Enhancing adoptive cell transfer by promoting a population of superior adaptive immune cells

Abstract

The present disclosure relates to mitochondrial-enhanced immune cells, their compositions and therapeutic uses.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 253,058, filed October 6, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Field The present invention relates to the field of biomedicine, specifically to methods useful for the treatment of cancer, infectious diseases, and autoimmune diseases. Specifically, the present invention relates to therapeutic treatments using mitochondria-enhanced immune cells, including but not limited to mitochondria-enhanced adaptive immune cells. The present invention relates to mitochondria-enhanced immune cells for use in the treatment of cancer, infectious diseases, and autoimmune diseases. Specifically, the immune cells of the present invention are immune cells, including but not limited to T immune cells or in vitro expanded T cells. More specifically, the immune cells of the present invention are immune cells, including but not limited to blood circulating CD8 immune cells, tumor infiltrating lymphocytes (TILs), engineered T cells, chimeric antigen receptor (CAR) T cells, blood circulating CD4 immune cells, immunosuppressive regulatory T cells (Treg cells), effector T cells, memory T cells, alpha-beta T cells (αβ T cells), and gamma-delta T cells (γδ T cells). The mitochondria-enhanced immune cells have improved persistence, higher survival rate, and / or differentiation capacity. More specifically, the present invention relates to mitochondria-enhanced memory T cells (e.g., memory T cells transplanted with exogenous mitochondria) with improved persistence, higher survival capacity, and / or higher differentiation capacity. The mitochondria-enhanced memory T cells of the present invention show increased efficacy in adoptive cell transfer therapy (ACT), for example, due to the enhanced proportion of highly persistent cells in a mixed bulk population. The present invention further provides mitochondria-enhanced immunosuppressive regulatory T cells (Treg) (e.g., Treg CD4 T cells transplanted with exogenous mitochondria) with improved survival capacity and / or higher differentiation capacity for the treatment of autoimmune diseases and transplanted organ or tissue rejection. The present invention relates to pharmaceutical compositions comprising mitochondria-enhanced immune cells.The present invention relates to the increased effectiveness of cell technologies resulting in the generation of a higher percentage of immune T cells or a higher percentage of immune T cells at a particular differentiation stage with improved persistence, higher viability, and / or higher differentiation potential. [Background technology]

[0003] Cancer and autoimmunity share a common origin, but exert powerful forces working in opposite directions. Both diseases result from the failure of the body's immune system. Cancer often develops because the immune system fails to recognize and / or attack defective and / or transformed cells, allowing them to divide and grow. Conversely, autoimmunity, a faulty immune response that leads to diseases such as colitis and lupus, occurs when the immune system mistakenly attacks healthy cells. Most parts of the body can be targeted by the immune system, including the heart, brain, nerves, muscles, connective tissues, skin, eyes, lungs, kidneys, digestive tract, blood cells, and blood vessels.

[0004] Cancer is one of the leading causes of death in developed countries, with an estimated 1.9 million new cases of cancer diagnosed in the United States in 2021 and 608,570 deaths due to cancer (https: / / www.cancer.org / content / dam / cancer-org / research / cancer-facts-and-statistics / annual-cancer-facts-and-figures / 2021 / cancer-facts-and-figures-2021.pdf). According to the World Health Organization (WHO), cancer is the leading cause of death worldwide, with approximately 10 million deaths in 2020 (Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Pineros M, et al. Global Cancer Observatory: Cancer Today. Lyon: International Agency for Research on Cancer; 2020 (https: / / gco.iarc.fr / today, accessed February 2020). The most common cancer incidences in 2020 (in terms of new cancer cases) were breast (2.26 million cases), lung (2.21 million cases), colon and rectal (1.93 million cases), prostate (1.41 million cases), skin cancer (non-melanoma) (1.2 million cases), and stomach (1.09 million cases). The most common causes of cancer deaths in 2020 were lung (1.8 million), colon and rectal (9.35 million), liver (0.83 million), stomach (7.69 million), and breast (6.85 million).

