Promoting training immunity with therapeutic nanobiologic compositions

Abstract

To provide a method for treating a patient having cancer or an infectious disease by promoting training immunity.SOLUTION: A nanobiological composition for promoting training immunity, comprising a nanoscale assembly, wherein the nanoscale assembly is a multi-component carrier composition comprising a phospholipid, a human apolipoprotein A-I (apoA-I), and a NOD2 activator such as bacterial peptidoglycan (e.g., MDP or MTP); wherein the nanobiological composition is a nanodisk or nanosphere having a diameter size of about 8 nm to 400 nm.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Patent Application No. 62 / 589,054, filed November 21, 2018, the entire contents of both of which are incorporated herein by reference.

[0002] (STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT) This invention was made with government support under Grant No. R01 HL118440 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] The present invention relates to therapeutic nanobiological compositions and methods for treating patients with cancer or infectious diseases by promoting trained immunity, which is a secondary long-term hyperresponsiveness as manifested by increased cytokine output caused by metabolic and epigenetic rewiring, by using the nanobiological compositions for stimulation of myeloid cells and their progenitor and stem cells in the bone marrow, spleen, and blood. [Background technology]

[0004] Current treatments for patients suffering from cancer can be inadequate. Patients with cancer need a treatment paradigm that is durable and does not cause as many problems with side effects as the first-line treatment itself.

[0005] Current cancer treatments may involve surgery, chemotherapy, hormonal therapy, and / or radiation therapy to eradicate neoplastic cells in patients (see, e.g., Stockdale, 1998, Medicine, Vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). More recently, cancer treatments have also included biological or immunotherapy. All of these approaches present numerous drawbacks to patients. For example, surgery may be contraindicated or unacceptable due to the patient's health.

[0006] Furthermore, surgery may not completely remove the neoplastic tissue. Radiation therapy is only effective if the neoplastic tissue is more sensitive to radiation than normal tissue. Radiation therapy often causes serious side effects. Hormone therapy is rarely given as a single agent. Although hormone therapy can be effective, it is often used to prevent or delay the recurrence of cancer after other treatments have removed most of the cancer cells. Biological therapy and immunotherapy have limited availability and may cause side effects such as rash or swelling or flu-like symptoms (such as fever, chills and fatigue, gastrointestinal problems, or allergic reactions).

[0007] Regarding chemotherapy, there are various chemotherapeutic agents available for the treatment of cancer. Most cancer chemotherapeutic agents act by directly or indirectly inhibiting DNA synthesis by inhibiting the biosynthetic pathway of deoxyribonucleotide triphosphate precursors, thereby preventing DNA replication and concomitant cell division. Gilman et al., Goodman and Gilman, The Pharmacological Basis of Therapeutics, 10th Edition (McGraw Hill, New York).

[0008] Despite the availability of various chemotherapeutic agents, chemotherapy has many drawbacks. (Stockdale, Medicine, Vol. 3, Rubenstein and Federman, eds., Chapter 12, Section 10, 1998) Nearly all chemotherapeutic agents are toxic, and chemotherapy causes serious and often dangerous side effects, such as severe nausea, bone marrow suppression, and immunosuppression. Furthermore, even with the use of combination chemotherapy agents, many tumor cells are resistant to or develop resistance to chemotherapeutic agents. In fact, cells resistant to a particular chemotherapeutic agent used in a treatment protocol often prove resistant to other drugs, even if those drugs act by mechanisms different from those of the drug used in the particular treatment. This phenomenon is called pleiotropic drug or multidrug resistance. Due to drug resistance, many cancers prove resistant to standard chemotherapy treatment protocols. Furthermore, there is a great need for safe and effective methods of treating, preventing, and managing cancer and other diseases and illnesses caused by defective trained immunity, particularly diseases that are resistant to standard treatments such as surgery, radiation therapy, chemotherapy, and hormone therapy, while reducing or avoiding the toxicity and / or side effects associated with conventional treatments.

[0009] Over the past few decades, our understanding of the immune system has led to several promising immunotherapeutic approaches that offer significant benefits to patients. Today's clinically relevant immunotherapies involve either effector molecules such as cytokines or the cellular phase of adaptive immunity. While anti-cytokine therapy can successfully neutralize bioactive cytokines in autoimmune and autoinflammatory diseases, the most intensively used immunotherapies in cancer patients involve the administration of checkpoint inhibitors. These checkpoint inhibitors release the brakes on T cells, allowing them to eliminate tumor cells. Antibodies specific for cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and antibodies against programmed cell death protein 1 (PD-1) and its ligand PD-L1 have made the most progress in clinical applications. Alternatively, adoptive T-cell therapy involves harvesting these cells from patients, expanding their numbers in culture, and reintroducing them into the body. T cells can also be genetically modified in culture to enhance their affinity for tumor cells. Dendritic cell therapy is another treatment that has attracted much interest. In this therapy, tumor-specific antigens are presented to dendritic cells either ex vivo or in vivo to induce tumor-specific T cell responses.

[0010] Although the immunotherapeutic approaches described above focus on T lymphocytes, cells from the adaptive immune system, improved treatments remain needed. Summary of the Invention

[0011] Therefore, to address these and other deficiencies in the prior art, in preferred embodiments of the present invention, the present invention provides nanobiological compositions involving the innate immune system, particularly myeloid cells and their stem and progenitor cells in the bone marrow, blood, and spleen, and methods of treating patients in need thereof with therapeutic agents to promote trained immunity.

[0012] Trained immunity is defined by a secondary, long-term enhanced responsiveness, as manifested by increased cytokine secretion caused by metabolic and epigenetic rewiring, of myeloid cells and their progenitor and stem cells in the bone marrow, spleen, and blood upon restimulation after a primary insult. Trained immunity (also called innate immune memory) is also defined by increased long-term responsiveness (e.g., elevated cytokine production) following restimulation with a secondary stimulus induced by a primary insult that stimulates myeloid innate immune cells or their progenitor and stem cells in the bone marrow and spleen, and mediated by epigenetic, metabolic, and transcriptional rewiring.

[0013] Treating cancer or sepsis In a preferred, non-limiting embodiment of the present invention, there is provided a method of treating a patient by inducing trained immunity to treat cancer or sepsis, comprising: (i) administering to said patient a nanobiological composition in an amount effective to promote a hyperresponsive innate immune response; The nanobiological composition (i) comprises nanoscale assemblies, and (ii) has an innate immune response-promoting drug incorporated into the nanoscale assemblies; the nanoscale assembly is a multicomponent carrier composition comprising (a) a phospholipid, (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; the nanobiological composition is a nanodisk or nanosphere having a diameter size of about 8 nm to 400 nm in an aqueous environment; The nanobiological composition is functionalized with molecular structures that activate or bind to pathogen-recognition receptors Dectin-1 or NOD2 to induce trained immunity in myeloid cells and their stem and progenitor cells in bone marrow, blood, and spleen, where the molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers, and the molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP), the nanoscale assemblies deliver the trained immune-stimulating molecular structures to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; thereby promoting a hyperresponsive innate immune response caused by trained immunity in the patient and treating cancer or sepsis. A method is provided which includes:

[0014] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0015] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0016] Improving the efficacy of checkpoint inhibitors In another non-limiting preferred embodiment of the present invention, the present invention provides a method of treating a patient by improving the efficacy of checkpoint inhibitor therapy by inducing trained immunity, comprising: (1) administering to said patient a nanobiological composition in an amount effective to promote a hyperresponsive innate immune response; The nanobiological composition (i) comprises nanoscale assemblies, and (ii) has an innate immune response-promoting drug incorporated into the nanoscale assemblies; the nanoscale assembly is a multicomponent carrier composition comprising (a) a phospholipid and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; the nanobiological composition is a nanodisk or nanosphere having a diameter size of about 8 nm to 400 nm in an aqueous environment; The nanobiological composition is functionalized with molecular structures that activate or bind to pathogen-recognition receptors Dectin-1 or NOD2 to induce trained immunity in myeloid cells and their stem and progenitor cells in bone marrow, blood, and spleen, where the molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers, and the molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP), the nanoscale assemblies deliver the trained immune-stimulating molecular structures to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; thereby promoting a hyperresponsive innate immune response in the patient caused by trained immunization; (2) administering to the patient a checkpoint inhibitor, thereby enhancing the efficacy of checkpoint inhibitor therapy by promoting a hyperresponsive innate immune response caused by trained immunity. and a method comprising:

[0017] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0018] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0019] Promoting long-term tumor remission In a preferred, non-limiting embodiment of the present invention, there is provided a method for promoting long-term tumor remission in a patient diagnosed with cancer, comprising: (1) administering to said patient a standard of care regimen specific to the patient's cancer, including chemotherapy, radiation therapy, immunotherapy, and therapeutically effective combinations thereof; (2) administering to said patient a nanobiological composition in an amount effective to promote a long-term hyperresponsive innate immune response; The nanobiological composition (i) comprises nanoscale assemblies, and (ii) has an innate immune response-promoting drug incorporated into the nanoscale assemblies; the nanoscale assembly is a multicomponent carrier composition comprising (a) a phospholipid, and (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I; The promoter has a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2, and examples of molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers, and examples of molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP), the nanobiological composition is a nanodisk or nanosphere having a diameter size of about 8 nm to 400 nm in an aqueous environment; the nanoscale assemblies deliver the stimulatory agent to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; thereby promoting a hyperresponsive innate immune response in the patient caused by trained immunization; (3) optionally administering to the patient a checkpoint inhibitor, thereby enhancing the efficacy of checkpoint inhibitor therapy by promoting a hyperresponsive innate immune response caused by trained immunity. A method is provided which includes:

[0020] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0021] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0022] Providing long-term natural and trained immunity In a non-limiting preferred embodiment of the present invention, there is provided a method of treating a patient affected by defective trained immunity (immunoparalysis) to promote a long-term hyperresponsive innate immune response in said patient, comprising: (1) administering to said patient a nanobiological composition in an amount effective to promote a hyperresponsive innate immune response; The nanobiological composition (i) comprises nanoscale assemblies, and (ii) has a facilitating agent incorporated into the nanoscale assemblies; The nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid and (b) apoA-I or a peptidomimetic of apoA-I; The promoter is a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow, blood, and spleen and their stem and progenitor cells; molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers; and molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP); the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; the nanoscale assemblies deliver the drug to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; and thereby promoting a hyperresponsive innate immune response in the patient; (2) optionally administering a checkpoint inhibitor to the patient after administration of the nanobiological composition; thereby enhancing the efficacy of checkpoint inhibitor therapy by promoting a hyperresponsive innate immune response caused by trained immunity. A method is provided which includes:

[0023] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0024] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0025] PET imaging of drug accumulation in the body In a non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for imaging the accumulation of the nanobiological composition in the bone marrow, blood and / or spleen of a patient affected by trained immunity, comprising: (i) comprising nanoscale assemblies, and (ii) having an enhancer incorporated into the nanoscale assemblies, and (iii) a positron emission tomography (PET) radioisotope incorporated into the nanoscale assemblies; The nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid and (b) apoA-I or a peptidomimetic of apoA-I; The promoter is a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow, blood, and spleen and their stem and progenitor cells; molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers; and molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP); PET imaging radioisotopes 89 Zr, 124 I, 64 Cu, 18 F and 86 Y, and the PET imaging radioisotope is complexed to the nanobiological composition using a suitable chelating agent to form a stable nanobiological composition-radioisotope chelate; the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; The nanoscale assemblies deliver stable nanobiological composition-radioisotope chelates to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen. A nanobiological composition is provided, characterized in that:

[0026] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0027] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0028] In a preferred, non-limiting embodiment of the present invention, there is provided a method for positron emission tomography (PET) imaging of the accumulation of a nanobiological composition in the bone marrow, blood, and / or spleen of a patient affected by trained immunity, comprising: (1) administering to said patient a nanobiological composition in an amount effective to promote a hyperresponsive innate immune response; The nanobiological composition (i) comprises nanoscale assemblies, and (ii) has a facilitating agent incorporated into the nanoscale assemblies, and (iii) a positron emission tomography (PET) radioisotope incorporated into the nanoscale assemblies; The nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid and (b) apoA-I or a peptidomimetic of apoA-I; The promoter is a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow, blood, and spleen and their stem and progenitor cells; molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers; and molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP); PET imaging radioisotopes 89 Zr,124 I, 64 Cu, 18 F and 86 Y, and the PET imaging radioisotope is complexed to the nanobiological composition using a suitable chelating agent to form a stable nanobiological composition-radioisotope chelate; the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; The nanoscale assemblies comprise a step of delivering a stable nanobiological composition-radioisotope chelate to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; (2) performing PET imaging of the patient to visualize the biodistribution of the stable nanobiological composition-radioisotope chelate within the bone marrow, blood, and / or spleen of the patient's body; A method is provided which includes:

[0029] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0030] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0031] In a non-limiting preferred embodiment, the method of radiopharmaceutical imaging comprises the further step of administering to said patient a checkpoint inhibitor along with the nanobiological composition, thereby enhancing the efficacy of checkpoint inhibitor therapy by promoting a hyperresponsive innate immune response caused by trained immunity.

[0032] In a non-limiting preferred embodiment of the present invention, a method for promoting a hyperresponsive innate immune response for at least 7 to 30 days is provided.

[0033] In a non-limiting preferred embodiment of the present invention, a method for promoting a hyperresponsive innate immune response for at least 30 to 100 days is provided.

