Formulations and methods for cell therapy involving chimeric antigen receptors

Abstract

The energy of chimeric antigen receptor (CAR)-containing cells can be enhanced by obtaining platelets from blood, obtaining mitolets from the platelets, and co-incubating the mitolets with CAR-containing cells. Incorporation of mitolets into CAR-containing cells results in enhanced CAR-containing cells with increased metabolic activity. The enhanced CAR-containing cells can be used to treat indications treatable by CAR-containing cells. Mitolets can include coated mitochondria or mitochondria enclosed in vesicles.

Description

[Background technology]

[0001] The present technology relates generally to formulations and methods for enhancing chimeric antigen receptor (CAR)-based technologies, and more specifically to formulations and methods for using mitlets to increase the energy and persistence of CAR-expressing cells in CAR-based therapies.

[0002] Clinical therapies based on cells expressing CARs have shown promising clinical outcomes for various cancer indications. CARs are synthetic receptors engineered to bind to specific antigens on the surface of target cells (e.g., cancer cells) and trigger a cytotoxic response that results in the death of the target cells. Typically, CARs are expressed in immunological cells (e.g., T cells and natural killer (NK) cells). CARs contain an extracellular domain (e.g., a single-chain variable fragment, scFV) that recognizes and binds to an antigen on the target cell, a transmembrane domain, a spacer domain (e.g., an IgG hinge region) that connects the extracellular and transmembrane domains, and a costimulatory domain.

[0003] In a typical CAR-T cell treatment, T cells are removed from the patient. These autologous T cells are sent to a laboratory where a gene for a chimeric tumor antigen-specific receptor is added to the T cells. This addition results in the expression of the gene in the T cells, thereby generating CAR-T cells. The CAR-T cells are expanded to reach a sufficient number for an effective dose. The laboratory freezes these expanded CAR-T cells and sends them back to a hospital or treatment center. At the hospital or treatment center, these frozen CAR-T cells are thawed and infused into the patient. While CAR-related therapies have shown promise for various cancer indications, there are still several issues with this type of therapy that limit its widespread adoption.

[0004] Some of the issues with CAR-based therapies include: (1) the development of cytokine release syndrome (CRS) and neurotoxicity, which is particularly prominent with CD19 CAR-T cell therapy; (2) on-target off-tumor effects that may be related to the recognition of molecular biomarkers expressed in healthy tissues (e.g., B-cell aplasia in anti-CD19 / CD20 CAR-T cell therapy); (3) antigen escape / defects that may lead to disease relapse (e.g., CD19-negative relapse in B-cell malignancies); (4) graft-versus-host disease (GVHD); (5) HLA restriction; (6) a scarce T-cell source; (7) slow and expensive growth and expansion cultures; and (8) limited persistence after infusion into patients. Lu H et al., "From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies," Front Oncol. 2021, August 6;11:720-501. The limited persistence may be due to weakening of the CAR-T cells due to the expansion and freeze-thaw process, which degraded mitochondrial quality, or the patient was at an age when immunological cells were already weakened. To address these issues, various formulations and methods are provided for incorporating mitochondria (vesicle-enclosed mitochondria; or coated mitochondria) into CAR-based therapies and during the generation and expansion of CAR-expressing cells used in such therapies. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 10,113,147 [Non-patent literature]

[0006] [Non-Patent Document 1] Lu H et al. “From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies” Front Oncol. August 6, 2021;11:720501 [Non-patent document 2] Wang X and Riviere I, "Clinical manufacturing of CAR T cells: foundation of a promising therapy," Mol Ther Oncolytics, 2016 Jun 15;3:16015 [Non-patent document 3] Moreau T et al. “Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.” Nat Commun. 2016;7:11208 [Non-patent document 4] Thon et al. “Platelet bioreactor: accelerated evolution of design and manufacture” Platelets July 2017; 28(5): 472-477 [Non-patent document 5] Morris EC et al. “Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy” Nat Rev Immunol. February 2022;22(2):85-96 [Non-patent document 6] Hirano T, "IL-6 in inflammation, autoimmunity and cancer" Int Immunol. March 1, 2021; 33(3): 127-148 [Non-Patent Document 7] Yang P et al. “Immunological Feature and Transcriptional Signaling of Ly6C Monocyte Subsets From Transcriptome Analysis in Control and Hyperhomocysteinemic Mice.” Front Immunol. February 25, 2021;12:632333 [Non-patent document 8] Fan X et al., "CD49b defines functionally mature Treg cells that survey skin and vascular tissues." J Exp Med. 2018, November 5;215(11):2796-2814 Summary of the Invention

[0007] The formulations and methods disclosed herein each have several aspects, no single one of which is solely responsible for its desirable properties. Without limiting the scope of the claims, some prominent features will now be briefly discussed. Numerous other embodiments are also contemplated, including embodiments having fewer, additional, and / or different components, steps, features, objects, benefits, and desirable properties. Components, aspects, and steps may also be arranged and ordered differently. After considering this discussion, and particularly after reading the section entitled "Detailed Description of the Invention," one will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

[0008] In some embodiments, a method for enhancing the activity of chimeric antigen receptor (CAR)-containing cells is provided, the method comprising obtaining platelets from blood; obtaining mitlets from the platelets; co-incubating the mitlets with CAR-containing cells, wherein uptake of the mitlets by the CAR-containing cells enhances the metabolic activity of the CAR-containing cells; and preparing the enhanced CAR-containing cells for treatment of an indication in a patient. In some embodiments, the indication comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the tumor comprises a non-solid tumor.

[0009] In some embodiments, the blood is from the patient. In some embodiments, the blood is from a donor. In some embodiments, the donor comprises a family member of the patient.

[0010] In some embodiments, obtaining platelets includes adding an anticoagulant and a buffer to blood to form a mixture; separating the mixture into a supernatant and platelet-rich plasma (PRP); and collecting the PRP. In some embodiments, the anticoagulant includes an acid citrate dextrose anticoagulant (ACD). In some embodiments, the technology described herein relates to a method of claim 9, wherein the buffer includes a Tyrode's buffer. In some embodiments, the Tyrode's buffer has a pH in the range of 6.0 to 7.0.

[0011] In some embodiments, the step of obtaining mitolets includes stimulating collected PRP, thereby releasing extracellular vesicles from platelets in the PRP; and collecting the extracellular vesicles, wherein the collected extracellular vesicles contain mitochondria, and the mitolets contain the collected extracellular vesicles. In some embodiments, the step of stimulating PRP includes exposing PRP to an immune complex in the presence of Ca. In some embodiments, the immune complex comprises heat-aggregated IgG. In some embodiments, the concentration of the heat-aggregated IgG is 0.1 mg / mL to 2.5 mg / mL.

[0012] In some embodiments, the method further comprises matching the mtDNA haplotype of the obtained mitochondria with the mtDNA haplotype of the patient.

[0013] In some embodiments, the step of obtaining mitolets further comprises expanding the mitolets in a bioreactor. In some embodiments, the method further comprises coating the mitolets after the expanding step. In some embodiments, the co-incubating step occurs during the production of the CAR-containing cells.

[0014] In some embodiments, the preparing step comprises adding a cryopreservation agent to the mitlet and enhanced CAR-containing cells; and freezing the mitlet and enhanced CAR-containing cells. In some embodiments, the cryopreservation agent is selected from the group consisting of sugars, oligosaccharides, and polysaccharides. In some embodiments, the cryopreservation agent comprises DMSO. In some embodiments, the cryopreservation agent comprises trehalose. In some embodiments, the cryopreservation agent comprises phosphate-buffered saline.

[0015] In some embodiments, the method further comprises administering a therapeutically effective amount of Mitlet to the patient after the preparing step. In some embodiments, the administering step further comprises administering a therapeutically effective amount of enhanced CAR-containing cells to the patient. In some embodiments, the administering step of CAR-containing cells precedes the administration of Mitlet. In some embodiments, the steps of administering CAR-containing cells and administering Mitlet to the patient occur simultaneously. In some embodiments, the administering step further comprises administering a follow-up amount of Mitlet at least two days after the initial administering step. In some embodiments, the administering step is repeated after every two days.

[0016] In yet another embodiment, a method of treating an indication is provided, the method comprising administering therapeutically effective amounts of mitlets and CAR-containing cells to a patient to treat the indication, wherein the mitlets are obtained from a source. In some embodiments, the indication comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the tumor comprises a non-solid tumor. In some embodiments, the source is placental tissue. In some embodiments, the source is bone marrow. In some embodiments, the source is adipose tissue. In some embodiments, the source is platelets obtained from blood. In some embodiments, the mitlets have been expanded in a bioreactor.

[0017] In some embodiments, the mitolet comprises a coating. In some embodiments, the coating comprises asialoglycoprotein (AsG). In some embodiments, the AsG comprises asialoorosomucoid (AsOR). In some embodiments, the coating further comprises poly-L-lysine linked to AsOR. In some embodiments, the coating is complexed with a conjugate. In some embodiments, the conjugate comprises AsOR. In some embodiments, the conjugate further comprises listeriolysin O (LLO).

[0018] In some embodiments, the step of administering Mitlet occurs before the step of administering CAR-containing cells. In some embodiments, during the administering step, Mitlet is co-administered with the CAR-containing cells. In some embodiments, the method further comprises administering a follow-up dose of Mitlet to the patient at least two days after the administering step. In some embodiments, the method further comprises administering a repeat dose of Mitlet to the patient at least every two days after the administering step.

[0019] In some embodiments, the CAR-containing cells comprise CAR-T cells, hi some embodiments, the CAR-containing cells comprise CAR-NK cells.

[0020] In yet another embodiment, a formulation for treating an indication is provided, the formulation comprising mitochondria-containing mitochondria; and cells expressing a CAR, wherein the mitolets are derived from a source. In some embodiments, the CAR-expressing cells are T cells. In some embodiments, the CAR-expressing cells are NK cells. In some embodiments, the indication comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the tumor comprises a non-solid tumor. In some embodiments, the source comprises placental tissue. In some embodiments, the source comprises bone marrow. In some embodiments, the source comprises adipose tissue. In some embodiments, the source comprises platelets. In some embodiments, the platelets are obtained from blood. In some embodiments, the mitolets are expanded in a bioreactor.

[0021] In some embodiments, the technology described herein relates to a formulation further comprising a cryopreservation agent. In some embodiments, the cryopreservation agent is from the group consisting of saccharides, oligosaccharides, and polysaccharides. In some embodiments, the cryopreservation agent comprises DMSO. In some embodiments, the mitolet and CAR-expressing cells are held in separate containers. In some embodiments, the container comprises a cryopreservation bag. In some embodiments, the container is made of fluorinated ethylene propylene (FEP). In some embodiments, the container is made of ethylene vinyl acetate (EVA). In some embodiments, the container is made of polyolefin.

[0022] The methods and formulations disclosed herein each have several aspects, no single one of which is solely responsible for its desirable properties. Without limiting the scope of the claims, some prominent features will now be briefly discussed. Numerous other examples are also contemplated, including examples having fewer, additional, and / or different components, steps, features, objects, benefits, and advantages. The components, aspects, and steps may also be arranged and ordered differently. One or more of the disclosed steps may be repeated any number of times. After considering this discussion, and particularly after reading the section entitled "Detailed Description of the Invention," one will understand how the features of the methods disclosed herein provide desirable properties over other known devices and methods.

[0023] It should be understood that any features of the methods disclosed herein may be combined together in any desired manner. It should further be understood that any combination of features of the methods may be used together and / or combined with any of the examples disclosed herein. It should further be understood that any feature or combination of features of any of the methods may be combined together and / or combined with any of the examples disclosed herein in any desired manner.

[0024] It should be appreciated that all combinations of the foregoing concepts, and the additional concepts described in more detail below, are contemplated as part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.