[0005] Cancer results from defective cells that acquire mutations that allow them to evade normal cell cycle checkpoints. Over time, the hyperproliferation of mutated cells creates a heterogeneous tumor mass composed of various cell types. Once cancer cells acquire metastatic characteristics, such as the ability to motility, invasiveness, and modulate the environment to favor their survival, they can spread throughout the body and give rise to distant metastases away from the original tumor bed.

[0006] Immune cells play a key role in detecting, controlling, and eradicating cancer cells and pathogens. T cell receptors (TCRs) on the surface of T lymphocytes recognize antigenic peptide fragments presented on major histocompatibility complex (MHC) molecules. During acute infections, naive T cells specific for invading pathogens are activated via their TCRs in the context of MHC / antigen presentation, clonally expand, and give rise to effector cells. Through direct killing, effector cells mediate the removal of infected cells from the body. Upon pathogen clearance, the mounted immune response contracts, resulting in apoptosis of the majority of activated CD8 T cells. A portion of antigen-specific T cells further differentiate to generate a memory T cell pool that provides long-term protection to the individual (Figure 1). Notably, memory cells have distinct characteristics such as enhanced persistence, self-renewal capacity, and efficient recall capacity upon reinfection by the encountered pathogen. In the case of a second infection with the same pathogen, the immune response induced and triggered by memory cells occurs faster and stronger compared to naive T cells (Vanja Lazarevic et al., “T-bet: a bridge between innate and adaptive immunity” Nat Rev Immunol. 2013 Nov;13(11):777-789).

[0007] Interestingly, it has been shown that the metabolism of naive, effector, and memory CD8 T cells differs. Naive CD8 T cells are quiescent and mostly depend on oxidative phosphorylation (OXPHOS) to supply their energy needs. Upon activation, effector CD8 T cells prioritize glycolysis to maintain their effector function and clonal proliferation. In fact, the breakdown of glucose molecules is responsible for the generation of building blocks important to meet the requirements of their high proliferation rate. Memory CD8 T cells, on the other hand, depend on OXPHOS and fatty acid oxidation (FAO). Memory cells have been shown to have more mitochondrial mass than naive T cells. The reliance of memory cells on mitochondria to sustain ATP production provides memory cells with a bioenergetic advantage. Indeed, memory CD8 T cells exhibit enhanced respiratory reserve, measured as the spare respiratory capacity (SRC) (Gerritje JW van der Windt et al.; Immunity, 2012 January 27;36(1):68-78; Guillermo O. Rangel Rivera et al., Front. Immunol., 18 March 2021 |https: / / doi.org / 10.3389 / fimmu.2021.645242).

[0008] Different subsets of memory CD8 T cells have complementary roles or localizations. Among others, stem cell-like memory, effector memory, central memory, and tissue-resident memory can be highlighted. Both stem cell-like and central memory T cells express CD62L, an L-selectin that mediates adhesion and allows homing to secondary lymphoid tissues. This unique ability to enter lymph nodes (LNs) results in optimized screening of antigen-presenting cells (APCs) bearing various antigens on their surface, and a strong recall response induced by central memory cells. Effector memory T cells are restricted in circulation to the bloodstream and display direct cytotoxic and effector functions upon reinfection by encountered pathogens. Conversely, tissue-resident memory cells do not circulate, but instead localize to peripheral tissues such as the skin, lung, and intestine, where they efficiently block pathogens at diverse sites of entry.

[0009] The immune system is educated to tolerate and not react against self, so that cancer cells may not be detected with the same intensity as invading pathogens. In cancer patients, T cells usually respond poorly or not at all to syngeneic transformed cells because of (i) poor antigenicity, (ii) the transformed cells are not phenotypically foreign, and (iii) the generalized immunosuppressive state often associated with cancer (Medler et al., 2015, “Immune response to cancer therapy: mounting an effective antitumor response and mechanisms of resistance”, Trends Cancer 1:66-75).