[0034] In a non-limiting preferred embodiment of the present invention, a method is provided for promoting a hyperresponsive innate immune response for more than 100 days and up to three years.

[0035] In a preferred, non-limiting embodiment of the present invention, a method is provided wherein the patient affected by trained immunization is suffering from cancer of the bladder, blood vessels, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lymph node, lung, mouth, neck, ovary, pancreas, prostate, rectum, skin, stomach, testis, throat, thyroid, urothelium, or uterus.

[0036] In a non-limiting preferred embodiment of the present invention, a method is provided in which the nanobiological composition is administered once and a hyperresponsive innate immune response is promoted for at least 30 days.

[0037] In a non-limiting preferred embodiment of the present invention, a method is provided in which the nanobiological composition is administered at least once daily on each day of a multiple dose regimen and promotes a hyperresponsive innate immune response for at least 30 days.

[0038] In non-limiting preferred embodiments of the present invention, the promoter is a drug or compound or polymer that activates or binds to MDP, MTP, β-glucan, a sugar polymer, ox-LDL, BCG, bacterial peptidoglycan, a viral peptide, Dectin-1 or NOD2, a promoter of an inflammasome, a promoter of a metabolic pathway, and / or a promoter of an epigenetic pathway in hematopoietic stem cells (HSCs), common myeloid progenitor cells (CMPs), or myeloid cells.

[0039] In a non-limiting preferred embodiment of the present invention, a method is provided wherein trained immunity is defined by a secondary hyperresponsiveness as manifested by increased cytokine output caused by metabolic and epigenetic rewiring to restimulation following administration of the nanobiological composition to cause primary injury of myeloid cells and their progenitor and stem cells in the bone marrow.

[0040] In a non-limiting preferred embodiment of the present invention, a method is provided in which trained immunity is induced following administration of the nanobiological composition to produce a primary insult that stimulates myeloid innate immune cells or their progenitor and stem cells in the bone marrow, and is characterized by increased long-term responsiveness due to high cytokine production following administration of the nanobiological composition to produce a secondary stimulation of these cells mediated by epigenetic, metabolic, and transcriptional rewiring.

[0041] In preferred, non-limiting embodiments of the present invention, the promoter is a NOD2 receptor promoter, an mTOR promoter, a ribosomal protein S6 kinase beta-1 (S6K1) promoter, a histone H3K27 demethylase promoter, a BET bromodomain blockade promoter, a promoter of histone methyltransferases and acetyltransferases, a promoter of DNA methyltransferases and acetyltransferases, an inflammasome promoter, a promoter of the serine / threonine kinase Akt, a promoter of hypoxia inducible factor 1-alpha, also known as HIF-1alpha, an inhibitor of histone and DNA demethylases and deacetylases, and mixtures of one or more thereof.

[0042] In a non-limiting preferred embodiment of the present invention, a method is provided wherein the patient has severe sepsis or is in septic shock.

[0043] In a preferred, non-limiting embodiment of the present invention, a method is provided wherein the patient has sepsis associated with a bacterial, viral, or fungal infection of the lungs, abdomen, kidneys, or bloodstream.

[0044] In a preferred, non-limiting embodiment of the present invention, a method is provided comprising administering the nanobiological composition to a patient in a treatment regimen comprising two or more doses to cause accumulation of the drug in myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood, and / or spleen.

[0045] In a preferred, non-limiting embodiment of the present invention, a method is provided that includes co-administering an anti-cancer agent as a combination therapy with the nanobiological composition.

[0046] Nanobiological Compositions In a non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for promoting trained immunity, comprising: (i) comprising nanoscale assemblies; and (ii) having an enhancer incorporated into the nanoscale assemblies; (i) the nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid, and (b) apoA-I or a peptidomimetic of apoA-I; The promoter is a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow, blood, and spleen and their stem and progenitor cells; molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers; and molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP); the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; the nanoscale assemblies deliver the drug to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; and thereby promoting a hyperresponsive innate immune response in patients Nanobiological compositions are provided.

[0047] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0048] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0049] In a non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for promoting trained immunity, wherein the promoter is MDP, MTP, β-glucan, a sugar polymer, ox-LDL, BCG, bacterial peptidoglycan, a viral peptide, Dectin-1, a promoter of inflammasomes, a promoter of metabolic pathways, and / or a promoter of epigenetic pathways in hematopoietic stem cells (HSCs), common myeloid progenitor cells (CMPs), or myeloid cells.

[0050] In a preferred, non-limiting embodiment of the present invention, there is provided a nanobiological composition for promoting trained immunity, wherein the promoter is a NOD2 receptor promoter, an mTOR promoter, a ribosomal protein S6 kinase beta-1 (S6K1) promoter, an HMG-CoA reductase promoter (statin), a histone H3K27 demethylase promoter, a BET bromodomain blockade promoter, a promoter of histone methyltransferases and acetyltransferases, a promoter of DNA methyltransferases and acetyltransferases, an inflammasome promoter, a promoter of the serine / threonine kinase Akt, a promoter of hypoxia inducible factor 1-alpha, also known as HIF-1alpha, and mixtures of one or more thereof.

[0051] Radiolabeled nanobiological compositions In a non-limiting preferred embodiment of the present invention, there is provided a nanobiological composition for imaging accumulation in bone marrow, blood, and spleen, comprising: (i) comprising nanoscale assemblies, and (ii) having a facilitating agent incorporated into the nanoscale assemblies, and (iii) a positron emission tomography (PET) imaging radioisotope incorporated into the nanoscale assemblies; The nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid and (b) apoA-I or a peptidomimetic of apoA-I; The promoter is a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow, blood, and spleen and their stem and progenitor cells; molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers; and molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide (MDP) and muramyl tripeptide (MTP); PET imaging radioisotopes 89 Zr, 124 I, 64 Cu, 18 F and 86 Y, and the PET imaging radioisotope is complexed to the nanobiological composition using a suitable chelating agent to form a stable nanobiological composition-radioisotope chelate; the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; The nanoscale assemblies deliver stable nanobiological composition-radioisotope chelates to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen. A nanobiological composition is provided, comprising:

[0052] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0053] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0054] Manufacturing Process In a preferred, non-limiting embodiment of the present invention, there is provided a process for producing a nanobiological composition for inhibiting trained immunity, comprising: incorporating an enhancer into the nanoscale assemblies, The nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid and (b) apoA-I or a peptidomimetic of apoA-I; The promoter has a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow and their stem and progenitor cells; the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; the nanoscale assemblies deliver the drug to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen; and thereby promoting a hyperresponsive innate immune response in the patient. A process is provided that includes:

[0055] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof.

[0056] In another non-limiting preferred embodiment of the present invention, the nanoscale assemblies also include (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and (d) cholesterol.

[0057] In a preferred, non-limiting embodiment of the present invention, the nanoscale assemblies also include a phospholipid bound to a radioisotope chelator.

[0058] In a preferred, non-limiting embodiment of the present invention, there is provided a process for producing a nanobiological composition for inhibiting trained immunity, wherein the promoter is MDP, MTP, β-glucan, a sugar polymer, ox-LDL, BCG, bacterial peptidoglycan, a viral peptide, Dectin-1, a promoter of an inflammasome, a promoter of a metabolic pathway, and / or a promoter of an epigenetic pathway in hematopoietic stem cells (HSCs), common myeloid progenitor cells (CMPs), or myeloid cells.

[0059] In a non-limiting preferred embodiment of the present invention, there is provided a process for producing a nanobiological composition for inhibiting trained immunity, characterized in that the assemblies are brought together using microfluidics, scaled-up microfluidizer technology, sonication, organic-to-aqueous infusion, or lipid membrane hydration.

[0060] For the purpose of illustrating the invention, there are shown in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0061] [Figure 1]Figure 1A shows the levels of cytokines IL-6 (Fig. 1A) and TNF-α (Fig. 1B) in human monocytes exposed to training immunity inducers (BCG, MDP, or MTP-HDL) for 24 hours, after which the cells were washed and allowed to restimulate for 5 days before restimulation with LPS. The increased cytokine production indicates the ability of MTP-HDL to induce training immunity. [Figure 2] Maximum intensity projection (MIP) PET images of mice intravenously injected with 89Zr-labeled MTP-HDL show high uptake in the bone marrow. [Figure 3] 1 is a graph of dose-response curves obtained in C57BL / 6 mice inoculated with B16F10 tumor cells in the flank to grow melanoma. The animals were treated with different doses of MTP-HDL (a muramyl tripeptide-functionalized HDL nanobiological composition) at different frequencies (one, two, or three times). Tumor volume as a function of time after tumor cell inoculation and as a function of different treatments is shown. [Figure 4] 1 is a graph of monocytes per mL of bone marrow over days following three intravenous MDP-HDL infusions versus controls. [Figure 5] 1 is a graph of FDG-PET imaging results of bone marrow showing control vs. MDP-HDL. FDG (sugar analog) uptake is expressed as standard uptake value (SUV). [Figure 6] Figure 1 shows a graph comparing tumor volume versus days after tumor inoculation for PD-1 inhibitors, MTP-HDL, and the combination of PD-1 inhibitors and MTP-HDL treatment. MTP-HDL was administered intravenously on days 8, 11, and 13 after tumor inoculation. Checkpoint inhibitors were administered on days 11 and 14. [Figure 7]

[0023] Figure 1 shows a graph comparing tumor volume versus days after tumor inoculation for CTLA-4 inhibitors, MTP-HDL, and the combination of CTLA-4 inhibitors and MTP-HDL treatment. MTP-HDL was administered intravenously on days 8, 11, and 13 after tumor inoculation. Checkpoint inhibitors were administered on days 11 and 14. [Figure 8]Figure 1 shows a graph comparing tumor volume versus days after tumor inoculation for PD-1 + CTLA-4 inhibitor, MTP-HDL, and the combination of PD-1 + CTLA-4 inhibitor and MTP-HDL treatment. MTP-HDL was administered intravenously on days 8, 11, and 13 after tumor inoculation. Checkpoint inhibitors were administered on days 11 and 14. [Figure 9] Figure 1 shows a comparison of PD-1 + CTLA-4 inhibitors, MTP-HDL, and the combination of PD-1 + CTLA-4 inhibitors and MTP-HDL treatment with continued MTP-HDL treatment, showing tumor volume versus days after tumor inoculation. MTP-HDL was administered intravenously on days 8, 11, 13, 15, and 17 after tumor inoculation. Checkpoint inhibitors were administered on days 11 and 14. [Figure 10] 1 is a graph of flow cytometry results 24 hours after the third injection of MTP-HDL showing the percentage of viable CD11b+ myeloid cells for various treatments and the PBS control. [Figure 11] 1 is a graph of flow cytometry results 24 hours after the third injection of MTP-HDL showing the percentage of viable bone marrow monocytes for various treatments and the PBS control. [Figure 12A] Figure 12 is a graph of flow cytometry results 24 hours after the third injection of MTP-HDL. Figure 12A shows the percentage of viable CD11b+ blood cells for various treatments and the PBS control. [Figure 12B] Figure 12 is a graph of flow cytometry results 24 hours after the third injection of MTP-HDL. Figure 12B shows the percentage of viable CD11b+ splenocytes for various treatments and the PBS control. [Figure 13A] Figure 13 is a graph of flow cytometry results 24 hours after the third injection of MTP-HDL. Figure 13A shows the percentage of viable blood monocytes for the various treatments and the PBS control. [Figure 13B]Figure 13 is a graph of flow cytometry results 24 hours after the third injection of MTP-HDL. Figure 13B shows the percentage of viable splenic monocytes for the various treatments and the PBS control. [Figure 14] Schematic representation of the processes controlling trained immunity at the epigenetic, cellular, and systems levels. The first identified "trainers" included the fungal PAMP β-glucan and the bacterial PAMP peptidoglycan / BCG. Trained immunity is epigenetically regulated to generate a stronger response to restimulation. Myeloid progenitor cells can be stimulated to produce "trained" myeloid cells over the long term, thereby providing a compelling framework for sustained therapeutic intervention. [Figure 15] Cellular diagram showing that trained immunity is regulated at the cellular level by bacterial, fungal, and metabolic pathways, resulting in epigenetic modifications that underlie cytokine secretion. [Figure 16] Schematic of the process showing that bone marrow-avid nanomaterials that inhibit (green) or promote (red) trained immunity can be used to stimulate the immune system and treat a variety of diseases ranging from cardiovascular disease and its clinical consequences, autoimmune disorders, to sepsis and infections, and cancer. [Figure 17] FIG. 1 shows that stimulation of the immune system's sensitivity to immune checkpoint blockade therapy can be achieved by promoting trained immunity. [Figure 18] FIG. 1 is a graphic representation of the radioisotope labeling process. [Figure 19] 1 is a graphic representation of PET imaging using radioisotopes delivered by nanobiological compositions, showing the accumulation of the nanobiological compositions in the bone marrow and spleen in mouse, rabbit, monkey, and pig models. DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention relates to nanobiological compositions for promoting trained immunity, methods for making such nanobiological compositions, methods for incorporating drugs into said nanobiological compositions, and prodrug formulations in which drugs are combined with functionalized linker moieties such as phospholipids, aliphatic chains, sterols, etc.

