver, iron oxide, calcium phosphate, or antioxidant particles. [Invention 1005] An immunotherapy construct of the present invention 1004, wherein the inorganic particles contain antioxidant particles containing cerium oxide. [Invention 1006] The delivery vehicle is an immunotherapy construct of the present invention 1004, comprising mesoporous silica particles. [Invention 1007] The delivery vehicle is fullerene, endohedral metal fullerene. An immunotherapy construct of Invention 1001 comprising one or more of the following: metallofullerene, trimetallic nitride-templated endohedral metallofullerene, single-walled carbon nanotubes and multi-walled carbon nanotubes, branched carbon nanotubes and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled boron / nitrate nanotubes 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 silica nanospheres, polymer microspheres and polymer nanospheres, silica shells, biodegradable PLGA microspheres and biodegradable PLGA 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. [Invention 1008] An immunotherapy construct according to any of Invention 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. [Invention 1009] An immunotherapy construct according to any of invention 1001 to 1008, having a hydrodynamic size of 5 nm to 999 nm. [Invention 1010] An immunotherapy construct according to any of the present invention 1001 to 1008, having a hydrodynamic size of 1 micron to 1000 microns. [Invention 1011] An immunotherapy construct of the present invention 1006, wherein the delivery vehicle contains mesoporous silica nanoparticles having a size of approximately 5 to approximately 200 nm. [Invention 1012] An immunotherapy construct of the present invention 1011, wherein mesoporous silica nanoparticles are coated with crosslinked polyethyleneimine and polyethylene glycol. [Invention 1013] An immunotherapy construct according to any of Invention 1001 to 1012, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-Cas9 element), oligonucleotide, polynucleotide, peptide, protein, chemotherapeutic agent, toxin, antioxidant, small molecule inhibitor, antibody, or radiotherapy agent. [Invention 1014] An immunotherapy construct of the present invention 1013, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, or DNA. [Invention 1015] An immunotherapy construct of the present invention 1014, wherein at least one therapeutic agent comprises siRNA. [Invention 1016] An immunotherapy construct of Invention 1015, comprising at least one therapeutic agent containing an siRNA that 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, BRAF, BRAF V600E, or MTDH. [Invention 1017] An immunotherapy construct of the present invention 1015 or 1016, wherein at least one therapeutic agent comprises an siRNA that inhibits the expression or activity of STAT3. [Invention 1018] An immunotherapy construct according to any one of the invention 1015 to 1017, wherein at least one therapeutic agent comprises siRNA that inhibits the expression of HER2 activity. [Invention 1019] An immunotherapy construct according to any of the Invention 1001 to 1012, wherein at least one 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, BRAF, BRAF V600E, or MTDH. [Invention 1020] An immunotherapy construct according to any of Invention 1001 to 1019, wherein at least one therapeutic agent comprises one or more anticancer agents selected from antibiotics, plant alkaloids, PLK1 inhibitors, mitotic kinase inhibitors, immune checkpoint inhibitors, platinum-based chemotherapeutic agents, HER2 small molecule inhibitors, anti-EGFR antibodies, and anti-HER2 antibodies. [Invention 1021] An immunotherapy construct of the present invention 1020, wherein at least one therapeutic agent comprises an immune checkpoint inhibitor, the immune checkpoint inhibitor being an antibody against PD-L1, PD1, or CTLA4. [Invention 1022] An immunotherapy construct of the present invention 1021, wherein the immune checkpoint inhibitor is an antibody against PD-L1. [Invention 1023] An immunotherapy construct according to any of invention 1001 to 1022, wherein at least one therapeutic agent comprises a PLK1 inhibitor. [Invention 1024] An immunotherapy construct of the present invention 1023, wherein the PLK1 inhibitor is volasertib. [Invention 1025] An immunotherapy construct according to any of Invention 1001 to 1024, wherein at least one therapeutic agent comprises one or more of docetaxel, mitoxantrone, or cabazitaxel. [Invention 1026] An immunotherapy construct according to any of invention 1001 to 1025, wherein at least one therapeutic agent comprises an anti-EGFR antibody. [Invention 1027] An immunotherapy construct of the present invention 1026, wherein the anti-EGFR antibody is cetuximab. [Invention 1028] An immunotherapy antibody according to any of Invention 1001 to 1025, wherein at least one therapeutic agent comprises an anti-HER2 antibody. [Invention 1029] The immunotherapy antibody of the present invention 1028, wherein the anti-HER2 antibody is trastuzumab. [Invention 1030] An immunotherapy construct according to any of the Invention 1001 to 1029, wherein the adjuvant has immunostimulatory activity and comprises one or more of the following: 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 adjuvants or surfactant-based adjuvants, lipopolysaccharides, plant extracts, or derivatives thereof. [Invention 1031] An immunotherapy construct according to any of Invention 1001 to 1030, wherein the adjuvant comprises a CpG oligonucleotide, imiquimod, reximod, gardikimod, poly-I:C, poly-ICLC, dSLIM, or EnanDIM. [Invention 1032] An immunotherapy construct according to any of invention 1001 to 1031, wherein the adjuvant contains a CpG oligonucleotide. [Invention 1033] An immunotherapy construct according to any of invention 1001 to 1032, At least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof A composition containing the following: [Invention 1034] A method for treating cancer, comprising the step of administering an effective amount of any immunotherapy construct according to Invention 1001 to 1032 or a composition according to Invention 1033 to a subject having cancer. [Invention 1035] The method of the present invention 1034, wherein the subject is a mammal. [Invention 1036] The method of the present invention 1035, wherein the mammal is a human. [Invention 1037] A method for treating cells that exhibit symptoms of cancer, A step of contacting the cells with a therapeutically effective amount of any immunotherapy construct according to invention 1001 to 1032 or a composition according to invention 1033. The method, including the method described above. [Invention 1038] A method for treating cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, A step of contacting the cells with a therapeutically effective amount of any immunotherapy construct according to invention 1001 to 1032 or a composition according to invention 1033. The method, including the method described above. [Invention 1039] A method for treating cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, A step of ex vivo contacting cells with an effective amount of an immunotherapy construct from any of the inventions 1001 to 1032 or a composition from the invention 1033. The method, including the method described above. [Invention 1040] The method of the present invention 1038 or 1039, wherein the cells are cancer cells. [Invention 1041] The method of the present invention 1038 or 1039, wherein the cells are not cancer cells. [Invention 1042] The method of the present invention 1041, wherein the cells are immune cells. [Invention 1043] A method according to invention 1038 or 1039, wherein cells are immortalized. [Invention 1044] A method according to any one of the present invention 1037 to 1043, further comprising the step of administering at least one treated cell back to the target. [Invention 1045] A method for treating subjects diagnosed with an overproliferative disease or overproliferative condition, or diagnosed as being at high risk of developing such a disease or condition, A step of administering an effective amount of the composition of the present invention 1033 to the subject. The method, including the method described above. [Invention 1046] The method of the present invention 1045, wherein the subject is a mammal. [Invention 1047] The method of the present invention 1046, wherein the mammal is a human. [Invention 1048] A method according to any one of items 1045 to 1047 of the present invention, wherein the hyperproliferative disorder or hyperproliferative condition includes one or more of cancer, precancerous conditions, or cancerous metastases. [Invention 1049] A method according to any one of the present invention 1045 to 1048, wherein the hyperproliferative disorder includes one or more of the following: melanoma, lung cancer, breast cancer, pancreatic cancer, brain cancer, prostate cancer, head and neck cancer, kidney cancer, colorectal cancer, lymphoma, gastric cancer, colon cancer, liver cancer, or rare cancers. [Invention 1050] The administration process is, Injection into or at the target tumor, Local injection into or at the target tumor, Systemic injection in the subject, Systemic injection into the subject, or Local application to the target Any method of the present invention 1045 to 1049, including the above. [Invention 1051] A method according to any one of the present invention 1045 to 1050, wherein the administration step includes the application of microneedles to the target. [Invention 1052] A method for enhancing the effect of anticancer therapy in subjects that require it, An effective amount of any immunotherapy construct according to Invention 1001 to 1032 or a composition according to Invention 1033, At least one anticancer drug and The process of administering it to the target that needs it. The method, including the method described above. [Invention 1053] The method of the present invention 1052, wherein the anticancer agent is a chemotherapeutic agent or a targeted therapy agent. [Invention 1054] A method for enhancing the effect of checkpoint block immunotherapy in subjects diagnosed with neoplasms, An effective amount of any immunotherapy construct according to Invention 1001 to 1032 or a composition according to Invention 1033, At least one immune checkpoint inhibitor and The process of administering it to the target that needs it. The method, including the method described above. [Invention 1055] A method for enhancing the effect of radiotherapy in a subject diagnosed with a neoplasm, An effective amount of any immunotherapy construct according to Invention 1001 to 1032 or a composition according to Invention 1033, At least one radiation therapy and The process of administering it to the target that needs it. The method, including the method described above. [Invention 1056] A method according to any one of items 1052 to 1055 of the present invention, wherein an immunotherapy construct or composition and an anticancer therapy are administered sequentially or simultaneously. [Invention 1057] Any method according to 1052 to 1056 of the present invention, wherein the subject is a mammal. [Invention 1058] The method of the present invention 1057, wherein the mammal is a human. [Invention 1059] An immunotherapy construct according to any of invention 1001 to 1032, At least one anticancer drug and A kit that includes this. [Invention 1060] A kit according to Invention 1059, wherein the anticancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor. [Brief explanation of the drawing]

[0022] [Figure 1] Cancer treatment. Targeted therapy has significantly improved cancer prognosis compared to nonspecific toxic chemotherapy. However, this effect is not sustained. Immune checkpoint inhibitors (ICIs) unleash the body's own immune system to attack cancer, potentially leading to a cure. However, only a small fraction 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 enhances the anti-tumor T cell repertoire by manipulating the tumor microenvironment (TME), thereby increasing the cure rate for cancer patients treated with ICIs. [Figure 2]The present inventors' novel in-situ tumor vaccination mechanism for their immunotherapy constructs. The immunotherapy construct AIRISE is injected intratumorally into only one of the tumors (e.g., melanoma or thoracic tumor). In one example, namely AIRISE-01 or CpG / DTX-NP, docetaxel (DTX), a chemotherapeutic agent also possessing adjuvant properties, kills local cancer cells and releases tumor antigens, while the CpG oligonucleotide (adjuvant) activates local antigen-presenting cells (APCs) (primarily dendritic cells (DCs)). In another example, namely AIRISE-02 or siSTAT3-CpG-NP, siSTAT3 can kill some cancer cells, and knocking down STAT3 reduces the immunosuppressive tumor microenvironment that interferes with the priming, activation, and function of the antitumor adaptive immune response. Tumor antigens already present in the tumor microenvironment (TME, including cancer cells, immune cells, etc.) or released by the inventors' treatment are taken up by AIRISE-activated APCs within the tumor and tumor-discharging lymph nodes. Next, the APC (antigen-mediated polycythemia) presents these antigens (cross-presentation) to prime tumor antigen-specific T cells. These activated cytotoxic (effector CD8+) T cells proliferate and enter systemic circulation. They specifically home to tumors that have the same set of antigens as the treated tumor, wherever they are located in the body (e.g., home to both treated tumors and untreated metastatic tumors). As more cancer cells are killed by cytotoxic T cells, 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 addition, in these embodiments, mesoporous silica nanoparticles possess antioxidant properties (Morry, J. Biomaterials, 66:41-52, 2015), and these antioxidant properties can also further modulate immunosuppressive TME and inhibit tumor-promoting activity. This vaccination, locally induced at the tumor site, elicits a systemic antitumor immune response throughout the body. [Figure 3]Superior activation of dendritic cells (MHCII+CD80+CD11c+ cells) after treatment with CpG-NP, surpassing that of CpG. CpG or CpG-NP was administered to mice by foot-plantar injection. One day after treatment, draining lymph nodes (DLNs) and non-draining lymph nodes (NDLNs) were collected and treated as single cells for flow cytometry analysis to identify the percentage of activated dendritic cells. *p<0.05, **p<0.01, ***p<0.0001. Unless otherwise specified, CpG ODN 1826 (SEQ ID NO:7) was used throughout the examples. [Figure 4A] The effect of CpG-NP administered to a melanoma mouse model, such as (Figure 4A, mice transplanted with bilateral tumors), on inducing in-situ tumor vaccination, as shown by inhibition of tumor growth curves in locally treated tumors (Figure 4B) and distally untreated tumors (Figure 4C), and extension of the mouse survival curve (Figure 4D). 250,000 and 100,000 B16F10 cells were transplanted into each mouse (C57BL / 6) to establish local and distal tumors, respectively. Eight days after tumor transplantation, the local tumors were injected intratumorally with either CpG-NP or saline every three days for a total of three injections. The doses (per injection) were 20 μg of CpG and 0.2 mg of NP. Tumor volume was plotted as mean and SEM. Statistical significance (*) between CpG-NP and saline was assessed. *p<0.05, ***p<0.001, ****p<0.0001. [Figure 4B] See the explanation in Figure 4A. [Figure 4C] See the explanation in Figure 4A. [Figure 4D] See the explanation in Figure 4A. [Figure 5A]The effect of CpG and / or docetaxel (DTX)-loaded NP (AIRISE-01), administered to a melanoma mouse model as shown in (Figure 5A), on inducing in-situ tumor vaccination, as indicated by inhibition of tumor growth curves in locally treated tumors (Figure 5B) and distally untreated tumors (Figure 5C), and extension of the mouse survival curve (Figure 5D). Mice (same model as in Figure 4) were treated with CpG-NP, CpG-DTX-NP, or saline. Dosage (per injection): CpG 20 μg; DTX 2 μg; NP 0.2 mg. Tumor volume is plotted as mean and SEM. * indicates a statistically significant difference between saline and CpG-NP. $ indicates a statistically significant difference between saline and CpG-DTX-NP. **p and $$p<0.01; ****p and $$$$p<0.0001. [Figure 5B] See the explanation in Figure 5A. [Figure 5C] See the explanation in Figure 5A. [Figure 5D] See the explanation in Figure 5A. [Figure 6] Increased cytotoxic CD8+ T cells in local and distal tumors, DLN in local (treated) and distal tumors, and non-DLN induced by CpG-DTX-NP (shown as NP in this figure). All conditions were the same as in Figure 5A. Seven days after the first injection, tumors and lymph nodes (tumor-discharged and non-discharged) were collected (see Figure 5A). Cells were stained using a panel of antibodies to assess lymphoid cell populations and activity. p-values: *p<0.05, **p<0.01, ***p<0.001. [Figure 7A]The effect of siSTAT3-CpG-NP (AIRISE-02) administered to a melanoma mouse model, as shown in (Figure 7A), on inducing in-situ tumor vaccination, as indicated by inhibition of tumor growth curves in locally treated tumors (Figure 7B) and distally untreated tumors (Figure 7C), and extension of the mouse survival curve (Figure 7D). Tumor volume is plotted as mean and SEM. Dosage (per injection): CpG 20 μg; siSTAT3 4 μg; NP 0.2 mg. Statistical significance (specified p-value, *) was assessed between CpG-NP and siSTAT3-CpG-NP (two best-responding groups). [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 8A] CD8-dependent effects of siSTAT3-CpG-NP (AIRISE-02). C57 / BL6 mice carrying B16F10 tumors were established and treated as shown in Figure 8A. CD8 depletion antibody (Clone 2.43, BioXcell, 200 μg / mouse, twice weekly, ip) was administered to the mouse group starting one day before the first intratumoral treatment with AIRISE-02 and continuing throughout the study. CD8 depletion was shown to reduce the effects of AIRISE-02 in inhibition of local tumors (Figure 8B), distal untreated tumors (Figure 8C), and extension of mouse survival (Figure 8D), indicating that the effects of AIRISE-02 are immune-dependent rather than directly cytotoxic. [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Figure 8D] See the explanation in Figure 8A. [Figure 9A]siSTAT3-CpG-NP (AIRISE-02) enhanced the effects of checkpoint inhibitors (PD1 antibody and CTLA4 antibody). C57 / BL6 mice carrying B16F10 tumors were established and treated as shown in Figure 9A. Checkpoint inhibitors (PD1 mAb 200 μg / mouse and CTLA4 mAb 100 μg / mouse, ip) were administered to two groups of mice: one group received intratumoral AIRISE-02 concurrently, and the other group received them 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 distal untreated tumors (Figure 9C) and extended mouse survival better than AIRISE-02 or the checkpoint inhibitor cocktail alone (Figure 9D). Five out of eight mice treated with the combination were cured (tumor-free). [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 9D] See the explanation in Figure 9A. [Figure 10] siSTAT3-CpG-NP (AIRISE-02) extended the survival time of mice carrying experimental metastatic lung tumors. 200,000 Lewis lung cancer (LLC-JSP) cells were injected (via the tail vein) into C57 / BL6 mice to establish lung cancer in the mice's lungs. The 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) evoke an in-situ vaccination effect. C57 / BL6 mice carrying B16F10 tumors were established and treated as shown in Figure 11A. The therapeutic construct reduced the treated tumor (Figure 11B) and distal tumor (Figure 11C), and extended the survival time of the mice (Figure 11D). Dosage (per injection): CpG 20 μg; siSTAT3 4 μg. The therapeutic construct had an average size of 1068 nm (1.1 microns) as measured by DLS. [Figure 11B] See the explanation in Figure 11A. [Figure 11C] See the explanation in Figure 11A. [Figure 11D] See the explanation in Figure 11A. [Figure 12A] Simultaneous NP-mediated delivery of siRNA and CpG to cancer cells and immune cells. B16F10 (Figure 12A) and J774 (Figure 12B) cells (both mouse cell lines) and (Figure 12C) dendritic cells (BMDCs) isolated from the bone marrow of C3H / HEJ mice were treated with NPs carrying siRNA or scrambled siRNA (siSCR) against STAT3, or CpG-loaded NPs (CpG-NPs). The dose of each siRNA was 50 nM, or 2.0 wt% of the NP, and the dose of CpG was 2 wt% of the NP for B16F10 and J774, and 4 wt% of the NP for BMDCs. mRNA was analyzed by qRT-PCR 48 hours after treatment. 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 significantly affected by CpG loaded on the NPs. Unless otherwise specified throughout the examples, "NP" refers to mesoporous silica nanoparticles coated with crosslinked 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 the explanation in Figure 12A. [Figure 12C] See the explanation in Figure 12A. [Figure 13]HCC1954 cells (human HER2+ cancer cells) were treated with trastuzumab conjugate NPs (T-NPs) carrying siRNA against HER2 or STAT3. The dose of each siRNA was 30 nM throughout the study, and 2.0 wt% of the NP. Protein analysis by Western blotting 72 hours after treatment showed that 80% knockdown of STAT3 was achieved. The data indicate that nanoparticles can deliver at least two siRNA sequences (e.g., siHER2 and siSTAT3) without loss of efficacy compared to a single siRNA. [Figure 14A] Preferential uptake of antibody-containing nanoparticles as homing target agents. One hour after exposure, EGFR antibody (cetuximab) conjugate nanoparticles (C-NP) were preferentially taken up by EGFR-overexpressing lung cancer cells (A549 and H460) rather than normal lung cells (NL20), as shown in Figure 14A. Figure 14B shows the levels of EGFR expression in these cell lines as measured by flow cytometry. Similarly, Figure 14C shows that HER2 antibody (trastuzumab) conjugate nanoparticles (T-siSCR-NP) were also preferentially taken up by HER2-overexpressing breast cancer cells (BT474, SKBR3) rather than MCF7 (which has low HER2 expression as shown by Western blot analysis (inset in Figure 14C)). The preferential effect was not observed with rituximab (CD20 antibody) conjugate nanoparticles (R-siSCR-T). siSCR indicates scrambled siRNA. [Figure 14B] See the explanation in Figure 14A. [Figure 14C] See the explanation in Figure 14A. [Figure 15]Adding CpG (SEQ ID NO:7) to nanoparticles containing a PLK1 inhibitor (p-iPLK1-NP) increased the therapeutic benefit, as shown by the Kaplan-Meier survival curve. 100K LLC-JSP cells (lung cancer cells) were injected into the right flank of C57BL / 6 mice, and 40K cells were injected into the left flank. Twelve days after tumor inoculation, the right (local) tumors of the mice were treated intratumorally 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 both 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] The effect of CpG and mitoxantrone (MTX)-loaded NPs, administered to a melanoma mouse model as shown in (Figure 16A), on inducing in-situ tumor vaccination, as indicated by inhibition of tumor growth curves in locally treated tumors (Figure 16B) and distally untreated tumors (Figure 16C), and extension of the mouse survival curve (Figure 16D). Mice were treated with CpG-MTX-NPs or saline. Dosage (per injection): CpG 20 μg; MTX 2 μg; NP 0.2 mg. Tumor volume is plotted as mean and SEM. For tumor volume, **p<0.01** for CpG-MTX-NPs versus saline. [Figure 16B] See the explanation in Figure 16A. [Figure 16C] See the explanation in Figure 16A. [Figure 16D] See the explanation in Figure 16A. [Figure 17A]AIRISE-02 enhanced CD8+ T cell proliferation in local (treated) and untreated tumors, as well as in their tumor-discharging lymph nodes (DLNs). The model, therapeutic dose, and schedule were shown in Figure 7. Seven days after the initial treatment, cells collected from the tumors and DLNs of both local (treated) and distal (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 of tumors (A) and DLNs (B), along with the proliferation status (Ki-67) of effector (CD44+) CD8+ T cells in lymph nodes (C). Unless otherwise specified in parentheses, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (n=3 / group) for AIRISE-02 versus saline. [Figure 17B] See the explanation in Figure 17A. [Figure 17C] See the explanation in Figure 17A. [Figure 18] NP cell uptake within TME. Alexa 488-siRNA-CpG-NP was injected intratumorally into mice (same model as Figure 7, B16F10 tumor size approximately 100 mm³, n=3 / group, plotted as mean and SD). Two hours after injection, cells within the treated tumors were profiled, and the presence of siRNA-CpG-NP(NP+) in each population was analyzed. [Figure 19] siSTAT3-NP can knock down STAT3 in multiple cells of multiple species. D-17 (canine osteosarcoma), BMDC (mouse-derived bone marrow dendritic cells), J774 (mouse macrophage), B16F10 (mouse melanoma), and HCC1954 (human breast cancer) cells were treated with siSTAT3-NP (50 nM) for 48 hours. qRT-PCR analysis was performed for STAT3 and HPRT mRNA using corresponding species primers. 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 resulted in a complete response in mice carrying bilateral CT26 tumors. 250K and 100K CT26 cells were transplanted into the bilateral abdomen of each mouse (Balb / c). Fifteen days after tumor transplantation, the mice were treated as outlined. Tumor growth curves for locally treated tumors and distally 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. Checkpoint inhibitors (PD1 mAb 200 μg / mouse and CTLA4 mAb 100 μg / mouse, ip) were administered to two groups of mice: one group concurrently with intratumoral AIRISE-02, and the other group alone. [Figure 21] AIRISE-02(siSTAT3-CpG-NP)+ICI is also effective against mice carrying high-grade 4T1 bilateral thoracic tumors. 100K and 40K 4T1 cells were transplanted into the bilateral mammary fat pads of each mouse (Balb / c). Eleven days after tumor transplantation, the mice were treated as outlined. Tumor growth curves for locally treated tumors and distally untreated tumors are plotted as spider plots (each line represents an individual mouse). The doses are the same as in Figure 20. [Figure 22] Mouse survival curves in Figure 21. [Figure 23] Safety profile of siSTAT3-CpG-NP (AIRISE-02). Three female Balb / c mice were intramuscularly administered AIRISE-02. The mice were depilated and injected once into the caudal thigh muscle. Images of the injection site were taken before injection, after injection, 24 hours after injection, and 72 hours after injection. Injection dose: CpG 16 μg; siSTAT3 5 μg; NP 0.25 mg. [Figure 24]Safety profile of AIRISE-02 in mice. Mice carrying bilateral MM3MG-HER2d16 tumors transplanted into the mammary fat pad (Balb / c) (as described in Tsao et al., JCI Insight, 4(24):e131882, 2019) were treated with AIRISE-02 by five intratumor injections into one of the two tumors in each mouse over a two-week period. Body weight was monitored as shown in (A). Mice were euthanized when the tumor exceeded 2 cm in diameter or when the mouse showed signs of pain or distress (15–55 days post-treatment). After euthanasia, blood was collected and processed into serum. Serum biomarkers were measured by Beckman AU680 (IDEXX BioAnalytics, West Sacramento, CA) and reported in (B). The dose was the same as in Figure 20. [Figure 25] A schematic diagram illustrating the safety profile of AIRISE-02 in cynomolgus monkeys. Three escalating doses of AIRISE-02 were administered subcutaneously to cynomolgus monkeys (approximately 2 years old, 3.1 ± 0.2 kg, n=3) as shown 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 response, PK, clinicopathology, cytokine levels, complement degradation products, and anti-drug antibodies were monitored. [Figure 26] Hydrodynamic sizes of cross-linked PEI and PEG(NP) coated mesoporous silica nanoparticles loaded with different amounts of siRNA and CpG, identified as weight % of the total construct. Average size (Z-mean) and polydispersity index (PDI) are shown from three measurements using a Malvern Zetasizer. [Figure 27]CpG-NPs can induce antigen-specific (adaptive) immune responses in the presence of antigens. The figure shows the percentage of IFNγ-activated CD8+ T cells after incubation in the presence of SF (SIINFEKL peptide). Cells were obtained from the 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. Dosage used: CpG 16 μg and SF 40 μg. The route of administration in mice was paw-plantar injection. [Figure 28] Hydrodynamic sizes of nanoparticles (MSNP-PEI-PEG) loaded with approximately 2 wt% and 9 wt% poly(I:C) as measured in PBS. [Figure 29A] AIRISE-02. (A) TEM image of the mesoporous silica nanoparticle core, (B) schematic diagram of AIRISE-02 containing crosslinked (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015), PEI-coated mesoporous silica nanoparticles, conjugated with PEG to yield a nanoparticle construct (NP). The NP was loaded with siSTAT3 and CpG by electrostatic interaction by mixing in PBS for 10-40 minutes. (C) Hydrodynamic size of AIRISE-02 ((2%)siSTAT3-(6%)CpG-NP). [Figure 29B] See the explanation in Figure 29A. [Figure 29C] See the explanation in Figure 29A. [Figure 30]Localized siRNA-NPs in porcine skin with and without microneedle roller pretreatment. Fluorescence images of porcine skin treated for 1 hour with a single local application of Dy677-siSCR-NP in Aquaphor, with and without pretreatment using a microneedle roller. siRNA signals are indicated by arrows. Tissue nuclei were also stained using Hoechst 33342. [Figure 31] Localized siRNA-NP / Tween-Aquaphor in mice with and without microneedle roller pretreatment. Fluorescence images of mouse skin treated for 1.5 hours with a single local application of Dy677-siSCR-NP in Tween / Aquaphor, with and without microneedle roller pretreatment. siRNA signals are indicated by arrows. Tissue nuclei were also stained using Hoechst 33342. [Figure 32] EGFR knockdown effect of localized siRNA-NPs using a microneedle roller versus injected siRNA-NPs. Mouse skin was collected 3 days after a single local 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 physiological saline (B). Skin tissue was fixed and stained with a fluorescently labeled EGFR antibody for EGFR signal quantification. 4-8 images (20x magnification) were taken per condition, and 3 animals were processed per group. [Figure 33] Dextran-based microneedles containing NPs loaded with Dy677-siRNA [Figure 34] Survival rates of (A) bone marrow-derived dendritic cells and (B) J774 cells of mice 2 days after treatment with AIRISE-02 containing different amounts of CpG and 2 wt% siSTAT3. Dose: siRNA 50 nM. [Figure 35]Co-delivery of non-targeted scrambled siRNA (siSCR) and CpG via NP or Dharmafect to dendritic cells isolated from C3H / HEJ mice. The dose of each siRNA was 50 nM and 2.0 wt% of the NP, and the dose of CpG was 4 wt% of the NP. siRNA-Dharmafect formulations were prepared according to the manufacturer's protocol. mRNA was analyzed by qRT-PCR 48 hours after treatment. "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 36] siSTAT3 / siCXCR4-CpG-NP+ICI is effective against mice carrying high-grade 4T1 thoracic tumors. 100K and 40K 4T1 cells were transplanted into the bilateral mammary fat pads of each mouse (Balb / c). Eight days after tumor transplantation, mice were treated with or without siSTAT3 / siCXCR4-CpG-NP using ICI (three doses; every three days) in the same manner as in Figures 20 and 21. Tumor growth curves for locally treated tumors and distally untreated tumors 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. [Modes for carrying out the invention]