[0025] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numbers correspond to similar, but possibly not identical, components. For the sake of brevity, reference numbers or features having previously described functionality may or may not be described with reference to other drawings in which they appear. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows a diagrammatic representation of the overview of CAR immunotherapy, from collection of immune cells from the patient to infusion of engineered autologous immune cells into the patient. [Figure 2] 1 is a flowchart illustrating some methods of embodiments. [Figure 3] 1 is a flowchart of the process for collecting and preparing mitolets for transplantation according to some embodiments of the present invention. [Figure 4A-B] 4A-4B show mitlets according to some embodiments of the present invention, where FIG. 4A shows the exclusion of mitlets from platelets and FIG. 4B shows mitlet uptake in various cells. [Figure 5] FIG. 1 illustrates a method for growing multiple types of mitochondria and coating thereof for transport and infusion according to some embodiments of the present invention. [Figure 6] FIG. 1 shows a diagrammatic representation of a combination therapy comprising administration of Mitlet at various stages of immunotherapy involving CAR cells according to some embodiments of the present invention. [Figure 7] FIG. 1 is a dot plot showing the mitolet population, where DsRed-labeled mitolets represent approximately 40% of total CD41+ cells. [Figure 8A-B] 8A and 8B are confocal fluorescence images showing mitlet uptake by immunological cells in some embodiments of the present invention, where FIG. 8A shows mitlet uptake after 60 minutes and FIG. 8B shows mitlet uptake after 24 hours. [Figure 9] 1 is a confocal fluorescence image showing mitolet uptake by bone marrow tissue over a period of just 15 minutes in accordance with some embodiments of the present invention. [Figure 10A-B] 10A and 10B are graphs showing cellular uptake of mitlet as determined by a fluorescence-based cell tracking assay according to some embodiments of the present invention, where FIG. 10A shows cellular uptake of mitlet in neutrophils and FIG. 10B shows cellular uptake of mitlet in leukocytes. [Figure 11]Confocal fluorescence images showing mitlet uptake by T cells. Blue-labeled elements in the image represent the nuclei of T cells. Green-labeled elements in the image represent the cell membrane. Red-labeled elements represent mitlet. [Figure 12A-B] 12A and 12B show the effect of Mitlet of some embodiments of the present invention on cytokine storm induced in a mouse model via H1N1 infection, where FIG. 12A shows the measured concentrations of IL-6 plasma levels before and after infection with H1N1 among various treatment groups, and FIG. 12B shows post-infection survival rates among various treatment groups. [Figure 13] FIG. 1 shows the survival rates of different treatment groups of mice that experienced induced sepsis according to some embodiments of the present invention. [Figure 14A-D] Figures 14A and 14B show the survival rates of various treatment groups in which mitolets isolated from young mice or isolated liver mitochondria from young mice were administered to aged recipient mice with induced sepsis infection. Figures 14A and 14B show the results of the mitolet administration experiment, and Figure 14C shows the combined results of Figures 14A and 14B. Figure 14D shows the results of administering isolated liver mitochondria. [Figure 15A-D] Figures 15A and 15B show results showing significantly reduced cytokine IL-6 and bacterial levels after Mitlet administration. Figure 15A shows bacterial levels after Mitlet administration in the first experiment. Figure 15B shows bacterial levels after Mitlet administration in the second experiment. Figure 15C shows bacterial levels from a combination of Figures 15A and 15B. Figure 15D shows plasma IL-6 levels in the first experiment. [Figure 16] Figure 1 shows the results of flow cytometry cell sorting of neutrophil isolation according to some embodiments of the present invention. After cell sorting, 66.4% of the sorted cells were CD11b+ and Ly6G+. [Figure 17] Figure 1 shows the results of flow cytometry cell sorting of monocyte isolation according to some embodiments of the present invention. After cell sorting, 84.5% of the sorted cells were CD11b+ and Ly6C+. [Figure 18] Figure 1 shows flow cytometry cell sorting of T cell isolation according to some embodiments of the present invention. After cell sorting, 80% of the sorted cells were CD3+. [Figure 19] Figure 1 shows flow cytometry cell sorting of CD3+ cells according to some embodiments of the present invention. After cell sorting, 64% of the sorted cells were CD4+ and 28% of the sorted cells were CD8+. [Figure 20] Figure 1 shows flow cytometry cell sorting of NK cell isolation according to some embodiments of the present invention. After cell sorting, 74.2% of the sorted cells were CD49+ and CD3-. [Figure 21A-B] 21A and 21B show the bioenergetic profiling of immune cells of some embodiments of the present invention after 6 hours of co-incubation with mitochondria, as measured by oxygen consumption rate (OCR), where FIG. 21A shows the OCR of bone marrow neutrophils and FIG. 21B shows the OCR of T cells. [Figure 22A-B] 22A and 22B show some embodiments of bioenergetic profiling measured by OCR of bone marrow neutrophils and T cells after vesicle or mitolet co-incubation, where FIG. 22A shows basal OCR and FIG. 22B shows maximum OCR. [Figure 23] FIG. 1 shows bioenergetic profiling of resting and activated T cells according to some embodiments of the present invention. [Figure 24A-B] Figures 24A and 24B show bioenergetic profiling of T cells measured by OCR, according to some embodiments of the present invention. Figure 24A shows resting T cells in the presence of various concentrations of mitlet (number of mitlets per cell) after 24 hours. Figure 24B shows activated T cells in the presence of various concentrations of mitlet (number of mitlets per cell) and mito. [Figure 25A-B]Figure 25 shows the bioenergetic profiling of resting and activated T cells after Mitlet uptake as measured by OCR, according to some embodiments of the present invention: Figure 25A shows basal OCR and Figure 25B shows maximum OCR. DETAILED DESCRIPTION OF THE INVENTION

[0027] All patents, applications, published applications, and other publications referenced herein are incorporated herein in their entirety by reference to the material to which they refer. Where a term or phrase is used herein in a manner contrary to or otherwise inconsistent with the definition set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the usage herein controls over the definition incorporated herein by reference.

[0028] Sepsis, COVID-19, cancer, and many other diseases tend to be more severe in older individuals because their immune systems weaken with age. A new treatment approach is being developed to reverse immune weakness by, at least temporarily, rejuvenating an older immune system. A recently discovered immune component called Mitlet is being infused into patients for the treatment of various diseases or conditions. Mitlet can be viewed as a kind of biological "battery pack" into which platelets, T cells, NK cells, neutrophils, and other immune components trade to conserve energy. "Young" Mitlet can be grown in specialized bioreactors for their growth and expansion and then infused into the body, where these immune cells readily take up the Mitlet and use its internal mitochondria. In earlier studies, mammalian models of disease receiving these infusions showed increased immunity, reduced cytokine storms, and dramatically improved survival against bacterial and viral infections. The energy boost provided by Mitlet persists for at least several weeks. ISET (Immune System Energetic Transplantation) therapy is considered comparable to other immunotherapies such as CAR-T, monoclonal antibodies, and checkpoint inhibitors.

[0029] ISET therapy replaces the immune system's "battery pack," allowing it to act faster and fight infection more effectively, essentially temporarily rejuvenating the aging immune system. In some embodiments, ISET therapy involves growing large numbers of specialized extracellular vesicles called mitlets in an external bioreactor and then injecting them into the patient's bloodstream. In some embodiments, ISET therapy involves harvesting large numbers of mitlets from platelets from a blood donor. The mitlets are almost immediately absorbed by other immune components floating in the blood, such as neutrophils, T cells, NK cells, and platelets, allowing them to enhance or improve the metabolic capacity of each of these components and improve their respective cellular capabilities.

[0030] ISET not only provides the immune system with additional mitochondria, but can also provide younger mitochondria to elderly individuals. In elderly individuals, mitochondria tend to have a declining metabolic capacity, thereby reducing their ability to fully meet the metabolic needs of cells. ISET therapy can propagate younger, more powerful mitochondria contained in mitochondria (obtained from seed material from young donors). When transplanted into the immune system of an elderly individual, these younger mitochondria are taken up by various immunological components of the elderly individual's immune system, thereby "combining" with existing mitochondria and enhancing the metabolic capacity of cells, thus making the immune system stronger (i.e., "younger").

[0031] As shown in Figure 1, the CAR T-cell therapy process begins with the collection of peripheral blood from a patient and leukapheresis to isolate and extract peripheral blood mononuclear cells (PBMCs), which contain white blood cells. More recently, umbilical cord blood (a product of childbirth) can be another source of immunological cells. After leukapheresis, T cells are isolated from the collected immunological cells (e.g., white blood cells) and then activated ex vivo using a variety of different techniques, including, but not limited to, cell-based activation, bead-based activation (e.g., antibody-coated magnetic beads, antibody-coated nanobeads, etc.), soluble and dissociated T cell stimulatory reagents (e.g., Expamer), and anti-CD3 antibodies. Wang X and Riviere I, "Clinical manufacturing of CAR T cells: foundation of a promising therapy," Mol Ther Oncolytics, 2016 Jun 15;3:16015. As shown in Figure 1, immunological cells are genetically modified to express a CAR after delivery by viral or non-viral delivery systems. Some examples of viral delivery systems include gamma retroviral vectors and lentiviral vectors, the latter being more commonly used due to their safer integration site profile. Some examples of non-viral delivery systems include transposon / transposase-based systems and mRNA electroporation.

[0032] Following CAR expression in immunological cells, the CAR-expressing immunological cells are grown and expanded. There are several platforms available for expanding these CAR-expressing cells. One non-limiting example is the expansion of CAR-containing cells using a bioreactor. Another non-limiting example is the Miltenyi CliniMACS Prodigy® system, which includes a cell washer, a magnetic cell separation system, and a cell culture device. CAR-T cells and CAR-NK cells isolated from immunological cells are expanded using the CliniMACS Prodigy® system. Another non-limiting example of the expansion of CAR-containing cells is repeated artificial antigen-presenting cell (AAPC) stimulation, whereby expanded CAR-containing cells generated by a transposon / transposase-based system are repeatedly stimulated with gamma-irradiated AAPC in the presence of stimulatory molecules such as cytokines (e.g., IL-2 and IL-21). In some examples, engineered cell lines such as K652 express a variety of stimulatory molecules, such as CD40, CD40L, CD70, CD80, CD83, CD86, CD137L, ICOSL, GITRL, and CD134L. Wang X and Riviere I, Mol Ther Oncolytics, 2016 Jun 15;3:16015. After expansion, CAR-containing cells are formulated using various detergents and formulations. Furthermore, formulated CAR-containing cells may be preserved via freezing (e.g., cryopreservation) for transportation. Examples of components used in cryopreservation media include dimethyl sulfoxide (DMSO).

[0033] To reduce some of the limitations associated with CAR-based therapy, a method for enhancing the activity of CAR-containing cells is provided, as shown in FIG. 2A , comprising obtaining mitolets as platelet-derived mitochondria in step 210; co-incubating the mitolets with CAR-containing cells in step 240, wherein uptake of the mitolets by the CAR-containing cells enhances the metabolic activity of the CAR-containing cells; and delivering an amount of CAR-containing cells with enhanced metabolic activity to a subject in step 280 for treatment of an indication. In some embodiments, the indication comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the tumor comprises a non-solid tumor.

[0034] As shown in FIG. 2B, in some embodiments, the process for obtaining platelets includes adding an anticoagulant and a buffer to blood to form a mixture in step 220; separating the mixture into a supernatant and platelet-rich plasma (PRP) in step 222; and collecting the PRP in step 224. In some embodiments, the process for obtaining mitolets includes stimulating the collected PRP in step 230, thereby releasing extracellular vesicles from the platelets in the PRP; and collecting the extracellular vesicles in step 232, wherein the collected extracellular vesicles contain mitochondria, and the mitolets contain the collected extracellular vesicles. As shown in FIG. 3, in some embodiments, platelets are obtained from the blood of a donor or patient. In some embodiments, the donor includes a family member of the patient. In some embodiments, the blood is derived from a mouse. Mouse blood is used to test the feasibility of a method for extracting mitolets. In some embodiments, the blood is derived from a human donor.