[0063] Inflammation is triggered by innate immune cells as a defense mechanism against tissue injury. An ancient mechanism of immune memory, termed trained immunity and also called innate immune memory, is induced by a primary insult that stimulates these cells or their progenitor and stem cells in the bone marrow, and is defined by increased long-term responsiveness (e.g., elevated cytokine production) after secondary restimulation of myeloid innate immune cells, mediated by epigenetic, metabolic, and transcriptional rewiring.

[0064] Trained immunity is defined by a secondary long-term hyperresponsiveness manifested by increased cytokine output caused by metabolic and epigenetic rewiring of myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood, and / or spleen to restimulation after primary insult.

[0065] The present invention relates to one preferred embodiment of myeloid cell-specific nanoimmunotherapy based on delivering nanobiological compositions carrying or having incorporated stimulatory agents that promote epigenetic and metabolic modifications that underlie trained immunity. The present invention relates to therapeutic nanobiological compositions and methods for treating patients with cancer by promoting trained immunity, i.e., increased long-term responsiveness that is the result of metabolic and epigenetic rewiring of myeloid cells and their stem and progenitor cells in the bone marrow, spleen, and blood, induced by a primary insult and characterized by increased cytokine excretion after restimulation with one or more secondary stimuli.

[0066] definition "Treat" or "treatment" The phrase "treating" or "treatment" of a condition, disorder, or disease (1) Preventing or delaying the onset of clinical symptoms of a condition, disorder, or disease in a person who is suffering from or susceptible to the condition, disorder, or disease but who has not yet experienced or exhibited clinical symptoms of the condition, disorder, or disease; (2) inhibiting a condition, disorder, or disease, i.e., arresting, reducing, or delaying the onset or recurrence (in the case of maintenance treatment) of a disease or at least one clinical symptom, sign, or test thereof; or (3) Relieving disease, i.e., causing the remission of a condition, disorder, or disease or at least one of its clinical or subclinical symptoms or signs. Includes:

[0067] Nanobiological Compositions The term "nanobiological composition" refers to a composition that (i) comprises nanoscale assemblies and (ii) has a promoter incorporated into the nanoscale assemblies, where the drug is a promoter of an inflammasome, a promoter of a metabolic pathway, and / or a promoter of an epigenetic pathway in hematopoietic stem cells (HSCs), common myeloid progenitor cells (CMPs), or myeloid cells.

[0068] Nanoscale assemblies The term "nanoscale assembly" (NA) refers to a multi-component carrier composition for carrying an active payload, e.g., a drug. The nanoscale assembly comprises the following subcomponents: (a) phospholipids, (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I, and optionally (c) a hydrophobic matrix. The nanoscale assembly may also optionally comprise (d) cholesterol.

[0069] The term "nanoscale assembly" (NA) also refers to a multi-component carrier composition comprising (a) phospholipids, (b) apolipoprotein AI (apoA-I) or a peptidomimetic of apoA-I, and (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, and sterol esters. The nanoscale assembly may also optionally comprise (d) cholesterol.

[0070] phospholipids The term "phospholipid" refers to an amphipathic compound consisting of two hydrophobic fatty acid "tails" and one hydrophilic "head" consisting of a phosphate group. The two components are linked together by a glycerol molecule. The phosphate group may be modified with simple organic molecules such as choline, ethanolamine, or serine.

[0071] Choline refers to an essential bioactive nutrient with the chemical formula R-(CH2)2-N-(CH2)4. When the R-moiety is phospho-, it is called phosphocholine.

[0072] Examples of suitable phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin or other ceramides, and phospholipid-containing oils such as lecithin oil. Combinations of phospholipids or mixtures of phospholipids with other substances may also be used.

[0073] Non-limiting examples of phospholipids that can be used in the present compositions include phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid / ester (PA) and lysophosphatidylcholine.

[0074] Specific examples include DDPC CAS-3436-44-0 1,2-didecanoyl-sn-glycero-3-phosphocholine, DEPA-NA CAS-80724-31-8 1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt), DEPC CAS-56649-39-9 1,2-dierucoyl-sn-glycero-3-phosphocholine, DEPE CAS-988-07-2 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine, DEPG-NA 1,2-dierucoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DLOPC CAS-998-06-1 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPA-NA 1,2-Dilauroyl-sn-glycero-3-phosphate (sodium salt), DLPC CAS-18194-25-7 1,2-Dilauroyl-sn-glycero-3-phosphocholine, DLPE 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine, DLPG-NA 1,2-Dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol...)(sodium salt), DLPG-NH4 1,2-Dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol...)(ammonium salt), DLPS-NA 1,2-Dilauroyl-sn-glycero-3-phosphoserine (sodium salt), DMPA-NA CAS-80724-3 1,2-Dimyristoyl-sn-glycero-3-phosphate (sodium salt), DMPC CAS-18194-24-6 1,2-Dimyristoyl-sn-glycero-3-phosphocholine, DMPE CAS-988-07-2 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPG-NA CAS-67232-80-8 1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DMPG-NH4 1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (ammonium salt), DMPG-NH4 / NA 1,2-Dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol...)) (sodium / ammonium salt), DMPS-NA 1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt), DOPA-NA 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), DOPC CAS-4235-95-4 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPE CAS-4004-5-1 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPG-NA CAS-62700-69-0 1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DOPS-NA CAS-70614-14-1 1,2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt), DPPA-NA CAS-71065-87-7 1,2-Dipalmitoyl-sn-glycero-3-phosphate (sodium salt), DPPC CAS-63-89-8 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, DPPE CAS-923-61-5 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPG-NA CAS-67232-81-9 1,2-Dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol...)(sodium salt), DPPG-NH4 CAS-73548-70-6 1,2-Dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol...)(ammonium salt), DPPS-NA 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt), DSPA-NA CAS-108321-18-2 1,2-Distearoyl-sn-glycero-3-phosphate (sodium salt), DSPC CAS-816-94-4 1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPE CAS-1069-79-0 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, DSPG-NA CAS-67232-82-0 1,2-Distearoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (sodium salt), DSPG-NH4 CAS-108347-80-4 1,2-Distearoyl-sn-glycero-3[phospho-rac-(1-glycerol...) (ammonium salt), DSPS-NA 1,2-distearoyl-sn-glycero-3-phosphoserine (sodium salt), EPC egg PC, HEPC hydrogenated egg PC, HSPC hydrogenated soy PC, LYSOPC myristic CAS-18194-24-6 1-myristoyl-sn-glycero-3-phosphocholine, LYSOPC myristic CAS-17364-16-8 1-palmitoyl-sn-glycero-3-phosphocholine, LYSOPC stearic CAS-19420-57-6 1-stearoyl-sn-glycero-3-phosphocholine, milk sphingomyelin, MPPC 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, MSPC 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, PMPC 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, POPC CAS-26853-31-6 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPG-NA CAS-81490-05-3 1-Palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)...] (sodium salt), PSPC 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, SMPC 1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, SOPC Examples include 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and SPPC 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine.

[0075] In some preferred embodiments, specific, non-limiting examples of phospholipids include dimyristoylphosphatidylcholine (DMPC), soy lecithin, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG ...oleoylphosphatidylglycerol (DOPC), dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylglycerol (DLPG), dilauroylphospha Dimyristoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl sphingomyelin (DPSP), distearoyl sphingomyelin (DSSP), and mixtures thereof.

[0076] In certain embodiments, when the composition comprises (consists essentially of, or consists of) two or more phospholipids, the weight ratio of the two phospholipids can be in the range of about 1:10 to about 10:1, about 2:1 to about 4:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 7:1 to about 9:1, or about 8:1 to about 9:1. For example, the weight ratio of the two phospholipids can be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0077] In one embodiment, the (a) phospholipids of the nanoscale assembly comprise (consist essentially of, or consist of) a mixture of double-chain diacyl phospholipids and single-chain acyl phospholipids / lysolipids.

[0078] In one embodiment, the (a) phospholipid is a mixture of phospholipids and lysolipids, namely (DMPC) and (MHPC).

[0079] The weight ratio of DMPC:MHPC may be in the range of about 1:10 to about 10:1, about 2:1 to about 4:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 7:1 to about 9:1, or about 8:1 to about 9:1. The weight ratio of DMPC:MHPC may be in the range of about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0080] In one embodiment, the (a) phospholipid is a mixture of phospholipids and lysolipids, i.e., (POPC) and (PHPC).

[0081] The weight ratio of POPC:PHPC may be in the range of about 1:10 to about 10:1, about 2:1 to about 4:1, about 1:1 to about 5:1, about 2:1 to about 5:1, about 6:1 to about 10:1, about 7:1 to about 10:1, about 8:1 to about 10:1, about 7:1 to about 9:1, or about 8:1 to about 9:1. The weight ratio of POPC:PHPC may be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1.

[0082] It should be noted that all phospholipids ranging from C4 to C30 chain length, saturated or unsaturated, cis or trans, unsubstituted or substituted with 1 to 6 side chains, and with or without lysolipid additions are contemplated for use in the nanoscale assemblies or nanoparticle / nanobiological compositions described herein.

[0083] Additionally, other synthetic variants and variants with other phospholipid head groups are contemplated.

[0084] As used herein, "lysolipid" includes, in non-limiting embodiments, (acyl-, single-chain) lysolipids such as 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC), 1-palmitoyl-2-hexadecyl-sn-glycero-3-phosphocholine (PHPC), and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (SHPC).

[0085] Apolipoprotein AI (apoA-I) (apoA1) The terms "apolipoprotein AI" or "apoA-I" and "apolipoprotein AI" or "apoA1" also refer to the protein encoded by the APOA1 gene in humans and, as used herein, also include peptide mimetics of apoA-I. Apolipoprotein AI (apoA-I) is a minor component (b) in the nanoscale assembly.

[0086] Hydrophobic matrix The term "hydrophobic matrix" refers to the core or filler or structural modifier of a nanobiological composition. Structural modifications include (1) using a hydrophobic matrix to increase or engineer the particle size of nanoscale assemblies made solely of (a) phospholipids and (b) apoA-I, (2) increasing or decreasing (engineering) the stiffness of nanoscale assembly particles, (3) increasing or decreasing (engineering) the viscosity of nanoscale assembly particles, and (4) improving or decreasing (engineering) the biodistribution properties of nanoscale assembly particles.

[0087] The particle size, rigidity, viscosity, and / or biodistribution of nanoscale assemblies can be modified by the amount and type of hydrophobic molecules added. In a non-limiting example, nanoscale assemblies made solely from (a) phospholipids and (b) apoA-I may have a diameter of 10 nm to 50 nm. Adding (c) hydrophobic matrix molecules, such as triglycerides, expands the nanoscale assemblies to a minimum of 10 nm to at least 30 nm. Adding more triglycerides can increase the diameter of the nanoscale assemblies to at least 50 nm, at least 75 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 300 nm, and up to 400 nm within the scope of the present invention.

[0088] Depending on the manufacturing method, uniformly sized nanoscale aggregate particles can be prepared, or a mixture of non-uniformly sized nanoscale aggregate particles can be prepared by not filtering or by preparing nanoscale aggregate particles of a range of different sizes and then recombining them in a post-manufacturing step. The larger the size of the nanoscale aggregate particles, the more drug can be incorporated. However, larger sizes (e.g., greater than 120 nm) may limit, prevent, or slow the diffusion of the nanoscale aggregate particles into the tissues of the patient being treated. Smaller nanoscale aggregate particles do not carry as much drug per particle, but can access the bone marrow, blood, or spleen, or other local tissues affected by trained immunity, such as the transplanted tissue, surrounding tissue, and atherosclerotic plaque (biodistribution). The use of a heterogeneous mixture of nanoparticle sizes in a single dose or regimen can result in an immediate reduction in innate immune hyperresponsiveness, as well as a sustained, long-term reduction in innate immune hyperresponsiveness that can last for days, weeks, months, and years, where the nanobiological composition reverses, modulates, or regulates metabolic, epigenetic, and inflammasome pathways in hematopoietic stem cells (HSCs), common myeloid progenitor cells (CMPs), and myeloid cells, such as monocytes, macrophages, and other short-lived circulating cells.

[0089] By adding other (c) hydrophobic matrix molecules such as cholesterol, fatty acid esters, hydrophobic polymers, sterol esters, and different types of triglycerides or specific mixtures thereof, nanoscale aggregate particles can be further engineered to emphasize specific desired properties for specific purposes. Size, stiffness, and viscosity can influence loading and biodistribution.

[0090] As a non-limiting example, the maximum loading capacity can be determined by dividing the internal volume of the nanoscale aggregate particle by the volume of the drug-loaded sphere.

[0091] Particle: Assuming a 100 nm spherical particle with a 2.2 nm to 3.0 nm phospholipid wall, a volume (L) of 4 / 3π(r)3 gives an inner diameter of 94 nm.

[0092] Drug: Assuming the stimulant as a 12 x 12 x 35 Angstrom or 1.2 x 1.2 x 3.5 nm cylinder, we can now assume that multiple (e.g., 7 or 9) drug molecule cylinders form 3.5 nm diameter spheres with a radius of 1.75 nm, with a Vol(small) of 4 / 3π(r).

[0093] Maximum loading capacity (calculated): approximately 487k 3.5nm spheres in a 100nm particle.

[0094] Biologically relevant lipids include fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides. A complete list of over 42,000 lipids can be found at https: / / www.lipidmaps.org.