[0023] Reference to sequence listings 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 complementary strands are understood to be included in the embodiments where appropriate. A computer-readable text file titled "51127-004WO2_Sequence Listing_07.13.20_ST25.txt", created on or around July 13, 2020, with a file size of 2KB, contains the sequence listing of 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): TIFF0007870011000001.tif3128 (The last two positions are deoxy bases).

[0025] SEQ ID NO:2 is a representative antisense sequence for STAT3-specific siRNA (siSTAT3): TIFF0007870011000002.tif3128 (The last two positions are deoxy bases).

[0026] SEQ ID NO:3 is a representative sense sequence for HER2-specific siRNA (siHER2): TIFF0007870011000003.tif3128

[0027] SEQ ID NO:4 is a representative antisense sequence for HER2-specific siRNA (siHER2): TIFF0007870011000004.tif3128

[0028] SEQ ID NO:5 is a representative sense sequence for SCR-specific siRNA (siSCR): TIFF0007870011000005.tif3128

[0029] SEQ ID NO:6 is a representative antisense sequence for SCR-specific siRNA (siSCR): TIFF0007870011000006.tif3128

[0030] SEQ ID NO:7 is the sequence of CpG ODN 1826 (mouse strain) used throughout the examples: TIFF0007870011000007.tif3128 This ODN contains a complete phosphorothioate skeleton 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: TIFF0007870011000008.tif3128 This ODN contains a complete phosphorothioate skeleton and is nuclease resistant.

[0032] Detailed explanation An immunotherapy approach for cancer treatment described herein, called AIRISE (Augmenting Immune Response and Inhibiting Suppressive Environment of Tumors) (Figures 1 and 2), utilizes the patient's own tumor as a depot (in-situ tumor vaccination) of a personalized set of tumor antigens. The provided immunotherapy construct carries at least one adjuvant (e.g., a CpG oligonucleotide) and one or more therapeutic agents / compounds (e.g., siRNA, antisense, oligonucleotide, drug, small molecule, antibody, etc.) that induce 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 immunotherapy 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) that present the antigens to naive T cells. Thereafter, T cells (against their tumor antigens) are primed and activated to become effector T cells (either in lymph nodes or within the tumor site), proliferate systemically, and consequently result in an increased and improved immune response against the treated tumor and against other tumors away from the initial administration site (e.g., metastatic sites). The effect on tumors away from the initial administration site is also known in this field as the abscopal effect.

[0034] These antitumor T cells, trained to recognize specific tumor antigens, control tumors both at the injection site and elsewhere in the body (see Figure 2). The cargo combination can be applied to any type of micro / nanoparticle, i.e., the form of the immunotherapy construct and its method of use is independent of the delivery vehicle. Specific exemplary delivery vehicles are described herein.

[0035] The present invention provides in-situ tumor vaccination effects using the immunotherapies described herein, exemplified by immunotherapy constructs loaded with CpG and siSTAT3 (siSTAT3-CpG-NP) or CpG and docetaxel (CpG / DTX-NP). In these two exemplary therapeutic activators, docetaxel (DTX) kills local cancer cells and releases tumor antigens, CpG activates local antigen-presenting cells (APCs, mainly DCs), while siSTAT3 kills some cancer cells, but its primary role is to reduce the immunosuppressive tumor microenvironment (TME) that interferes with the priming and action of the antitumor adaptive immune response. It should be noted that siSTAT3-CpG-NP is designed to be taken up by both cancer and APCs. While 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 (either already present in the TME or released by the treatment provided) are taken up by CpG-activated APCs or AIRISE-activated APCs within the tumor and tumor dissipation lymph nodes. The APCs then (cross-)present these antigens to prime tumor antigen-specific T cells. These activated cytotoxic (effector) T cells proliferate and enter systemic circulation. They specifically home to tumors that possess the same set of antigens as the treated tumor, wherever they are located in the body (e.g., home to both the treated tumor and distal (untreated) tumors). Increased cancer cell death by cytotoxic T cells leads to the release of even more tumor antigens, 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 locally immunosuppressive TME and inhibit tumor-promoting activity. This locally induced vaccination at the tumor site generates systemic anti-tumor immunity throughout the body.

[0036] In certain embodiments, the delivery vehicle comprises an MSNP core for drug loading (e.g., about 50 nm) coated with an in vivo reducing crosslinked cationic polymer, such as polyethyleneimine (PEI), for oligo loading and endosomal extrusion, and a stabilizer, such as polyethylene glycol (PEG), which prevents nanoparticle aggregation, protects the oligo cargo from degradation by blood enzymes (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015), shields the charge of PEI, and enhances safety. The oligo (siRNA and / or CpG) is loaded into the construct last and mixed in PBS at room temperature for several minutes (e.g., 5 minutes). The oligonucleotides (siRNA and / or CpG) are electrostatically bound to PEI in an oligo-sequence-independent manner and protected from enzymatic degradation under the PEG layer (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). The resulting nanoparticles (NPs) are highly optimized for siRNA delivery efficacy with respect to MSNP size, molecular weight and composition of PEI and PEG, PEI crosslinking conditions (to enhance buffering capacity and reduce charge), and oligonucleotide and (optionally) antibody loading (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). This embodiment of siRNA-NPs has a rigid MSNP core size of 50 nm (by TEM) and a hydrodynamic size of 100 nm with a narrow size distribution (NPs with polymer coating). This embodiment of the siRNA-NP comprises 13.5 wt% PEI and 18.2 wt% PEG, and can be loaded with 2–4 wt% siRNA or up to 10 wt% CpG oligo. The drug (e.g., taxane) may be loaded in 0.5–3 wt% within the MSNP core or on the polymer. 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 embodiment, an immunotherapy construct is provided comprising a delivery system and at least one therapeutic agent, 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, which induces tumor antigen release and / or modulates an immunosuppressive tumor microenvironment, and at least one adjuvant compound, for example, attached to the surface of the delivery system, bound to the delivery system, encapsulated within the delivery system, or contained within the delivery system.

[0038] In this embodiment, the delivery system includes liposomes, lipid particles, polymer particles, inorganic particles, polymer or lipid-coated inorganic particles, or hybrids thereof (or, in other embodiments, the delivery system is liposomes, lipid particles, polymer particles, inorganic particles, polymer or lipid-coated inorganic particles, or hybrids thereof).

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

[0040] In many more examples of immunotherapy constructs, the delivery vehicle is fullerene, endohedral metal fullerene. The material comprises one or more of the following: metallofullerene, trimetallic nitride-templated endohedral metallofullerene, single-walled carbon nanotubes and multi-walled carbon nanotubes, branched carbon nanotubes and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled boron / nitrate nanotubes 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 silica nanospheres, polymer microspheres and polymer nanospheres, silica shells, biodegradable PLGA microspheres and biodegradable PLGA nanospheres, gold particles, cerium oxide particles, zinc oxide particles, silver particles, aluminum particles, carbon particles, iron particles, iron oxide particles, adjuvant particles (e.g., viromosomes or other virus-like particles), and / or modified micelles. Optionally, the delivery vehicle comprises polymers. In certain examples, the polymer particles include one or more of PLGA, PLL, polyarginine, PEG, PEI, or chitosan.

[0041] In any embodiment of the immunotherapy constructs provided, for example, the constructs are intended to be nanoparticles having a hydrodynamic size of 5 nm to 999 nm (e.g., approximately 80 nm to approximately 200 nm or approximately 90 nm to approximately 150 nm) when measured in an aqueous medium (e.g., PBS, Tris buffer, or water). In some embodiments, the immunotherapy constructs have a hydrodynamic size of less than 150 nm when measured in an aqueous medium (e.g., PBS, Tris buffer, or water). In yet another example, the immunotherapy constructs are microparticles having a hydrodynamic size of 1 micron to 1000 microns (e.g., 1 micron to 50 microns) when measured in an aqueous medium (e.g., PBS, Tris buffer, or water).

[0042] In various embodiments of the immunotherapy constructs described, the therapeutic agent includes siRNA, miRNA, antisense oligonucleotides, mRNA, DNA, sgRNA (CRISPR-Cas9 element), other oligonucleotides, other polynucleotides, peptides, proteins, chemotherapeutic agents, toxins, antioxidants, small molecule inhibitors, antibodies, or radiotherapy agents. Specific examples include the therapeutic agent inhibiting 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 further embodiments of immunotherapy constructs, the therapeutic agent is an anticancer agent comprising one or more of the following: antibiotics (e.g., docetaxel, doxorubicin, or mitoxantrone), plant alkaloids (e.g., cabazitaxel), PLK1 inhibitors, mitotic kinase inhibitors, immune checkpoint inhibitors (such as antibodies against PD-L1, PD1, or CTLA4), platinum-based chemotherapeutic agents, small molecule HER2 inhibitors, or HER2-specific antibodies. Examples of mitotic kinase inhibitors include, but are not limited to, inhibitors of at least one of the following: 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 inhibitors include one or more of the following: GSK461364, BI2536, Tak960, NMS-P937, BI6727 (voracertib), Chk 1 kinase inhibitors LY2603618, AU14022, YK-4-279, or PMN.

[0044] In any embodiment of an immunotherapy construct, the adjuvant compound has immunostimulatory activity. For example, the adjuvant compound may include one or more of the following: 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. Specific examples include CpG oligonucleotides, imiquimod, reximod, gardikimod, polyIC, polyICLC, dSLIM, or EnanDIM.

[0045] In this specification, it is particularly intended that none of the embodiments of the immunotherapy constructs provided may contain tumor-specific antigens or ovalbumin.

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

[0047] Methods of using the immunotherapy constructs described herein, for example, methods for treating or preventing cancer or other hyperproliferative diseases, are also provided.

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

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

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

[0051] Another embodiment provided is a method for treating a subject diagnosed with or at high risk of developing a hyperproliferative disorder or hyperproliferative condition, comprising the step of administering an effective amount of a composition comprising at least one immunotherapy construct described herein to the subject. For example, in various embodiments, the hyperproliferative disorder or hyperproliferative condition is intended to include one or more of cancers, precancerous conditions, or cancerous metastases. For example, the hyperproliferative disorder may include, in some cases, 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] Methods are also provided for combining the administration of an immunotherapy construct with at least one other treatment, such as the treatment of cancer or another hyperproliferative disease or hyperproliferative condition. In a first example of such a combination method, a method is provided which involves administering to a subject in need an effective amount of one of the described embodiments of an immunotherapy construct, or a composition containing an immunotherapy construct, and at least one anticancer agent (e.g., a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor) to enhance the effect of anticancer therapy in the subject in need. In another exemplary combination method, a method is provided which involves administering to a subject in need an effective amount of one of the described embodiments of an immunotherapy construct, or a composition containing an immunotherapy construct, and at least one immune checkpoint inhibitor to enhance the effect of checkpoint blockade immunotherapy in a subject diagnosed with a neoplasm. Yet another combination method is a method for enhancing the effect of radiotherapy in a subject diagnosed with a neoplasm, and is provided which involves administering to a subject in need an effective amount of one of the described embodiments of an immunotherapy construct, or a composition containing an immunotherapy construct, and at least one radiotherapy. In any embodiment of the combination method, examples are provided in which an immunotherapy construct or composition and a second agent (generally an anticancer agent or anticancer therapy) are administered sequentially or concurrently. As used herein, the term “enhances” with respect to the therapeutic effect of an anticancer therapy means an increase in the therapeutic effect of an anticancer therapy (e.g., treatment with an anticancer agent, radiotherapy, or checkpoint immunotherapy) that exceeds the therapeutic effect that would normally be obtained if the anticancer therapy were administered without the immunotherapy construct of the present invention. “Enhanced therapeutic effect” occurs when there is an acceleration and / or increase in intensity and / or degree of the therapeutic effect obtained by the anticancer therapy. “Enhanced therapeutic effect” also includes an extension of the useful duration of the therapeutic benefit. “Enhanced therapeutic effect” may also occur when, when administered concurrently with the immunotherapy construct provided by the present invention, a relatively low dose, frequency, or duration of the anticancer therapy is required to obtain the same benefit and / or effect as when the anticancer therapy is administered alone, even if the relatively high dose, frequency, or duration of the anticancer therapy is used.The enhancing effect, while not always the case, preferably results in the treatment of acute symptoms that are ineffective or poorly therapeutically treated with anticancer therapy alone. When the immunotherapy construct of the present invention is administered concurrently with anticancer therapy, enhancement is achieved when the therapeutic effect increases by at least 10% (e.g., at least 25%, at least 50%, at least 75%, or at least 100%) compared to administration of anticancer therapy alone.

[0053] In any of the described methods, the step of administering the immunotherapy construct may include one or more of the following: direct injection into or around the target tumor, lesion or excised tumor area; systemic injection into the target; local application to the target; inhalation; implantation device; or microneedle application to the target.

[0054] In any example of the method embodiments provided, the subject (being treated, administered a construct or composition, or from which cells are obtained) is a mammal. For example, in certain embodiments, the mammal is a human.

[0055] Kits comprising the immunotherapy constructs described herein and at least one anticancer agent are also provided herein. In some embodiments, the anticancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor.

[0056] To support the teachings of this disclosure, aspects of this disclosure are described below with additional details and options: (I) immunotherapy constructs; (II) therapeutic agents (to induce tumor antigen release and / or modulate the immunosuppressive tumor microenvironment); (III) adjuvant compounds; (IV) any additional components; (V) delivery systems; (VI) pharmaceutical compositions and dosage formulations; (VII) exemplary uses; (VIII) kits; (IX) exemplary embodiments; and (X) examples.

[0057] (I) Immunotherapy construct A novel class of immunotherapeutic agents (generally referred to as “immunotherapy constructs”) comprising engineered particles that simultaneously deliver an adjuvant and a therapeutic activator to cancer cells is described herein. Embodiments provide therapeutic activators that induce antigen release (specifically, tumor antigen release) and / or modulate an immunosuppressive environment (such as the tumor microenvironment). These immunotherapy constructs enhance the CD8+ T cell repertoire and induce systemic antitumor immunotherapy effects without requiring knowledge or identification of which antigens are associated with the treated cancer.