[0035] In some embodiments, the step of adding an anticoagulant and buffer to the blood prevents the blood from thickening and solidifying. In some embodiments, the anticoagulant is ACD (20%). In some embodiments, the buffer is 40% Tyrode's buffer, pH 6.5. After adding the anticoagulant and buffer to the blood, the mixture is then separated into PRP. In some embodiments, the separation is by centrifugation. Plasma is the liquid portion of whole blood. Plasma is composed mostly of water and proteins and provides a medium for red blood cells, white blood cells, and platelets to circulate throughout the body. Platelets are the blood cells responsible for blood clots and other necessary growth and healing functions. After centrifugation of the mixture, the blood cells form a pellet that accumulates at the bottom of the tube. The pellet, called PRP, contains concentrated platelets.

[0036] In some embodiments, a buffer is then added to the collected PRP, and the platelets are resuspended. The platelets are then activated or stimulated. In some embodiments, any number of substances may be used to activate the platelets, including radioactive carbon isotopes, prostaglandins, serotonin, adenosine triphosphate, collagen, L-lactate dehydrogenase, thrombin, magnesium, adenosine, calcium, heat-aggregated antibodies, or any combination thereof. In some embodiments, the platelets are activated by freeze-thaw cycling. As used herein, the term "freeze-thaw cycling" refers to freezing the mitochondria of some embodiments to a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a defined period of time, and thawing the mitochondria to room temperature, body temperature, or any temperature above 0°C. The term "room temperature," as used herein, refers to a temperature between 18°C ​​and 25°C. The term "body temperature," as used herein, refers to a temperature between 35.5°C and 37.5°C, preferably 37°C.

[0037] Finding a source of mitochondria for transplantation is a challenge. As with any donated organ, mitochondria from young, healthy donors are in short supply. While some diseases or injuries can be cured with autologous mitochondria harvested from a patient's own leg muscles, for example, for many other diseases, "patients" have poor-quality mitochondria due to age or mitochondrial DNA (mtDNA) mutations. For these patients, donated mitochondria are a preferable solution. Furthermore, freshly isolated mitochondria rapidly die within minutes of isolation and can also elicit immune reactivity if they enter the bloodstream intact, thereby reducing their effectiveness as a therapy. To overcome these obstacles, it would be advantageous to find a readily available source of mitochondria that are easy to donate and, at the same time, coated or encapsulated in vesicles that reduce detection or reactivity by the immune system.

[0038] Platelets from human blood contain an average of 4-5 mitochondria, which are released into extracellular vesicles upon platelet activation. In some embodiments, platelet-derived mitochondria-containing extracellular vesicles are referred to herein as mitlets. These mitlets are typically larger (>400 nM) than those in other platelet extracts or lysates (30-100 nM), and other less well-known sizes may be present as well.

[0039] Mitlet has been shown to donate mitochondria to nearby cells and increase the respiratory activity of cells that have absorbed mitlet, thereby improving the cellular respiration (and metabolic capacity) of cells that take up mitlet. Platelet-derived mitlet contains several desirable properties, any one or a combination of which makes it suitable for rapid commercialization: mitlet can be extracted from platelets that have "expired" and would otherwise have to be discarded; mitlet represents another good, medically relevant use for platelets that would otherwise be wasted; mitlet can be collected in most blood banks that are already in close proximity to hospitals, which already have all the necessary skilled personnel, clean handling experience, and necessary equipment, making mitlet products potentially available for global use very soon. In some embodiments, mitlet can be included in a variety of platelet transfusions, making it easy to incorporate and test by medical professionals already familiar with transfusion therapy.

[0040] In some embodiments, the blood is from a mammalian subject. In some embodiments, the mammalian subject is a human subject. In some embodiments, the mammalian subject is selected from the group consisting of a human, a horse, a dog, a cat, a mouse, a rat, a cow, and a sheep. Each possibility represents a separate embodiment of the present invention.

[0041] In some embodiments, the mitlets comprise mitochondria and mitochondrial coatings. In some embodiments, the mitlets are derived from mammalian cells. In some embodiments, the mammalian cells are human cells. In some embodiments, the mitlets are derived from cells in culture. In some embodiments, the mitlets are derived from tissue. In some embodiments, the mitlets are derived from cells or tissue selected from the group consisting of human placenta, human placental cells grown in culture, and human blood cells. In some embodiments, the mitlets are derived from cells or tissue selected from the group consisting of placenta, placental cells grown in culture, and blood cells. In some embodiments, the mitlets are derived from adipose tissue.

[0042] In some embodiments, obtaining mitochondria includes selecting a source comprising mitochondria and stem cells. In some embodiments, the source includes placental tissue comprising mitochondria and stem cells. In some embodiments, the source includes bone marrow cells comprising mitochondria and stem cells. In some embodiments, the source includes adipose tissue comprising mitochondria and stem cells.

[0043] In some embodiments, the step of obtaining mitochondria further comprises extracting stem cells and mitochondria from a source and separating the stem cells and mitochondria into a first and a second pool, respectively. In some embodiments, the extraction step comprises separating the stem cells and miscellaneous mitochondria and isolating these cells into two different pools of material. In some embodiments, the first pool of material comprises stem cells. In some embodiments, the stem cells are isolated before the miscellaneous mitochondria. In some embodiments, the second pool comprises miscellaneous mitochondria extracted from the remainder of the tissue derived from the previously extracted stem cells. In some embodiments, the remainder of the tissue is disrupted prior to the step of extracting the miscellaneous mitochondria. In some embodiments, the tissue comprises umbilical cord blood. In some embodiments, the tissue comprises umbilical cord. In some embodiments, the tissue comprises bone marrow. In some embodiments, the tissue comprises adipose tissue. In some embodiments, the tissue comprises any tissue associated with stem cells. In some embodiments, the tissue comprises any stem cell-containing tissue.

[0044] In some embodiments, as shown in FIG. 5 , the step of obtaining mitochondria further comprises expanding the mitochondria in a bioreactor, and expanding the mitochondria further comprises incubating (i.e., packing) the heterogeneous mitochondria into stem cells and expanding the packed stem cells in a bioreactor. In some embodiments, the heterogeneous mitochondria not used by the stem cells may be used for therapy. In some embodiments, the step of obtaining mitolets further comprises expanding the stem cells packed with the heterogeneous mitochondria at the fastest rate allowed by the bioreactor. In some embodiments, the step of obtaining mitolets further comprises adjusting selection conditions to favor high-quality mitochondria. In some embodiments, selection conditions include hypoxia, glucose starvation, use of the TreeFrog method, or any combination thereof. In some embodiments, the step of expanding mitochondria in a bioreactor further comprises differentiating the remaining stem cells into megakaryocytes, thereby increasing the number of mitochondria produced by 100-fold or even 1000-fold compared to traditional cell culture techniques. Megakaryocytes are polyploid cells derived from hematopoietic stem cells found in bone marrow. Their role includes participating in localized clot formation to block bleeding and producing platelets. Megakaryocytes expanded in bioreactors can be derived from hematopoietic stem cells found in bone marrow as well as from other sources.

[0045] In some embodiments, megakaryocytes are generated / induced from stem cells sourced from adipose tissue. In some embodiments, the adipose tissue comprises subcutaneous adipose tissue. In some embodiments, the method further comprises obtaining a source. In some embodiments, the source comprises adipose tissue, wherein the adipose tissue is obtained by any known technique in the art. In some embodiments, after obtaining the adipose tissue, the method further comprises digesting the adipose tissue with a digestive agent. In some embodiments, the digestive agent is type II collagenase. In some embodiments, the method further comprises centrifuging the digested adipose tissue to produce adipose-derived mesenchymal stromal stem cells (ASCL). In some embodiments, the method further comprises treating the ASCL with a megakaryocyte lineage induction medium to produce megakaryocytes. In some embodiments, the megakaryocytic lineage induction medium includes 2 mM L-glutamine in Iscove's modified Dulbecco's medium (IMDM); 100 U / mL penicillin-streptomycin solution; 0.5% bovine serum albumin (BSA); 4 μg / mL LDL cholesterol; 200 μg / mL iron-saturated transferrin; 10 μg / mL insulin; 50 μM 2-β mercaptoethanol, nucleotides (about 20 μM each of ATP, UTP, GTP, and CTP), and 50 ng / mL thrombopoietin (TPO). See also U.S. Patent No. 10,113,147.

[0046] In some embodiments, megakaryocytes are generated / derived from stem cells originating from pluripotent stem cells (PSCs). In some embodiments, obtaining mitochondria further comprises obtaining PSCs. In some embodiments, the method further comprises transducing expression of a transcription factor in the PSCs via a vector. In some embodiments, the transcription factor is cloned into a vector backbone. In some embodiments, the vector comprises a lentiviral vector. In some embodiments, the transcription factor includes GATA-binding protein 1 (GATA1); friend leukemia integration 1 (FLI1); and T-cell acute lymphocytic leukemia protein 1 (TAL1). See also Moreau T et al., "Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming." Nat Commun. 2016;7:11208. In some embodiments, the method further comprises maintaining the transduced PSCs in PSC medium for about 2 days. In some embodiments, the PSC medium comprises fibroblast growth factor (FGF2) and activin A. In some embodiments, the method further comprises maintaining the transduced PSCs in megakaryocyte medium. In some embodiments, the megakaryocyte medium comprises TPO and SCF for at least 5 days after the step of maintaining in PSC medium.

[0047] In some embodiments, the method further comprises incubating / transferring the extracted mitochondria to the extracted stem cells to produce encased stem cells. In some embodiments, the method further comprises expanding the encased stem cells in a bioreactor. In some embodiments, the method further comprises adjusting the environmental conditions of the bioreactor to favor the proliferation of encased stem cell mitochondria. In some embodiments, the method further comprises converting the encased stem cells into megakaryocytes. In some embodiments, the method further comprises isolating mitochondria from megakaryocytes. In some embodiments, the method further comprises applying a coating to the mitochondria after isolation of the mitochondria from the megakaryocytes.

[0048] In some embodiments, the method further comprises matching the mtDNA haplotype of the obtained mitolet with the mtDNA haplotype of the patient, at least to the extent that the patient does not develop an acute immune response to the mitolet.

[0049] In some embodiments, the step of obtaining mitolets further comprises a step of expanding the mitolets in a bioreactor. In some embodiments, the method further comprises a step of coating the mitolets. In some embodiments, the coating step occurs after the expansion step. In some embodiments, the coating comprises asialoorosomucoid (AsOR). In some embodiments, the coating further comprises poly-L-lysine (PL). In some embodiments, the method further comprises a step of complexing the coated mitolets with a conjugate. In some embodiments, the conjugate comprises AsOR. In some embodiments, the conjugate further comprises listeriolysin O (LLO).

[0050] In some embodiments, the step of administering Mitlet occurs before the step of administering CAR-containing cells. In some embodiments, during the administering step, Mitlet is co-administered with the CAR-containing cells. In some embodiments, the method further comprises administering a follow-up dose of Mitlet to the patient at least two days after the administration of the CAR-containing cells. In some embodiments, the method further comprises administering a repeat dose of Mitlet to the patient at least every two days after the administering step.