[0095] triglycerides The term "triglyceride" and similar terms refer to an ester derived from one molecule of glycerol and three molecules of fatty acids. The notation used herein to describe triglycerides is the same as that used below to describe fatty acids. Triglycerides can contain glycerol with any combination of the following polyunsaturated and saturated fatty acids: C18:1, C14:1, and C16:1. The fatty acids can be attached to the glycerol molecule in any order; for example, any fatty acid can react with any of the hydroxyl groups on the glycerol molecule to form an ester bond. A triglyceride of C18:1 fatty acids simply means that the fatty acid component of the triglyceride is derived from or based on a C18:1 fatty acid. That is, a C18:1 triglyceride is an ester of glycerol with three fatty acids, each of 18 carbon atoms, with each fatty acid having one double bond. Similarly, a C14:1 triglyceride is an ester of glycerol with three fatty acids of 14 carbon atoms each, with each fatty acid having one double bond. Similarly, a C16:1 triglyceride is an ester of glycerol with three fatty acids of 16 carbon atoms each, with each fatty acid having one double bond. A triglyceride of C18:1 fatty acids combined with C14:1 and / or C16:1 fatty acids means (a) that the C18:1 triglyceride is mixed with C14:1 triglycerides or C16:1 triglycerides or both, or (b) that at least one of the fatty acid components of the triglyceride is derived from or based on a C18:1 fatty acid, while the other two are derived from or based on a C14:1 fatty acid and / or a C16:1 fatty acid.

[0096] fatty acid The term "fatty acid" and similar terms refer to a carboxylic acid with a long aliphatic tail, either saturated or unsaturated. Fatty acids may be esterified into phospholipids and triglycerides. As used herein, fatty acid chain lengths include saturated or unsaturated, cis or trans, and C4 to C30, unsubstituted or substituted with one to six side chains. Unsaturated fatty acids have one or more double bonds between carbon atoms. Saturated fatty acids contain no double bonds. The notation used herein to describe fatty acids includes a capital "C" for the carbon atom, followed by a number describing the number of carbon atoms in the fatty acid, followed by a colon and another number for the number of double bonds in the fatty acid. For example, C16:1 refers to a 16-carbon fatty acid with one double bond, such as palmitoleic acid. The number after the colon in this notation does not indicate the configuration of the double bonds in the fatty acid or whether the hydrogen atoms attached to the carbon atoms of the double bonds are cis to each other. Other examples of this designation include C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:3 (alpha-linolenic acid) and C20:4 (arachidonic acid).

[0097] Sterols and sterol esters The term "sterol," including but not limited to cholesterol, can also be used in the methods and compounds described herein. Sterols are animal or plant steroids containing only a hydroxyl group at C-3 and no other functional groups. Generally, sterols contain 27-30 carbon atoms and one double bond at the 5 / 6 position, and sometimes at the 7 / 8, 8 / 9, or other positions. In addition to these unsaturated species, other sterols are saturated compounds obtained by hydrogenation. One example of a suitable animal sterol is cholesterol. Typical examples of suitable phytosterols that are preferred from an application standpoint are ergosterol, campesterol, stigmasterol, brassicasterol, preferably sitosterol or sitostanol, more particularly β-sitosterol or β-sitostanol. In addition to the above phytosterols, their esters are preferably used. The acid component of the esters is represented by the formula (I): R1CO-OH(I) wherein RCO is an aliphatic, straight-chain or branched-chain acyl group containing 2 to 30 carbon atoms and 0 and / or 1, 2 or 3 double bonds. Typical examples are acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, 2-ethylhexanoic acid, capric acid, lauric acid, isotridecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselinic acid, linoleic acid, conjugated linoleic acid (CLA), linolenic acid, eleostearic acid, arachidic acid, gadoleic acid, behenic acid and erucic acid. can be converted back to the corresponding carboxylic acid.

[0098] Hydrophobic polymer The one or more hydrophobic polymers used to construct the matrix may be selected from the group of polymers approved for human use (i.e., biocompatible and FDA approved).

[0099] Examples of such polymers include, but are not limited to, the following polymers: polyalkenedicarboxylates, polyanhydrides, poly(aspartic acid), polyamides, polybutylene succinate (PBS), copolymers of polybutylene succinate and adipate (PBSA), poly(ε-caprolactone) (PCL), polycarbonates such as polyalkylene carbonate (PC), polyesters such as aliphatic polyesters and polyesteramides, polyethylene succinate (PES), polyglycolic acid, PEG, PEG-10, PEG-12, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-34, PEG-35, PEG-36, PEG-37, PEG-38, PEG-39, PEG-40, PEG-41, PEG-42, PEG-43, PEG-44, PEG-45, PEG-46, PEG-47, PEG-48, PEG-49 ...9, PEG-49, PEG-49, PEG-49, PEG-49, PEG-49, PEG-49, PEG Examples of suitable polymers include poly(propylene glycolide) (PGA), polyimines and polyalkyleneimines (PI, PAI), polylactic acids (PLA, PLLA, PDLLA), copolymers of polylactic and glycolic acid (PLGA), poly(l-lysine), polymethacrylates, polypeptides, polyorthoesters, poly-p-dioxanone (PPDO), (hydrophobically) modified polysaccharides, polysiloxanes and polyalkylsiloxanes, polyureas, polyurethanes and polyvinyl alcohols, derivatives of such polymers, copolymers, block copolymers, branched polymers and polymer blends.

[0100] Prodrug As used herein, unless otherwise specified, the term "prodrug" refers to a derivative of a compound that hydrolyzes, oxidizes, or otherwise reacts under biological conditions (in vitro or in vivo) to provide the compound. Examples of prodrugs include, but are not limited to, derivatives of the nanobiological compositions of the invention that contain biohydrolyzable moieties, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. Other examples of prodrugs include derivatives of the nanobiological compositions of the invention that contain -NO, -NO2, -ONO, or -ONO2 moieties. Prodrugs can typically be prepared using well-known methods, such as those described in 1 Burger's Medicinal Chemistry and Drug Discovery, 172-178, 949-982 (Manfred E. Wolff, ed., 5th ed. 1995) and Design of Prodrugs (H. Bundgaard, ed., Elselvier, NY 1985).

[0101] Increasing the compatibility of drugs with nanobiological compositions can be achieved using the following strategies: Covalently linking drugs to hydrophobic moieties such as cholesterol. If necessary, a prodrug approach can be achieved by, for example, a labile bond resulting in an enzymatically cleavable prodrug.

[0102] The derivatized drug is then incorporated into lipid-based nanobiological compositions used for in vivo drug delivery. The primary goal of drug derivatization is to form drug conjugates with higher hydrophobicity compared to the parent drug. As a result, retention of the drug conjugate within the nanobiological composition is enhanced compared to that of the parent drug, resulting in reduced binding and improved delivery to target tissues. In the case of prodrug strategies, different types of hydrophobic moieties can result in different in vivo cleavage rates, thereby affecting the rate at which the active drug is generated and, therefore, the overall therapeutic efficacy of the nanobiological composition-drug construct.

[0103] biohydrolyzable As used herein, unless otherwise specified, the terms "biohydrolyzable amide," "biohydrolyzable ester," "biohydrolyzable carbamate," "biohydrolyzable carbonate," "biohydrolyzable ureide," and "biohydrolyzable phosphate" mean, respectively, 1) a compound that does not interfere with the biological activity of a compound but can be incorporated into the compound to impart advantageous properties in vivo, such as duration of action or onset of action, or 2) an amide, ester, carbamate, carbonate, ureide, or phosphate of a compound that is biologically inactive but is converted in vivo to a biologically active compound. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, lower acyloxyalkyl esters (such as acetoxylmethyl, acetoxyethyl, aminocarbonyloxymethyl, pivaloyloxymethyl, and pivaloyloxyethyl esters), lactonyl esters (such as phthalidyl and thiophthalidyl esters), lower alkoxyacyloxyalkyl esters (such as methoxycarbonyloxymethyl, ethoxycarbonyloxyethyl, and isopropoxycarbonyloxyethyl esters), alkoxyalkyl esters, choline esters, and acylaminoalkyl esters (such as acetamidomethyl esters). Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, α-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, amino acids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines.

[0104] Method for producing nanoscale assemblies The methods are described below and variations on these methods exist.

[0105] Method 1 A. Phospholipids, drug (prodrug), and any triglycerides or polymers are dissolved (typically in chloroform, ethanol, or acetonitrile). The solution is then evaporated under vacuum to form a film of the components. A buffer solution is then added to hydrate the film, producing a vesicle suspension. B. The phospholipid, drug (prodrug), and any triglycerides or polymers are dissolved (typically in chloroform, ethanol, or acetonitrile). This solution is poured or added dropwise with stirring to a gently heated buffer solution until the organic solvent has completely evaporated and a vesicle suspension is formed.

[0106] Apolipoprotein AI (apoA-I) (which may already be present in B) was added dropwise to the vesicle suspension generated using either A or B to avoid denaturation. The resulting mixture was thoroughly cooled using an external ice-water bath and sonicated for 30 minutes using a tip sonicator. The resulting solution containing the nanobiological composition and other by-products was transferred to a Sartorius Vivaspin tube with a molecular weight cutoff corresponding to the estimated size of the nanobiological composition (typically, Vivaspin tubes with a cutoff of 10,000-100,000 kDa are used). These tubes were centrifuged until approximately 90% of the solvent volume passed through the filter. A volume of buffer approximately equal to the volume of the remaining solution was then added, and the tubes were spun again until approximately half of the volume passed through the filter. This process was repeated twice, after which the remaining solution was passed through a 0.22 μm polyethersulfone syringe filter to obtain the final nanobiological solution.

[0107] Method 2 In another approach, the phospholipid, drug (prodrug) and any triglycerides or polymers are dissolved (typically in ethanol or acetonitrile) and loaded into a syringe.

[0108] A second syringe is filled with a solution of apolipoprotein AI (apoA-I) in phosphate-buffered saline. The contents of both syringes are mixed using a microfluidic pump and a microvortex platform. The resulting solution containing the nanobiological composition and other by-products is transferred to a Sartorius Vivaspin tube with a molecular weight cutoff corresponding to the estimated particle size (typically, Vivaspin tubes with a cutoff of 10,000-100,000 kDa are used). These tubes are centrifuged until approximately 90% of the solvent volume passes through the filter. A volume of phosphate-buffered saline approximately equal to the volume of the remaining solution is then added, and the tubes are spun again until approximately half of the volume passes through the filter. This is repeated twice, after which the remaining solution is passed through a 0.22 μm polyethersulfone syringe filter to obtain the final nanobiological solution.

[0109] Microfluidizer method Another preferred method according to the present invention uses microfluidizer technology to prepare the nanoscale assemblies and the final nanobiological composition.

[0110] A microfluidizer is a device for preparing small particle size materials that works on the submerged jet principle. To obtain nanoparticles, a microfluidizer operates by forcing a premix stream through a so-called interaction chamber, which consists of a system of channels in a ceramic block that splits the premix into two streams. During microfluidization, precisely controlled shear, turbulence, and cavitation forces are generated in the interaction chamber. The two streams are then recombined at high speed to generate shear forces. The resulting product can be recycled through the microfluidizer to obtain even smaller particles.

[0111] The advantages of microfluidization over conventional milling processes include significantly reduced contamination of the final product and easier scale-up of manufacturing.

[0112] Combination Therapy - Delivery of Nanobiological Compositions in Combination with Checkpoint Inhibitors Combination therapy with checkpoint inhibitors and trained immune-inducing nanobiological compositions is also contemplated within the scope of the present subject matter.

[0113] Checkpoint inhibitors Checkpoint inhibitors refer to a class of drugs that block specific proteins made by some types of immune system cells, such as T cells, and some cancer cells. These proteins help keep the immune response in check and can prevent T cells from killing cancer cells. When these proteins are blocked, the "brakes" on the immune system are released, allowing T cells to better kill cancer cells. Examples of checkpoint proteins present on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. Some immune checkpoint inhibitors are used to treat cancer.

[0114] Background on checkpoint inhibitors Immune checkpoints regulate T cell function in the immune system. T cells play a central role in cell-mediated immunity. Checkpoint proteins interact with specific ligands that signal T cells, essentially shutting down or inhibiting T cell function. Cancer cells exploit this system by promoting high levels of checkpoint proteins on their surface, thereby controlling T cells that express checkpoint proteins on their surface that enter the tumor microenvironment, thus suppressing anti-cancer immune responses. Therefore, inhibition of checkpoint proteins results in full or partial restoration of T cell function and the immune response to cancer cells. Examples of checkpoint proteins include, but are not limited to, CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (which belongs to the CD2 family of molecules and is expressed on all NK, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands.

[0115] Types of checkpoint inhibitors Checkpoint inhibitors include any agent that blocks or inhibits an inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors or antibodies that bind to and block or inhibit immune checkpoint receptor ligands.

[0116] Exemplary checkpoint molecules that can be targeted for blocking or inhibiting to reactivate the immune response include, but are not limited to, CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (a member of the CD2 family of molecules and expressed on all NK, gamma delta, and memory CD8+ (alpha beta) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands, including, but not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7.

[0117] Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049.