[0058] This strategy has many important features. They are effective, personalized, safe due to local delivery, permanent because it trains and utilizes the body's immune cells to attack cancer through a memory effect, inexpensive (e.g., requiring low doses and few doses), and applicable to many types of cancer.

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

[0060] (II) Therapeutic agents Examples of therapeutic activators (e.g., therapeutic oligonucleotides including siRNA, miRNA, antisense oligonucleotides, sgRNA-cas9, DNA and mRNA, as well as small molecule inhibitors, chemotherapeutic agents, antibodies, and chemicals) delivered to cancer cells and / or immune cells by the provided engineered immunotherapy constructs induce tumor antigen release and / or modulate the immunosuppressive tumor microenvironment. In specific embodiments, the activator kills cancer cells, thereby releasing tumor antigens, while simultaneously, a co-delivered adjuvant (e.g., CpG, R848, poly(I:C, etc.) prims 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 far from the local delivery of the immunotherapy construct), and can also reduce or even prevent the spread, recurrence, or development of new tumors having 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 may be adjusted to reduce toxicity to beneficial immune cells. In certain embodiments, therapeutic agents that influence cancer survival rates without harming immune cells are utilized on immunotherapy constructs. In other examples, therapeutic activators (e.g., siRNA, inhibitors, or other drugs against STAT3, CD39, CD73, IDO-6, PD-L1, TGF-β, antioxidants, etc.) can also be loaded onto / into a delivery vehicle (e.g., particles) to modulate an immunosuppressive tumor microenvironment, enabling priming and activation of immune cells to effectively attack cancer cells using antigens already present in the tumor, or antigens whose release is triggered by the immunotherapy construct.Examples of therapeutic agents 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, β-catenin, 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 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. Immunological checkpoints that, when inhibited, are beneficial for cancer treatment include, but are not exhaustive, PD-L1, PD-1, CTLA-4, LAG-3, TIM-3, B7-H3, VISTA, A2AR, and IDO (Khair et al., Frontiers Immunology, 10:453, 2019). In summary, immunotherapy constructs induce long-lasting immune-mediated anticancer effects. Memory-adaptive immunity can also be established.

[0061] In certain embodiments, immunotherapy constructs are used to activate an immune response. Such embodiments are not limited to any particular manner in which an immune response is activated.

[0062] Therapeutic oligonucleotides. Various types of therapeutic oligonucleotides can be used, and these may include, but are not exhaustive, 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 art as long as it can specifically regulate (interfere with or enhance) the action or synthesis of specific genes and proteins. Each particular oligonucleotide may have one or more targets. Examples of gene / protein targets of interest to the present invention include immune checkpoints, transcription factors, phosphatases, kinases, and the like. 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. These include 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 immunosuppressive 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, would be beneficial for cancer treatment include, but are not exhaustive, 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., siRNA against BLC2 and AKT1, siRNA against AR and MYC). They can also contain immunostimulatory sequences / elements that can simultaneously enhance the immune response and modulate the expression of target genes. They can also be designed to target the aforementioned genes with mutations.

[0063] In certain embodiments, immunotherapy constructs contain RNA interference-mediated oligonucleotides as activators. RNA interference is a highly conserved mechanism caused by double-stranded RNA (dsRNA) that can downregulate the transcript of a gene homologous to the dsRNA. dsRNA is initially processed by a dicer into short double-stranded molecules of 21-23 nucleotides called small interfering RNA (siRNA). Once incorporated into the RNA-induced silencing complex (RISC), dsRNA can mediate gene silencing by cleaving target mRNA. "siRNA," or "small interfering ribonucleic acid," refers to two strands of ribonucleotides that hybridize along complementary regions under physiological conditions. The siRNA molecule contains a double-stranded region substantially identical to the region of the target gene's mRNA. A region with 100% identity to the corresponding sequence of the target gene is suitable. This state is called "perfectly complementary." However, this region may also contain one, two, or three mismatches compared to the corresponding region of the target gene, depending on the length of the targeted mRNA region, and therefore may not be perfectly complementary. Methods for analyzing and identifying siRNAs with sufficient sequence identity to effectively inhibit the expression of a specific target sequence are known in the art. The coding region is considered a suitable mRNA target region. Untranslated regions, such as the 5'-UTR, 3'-UTR, and splice junctions, are also suitable, insofar as those regions are specific to the mRNA target.

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

[0065] In certain embodiments, it is intended that the development of any gene of interest can be inhibited using an siRNA delivery method (e.g., by loading siRNA onto the immunotherapy construct) that uses the immunotherapy constructs disclosed herein. Specific targets include genes known as drivers in cancer and other diseases, such as 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 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, siRNA is specifically intended to target mutant or mutated genes rather than wild-type genes.

[0066] Those skilled in the art will understand how to utilize representative sequences of these targets, which are readily available in public sequence databases. The following table provides sample sequence information: TIFF0007870011000009.tif125162TIFF0007870011000010.tif249162TIFF0007870011 000011.tif253162TIFF0007870011000012.tif249162TIFF0007870011000013.tif54162

[0067] Such embodiments are not limited to specific methods for evaluating the delivery profile of siRNA in vitro and / or in vivo. In some embodiments, labeling the siRNA molecule with imaging agents (e.g., fluorescent dyes FITC, RITC, Cy® dye, Dylight® dye, or Alexa Fluor® dye) or radiotraceers allows for visualization of the in vivo distribution and intracellular delivery profile of siRNA molecules at the organ level. In some embodiments, RT-PCR, FISH, IHC, flow cytometry, and Western blotting are used to analyze target proteins at the mRNA level and protein level, respectively.

[0068] In certain embodiments, the disclosure provides a method for inhibiting an intracellular target gene, comprising the step of introducing an siRNA capable of inhibiting the target gene by RNA interference into cells, wherein the siRNA comprises two complementary RNA strands, and the siRNA is loaded onto an immunotherapy construct. In some embodiments, the siRNA is modified with cholesterol at its 3' sense strand. In some embodiments, the cells are located within a human or animal subject (e.g., a horse, dog, cat, or other domesticated animal, livestock, or other animal with cancer).

[0069] MicroRNAs (miRNAs), or miRNA mimes, are short non-coding RNAs that can target and effectively silence protein-coding genes via their 3'-UTR elements. While the crucial roles of miRNAs in numerous biological processes are well-established, a comprehensive analysis of miRNA function in complex diseases is lacking. miRNAs are initially transcribed as primary miRNAs (pri-miRNAs), which are then cleaved by nuclear RNAse Drosha and Pasha to yield precursor miRNAs (pre-miRNAs). These precursors are further processed by cytoplasmic RNAse III dicers to form short double-stranded miR-miR* double helices, one of which (miR) is then incorporated into an RNA-induced silencing complex (RISC) containing the enzyme dicer and Argonaut (Ago). Mature miRNAs (approximately 17–24 nt) direct the RISC to a specific target site located within the 3'UTR of the target gene. Upon binding to a target site, miRNAs repress translation through mRNA degradation, translation inhibition, and / or sequestering to a processing body (P body) (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 have 3'-UTR miRNA target sites (Friedman et al., Genome Res., 19:92-105, 2009).In this regard, miRNAs act as important regulators of diverse processes, such 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 adipogenesis (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, studies of miRNA expression in chronic lymphocytic leukemia (Calin et al., Proc. Natl. Acad. Sci. USA, 105:5166-5171, 2008), colonic 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 infections (Pfeffer et al., Science, 304:734-736, 2004) suggest important associations between miRNAs and numerous diseases.

[0070] miRNAs observed to date are typically 21–22 nucleotides long and arise from longer precursors transcribed from non-protein-coding genes, as outlined in Carrington and Ambros (Science, 301(5631):336–338, 2003). These precursors form structures that fold over each other within self-complementary regions. They are then processed by nuclease dicers in animals (or DCL1 in plants). miRNA molecules interrupt translation through accurate or inaccurate base pairing with their targets. In some embodiments, miRNAs can be used as components of immunotherapy constructs that are therapeutically provided to or administered to subjects, such as human patients, to treat diseases, such as cancer. Alternatively, in some embodiments, a nucleic acid complementary to miRNA may be therapeutically administered to a subject in vivo or used in vitro to produce a desired therapeutic outcome (e.g., miRNA-142-3p, miRNA-142-3p, miRNA-124, or miRNA-138). Thus, a complementary nucleic acid may be used as a template to produce a desired therapeutic miRNA (e.g., miRNA-142-3p, miRNA-142-3p, miRNA-124, or miRNA-138).

[0071] Embodiments are particularly intended in which therapeutic oligonucleotides target or are specific to STAT3. STAT3 refers to the protein “signaling transcription factor 3” and its homologs. This term includes human proteins, whether wild-type or mutant. In embodiments, “STAT3” refers to proteins associated with Entrez Gene 6774, OMIM 102582, UniProt P40763 and / or RefSeq(protein)NP 003141 (which refers to proteins and associated nucleic acids known as of the filing date of this application). “Phosphorylated STAT3” refers to the STAT3 protein that is phosphorylated and activated by phosphorylation. In embodiments, phosphorylated STAT3 is phosphorylated on tyrosine 705 or on the residue corresponding to tyrosine 705 in the homolog. In embodiments, activation of STAT3 means that STAT3 can activate the transcription of other genes. In some embodiments, activated STAT3 is phosphorylated on tyrosine 705 or a corresponding residue to form a dimer (e.g., a homodimer or heterodimer), translocate to the nucleus, and / or activate transcription. In some embodiments, activated STAT3 forms a homodimer. Examples of proteins that phosphorylate STAT3 and thereby activate it include JAK2, EGFR, c-MET, and PDGF-R.

[0072] Anticancer agents. The term anticancer agent is used according to its simple, ordinary meaning and refers to a composition (e.g., compound, drug, antagonist, inhibitor, modulator) that has antineoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, an anticancer agent is a chemotherapeutic agent. In some embodiments, an anticancer agent is a targeted therapy agent. In some embodiments, an anticancer agent is an immune checkpoint inhibitor. In some embodiments, an anticancer agent is a drug identified herein that is useful in a method of treating cancer. In some embodiments, an anticancer agent is a drug approved by the FDA or a similar regulatory authority in a country other than the United States for the treatment of cancer.

[0073] Examples of anticancer drugs 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, e.g., nitrogen mustards (e.g., mechloretamine, cyclophosphamide, uramustine, chlorambucil, melphalan, ifosfamide), ethyleneimines and methylmelamines (e.g., hexamethylmelamine and thiotepa), alkyl sulfonates (e.g., busulfan and hepsulfam), nitrosoureas (e.g., carmustine, lomusitine, semustine and streptozocin), and triazenes (e.g., decarbazine); antimetabolites, e.g., folic acid analogs (e.g., methotrexate, leucovorin, larcitrexed and pemetrexed), pyrimidines Plant alkaloids (e.g., fluorouracil, floxouridine, cytarabine, capecitabine, and gemcitabine) and purine analogs (e.g., mercaptopurine, thioguanine, pentostatin, fludarabine, and 5-azathiopurine); plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, cabazitaxel, and homohalintinine); topoisomerase inhibitors, e.g., camptothecin derivatives (e.g., irinotecan and topotecan), amsacrin, and epipodophyllotoxin (e.g., etoposide (VP16), etoposide phosphate, and teniposide);Antibiotics, such as anthracendions (e.g., mitoxantrone), anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and fluorodaunorunicin hydrochloride), (e.g., hydrochloride), Streptomyces-derived antibiotics or their derivatives (e.g., dactinomycin, bleomycin, mitomycin, geldanamycin, plicamycin and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin), clofazimine and β-lactam derivatives; platinum-based chemotherapeutic agents (e.g., cisplatin, oxaliplatin, carboplatin); substituted ureas (e.g., hydroxyurea); methylhydrazine derivatives (e.g., procarbazine); adrenal cortical inhibitors (e.g., mitotane and aminoglutethimide); angiogenesis inhibitors (e.g., L-asparaginase and arginine deiminase); PI3K inhibitors (e.g., wartmannin and LY294002); mTOR inhibitors (e.g., sertraline); DNA methyltransferase inhibitors (e.g., 5-aza-2'-deoxycytidine); antisense oligonucleotides; apoptosis gene modulators; apoptosis Cis-regulatory factors (e.g., deoxyadenosine and tryptolide); BCR / ABL antagonists; bFGF inhibitors; casein kinase inhibitors (ICOS); gallium nitrate; gelatinase inhibitors; glutathione inhibitors (e.g., etanidazole); immunostimulant peptides; insulin-like growth factor 1 receptor inhibitors; leukemia inhibitors; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIF inhibitors; mismatch 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; signaling inhibitors / modulators (e.g., itraconazole); single-stranded antigen-binding proteins;Stem cell inhibitors; stromelysin inhibitors; synthetic glycosaminoglycans; telomerase inhibitors; thyroid-stimulating hormone; translation inhibitors; urokinase receptor antagonists; gonadotropin-releasing hormone agonists (GnRH), e.g., goserelin and leuprolide (leuprorelin); steroids, e.g., corticosteroids (e.g., prednisone and dexamethasone); progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate); antiprogestrogens (e.g., mifepristone); estrogens (e.g., diethylstilbestrol and ethinylestradiol); antiestrogenic drugs, e.g., aromatase inhibitors (e.g., exemestane, fadrozol, letrozole, pentrozole and anastrozole); selective estrogen receptor modulators (e.g., tamoxifen analogs, tamoxifen Methiozide analogs, panomiphene analogs, and clomiphene analogs); androgens (e.g., testosterone propionate and fluoxymesterone); antiandrogenic drugs (e.g., flutamide, finasteride, and bicalutamide); immunostimulants, levamisol, 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-calitiamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas aeruginosa exotoxin conjugate, etc.); radioimmunotherapy agents (e.g., 111 In, 90 Y or 131 Anti-CD20 monoclonal antibodies conjugated to 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, e.g., one or more inhibitors 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, Peritinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626, Sorafenib, Imatinib ( Examples include Gleevec®, sunitinib and dasatinib; immune checkpoint inhibitors (e.g., anti-CTLA4 antibody, anti-PD1 / L1 antibody); PLK1 inhibitors (GSK461364, BI2536, Tak960, NMS-P937, boracertib), etc., or mixtures thereof (e.g., leuprolide + estrogen + progesterone).

[0074] Furthermore, the immunotherapy constructs described herein include, without limitation, immunostimulants (e.g., Bacille Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), therapeutic monoclonal antibodies (e.g., anti-CD20 monoclonal antibody, anti-HER2 monoclonal antibody, anti-CD52 monoclonal antibody, anti-HLA-DR monoclonal antibody, and anti-VEGF monoclonal antibody), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), immune checkpoint inhibitors (e.g., anti-CTLA4 antibody, anti-PD1 antibody, anti-PD-L1 antibody), and radioimmunotherapy (e.g., 111 In, 90 Y or 131 anti-CD20 monoclonal antibody conjugated to I, etc.) and can be co-administered with conventional immunotherapeutic agents. These immunotherapeutic agents can also be directly loaded onto the immunotherapy constructs to enhance their therapeutic effects, reduce toxicity, and shorten the administration time.

[0075] In a further aspect, the immunotherapy constructs described herein include, without limitation, 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 Bi, etc.) radionuclides and can be co-administered with conventional radiotherapy agents. These radiotherapy agents can also be directly loaded onto the immunotherapy constructs to enhance the therapeutic effects, reduce toxicity, and shorten the administration time.

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

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

[0078] Generally, an adjuvant is any substance that, when mixed with a vaccine composition, increases or otherwise modifies the immune response to a (cancer) antigen. Adjuvants with immunostimulatory activity are particularly intended. Adjuvants induce nonspecific activation of the immune system unless they are 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 the immune-mediated response or a reduction in disease symptoms. For example, an increase in humoral immunity typically manifests as a significant increase in the titer of antibodies induced against the antigen, and an increase in T cell activity typically manifests as an increase in antigen-specific T cell proliferation, target cell death, or cytokine secretion. Adjuvants can also alter the immune response, for example, by changing a primarily humoral or Th2 response to a primarily 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 conjugate 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 potassium aluminum sulfate); anti-CD40 antibodies (e.g., CP-870, 893); cytokines (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF)); small molecule TLR agonists (e.g., imiquimod, reximod, gardikimod, and 3M-052); fusion proteins (e.g., ImuFact IMP321, CyaA, and ONTAK); oil-based or surfactant-based adjuvants (e.g., MF59, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, and Montanide ISA-51); plant extracts (e.g., QS21 stimulon derived from saponins (Aquila) Biotech, Worcester, Mass., USA); includes mycobacterial extracts and synthetic bacterial cell wall mimics, e.g., lipopolysaccharides (e.g., monophosphoryl lipid A, OM-174, OM-197-MP-EC, and Pam3Cys); xanthenon derivatives (e.g., badi mezan); mixtures thereof (e.g., AS-15); and other proprietary adjuvants, e.g., 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 are directly involved in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), promoting the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (US Patent 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). Additionally, Toll-like receptors (TLRs), or agents that activate TLRs, may be used as adjuvants and are important members of the pattern recognition receptor (PRR) family, which recognize conserved motifs shared by many microorganisms, known as "pathogen-associated molecular patterns" (PAMPS).

[0081] In some embodiments, adjuvants include CpG oligonucleotides. CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. While not bound by any specifically mechanistic theory, CpG oligonucleotides function at least partially by activating the innate (maladaptive) 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 dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates, in both prophylactic and therapeutic vaccines. More importantly, CpG-induced TLR9 activation promotes dendritic cell maturation and differentiation, even without the help of CD4 T cells. H It enhances the activation of single cells and generates potent cytotoxic T lymphocytes (CTLs). TLR9 stimulation induces T H 1 bias is usually T H2. Bias is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA). CpG oligonucleotides exhibit even greater adjuvant activity when formulated, administered co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce a strong response when the antigen is relatively weak. They also accelerate the immune response and, in some experiments, have allowed for a two-order-of-magnitude reduction in antigen dose while obtaining an antibody response comparable to a full-dose vaccine without CpG (Krieg, Nature Reviews, Drug Discovery, 5:471-484, 2006). U.S. Patent No. 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleoside adjuvants, and antigens to induce an antigen-specific immune response. A commercially available CpG TLR9 agonist is dSLIM (double Stem Loop Immunomodulator) manufactured by Mologen (Berlin, Germany). Other TLR-binding molecules, such as RNA-binding TLR 7, RNA-binding TLR 8, and / or RNA-binding TLR 9, may also be used.

[0082] For example, xanthenone derivatives such as badimezan or AsA404 (also known as 5,6-dimethylxanthenone-4-acetic acid (DMXAA)) may also be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives may also be administered in parallel with the vaccine of the present invention, for example, via systemic delivery or intratumoral delivery, to stimulate immunity at the tumor site. Although not bound by theory, such xanthenone derivatives are thought to function by stimulating interferon (IFN) production via stimulators of the IFN gene 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 non-CpG bacterial RNA, as well as immunoactive 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 can 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 those skilled in the art without the use of excessive experimentation. Additional adjuvants include colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF, salglamostim).

[0083] Poly-ICLC is a synthetically prepared double-stranded RNA containing a poly-I chain and a poly-C chain with an average length of 5000 nucleotides, stabilized against thermal 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, resulting in the activation of DC and natural killer (NK) cells, as well as the production of a “natural mixture” of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct, broad-spectrum host-targeting anti-infective and possibly antitumor effects mediated by two IFN-inducible nuclear enzyme systems, namely 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 conjugated to immunotherapy constructs include TLR ligands, type C lectin receptor ligands, NOD-like receptor ligands, RLR ligands, and RAGE ligands. TLR ligands may 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 may also include TLR3 ligands (e.g., polyinosine-polycytidic acid (Poly(I:C))), TLR7 ligands (e.g., imiquimod and reciquimod), and TLR9 ligands.