[0010] Interestingly, targeting specific immune suppressive mechanisms by checkpoint blockade therapy enhances the immune response elicited against cancer. Furthermore, metabolic competition may exist at the tumor site between cancer cells and infiltrating immune cells. A high dependency of cancer cells on glycolysis is often observed, leading to a reduction in glucose, one fuel source, from the intra-tumoral environment. The inability to engage in glycolysis in effector CD8 T cells has a dramatic negative impact on their effector function and killing capacity.

[0011] T cell-based immunotherapy uses the cancer patient's immune system to target the tumor mass itself. Immune cells are extracted directly from the tumor, such as tumor-infiltrating lymphocytes (TILs), or directly from the blood, such as peripheral blood mononuclear cells (PBMCs). TILs with the correct anti-tumor specificity can be selected by cell culture methods and effective killing ability. CD8 T cells extracted from the blood can be modified to acquire tumor reactivity, such as by chimeric antigen receptors (CARs). TILs or CAR-T cells are cultured in vitro, for example in the presence of high doses of IL-2, which promotes robust proliferation, before being reinfused into the patient, a method called adoptive cell transfer (ACT) (Rohaan, MW, Wilgenhof, S. & Haanen, JBAG, “Adoptive cellular therapies: the current landscape”, Virchows Arch 474, 449-461 (2019). One advantage of ACT is its high specificity compared to conventional therapies such as chemotherapy, radiotherapy, and surgery. Moreover, in some cancers, such as melanoma and lung cancer, ACT with autologous TILs represents the most efficient way to treat patients.Patients with advanced melanoma treated with conventional chemotherapy have an overall survival rate of 10%, while after ACT, the overall survival rate increases to 41% (Larkin, James et al. “Overall Survival in Patients With Advanced Melanoma Who Received Nivolumab Versus Investigator's Choice Chemotherapy in CheckMate 037: A Randomized, Controlled, Open-Label Phase III Trial.” Journal of clinical oncology: official journal of the American Society of Clinical Oncology vol. 36, 4 (2018): 383-390. doi: 10.1200 / JCO.2016.71.8023; Dafni, U et al. “Eficacy of acoptive therapy with tumor-infiltrating lymphocytes and recombinant interleukin-2 in advanced cutaneous melanoma: a systematic review and meta-analysis.” Annals of Oncology: official journal of the European Society for Medical Oncology vol.30,12(2019):1902-1913.doi:10.1093 / annonc / mdz398).Similarly, CAR therapy targeting CD19 expression has consistently demonstrated high antitumor efficacy in children and adults with relapsed B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (B-CLL), and non-Hodgkin lymphoma (NHL), with complete remission rates ranging from 70 to 94% in different clinical trials (Wang et al., 2017, “New development in CAR-T cell therapy”, J Hematol Oncol 10:53; Morotti, M., Albukhari, A., Alsaadi, A. et al., “Promises and challenges of adoptive T-cell therapies for solid tumours”, Br J Cancer 124, 1759-1776 (2021)). ACT has yet to realize its potential for treating a wide variety of diseases, including cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immune disorders. Nevertheless, obstacles remain to be overcome for ACT therapy. Patients who exhibit no or very low amounts of tumor-infiltrating T cells will not benefit from this therapy. Tumors can be classified as "hot" tumors, i.e., tumors with elevated levels of T cell infiltration, and "cold" tumors, i.e., tumors with low levels of T cell infiltration. "Hot" tumors would be favorable for harvesting sufficient amounts of CD8 T cells for in vitro expansion before reinfusion. Furthermore, cultured TILs must maintain effector function, proliferation capacity, and self-renewal capacity during ACT to induce a strong anti-tumor response. As a result, infusion of terminally differentiated TILs, such as cells with limited proliferation and self-renewal capacity, is detrimental with respect to the clinical outcome of patients. Importantly, selection of TILs exhibiting a memory-like phenotype improves anti-tumor responses after transfer to patients.Current methods of targeting one subset within extracted TILs are time consuming, can be metabolically challenging for the cells, and can require surface fluorescent labeling, such as sorting by flow cytometry.