[0118] Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1, MEDI4736), MK-3475 (a PD-1 blocker), nivolumab (an anti-PD-1 antibody), CT-011 (an anti-PD-1 antibody), BY55 monoclonal antibody, AMP224 (an anti-PD-L1 antibody), BMS-936559 (an anti-PD-L1 antibody), MPLDL3280A (an anti-PD-L1 antibody), MSB0010718C (an anti-PD-L1 antibody), and yervoy / ipilimumab (an anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0119] Checkpoint inhibitors that block PD-1 include nivolumab (Opdivo) and pembrolizumab (Keytruda). Nivolumab and pembrolizumab are treatments for some people with melanoma skin cancer, Hodgkin's lymphoma, non-small cell lung cancer, and cancer of the urinary tract (urothelial carcinoma). The urinary tract includes the center of the kidney (renal pelvis), the tubes that carry urine from the kidney to the bladder (ureters), the bladder, and the tube that drains urine from the bladder to the body (urethra).

[0120] Checkpoint inhibitors that block CTLA-4 include ipilimumab (Yervoy), which is used as a treatment for advanced melanoma.

[0121] Checkpoint inhibitors that block PD-L1 include atezolizumab (also known as MPDL3280A), a treatment for some people with lung cancer and urothelial carcinoma. It is in clinical trials for other cancers, including breast cancer.

[0122] Programmed cell death protein 1 (PD-1) is a 288-amino acid cell surface protein molecule expressed on T cells and pro-B cells and plays a role in their fate / differentiation. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-1 plays a role in tumor-specific escape from immunosurveillance. PD-1 is upregulated in melanoma infiltrating T lymphocytes (TILs) (Doth (2009) Blood 114(8):1457-58). Tumors have been shown to express PD-1 ligands (PD-L1 and PD-L2), which, when combined with upregulation of PD-1 in CTLs, can lead to loss of T cell function and the inability of CTLs to mediate effective antitumor responses.

[0123] Clinical trials in melanoma have demonstrated robust antitumor responses with anti-PD-1 blockade. Significant benefits of PD-1 blockade have also been documented in advanced melanoma, ovarian cancer, non-small cell lung cancer, prostate cancer, renal cell carcinoma, and colorectal cancer. Studies in mouse models have applied this evidence to glioma therapy. Anti-PD-1 blockade, as an adjuvant to radiation-promoted cytotoxic T cell populations and associated prolonged survival, has been beneficial in mice bearing glioma tumors.

[0124] In view of the results provided herein, embodiments of the present disclosure include the combination treatment of any solid tumor with any checkpoint inhibitor combined with one or more of the trained immune-inducing nanobiological compositions such as MDP-HDL, MTP-HDL, PG-HDL, BG-HDL and UA-HDL.

[0125] antibody checkpoint inhibitors One aspect of the present disclosure provides checkpoint inhibitors that are antibodies that function as PD-1 inhibitors, thereby modulating immune responses controlled by PD-1. In one embodiment, the anti-PD-1 antibody may be an antigen-binding fragment. The anti-PD-1 antibodies disclosed herein can bind to human PD-1 and affect the activity of PD-1, thereby inhibiting the function of immune cells expressing PD-1. Examples of PD-1 and PD-L1 blockers are described in U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT patent applications WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699.

[0126] There are several PD-1 inhibitors currently being tested in clinical trials. CT-011 is a humanized IgG1 monoclonal antibody against PD-1. A phase II clinical trial in subjects with diffuse large B-cell lymphoma (DLBCL) who underwent autologous stem cell transplantation was recently completed. Preliminary results demonstrated that 70% of subjects were progression-free at the end of the follow-up period compared to 47% in the control group, and 82% of subjects were alive compared to 62% in the control group. The trial demonstrated that CT-011 not only blocks PD-1 function but also enhances the activity of natural killer cells, thus enhancing the anti-tumor immune response.

[0127] BMS-936558 is a fully human IgG4 monoclonal antibody that targets PD-1. In a Phase I study, biweekly administration of BMS-936558 in subjects with advanced, refractory malignancies demonstrated durable partial or complete regressions. The most significant response rates were observed in subjects with melanoma (28%) and renal cell carcinoma (27%), but significant clinical activity was also observed in subjects with non-small cell lung cancer (NSCLC), with some responses lasting more than one year.

[0128] BMS-936559 is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand PD-L1. Phase I results showed that biweekly administration of the drug resulted in durable responses, particularly in subjects with melanoma. Objective response rates ranged from 6% to 17% in subjects with advanced NSCLC, melanoma, RCC, or ovarian cancer, depending on the cancer type, with some subjects experiencing responses lasting for more than a year.

[0129] MK-3475 is a humanized IgG4 anti-PD-1 monoclonal antibody in Phase III investigational therapy alone or in combination with chemotherapy as first-line treatment for advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. MK-3475 is currently undergoing multiple global Phase III clinical trials.

[0130] MPDL-3280A (atezolizumab) is a monoclonal antibody that also targets PD-L1. MPDL-3280A has received Breakthrough Therapy Designation from the U.S. Food and Drug Administration (FDA) for the treatment of people whose NSCLC expresses PD-L1 and has progressed on or after standard therapy.

[0131] AMP-224 is a fusion protein of the extracellular domain of IgG1 and a second PD-1 ligand, PD-L2, with the potential to block the PD-L2 / PD-1 interaction. AMP-224 is currently in Phase I trials as a monotherapy in subjects with advanced cancer.

[0132] MEDI-4736 is an anti-PD-L1 antibody that demonstrated an acceptable safety profile and sustained clinical activity in this dose-escalation study. MEDI-4736 is currently being developed as a monotherapy and in combination with other anti-PD-L1 antibodies for the treatment of multiple cancers.

[0133] Thus, in certain embodiments, PD-1 blockers include anti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX-1106, BMS-936558, ONO-4538) (a fully human IgG4 antibody that binds to PD-1 and blocks its activation by its ligands PD-L1 and PD-L2), pembrolizumab / lambrolizumab (MK-3475 or SCH-900475) (a humanized monoclonal IgG4 antibody against PD-1), CT-011 (a humanized antibody that binds to PD-1), AMP-224 (a fusion protein of B7-DC), antibody Fc portions, and BMS-936559 (MDX-1105-01) for PD-L1 (B7-H1) blockade. Other immune checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors, particularly the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). TIM3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors are also included (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94).

[0134] Combination therapy - delivery of nanobiological compositions in combination with anticancer drugs Examples of anti-cancer drugs include, but are not limited to, acivicin, aclarubicin, acodazole hydrochloride, acronine, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate dimesylate), bizelesin, bleomycin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, celecoxib (COX-2 inhibitor), chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziconazole, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflomithine hydrochloride hydrochloride), elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride hydrochloride), estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etopurine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, foskidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, iproplatin, irinotecan, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate,Melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, meturedepa, mitindomide, mitocalcin, mitochromin, mitogillin, mitomarcine, mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, pegaspargase Peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurin, safingol, safingol hydrochloride, Sems cin, simtrazene, sparfosate sodium, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium, taxotere, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teloxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, toremifene citrate, trestron acetate , triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride.

[0135] Other anti-cancer agents include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecipenol, adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogens (prostate cancer treatments), antiestrogens, antineoplastic agents, antisense oligonucleotides, aphidicolin glycinate, apoptotic gene regulators, apoptosis modulators, apurinic acid, ara-CDP- DL-PTBA, arginine deaminase, aslaculin, atamestane, atlimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, beta-clamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistraten A, bizelesin, breflate, bropirimine, budotitanium, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives, capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, CaRest M3, CARN 700, cartilage-derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin (chlorln), chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogue, clotrimazole, colismycin A, colismycin B, combretastatin A4, combretastatin analogue, conagenin, clambecidin 816, crisnatol, cryptophycin 8, cryptophycin A derivative, curacin A, cyclopentaquinone, cycloplatam,Cytarabine ocfosfate, cytolytic agents, cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexphosphamide, dexrazoxane, dexverapamil, diazicon, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, 9-dihydrotaxol, dioxamycin, diphenylspiromustine, docetaxel, docosanol, dolasetron, doxifluridine, doxorubicin, droloxifene, dronabinol, duocarmycin SA, ebselen, Ecomus tin, edelfosine, edrecolomab, eflomitine, elemene, emiteflu, epirubicin, epristeride, estramustine analogues, estrogen agonists, estrogen antagonists, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunornithine hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin texaphyrin), gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imatinib (e.g., Gleevec®), imiquimod, immunostimulating peptides, insulin-like growth factor-1 receptor inhibitors, interferon agonists, interferons, interleukins, iobenguane, Iododoxorubicin, 4-ipomeanol, ilopract, irsogladine, isobengazole, isohomohalichondrin B, itasetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprolide + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analogues, lipophilic disaccharide peptides, lipophilic platinum compounds,Lisoclinamide 7, lobaplatin, lombricin, lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan, lutetium texaphyrin, lisofylline, lytic peptides, maytansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase inhibitors, menogaril, mervalone, meterelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, mirimostim, mitoguazone, mitolactol, mitomycin analogs, mito Nafide, myotoxin fibroblast growth factor-saporin, mitoxantrone, mofalotene, molgramostim, erbitux, human chorionic gonadotropin, monophosphoryl lipid A + myobacterium cell wall sk, mopidamol, mustard anticancer drug, mycaperoxide B, myobacterium cell wall extract, myriaporone, N-acetyldinaline, N-substituted benzamide, nafarelin, nagressip, naloxone + pentazocine, napavine, naphterpine, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamayci nitriloacetate, nitric oxide regulators, nitric oxide antioxidants, nitrulline, oblimersen (Genasense®), 06-benzylguanine, octreotide, oxenon, oligonucleotides, onapristone, ondansetron, ondansetron, oracin, oral cytokine inducers, ormaplatin, osateron, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin, pamidronate, panaxytriol, panomyphen, paraben tin, pazelliptin, pegaspargase, perdecin, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitors, picibanil, pilocarpine hydrochloride, pirarubicin, piritrexim, prasetin A, prasetin B, plasminogen activator inhibitors, platinum complexes, platinum compounds, platinum-triamine complexes, porfimer sodium, porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2,Proteasome inhibitors, protein A-based immunomodulators, protein kinase C inhibitors, protein kinase C inhibitors (microalgae), protein tyrosine phosphatase inhibitors, purine nucleoside phosphorylase inhibitors, purpurins, pyrazoloacridines, pyridoxylated hemoglobin polyoxyethylene conjugates, raf antagonists, raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors, ras inhibitors, ras-G AP inhibitors, demethylated leteriptin, rhenium Re186 etidronate, rhizoxin, ribozyme, RII retinamide, rohitukin, romurtide, roquinimex, rubiginone B1, ruboxil, safingol, saintpin, SarCNU, sarcophytol A, sargramostim, Sdi1 mimetic, semustine, senescence-derived inhibitor 1, sense oligonucleotides, signal transduction inhibitors, sizofiran, sobuzoxane, sodium borocaptate (sodium borocaptate), sodium phenylacetate, sorberol, somatomedin-binding protein, sonermin, sparfosic acid, spicamycin D, spiromustine, splenopentin, spongistatin 1, squalamine, stypamide, stromelysin inhibitors, sulfinosine, superactive vasoactive intestinal peptide antagonists, sulagist, suramin, swainsonine, talimustine, tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium, tegafur, tellapyrylium, telomerase inhibitors, temoporfin, teniposide, tetrachlorodecaoxide, tetrazomine, thaliblastine, thiocoraline, Thrombopoietin, thrombopoietin mimetics, thymalfasin, thymopoietin receptor agonist, thymotrinan, thyroid-stimulating hormone, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, topsentin, toremifene, translation inhibitors, tretinoin, triacetyluridine, triciribine, trimetrexate, triptorelin, tropisetron, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex, urogenital sinus-derived growth inhibitor, urokinase receptor antagonist, vapreotide, variolin B, veraresol, veramine, verudin, verteporfin,These include vinorelbine, vinxartin, vitaxin, vorozole, zanoterone, zeniplatin, zilascorub, and zinostatin stimalamer.

[0136] Second-line small molecule drugs Small molecule drugs that can be used in combination therapy with the nanobiological compositions of the present invention include acetaminophen, acetylsalicylic acid, adriamycin, azathioprine, Biaxin, bisphosphonates, busulfan, capecitabine, carboplatin, celecoxib, chloroquine, cisplatin, cyclophosphamide, cyclosporine, cytarabine, d-penicillamine, dacarbazine, daunorubicin, dexamethasone, diflunisal, docetaxel, doxorubicin, estramustine phosphate sodium, etoposide, etoricoxib, fenoprofen, fludarabine, flufenamic acid, fluorouracil, flurbiprofen, ganciclovir, gemcitabine, gliadel, GM-CSF, hydroxychloroquine ibuprofen, IL-2, indomethacin, interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-14), interleukin-16 (IL-14), interleukin-17 (IL-14), interleukin-18 (IL-14), interleukin-19 (IL-14), interleukin-20 (IL-14), interleukin-21 (IL-14), interleukin-22 (IL-14), interleukin-33 (IL-14), interleukin-34 (IL-14), interleukin-15 (IL-1 Interferon alfa, irinotecan, ketoprofen, leflunomide, leucovorin, lumiracoxib, meclofenamate, mefenamic acid, melphalan, methylprednisolone, methotrexate, naproxen, nimesulide, oblimersen, oxaprozin, pacilitaxel, palmitronate, parecoxib, pegylated interferon alfa, phenylbutazone, piroxicam, prednisone, prednisolone, procarbazine, Remicade, rofecoxib, steroids, sulfasalazine, sulindac, tamoxifen, taxol, taxotere, temodar, temozolomide, tenoxicam, thiotepa, topotecan, valdecoxib, vinblastine, vincristine, vinorelbine, and zoledronic acid.