[0085] As used herein, the term “TLR-binding DNA substituent” refers to a substituent or moiety that can bind to a Toll-like receptor (“TLR”) and comprises at least one deoxyribonucleic acid. In some embodiments, the TLR-binding DNA substituent is a nucleic acid. In some embodiments, the TLR-binding DNA substituent comprises at least one nucleic acid analog. In some embodiments, the TLR-binding DNA substituent comprises at least one nucleic acid analog having an alternative skeleton (e.g., phosphodiester derivatives (e.g., phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boronphosphonate, or O-methylphosphoramidite), peptide nucleic acid skeleton, LNA, or binding). In some embodiments, the TLR-binding DNA substituent comprises DNA. In some embodiments, any nucleotide sugar in the TLR-binding DNA substituent is deoxyribose (e.g., any nucleotide is DNA). In some embodiments, the TLR-binding DNA substituent includes or is DNA having internucleotide bonds selected from phosphodiesters and phosphodiester derivatives (e.g., phosphoramides, phosphorodiamidates, phosphorothioates, phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methylphosphonates, boronphosphonates, O-methylphosphoramidites, or combinations thereof). In some embodiments, the TLR-binding DNA substituent includes DNA having internucleotide bonds selected from phosphodiesters and phosphorothioates. In some embodiments, the TLR-binding DNA substituent includes or is DNA having skeletal bonds selected from phosphodiesters and phosphorodithioates. In some embodiments, the TLR-binding DNA substituent includes or is DNA containing phosphodiester skeletal bonds. In some embodiments, the TLR-binding DNA substituent includes or is DNA containing phosphorothioate skeletal bonds. In some embodiments, the TLR-binding DNA substituent includes or is DNA containing phosphorodithioate skeletal bonds. In this embodiment, the TLR-binding DNA substituent preferentially binds to TLR9 over other TLRs.In one embodiment, the TLR-binding DNA substituent specifically binds to TLR9. In another embodiment, the TLR-binding DNA substituent specifically binds to TLR3. In another embodiment, the TLR-binding DNA substituent specifically binds to TLR7. In another embodiment, the TLR-binding DNA substituent specifically binds to TLR8. In another embodiment, the TLR-binding DNA substituent specifically binds to a cell subcompartment (e.g., endosome) associated TLR (e.g., TLR3, TLR7, TLR8, or TLR9). In another embodiment, the TLR-binding DNA substituent contains or is a G-rich oligonucleotide. In another embodiment, the TLR-binding DNA substituent contains a CpG motif, where C and G are nucleotides and p is a phosphate linking C and G. In another embodiment, the CpG motif is unmethylated. In another embodiment, the TLR-binding DNA substituent is a class A CpG oligodeoxynucleotide (ODN). In one embodiment, the TLR-binding DNA substituent is a class B CpG oligodeoxynucleotide (ODN). In another embodiment, the TLR-binding DNA substituent is a class C CpG oligodeoxynucleotide (ODN). In another embodiment, the TLR-binding DNA substituent (e.g., TLR9-binding DNA substituent) comprises a deoxyribonucleic acid having an A base, a G base, a C base, or a T base and a phosphodiester bond and / or a phosphodiester derivative bond (e.g., a phosphorothioate bond).

[0086] The term "CpG motif" refers to a 5'C nucleotide linked to a 3'G nucleotide via a phosphodiester nucleotide bond or a phosphodiester derivative nucleotide bond. In some embodiments, the CpG motif includes a phosphodiester nucleotide bond. In some embodiments, the CpG motif includes a phosphodiester derivative nucleotide bond.

[0087] As used herein, the terms “Class A CpG ODN,” “Class A CpG ODN,” “Type D CpG ODN,” or “Class A CpG DNA sequence” are used in accordance with their general meanings in biological and chemical sciences and refer to a CpG motif containing an oligodeoxynucleotide, which includes a poly-G sequence at the 5', 3', or both ends; an internal palindromic sequence containing a CpG motif; or one or more phosphodiester derivatives that conjugate a deoxynucleotide. In some embodiments, a Class A CpG ODN comprises a poly-G sequence at the 5', 3', or both ends; an internal palindromic sequence containing a CpG motif; and one or more phosphodiester derivatives that conjugate a deoxynucleotide. In some embodiments, the phosphodiester derivative is a phosphorothioate. Examples of Class A CpG ODNs include ODN D19, ODN 1585, ODN 2216, and ODN 2336.

[0088] The terms “Class B CpG ODN,” “Class B CpG ODN,” “Type K CpG ODN,” or “Class B CpG DNA sequence” are used according to their general meanings in biological and chemical sciences and refer to a CpG motif containing a hexameric motif containing a CpG motif; and a CpG motif containing an oligodeoxynucleotide containing one or more phosphodiester derivatives that bind any deoxynucleotide. In aspects, a Class B CpG ODN comprises a hexameric motif containing a CpG motif and one or more copies of a phosphodiester derivative that binds any deoxynucleotide. In aspects, the phosphodiester derivative is a phosphorothioate. In aspects, a Class B CpG ODN comprises one hexameric motif containing a CpG motif. In aspects, a Class B CpG ODN comprises two copies of a hexameric motif containing a CpG motif. In aspects, a Class B CpG ODN comprises three copies of a hexameric motif containing a CpG motif. In some embodiments, a class B CpG ODN contains four copies of a hexamer motif that includes a CpG motif. Examples of class B CpG ODNs include ODN 1668, ODN 1826, ODN 2006, and ODN 2007.

[0089] The terms "Class C CpG ODN," "Class C CpG ODN," or "Type C CpG DNA sequence" are used according to their general meanings in biological and chemical sciences and refer to oligodeoxynucleotides containing a palindromic sequence with a CpG motif and a phosphodiester derivative (phosphorothioate) that binds any deoxynucleotide. Examples of Class C CpG ODNs include ODN 2395 and ODN M362.

[0090] (IV) Any additional components targeting part One or more targeting moieties (also known as targeting molecules) may be included, for example, loaded into a delivery vehicle, attached to the surface of a delivery vehicle, and / or encapsulated within a delivery vehicle. In some embodiments, the targeting moieties are presented on the outer surface of the delivery vehicle. In certain embodiments, such targeting moieties enable specific cell targeting (e.g., prevention of delivery of toxic therapeutic agents to immune cells, enrichment / targeting of delivery of therapeutic agents with cell-specific function). In certain embodiments, the targeting moieties may also have therapeutic activity and therefore function as therapeutic agents (e.g., PD-L1 antibodies) in the immunotherapy 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, aphibody molecules, ligands, small molecules, or polysaccharides that bind to one or more targets associated with organs, tissues, cells or extracellular matrix, or specific types of tumors or infected cells. The degree of specificity to which the delivery vehicle is targeted can be modulated by selecting targeting molecules with appropriate affinity and specificity. Antibodies, for example, are highly specific. They can be polyclonal, monoclonal, fragment, recombinant, or single-stranded, many of which are commercially available or readily obtainable using standard techniques. T cell-specific molecules, antigens, and tumor targeting molecules can be conjugated to the surface of immunotherapy constructs. Targeting molecules can be conjugated to the ends of one or more PEG chains present on the particle surface.

[0092] In some embodiments, the targeting moiety is an antibody or its antigen-binding fragment (e.g., a single-strand variable fragment) that specifically recognizes cells or tumor markers that are exclusively or in high concentrations on target cells, such as malignant cells (e.g., tumor antigens). Suitable targeting molecules that can be used to direct immunotherapy constructs to cells and tissues of interest, as well as methods for conjugating target molecules into 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, the therapeutic agent may be toxic to both cancer cells and immune cells, resulting in suboptimal effects. Therefore, in certain embodiments, the immunotherapy construct can be conjugated with a targeting moiety to enrich the delivery of the therapeutic agent and adjuvant to cancer cells only. Examples include antibodies against HER2, EGFR, PSMA, PD-L1, etc., that are expressed or optionally overexpressed on cancer cells. In some embodiments, the immunotherapy construct can be conjugated with a targeting portion to enrich the delivery of therapeutic agents and adjuvants to immune cells only.

[0093] Targeting molecules may include neuropilins and endothelial targeting molecules, integrins, selectins, adhesion molecules, bone targeting molecules such as zoledronic acid and alendronate (for example, to target cancer that has metastasized to the bone), stroma, and fibroblast targeting molecules.

[0094] In some embodiments, the targeting portion targets the immunotherapy 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 immunotherapy construct enhances the activity of DCs processing tumor antigens, thereby more effectively inducing in-situ tumor vaccination. Targeted delivery to DCs may be performed. Targeting exogenous antigens to internalized surface molecules on antigen-presenting cells distributed throughout the body facilitates particle uptake and can overcome the 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 presented 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 2 to 4 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, the antigen was fused to the anti-DEC205 heavy chain, and recombinant antibody molecules were used for immunization.

[0096] Various other endocytosis receptors, including mannose-specific lectins (mannose receptors) and IgG Fc receptors, have also been targeted in this manner, resulting in similarly improved 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. For preferential uptake by immune cells expressing these receptors, targeting moies to these receptors can be attached to immunotherapy constructs. An example is mannose attached to an immunotherapy construct for targeted delivery to macrophages and DCs with 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, signaling internally and thereby potentially increasing DC antigen uptake, maturation, and T cell stimulation capabilities. PAMPs that can be conjugated or co-encapsulated on the particle surface include unmethylated CpG DNA (bacterial), double-stranded RNA (viral), lipopolysaccharide (bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacterial), flagellin (bacterial), poly(inosine-cytidylic acid) (bacterial), lipoteichoic acid (bacterial), or imidazoquinoline (synthetic).

[0098] The targeted molecule can be covalently bonded to the delivery vehicle using various methods known in the art. In a preferred embodiment, the targeted moiety is attached to the delivery vehicle by PEGylation or biotin-avidin crosslinking.

[0099] CD40 agonist. In certain embodiments, the targeting moiety targets CD40. This moiety may 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 indirect immune activation effects and direct cytotoxic effects on tumors, resulting in a "2:1" mechanism of action for CD40 agonists. CD40 agonists are known in the art and are outlined in Vonderheide (Clin Cancer Res, 13(4):1083-1088, 2007). Exemplary agonists include recombinant CD40L (recombinant human trimer), CD-870, 893 (full human IgG2 mAb), SGN-40 (humanized IgG1), and HCD 122 (full human IgG1 mAb). Soluble agonist CD40 antibodies have been shown to replace T cell support provided by CD4+ lymphocytes in mouse models 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 integrins. Studies have shown that integrins can function as markers to distinguish tumor cells from normal cells because they are overexpressed on the surface of tumor cells. Certain integrins also activate TGF-β via extracellular pathways. Latent TGF-β, after being released from tumor cells, binds to integrins on the surface of tumor cells, resulting in the activation of latent TGF-β. Increased TGF-β concentration within the tumor microenvironment supports immunosuppression and mobilizes regulatory T cells into the tumor environment.

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

[0102] A T cell receptor that recognizes the p53 antigen. In certain embodiments, the targeting portion is a T cell receptor (TCR) that recognizes the p53 antigen in relation to human MHC. A T cell receptor recombinant protein derived from bacterial, eukaryotic, or yeast cells, containing a T cell receptor (α / β TCR or γ / Δ TCR) composed of an alpha, beta, or gamma / delta chain.

[0103] IL-15 / IL-15Rα. In another embodiment, the targeting moiety is the IL-15 / IL-15Rα complex. Interleukin-15 (IL-15) is a cytokine that shares a specific receptor subunit with IL-2 and therefore has several 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, for example, natural killer (NK) cells with nanoparticle compositions.

[0104] (V) Delivery System The embodiments of immunotherapy constructs provided herein are independent of the delivery system used for the delivery of the therapeutic agent and adjuvant. Therefore, in various embodiments, the delivery system may use or be based on any known or planned-to-be-developed particulate delivery vehicle. These include nanoparticles, fullerenes, endohedral metal fullerenes, trimetal nitride-templated endohedral metal fullerenes, single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled boron / nitrate nanotubes and multi-walled boron / nitrate nanotubes, carbon nanotube peapods, carbon nanohorns, carbon nanohorn peapods, liposomes, lipid nanoparticles, lipoplexes, polymer nanoparticles, calcium phosphate particles, aluminum salt particles, polyplexes, nanoshells, dendrimers, microparticles, quantum dots, superparamagnetic nanoparticles, nanorods, cellulose nanoparticles, glass microspheres and glass nanospheres, and polymer microspheres and polymer nanospheres, biodegradable PLGA microspheres and biodegradable PLGA nanospheres, gold nanoparticles, adjuvant particles (e.g., viromosomes or other virus-like particles), silver nanoparticles, carbon nanoparticles, iron nanoparticles, porous silica nanoparticles 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. The particles can be of any shape, structure and porosity. Therapeutic agents, adjuvants and any additional compounds may 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 may 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 includes mesoporous silica nanoparticles (MSNPs), such as those described in U.S. Patent Application Publication US2017 / 0173169 or U.S. Patent Application Publication US2017 / 0172923, which are incorporated herein by reference.

[0106] In some embodiments, the average particle size of the mesoporous nanoparticles (or different nanoparticles) is approximately 5 nm to 200 nm, approximately 5 nm to 90 nm, approximately 5 nm to 20 nm, approximately 30 nm to 100 nm, approximately 30 nm to 80 nm, approximately 30 nm to 60 nm, approximately 40 nm to 80 nm, approximately 70 nm to 90 nm, or approximately 5 nm, approximately 10 nm, approximately 20 nm, approximately 30 nm, approximately 40 nm, approximately 50 nm, approximately 60 nm, approximately 70 nm, approximately 80 nm, approximately 90 nm, or approximately 100 nm. In some embodiments, the mesoporous silica nanoparticles are coated with a cationic polymer or other compound. The cationic polymer can be bonded to the surface of the nanoparticles using any suitable means. In some embodiments, the cationic polymer is bonded to the nanoparticles via electrostatic interactions. Cationic polymers are any positively charged polymers, for example, without limit, 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 are obvious 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] Cationic polymers can be linear or branched. In some embodiments, cationic polymers can be in the size range of about 500 Da to about 25 kDa and can be branched or linear. For example, branched PEI having an average size of 1.8 kDa to 10 kDa may be loaded onto the nanoparticle core. The ratio of cationic polymer to nanoparticles can vary depending on the desired result. Cationic polymers can be present in 1 to 50 wt% of the nanoconstruction, e.g., 5 to 40 wt%, 10 to 30 wt%, 20 to 30 wt%, 5 to 15 wt%, 5 to 20 wt%, 5 to 25 wt%, 5 to 30 wt%, 10 to 20 wt%, 10 to 25 wt%, or 25 to 40 wt%, e.g., about 5, about 10, about 15, about 20, about 25, about 30, or about 35 wt%. In some embodiments, cationic polymers are present in 10 to 20 wt%.

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

[0109] The stabilizer may be conjugated to MSNPs (or different nanoparticles) and / or cationic polymers by any suitable means, for example. In some embodiments, the stabilizer is conjugated to amine groups or other reactive groups of a crosslinked cationic polymer coated on nanoparticles (e.g., MSNPs). Exemplary stabilizers include, but are not limited to, PEG, dextran, polysialic acid, hyaluronic acid, polyvinylpyrrolidone, polyvinyl alcohol, and polyacrylamide or combinations thereof.

[0110] Stabilizers may have multiple chemically reactive groups for attachment to, for example, nanoparticles, cationic polymers, and / or other components. For example, reactive stabilizers, such as PEG derivatives, may have maleimide-PEG-N-hydroxysuccinimidyl ester (Mal-PEG-NHS) containing both two electrophilic moieties, e.g., a Michael acceptor and an activating ester. Stabilizers used in conjunction with the compositions and methods of the present invention, such as PEG, generally have molecular weights in the range of 500 Da to 40 kDa, for example, 2 to 10 kDa. Stabilizers may be present in 1 to 50% by weight of the nanoconstruction, for example, 5 to 30% by weight, 10 to 20% by weight, 10 to 25% by weight, 5 to 15% by weight, 5 to 20% by weight, 5 to 25% by weight, or 1 to 10% by weight, for example, 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 statistically average particle size (diameter) of particles within a particle population. For essentially spherical particles, the diameter may refer to either the physical diameter or the hydrodynamic diameter. For non-spherical particles, the diameter may preferentially refer to the hydrodynamic diameter. As used herein, the diameter of non-spherical particles may refer to the maximum straight-line distance between two points on the particle's surface. The average hydrodynamic particle size can be measured using methods known in the art, such as dynamic light scattering.

[0112] "Monodisperse" and "uniform size distribution" are used without distinction herein and refer to a collection of nanoparticles or microparticles in which all particles are the same or nearly the same size. As used herein, 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, “nanoparticles” generally refer to particles 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.” This disclosure is not limited to any particular type or species of nanoparticles for forming complexes with adjuvants and therapeutic agents configured to treat or prevent cancer and associated hyperproliferative disorders.

[0114] Examples of nanoparticles include fullerenes (also known as C 60 , C 70 , C 76 , C 80 , C 84 Examples include endohedral metallic fullerenes (EMIs) containing additional atoms, ions, or clusters inside their fullerene cages, trimetallic nitride template endohedral metallic fullerenes (TNT EMEs, which are highly symmetrical tetraatomic molecular cluster endohedrals formed within a trimetallic nitride template in a carbon cage), single-walled and multi-walled carbon nanotubes, branched and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled boron / nitrate nanotubes and multi-walled boron / nitrate nanotubes, carbon nanotube peapods (nanoparticles with internal metallic fullerenes and / or other internal chemical structures), carbon nanohorns, carbon nanohorn peapods, lipid particle liposomes, lipoplexes, polymer nanoparticles, polyplexes, nanoshells, dendrimers, quantum dots, superparamagnetic nanoparticles, nanorods, adjuvant particles (e.g., viromosomes or other virus-like particles), and cellulose nanoparticles. Other exemplary nanoparticles include glass microspheres and glass nanospheres, polymer microspheres and polymer nanospheres, biodegradable PLGA microspheres and biodegradable PLGA 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 herein, the term “hydrophobic polymer block” refers to a segment of a polymer that is hydrophobic in itself. As used herein, the term “micelle” refers to an aggregate of molecules dispersed in a liquid. A typical micelle in an aqueous solution forms an aggregate with a hydrophilic “head” region in contact with the surrounding solvent, isolating a hydrophobic single tail region at the center of the micelle. In some embodiments, the head region may be, for example, a surface region of the polyol polymer, and the tail region may 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 relatively small, preferably about 1 to 50 micrometers in size. For ease of explanation, the use of “nanoparticles” as used herein encompasses true nanoparticles (size 1 nm to 1000 nm), microparticles (e.g., 1 micrometer to 50 micrometers), or both.

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

[0118] Disclosed are immunotherapy construct-containing compositions comprising one or more activators and one or more adjuvant compounds, each loaded within a delivery vehicle, attached to the surface of a delivery vehicle, and / or encapsulated within a delivery vehicle. Nanoparticle compositions offer several advantages beyond delivering one or more activators to target cells in solution. For example, nanoparticle compositions present one or more activators at local concentrations on or within nanoparticles, increasing binding activity when the nanoparticles encounter target cells. Nanoparticle compositions can also function as depots for activators with adjustable release kinetics that can be extended over several days to extend the effective systemic half-life and efficacy of one or more drugs.

[0119] Typically, two or more activators (including one therapeutic agent and one adjuvant) are loaded into a delivery vehicle, attached to the surface of the delivery vehicle, and / or encapsulated within the delivery vehicle. The relative concentrations of each of the two or more activators and their positions on or within the delivery vehicle can be manipulated during the composition's preparation to adapt to the preferred dosage and presentation received by target cells. Loading two or more activators into or on the same delivery vehicle makes it possible to present two or more activators simultaneously to target cells or the same tumor microenvironment, or to present them to target cells in other predetermined orders.

[0120] The delivery vehicle may be, for example, a nanolipogel, polymer particles, silica particles, liposomes, or multilayer vesicles. In certain embodiments, the microparticle delivery vehicle is a nanoscale composition, e.g., from 10 nm to less than 1 micron. However, in some embodiments and applications, it will be understood that the particles may be even smaller or larger (e.g., microparticles). The exemplary immunotherapy constructs disclosed herein may be called nanoparticle compositions, but in some embodiments and applications, it will be understood that the microparticle compositions may be somewhat larger than nanoparticles. For example, the microparticle compositions may also be from 1 micron to 1000 microns. Such compositions may be called fine particle compositions.

[0121] In embodiments for treating cancer, it is desirable that the particles are of a size suitable for reaching the tumor microenvironment. In certain embodiments, the particles are of a size suitable for reaching the tumor microenvironment and / or tumor cells through enhanced permeability and retention (EPR) effects. EPR refers to the property that molecules of a certain size (e.g., the particulate matter compositions described herein) tend to accumulate in tumor tissue in much greater quantities than in normal tissue. Therefore, in compositions for treating cancer, the delivery vehicle is preferably in the range of 25 nm to 500 nm, and more preferably in 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 ingredients. 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 and provides biocompatibility and a surface for functionalization by targeted molecules. The outer shell encapsulates components so they remain hidden until desired, for example, in response to environmental conditions or stimuli, creating monodisperse, reproducible particle populations and mediating internalization into desired cell types. The inner core may be a dendrimer or other polymer and has distinct, additional functions relative to the outer shell. For example, the inner shell may enable secondary deposition of drugs, vaccines, or imaging agents, increase the loading of components with different physicochemical properties into the particles, allow for controllable release of contents from the particles, and increase the cytosolic availability of DNA / RNA, drugs, and / or proteins by disrupting endosomes, any of which 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 crosslinked block copolymer comprising 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 the polymer matrix or covalently bonded to the polymer matrix. The hydrogel core is surrounded by a liposome shell.

[0125] Nanolipogels can be constructed to incorporate various activators that can be subsequently released in a controlled manner. Activators can be dispersed within a hydrogel matrix, dispersed within liposome shells, covalently bound to liposome shells, and combinations thereof. Activators can be selectively incorporated into each of these locations within the nanolipogel. Furthermore, the release rate of the activator from each of these locations can be independently controlled. Because each of these locations has different properties, including size and hydrophobic / hydrophilicity, the chemical components independently incorporated into each of these locations can differ dramatically in terms of size and composition. For example, a nanolipogel can be loaded with one or more compounds dispersed within a polymer matrix, a bound small molecule hydrophobic drug, and an adjuvant. Nanolipogels can result in the simultaneous and sustained release of drugs with vastly different chemical compositions and molecular weights.