[0012] Despite demonstrating high efficacy against hematological malignancies, strong antitumor responses induced by CAR-T cells are often accompanied by toxicity (i.e., severe cytokine release syndrome and neurotoxicity), while patients with poor CAR-T proliferation and persistence show reduced rates of sustained remission. In summary, the selection of memory-like TILs or CAR-T cells in a simple and efficient manner would dramatically benefit clinical outcomes by potentially improving the duration of mediated responses.

[0013] Autoimmunity, a failure of the immune response that results in diseases such as colitis and lupus, occurs when the immune system mistakenly attacks healthy cells. Most parts of the body can be targeted by the immune system, including the heart, brain, nerves, muscles, connective tissue, skin, eyes, lungs, kidneys, gastrointestinal blood cells, and blood vessels. There is a wide range of autoimmune diseases, as they vary depending on the part of the body that is targeted by the immune system. Common autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune vasculitis, myasthenia gravis, pernicious anemia, Hashimoto's thyroiditis, type 1 diabetes, inflammatory bowel disease (IBS), Addison's disease, Graves' disease, Sjogren's syndrome, psoriasis, and celiac disease. To date, the American Autoimmune Related Disease Association (AARDA) has classified over 100 autoimmune diseases, making it the third most common type of disease in the United States. In fact, autoimmune diseases affect 5-10% of the world's population, especially women, who are 2-10 times more likely to suffer from autoimmune diseases than men. Although most diseases can occur at any age, some diseases occur primarily during childhood and adolescence (e.g., type 1 diabetes), mid-adulthood (e.g., myasthenia gravis, multiple sclerosis), or older adults (e.g., rheumatoid arthritis, primary systemic vasculitis) (Wang et al., 2015, "Human autoimmune diseases: a comprehensive update", J Intern Med 278:369-95).

[0014] Regulatory T cells (Tregs) belong to the CD4 T cell compartment and play a key role in controlling, reducing, or treating autoimmune diseases. They depend on mitochondria to support their function and energy needs. Tregs balance the triggered immune response, allow an appropriate response to invading pathogens, and act to avoid or limit tissue damage targeted by the immune system. They mediate their role in dampening immune responses (i) by direct binding to immune cells, (ii) by producing anti-inflammatory cytokines such as IL-10 and IL-35, and (iii) by competing with higher affinity for the survival signal IL-2. Polyclonal Treg therapy uses the same principle as ACT, whereby extracted autologous Tregs are expanded in vitro and then reinjected into the patient with the aim of restoring balance to the immune response (Peter J. Eggenhuizen et al., “Treg Enhancing Therapies to Treat Autoimmune Diseases”, Int J Mol Sci. 2020 Oct; 21(19): 7015).

[0015] Thus, the technical problem underlying the present invention is to provide novel treatments and therapeutic strategies for the selection of persistent or memory-like TIL and CD8 T cells from blood, or Tregs from the CD4 compartment. The present invention aims to increase the proportion of CD8 T cells with higher viability after in vitro expansion, which are used in conjunction with ACT or Tregs selected for Treg therapy. The solution of the above technical problem is achieved by providing the embodiments characterized in the claims. Summary of the Invention

[0016] The present disclosure relates to mitochondrial-enhanced immune cells, compositions and therapeutic uses thereof.

[0017] The present disclosure provides immune cells, which are treated with isolated viable mitochondria or exogenous isolated viable mitochondria in an amount effective to enhance survival and / or promote selection of adaptive immune cells, e.g., human adaptive immune cells, such as B cells or T cells, preferably T cells, such as CD4 immune T cells or CD8 immune T cells, compared to adaptive immune cells, e.g., human adaptive immune cells, that are not treated with isolated viable mitochondria or exogenous viable mitochondria. In some embodiments, the mitochondria of the present disclosure enhance survival and / or promote selection of memory CD8 T cells, such as central memory CD8 T cells and effector memory CD8 T cells. In some other embodiments, the viable mitochondria are in an amount effective to enhance survival and / or promote selection of regulatory T (Treg) cells, such as Treg CD4 cells.

[0018] Provided herein is a composition of isolated viable mitochondria or immune cells treated with exogenous isolated viable mitochondria. The composition may further comprise one or more pharma- ceutically acceptable carriers.