[0137] dosage Dosages generally range from 5 μg to 100 mg / kg of recipient (mammal) body weight per day, more usually from 5 μg to 10 mg / kg of body weight per day. This amount may be administered in a single dose per day, or more usually in subdoses administered several times per day (e.g., two, three, four, five, or six times) to provide the same total daily dose. The effective amount of the salt or solvate can be determined as a percentage of the effective amount of the compound in a nanobiological composition containing the enhancer, where the enhancer or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, or prodrug thereof is formulated as a nanobiological composition using nanoscale assemblies (IMPEPi-NA).

[0138] cancer The term "cancer" as used herein includes, but is not limited to, solid tumors and blood-borne tumors. The term "cancer" refers to diseases of skin tissue, organs, blood and blood vessels, including, but not limited to, cancer of the bladder, blood vessels, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovary, pancreas, prostate, rectum, skin, stomach, testicles, throat, thyroid, urothelium and uterus.

[0139] Specific cancers include, but are not limited to, aggressive malignant tumors, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastases, glioblastoma multiforme, glioblastoma, brainstem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C and D colorectal cancer, unresectable colorectal cancer, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karyotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, cutaneous B-cell lymphoma, diffuse large B-cell lymphoma, low-grade follicular lymphoma, malignant These include melanoma, malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, peritoneal cancer, papillary serous adenocarcinoma, gynecological sarcoma, soft tissue sarcoma, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressiva, hormone-refractory prostate cancer, resected high-risk soft tissue sarcoma, unresectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube cancer, androgen-independent prostate cancer, androgen-dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma. In a specific embodiment, the cancer is metastatic. In another embodiment, the cancer is refractory or resistant to chemotherapy or radiation, particularly thalidomide.

[0140] General Pharmaceutical Definitions As used herein, a "prophylactically effective" amount refers to an amount of a substance that is effective in preventing or delaying the onset of a given disease state in a subject to which the substance is administered. A prophylactically effective amount refers to an amount that is effective at the dosage and duration necessary to achieve the desired preventive result. Typically, a prophylactic dose is used in a subject before or at an early stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

[0141] As used herein, a "therapeutically effective" amount is an amount of a substance effective to treat, ameliorate, or alleviate the symptoms for which the substance is administered or the cause thereof in a subject suffering from a given condition.

[0142] In one embodiment, the therapeutically or prophylactically effective amount is from about 1 mg of agent / kg subject to about 1 g of agent / kg subject per administration. In another embodiment, the therapeutically or prophylactically effective amount is from about 10 mg of agent / kg subject to about 500 mg of agent / kg subject. In a further embodiment, the therapeutically or prophylactically effective amount is from about 50 mg of agent / kg subject to about 200 mg of agent / kg subject. In a further embodiment, the therapeutically or prophylactically effective amount is about 100 mg of agent / kg subject. In yet a further embodiment, the therapeutically or prophylactically effective amount is selected from 50 mg of agent / kg subject, 100 mg of agent / kg subject, 150 mg of agent / kg subject, 200 mg of agent / kg subject, 250 mg of agent / kg subject, 300 mg of agent / kg subject, 400 mg of agent / kg subject, and 500 mg of agent / kg subject.

[0143] The pharmaceutical compositions of the present invention may be adapted for administration by any suitable route, for example, oral (including buccal or sublingual), inhalation, nasal, ocular, or parenteral (intravenous and intramuscular) routes. Such compositions may be prepared by any method known in the art of pharmacy, for example, by bringing the active ingredient into association with the carrier or excipient. Parenteral dosage forms are preferred.

[0144] Parenteral dosage forms can be administered to patients by various routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready for solution or suspension in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[0145] Suitable vehicles that can be used to provide the parenteral dosage form of the present invention are well known to those skilled in the art. Examples include, but are not limited to, aqueous vehicles such as water for injection USP, sodium chloride injection, Ringer's solution, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's solution; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0146] Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention. For example, cyclodextrin and its derivatives can be used to increase the solubility of the nanoscale particles and derivatives of the invention.

[0147] The pH of a pharmaceutical composition or dosage form may be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. To improve delivery, compounds such as stearic acid can be added to a pharmaceutical composition or dosage form to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients. In this regard, stearic acid can function as a lipid vehicle for the formulation, as an emulsifier or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates, or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

[0148] Radiolabeling for PET imaging of drug accumulation in the body In a preferred, non-limiting embodiment of the present invention, there is provided a radiopharmaceutical composition and a method for radiopharmaceutical imaging of the accumulation of said nanobiological composition in the bone marrow, blood and / or spleen of a patient affected by trained immunity, comprising: (i) administering to said patient a nanobiological composition in an amount effective to promote a hyperresponsive innate immune response; The nanobiological composition (i) comprises nanoscale assemblies, and (ii) has a facilitating agent incorporated into the nanoscale assemblies and (iii) a positron emission tomography (PET) imaging agent incorporated into the nanoscale assemblies; the nanoscale assembly is a multi-component carrier composition comprising (a) a phospholipid and (b) apoA-I or a peptidomimetic of apoA-I, and optionally (c) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or combinations thereof, and optionally (d) cholesterol; The promoter is a molecular structure that activates or binds to the pathogen recognition receptor Dectin-1 or NOD2 to induce trained immunity in myeloid cells in the bone marrow, blood, and spleen and their stem and progenitor cells; molecular structures that activate or bind to Dectin-1 include, but are not limited to, β-glucan and its derivatives such as 11-13 gluco-oligomers; and molecular structures that activate or bind to NOD2 include, but are not limited to, peptidoglycan and its derivatives such as muramyl dipeptide and muramyl tripeptide; PET contrast agents 89 Zr, 124 I, 64 Cu, 18 F and 86 Y, and the PET imaging agent is complexed to the nanobiological composition using a suitable chelating agent to form a stable drug-contrast agent chelate; the nanobiological composition self-assembles in an aqueous environment into nanodiscs or nanospheres having a diameter size of about 8 nm to 400 nm; delivering the nanoscale assemblies to myeloid cells, myeloid progenitor cells, or hematopoietic stem cells in the patient's bone marrow, blood, and / or spleen, the stable drug-imaging agent chelate; (ii) performing PET imaging of the patient to visualize the biodistribution of the stable drug-imaging agent chelate within the bone marrow, blood, and / or spleen of the patient's body; and a method comprising:

[0149] In a non-limiting preferred embodiment, the method of radiopharmaceutical imaging comprises the further step of administering a checkpoint inhibitor to said patient simultaneously with or for a specified period of time after the nanobiological composition, thereby enhancing the efficacy of checkpoint inhibitor therapy by promoting a hyperresponsive innate immune response caused by trained immunity.

[0150] 89 An exemplary protocol using Zr is described in Example 5.

[0151] Additionally, ex vivo methods can be used to detect chromatin fragments using gamma counting or autoradiography. 89 The tissue uptake of Zr-labeled nanoparticles may be quantified to confirm the imaging results.

[0152] This also provides a novel approach to autoradiography-based histological examination, which allows for the assessment of the local distribution of nanomaterials within the tissue of interest by comparing the radioactive deposition pattern obtained by autoradiography with histological and / or immunohistochemical staining on the same or adjacent sections.

[0153] Currently, the most commonly used methods for assessing the in vivo behavior of nanotherapeutics rely on fluorescent dyes. However, these techniques are not quantitative due to autofluorescence, quenching, FRET, and the high sensitivity of fluorophores to their environment (e.g., pH or solvent polarity). The incorporation of magnetic resonance imaging contrast agents as nanoparticle labels has been attempted, but requires high payloads and administration, which compromises the integrity of the nanoparticle formulation. Nuclear contrast agents do not suffer from these drawbacks. 89Zr is particularly suitable for PET imaging due to its positron emission and its relatively long physical half-life (78.4 hours), which allows for longitudinal studies of slowly cleared materials and obviates the need for a nearby cyclotron.

[0154] The techniques described herein include: 89 This provides an excellent method for functionalizing nanobiological compositions with Zr. DSPE-DFO represents a stable method for immobilizing the DFO chelator in lipid monolayers or bilayers. Furthermore, because DFO is present outside the nanoparticle platform, nanoparticles can be labeled after formulation. This eliminates the need to formulate them under radiation-shielding conditions and reduces the amount of active agent that needs to be used. DSPE-DFO is first incorporated, and then 89 The mild conditions under which Zr is incorporated are compatible with a wide variety of nanoparticle types and formulation methods.

[0155] In yet another preferred embodiment of the invention where additional stability is desired in the formulation, the present invention provides a C 34 -DFO, 6 A lipophilic DFO derivative named

[0156] In yet a further non-limiting preferred embodiment of the present invention, the present invention provides a method for preparing a polymerizable composition comprising first formulating particles, then functionalizing the protein component with commercially available p-NCS-Bz-DFO, and finally preparing a polymerizable composition using our general procedure. 89 These include nanoparticles coated with radiolabeled proteins prepared by introducing Zr.

[0157] trained immunity Figure 14 shows a current schematic of the processes controlling trained immunity at the epigenetic, cellular, and systems levels. The first identified "trainers" include the fungal PAMP β-glucan and the bacterial PAMP peptidoglycan / BCG. Trained immunity is epigenetically regulated, resulting in a stronger response to restimulation. Myeloid progenitor cells can be stimulated to produce "trained" myeloid cells over the long term, thereby providing a compelling framework for sustained therapeutic intervention.

[0158] In vitro models in which human monocytes were exposed to either Candida albicans (C. albicans) or β-glucan demonstrated genome-wide changes in epigenetic marks, including H3K4me1, H3K4me, and H3K27Ac (Figure 14, top). Other studies have identified BCG and peptidoglycan as inducers of these training immune-related epigenetic modifications, albeit via NOD2-dependent pathways. In addition to these epigenetic modifications, cellular metabolic pathways are simultaneously upregulated. Indeed, these metabolic changes enhance the cell's ability to regulate the function of specific epigenetic enzymes. β-glucan training induces the Dectin-1 / Akt / mTOR / HIF-1α pathway to shift cellular metabolism from oxidative phosphorylation to glycolysis, which is associated with a decreased basal respiratory rate, increased glucose consumption, and higher lactate production.

[0159] These epigenetic and metabolic changes perfectly represent the enhanced response of individual myeloid cells to secondary insults, but until recently, it remained unclear how this innate immune memory is preserved over the long term. While monocytes have a lifespan of only a few days, the protective function of trained immunity is preserved for longer periods in patients, up to several months or even a year. Recent insights have revealed that, at a systems level, trained immunity is a functional program induced in specific hematopoietic stem and progenitor cells (Figure 14, bottom). When mice were administered β-glucan, more myeloid-biased multipotent progenitor cells (MPPs) and long-term hematopoietic stem cells (LT-FlSCs) were observed in the bone marrow. Various cell proliferation-related pathways, including cell cycle genes, cholesterol biosynthesis pathways, and glycolysis, were upregulated, and these increases were identified as IL-1β- and granulocyte / macrophage colony-stimulating factor (GM-CSF)-dependent. The longevity of these effects was found to last up to one month, and transplantation of hematopoietic stem cells from β-glucan-trained mice induced myelopoiesis in untrained recipients. Similar observations were made after administration of BCG.

[0160] Because trained immunity is a property of myeloid-biased progenitor cells, nanomaterials designed to accumulate in myeloid progenitor cells are illustrated to induce long-term therapeutic effects that target trained immunity.

[0161] FIG. 15 shows that cells exhibiting trained immunity are regulated at the cellular level by bacterial, fungal, and metabolic pathways, resulting in epigenetic modifications that underlie cytokine secretion.

[0162] Immunological signaling events leading to a trained immune phenotype The induction of trained immunity by microbial ligands is driven by specific receptor signaling pathways, which subsequently activate metabolic, epigenetic, and transcriptional events. An overview of the most important pathways currently identified is provided in the figure.

[0163] Dectin-1-dependent fungal pathway Innate immune cells initiate nonspecific immune responses against foreign pathogens after recognizing β-glucans. β-glucans present in fungal cell walls are glucose polymers that are recognized by macrophages as PAMPs via the C-type lectin receptor dectin-151. Macrophage activation via dectin-1 induces specific epigenetic marks that result in trained immunity (Figure 15, red pathway). This activation pathway, which can be exploited for therapeutic intervention, is typical of fungal infections, such as nonlethal infection with Candida albicans. As mentioned in the introduction, Candida albicans has been shown to protect mice from lethal candidiasis through monocyte-dependent trained immunity.

[0164] NOD2-dependent bacterial pathway Peptidoglycan is a PAMP that cooperates with endotoxin to trigger the release of proinflammatory cytokines. The minimal bioactive motif of peptidoglycan shared by all bacteria is muramyl dipeptide (MDP). MDP-mediated activation of innate immune cells involves the cytoplasmic PRR nucleotide-binding oligomerization domain 2 (NOD2). NOD2 activation and signaling via NF-κB stimulates epigenetic rewiring of macrophages and induces trained immunity (Figure 15, green pathway). This trained immune activation pathway is characterized by bacterial infection, such as BCG vaccination, which results in proinflammatory cytokine production. The nonspecific protective effect of BCG has been exploited as an immunotherapy for noninvasive bladder cancer.