[0051] In some embodiments, Mitlet is added during the method for producing CAR-containing cells, as shown in Figure 6 as a co-incubating step. In some embodiments of the method, the co-incubating step occurs during the production of CAR-containing cells. In some embodiments of the method, the co-incubating step occurs during the growth and expansion of cells expressing a CAR gene, as shown in Figure 6. In some embodiments of the method, the co-incubating step occurs during activation of T cells. In some embodiments of the method, the co-incubating step occurs while engineering T cells with a CAR gene.

[0052] In some embodiments, the preparing step comprises adding a cryopreservation agent to the mitlets and the enhanced CAR-containing cells. In some embodiments, the cryopreservation agent is selected from the group consisting of sugars, oligosaccharides, and polysaccharides. In some embodiments, the cryopreservation agent comprises DMSO. In some embodiments, the cryopreservation agent comprises trehalose. In some embodiments, the preparing step further comprises freezing the mitlets and the enhanced CAR-containing cells.

[0053] In some embodiments, the method further comprises administering a therapeutically effective amount of Mitlet to the patient after the preparing step. In some embodiments, the administering step further comprises administering a therapeutically effective amount of enhanced CAR-containing cells to the patient. In some embodiments, the administering step of CAR-containing cells precedes the administration of Mitlet. In some embodiments, the steps of administering CAR-containing cells and administering Mitlet to the patient are performed simultaneously. In some embodiments, the administering step further comprises administering a follow-up dose of Mitlet at least two days after the initial administering step. In some embodiments, the administering step is repeated every two days after the initial administering step.

[0054] In another embodiment, a method of treating an indication is provided, the method comprising administering a therapeutically effective amount of mitochondria and a therapeutically effective amount of CAR-containing cells to a patient to treat the indication. In some embodiments, the indication comprises a tumor. In some embodiments, the tumor comprises a solid tumor. In some embodiments, the tumor comprises a non-solid tumor.

[0055] In some embodiments, the mitolets are obtained from a source. In some embodiments, the source is placental tissue. In some embodiments, the source is bone marrow. In some embodiments, the source is adipose tissue. In some embodiments, the source is platelets obtained from blood.

[0056] In some embodiments, mitolets are harvested from expanded cells or cell fragments in a bioreactor. Platelets are produced by megakaryocytes in the bone marrow. Mitolets may also be harvested from vesicles released from activated platelets, and platelet growth and expansion in a bioreactor provides the ability to grow and harvest mitolets on a large scale. An example of using primary platelets for platelet production in a bioreactor has been described. Thon et al., "Platelet bioreactor: accelerated evolution of design and manufacture," Platelets 2017 July;28(5):472-477.

[0057] In some embodiments, the CAR-containing cells comprise CAR-T cells, hi some embodiments, the CAR-containing cells comprise CAR-NK cells.

[0058] In some embodiments, the CAR-containing cells and mitlets are stored in separate containers. In some embodiments, the CAR-containing cells and mitlets are combined in the same container before administration to a patient. In some embodiments, the mitlets and CAR-containing cells are co-administered to the same administration site in a patient. In some embodiments, the administration of the CAR-containing cells and mitlets is staggered so that they do not overlap at the same administration site, where the CAR-containing cells are administered before the mitlets (or vice versa). In some embodiments, the administration sites of the CAR-containing cells and mitlets are different from each other. In some embodiments, the container comprises a bag. In some embodiments, the bag is a cryopreservation bag. In some embodiments, the bag comprises fluorinated ethylene propylene (FEP). In some embodiments, the bag comprises ethylene vinyl acetate (EVA). In some embodiments, the bag comprises a polyolefin.

[0059] The following examples are presented to provide a more complete understanding of the present invention. The specific techniques, conditions, materials, proportions, and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention.

[0060] Example 1 This example describes how to collect vesicles and mitochondria, which contain vesicles and vesicle-encapsulated mitochondria, from mammalian blood, according to some embodiments.

[0061] The following steps were carried out:

[0062] 1. Blood was collected from a mouse donor. In this particular protocol, the donor was a male DsRed mouse, a transgenic mouse expressing the red fluorescent protein variant DsRed. MST was expressed under the control of the chicken β-actin promoter linked to the cytomegalovirus (CMV) immediate-early enhancer. Three 1 mL volumes of blood (1 mL per mouse) were used.

[0063] 2. ACD (20%) was added as an anticoagulant, and 40% Tyrode's buffer (TB) pH 6.5 was also added to the blood. The blood mixture (20% ACD + 40% Tyrode's buffer (TB) pH 6.5) was then centrifuged at 500 g for 3 minutes. The PRP and buffy coat were then collected and centrifuged at 300 g for 2 minutes.

[0064] 3. PRP was collected and 20% ACD + 10 mM EDTA was added before a centrifugation step at 1300 g for 5 minutes.

[0065] 4. Each pellet was suspended in 0.1 mL TB pH 6.5 and 0.9 mL TB pH 7.4 was added.

[0066] 5. Platelets were pooled, counted using a Cellometer and diluted to 10e8 / mL in TB 7.4.

[0067] 6. A total of 900 million platelets were obtained and 5 mM CaCl2 was added before stimulation.

[0068] 7. Platelets were co-stimulated with 0.5 mg / mL heat-aggregated IgG at room temperature overnight (16 hours). Heat-aggregated IgG was prepared by agglutinating human IgG (25 mg / mL, MPBIO) at 62°C for 1 hour.

[0069] 8. 10 mM EDTA was added to stop the stimulation.

[0070] 9. The stimulated platelets were centrifuged at 300 g for 5 minutes to remove any remaining platelets or cells.

[0071] 10. The supernatant was collected and the mitolets were analyzed using a flow cytometer.

[0072] 11. Evaluation of the remaining platelets showed less than 1% contamination.

[0073] 12. The obtained mitolets were diluted 3-fold with PBS and centrifuged at 18,000 g for 90 minutes at 18°C.

[0074] 13. The pellet was resuspended in 0.3 mL PBS and the mitolets were counted by flow cytometry at a concentration of 1.5 x 10 9 It was estimated that there were 100 Mytretinoin / ml.

[0075] 14. Mitlets can be tagged with a CD41 tag, allowing them to be counted by flow cytometry. When tagged in this way, mitlets accounted for approximately 40% of all CD41+ PEVs. A dot plot representing the mitlet population is illustrated in Figure 7. (DsRed = mitlet).

[0076] Example 2 Mouse monocytes were isolated from bone marrow and incubated with mouse mitolets for at least 24 hours. Mouse mitolets were generated from platelet activation in transgenic mice expressing a fluorescent protein (e.g., DsRed) in mitochondria. The results shown in Figure 8A show that isolated monocytes absorbed mitolets in just 60 minutes. As shown in Figure 8B, at 24 hours, isolated monocytes absorbed significant amounts of mitochondria from mitolets.

[0077] When Mitlet was injected into mice in vivo, it was rapidly taken up by platelets, white blood cells, bone marrow, and spleen. As shown in Figure 9, uptake of Mitlet by bone marrow tissue in vivo occurred within 15 minutes (as shown) or faster. Other cells that readily take up Mitlet include neutrophils, white blood cells, and bone marrow and spleen cells. With regard to neutrophils, as shown in Figure 10A, these cells readily take up Mitlet within a 2-minute incubation. Similar to white blood cells, Mitlet was observed to be taken up by these important T cells within a 2-minute incubation with Mitlet. As shown by these data, various cell types vigorously take up Mitlet within just a few minutes (or less) of exposure, thereby rapidly enhancing not only the number but also the quality of mitochondria contained within them.

[0078] As visualized by the confocal images in Figure 11, mitlet was shown to be internalized by T cells in vitro. The nuclei of the cells in the image are represented by blue-labeled cellular structures. The cell membranes in the image are represented by green-labeled cellular structures; in the nearby T cells, the cell membranes appear dim because they are slightly outside the focal plane of the image. Finally, mitlet is represented by red-labeled cellular structures. As indicated by the arrows in Figure 11, mitlet is shown to be successfully taken up by T cells.

[0079] Example 3 CRS is a major drawback of CAR-T cell therapy, and (in severe cases) CRS manifests along with other features of a systemic inflammatory response, including hypotension, hypoxia, and / or organ failure, the latter involving the cardiac, pulmonary, hepatic, renal, and gastrointestinal systems. Morris EC et al., "Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy," Nat Rev Immunol. 2022 Feb;22(2):85-96. To evaluate the ability of Mytret® to alleviate CRS and / or its effects, a mouse model of H1N1 infection, specifically the H1N1(PR8) influenza infection model, was used. The groups included in the study were: (1) healthy controls without exposure to H1N1, n = 5; (2) untreated H1N1-exposed controls; (3) those receiving Mitlet, n = 10 (five received Mitlet frozen in phosphate-buffered saline (PBS) and five received Mitlet frozen in cryopreservative); and (4) those receiving fresh liver mitochondria, n = 10. Groups (2) through (4) were infected with H1N1. Plasma cytokine levels, particularly interleukin 6 (IL-6) (pg / mL), were measured at various time points: 1 day before infection, and 3 and 7 days after infection, and the results are shown in Figure 12A. IL-6 is a pleiotropic inflammatory cytokine that is often involved in the fatal CRS seen with CAR-T cell treatment, symptoms of which can begin to appear within days of T cell infusion. Hirano T, "IL-6 in inflammation, autoimmunity and cancer" Int Immunol. March 1, 2021; 33(3): 127-148.

[0080] As shown in Figure 12A, healthy controls not exposed to H1N1 exhibited low baseline plasma concentrations of IL-6. However, in the group exposed to H1N1 without treatment, IL-6 levels reached approximately 70 pg / mL. When treated with frozen mitochondria, IL-6 levels only reached approximately 30 pg / mL, more than 50% lower than the group exposed to H1N1 without treatment. Mice administered fresh liver mitochondria did not exhibit lower levels of IL-6 concentration in plasma. However, the attenuation of IL-6 release from mice receiving fresh liver mitochondria was not as strong as that from frozen mitochondria.

[0081] Referring to Figure 12A, blood was collected from each animal and processed to plasma for cytokine analysis by Luminex (n = 5-10 per group). Samples were analyzed in one single analyte at 4-fold dilutions. Each point represents the mean ± SEM for the group at each time point. The functional lower limit of quantitation (LLOQ) (3.34 pg / mL) is plotted as a black dotted line. Values ​​below the LLOQ are plotted as 0 pg / mL.

[0082] As shown in Figure 12B, survival of each group was measured using Kaplan-Meier survival estimation. On day 7, the group exposed to H1N1 but not receiving treatment had the lowest chance of survival of all groups, with a survival rate of 40% by day 7. The H1N1 group receiving fresh liver mitochondria had the next lowest survival rate by day 7. For the H1N1 group receiving pre-frozen Mitlet, survival rate by day 7 was just under 60%, but still significantly higher than the H1N1 group without treatment. These results indicate that Mitlet administration can reduce cytokine release (and therefore reduce the severity of the cytokine storm) while improving or boosting the immune system in fighting off infection.

[0083] Example 4 The effects of Mitlet on survival and bacterial burden in a mouse model of sepsis were investigated. The results are shown in Figure 13. The control group included 13-month-old mice (n=8). The treatment group included 13-month-old mice (n=8) treated with fresh Mitlet. Mitlet was isolated from 1-month-old mice. Essentially, a young source of mitochondria is administered to middle-aged mice. The treatment schedule included injection of Mitlet 1 day before sepsis and 1, 2, and 3 days after sepsis. Bacterial counts and cytokine levels were collected 2 days before sepsis and 1 and 3 days after sepsis. Survival data, shown in Figure 13, were also collected.