[0019] Also provided herein are methods of enhancing the viability and / or facilitating the selection of an immune cell or population of immune cells, such as adaptive immune cells, e.g., human T cells, comprising (a) activating the immune cells in vitro in a cell-free medium comprising a specific activating receptor agonist antibody capable of driving adaptive cell (such as T cell) activation, and (b) exposing the immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days. In some embodiments, the methods of enhancing the viability and / or facilitating the selection of an immune cell or population of immune cells alternatively comprise (a) activating the immune cells in vitro in a cell-free medium with coated CD3 / CD28 beads, optionally in the presence of a recombinant interleukin, such as IL-2, and (b) exposing the immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days.

[0020] Also provided herein is an immune cell, e.g., a human immune cell such as a human T cell, or population of immune cells, treated with isolated viable mitochondria, or consisting of exogenous isolated viable mitochondria, for use in a method of treating a subject in need thereof. [Brief description of the drawings]

[0021] [Figure 1] It is an immune response elicited in response to an acute infection. [Diagram 2] FIG. 1 is a schematic diagram of one exemplary protocol for isolating mitochondria from tissues or cultured cells. [Figure 3A]Increase in the percentage of central and effector memory CD8+ T cells 9 days after mitochondria transplantation. Percentage of fold change of central memory CD8+ T cells upon mitochondria transplantation. CD8+ T cells from bulk populations were transplanted with exogenous mitochondria at dose levels of 30 μg and 100 μg mitochondria per million CD8+ T cells, measured using Qubit™ Protein Assay, 12 days after activation. At 9 days after transplantation, CD8+ T cells were stained and analyzed by flow cytometry using FACSLyric (BD Biosciences) and classified as central memory (CD62L+, CD45RA-, CD45RO+) and effector memory (CD62L-, CD45RA-, CD45RO+). Data represent three independent experiments presented as mean ± SD of three donors. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3B] Increase in the percentage of central and effector memory CD8+ T cells 9 days after mitochondria transplantation. Percentage of fold change of effector memory CD8+ T cells upon mitochondria transplantation. CD8+ T cells from bulk populations were transplanted with exogenous mitochondria at dose levels of 30 μg and 100 μg mitochondria per million CD8+ T cells, measured using Qubit™ Protein Assay, 12 days after activation. At 9 days after transplantation, CD8+ T cells were stained and analyzed by flow cytometry using FACSLyric (BD Biosciences) and classified as central memory (CD62L+, CD45RA-, CD45RO+) and effector memory (CD62L-, CD45RA-, CD45RO+). Data represent three independent experiments presented as mean ± SD of three donors. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Detailed Description of the Invention

[0022] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference to what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodology, and are generally understood and commonly employed by those skilled in the art using conventional methodology, and are generally understood and commonly employed by those skilled in the art using conventional methodology, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4 th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise noted.

[0023] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Terms such as "include," "such as," and the like are intended to convey inclusion without limitation unless specifically stated otherwise.

[0024] As used herein, the term "comprising" also specifically includes embodiments that "consist of," and "consisting essentially of," the recited elements, unless specifically stated otherwise.

[0025] The term "about" refers to and encompasses the indicated value and a range above and below that value. In certain embodiments, the term "about" refers to the indicated value ±10%, ±5%, or ±1% of the indicated value. In certain embodiments, the term "about" refers to the indicated value ±1 standard deviation of the indicated value.

[0026] The term "isolated" means changed or removed from a natural state or environment. For example, a nucleic acid or peptide that is naturally present in a living animal or cell is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated."

[0027] The term "mitochondria" or "mitochondrion" as used herein refers to viable mitochondria that are (essentially) free of eukaryotic cellular material, e.g., foreign eukaryotic cellular material isolated / purified from cells or cell culture. Thus, only minimal amounts of cellular components (other than mitochondria) are present in the (composition of) mitochondria used herein. Preferably, no cellular components other than mitochondria are present in the (composition of) mitochondria used herein. Isolated mitochondria are preferably present in a substantially purified form, e.g., partially or completely separated from coexisting materials in their native state. In this sense, "mitochondria" as used herein is "isolated mitochondria", and the terms "mitochondria" and "isolated mitochondria" can be used interchangeably. Any technique known in the art can be used to isolate mitochondria, e.g., subcellular fractionation by repeated differential centrifugation (DC) or density gradient centrifugation (DGC). Thus, the mitochondria of the present invention are preferably alive or viable and have a negative membrane potential. In the sense of the present invention, "being alive" means having or maintaining a metabolic or another biological function or structure.