[0165] Oxidized low-density lipoprotein Lipid metabolism can lead to the induction of trained immunity. Oxidized low-density lipoprotein (oxLDL) is a DAMP that binds to the cell surface receptor CD36. Upon internalization and release into the cytoplasm, oxLDL can lead to the formation of cholesterol crystals, thereby activating the NLRP3 inflammasome. A recent report using Ldlr- / - mice highlighted the crucial role of NRLP3 activation by consumption of a Western diet and established a mechanistic link between oxLDL-induced trained immunity and cardiovascular disease via inflammasome activation. oxLDL induces a long-lasting proinflammatory phenotype in monocytes and accelerates atherosclerosis, whereas the histone methyltransferase inhibitor methylthioadenosine completely abolished oxLDL-induced training.

[0166] Metabolic and epigenetic rewiring during induction of trained immunity One of the most important effects of trained immunity is the rewiring of innate immune cell metabolism. A key part of this rewiring is the metabolic switch from oxidative phosphorylation to aerobic glycolysis, which results in innate immune cell activation and proinflammatory cytokine secretion. Candida albicans and β-glucan induce this specific metabolic process via the AKT / mTOR / Hif-1α pathway. BCG vaccination also induces immune metabolic activation and epigenetic remodeling, which involves the inhibition of glycolysis by 2-deoxyglucose (2-DG) during BCG training, which suppresses the increase in cytokine production (Figure 15, purple pathway). Pharmacological modulation of rate-limiting glycolytic enzymes disrupts the histone marks H3K4me3 and H3K9me3, which underlie both β-glucan- and BCG-induced trained immunity.

[0167] Another important metabolic event in trained monocytes is the anabolic repurposing of the Krebs cycle to synthesize cholesterol and phospholipids from citrate and acetyl-CoA. The cholesterol synthesis pathway is upregulated after β-glucan training due to the restriction of cholesterol synthesis by fluvastatin, which downregulates H3K4me3 and prevents proinflammatory cytokine production and trained immunity. Synthesizing the cholesterol metabolite mevalonate is crucial in this process, as trained immunity is prevented by enzyme inhibitors downstream of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA)-reductase (Figure 15, yellow pathway). Inhibiting glycolysis with 2-DG, inhibiting the mTOR pathway with rapamycin, and inhibiting histone methylation with methylthioadenosine (MTA, a methyltransferase inhibitor) prevents mevalonate-induced trained immunity, demonstrating the delicate balance between molecular, metabolic, and epigenetic regulation in trained macrophages.

[0168] The Krebs cycle is replenished by glutaminolysis. Interestingly, this leads to the accumulation of succinate, particularly fumarate, which is a cofactor for a family of important epigenetic enzymes. In this regard, succinate represses JMJD3, resulting in H3K27 trimethylation of specific genes (e.g., those associated with the M2 phenotype). However, JMJD enzyme expression was not different in trained monocytes. In contrast, fumarate inhibits the KDM5 histone demethylase. Both KDM5 expression and function have been found to be blocked / disturbed in trained monocytes. Because KDM5 is a demethylase for H3K4 methylation, its repression allows the long-term stability of this important mark on open chromatin, thus promoting gene transcription.

[0169] Promoting trained immunity BCG induced trained immunity through the NOD2-dependent bacterial pathway. NOD2 is an intracellular PRR activated by peptidoglycan, a polymeric structure composed of sugars and amino acids that is essential for bacterial cell walls. The smallest molecular structure capable of inducing a NOD2-dependent immune response is muramyl dipeptide (MDP). MDP is a synthetic peptide complex consisting of a short amino acid chain of N-acetylmuramic acid and L-alanine-D-isoglutamine dipeptide.

[0170] Alternatively, trained immunity can be induced by fungal pathogens via the dectin-1 pathway. Dectin-1 is a C-type lectin transmembrane signaling receptor that can be activated by polysaccharides rich in β1,3-linked glucose or both β1,3- and β1,6-linked glucose, known as β-glucans. Palma and colleagues extensively studied other dectin-1-activating polysaccharides, including liposomal formulations, and found that 1,3-linked glucose oligomers with a minimum length of 10 or 11 mers were required for dectin-1 binding. Therefore, unlike NOD2 binding, small molecule ligands are not available for dectin-1-dependent trained immunity induction.

[0171] In addition to PAMP-related mechanisms, metabolic "trainers" such as uric acid and oxLDL have been shown to induce trained immunity through mTOR signaling and protein kinase B (AKT) phosphorylation. This indicates that uric acid itself can be used to induce trained immunity. While the exact mechanism by which oxLDL induces training remains a subject of investigation, Christ and colleagues have provided compelling evidence for the importance of the NLRP3 inflammasome and downstream IL-1R signaling pathways, highlighting the crucial role of IL-1β. Also of interest in the context of oxLDL is the recently discovered role of mevalonate, a cholesterol synthesis intermediate. Bekkering and colleagues found that mevalonate induces training through activation of the IGF-1 receptor (IGF-1R) and mTOR, and subsequent histone modification 6l. Therefore, mevalonate, further enhanced by 6-fluoromevalonate, can be used pharmacologically to induce trained immunity. As research progresses, currently unknown pathways and molecular structures, including other bacterial and fungal derivatives and viral PAMPs, that promote trained immunity will likely be identified.

[0172] Figure 16 shows a schematic of the process, demonstrating that bone marrow-specific nanomaterials that inhibit (green) or promote (red) trained immunity can be used to stimulate the immune system and treat a variety of diseases ranging from cardiovascular disease and its clinical consequences, autoimmune disorders, to sepsis and infections, and cancer.

[0173] Nanoparticle delivery vehicles can increase the percentage of drugs that reach their intended targets and improve the toxicity profile of therapeutic agents. Furthermore, nanoparticle delivery vehicles can facilitate cellular internalization of drugs, which is particularly relevant to nucleotide therapeutics. Furthermore, nanoparticles can protect drugs from premature metabolism or degradation.

[0174] FIG. 17 illustrates that stimulating the immune system's sensitivity to immune checkpoint blockade therapy can be achieved by promoting trained immunity.

[0175] For example, it is becoming increasingly clear that checkpoint blockade immunotherapy benefits only a subset of patients in certain tumor types. A pooled analysis of the KEYNOTE-001127 trial found that approximately 34% of patients with late-stage melanoma had an objective response, with 6% of these patients being complete responders. Furthermore, in a variety of other malignancies, including prostate and ovarian cancer, the therapeutic effect of checkpoint inhibitors has been very low.

[0176] A recent study of peripheral blood from patients using high-dimensional single-cell mass cytometry and a bioinformatics pipeline revealed that the frequency of classically activated monocytes predicts treatment response. Furthermore, high levels of immunosuppressive myeloid cells result in impaired T cell function and a failure to respond to checkpoint blockade immunotherapy. We predict that training immunostimulatory therapy can promote systemic and tumor accumulation of classically activated monocytes, thereby enhancing sensitivity to checkpoint inhibitors, as outlined in Figure 17. [Example]

[0177] The following examples are included to demonstrate embodiments of the present disclosure. The following examples are provided by way of illustration only and to aid those of ordinary skill in the art in using the present disclosure. The examples in no way otherwise limit the scope of the present disclosure. Those of ordinary skill in the art should, in light of the present disclosure, understand that many variations are possible in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0178] Example 1 - Microfluidizer Assembly 1 This example demonstrates the preparation of a pharmaceutical composition containing a stimulant and nanoscale aggregates, where the stimulant concentration is 4-8 mg / mL in the nanoscale aggregate / emulsion and the formulation is prepared at a 300 mL scale. The stimulant (2400 mg) is dissolved in 12 mL of chloroform / t-butanol. This solution is then added to 288 mL of a nanoscale aggregate solution (3% w / v) containing a POPC / PHPC phospholipid mixture, apoA-I, tricaprylin, and cholesterol. The mixture is homogenized (Vitris homogenizer model Tempest IQ) at 10,000-15,000 rpm for 5 minutes to form a crude emulsion, then transferred to a high-pressure homogenizer. The emulsification is performed at 20,000 psi with emulsion recirculation. The resulting system is transferred to a Rotavap, and the solvent is rapidly removed at 40°C and reduced pressure (25 mm Hg). The resulting dispersion is clear. This dispersion is filtered sequentially through multiple filters, with the size of the filtered formulation ranging from 8 to 400 nm.

[0179] Example 2 - Microfluidizer Assembly 2 This example demonstrates the preparation of a pharmaceutical composition containing a stimulator and nanoscale assemblies, where the stimulator concentration is 4-8 mg / mL in the nanoscale assemblies / emulsion and the formulation is prepared at a 300 mL scale. The stimulator (2400 mg) is dissolved in 12 mL of chloroform / t-butanol. This solution is then diluted with a mixture of POPC / PHPC phospholipids, a peptidomimetic of apoA-I, C 16 ~C 20 A 288 mL nanoscale aggregate solution (3% w / v) containing a mixture of triglycerides, a mixture of cholesterol and one or more sterol esters, and a hydrophobic polymer was added. The mixture was homogenized (Vitris homogenizer model Tempest IQ) at 10,000-15,000 rpm for 5 minutes to form a crude emulsion, then transferred to a high-pressure homogenizer at 20,000 psi with recirculation of the emulsion.

[0180] The resulting system is transferred to a Rotavap and the solvent is rapidly removed at 40°C and reduced pressure (25 mm Hg). The resulting dispersion is transparent. This dispersion is then filtered sequentially through multiple filters. The size of the filtered formulation is between 35 and 100 nm.

[0181] Example 3 - Freeze-drying of the nanobiological compositions of Examples 1 and 2 A nanobiological composition is formed as in any of the above examples. The dispersion is then freeze-dried for an additional 60 hours (FTS Systems, Dura-Dry μP, Stone Ridge, NY). The resulting freeze-dried cake can be easily reconstituted to the original dispersion by adding sterile water or 0.9% (w / v) sterile saline. The particle size after reconstitution is the same as before freeze-drying.

[0182] Example 4 - Nanobiological composition treatment alone or in combination with checkpoint inhibitors was effective in reducing tumor size and enhancing trained immunity The MTP-HDL nanobiological composition was formulated from the phospholipids DMPC, cholesterol, and muramyl tripeptide phosphatidylethanolamine (MTP-DSPE) as described herein.

[0183] In vitro assays in which monocytes were exposed to each "training" agent (β-glucan, MDP, or MTP-HDL) for 24 hours, then restimulated with LPS, showed that MTP-HDL induced training immunity in human monocytes in vitro, as evidenced by increased IL-6 and TNF-α secretion (Figure 1). In vivo PET-CT revealed that MTP-HDL induced training immunity in human monocytes in vitro. 89 The in vivo behavior of the Zr-labeled MTP-HDL nanobiological composition was quantitatively and non-invasively studied. High binding activity to bone marrow (Figure 2) and the presence of MTP-HDL in hematopoietic stem cells and myeloid progenitor cells were observed.

[0184] A dose-response study was conducted in C57BL / 6 mice bearing B16F10 melanoma tumors, using different regimens: low dose (0.375 mg / kg MTP), high dose (1.5 mg / kg MTP), and a non-functionalized HDL control group, with one, two, or three injections. Dose- and regimen-dependence was observed without any adverse effects (Figure 3). As shown in Figure 3, all doses of MTP-HDL reduced tumor volume most effectively, with the higher dose of 1.5 mg / kg administered two or three times.

[0185] The most effective regimen, consisting of three intravenous MTP-HDL injections at 1.5 mg / kg (MTP), was administered to normal C57BL / 6 mice. At several time points after the final MTP-HDL injection, mice were sacrificed and monocyte counts were quantified. A clear increase in monocyte counts was observed as a result of MTP-HDL treatment (Figure 4).

[0186] In a separate set of experiments, typical C57BL / 6 mice were given three intravenous MTP-HDL injections at 1.5 mg / kg (MTP) and subsequently subjected to FDG-PET imaging of the bone marrow. Because FDG is a sugar analog, its uptake is proportional to metabolic activity, which was found to be higher in the bone marrow of mice treated with MTP-HDL (Figure 5). In vivo treatment studies were performed using MTP-HDL in combination with different checkpoint blockade immunotherapies. Treatment groups consisted of a 200 μg checkpoint inhibitor dose of anti-CTLA-4 (Figure 6), anti-PD-1 (Figure 7), or a combination of both (Figures 8 and 9), with or without the simultaneous induction of training immunity with MTP-HDL. The combination of checkpoint blockade with MTP-HDL-induced training immunity resulted in significantly enhanced antitumor activity compared to several controls.

[0187] Flow cytometry analysis of cells in blood, bone marrow, and spleen revealed that not only did MDP-HDL alone increase both monocytes and CD11b+ cells in all tissues more than control and combined anti-CTLA4 and anti-PD-1 therapy, but the combination of all three was most effective in increasing both cell types in all tissues (see Figures 10-13).

[0188] Example 5 - Radiopharmaceutical labeling of training immunostimulants In a non-limiting example, radiopharmaceutical labeling of training immunostimulants / molecules can be achieved via various chelators, primarily 3-hydroxamate groups. 89 This can be achieved with deferoxamine B (DFO), which can form a stable chelate with Zr.

[0189] Generally, a phospholipid is conjugated with a chelator compound, a nanobiological composition is prepared with the enhancing drug or molecule, and finally, a radioisotope is complexed to the nanobiological composition (which already has the chelator attached).