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

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

[0128] Hydrophobic activators such as proteins may be covalently bound to the surface of the nanolipogel, whereas hydrophilic activators 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 activators may be conjugated to the ends 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, which allows for the attachment of a biotinylated targeting moiety, a detectable label, or other activator thereto, if desired.

[0130] In certain embodiments, one or more activators are covalently bound to the surface of the nanolipogel via a linking group that is cleaved in response to an external chemical or physical stimulus, such as a change in ambient pH, to cause release of the activator at a desired physiological location.

[0131] Core. The nanolipogel core is formed from a polymeric matrix. The matrix can include one or more host molecules, as will be described in more detail below. The nanolipogel core can further include one or more activators. The activators may be complexed with the host molecules, dispersed within the polymeric matrix, or a combination thereof.

[0132] The polymeric matrix of the nanolipogel can be formed from one or more polymers or copolymers. By varying the composition and morphology of the polymeric matrix, various controlled release characteristics can be achieved, enabling the delivery of one or more activators at a moderate constant dose over a long period of time.

[0133] The polymer matrix may be formed from a non-biodegradable polymer or a biodegradable polymer. However, the polymer matrix is ​​preferably biodegradable. The polymer matrix may 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. To increase the molecular weight of the polymer, a biodegradable crosslinking agent may be used, which can be removed from the body as small fragments after the crosslinking agent has degraded.

[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 poly-3-hydroxybutyrate or poly-4-hydroxybutyrate; polycaprolactone; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); poly(glycolide-co-caprolactone); polycarbonates, such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), polypeptides and poly(amino acids); polyesteramides; other biocompatible polyesters; poly(dioxanone); poly(alkylene alkyleneates); hydrophilic polyethers; polyurethanes; polyether esters; polyacetals; polycyanoacrylates; polysiloxanes; poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acid), polyvinyl alcohol, polyvinyl pyrrolidone; 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 their copolymers or derivatives containing 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 their derivatives, copolymers and blends are included.

[0135] As used herein, “derivative” includes polymers having substitutions, additions of chemical groups, and other modifications to the polymer backbone commonly performed by those skilled in the art. Natural polymers, including proteins such as albumin, collagen, gelatin, prolamin, e.g., zein, and polysaccharides such as alginates and pectin, may be incorporated into the polymer matrix. A variety of polymers may be used to form the polymer matrix, but generally the resulting polymer matrix is ​​a hydrogel. In certain cases, if the polymer matrix contains natural polymers, the natural polymers are biopolymers that degrade by hydrolysis, e.g., polyhydroxyalkanoates.

[0136] The polymer matrix may optionally contain one or more crosslinkable polymers. Preferably, the crosslinkable polymer contains one or more photopolymerizable groups to enable crosslinking of the polymer matrix after nanolipogel formation. Examples of suitable photopolymerizable groups include vinyl groups, acrylate groups, methacrylate groups, and acrylamide groups. If present, the photopolymerizable groups may be incorporated into the backbone of the crosslinkable polymer, into one or more of the side chains of the crosslinkable polymer, into one or more of the ends of the crosslinkable polymer, or into a combination thereof.

[0137] Polymer matrices can be formed from polymers with varying molecular weights to form nanolipogels having properties, including optimal drug release rates for specific applications. Generally, the polymers constituting the polymer matrix have an average molecular weight in the range of 500 Da to 50 kDa. When the polymer matrix is ​​formed from non-crosslinkable polymers, the polymers typically have an average molecular weight in the range of 1 kDa to 50 kDa, more preferably 1 kDa to 70 kDa, and most preferably 5 kDa to 50 kDa. When the polymer matrix is ​​formed from crosslinkable polymers, the polymers typically have an even lower average molecular weight in the range of 500 Da to 25 kDa, more preferably 1 kDa to 10 kDa, and most preferably 3 kDa to 6 kDa. In certain embodiments, the polymer matrix is ​​formed from a crosslinkable 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, 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 a polymer matrix with tunable hydrophobic / hydrophilic properties and / or desired in vivo degradation properties.

[0141] The degradation rate of polyester segments, and often the corresponding drug release rate, can be varied from a few days (for pure PGA) to several months (for pure PLA), and this can be easily manipulated by changing the ratio of PLA to PGA within the polyester segment. Furthermore, poly(alkylene oxides), such as PEG, as well as 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 any 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 in the range of 500 Da to 10,000 Da, more preferably 1,000 Da to 7,000 Da, and most preferably 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 in the range of 100 Da to 3,500 Da, more preferably 100 Da to 1,000 Da, and most preferably 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 alginates, cellulose derivatives, and polyhydroxyalkanoates, such as polyhydroxybutyrate. The in vivo stability of microparticles can be controlled during manufacturing 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 time these materials circulate.

[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 alginates produced by conventional ion 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 formation apparatus that uses a stream of nitrogen gas to potentially break up the droplets. An ion curing bath, which is slowly agitated (e.g., 100-170 RPM), is placed below the extruder to capture the formed microdroplets. 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 extruders of various sizes 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 (e.g., alginates, CMCs), positively charged ligands of different molecular weights (e.g., polylysine, polyethyleneimine) can be ionically bonded to them.

[0148] Perhaps the most widely used are aliphatic polyesters, particularly the hydrophobic poly(lactic acid) (PLA), the relatively hydrophilic poly(glycolic acid) (PGA), and their copolymers, 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. Secondly, the physiological compatibility of PLGA and its homopolymers PGA and PLA is 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 various ways to improve pharmacokinetics and biodistribution to target tissues, either through passive or active targeting. Microparticles are designed to release encapsulated or attached molecules over a period of days to weeks. Factors influencing the 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 the polymer composition. Aliphatic polyesters exhibit different hydrophobic properties, which affects their degradation rates. Specifically, hydrophobic poly(lactic acid) (PLA), relatively hydrophilic poly(glycolic acid) (PGA), and their copolymers, poly(lactide-co-glycolide) (PLGA), have varying release rates. The degradation rates of these polymers, and often the corresponding drug release rates, can range from a few days (PGA) to several months (PLA) and can be easily manipulated by changing the ratio of PLA to PGA.

[0149] Shell components. A nanolipogel contains a liposome shell composed of one or more concentric lipid monolayers or lipid bilayers. The shell may further contain one or more activators, targeting molecules, or combinations thereof.

[0150] The nanolipogel comprises a liposome shell composed of one or more concentric lipid monolayers or lipid bilayers. The composition of the liposome shell may be modified to affect the release rate of one or more activators in vivo. Alternatively, the lipids may be covalently crosslinked to alter in vivo drug release, if desired.

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

[0152] Suitable neutral and anionic lipids include sterols and lipids, such as cholesterol, phospholipids, lysolipids, lysophospholipids, and sphingolipids. Neutral and anionic lipids include phosphatidylcholine (PC) containing 1,2-diacyl-glycero-3-phosphocholine (e.g., egg PC, soy PC); phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids, such as sphingomyelin; sphingoglycolipids (also known as 1-ceramidyl glucosides), such as ceramide galactopyranoside, ganglioside, and cerebroside; fatty acids, sterols containing carboxylic acid groups, such as cholesterol. or their derivatives; and 1,2-diacyl-sn-glycero-3-phosphoethanolamine, which includes 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-dioreollylglycerylphosphatidylethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine (DHPE), 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), and 1,2-dimiristoylphosphatidylcholine (DMPC). Natural derivatives of these lipids (e.g., tissue-derived L.-α-phosphatidyl: egg yolk, heart, brain, liver, soybean) and / or synthetic derivatives (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholine, 1-acyl-2-acyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-SN-glycero-3-phosphocholine) are also suitable.

[0153] Suitable cationic lipids include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salt, 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-dioloyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), and 1,2-dialkyloxy-3 -Dimethylammonium propane, dioctadecylamide glycylspermine (DOGS), 3-[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol); 2,3-Dioleoyloxy-N-(2-(sperminecarboxamide)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), β-alanylcholesterol, cetyltrimethylammonium bromide (CTAB), diC 14- Amidine, N-tert-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine, N-(α-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonioacetyl)diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N,N,N',N'-tetramethyl-,N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy C-1,4-butanediammonium iodide, 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives, for example, 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), Furthermore, 2,3-dialkyloxypropyl quaternary ammonium derivatives containing a hydroxyalkyl moiety on a quaternary amine, for example, 1,2-dioleyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropylammonium bromide (DORIE-HP), 1,2-dioleyl-oxypropyl-3-dimethyl-hydroxypropyl This includes ammonium 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 above neutral lipids, anionic lipids, and cationic lipids. By incorporating one or more PEGylated lipid derivatives into the lipid shell, a nanolipogel presenting polyethylene glycol chains on its surface can be obtained. The resulting nanolipogel can have increased stability and circulation time in vivo compared to nanolipogels lacking PEG chains on their surface. Examples of suitable PEGylated lipids include distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG) including DSPE PEG(2000MW) and DSPE PEG(5000MW), dipalmitoyl-glycero-succinic acid 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 specific 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 one or more PEGylated lipids to 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 specific embodiments, the molar ratio of one or more PEGylated lipids to 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 one or more phospholipids to one or more additional lipids or sterols is in the range of 1:1 to 6:1, more preferably 2:1 to 6:1, and most preferably 3:1 to 5:1. In certain embodiments, the molar ratio of one or more phospholipids to 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 may also be polymer particles, e.g., microparticles or nanoparticles. The particles may be biodegradable or non-biodegradable. Exemplary polymers that may be used to manufacture polymer particles are described above with respect to the polymer matrix components of nanolipogels.

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

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

[0162] In some embodiments, the particles are composed of PLGA, a safe, FDA-approved polymer. PLGA particles are advantageous because they can protect the activator (i.e., act as an encapsulant), facilitate long-term release, and are suitable for adding a targeted moiety.

[0163] The particles may contain one or more polymer conjugates comprising an end-to-end bond between the polymer and a targeting moiety, a detectable label, or other activator. For example, the modified polymer may be PLGA-PEG-phosphonate. In another example, the particles may be modified to include an avidin moiety, to which a biotinylated targeting moiety, a detectable label, or other activator can be conjugated.

[0164] Examples of preferred natural polymers include proteins, such as albumin, collagen, gelatin, and prolamins, such as zein, as well as polysaccharides, such as alginates, cellulose derivatives, and polyhydroxyalkanoates, such as polyhydroxybutyrate. The in vivo stability of the particles can be adjusted during manufacturing 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 time these materials circulate.

[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 the 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 whose properties can be tuned to facilitate different release rates and which can encapsulate drugs by distal loading. The release rate is tuned 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 a drug either before, during, or after formation, and then function as a controlled-release vehicle for the drug. Multiple drugs may be loaded into the nanolipogel so that controlled release of multiple drugs is subsequently achieved.

[0168] Nanolipogels are loaded with one or more therapeutic agents and / or adjuvants by a rehydration process of the nanolipogel in the presence of the drugs during and / or after formation. For example, a nanolipogel is loaded with a molecule that functions as an adjuvant, and the nanolipogel then incorporates one or more anticancer agents after formation to deliver and release the adjuvant along with the anticancer agents.

[0169] Polymer nanoparticles Emulsion method. In some embodiments, polymer nanoparticles are prepared using the emulsion solvent evaporation method. For example, the polymer 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 may be one or more of chloroform, dichloromethane, and acyl acetate. The drug may be dissolved in one or more of acetone, ethanol, methanol, isopropyl alcohol, acetonitrile, and dimethyl sulfoxide (DMSO). Then, an aqueous solution is added to the resulting mixture solution to obtain an emulsion solution by emulsification. The emulsification technique may be probe sonication or homogenization by homogenizer. The peptide or fluorophore or drug may 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, polymer nanoparticles are prepared using a nanoprecipitation method or a microfluidic device. The polymer 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. Particles can be produced from various polymers using various methods that can be selected based on criteria including the polymer composition of the particles, and the drug is loaded into or bound to 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 (dispersed soluble or 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 has evaporated, leaving solid particles. The resulting particles are washed with water and dried overnight in a freeze-dryer. This method can yield particles of 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, carried out in a completely anhydrous organic solvent, 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 5°C above the melting point of the polymer while continuously 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 fluid powder. This method yields particles with a size of 0.5 to 1000 microns. The outer surface of spheres prepared by this technique is usually smooth and dense. This procedure is used to prepare particles made from polyesters and polyanhydrides. However, this method is limited to polymers with a molecular weight of 1,000 to 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 suspended by stirring in an organic oil (e.g., silicone oil) to form an emulsion. Unlike solvent evaporation, this method can be used to produce 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 heavily 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 / hour, and nozzle diameter = 0.5 mm. Microparticles ranging from 1 to 10 microns are obtained in a form depending on the type of polymer used.

[0177] Hydrogel particles. Particles made from gel-type polymers such as alginates are produced by conventional ion 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 formation apparatus that uses a stream of nitrogen gas to break up droplets, if applicable. An ion curing bath, slowly agitated (e.g., 100-170 RPM), is placed below the extruder to capture the formed microdroplets. 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 extruders of various sizes or by varying the flow rate of nitrogen gas or polymer solution. Chitosan particles can be prepared by dissolving the polymer in an acidic solution and crosslinking it 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., alginates, CMC), positively charged ligands of different molecular weights (e.g., polylysine, polyethyleneimine) can be ionically bonded.

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

[0179] The delivery vehicle may be silica particles. Suitable silica particles useful for preparing the disclosed nanoparticle 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) may be used to deliver adjuvants and other therapeutic agents to cells. The solubility of silica or silicon in the body provides the sustained release capability of the drug carried by the particles. Furthermore, silica particles or silicon particles can be modified using biodegradable polymers or bio-reducing crosslinking agents to provide sustained release capabilities.

[0180] (VI) Pharmaceutical compositions and dosage forms Compositions for use in the treatment of cancer, precancerous and other proliferative disorders are provided herein. The composition comprises at least two active ingredients / agents, one of which is a therapeutic activator that (1) induces tumor antigen release and / or (2) modulates the immunosuppressive tumor microenvironment, and the other is an adjuvant. As described herein, the activator is conjugated to a delivery / delivery vehicle in a delivery vehicle, such as liposomes, organic (nano or micro) particles, or inorganic (nano or micro) particles.

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

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

[0183] As used herein, the term “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, solvate, or prodrug, such as an ester, of a desired activator that, when administered to a recipient, can provide (directly or indirectly) the desired activator or its active metabolite or residue. Such derivatives are recognizable to those skilled in the art without excessive experimentation. Nevertheless, refer to the teachings in 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 a composition for treatment can be used as is, it may be preferable to administer it in a pharmaceutical formulation by mixing it with a suitable pharmaceutical excipient, diluent, or carrier selected, for example, in relation to the intended route of administration and standard pharmacopoeia. Thus, in one aspect, a pharmaceutical composition or pharmaceutical formulation comprises at least one active composition or a pharmaceutically acceptable derivative thereof, in combination with pharmaceutically acceptable excipients, diluents, and / or carriers. The excipients, diluents, and / or carriers are "acceptable" in the sense that they are compatible with the other components of the formulation and not significantly harmful to its recipient.

[0185] Any compositional formulation disclosed herein may favorably include any other pharmaceutically acceptable carrier, including any carrier that does not produce significantly adverse allergic reactions or other undesirable reactions that outweigh the benefits of administration, whether for research, prophylactic, and / or therapeutic purposes. 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 edit. 2005) and Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, formulations may be prepared to meet the sterility, pyrogenicity, general safety, and purity standards required by the U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory authorities. Pharmaceutical excipients, pharmaceutical diluents, and pharmaceutical carriers may be selected with respect to the intended route of administration and standard pharmaceutical practices.

[0186] Such pharmaceutical formulations may be provided for use in the conventional manner with one or more suitable excipients, diluents, and carriers. Pharmaceutically acceptable excipients include diluents, binders, lubricants, flow enhancers, disintegrants, colorants, and other components that assist or enable the formation of dosage forms for bioactive materials. Preservatives, stabilizers, dyes, and even flavorings may be provided in the pharmaceutical composition. Examples of preservatives include esters of sodium benzoate, ascorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may 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 all kinds of fillers or bulking agents, solvents or co-solvents, dispersions, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption retarders, salts, stabilizers, buffers, chelating agents (e.g., EDTA), gels, binders, disintegrants, and / or lubricants.

[0188] Examples of buffers include citrate buffers, succinate buffers, tartarate 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] Examples of isotonic agents include polyhydric sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0191] Examples of 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] The “therapeutic dose” or “therapeutic amount” means the amount of a compound sufficient to achieve a condition, disorder, or pathology when administered to a subject to treat such a condition, disorder, or pathology. The “therapeutic dose” varies depending on the compound, the disease and its severity, as well as the age, weight, physical condition, and responsiveness of the mammal being treated. The exact dose and formulation depend on the therapeutic purpose and can be determined by those skilled 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, “therapeutic dose” is used to mean the amount or dose sufficient to adjust, for example, 10%, 50%, or 90% of the desired activity, for example, by increasing or decreasing it. Generally, the therapeutically effective dose is sufficient to produce 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 administered to a specific subject may be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, stage of cancer, type of cancer, previous or concurrent therapeutic interventions, the subject's idiopathic disease, and physical, physiological, and psychological factors, including the route of administration.

[0194] The effective dose for this use depends on the severity and location of the disease, as well as the weight and general condition of the patient being treated, especially when metastatic sites are involved. Generally, the dose ranges from 0.01 mg / kg to 100 mg / kg host body weight per day of the immunotherapy construct, with doses of 0.1 mg / kg to 10 mg / kg per day, for example, 3 to 7 mg / kg, being more commonly used. Long-term maintenance doses may be adjusted as needed. However, the dose may vary depending on the patient's requirements, the severity of the condition being treated, and the compound used. For example, the dose may be determined empirically in a particular patient, taking into account the type and stage of cancer diagnosed. In the context of this invention, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response over time. The size of the dose is also determined by 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 dose for a particular situation is within the scope of the practitioner's skill. Generally, treatment is initiated with a relatively low dose, below the optimal dose of the compound. Subsequently, the dosage should be gradually increased until the optimal effect is achieved under the given circumstances. For convenience, if desired, the total daily dose may be divided and administered in installments throughout the day.

[0195] The selected dosage may be influenced by the desired therapeutic effect, route of administration, desired treatment duration, and the specific immunotherapy complex used. Generally, immunotherapy constructs may be administered in doses ranging from 0.001 mg / kg to 100 mg / kg (for example, daily; or two, three, four, five or more times per week; or two, three, four, five or more times per month, as will be further detailed below). The route of administration may also be considered when determining the dosage. For example, in certain embodiments, the immunotherapy construct is administered via an intravenous or intraperitoneal route in doses ranging from 0.01 mg / kg to 100 mg / kg (e.g., daily; or two, three, four, five or more times per week; or two, three, four, five or more times per month, etc.), or via a subcutaneous route (e.g., local injection into the tumor or TME, or local injection adjacent to the tumor or TME) in doses ranging from 0.0001 mg / kg to 1 mg / kg (e.g., daily; or two, three, four, five or more times per week; or two, three, four, five or more times per month, etc.). Further exemplary doses are described below.

[0196] A suitable dose may range from 0.01 mg / kg to 100 mg / kg body weight per day, week, or month. An exemplary dose may include 0.05 mg / kg to 10.0 mg / kg of the active compound (immunotherapy construct) disclosed herein. The total daily dose may be 0.05 mg / kg to 30.0 mg / kg of the drug administered to the subject one to three times a day, including total daily doses of 0.05 to 3.0, 0.1 to 3.0, 0.5 to 3.0, 1.0 to 3.0, 1.5 to 3.0, 2.0 to 3.0, 2.5 to 3.0 and 0.5 to 3.0 mg / kg / day of drug administration forms using oral administration over 60 minutes, intravenous administration, or other methods. In a specific example, the dose may be administered to the subject as 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 containing up to 92-98% wt / v of the compounds disclosed herein, either as a QD or BID.

[0197] Additional useful doses may often be in the range of 0.1–5 μg / kg or 0.5–1 μg / kg. In other examples, doses may 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 may 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 higher.

[0198] The treatment materials of this disclosure may be used in serious disease conditions, i.e., life-threatening or potentially life-threatening situations. In such cases, it may be possible to administer these compositions in substantially excessive amounts, as may be deemed desirable by the treating physician.

[0199] As will be understood by those skilled in the art, a particular dosage is affected by the pharmacokinetics of the active compound. For administration, a therapeutically effective dose (also referred to herein as dose) can be initially estimated based on results obtained from in vitro assays and / or animal model studies. Such information can be used to more accurately determine a useful dose for the subject of interest. Useful preclinical studies include pharmacodynamic analyses, toxicological analyses, and the like.

[0200] The therapeutically effective dose can be achieved by administering single or multiple doses during the course of the treatment regimen (for example, every hour, every two hours, every three hours, every four hours, every six hours, every nine hours, every twelve hours, every eighteen hours, daily, every other day, every three days, every four days, every five days, every six days, weekly, every two weeks, every three weeks, or monthly).

[0201] The effective dose of a compound containing an active agent includes the dose that partially or completely achieves the desired therapeutic, prophylactic, and / or biological effect. The actual effective dose for a particular use depends on the condition being treated and the route of administration. Effective doses for use in humans can be determined from animal models. For example, doses for humans may be formulated to achieve local (e.g., intratumoral) or circulating levels that have been found to be effective in animals.