[0084] The results in Figure 10 show that the Mitlet-treated group exhibited a higher survival rate each day after sepsis induction compared to the controls. One day after sepsis induction, the Mitlet-treated group exhibited a survival rate of approximately 85%, while all of the controls had died by this time. The survival rate of the Mitlet-treated group continued to decline over six days, as shown in Figure 13, but the survival rate of the Mitlet-treated group was still significantly higher than that of the controls. Furthermore, this may suggest a more aggressive Mitlet treatment compared to the treatment schedule used in this example.

[0085] Example 5 Mitlets isolated from young mice and transplanted into aged recipient mice with septic infection significantly improved survival, as shown in Figures 14A-14C, and reduced bacterial counts, as shown in Figures 15A-15C. Mitlets were isolated from 2-month-old mice and administered to 13-month-old mice with sepsis. To isolate mitlets, blood from 2-month-old C57BL / 6 mice stored on ice at 20% ACD was purchased. Platelets were isolated from the blood, washed with Tyrode's buffer, pH 7.4, and diluted to 1 x 10 in Tyrode's buffer, pH 7.4, containing CACl2. 8Platelets were resuspended at 1000 cells / mL. Platelets were stimulated with thrombin overnight (16 hours) at room temperature. To stop the stimulation, 10 mM EDTA was added, and the stimulated platelets were centrifuged at 300 g for 5 minutes to remove any remaining platelets or cells. The resulting mixture was concentrated by ultracentrifugation at 18,000 g for 60 minutes at 18°C, and approximately 98% of the mitolets were isolated according to protein quantification. The mitolet pellet was resuspended in filtered PBS, pH 7.4. The resulting mitolets were chilled to 4°C for up to one week before administration.

[0086] To induce sepsis (polymicrobial abdominal sepsis), 13-month-old mice were injected with a bolus of fecal suspension (CS). Each mouse was monitored multiple times daily for 14 days for survival and health. Twelve hours after sepsis induction, a small blood sample was collected aseptically from the tail vein of each mouse. Each blood sample (10 μL) was diluted with 9 volumes (90 μL) of 1X citric acid solution (0.32% sodium citrate in 0.9% sodium chloride) and placed on an agar plate. After incubating the agar plate for 2 days, anaerobic bacterial colonies were counted.

[0087] Isolated liver mitochondria were obtained from healthy, 2-month-old C57BL / 6 mice. Naive, isoflurane-anesthetized mice were euthanized by cervical dislocation, and the livers were removed and placed in ice-cold homogenization buffer. The livers were transferred to a tissue homogenizer with 5 ml of ice-cold homogenizing buffer (300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA (pH 7.2)). The tissue was homogenized for 60 seconds. 250 μL of subtilisin A (96.61 μM) was added to the homogenate and mixed by inversion. The mixture was then incubated on ice for 10 minutes. The chilled homogenate was filtered using a pre-wetted 40 μm filter into a 50 mL Falcon tube on ice. This filtration step was repeated with another 40 μm filter, followed by a 10 μm filter. The filtrate was then transferred to a 1.5 mL Eppendorf tube and centrifuged at 9,000 x g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of ice-cold PBS. The concentrated filtrate was then filtered through 1.2 μm and 0.8 μm filters. The concentration of isolated liver mitochondria was confirmed by protein assay. The concentration of isolated liver mitochondria was adjusted to 10 mg / kg for administration.

[0088] The Mitlet administration schedule for 13-month-old mice is summarized in Table 1 below. In the first experiment, Mitlet was administered on days -1, 1, 2, and 3. Bacterial counts, shown in Figures 15A-15D, were determined on days -2, 1, and 3. In the second experiment, three additional treatments were administered on days 3, 4, and 5.

[0089] [Table 1]

[0090] Referring to Figures 14A-14C, Mitlet treatment significantly prevented early death one week after sepsis induction (P<0.01). However, Mitlet treatment (by days 2-5) delayed but did not prevent later death. Survival curves in Figures 14A-14D were analyzed using the Kaplan-Meier log-rank test. Data for two-group comparisons were analyzed using the Student's t-test. For multiple comparisons, a Shapiro-Wilk normality test was performed. If data passed the normality test, data were analyzed using a one-way ANOVA with a Holm-Sydak post-hoc test. If data were not normally distributed, a Kruskal-Wallis test and a Dunn post-hoc test were used. When a single group was evaluated multiple times (i.e., bacterial load), a one-way ANOVA with repeated measures was used. When multiple groups were evaluated multiple times (i.e., body temperature data), a two-way ANOVA with a Holm-Sydak post-hoc test was used. Data shown in Figures 14A-14D are expressed as mean and standard deviation, where p<0.05 was considered statistically significant.

[0091] In contrast to the Mitlet-treated group, the group receiving isolated liver mitochondria showed no protective effect, as shown in Figure 14D. Sepsis-induced mice receiving isolated liver mitochondria died at a similar time as the negative control.

[0092] 15A-15C, bacterial counts in the first and second experiments were lower in Mitlet-treated mice compared to sepsis controls. While Mitlet treatment did not completely eliminate the circulating bacteria analyzed, Mitlet treatment reduced bacterial counts several-fold. As shown in Figure 15D, Mitlet treatment reduced IL-6 cytokine levels by approximately 60%.

[0093] Example 6 To investigate the immunomodulatory function of mitochondrial injection, we first studied the isolation of immune cells. Two mice were anesthetized and then sacrificed. Spleens and hind limbs were obtained from these mice. The spleens were crushed to obtain either T cells or NK cells. The bone marrow from one pair of limbs (tibia, femur, and iliac crest) was flushed to isolate neutrophils. The other pair of limbs was flushed to isolate monocytes. The crushed tissue samples were sorted to recover specific cell types, and the results are shown in Table 2 below.

[0094] [Table 2]

[0095] As shown in Figure 16, after cell sorting, 66.4% of the sorted cells were CD11b + and Ly6G + CD11b binds to integrin α M Also known as CD11b, it is a protein subunit that binds to β2-integrin (CD18) to form the complement receptor (CD3). CD11b is also a cell surface antigen expressed by various immunological cells. Ly6G (lymphocyte antigen 6 complex, G locus) is a glycosylphosphatidylinositol (GPI)-linked differentiation antigen expressed by myeloid-derived cells. Monocytes transiently express Ly6G during myeloid development. Granulocytes and neutrophils express Ly6G, and Ly6G, along with CD11b, is used as a marker for these cells. Based on the results shown in Figure 16, CD11b + Neutrophils can be isolated from a tissue sample.

[0096] As shown in Figure 17, after cell sorting, 84.5% of the sorted cells were CD11b + and Ly6C +Ly6C (lymphocyte antigen 6 complex, C locus) is used as a surface marker for mouse monocytes. Yang P et al., "Immunological Feature and Transcriptional Signaling of Ly6C Monocyte Subsets From Transcriptome Analysis in Control and Hyperhomocysteinemic Mice." Front Immunol. 2021 Feb. 25;12:632-333. Based on the results shown in Figure 17, CD11 + Monocytes can be isolated from a tissue sample.

[0097] As shown in Figure 18, after cell sorting, 80% of the sorted cells were CD3 + CD3 is a multimeric protein complex containing four distinct polypeptide chains: delta (δ), epsilon (ε), gamma (γ), and zeta (ζ). These chains assemble into three pairs of dimers (e.g., δε, γε, ζζ). CD3 functions as a signaling component of the T cell receptor and is widely used as a T cell marker. Based on the results shown in Figure 18, CD3 + T cells can be isolated from a tissue sample.

[0098] As shown in Figure 19, CD3 + After cell sorting, 64% of the cells were CD4 + and 28% were CD8 + CD4 is an adhesion molecule that binds to MHC class II molecules and is involved in signal transduction. CD8 is also an adhesion molecule that binds to MHC class I molecules; similarly, CD8 is involved in signal transduction.

[0099] As shown in Figure 20, after cell sorting, 74.2% of the sorted cells were CD49b + and CD3 -CD49b, also known as integrin α2, heterodimerizes with CD29 (integrin β1) to form the very late antigen 2 (VLA-2) complex, a receptor for fibrillar collagen and other extracellular matrix (ECM) proteins. Fan X et al., "CD49b defines functionally mature Treg cells that survey skin and vascular tissues." J Exp Med. 2018 Nov. 5;215(11):2796-2814. CD49b is a well-known marker for NK cells. Based on the results shown in Figure 20, NK cells can be isolated from tissue samples.

[0100] Example 7 The oxygen consumption rate (OCR) of bone marrow neutrophils (FIG. 21A) and T cells (FIG. 21B) was measured by the Seahorse XF assay 6 hours after the cells were incubated with mitolets. The Seahorse XF assay determines the mitochondrial stress profile for each of these cell types. During the assay, the OCR was measured for bone marrow neutrophils and T cells at different mitolet incubation levels, as shown in FIGS. 21A and 21B, respectively. The mitolet levels analyzed in this assay were 3, 10, or 30 mitolets per cell. The control group of cells was not incubated with mitolets and was designated "0 mitolets," as shown in FIGS. 21A and 21B.

[0101] For the Seahorse XF assay in this example, basal respiration was measured during the first 25 minutes of the assay. Basal respiration measures the oxygen consumption used to satisfy cellular ATP demand, resulting from mitochondrial proton leak, which illustrates the energy demand of bone marrow neutrophils (Figure 21A) and T cells (Figure 21B) under baseline conditions. As shown in Figure 21A, the energy demand of bone marrow neutrophils increased in the groups incubated with mitolets, particularly in the group receiving 3 mitolets / cell, as also depicted in Figure 22A. As shown in Figure 21B, the energy demand of T cells also increased after incubation with mitolets in the groups receiving 3 and 10 mitolets per cell, showing the greatest increase relative to the control.

[0102] As shown in Figure 21A (neutrophils) and Figure 21B (T cells), the decrease in OCR caused by the injection of the ATP synthase inhibitor oligomycin at 25 minutes represents the portion of basal respiration that drives ATP production to meet the energy needs of each cell. Furthermore, these results are indicative of increased ATP production in neutrophils and T cells, occurring even 6 hours after mitochondria uptake. This increase in ATP production represents a boost in the energetic capacity of neutrophils and T cells that received mitochondria from mitochondria.

[0103] As shown in Figures 21A and 22B for both cell types, this boost in neutrophil and T cell cellular energy is further evidenced by maximal respiration readings revealing maximum oxygen consumption in analyzed cells after addition of FCCP (an uncoupler of mitochondrial oxidative phosphorylation) around 50 minutes. FCCP mimics physiological energy demands, causing the respiratory chain to operate at maximum capacity. Addition of FCCP causes rapid oxidation of substrates (e.g., sugars, fats, and amino acids) to meet metabolic demands and achieve a maximum rate of respiration for each cell.

[0104] Example 7 The presence of mitolets in resting (inactivated) and activated T cells was assessed for their respective cellular respiration. Mitolets were generated by stimulating blood from two transgenic mice expressing a fluorescently labeled marker. Stimulation was performed overnight with thrombin (0.5 U / ml), followed by enrichment of mitolets by ultracentrifugation. T cells were extracted from the spleens of wild-type mice, yielding higher-than-normal numbers of T cells. A subset of the extracted T cells was activated overnight with αCD3 / αCD28 beads in the presence of 0, 3, or 30 mitolets / cell. Furthermore, naked mitochondria ("mito") were also examined in activated T cells. Mitochondrial respiration was examined using the Seahorse XF assay, which measures OCR for various parameters involved in cellular respiration. Uptake was measured using flow cytometry and microscopy.

[0105] As shown by the Seahorse assay results in Figure 23, resting and activated mitochondria (both mitolet and no mitolet groups) showed different levels of oxygen consumption, as expected. Activated T cells showed significantly increased OCR after 24 hours, as expected, particularly for basal respiration (before oligo injection), ATP production (between oligo injection and FCCP injection), and maximal respiration (between FCCP injection and rotenone and antimycin A injection) readings of the assay.