[0028] As used herein, the term "viable mitochondria" is used herein to describe viable mitochondria that are intact, active, functional, and capable of respiration. According to some embodiments, "viable mitochondria" refers to mitochondria that exhibit biological functions, such as, for example, respiration, and ATP and / or protein synthesis.

[0029] As used herein, the term "intact mitochondria" is used throughout the present specification to describe mitochondria that contain integer outer and inner membranes, integer intermembrane space, integer crista (formed by the inner membrane), and integer matrix. Alternatively, intact mitochondria are mitochondria that preserve their structure and ultrastructure. In another embodiment, intact mitochondria contain an active respiratory chain complex IV embedded in the inner membrane and maintain the membrane potential and the ability to synthesize ATP.

[0030] As used herein, the term "transplantation" is used throughout the present specification as a general term to describe the process of transplanting an organ, tissue, cell mass, individual cell, or organelle into a recipient. The term "cell transplantation" is used throughout the present specification as a general term to describe the process of transplanting at least one cell, such as the enhanced immune cells described herein, into a recipient. The term includes all categories of transplantation known in the art, including blood transfusion. Transplants are classified by the site and genetic relationship between the donor and the recipient. The term includes, for example, autotransplantation (removal and transplantation of cells or tissue from one location in a patient to the same or another location in the same subject), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantation between members of different species).

[0031] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds that contain amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limit is placed on the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0032] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. The term also includes non-immunoglobulin antigen-binding protein molecules, so-called antibody mimetics. Antibodies specifically include intact antibodies (e.g., intact immunoglobulin G, IgG), antibody fragments (e.g., Fab fragments, single-chain Fvs (scFvs), single domain antibodies, VFvs ... H , V L , V HH , NAR, tandem scFv, diabody, single chain diabody, DART, tandAb, minibody, single domain antibody (e.g. camelid V HH), other antibody fragments or formats known to those of skill in the art, and antibody mimetics (e.g., adnectins, affibodies, affilins, anticalins, avimers, DARPins, knottins, etc.). Antibodies can be monospecific, bispecific, and multispecific.

[0033] The term "antigen-binding domain" refers to a portion of an antibody or T cell receptor that can specifically bind to an antigen or epitope via the variable domain. As used herein, "variable domain" refers to a variable nucleotide sequence resulting from a recombination event and can include, for example, the V, J, and / or D regions of a T cell receptor (TCR) sequence from a T cell, such as an activated T cell, or can include the V, J, and / or D regions of an antibody. The term "antigen-binding fragment" refers to at least a portion of an antibody or TCR or a recombinant variant thereof that contains the antigen-binding domain, i.e., the variable domain and the hypervariable loops, the so-called complementarity determining regions (CDRs), sufficient to confer recognition and specific binding of the antigen-binding fragment to a target, such as an antigen and its defined epitope. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments, single-chain (sc)Fv ("scFv") antibody fragments, linear antibodies, single domain antibodies (abbreviated as "sdAb") (V, V ... L or V H Camelidae V HH Exemplary antibody and antibody fragment formats include, but are not limited to, domains (nanobodies), multispecific antibodies generated from antibody fragments, and TCR fragments. Exemplary antibody and antibody fragment formats are described in detail in Brinkmann et al. (MABS, 2017, Vol. 9, No. 2, 182-212), which is incorporated herein by reference for all that it teaches.

[0034] The term "scFv" refers to the assembly of an antibody light chain (V) via a flexible polypeptide linker.L ) linked at the N-terminus and C-terminus of the variable fragment of an antibody heavy chain (V H scFv refers to a fusion protein that contains a variable fragment of an antibody (such as a ribosome-specific antibody) and can be expressed as a single polypeptide chain, whereas scFv retains the specificity of the intact antibody from which it is derived.