[0202] The composition can be administered with one or more anesthetics, 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, nalbufine, tramadol, benzocaine, dibucaine, ethyl chloride, lidocaine, and / or phenazopyridine.

[0203] In certain embodiments, including treating or preventing cancer (e.g., cancer metastasis), the compositions disclosed herein may be used in combination with other cancer treatments such as chemotherapeutic agents, radiotherapy, and / or immunotherapy. The compositions described herein may be administered concurrently with or consecutively with another treatment within a selected time frame, for example, within a time frame of 10 minutes, 1 hour, 3 hours, 10 hours, 15 hours, 24 hours, or 48 hours, or within a clinically relevant treatment frame for the complementary treatment.

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

[0205] In some embodiments, the composition is administered systemically in an amount effective for delivery of the composition to target cells, for example, by intravenous or intraperitoneal administration. Other routes include intravenous infusion or mucosal administration.

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

[0207] For example, in certain embodiments, immunotherapy constructs may be administered locally to easily accessible tumors, such as melanoma, head and neck cancer, breast cancer, and lymphoma, or systemically to other cancers, such as lung cancer, liver cancer, pancreatic cancer, prostate cancer, and metastatic cancer.

[0208] Accordingly, the therapeutic compositions described herein may be administered (alone or as part of a combination therapy) by various routes, including any convenient method for use in human or veterinary medicinal products. A therapeutically effective amount of the desired activator can be formulated into a pharmaceutical composition to be delivered parenterally, transmucosally (e.g., orally, nasally, or rectally), or percutaneously. In some embodiments, administration is, for example, intravenous injection, or parenterally via arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intracerebroventricular, and intracranial administration. Administration may be as a bolus, by continuous infusion over a period of time, or by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intra-articular bursal, intrathecal, oral, local, or inhalation routes. In certain embodiments, the immunotherapy constructs provided herein may also be delivered locally to non-cutaneous tumors, for example, using a hepatic artery infusion pump or an implantable system that enables convective-enhanced delivery. In certain embodiments, for example, in embodiments involving the treatment of inflammatory conditions affecting joints, the pharmaceutical composition may be administered directly to the synovial membrane, synovial fluid, or joint capsule, preferably by injection using a syringe. Administration may be topical or systemic. The choice may be influenced by the condition being treated, as well as the activator and composition being administered.

[0209] For injection, the composition can be prepared as an aqueous solution in a buffer solution such as Hanks' solution, Ringer's solution, or physiological saline. The solution may contain compounding agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the composition may be in lyophilized and / or powder form to be prepared using a suitable vehicle, such as sterile pyrogen-free water, before use.

[0210] Compositions containing immunotherapy constructs may be administered in aqueous solution by parenteral injection. Injectable formulations may be in the form of suspensions or emulsions and may optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such injectable compositions may include diluents, e.g., sterile water, various buffer-containing substances (e.g., Tris-HCl, acetate, phosphate), buffered saline of pH and ionic strength, and optionally additives, e.g., 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, e.g., olive oil and corn oil, gelatin, and injectable organic esters, e.g., ethyl oleate. The injectable formulation may be freeze-dried and resuspended, for example, immediately before use. The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0211] In other embodiments, the immunotherapy construct-containing composition is applied topically or by intravenous infusion. Topical administration may include application to the lungs, nose, oral cavity (sublingual, buccal), vagina, or rectal mucosa. These administration methods can be carried out by formulating a shell or coating of the delivery vehicle using a mucosal transport element. When the composition is delivered as either an aerosol or spray-dried particles having an aerodynamic diameter of less than 5 microns, it may be delivered to the lungs while traversing the pulmonary epithelium and moving into the bloodstream during inhalation.

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

[0213] Formulations for administration to mucous 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 prescriber.

[0214] Transdermal formulations may be prepared. These are typically ointments, lotions, sprays, or patches, all of which can be prepared using standard techniques. Transdermal formulations may contain penetration enhancers. In combination with this method, chemical enhancers, as well as physical methods including electroporation and microneedling, may function.

[0215] Microneedles (MNs) are micron-sized needles with a height of 10–2000 μm and a width of 10–50 μm that can penetrate directly through the epidermis to the dermis with minimal or no pain (Hao et al., J Biomed Nanotechnol, 13(12):1581-1597, 2017). Several types of microneedles can be used. In some embodiments, a metal or plastic microneedle roller can be used to physically disrupt the skin surface to facilitate the penetration of the applied topical agent (in this case, an immunotherapy construct). In some embodiments, biodegradable and soluble microneedles can contain the immunotherapy construct. When administered to the skin, the microneedles can dissolve and release the construct deep within the skin layers. In some embodiments, non-biodegradable microneedles may be coated with the immunotherapy construct to deliver the coated construct deep into the skin layers. Microneedles can be fabricated from a wide range of materials, including, but not limited to, polymers, sugars, polysaccharides, peptides, proteins, metals, and inorganic compounds (Ye et al., Adv Drug Deliv Rev, 127:106-118, 2018). Any materials and fabrication methods known in the art for microneedle technology are applicable to enhance the delivery of this immunotherapy construct.

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

[0217] (VII) Exemplary usage: By providing herein an immunotherapy construct comprising at least one adjuvant and at least one therapeutic activator capable of inducing antigen release and / or modulating an immunosuppressive environment (such as a tumor microenvironment), a method for treating and / or preventing hyperproliferative disorders, hyperproliferative disorders, or hyperproliferative conditions, including cancer, cancer symptoms, cancer progression (including from precancerous to cancerous), and cancer metastasis. Specific examples of hyperproliferative disorders, hyperproliferative disorders, or hyperproliferative conditions include cancer. In some embodiments, cancer can suppress the immune system of a subject or individual having cancer. In some embodiments, the immunotherapy constructs provided herein can suppress or reverse cancer-mediated immunosuppression, enabling immune recognition and elimination of malignant tumors.

[0218] As used herein, the term “treatment” or “to treat” refers to any improvement in cancer that occurs in a treated subject compared to an untreated subject. Such improvement may be prevention of cancer progression or worsening (e.g., improvement in progression-free survival). Furthermore, such improvement may also be a reduction or cure of cancer or its associated symptoms (e.g., 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 longer), prevention of cancer recurrence or relapse, reduction of metastasis, or reduction in the number of tumors or lesions). It will be understood that a treatment may not be successful in 100% of the treated subject, but may be successful in some individuals compared to other treatments received by the individual, as determined by a person skilled in the art (e.g., a physician). As used herein, the term “prevent” refers to avoiding the development of cancer or its associated syndromes as used herein. Prevention will be understood to mean avoiding the development of cancer within a future timeframe. The time frame preferably begins at the time of administration of the compound in the sense of the present invention and lasts for at least one month, at least six months, at least nine months, at least one year, at least two years, at least five years, at least ten years, or even for the remainder of the subject's physiological lifespan. It will be understood that prevention may not be successful in 100% of the subjects being treated, but may be successful in some individuals compared to other treatments received by the individual, as determined by those skilled in the art (e.g., physicians). Prevention may also be associated with the recurrence of cancer after remission, as measured, for example, by a reduction in the probability of recurrence within a population. Prevention also refers to the elimination of cancer cells in any part of the body that would otherwise regrow and subsequently exhibit a positive prognosis and disease symptoms.

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

[0220] In mature animals, a balance is usually maintained between cell regeneration and cell death in most organs and tissues. Various types of mature cells in the body have a given 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 the normal proliferation control mechanisms. These cells can grow to considerable size, producing clones of cells that can give rise to tumors or neoplasms. Tumors that cannot grow uncontrollably and do not extensively invade healthy surrounding tissues are benign. Tumors that continue to grow and gradually become 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 detach from the tumor, invade blood vessels or lymphatic vessels, and are carried to other tissues where they continue to grow. Thus, a primary tumor in one site can give rise to secondary tumors in another site.

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

[0222] Malignant tumors can be classified according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors arising from endodermal or ectodermic tissue, such as skin, or the inner lining of the epithelium of viscera and glands. Sarcomas, though less frequently, originate from mesodermal connective tissue, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemia grows as single cells, while lymphoma tends to grow as a tumor mass. Malignant tumors can appear in multiple organs or tissues of the body, establishing cancer.

[0223] The types of cancer that can be treated with the provided compositions and methods include, but are not limited to, vascular cancers such as multiple myeloma, as well as 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 may also be used to treat metastases or tumors at multiple locations.

[0224] Administration is not limited to the treatment of existing tumors, but may also be used to prevent or reduce the risk of an individual developing such a disease, i.e., for prophylactic use, and, for example, to reduce the spread of cancer by metastasis. Potential candidates for prophylactic vaccination include individuals at high risk of developing cancer, i.e., those with a personal or family history of certain types of cancer.

[0225] In one embodiment, an immunotherapy construct comprising the adjuvant CpG, siRNA against BRAFV600E to kill melanoma and progenitor cells (melanocytes in the nevus) and induce antigen release, and siRNA against STAT3 to mitigate the immunosuppressive environment can be used to induce adaptive immunity in subjects at high risk of developing melanoma and in patients with melanoma. The immunotherapy construct not only prevents and treats melanoma but also provides protection from future recurrence or relapse after surgery in patients with late-stage melanoma. Along with the targeting agent, siRNA against other genes may be incorporated on or within the nanoparticles / construct.

[0226] Various modes of treatment, including systemic immunotherapy, chemotherapy, and biochemotherapy, have been tested in adjuvant settings, but they also impose systemic toxicity and side effects that can be overcome by local treatment with immunotherapy constructs described herein. In certain embodiments, breast cancer can be treated in an adjuvant setting by intratumoral injection prior to surgical removal of the primary thoracic tumor using immunotherapy constructs (e.g., containing CpG or another adjuvant along with chemotherapeutic agents, targeted therapies, and / or siRNA against STAT3), 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 with the compounds, pharmaceutical compositions, or methods provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, and 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). This includes cancers such as ovarian cancer, pancreatic cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer, adenocarcinoma, large cell lung cancer, small cell lung cancer, 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 esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include cancers of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovarian, 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 tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, and genitourinary tract cancer. Examples include cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, neoplasms of the endocrine or exocrine parts of the pancreas, medullary thyroid carcinoma, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, papillary Paget's disease, phyllodes tumor, lobular carcinoma, ductal carcinoma, stellate cell carcinoma of the pancreas, stellate cell carcinoma of the hepatic thyroid, or prostate cancer. As used herein, the term "precancerous" 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" broadly refers to a progressive malignant disease of the hematopoietic organs, generally characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemia is generally clinically classified based on (1) the duration and characteristics of the acute or chronic disease; (2) the type of cells involved; myeloid, lymphoid, or monocytic; and (3) an increase or absence of the number of abnormal cells, whether hematological leukemic or aleukemic (subleukemic). Exemplary leukemias that can be treated with the compounds, pharmaceutical compositions, or methods provided herein include, for example, acute non-lymphocytic 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, cutaneous leukemia, embryocellular leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic cell leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, and lymphocytic leukemia. Leukemia includes lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloid leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, or anaplastic cell leukemia.

[0229] The term "sarcoma" generally refers to a tumor composed of a substance such as embryonic connective tissue, and generally consisting of densely packed cells embedded in a fibrous or homogeneous material. Sarcomas that can be treated with the compounds, pharmaceutical compositions or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma, liposarcoma, hydatidiform soft tissue sarcoma, amelosarcoma, rhabdomyosarcoma, and green sarcoma (chloroma). This includes sarcomas, 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, leukemosarcoma, malignant mesenchymal sarcoma, paraosteal osteosarcoma, reticulocytic sarcoma, Rous sarcoma, serous cystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

[0230] The term “melanoma” is interpreted to mean tumors arising from the melanocyte system of the skin and other organs. Melanomas that can be treated with the compounds, pharmaceutical compositions or methods provided herein include, for example, acral lentiginous melanoma, achromosomal malignant melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passé melanoma, juvenile melanoma, lentigo malignant melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0231] The term "carcinoma" refers to a malignant neoplasm composed of epithelial cells that tends to invade surrounding tissues and cause metastasis. Exemplary carcinomas that can be treated with the compounds, pharmaceutical compositions, or methods provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, lobular carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma (carcinoma adenomatosum), adrenal cortical carcinoma, alveolar carcinoma, alveolar epithelial carcinoma, basal cell carcinoma, basocellular carcinoma, basosquamous cell carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic lung carcinoma, cerebriform carcinoma, cholangiocarcinoma, chorionic carcinoma, gelatinous carcinoma, comedone carcinoma, corpus carcinoma, cribriform carcinoma, armory carcinoma, skin carcinoma, and cylindrical carcinoma. Carcinoma, cylindrical cell carcinoma, adenocarcinoma, ductal carcinoma, carcinoma durum, embryonic carcinoma, medullary carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma (carcinoma epitheliale adenoides), exotropic carcinoma, ulcerative carcinoma, fibrous carcinoma (carcinoma fibrosum), gelatinous carcinoma (gelatiniforni carcinoma), colloidal carcinoma, giant cell carcinoma, giant cell carcinoma (carcinoma gigantocellulare), adenocarcinoma (glandular carcinoma), granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Haasle cell carcinoma, hyaline carcinoma, adrenal carcinoma, infant embryonic carcinoma, carcinoma in situ, carcinoma in epidermis, carcinoma in situ carcinoma, Krompecher's carcinoma, Kurticsky cell carcinoma, large cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinomaCarcinoma, lobular carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, black carcinoma, molle carcinoma, mucinous carcinoma, mucinous secretory carcinoma, mucocellular carcinoma, mucoepidermoid carcinoma, mucinous carcinoma, mucinous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossificans carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, pre-invasive carcinoma, squamous cell carcinoma, pultaceous carcinoma, renal cell carcinoma of the kidney, pre-cell carcinoma, sarcomatoid carcinoma This includes sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spheroid cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, nodular carcinoma (carcinoma tuberosum), tubular carcinoma, tubeous carcinoma, verrucous carcinoma, or choriocarcinoma (carcinoma villosum).

[0232] (VIII) kit In particular, an active ingredient comprising at least one described therapeutic construct (containing at least one therapeutic agent and at least one adjuvant, or a delivery vehicle conjugated thereto) may be provided as a kit. The kit may optionally include one or more containers containing (containing) one or more compounds or complexes described herein (e.g., anticancer agents) together with one or more additional agents for use in treatment. For example, some kits may contain a certain amount of at least one additional anticancer 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, but this is not mandatory. A particular kit is expected to contain multiple doses.

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

[0235] Preferred methods, materials, and examples used for carrying out and / or testing aspects of the disclosed invention are described herein. Such methods and materials are illustrative and not intended to limit the scope. Other methods, materials, and examples similar to or equivalent to those described herein may be used.

[0236] The following exemplary embodiments and examples are included to demonstrate specific embodiments of the Disclosure. Those skilled in the art should recognize in light of the Disclosure that many modifications can be made to specific embodiments disclosed herein without departing from the spirit and scope of the Disclosure, and similar or comparable results can still be obtained.

[0237] (IX) Exemplary aspects 1. A delivery system and at least one therapeutic agent, 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, which induces tumor antigen release and / or modulates the immunosuppressive tumor microenvironment. For example, at least one adjuvant compound that is attached to the surface of the delivery system, bound to the delivery system, encapsulated within the delivery system, or contained within the delivery system. An immunotherapy construct including, The immunotherapy construct, which does not contain tumor-specific antigens or ovalbumin. 2. An immunotherapy construct according to embodiment 1, wherein the delivery system comprises liposomes, lipid-based particles, polymer particles, inorganic particles, inorganic particles coated with polymers or lipids, or hybrids thereof. 3. An immunotherapy construct according to embodiment 2, wherein the delivery vehicle is a liposome, a lipid-based particle, a polymer particle, an inorganic particle, or an inorganic particle coated with a polymer or lipid. 4. An immunotherapy construct according to embodiment 3, wherein the delivery vehicle is an inorganic particle comprising one or more of mesoporous silica, gold, aluminum, iron oxide, calcium phosphate, or antioxidant particles. 5. An immunotherapy construct according to embodiment 4, wherein the inorganic particles include antioxidant particles containing cerium oxide. 6. An immunotherapy construct according to embodiment 4, wherein the delivery vehicle comprises mesoporous silica particles. 7. The delivery vehicle is fullerene, endohedral metal fullerene. An immunotherapy construct according to Embodiment 1, comprising one or more of the following: metallofullerene, trimetallic nitride-templated endohedral metallofullerene, single-walled carbon nanotubes and multi-walled carbon nanotubes, branched carbon nanotubes and dendritic carbon nanotubes, gold nanorods, silver nanorods, single-walled boron / nitrate nanotubes 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 silica nanospheres, polymer microspheres and polymer nanospheres, silica shells, biodegradable PLGA microspheres and biodegradable PLGA 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. An immunotherapy construct in any one of embodiments 1 to 7, wherein the delivery vehicle is a polymer particle comprising one or more of PLGA, PLL, polyarginine, PEG, PEI, or chitosan. 9. An immunotherapy construct, any one of embodiments 1 to 8, which is a nanoparticle having a hydrodynamic size of 5 nm to 999 nm. 10. An immunotherapy construct, any one of embodiments 1 to 8, which is a nanoparticle having a hydrodynamic size of 1 micron to 1000 microns. 11. An immunotherapy construct according to embodiment 6, wherein the delivery vehicle comprises mesoporous silica nanoparticles having a size of approximately 5 to approximately 200 nm. 12. An immunotherapy construct according to embodiment 11, wherein mesoporous silica nanoparticles are coated with crosslinked polyethyleneimine and polyethylene glycol. 13. An immunotherapy construct in any one of embodiments 1 to 12, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-Cas9 element), oligonucleotide, polynucleotide, peptide, protein, chemotherapeutic agent, toxin, antioxidant, small molecule inhibitor, antibody, or radiotherapy agent. 14. An immunotherapy construct according to embodiment 13, wherein at least one therapeutic agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, or DNA. 15. An immunotherapy construct according to embodiment 14, wherein at least one therapeutic agent comprises siRNA. 16. An immunotherapy construct according to embodiment 15, wherein at least one therapeutic agent comprises an siRNA that 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, BRAF, BRAF V600E, or MTDH. 17. An immunotherapy construct according to embodiment 15 or 16, wherein at least one therapeutic agent comprises an siRNA that inhibits the expression or activity of STAT3. 18. Any one of embodiments 15 to 17, wherein at least one therapeutic agent comprises an siRNA that inhibits the expression of HER2 activity. 19. An immunotherapy construct in any one of embodiments 1 to 12, wherein at least one 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. 20. An immunotherapy construct in any one of embodiments 1 to 19, wherein the therapeutic agent is an anticancer agent comprising one or more of the following: antibiotics, plant alkaloids, PLK1 inhibitors, mitotic kinase inhibitors, immune checkpoint inhibitors, platinum-based chemotherapeutic agents, HER2 small molecule inhibitors, or HER2-specific antibodies. 21. An immunotherapy construct according to embodiment 20, wherein the therapeutic agent is a checkpoint inhibitor, and the checkpoint inhibitor is an antibody against PD-L1, PD1, or CTLA4. 22. An immunotherapy construct according to embodiment 21, wherein the checkpoint inhibitor is an antibody against PD-L1. 23. An immunotherapy construct of any one of embodiments 1 to 22, wherein at least one therapeutic agent comprises a PLK1 inhibitor. 24. An immunotherapy construct according to embodiment 23, wherein the PLK1 inhibitor is volasertib. 25. An immunotherapy construct in any one of embodiments 1 to 24, wherein at least one therapeutic agent comprises one or more of docetaxel, mitoxantrone, or cabazitaxel. 26. An immunotherapy construct of any one of embodiments 1 to 25, wherein at least one therapeutic agent comprises an anti-EGFR antibody. 27. An immunotherapy construct according to embodiment 26, wherein the anti-EGFR antibody is cetuximab. 28. An immunotherapy construct of any one of embodiments 1 to 25, wherein at least one therapeutic agent comprises an anti-HER2 antibody. 29. An immunotherapy construct according to embodiment 28, wherein the anti-HER2 antibody is trastuzumab. 30. An immunotherapy construct in any one of embodiments 1 to 29, wherein the adjuvant has immunostimulatory activity and comprises one or more of the following: 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 adjuvants or surfactant-based adjuvants, lipopolysaccharides, plant extracts, or derivatives thereof. 31. An immunotherapy construct of any one of embodiments 1 to 30, wherein the adjuvant compound comprises a CpG oligonucleotide, imiquimod, reximod, gardikimod, poly-I:C, poly-ICLC, dSLIM, or EnanDIM. 32. An immunotherapy construct according to any one of embodiments 1 to 31, wherein the adjuvant compound comprises a CpG oligonucleotide. 33. An immunotherapy construct from any one of embodiments 1 to 32, At least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof A composition containing the following: 34. A method for treating cancer, comprising the step of administering to a subject having cancer an effective amount of any one immunotherapy construct of embodiments 1 to 32 or a composition of embodiment 33 in order to reduce one or more symptoms of cancer. 35. The method of embodiment 34, wherein the subject is a mammal. 36. The method of aspect 35, wherein the mammal is a human. 37. A method for treating cells exhibiting symptoms of cancer, The step of bringing the cells into contact with a therapeutically effective amount of any one immunotherapy construct from embodiments 1 to 32 or the composition of embodiment 33. The method, including the method described above. 38. A method for treating cells obtained from a subject exhibiting symptoms of cancer, The step of bringing the cells into contact with a therapeutically effective amount of any one immunotherapy construct from embodiments 1 to 32 or the composition of embodiment 33. The method, including the method described above. 39. A step of ex vivo contacting cells with an effective amount of an immunotherapy construct from any one of embodiments 1 to 32 or the composition of embodiment 33. Methods that include... 40. The method according to aspect 38 or 39, wherein the cells are cancer cells. 41. The method according to aspect 38 or 39, wherein the cells are not cancer cells. 42. The method according to embodiment 41, wherein the cells are immune cells. 43. A method according to aspect 38 or 39, wherein cells are immortalized. 44. Any one of embodiments 37 to 43, further comprising the step of administering at least one treated cell back to the target. 45. A method for treating a subject diagnosed with an overproliferative disease or overproliferative condition, or diagnosed as being at high risk of developing such a disease or condition, A step of administering an effective amount of the composition of embodiment 33 to the subject. The method, including the method described above. 46. ​​The method of aspect 45, wherein the subject is a mammal. 47. The method of aspect 46, wherein the mammal is a human. 48. Any one of the methods described in aspects 45 to 47, wherein the hyperproliferative disorder or hyperproliferative condition includes one or more of cancer, precancerous conditions, or cancerous metastases. 49. The method according to aspects 45-48, wherein the hyperproliferative disease includes 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. 50. The administration process is, Direct injection into tumors in the target population. Systemic injection in the subject, or Local application to the target One of the methods described in embodiments 45 to 49, which includes one or more of the above. 51. Any one of embodiments 45 to 50, wherein the administration step includes the application of microneedles to the target. 52. A method for enhancing the effect of anticancer therapy in a subject that requires it, An effective amount of any one immunotherapy construct from embodiments 1 to 32 or the composition of embodiment 33, At least one anticancer drug and The process of administering it to the target that needs it. The method, including the method described above. 53. The method according to embodiment 52, wherein the anticancer agent is a chemotherapeutic agent or a targeted therapy agent. 54. A method for enhancing the effect of checkpoint block immunotherapy in subjects diagnosed with neoplasms, An effective amount of any one immunotherapy construct from embodiments 1 to 32 or the composition of embodiment 33, At least one immune checkpoint inhibitor and The process of administering it to the target that needs it. The method, including the method described above. 55. A method for enhancing the effect of radiotherapy in a subject diagnosed with a neoplasm, An effective amount of any one immunotherapy construct from embodiments 1 to 32 or the composition of embodiment 33, At least one radiation therapy and The process of administering it to the target that needs it. The method, including the method described above. 56. Any one of embodiments 52 to 55, wherein an immunotherapy construct or composition and an anticancer therapy are administered sequentially or simultaneously. 57. Any one of the methods described in embodiments 52 to 56, wherein the subject is a mammal. 58. The method of aspect 57, wherein the mammal is a human. 59. An immunotherapy agent from any one of the embodiments 1 to 32, Anticancer drugs and A kit that includes this. 60. A kit according to embodiment 59, wherein the anticancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor. [Examples]