[0106] Figures 24A and 24B show the cellular respiration of resting and activated T cells in the presence of mitolets, respectively, after 24 hours. Figure 24A shows how, after 24 hours, resting T cells in the presence of mitolets showed little difference other than a small increase in basal respiration compared to the control (0 mitolets / cell). Figure 24B shows how activated T cells showed an increase in basal respiration in the presence of as few as 3 mitolets / cell. Furthermore, activated T cells tended to show an increase in maximal respiration with as few as 3 mitolets / cell. Furthermore, activated T cells in the presence of mitolets (10 mitolets / cell) did not show the increase in basal respiration seen with mitolets (3 mitolets / cell).

[0107] Figures 25A and 25B show basal and maximal respiration of resting and activated T cells in the presence of mitlet after 24 hours. For basal respiration shown in Figure 25A, a small increase in respiration of resting T cells was observed, but the increase in basal respiration of activated T cells in the presence of mitlet (3 mitlets / cell) was even greater. For maximal respiration shown in Figure 25B, an increase was only observed in activated T cells in the presence of mitlet (3 mitlets / cell).

[0108] These results indicate that activated T cells readily internalize Mitlet, whereas resting T cells appear to have a reduced propensity to do so. This internalization is a regulated process that depends on the activated state of T cells. Furthermore, this internalization activity is essentially the same as that observed in neutrophils and other immunological cell types. These findings suggest that Mitlet is a form of "battery park" that is compatible with all types of immune cells.

[0109] Example 8 After mitochondrial expansion, a coating process is performed to obtain mitotes containing mitochondria and a coating applied to the mitochondria. The coating process involves the preparation of AsOR. To prepare AsOR, 10 mg of alpha-acid glycoprotein (orosomucoid derived from human plasma, OR, Sigma catalog number G9885) is dissolved in 5 ml of MilliQ water. An equal volume of 5.0 ml of 0.2 N H2SO4 is then added. This mixture is heated at 80°C in a water bath for 1 hour. The heated mixture is dialyzed against 1 L of sterile MilliQ water (10 changes) over 48 hours at 4°C through a presoaked dialysis membrane (10 kDa molecular weight cutoff). The dialyzed protein is then stored frozen in 1 ml aliquots or lyophilized.

[0110] AsOR-Dylight was prepared by adding 200 μg (200 μl of 1 M NaHCO3, Alfa Aesar, catalog no. 14707) to 1 mg (2 ml of AsOR, 0.5 mg / ml). 50 μg of Dylight 488 NHS ester (ThermoFisher, catalog no. 46403) freshly dissolved in 50 μl of DMF (Sigma-Aldrich, catalog no. 227056-1L) was then immediately added to the AsOR solution, capped, and placed on a rocker platform (gentle shaking) at room temperature for 1 hour. Meanwhile, 1 L of MilliQ water was pre-chilled to 4°C. After shaking was complete, the reaction mixture was placed in presoaked dialysis tubing (ThermoFisher, catalog no. 88243, 10 kDa molecular weight cutoff membrane) and dialyzed against three changes of 1 L of MilliQ water at 4°C for 24 hours. If a subsequent conjugation reaction is to be performed, the sample is dialyzed twice against 150 ml of 0.1 M MES pH 6.0 (prepared from 1 M stock, Alfa Aesar, Cat. No. J61656) for buffer exchange.

[0111] To prepare AsOR-PL, 500 μg of poly-L-lysine (PL) (50 μl of 1 mg / 100 μl in 0.1 M MES pH 6) (Sigma-Aldrich, Catalog No. P0879) is added to 1 mg AsOR-Dylight in approximately 2.0 ml of MES pH 6 and mixed. 500 μg of fresh EDC (50 μl of 1 mg / 100 μl EDC stock in 0.1 M MES pH 6) is added to the AsOR-Dylight and PL mixture and incubated for 1 hour at room temperature on a rocking platform (gentle shaking). The reaction mixture is concentrated and buffer exchanged using 8 volumes of 0.5 ml 0.1 M MES pH 6.0 using a spin filter with a 10 kD molecular weight cutoff (Pierce, Catalog No. 88513, 0.5 ml capacity) according to the supplier's protocol. 230 Check the filtrate until the concentration is the same as the background. Sterilize the filtrate (0.45 μl) and store at 4°C or lyophilize.

[0112] To prepare AsOR-LLO, 1 mg of AsOR + 150 mM NaCl + 1 mM EDTA in 200 μl of PBS, pH 7.4, was reacted with 1 mg of SPDP (freshly prepared in 50 μl of DMSO) and brought to a final volume of 2000 μl with 1X PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA. The reaction mixture was then incubated for 1 hour. After incubation, the reaction was concentrated and washed to remove free SPDP using a spin filter (10 kDa molecular weight cutoff, 0.5 ml volume) by washing eight times with 0.5 ml of PBS pH 7.4 + 150 mM NaCl + 1 mM EDTA. 260 Check the filtrate until it is at baseline (for SPDP).

[0113] 240 μg of purified His-LLO (240 μg / 1 ml PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA) is treated with 0.5 ml of 150 mM DTT (freshly prepared in PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA). Final DTT concentration is 50 mM. The reaction is incubated for 1 hour at room temperature with gentle shaking. After the 1 hour incubation, the DTT-treated His-LLO is concentrated and washed four times with 0.5 ml PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA and a spin filter with a 10 kDa molecular weight cutoff to obtain a final volume of 130 μl. 240 μg of His-LLO is passed through a buffer-saturated 0.5 ml Zeba Spin molecular sieve column with a 7 kDa molecular weight cutoff to remove any remaining traces of DTT. A final 240 μg His-LLO in 130 μl is reacted with 360 μg AsOR-SPDP in a final volume of 1 ml PBS, pH 7.4, 150 mM NaCl, and 1 mM EDTA. The final LLO:AsOR molar ratio is 1:1.5. The reaction mixture is then placed on a rocking platform set at low speed for overnight incubation in a 4°C cold room.

[0114] After overnight incubation, sample aliquots were removed and analyzed by A280 nm absorbance to measure protein-bound PDP and A343 nm absorbance to measure 2-pyridinedione released by adding DTT. Buffer alone or with DTT as a blank, respectively. After overnight incubation, the reaction mixture was either purified as described below or placed in presoaked dialysis tubing with a 10 kD molecular weight cutoff, dialyzed against 20 L MilliQ water three times over 24 hours, and sterilized for storage at 4°C.

[0115] To prepare histidine-tagged LLO (His-LLO), bacterial stock (strain plasmid no. DP-E3570) was streaked onto LB / Km 100 μg / ml agar plates. The plates were incubated at 37°C for 24 hours. A single colony from the plate was inoculated into 25 ml of LB / Km (100 μg / ml working concentration) broth in a 125 ml culture flask and placed on an orbital shaker at 225 rpm for 24 hours at 37°C. The culture was diluted 1:100 into 1000 ml of fresh LB / Km (50 μg / ml working concentration) broth per 2 L flask. The flask was incubated at 37°C for 3 hours with shaking at 225 rpm. After 3 hours of growth, IPTG was added to the bacterial culture to a final concentration of 0.5. The culture is grown for an additional 5 hours before harvesting the cells by centrifugation at 8,500 rpm (6,540 x g) for 5 minutes at 4°C. The bacterial cells are washed once by resuspending the cell pellet in 40 ml of cold 1X PBS, pH 7.4, centrifuging the cell suspension at 8,500 rpm (6,540 x g) for 5 minutes at 4°C, and carefully discarding the clear supernatant into a flask containing commercial bleach. The cell pellet is stored at -80°C until use for protein extraction.

[0116] For protein extraction, the bacterial cell pellet was resuspended in 5 ml of lysis buffer (50 mM sodium phosphate buffer pH 8 containing 1 M sodium chloride, 10 mM 2-mercaptoethanol, 20 mM imidazole, and 1 mM PMSF). Cells were lysed on ice by mechanical disruption using an MP-Biomedical FastPrep-24 Classic system with a speed setting of 6.0 m / s and 40-second pulses (6 pulses with a 1-minute interval after each pulse). The cell lysate was spun at 8500 rpm (6,540 × g) for 10 minutes at 4 °C. The cleared lysate was then passed twice through a cobalt agarose column pre-equilibrated with lysis buffer. The final flow-through fraction was collected and set aside. The column is washed four times with 3 ml of lysis buffer and twice with wash buffer (1 M sodium chloride, 10 mM 2-mercaptoethanol, 20 mM imidazole, 1 mM PMSF, 5% glycerol, 50 mM sodium phosphate buffer pH 6 in 0.1% Tween 20). His-LLO is eluted with 3 ml of elution buffer (50 mM sodium phosphate buffer pH 6 containing 1 M sodium chloride, 10 mM 2-mercaptoethanol, 800 mM imidazole, 1 mM PMSF). Elution is performed with eluate A. 280 The His-LLO is concentrated using a Pierce spin filter with a 30 kDa molecular weight cutoff and buffer exchanged into storage buffer (50 mM sodium phosphate buffer pH 6 containing 1 M sodium chloride, 10 mM 2-mercaptoethanol, 20 mM imidazole, 1 mM PMSF, and 1 mM EDTA). 280 Protein concentration was estimated using a NanoDrop UV-Vis spectrophotometer by measuring the .mu.m.

[0117] To prepare and purify poly-L-lysine-Dylight-LLO, first add 0.4 mg of SPDP (dissolved in 40 μl of DMSO) to 1 mg of PL (5-10 kDa Sigma-Aldrich) in 1.0 ml of PBS pH 7.4 + 150 mM NaCl + 1 mM EDTA, bringing the final reaction volume to 1.0 ml with buffer, and then incubate the reaction at room temperature for 1 hour. After incubation, the sample was concentrated and washed three to four times with 0.5 ml of buffer (PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA) using a Pierce spin filter (Thermoscientific catalog number 88513) to concentrate the sample to 130 μl, and then passed through one 0.5 ml Zeba Spin molecular sieve column, 7 kD molecular weight cutoff (Thermoscientific catalog number 89882) to remove any remaining unreacted SPDP. 0.5 mg of PL-SPDP in 1.0 ml PBS [pH 7.4 + 150 mM NaCl + 1 mM EDTA] was reacted with 50 mg of Dylight 488 (dissolved in 50 μl of DMF) to a final reaction volume of 1.0 ml. The reaction volume was incubated at room temperature for 1.0 h.

[0118] After incubation, the sample is concentrated and washed four times with 0.5 ml PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA using a 0.5 ml spin filter with a 10 kD molecular weight cutoff. The sample is passed through two 0.5 ml Zeba molecular sieve spin columns with a 7 kD molecular weight cutoff to concentrate the sample to 130 μl. 0.38 mg of His-LLO (cobalt agarose column purified) is mixed with 11.5 mg dithiothreitol (DTT), 0.5 ml of 23 mg / ml dissolved in PBS pH 7.4. The final reaction volume is brought to 1 ml with PBS pH 7.4 and incubated at room temperature for 1.0 hour.