[0190] This protocol describes the reaction of the phospholipid DSPE with an isothiocyanate derivative of the chelating agent DFO (p-NCS-Bz-DFO), its formulation into nanobiological compositions, and the generation of nanoemulsions, followed by the dissolution of these nanoformulations. 89 This involves the synthesis of DSPE-DFO obtained by radiolabeling with Zr.

[0191] radioactive isotope 89 Zr was chosen due to its 3.3 day physical decay half-life, which eliminates the need for a nearby cyclotron and allows for the study of drugs that are slowly cleared from the body, such as antibodies. 89 Due to the relatively low positron energy of Zr 124 This allows for higher imaging resolution compared to other isotopes such as I.

[0192] Nanotherapeutics 89Zr labeling allows noninvasive study of in vivo behavior by positron emission tomography (PET) imaging in patients.

[0193] The protocol is: conjugating the chelator deferoxamine B (DFO) to the phospholipid DSPE, thereby forming a lipophilic chelator (DSPE-DFO) that is easily incorporated into different lipid nanoparticle platforms (approximately 0.5 wt%); preparing a nanoscale aggregate formulation incorporating DSPE-DFO (using sonication, hot dropwise nanoemulsion generation, or microfluidics); The nanoparticles were incubated in PBS at pH 7 at 30-40°C. 89 DSPE-DFO containing lipid nanoparticles by mixing with Zr-oxalate for 30–60 min. 89 A process of labeling with Zr Includes:

[0194] Furthermore, purification and characterization methods were used to obtain radiochemically pure 89 Zr-labeled lipid nanoparticles are obtained. Purification is typically achieved using either centrifugal filtration or a PD-10 desalting column, followed by evaluation using size-exclusion radio-HPLC. Radiochemical yields are typically greater than 80%, and radiochemical purities of greater than 95% are routinely achieved.

[0195] PET / CT or PET / MRI using common imaging strategies 89 To study the in vivo behavior of Zr-labeled nanobiological compositions.

[0196] FIG. 19 shows PET imaging using a radioisotope delivered by the nanobiological composition and demonstrates the accumulation of the nanobiological composition in the bone marrow and spleen of mouse, rabbit, monkey and pig models.

[0197] Example 6 - Synthesis of nanobiological compositions containing prodrugs material and method All chemicals were purchased from Sigma-Aldrich, Medchem Express, or Selleckchem, and PES syringe filters were obtained from Celltreat. A NE-1002X model microfluidic pump from World Precision Instruments was used in combination with a Zeonor herringbone mixer (#14-1038-0187-05) from Microfluidic-chipshop. Particles were purified using a 20 mL Vivaspin centrifugal filter with a 100 kDa MWCO. Dialysis bags were from Thermo Scientific. ApoA-I protein was purified in-house using a previously published procedure. Spectroscopic quantification of ApoA-I was performed on a BioTek Cytation 3 imaging plate reader using the Bradford assay. DLS and zeta potential measurements were performed on a Brookhaven Instrument ZetaPals analyzer, and particle size was determined by averaging the number distribution. A Bruker 600 Ultrashield magnet connected to a Bruker Advance 600 console was used. 1 H and 13 C NMR samples were analyzed and data processed using Topspin version 3.5 pl7.

[0198] Quantitative analysis of all drugs was performed using C 18 The HPLC analysis was performed using a Shimadzu UFLC system equipped with either a CN or CN column. Acetonitrile and water were used as the mobile phase, and compounds were detected with an SPD-M20a diode array detector.

[0199] Synthesis of nanobiological compositions of approximately 35 nm From a 10 mg / ml chloroform stock solution, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 250 μL), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC, 65 μL), cholesterol (15 μL), tricaprylin (1000 μL), and drug or prodrug (65 μL) were combined in a 20 ml vial and vacuum-dried. The resulting membrane was redissolved in an acetonitrile:methanol mixture (95%:5%, 3 ml total volume). Separately, a solution of ApoA-I protein in PBS (0.1 mg / ml) was prepared. Both solutions were simultaneously injected into a herringbone mixer using a microfluidic setup at a flow rate of 0.75 ml / min for the lipid solution and 6 ml / min for the ApoA-I solution. The resulting solution was concentrated by centrifugal filtration at 4000 rpm using a 100 MWCO Vivaspin tube to obtain a volume of 5 mL. PBS (5 mL) was added, and the solution was concentrated to 5 mL. PBS (5 mL) was added again, and the solution was concentrated to approximately 3 mL. The remaining solution was filtered through a 0.22 μm PES syringe filter to obtain the final nanobiological composition solution. To obtain the nanobiological composition for FACS measurement, 3,3'-dioctadecyloxacarbocyanine perchlorate (DIO-C) was added. 18 , 0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to an acetonitrile solution (prepared in-house). To scale up the synthesis of the nanobiological composition, the above procedure was simply repeated until a sufficient amount was produced.

[0200] Synthesis of this nanobiological composition (approximately 15 nm) For the synthesis of 15 nm-sized nanoparticles, a microfluidic procedure similar to that for 35 nm-sized particles was used. The acetonitrile mixture here contained POPC (250 μL) (again from a 10 mg / mL stock solution), PHPC (15 μL), cholesterol (13 μL), and the drug or prodrug (65 μL). The acetonitrile solution was injected at a flow rate of 0.75 mL / min. ApoA-I solution (0.1 mg / mL in PBS) was injected at 3 mL / min. To obtain the nanobiological composition for FACS measurement, DiO-C was injected at a flow rate of 0.75 mL / min. 18 (0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to the acetonitrile solution.

[0201] Synthesis of this nanobiological composition (approximately 65 nm) For the synthesis of 65 nm-sized nanoparticles, a microfluidic procedure similar to that for 35 nm-sized particles was used. The acetonitrile mixture here contained POPC (250 μL) (again from a 10 mg / mL stock solution), cholesterol (12 μL), tricaprylin (1400 μL), and the drug or prodrug (65 μL). The acetonitrile solution was infused at a flow rate of 0.75 mL / min. ApoA-I solution (0.1 mg / mL in PBS) was infused at 4 mL / min. To obtain the nanobiological composition for FACS measurement, DiO-C was used. 18 (0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to the acetonitrile solution.

[0202] Synthesis of this nanobiological composition (approximately 120 nm) For the synthesis of 120 nm-sized nanoparticles, a microfluidic procedure similar to that for 35 nm-sized particles was used. The acetonitrile mixture here contained POPC (100 μL) (again from a 10 mg / mL stock solution), cholesterol (10 μL), tricaprylin (4000 μL), and the drug or prodrug (65 μL). The acetonitrile solution was infused at a flow rate of 0.75 mL / min. ApoA-I solution (0.1 mg / mL in PBS) was infused at 1.5 mL / min. To obtain the nanobiological composition for FACS measurement, DiO-C was used. 18 (0.25 mg) was added to the acetonitrile solution. 89 To obtain the nanobiological composition for Zr labeling, DSPE-DFO (50 μg) was added to the acetonitrile solution.

[0203] Size stability of four different sized nanoparticles Aliquots (10 μL) of the final particle solution were dissolved in 1 mL of PBS, filtered through a 0.22 μm PES syringe filter, and analyzed by DLS to determine the mean of the number-average size distribution. Samples were analyzed immediately after particle synthesis and 2, 4, 6, 8, and 10 days later.

[0204] The embodiments herein and their various features and advantageous details are more fully described with reference to the non-limiting embodiments illustrated in the accompanying drawings and detailed in the above description. Descriptions of well-known components and processing techniques are omitted so as not to unnecessarily obscure the embodiments herein. The examples used herein are merely intended to facilitate understanding of how the embodiments herein can be implemented and to further enable those skilled in the art to implement the embodiments herein. Therefore, the examples should not be construed as limiting the scope of the embodiments herein.

[0205] Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0206] The terms used herein are merely for the purpose of describing particular embodiments and do not limit the overall scope of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, it will be understood that the terms "comprise" and / or "comprising," when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups.

[0207] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Nothing in this disclosure should be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used herein, the term "comprising" means "including, but not limited to."

[0208] As will be apparent to those skilled in the art, many modifications and variations can be made without departing from the spirit and scope of the invention. Functionally equivalent methods and apparatuses within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to particular methods, reagents, compounds, compositions, or biological systems, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the invention. With respect to virtually any plural and / or singular terms herein, those of skill in the art can translate from the plural to the singular and / or from the singular to the plural where appropriate for context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0209] It will be understood by those skilled in the art that, in general, the terms used in this specification, and in the appended claims in particular (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., "including" should be interpreted as "including, but not limited to," "having" should be interpreted as "having at least," "include" should be interpreted as "including, but not limited to," etc.). It will be further understood by those skilled in the art that substantially any disjunctive word and / or disjunctive phrase presenting two or more other terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of those terms, either of those terms, or both of those terms. For example, the phrase "A or B" should be understood to include the possibilities of "A" or "B" or "A and B."

[0210] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is thereby also described with respect to any individual member or subgroup of members of the Markush group.

[0211] As will be understood by those skilled in the art, for any and all purposes, including for purposes of providing this specification, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. Any listed range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal subportions. As will be understood by those skilled in the art, a range includes each individual member.

[0212] Some or all of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements thereon may be subsequently devised by those skilled in the art, each of which is intended to be encompassed by the disclosed embodiments.

[0213] While embodiments of the invention have been described herein, it should be noted that modifications and variations are possible for those skilled in the art in light of the above teachings. It is therefore understood that variations are possible in the particular embodiments of the invention disclosed which are within the scope and spirit of the invention as defined by the appended claims. Having described the invention above with the detail and particularity required by the Patent Laws, what is claimed and what is desired to be protected by Letters Patent is set forth in the appended claims.

Claims

1. A nanobiological composition for promoting trained immunity, comprising nanoscale assemblies, The nanoscale assembly is Phospholipids and human apolipoprotein AI (apoA-I); a NOD2 activator; and a multi-component carrier composition comprising: The nanobiological composition is a nanodisc or nanosphere having a diameter size of about 8 nm to 400 nm. Nanobiological compositions.

2. 10. The nanobiological composition of claim 1, wherein said NOD2 activator is bacterial peptidoglycan.

3. 3. The nanobiological composition of claim 2, wherein the bacterial peptidoglycan is muramyl dipeptide (MDP) or muramyl tripeptide (MTP).

4. 3. The nanobiological composition of claim 2, wherein said bacterial peptidoglycan is MDP.

5. 5. The nanobiological composition of claim 4, wherein the MDP is derivatized with a phospholipid, an aliphatic chain, or a sterol.

6. The nanobiological composition of claim 2 , wherein said bacterial peptidoglycan is MTP.

7. 7. The nanobiological composition of claim 6, wherein the MTP is derivatized with a phospholipid, an aliphatic chain, or a sterol.

8. 8. The nanobiological composition of any one of claims 1 to 7, wherein the phospholipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and mixtures thereof.

9. 9. The nanobiological composition of claim 8, wherein said phospholipid is DMPC.

10. 10. The nanobiological composition of any one of claims 1 to 9, wherein the nanobiological composition is a nanosphere comprising a hydrophobic matrix core, the nanosphere having a diameter of about 30 nm to about 150 nm.

11. 11. The nanobiological composition of claim 10, wherein said hydrophobic matrix core comprises one or more triglycerides, fatty acid esters, cholesterol, or combinations thereof.

12. 12. The nanobiological composition of claim 11, wherein said hydrophobic matrix core comprises one or more triglycerides.

13. 13. The nanobiological composition of claim 12, wherein said triglyceride is tricaprylin.

14. The nanobiological composition of any one of claims 1 to 9, wherein the nanobiological composition is a nanodisc having a diameter of about 8 nm to about 35 nm.

15. The nanoscale assembly is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), human apoA-I, a NOD2 activator; and Including, The NOD2 activator is MDP or MTP. The nanobiological composition of claim 1.

16. The nanobiological composition of any one of claims 1 to 15, adapted for intravenous administration.

17. The nanobiological composition of any one of claims 1 to 16, adapted for administration to a human.

18. 18. The nanobiological composition of any one of claims 1 to 17, wherein said nanoscale assemblies deliver said NOD2 activator to myeloid progenitor cells, said cells located in bone marrow.

19. The nanobiological composition of any one of claims 1 to 18 for treating cancer or sepsis in a patient in need thereof.

20. 20. The nanobiological composition of any one of claims 1 to 19 for promoting tumor remission in a patient in need thereof, comprising: (1) the patient is receiving chemotherapy, radiation therapy, immunotherapy, or a combination thereof; (2) the patient is administered the nanobiological composition; Nanobiological compositions.

21. 21. The nanobiological composition of claim 20, wherein the patient is receiving immunotherapy, and the immunotherapy is a checkpoint inhibitor.

22. 20. The nanobiological composition of claim 19, wherein the cancer is a cancer of the bladder, blood vessels, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lymph node, lung, mouth, neck, ovary, pancreas, prostate, rectum, skin, stomach, testicle, throat, thyroid, urothelium, or uterus.

23. 20. The nanobiological composition of claim 19, wherein the patient has severe sepsis or is in septic shock.

24. 20. The nanobiological composition of claim 19, wherein the patient has sepsis associated with a bacterial, viral, or fungal infection of the lungs, abdomen, kidneys, or bloodstream.

25. 20. The nanobiological composition of claim 19, wherein the patient is co-administered an anti-cancer agent as a combination therapy with the nanobiological composition.

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