[0238] (X) Example Example 1: Use of nanotechnology to manipulate tumors to serve as depots for cancer vaccines that prime systemic antitumor immunity. Immune checkpoint inhibitors (ICIs), such as those targeting PD-L1 / PD-1 and CTLA-4, have shown excellent results in clinical settings (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 immune memory, and result in a long-lasting immune response even after treatment has stopped. Therefore, in advanced cancer, ICIs can produce a more permanent response than chemotherapy and targeted therapy, as shown in Figure 1. However, this treatment is effective for only a portion of patients (approximately 10-40%) (Ribas, Update Cancer Therapeutics, 2(3):133-139, 2007; Topalian et al., N Engl J Med, 366(26):p.2443-54, 2012). The lack of response is typically due to a lack of pre-existing antitumor immunity (e.g., effector (CD8+) T cells against the tumor) (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 are locally killed in the presence of immune stimulation, releasing tumor antigens, which 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 avoids the need to pre-identify tumor antigens in conventional cancer vaccine development. It is also a personalized therapy because a unique set of tumor antigens is released, priming specific immunity for each patient.

[0240] The cancer immunotherapy approaches provided herein utilize the patient's own tumor as a depot (in-situ tumor vaccination) of 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 in combination 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.) (e.g., docetaxel, siRNA against STAT3) that trigger antigen release and / or modulate the immunosuppressive tumor microenvironment. When a tumor site is treated with the provided immunotherapy constructs (e.g., via local intratumor injection or via tumor homing by systemic delivery), tumor antigens are released in the presence of immune stimulation (provided by the adjuvant), and adaptive immunization is initiated. Simultaneously, compounds that modulate the immunosuppressive tumor microenvironment can be delivered onto the same nanoparticles to maximize the effect of in-situ tumor vaccination. Tumor antigens can be taken up by AIRISE-activated antigen-presenting cells (APCs) that 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 within the tumor site) and proliferate throughout the body. Effector T cells specifically home to tumors that share the same tumor antigens (e.g., both locally treated tumors and untreated metastatic tumors elsewhere in the body), regardless of their location in the body. Death of cancer cells by cytotoxic T cells releases even more tumor antigens, amplifying the process of anti-tumor T cell generation in a positive feedback loop. In particular, even if the treatment is local (e.g., intratumoral injection), locally induced vaccination effects at the tumor site produce a systemic, long-lasting anti-tumor immune response (Figure 2).For example, AIRISE can be injected directly into melanoma lesions, and the treatment can affect both the injected tumor and untreated metastatic melanoma tumors within the lung or liver.

[0241] These antitumor T cells, trained to recognize specific tumor antigens, control tumors both at the injection / treatment site and elsewhere in the body (Figure 2). The cargo combination can be applied to any type of micro / nanoparticle to construct AIRISE. The following examples utilize established mesoporous silica nanoparticles (US Patent Application Publication 2017 / 0172923) as proof of concept. In another example shown in Figure 11, cationic lipid particles with a hydrodynamic size of 1.1 microns were used for in-situ vaccination with CpG and siSTAT3, yielding results similar to Figure 7, suggesting that various types and sizes of particles can be used.

[0242] Methods and materials Synthesis and Characterization of Nanoparticles: As previously described, mesoporous silica nanoparticles were synthesized (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015; Ngamcherdtrakul et al., International J Nanomed, 13:4015-4027, 2018). In summary, mesoporous silica nanoparticles (MSNPs) were synthesized by sol-gel synthesis. The MSNP cores were coated layer by layer with polyethyleneimine (PEI) and polyethylene glycol (PEG). Furthermore, as previously described, the PEI on the MSNPs was crosslinked to enhance the efficacy and safety of oligonucleotide delivery (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015). The crosslinked PEI and PEG-coated MSNPs will hereafter be referred to as "NP" 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 an aqueous solution (e.g., PBS) at room temperature for 4 hours. Loading was performed in a complete binding mode, as confirmed by the absence of free cargo molecules in the supernatant when separating the cargo-loaded NPs by centrifugation. Cargo content was measured by spectrophotometry. siRNA was conjugated with the Dy677 dye (Dharmacon) and quantified by fluorescence signal. CpG was measured using Nanodrop Spectrophotometry. Unbound siRNA and CpG could also be measured by gel electrophoresis. Mitoxantrone (MTX) was quantified by absorbance measurement at 658 nm. Zetasizer was used to characterize the final NPs, including CpGs and siRNAs, in PBS based on their hydrodynamic size (Figures 26 and 29).

[0244] Docetaxel was overloaded onto the nanoparticles before PEI binding. In summary, MSNPs were mixed overnight with an ethanol solution of docetaxel before PEI binding. Unbound docetaxel and PEI were washed in PBS. Then, PEI-NPs (DTX) were conjugated with PEG according to a previous method (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 Mouse Melanoma Tumor Model: Six-week-old female C57BL / 6 mice were obtained from the Charles River NCI colony (Wilmington, MA). B16F10 cells were intradermally transplanted into the left shoulder (local, 250,000 cells) and right shoulder (distal, 100,000 cells) of each mouse. On day 8 post-transplantation, the test compound / construct was injected intratumorally into the left (local) tumor only, leaving the right (distal) tumor untreated. Unless otherwise specified, the test compound / construct was administered three times every three days. The load of both the local and distal tumors in the mice was measured every 1-2 days using a Vernier Caliper, with V = 0.5 × length × width. 2 Tumor volume was calculated using [method / tool]. Survival was also monitored. Total tumor load was 2000 mm. 3 When the number of mice exceeded a certain limit, they were euthanized.

[0246] For a study 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 on the same day as intratumoral treatment with the NP compound (3 doses every 3 days).

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

[0248] For immunoprofiling, local and distal tumors, as well as their respective draining lymph nodes, were collected and treated as single cells according to established protocols in the art. The collected cells were then stained with a set of fluorescently labeled antibodies 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) to identify various immune cell populations and their states together. Staining for specific intracellular proteins (e.g., Ki67, FoxP3, STAT3) is also possible according to the manufacturer's protocol (BD Biosciences). Typically, flow cytometry was performed against two separate antibody panels (lymphoid and myeloid) using a BD Fortessa (four lasers, up to 18 parameters). To ensure the robustness of the multicolor flow cytometry analysis, fluorescence compensation was performed according to established protocols known in the art. For the AIRISE uptake test in the tumor microenvironment (Figure 18), mice carrying bilateral B16F10 tumors were injected intratumorally with AIRISE-02 (loaded with Alexa488 dye conjugate siSCR instead of siSTAT3) as described above. Two hours after treatment, tumors (treated and untreated) were harvested, treated into single cells, and surface-stained using a panel of antibodies described herein. Flow cytometry was used to analyze the presence of AIRISE-02 in various cell populations within the tumors.

[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, the mice were randomly divided into groups and treated a total of four times with the test compound / construct every three days. In this model, AIRISE-02 was injected intravenously via the tail vein, not into the tumor.

[0250] Bilateral ectopic tumor model of CT26. 250K and 100K CT26 (mouse colorectal cancer) cells were transplanted into both flanks of each mouse (Balb / c). Fifteen days after tumor transplantation, the mice were treated with the test compound / construct.

[0251] 4T1 bilateral orthotopic tumor model. 100K and 40K 4T1 cells were transplanted into the bilateral mammary fat pads of each mouse (Balb / c). Eight or 11 days after tumor transplantation (as specified), mice were treated with the test compound / construct.

[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 a nanoparticle into one foot. 24 hours after injection, local draining lymph nodes (DLNs) and non-draining lymph nodes (NDLNs) were collected and analyzed for CD11c, MHCII, and CD80 expression by flow cytometry. CpG-loaded nanoparticles activated dendritic cells in local DLNs significantly better than free CpG (Figure 3). CpG nanoparticles also increased the potential for co-delivering several therapeutic cargoes to the same site. Furthermore, exemplary nanoparticles were highly optimized for the delivery of siRNAs capable of modulating tumor immunosuppressive properties at the mRNA level. To benefitfully prime the immunotherapy effect, co-delivery of CpG (or another adjuvant) with siRNAs or other target molecules addressing several characteristics of tumor immunosuppression has been proposed.

[0253] Adjuvant-loaded nanoparticles induce in-situ tumor vaccination. To evaluate the ability of CpG-NPs to induce in-situ tumor vaccination, mice carrying two (bilateral) melanoma tumors were used. The treatment was injected intratumorally into only one of the tumors, while the other tumor was left untreated. Growth of both tumors was monitored. Intratumoral treatment with CpG-NPs successfully primed the systemic immune response and induced a potent abscopal effect (inhibition of both the locally treated tumor and the distally untreated tumor) (Figure 4). As a result, survival was also extended. The immunotherapy construct techniques described herein do not require loading tumor antigens onto or within either of the conjugates to induce in-situ vaccination.

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

[0255] Surprisingly, simultaneous delivery of the chemotherapeutic agents docetaxel (DTX) and CpG (CpG-DTX-NP) on the same nanoparticles did not impair the in-situ vaccination efficacy of CpG-NP, despite the potential chemotherapeutic toxicity to immune cells (Figure 5). In fact, CpG-DTX-NP can control locally treated tumors better than CpG-NP. At the same time, CpG-DTX-NP still induces a slightly better in-situ vaccination efficacy than CpG-NP, as demonstrated by distal tumor control and long-term survival of mice. Furthermore, DTX-NP does not exhibit significant activity.

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

[0257] Immunotherapy analysis shows a significant increase in activated CD8+ T cells in tumors and lymph nodes. Post-treatment (Figure 5A), T cell status in local and distal tumors was characterized 7 days after the initial administration. Compared to saline, treatment with CpG-DTX-NP (abbreviated as "NP" in Figure 6) resulted in the following desirable characteristics of cytotoxic CD8+ T cells: relatively low PD1 expression in treated and distal tumors (Figure 6A), relatively high proliferation of CD8+ T cells in treated tumors (Figure 6B), relatively low depletion of CD8+ T cells in treated tumors (Figure 6C), and a higher ratio of CD8+ T cells to regulatory T cells (Figure 6D). Similarly, more CD8+ T cells were observed in the draining lymph nodes (DLNs) of treated tumors (Figure 6E), and these CD8+ T cells were more active (Figure 6F). In the DLNs of both tumors, activated CD8+ T cells were more proliferative (according to the Ki67 marker) (Figure 6G). The observation of relatively proliferative and active CD8+ T cells in non-DLN areas (Figure 6H) suggests that T cells were transported outside the local lymph node (e.g., into the bloodstream) and contributed to the abscopal effect of the treatment. These T cell properties indicate that the nanoparticles described herein can increase the beneficial antitumor T cell repertoire (non-wasted state) and induce systemic immunity. DTX on our NPs killed tumor cells and released tumor antigens, but did not harm CD8+ T cells; rather, it increased their proliferation.

[0258] Co-delivery of siRNA and CpG on the same NP can induce effective in-situ tumor vaccination. This was the first use of a single nanoparticle (NP) for co-delivery of an adjuvant and siRNA via intratumor injection. While delivery of immunogenic chemotherapeutic agents, tumor antigens, or adjuvants via nanoparticles has been performed previously, siRNA had never been co-delivered with an adjuvant on a nanoparticle.

[0259] Co-delivery of CpG oligonucleotides and siSTAT3 on NPs into tumors induced a systemic antitumor immune response in a melanoma mouse model. siSTAT3 (2 wt%) was loaded into the mesoporous silica core of the NP, and CpG oligo (10 wt%) was loaded onto the outer surface (bound to a cationic polymer layer but protected from enzymatic degradation under PEG). It has been proposed that tumor antigens (already present in the tumor or released by cancer death at the time of treatment) prime antitumor immunity in the presence of CpG-mediated immune stimulation. siSTAT3 modulates an immunosuppressive tumor environment and amplifies the immunotherapy response. To demonstrate this, siSTAT3-CpG-NP (AIRISE-02) was evaluated in a bilateral B16F10 melanoma tumor model in mice (Figure 7A). Eight days after tumor transplantation, AIRISE-02 was injected into one of the tumors (locally) a total of three times, every three days. AIRISE-02 significantly improved mouse survival (Figure 7D), reduced both local tumors (treated, Figure 7B) and distal tumors (untreated, Figure 7C), suggesting successful in-situ tumor vaccination and an abscopal effect of the treatment (effect beyond the treatment / injection site). Figures 7B–7C also show that siSTAT3-CpG-NP is superior to CpG-NP and siSTAT3-NP.

[0260] In another experiment, mice were treated with AIRISE-02 (siSTAT3-CpG-NP) using the same method as in Figure 7. Mice were euthanized one day after the third dose (or seven days after the first dose). Tumors and associated dissipating lymph nodes (DLNs) were collected and subjected to immunoprofiling by multicolor flow cytometry. Figure 17 shows that AIRISE-02 resulted in a significantly higher CD8 / Treg ratio in both localized tumors (treated) and distal tumors (untreated) as well as associated DLNs (p<0.05 for AIRISE-02 vs. saline), confirming the success of in-situ tumor vaccination. In patient tumors, regulatory T cells (Tregs) are typically elevated, suppressing the anti-tumor immune response, including CD8+ T cell activity. Therefore, an even higher intratumoral CD8 / Treg ratio is desirable and is one indicator of long-term survival in cancer patients. Consistent with the lower effect of AIRISE-02 in Figure 7, CpG-NP did not significantly increase CD8 / Treg cells either intratumor or in lymph nodes at this point. Furthermore, in AIRISE-02-treated mice, effector CD8+ T cells in 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-NP (AIRISE-02) (similar model to Figure 7). The siRNA was tagged using Alexa-488. Figure 18 shows that siRNA-CpG-NP was taken up by 15% of cells in the TME two hours after intratumoral injection, compared to a previous study which reported that CpG-siSTAT3 conjugates were taken up by only 2% of cells in the TME one and three hours after intratumoral injection (Kortylewski et al., Nature biotechnology, 27(10):925-932, 2009). Following our finding that the TME consists of 80-90% cancer cells, of the cells that took up siRNA-CpG-NP, 80% were cancer cells (CD45-) and 20% were immune cells (CD45+). In contrast, the aforementioned CpG-siSTAT3 conjugate is CpG-dependent in its treatment and is primarily taken up only by TLR9+ cells, not TLR9- cancer cells. Delivery of siSTAT3 and CpG to both cancer cells and immune cells is preferable. Among immune cells, myeloid cells (CD45+CD3-CD19-) took up the most siSTAT3-CpG-NPs. These included macrophages (F4 / 80+) and DCs (CD11c+MHCII+). NPs were not detected in distal untreated tumors (not shown), and no exfiltration of NPs into untreated tumors was observed.

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

[0263] Furthermore, since NP treatment generates a relatively large CD8+ T cell repertoire, it has been suggested that this treatment can be beneficially combined with checkpoint inhibitor treatment to enhance the anticancer effect. This was tested by using siSTAT3-CpG-NP (AIRISE-02, Figure 9A) together with two checkpoint inhibitors currently used in clinical practice (anti-PD-1 antibody and anti-CTLA4 antibody). NP treatment (intratumor to only one of the two tumors) had a similar effect on survival as the checkpoint inhibitor cocktail (intraperitoneal administration) (Figure 9D). The combination of AIRISE-02 and the checkpoint inhibitor cocktail substantially improved the effect on controlling local tumors (Figure 9B) and distal tumors (Figure 9C), as well as mouse survival rate (Figure 9D). Notably, complete cure was achieved in 5 out of 8 mice (tumor-free for the past 10 months), whereas mice were not cured with AIRISE-02 or ICI alone (Figure 10C). The curative effect on the B16F10 model is considered superior, as this model is known in the literature to be highly malignant, and previous CpG-based vaccines + ICI have not shown a curative effect when treatment was initiated one week after tumor transplantation (although a curative effect may be reported in prophylactic situations). In another group of cured mice, tumor reloading was performed by transplanting B16F10 cells three months after the last treatment. Cancer growth was rejected in the cured mice, suggesting the success of the long-term sustained antitumor effect (memory effect) of AIRISE + ICI.

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

[0265] Simultaneous intratumoral delivery of CpG oligos and siSTAT3 via cationic lipids also induces in situ tumor vaccination. siSTAT3 and CpG were mixed with a cationic lipid (Dharmafect from Dharmacon) to form lipid nanoparticles, which were administered to mice in the same manner as in Figure 7. Figures 11A–11C show that a response very similar to that observed with mesoporous silica nanoparticles (Figure 7) was achieved using cationic lipids. This indicates that therapeutic agents can be prepared using various types of nanoparticles and the cargo combinations described herein for intratumor injection.

[0266] NPs can deliver siRNA along with CpG to both cancer cells and immune cells, resulting in knockdown of target genes. As shown in Figure 12A for B16F10 cancer cells, Figure 12B for J774 macrophages, and Figure 12C for primary mouse DCs, NPs can deliver siRNA to both cancer cells and immune cells and knock 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 not altered compared to the untreated control (Figure 34) and STAT3 mRNA was normalized by housekeeping mRNA. While not bound by any explanation, it has been suggested 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 from Horizon Discovery based on cationic lipids (non-antioxidant)) was found to increase STAT3 expression in DCs (Figure 35), potentially leading to undesirable immunosuppressive TME. This suggests that the use of the antioxidant mesoporous silica nanoparticle platform described by Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015, may be more advantageous than lipid nanoparticles for controlling the STAT3-mediated pathway. Figure 19 also shows that STAT3 is conserved to such an extent that the same siSTAT3 sequence can knock down STAT3 in canine, mouse, and human cells, facilitating direct conversion from mouse 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 the patient for therapeutic effect and immune response (e.g., killing cancer cells). The cells may originate from the patient being treated or from a different healthy donor (e.g., stem cells and their derivatives (Senju et al., Int J Hematol, 91(3):392-400, 2010)). To produce a whole-cell cancer vaccine, cancer cells may be treated ex vivo (with or without additional agents) using our immunotherapy constructs (Keenan et al., Int J Hematol, 91(3):392-400, 2010; Goldstein et al., Int J Hematol, 117:118-127, 2011).