[0119] After incubation, the sample is concentrated and washed four times with 0.5 ml PBS, pH 7.4 + 150 mM NaCl + 1 mM EDTA using a 0.5 ml spin filter with a 10 kD molecular weight cutoff. The retentate is concentrated to 130 μl and passed through two 0.5 ml Zeba molecular sieve spin columns with a 7 kD molecular weight cutoff to eliminate DTT. 0.38 mg of activated His-LLO was reacted with 0.5 mg of PL-Dylight-SPDP in 130 μl at a molar ratio of 1:3 LLO:PL in a final reaction volume of 1 ml in buffer pH 7.4 (PBS + 150 mM NaCl + 1 mM EDTA). The reaction is incubated overnight at 4°C with gentle shaking. To the 900 μl reaction mixture, add 900 μl of 2X Binding Buffer A pH 8 (100 mM sodium phosphate + 2.0 M sodium chloride) and mix. The reaction mixture is loaded onto a 5 ml column (Qiagen, catalog number 34964) containing 2.5 ml of Ni-NTA-agarose (Qiagen, catalog number 30210) pre-saturated with Binding Buffer B pH 8 (50 mM sodium phosphate, 1.0 M sodium chloride, 20 mM imidazole, and 1.0 mM PMSF). The flow-through fraction is collected, and the column is washed once more with the same buffer and collected. The column is washed three times with 3 ml of Binding Buffer B. The column is washed twice with 3 ml of buffer (50 mM sodium phosphate, pH 6, 1.0 M sodium chloride, 20 mM imidazole, 1.0 mM PMSF, 5% glycerol, and 0.1% Tween 20).

[0120] HIS-LLO is finally eluted with 3 ml of elution buffer, pH 6 (50 mM sodium phosphate, 1.0 M sodium chloride, 800 mM imidazole, 1.0 mM PMSF, 1 mM EDTA). Elution is repeated with 1 ml of elution buffer, and the eluates are pooled, resulting in a 4 ml eluate. The 4 ml spin filter eluate is passed through a 0.5 ml Pierce spin filter with a 10 kD molecular weight cutoff pre-equilibrated with storage / transport buffer, pH 6 (50 mM NaH2PO4, 1 mM EDTA, 2.7 mM KCl, 5% (v / v) glycerol, and 0.5 M NaCl), collecting the entire retentate in 400 μl to eliminate free PL-SPDP. Optionally, to exclude higher molecular weight products, the retentate is passed through a 0.5 ml Pierce spin filter with a 100 kD molecular weight cutoff, collecting the retentate. Samples can be stored at -20°C until used for further analysis.

[0121] To titrate AsOR-Dylight-PL (AsOR-PL) conjugates with mitochondria (e.g., mitochondria grown in a bioreactor), 128 μg of mitochondria, fresh or thawed on ice, were pooled into one tube and washed twice with 1 ml of PBS, pH 7.4, by spinning at 12,000 rcf for 10 min at 4°C. The final pellet was resuspended in 250 μl of PBS. Protein concentration was measured using a bicinchoninic acid (BCA) or Bradford assay. Increasing amounts of AsOR-PL (0.25–2.0 μg) were added to a 128 μg aliquot of mitochondria, and the final volume was adjusted to 50 μl by adding PBS. These conjugates were incubated on ice for 45 min to allow binding of AsOR-PL to mitochondria, thereby coating the mitochondria with AsOR-PL and yielding mitochondrial pellets. After incubation, the mitolets are separated by centrifugation at 12,000 rcf for 10 minutes at 4°C. The supernatant is removed and the mitolet pellet is resuspended in 50 μl of fresh PBS. The pellet and supernatant are applied separately to agarose gels or HPAGE along with controls.

[0122] definition The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0123] 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. The use of the term "including" and other forms such as "include," "includes," and "included" is not limiting. The use of the term "having" and other forms such as "have," "has," and "had" is not limiting. As used herein, whether in transitional phrases or in the body of a claim, the terms "comprise(s)" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted synonymously with the phrases "having at least" or "including at least." For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0124] As used herein, the phrases "culture-grown cells" or "culture-grown tissues" refer to a number of cells or tissues, respectively, grown in a liquid, semi-solid, or solid medium outside the organism from which the cells or tissues originate. In some embodiments, cells grown in culture are cells grown in a bioreactor. By way of non-limiting example, cells may be grown in a bioreactor prior to isolation of mitolets from the cells.

[0125] As used herein, "therapeutically effective amount," "therapeutic amount," and the like refer to an amount of a compound, composition, or mixture that elicits a desired biological or medical response in a tissue, system, animal, or human, e.g., the response is to a disease or disorder, and the amount produces a response that reflects at least a partial amelioration of the disease or disorder, or symptoms associated with the disease or disorder.

[0126] As used herein, "administering" or "administration of" refers to providing a composition in a therapeutically effective amount to a subject (including a patient) in need of treatment. The route of administration can be topical, enteral, or parenteral.

[0127] As used herein, "mitlets" include mitochondria that are encapsulated in or coated with vesicles. In some examples, mitolets include vesicles and mitochondria encapsulated in the vesicles. In some examples, mitolets (including vesicles and mitochondria) are released or excreted by platelets. In some examples, mitolets are derived from various sources and grown and expanded in bioreactors. In some examples, vesicles include receptors that target a specific cell type. In some examples, the specific cell type includes immunological cells. In some examples, mitolets include coated mitochondria. In some examples, the coating includes asialoglycoprotein (AsG). In some examples, the AsG includes asialoorosomucoid (AsOR). In some examples, the coating further includes poly-L-lysine linked to AsOR. In some examples, the coated mitochondria are complexed with a conjugate. In some examples, the conjugate includes AsOR. In some embodiments, the conjugate further includes listeriolysin O (LLO).

[0128] As used herein, the term "bioreactor" includes an apparatus or device configured to support the growth and proliferation of biological entities under controlled and regulated environmental conditions. A bioreactor includes a containment chamber constructed of biocompatible materials that minimize any negative interactions with the contained biological entities. This chamber effectively prevents contamination from external sources and provides an isolated and controlled environment that promotes optimal growth conditions for the biological entities to grow or proliferate in the presence of an appropriate medium. A "bioreactor" may further include a series of sensors designed to continuously monitor and record critical parameters, including, but not limited to, temperature, pH, oxygen and carbon dioxide concentrations, and nutrient and waste levels. Feedback from these sensors is essential to inform adjustments to the bioreactor's internal environment, thereby maintaining optimal growth conditions. A bioreactor may further include a control system operably linked to these sensors and the containment chamber. The control system interprets data from the sensors and adjusts the environmental conditions within the containment chamber in response. Controlled manipulation of these parameters allows fine tuning of the environment of the biological entities, leading to enhanced growth and productivity. A "bioreactor" may be configured as an input / output system structured for the introduction of new medium necessary to supply nutrients and the removal of spent medium, important for the discharge of waste products. This system ensures a dynamic environment within the containment chamber, supporting the maintenance of viability and optimal productivity of the biological entities.

[0129] Additional Notes The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0130] It should be recognized that all combinations of the foregoing concepts, and additional concepts described in more detail below, are contemplated as part of the inventive subject matter disclosed herein (to the extent such concepts are not mutually inconsistent). In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as part of the inventive subject matter disclosed herein. It should also be recognized that terms expressly employed herein, which may appear in any disclosure incorporated by reference, should be given the meaning most consistent with the particular concepts disclosed herein.

[0131] Throughout this specification, references to "one example," "another example," "an example," etc. mean that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with that example is included in at least one example described herein and may or may not be present in other examples. Also, unless expressly specified otherwise, it should be understood that the described elements for any example may be combined in any suitable manner in various examples. While several examples have been described in detail, it should be understood that the disclosed examples may be modified. Accordingly, the above description should be considered non-limiting.

[0132] It should be understood that a feature, substance, characteristic, or group described in connection with a particular embodiment or example is applicable to any other embodiment or example described in this section or elsewhere in this specification, unless inconsistent. All features disclosed herein (including any accompanying claims, abstract, and drawings) and / or all method or process steps disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Protection is not limited to the details of any of the foregoing examples. Protection extends to any novel or any novel combination of features disclosed herein (including any accompanying claims, abstract, and drawings) or any novel or novel combination of method or process steps disclosed.

[0133] Furthermore, certain features that are described in this disclosure in the context of separate implementations may also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation may also be implemented in multiple implementations separately or in any suitable combination. Furthermore, while features may be described above as acting in a particular combination, one or more features from a claimed combination may, in some cases, be separated from the combination, and the combination may be claimed as a component of the combination or a variation of the components of the combination.

[0134] Furthermore, while operations may be depicted in the figures or described in the specification in a particular order, such operations need not be performed in the particular order shown or sequentially, or all operations may be performed to achieve desired results. Other operations not depicted or described may be incorporated into the example methods and processes. For example, one or more additional operations may be performed before, after, simultaneously with, or between any of the described operations. Furthermore, operations may be rearranged or resequenced in other implementations. Those skilled in the art will recognize that in some examples, the actual steps taken in the illustrated and / or disclosed methods may differ from those shown in the figures. In some examples, certain of the steps described above may be removed or others may be added. Furthermore, the features and characteristics of the specific examples disclosed above may be combined in various ways to form additional examples, all of which are within the scope of the present disclosure.

[0135] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not all such advantages may necessarily be achieved in accordance with a particular example. Thus, for example, one skilled in the art will recognize that the present disclosure may be embodied and performed in a manner that achieves one advantage or group of advantages as taught herein, without necessarily achieving other advantages that may be taught or suggested herein.

[0136] Conditional language such as "can," "could," "might," or "may," unless otherwise stated or understood within the context in which it is used, is generally intended to convey that a particular example includes certain features, elements, and / or steps even when other examples do not. Thus, such conditional language is generally not intended to imply that features, elements, and / or steps are somehow required for one or more examples, or that one or more examples necessarily include logic for determining, with or without user input or prompting, that those features, elements, and / or steps are included or performed in any particular example.

[0137] Conditional language such as the phrase "at least one of X, Y, and Z," unless otherwise stated, is generally understood in context to be used to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conditional language is generally not intended to imply that a particular instance of s requires the presence of at least one of X, at least one of Y, and at least one of Z.

[0138] As used herein, language of degree, such as the terms "approximately," "about," "generally," and "substantially," denotes a value, amount, or characteristic that is near a stated value, amount, or characteristic that still performs a desired function or achieves a desired result.

[0139] The scope of the present disclosure is not intended to be limited by the specific disclosed or preferred examples in this section or elsewhere herein, but may be defined by the claims, as set forth or hereafter set forth in this or other sections herein. Claim language is to be construed broadly based on the language employed in the claims, and not limited to the examples described herein or during prosecution of an application, which examples are to be construed as non-exclusive.

Claims

1. 1. A method of treating a subject with chimeric antigen receptor (CAR)-containing cells, wherein the subject has an indication treatable by CAR-containing cells, obtaining mitochondria as platelet-derived mitochondria; co-incubating the mitolets with the CAR-containing cells such that the CAR-containing cells incorporate the mitolets into the CAR-containing cells in an amount effective to enhance the metabolic activity of the CAR-containing cells; and delivering a quantity of CAR-containing cells with enhanced metabolic activity to a subject A method comprising:

2. The method of claim 1 , wherein the indication comprises a tumor.

3. The method of claim 2, wherein the tumor comprises a solid tumor.

4. The method of claim 2, wherein the tumor comprises a non-solid tumor.

5. 5. The method of any one of claims 1 to 4, wherein the platelets are obtained from a subject.

6. 5. The method of any one of claims 1 to 4, wherein the platelets are obtained from a donor other than the subject.

7. The method of claim 6, wherein the donor comprises a family member of the patient.

8. Platelets are adding an anticoagulant and a buffer to the blood to form a mixture; Separating the mixture into a supernatant and platelet-rich plasma (PRP); and Collecting PRP containing platelets 8. The method according to any one of claims 1 to 7, obtained by a method comprising:

9. 9. The method of claim 8, wherein the anticoagulant comprises an acid citrate dextrose anticoagulant (ACD).

10. 10. The method of claim 8 or claim 9, wherein the buffer comprises Tyrode's buffer.

11. 11. The method of claim 10, wherein the Tyrode's buffer has a pH in the range of 6.0 to 7.

0.