[0268] Furthermore, AIRISE-02 (siSTAT3-CpG-NP) was found to be effective in other tumor models, including colon cancer (Figure 20) and breast cancer (Figures 21 and 22). In particular, the inventors show that in these two models, combining ICI (systemic intracellular therapy) with AIRISE administered locally to one of the two tumors in each mouse yields better results than either AIRISE alone 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). Compared to treatment with physiological saline, there were no changes in body weight or serum biomarkers of renal and hepatic function when mice were treated with AIRISE-02 (Figure 24). AIRISE-02 was also tested in monkeys and found to be safe. Specifically, AIRISE-02 was subcutaneously administered to cynomolgus monkeys in a dose-escalation pattern every week (1, 2.8, and 9.5 mg / kg, n=3 animals / group, Figure 25). All three animals survived until the end of the planned study. There were no drug-related effects on clinical findings. Regarding skin findings, no drug-related skin findings were observed at low doses. At medium doses, test-related edema (grade 2–3) was observed at 48 and / or 72 hours post-administration but resolved after 7 days. At high doses, test-related edema (grade 1–3) and erythema (grade 1) were observed in all animals, starting as early as 24 hours post-administration and continuing for 7 days (last observation), but were not of concern. There were no test-related effects on body weight. There were no test-related effects on hematological parameters. There were no test-related effects on coagulation parameters. There were no test-related effects on clinical chemistry parameters.

[0270] Example 2: It is also possible to load two types of siRNA simultaneously onto an immunotherapy construct (such as NP) without losing efficacy. The immunotherapy constructs described herein can also deliver multiple siRNAs simultaneously, in addition to the simultaneous delivery of siRNA and CpG. For example, Figure 13 shows that when two individual siRNAs against HER2 or STAT3 were loaded onto an NP (essentially prepared as described in Example 1), specific knockdown of each protein was achieved. Figure 13 also shows that when both siRNAs were loaded into the same NP vial (see T-siHER2 / siSTAT3-NP), similar knockdown of the two proteins was achieved as when they were 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 against a melanoma model.

[0271] This demonstrates the versatility of the immunotherapy construct particles described herein when loading multiple types of oligonucleotides without loss of efficacy. The nanoparticle delivery described herein is also beneficial because it allows for the loading of multiple siRNAs, each capable of killing cancer cells and / or modulating 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 any combination of these features, such as exerting a cytotoxic effect on cancer cells, disrupting the immunosuppressive tumor microenvironment, containing immunostimulatory sequences, or otherwise.

[0273] Example 3: Cell type-specific / targeted immunotherapy constructs siRNA has great potential because it can precisely and effectively modulate any gene. Immunotherapy constructs (AIRISEs) can address different immunosuppressive pathways or different immune cell populations by utilizing antibodies (or other targeting agents) on our nanoparticles for specific delivery. Immunotherapy constructs comprising NPs provided herein can be conjugated with targeting agents for targeted delivery to specific cell populations, e.g., specific cells within a tumor. 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 (anti-EGFR antibody) show preferential uptake by EGFR+ cells rather than low-EGFR cells (Figures 14A–14B). Similarly, NPs conjugated with trastuzumab (anti-HER2 antibody) show preferential uptake by HER2+ cells rather than low-HER2 cells (Figure 14C) (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015).

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

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

[0277] method Before PEI binding, borasertib (a PLK1 inhibitor; iPLK1) was overloaded onto the nanoparticles. In summary, MSNPs were mixed overnight with an ethanol / DMSO solution of borasertib before PEI binding. Unbound docetaxel or borasertib and PEI were washed off in PBS. Then, PEI-NPs (iPLK1) were conjugated with PEG according to a previously published method (Ngamcherdtrakul et al., Advanced Functional Materials, 25(18):2646-2659, 2015; Ngamcherdtrakul et al., International J Nanomed, 13:4015-4027, 2018). The final product contained 0.5–2 wt% iPLK1. They had a DLS size of 100 nm. For the constructs (p-NPs) containing PD-L1 antibody, the PD-L1 antibody was thiolated and conjugated to the ends of the PEG layer on the nanoparticles according to a method previously published by the inventors (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). LLC-JSP cells were subcutaneously injected into the left flank (local, 100,000 cells) and right flank (distal, 40,000 cells) of each mouse. On post-transplant day 12, the test compound / construct was injected intratumorally into the left (local) tumor only, leaving the right (distal) tumor untreated. Unless otherwise specified, the test compound / construct was administered three times every three days. The load of both the local and distal tumors in the mice was measured every 1-2 days using a Vernier Caliper, with V = 0.5 × length × width. 2 Tumor volume was calculated using [method / tool]. Survival was also monitored. Total tumor load was 2000 mm. 3When the number of mice exceeded a certain limit, they were euthanized.

[0279] result Twelve days after tumor inoculation, left (local) tumors in mice were treated intratumorally 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) (distal tumors remained untreated). 0.5 mg of NPs (iPLK1 2.5 μg, PD-L1 antibody 20 μg, CpG 20 μg) in 50 μl was administered every three days for a total of three doses. The immunotherapy constructs extended the survival time of mice more than the same immunotherapy construct without CpG (Figure 15). The immunotherapy constructs were also far more effective than free PD-L1 antibody and volacertib administered at five times the concentration of those on the nanoparticles. Survival rates were further improved by incorporating an adjuvant (CpG) onto the same NP. For example, the inventors found that incorporating CpG onto p-iPLK1-NP (called p-iPLK1-NP-CpG) significantly improved the survival rate of 2 out of 7 mice, and one mouse was completely tumor-free.

[0280] Example 5: Application of topical formulation and AIRISE The immunotherapy constructs disclosed herein can be formulated into topical formulations. Several vehicles known in the art, such as Aquaphor (ointment type) and Carbopol (gel type), can be mixed with the constructs. Heat or a surfactant (e.g., polysorbate 80 (Tween 80) as an emulsifier) ​​can be used to enable better mixing of the vehicle with the aqueous suspension of AIRISE. As an example, 10% by weight of Tween-80 was confirmed not to cause premature leakage of siRNA from nanoparticles. It was also shown that 2.5% by weight of Tween-80 was sufficient to enhance the mixing of siRNA-NPs and Aquaphor when the mixture was heated to 55°C.

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

[0282] Figure 30 shows that the microneedle roller enhances the penetration of the siRNA nanoparticle construct when tested on porcine skin, which is similar in thickness to human skin. Pig skin was incubated with the formulation (Aquaphor solution of Dy677-siSCR-NP) 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. A significant enhancement of skin penetration with the microneedle roller was observed. When the Aquaphor solution of siRNA-NP was administered without the roller, the siRNA signal (arrow) was limited to the outer surface of the pig skin, whereas we observed the siRNA signal (arrow) extending beyond the epidermis to the dermis before the application of the microneedle roller (Figure 30).

[0283] Figure 31 shows that the microneedle roller enhances the local delivery of siRNA-NPs. First, mice were shaved one day before treatment. Dy677-siSCR-NP (0.72 nmol siRNA) was mixed with 100 μL of 2.5% Tween-Aquaphor (per application). Immediately before treatment, a dermal microroller was applied to only one side of the dorsal region in four directions, while the other side was not pretreated. For comparison, the mixture was applied to the shaved area (approximately 2 cm²) with and without microneedle pretreatment. 2 The treatment was applied to the specified application area. After 1.5 hours of processing time, the treated skin samples were collected and processed for imaging.

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

[0285] Microneedle morphology of AIRISE-02. As shown in Figure 33, for microneedle manufacturing, the use of soluble microneedles based on dextran, amylopectin, PVP, PEG, methylcellulose, chitosan, or other polymers or compounds known in the art was investigated. The use of these soluble microneedles enables painless home treatment and high needle density (100 needles / 1cm²). 2 This is highly effective in delivering AIRISE-02 due to the following. As an example (Figure 33), a dextran solution (300 mg / ml aqueous solution) containing NPs loaded with Dy677 conjugate siRNA was cast onto a microneedle mold. The solution was centrifuged or vacuumed to densely fill the mold. The microneedles were dried by air, desiccator, vacuum oven, refrigerator, or a combination thereof and removed from the mold. The needle height was varied from 300 to 800 microns depending on the mold and optimization. siRNA-NPs were successfully loaded onto these needle arrays (at approximately 0.5 nmol siRNA / array), and the needles dissolved completely within 5 minutes after application to porcine skin. Different dissolution times can be manipulated by changing the composition of the microneedles. Different shapes and forms of microneedle patches can also be manufactured using different molds.

[0286] Example 6: By using different nanoparticle materials, the disclosed cargo combinations can be delivered to 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. It was found that CpG and siSTAT3 delivered together with the cationic lipid particles also produced an in-situ tumor vaccination / immunostimulatory effect (Figure 11). Similar results were obtained using jetPEI (a commercially available PEI-based transfection agent that has reached the clinical stage) as the delivery system. This demonstrates the versatility of the disclosed cargo combination and its platform-independent nature. However, while lipid platforms are generally effective in delivering siRNA to cancer cells, the fact that layer-by-layer functionalized mesoporous silica nanoparticles (as described herein) exhibit better siRNA knockdown activity than lipid counterparts in immune cells (e.g., primary dendritic cells) is not of value (Figure 12).

[0287] As will be understood by those skilled in the art, each embodiment disclosed herein includes, is essentially, or may include its particular described element, process, component, or constituent. Therefore, the terms “include” or “including” should be interpreted as enumerating “comprise,” “consist of,” or “consist essentially of.” The transitional terms “comprise” or “comprises” mean including, and enabling, an unspecified number of elements, processes, components, or constituents. The transitional phrase “consisting of” excludes any elements, processes, components, or constituents not specified. The transitional phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, processes, components, or constituents, and those that do not substantially affect the embodiment. A significant effect, in this context, is a measurable reduction in the biological effects of an immunotherapy construct (such as anti-cancer effects).

[0288] Unless otherwise specified, all figures used in this specification and the claims, such as quantities of components, molecular weights and other properties, and reaction conditions, should be understood to be modified in all cases by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties 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 claims, each numerical parameter should be interpreted in light of the number of significant figures reported and by applying common rounding techniques. If further clarification is needed, the term “about” has a meaning reasonably given to a person skilled in the art when used in conjunction with a stated number or range, namely, that it is somewhat greater or somewhat less than the stated value or range, and within the range of ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the stated value.

[0289] Even though the numerical ranges and parameters representing the broad scope of the present invention are approximations, the numerical values ​​shown in specific embodiments are reported with a feasible degree of accuracy. However, any numerical value inherently includes certain errors that inevitably result from the standard deviation found in each test measurement.

[0290] In the context describing the present invention (particularly in the context of the appended claims), the terms “a,” “an,” “the,” and similar references should be interpreted as encompassing both singular and plural forms unless otherwise specified herein or unless clearly inconsistent with the context. The enumeration of value ranges herein is intended merely as a way of referring individually to each distinct value that falls within that range. Unless otherwise specified herein, each individual value is incorporated herein as if it were described individually. All methods described herein can be performed in any preferred order unless otherwise specified herein or unless clearly inconsistent with the context. The use of any and all examples or exemplary language provided herein (e.g., “etc.”) is intended merely to further illustrate the present invention and does not impose any limitation on the scope of the invention as otherwise claimed. Nothing in this specification should be interpreted as indicating any unclaimed element essential to the practice of the invention.

[0291] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Each group component may be referenced and claimed individually or in any combination with other components of the group or other elements found herein. For convenience and / or patentability reasons, it is anticipated that one or more components of a group may be included in or removed from a group. In the event of any such inclusion or removal, this specification shall be deemed to include the modified groups and thus satisfy the described description of all Markush groups used in the appended claims.

[0292] Certain aspects of the Invention are described herein, including the best mode known to the inventors for carrying out the Invention. Needless to say, modifications of these described aspects will be apparent to those skilled in the art by reading the foregoing description. The inventors anticipate that those skilled in the art will appropriately use such modifications, and they intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, to the extent permitted by applicable law. Furthermore, any combination of the elements described above in all possible modifications thereof is incorporated herein unless otherwise specified herein or unless it is clearly inconsistent with the context.

[0293] Furthermore, numerous patents, printed publications, scholarly articles, and other documents (materials referenced herein) are referenced throughout this specification. Each of these referenced materials is incorporated into this specification in its entirety by reference with respect to the teachings it references.

[0294] It should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be used are within the scope of the invention. For this reason, alternative configurations of the present invention may be used, without limitation, as examples, in accordance with the teachings herein. Accordingly, the present invention is not limited to those shown and described herein.

[0295] The details provided herein are illustrative and are intended only to illustrate preferred embodiments of the invention and are presented to provide what is considered to be the most useful and readily understandable explanation of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt has been made to provide structural details of the invention in more detail than is necessary for a basic understanding of the invention, and the description using drawings and / or embodiments will make it clear to those skilled in the art how some embodiments of the invention can actually be embodied.

[0296] The definitions and descriptions used in this disclosure are intended to control any future configurations unless expressly and uniquely altered in the examples, or unless the application of the meaning makes any configuration meaningless or essentially meaningless. Definitions should be interpreted in accordance with 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), where the construction of the term makes it meaningless or essentially meaningless.

Claims

1. 1) Delivery vehicle including the following: Silica, gold, or iron oxide nanoparticles; At least 10% by weight of polyethyleneimine (PEI) electrostatically coating the outer surface of the nanoparticles; and PEI or polyethylene glycol (PEG) bonded to nanoparticles, 2) At least one anticancer agent, which is a nucleic acid or small molecule inhibitor or chemotherapeutic agent non-covalently loaded onto the PEI coating of nanoparticles, or an antibody covalently bound to the PEI coating of nanoparticles, and 3) At least one nucleic acid adjuvant non-covalently attached to PEI and / or PEG on a delivery vehicle, which is not conjugated to an anticancer drug. An immunotherapy construct including, It does not contain tumor-specific antigens or ovalbumin, and Having a hydrodynamic size of less than 500 nm, The aforementioned immunotherapy construct.

2. The immunotherapy construct according to claim 1, wherein PEI is crosslinked.

3. The immunotherapy construct according to claim 1 or 2, wherein the nanoparticles are mesoporous silica nanoparticles.

4. The immunotherapy construct according to claim 1 or 2, wherein the delivery vehicle comprises mesoporous silica nanoparticles having a size of about 5 to about 200 nm.

5. An immunotherapy construct according to any one of claims 1 to 4, wherein at least one anticancer agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-Cas9 element), oligonucleotide, polynucleotide, peptide, protein, chemotherapeutic agent, small molecule inhibitor, or antibody.

6. The immunotherapy construct according to claim 5, wherein at least one anticancer agent comprises siRNA, miRNA, antisense oligonucleotide, mRNA, or DNA.

7. The immunotherapy construct according to claim 6, wherein at least one anticancer agent comprises siRNA.

8. An immunotherapy construct according to claim 7, wherein at least one anticancer agent comprises an siRNA that 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, AR, MYC, BRAF, BRAF V600E, or MTDH.

9. An immunotherapy construct according to claim 7 or 8, comprising at least one anticancer agent which siRNA inhibits the expression or activity of STAT3.

10. An immunotherapy construct according to any one of claims 7 to 9, wherein at least one anticancer agent comprises an siRNA that inhibits the expression or activity of HER2.

11. An immunotherapy construct according to any one of claims 1 to 4, wherein at least one anticancer 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, AR, MYC, BRAF, BRAF V600E, RAS, PI3K, or MTDH.

12. An immunotherapy construct according to any one of claims 1 to 11, wherein at least one anticancer agent is selected from antibiotics, plant alkaloids, PLK1 inhibitors, mitotic kinase inhibitors, immune checkpoint inhibitors, platinum-based chemotherapeutic agents, HER2 small molecule inhibitors, anti-EGFR antibodies, anti-HER2 antibodies, volasertib, cetuximab, trastuzumab, anti-PD-L1 antibodies, anti-PD1 antibodies, anti-CTLA4 antibodies, docetaxel, paclitaxel, doxorubicin, mitoxantrone, or cabazitaxel.

13. An immunotherapy construct according to any one of claims 1 to 12, wherein the adjuvant comprises an immunostimulatory activity comprising a CpG oligonucleotide, a DNA TLR agonist containing a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, or a derivative thereof; or one or more of a STING agonist, polyI:C, polyICLC, dSLIM, or EnanDIM.

14. An immunotherapy construct according to any one of claims 1 to 13, comprising PLK1 siRNA and an anti-EGFR antibody.

15. An immunotherapy construct according to any one of claims 1 to 14, At least one pharmaceutically acceptable carrier, excipient, diluent, or mixture thereof A composition containing the following:

16. An immunotherapy construct according to any one of claims 1 to 14 or a composition according to claim 15 for treating cancer by administering an effective amount of the immunotherapy construct or composition to a subject having cancer.

17. The immunotherapy construct or composition according to claim 16, wherein the target is a mammal.

18. The immunotherapy construct or composition according to claim 17, wherein the mammal is a human.

19. An immunotherapy construct according to any one of claims 1 to 14 or a composition according to claim 15 for treating cells exhibiting symptoms of cancer by contacting said cells with a therapeutically effective amount of the immunotherapy construct or composition.

20. A method for treating cells obtained from a subject diagnosed with cancer or another hyperproliferative disorder, cells obtained from a subject diagnosed with a high risk of developing such a disorder, or cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, the method comprising the step of contacting the cells with a therapeutically effective amount of the immunotherapy construct according to any one of claims 1 to 14 or the composition according to claim 15.

21. A method for treating cells obtained from a subject diagnosed with cancer or another hyperproliferative disorder, cells obtained from a subject diagnosed with a high risk of developing such a disorder, cells obtained from a subject exhibiting symptoms of cancer or another hyperproliferative disorder, or cells obtained from a healthy donor, the method comprising the step of ex vivo contacting the cells with a therapeutically effective amount of the immunotherapy construct according to any one of claims 1 to 14 or the composition according to claim 15.

22. The method according to claim 20 or 21, wherein the cells are cancer cells.

23. The method according to claim 20 or 21, wherein the cells are not cancer cells.

24. The method according to claim 23, wherein the cells are immune cells.

25. The method according to claim 20 or 21, wherein cells are immortalized.

26. An immunotherapy construct or composition according to claim 19 for administering at least one treated cell back to a subject diagnosed with cancer or another hyperproliferative disorder or a subject exhibiting symptoms of cancer.

27. The composition according to claim 15, for treating a subject diagnosed with an overproliferative disease or overproliferative condition, or a subject diagnosed with a high risk of developing such a disease or condition, by administering an effective amount of the composition to the subject.

28. The composition according to claim 27, wherein the target is a mammal.

29. The composition according to claim 28, wherein the mammal is a human.

30. The composition according to any one of claims 27 to 29, wherein the hyperproliferative disorder or hyperproliferative condition comprises one or more of cancer, precancerous conditions, or cancerous metastases.

31. The composition according to any one of claims 27 to 30, wherein the hyperproliferative disease comprises one or more of the following: melanoma, lung cancer, breast cancer, pancreatic cancer, brain cancer, prostate cancer, head and neck cancer, kidney cancer, colorectal cancer, lymphoma, gastric cancer, colon cancer, liver cancer, sarcoma, or rare cancers.

32. The administration is Injection into or at the target tumor, Local injection into or at the target tumor, Systemic injection in the subject, Systemic injection into the subject, or Local application to the target A composition according to any one of claims 27 to 31, comprising:

33. The composition according to any one of claims 27 to 32, wherein administration includes the application of microneedles to a subject.

34. An immunotherapy construct according to any one of claims 1 to 14 or a composition according to claim 15 for enhancing the effect of anticancer therapy in a subject requiring such enhancement, wherein an effective amount of the immunotherapy construct or composition is administered to the subject in combination with at least one anticancer agent as an anticancer therapy to enhance the effect of anticancer therapy.

35. The immunotherapy construct or composition according to claim 34, wherein the anticancer agent is a chemotherapeutic agent or a targeted therapy agent.

36. An immunotherapy construct according to any one of claims 1 to 14 or a composition according to claim 15 for enhancing the effect of checkpoint blockade immunotherapy in a subject diagnosed with a neoplasm, wherein an effective amount of the immunotherapy construct or composition is administered to a subject in need in combination with at least one immune checkpoint inhibitor as an anticancer therapy to enhance the effect of checkpoint blockade immunotherapy.

37. An immunotherapy construct according to any one of claims 1 to 14 or a composition according to claim 15 for enhancing the effect of radiotherapy in a subject diagnosed with a neoplasm, wherein an effective amount of the immunotherapy construct or composition is administered to a subject in need in combination with at least one radiotherapy as an anticancer therapy to enhance the effect of radiotherapy.

38. An immunotherapy construct or composition according to any one of claims 34 to 37, wherein the immunotherapy construct or composition and anticancer therapy are administered sequentially or simultaneously.

39. An immunotherapy construct or composition according to any one of claims 34 to 38, wherein the target is a mammal.

40. The immunotherapy construct or composition according to claim 39, wherein the mammal is a human.

41. A kit comprising an immunotherapy construct according to any one of claims 1 to 14 or a composition according to claim 15, and at least one additional anticancer agent.

42. The kit according to claim 41, wherein the anticancer agent is a chemotherapeutic agent, a targeted therapy agent, or an immune checkpoint inhibitor.

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