12. The process of obtaining Maitreya is as follows: stimulating the collected PRP, thereby releasing extracellular vesicles from the platelets in the PRP; and Collecting extracellular vesicles Including, The collected extracellular vesicles encapsulate mitochondria, Further comprising extracellular vesicles in which mitochondria are collected.

12. The method according to any one of claims 8 to 11.

13. The PRP stimulation step involves Ca 2+ 13. The method of claim 12, comprising exposing the PRP to the immune complex in the presence of

14. The method of claim 13, wherein the immune complex comprises heat-aggregated IgG.

15. 15. The method of claim 14, wherein the concentration of the heat-aggregated IgG is 0.1 mg / mL to 2.5 mg / mL.

16. The method of any one of claims 1 to 15, further comprising a step of matching the mtDNA haplotype of the obtained mitochondria with the mtDNA haplotype of the patient.

17. 17. The method of any one of claims 1 to 16, wherein the effective amount of mitolets is at least 3 mitolets per CAR-containing cell.

18. 18. The method of any one of claims 1 to 17, wherein the effective amount of mitolets is at least 10 mitolets per CAR-containing cell.

19. 19. The method of any one of claims 1 to 18, wherein the effective amount of mitolets is at least 30 mitolets per CAR-containing cell.

20. 20. The method of any one of claims 1 to 19, wherein obtaining the platelets further comprises expanding the platelets in a bioreactor.

21. 17. The method of claim 1, wherein the step of obtaining mitolets further comprises the step of growing the mitolets in a bioreactor.

22. The method according to any one of claims 1 to 7, wherein the step of obtaining mitolets further comprises a step of expanding the mitolets in a bioreactor.

23. The method of claim 22, further comprising the step of coating the mitolets after the growing step.

24. 24. The method of any one of claims 1 to 23, wherein the co-incubating step occurs during the manufacture of the CAR-containing cells.

25. Maitreya, adding a cryopreservative to the mitolets and the enhanced CAR-containing cells; and Freezing Mitret and Enhanced CAR-Containing Cells 25. The method of any one of claims 1 to 24, wherein the cell is preserved by a method comprising:

26. 26. The method of claim 25, wherein the cryopreservation agent is selected from the group consisting of sugars, oligosaccharides and polysaccharides.

27. 26. The method of claim 25, wherein the cryopreservation agent comprises DMSO.

28. 26. The method of claim 25, wherein the cryopreservation agent comprises trehalose.

29. 26. The method of claim 25, wherein the cryopreservation agent comprises phosphate buffered saline.

30. 30. The method of any one of claims 1 to 29, further comprising administering a therapeutically effective amount of Mitret to a patient after the preparing step.

31. 31. The method of claim 30, wherein the administering step further comprises administering a therapeutically effective amount of the enhanced CAR-containing cells to the patient.

32. 32. The method of claim 31 , wherein the step of administering the CAR-containing cells precedes the administration of the mitochondria.

33. The method of claim 31 , wherein the steps of administering the CAR-containing cells and administering the mitolet to the patient occur simultaneously.

34. 31. The method of claim 30, wherein the administering step further comprises administering a follow-up dose of Mytret at least two days after the initial administering step.

35. 31. The method of claim 30, wherein the administering step is repeated every two days.

36. 1. A method of treating a subject with chimeric antigen receptor (CAR)-containing cells, wherein the subject has an indication treatable by CAR-containing cells, Obtaining mitochondria; Administering the mitochondria and CAR-containing cells to a subject A method comprising:

37. 37. The method of claim 36, wherein the mitochondria are obtained from placental tissue.

38. 37. The method of claim 36, wherein the mitochondria are obtained from bone marrow.

39. 37. The method of claim 36, wherein the mitochondria are obtained from adipose tissue.

40. 40. The method of any one of claims 36 to 39, wherein the mitolet comprises a coating.

41. 41. The method of claim 40, wherein the coating comprises asialoglycoprotein (AsG).

42. 42. The method of claim 41, wherein the AsG comprises asialoorosomucoid (AsOR).

43. 43. The method of claim 42, wherein the coating further comprises poly-L-lysine linked to the AsOR.

44. The method of claim 42 or 43, further comprising the step of complexing the coated mitolets with a conjugate.

45. The method of claim 40, wherein the coated mitolets are complexed with a conjugate.

46. 46. ​​The method of claim 45, wherein the conjugate comprises an AsOR.

47. 46. ​​The method of claim 45, wherein the conjugate further comprises listeriolysin O (LLO).

48. 48. The method of any one of Claims 36-47, wherein the step of administering the mitochondria occurs before the step of administering the CAR-containing cells.

49. The method of any one of claims 36 to 47, wherein during the administering step, the mitolets are co-administered with the CAR-containing cells.

50. 48. The method of any one of claims 36 to 47, further comprising administering a follow-up dose of Mytret to the patient at least two days after the administering step.

51. 48. The method of any one of claims 36 to 47, further comprising administering repeat doses of Mytret to the patient at least every two days after the administering step.

52. 52. The method of any one of claims 1 to 51, wherein the CAR-containing cells comprise CAR-T cells.

53. 53. The method of any one of claims 1 to 52, wherein the CAR-containing cells comprise CAR-NK cells.

54. A formulation comprising mitochondria obtained from a biological source and CAR-expressing cells for the treatment of an indication treatable with CAR-containing cells.

55. 55. The formulation of Claim 54, wherein the CAR-expressing cells comprise T cells.

56. 55. The formulation of Claim 54, wherein the CAR-expressing cells comprise NK cells.

57. 55. The formulation of claim 54, wherein the indication comprises a tumor.

58. 55. The formulation of claim 54, wherein the tumor comprises a solid tumor.

59. 55. The formulation of claim 54, wherein the tumor comprises a non-solid tumor.

60. 60. The formulation of any one of Claims 54 to 59, wherein the source comprises placental tissue.

61. 60. The formulation of any one of Claims 54 to 59, wherein the source comprises bone marrow.

62. 60. The formulation of any one of Claims 54 to 59, wherein the source comprises adipose tissue.

63. 60. The formulation of any one of Claims 54 to 59, wherein the source comprises platelets.

64. 64. The formulation of claim 63, wherein the platelets are obtained from blood.

65. 65. A formulation according to any one of claims 54 to 64, wherein the mitolets are expanded in a bioreactor.

66. 66. The formulation of any one of claims 54 to 65, further comprising a cryopreservative.

67. 67. The formulation of claim 66, wherein the cryopreservative is from the group consisting of saccharides, oligosaccharides and polysaccharides.

68. 67. The formulation of claim 66, wherein the cryopreservative comprises DMSO.

69. The formulation of any one of claims 54 to 68, wherein the mitolet and the cells expressing CAR are maintained in separate containers.

70. 70. The formulation of claim 69, wherein the container comprises a cryopreservation bag.

71. 71. The formulation of claim 70, wherein the bag comprises fluorinated ethylene propylene (FEP).

72. 71. The formulation of claim 70, wherein the bag comprises ethylene vinyl acetate (EVA).

73. 71. The formulation of claim 70, wherein the bag comprises a polyolefin.

74. A formulation comprising CAR-containing cells with enhanced metabolic activity, in which the CAR-containing cells are co-incubated with mitochondrial cells derived from platelets, for use in treating an indication treatable by CAR-containing cells.

75. 75. The formulation of claim 74, wherein the indication comprises a tumor.

76. 76. The formulation of claim 75, wherein the tumor comprises a solid tumor.

77. 76. The formulation of claim 75, wherein the tumor comprises a non-solid tumor.

78. 78. The formulation of any one of Claims 74 to 77, wherein the platelets are obtained from a subject.

79. 78. The preparation of any one of Claims 74 to 77, wherein the platelets are obtained from a donor other than the subject.

80. 80. The formulation of claim 79, wherein the donor comprises a family member of the patient.

81. The platelets from which Mytret is derived are adding an anticoagulant and a buffer to the blood to form a mixture; Separating the mixture into a supernatant and platelet-rich plasma (PRP); and Collecting PRP containing platelets 81. The formulation of any one of claims 74 to 80, obtained by a process comprising:

82. 82. The formulation of claim 81, wherein the anticoagulant comprises an acid citrate dextrose anticoagulant (ACD).

83. 83. The formulation of claim 81 or 82, wherein the buffer comprises Tyrode's buffer.

84. 84. The formulation of claim 83, wherein the Tyrode's buffer has a pH in the range of 6.0 to 7.

0.

85. Maitreya, stimulating the collected PRP, thereby releasing extracellular vesicles from the platelets in the PRP; and Collecting extracellular vesicles and The collected extracellular vesicles encapsulate mitochondria, Further comprising extracellular vesicles in which mitochondria are collected.

85. The formulation of any one of claims 81 to 84.

86. PRP stimulation causes Ca 2+ 86. The formulation of Claim 85, comprising exposing PRP to an immune complex in the presence of

87. 87. The formulation of claim 86, wherein the immune complex comprises heat-aggregated IgG.

88. 88. The formulation of claim 87, wherein the concentration of the thermally aggregated IgG is 0.1 mg / mL to 2.5 mg / mL.

89. A formulation described in any one of claims 74 to 88, wherein the mtDNA haplotype of the mitolot matches the mtDNA haplotype of the patient.

90. 90. The formulation of any one of claims 74 to 89, comprising an effective amount of mitolets of at least 3 mitolets per CAR-containing cell.

91. 90. The formulation of any one of claims 74 to 89, comprising an effective amount of mitolets of at least 10 mitolets per CAR-containing cell.

92. 90. The formulation of any one of claims 74 to 89, comprising an effective amount of mitolets of at least 30 mitolets per CAR-containing cell.

93. 93. The preparation of any one of claims 74 to 92, wherein the platelets from which the mitolets are obtained are expanded in a bioreactor.

94. A formulation described in any one of claims 74 to 93, wherein the mitochondria are expanded in a bioreactor.

95. 95. The formulation of any one of claims 74 to 94, wherein the mitolet is coated.

96. Maitreya, adding a cryopreservative to the mitolets and the enhanced CAR-containing cells; and Freezing Mitret and Enhanced CAR-Containing Cells 96. The formulation of any one of claims 74 to 95, preserved by a method comprising:

97. 97. The formulation of claim 96, wherein the cryopreservation agent is selected from the group consisting of saccharides, oligosaccharides, and polysaccharides.

98. 97. The formulation of claim 96, wherein the cryopreservative comprises DMSO.

99. 97. The formulation of claim 96, wherein the cryopreservative comprises trehalose.

100. 97. The formulation of claim 96, wherein the cryopreservative comprises phosphate buffered saline.

101. 101. The formulation of any one of claims 74 to 100, further comprising a therapeutically effective amount of Mitlet.

102. 71. The formulation of any one of Claims 54 to 70, wherein the mitochondria are obtained from placental tissue.

103. 103. The formulation of claim 102, wherein the mitochondria are obtained from bone marrow.

104. 103. The formulation of claim 102, wherein the mitochondria are obtained from adipose tissue.

105. 105. The formulation of any one of Claims 102 to 104, wherein the mitochondria comprise a coating.

106. 106. The formulation of claim 105, wherein the coating comprises asialoglycoprotein (AsG).

107. The formulation of claim 106, wherein the AsG comprises asialoorosomucoid (AsOR).

108. The formulation of claim 105, wherein the coating further comprises poly-L-lysine linked to the AsOR.

109. 106. The formulation of claim 105, wherein the coated mitochondria are complexed with a conjugate.

110. 110. The formulation of claim 109, wherein the conjugate comprises AsOR.

111. 111. The formulation of claim 110, wherein the conjugate further comprises listeriolysin O (LLO).

112. 112. The formulation of any one of Claims 102-111, wherein the CAR-containing cells comprise CAR-T cells.

113. 113. The formulation of any one of Claims 102-112, wherein the CAR-containing cells comprise CAR-NK cells.

Images