[0035] The terms "linker" and "flexible polypeptide linker" as used in the context of scFvs refer to a peptide linker consisting of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, having the amino acid sequence (Gly-Gly-Gly-Gly-Ser). n In one embodiment, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)3 or (Gly4Ser)4. In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser). Also included within the scope of the present invention are the linkers described in WO2012 / 138475 (hereby incorporated by reference).

[0036] The "heavy chain variable region" or "V H (or, in the case of camelid single domain antibodies, e.g., nanobodies, “V HH ") refers to a fragment of a heavy chain containing the three CDRs inserted between flanking stretches known as framework regions (FRs), which are usually more conserved than the CDRs and form a scaffold that supports the CDRs.

[0037] Unless otherwise specified, as used herein, an scFv can be, for example, a VFv polypeptide, in any order with respect to the N-terminus and C-terminus of the polypeptide. L and V H The scFv may have a variable region, L -Linker-V H or V H -Linker-V L may include.

[0038] The term "antibody heavy chain" refers to the larger of two polypeptide chains that exist in antibody molecules in naturally occurring conformations and generally determines the immunoglobulin class to which the antibody belongs.

[0039] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in the naturally occurring conformations in antibody molecules. Kappa ("κ") and lambda ("λ") light chains refer to the two major antibody light chain isotypes.

[0040] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, e.g., an antibody expressed by a bacterial, yeast, plant, or mammalian cell. The term should also be taken to mean an antibody produced by the synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology that is available and well known in the art.

[0041] The term "antigen" or "ag" refers to a molecule foreign to the body, such as that present on a pathogen that can be bound by an antibody or T cell receptor. Antigens can be presented by antigen-presenting cells (APCs) and can elicit an immune response under certain circumstances.

[0042] Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Additionally, the antigen can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as used herein. Additionally, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be appreciated that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, which nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Additionally, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be appreciated that an antigen can be produced by chemical synthesis, can be derived from a biological sample, or can be a macromolecule other than a polypeptide, such as a lipid or carbohydrate. Such biological samples include, but are not limited to, tissue samples, tumor samples, cells, or fluids having other biological components.

[0043] The term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell viability, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested primarily by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in preventing the development of tumors.

[0044] The term "immunosuppressive effect" refers to a biological effect that can inhibit or interfere with normal immune function.

[0045] A "human antibody" or "human TCR" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or is derived from a non-human source that utilizes a human antibody or TCR repertoire or human antibody / TCR coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies and TCRs specifically exclude humanized antibodies and TCRs, respectively.

[0046] With respect to the binding of an antibody, TCR, or antigen-binding fragment thereof to a target molecule, the terms "bind," "specific binding," "specifically binds to," "specific for," "selectively binds," and "selectively for" to a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refer to binding that is measurably different from non-specific or non-selective interactions (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Specific binding is then indicated if binding of the antibody, TCR, or antigen-binding fragment thereof to the target molecule is competitively inhibited by the control molecule. Specific binding, as used herein, is defined as the binding rate of the antibody, TCR, or antigen-binding fragment thereof to the target molecule that is competitively inhibited by the control molecule. D A value of 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10It may refer to an affinity that is less than M. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0047] The term "autologous" refers to any material derived from the same individual that is later reintroduced into the individual.

[0048] The term "allogeneic" refers to any material derived from a different animal or different patient of the same species as the individual into whom the material is introduced. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical. In some embodiments, allogeneic material derived from individuals of the same species may be sufficiently genetically different to interact antigenically.

[0049] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0050] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease or disorder in a subject in need thereof. Treatment can be performed prophylactically and during the course of clinical pathology. Desirable effects of treatment include prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis.

[0051] As used herein, a "therapeutically effective amount" is an amount of a composition or its active ingredients sufficient to provide a beneficial effect or reduce otherwise adverse non-beneficial events in an individual to whom the composition is administered. A "therapeutically effective dose" herein means a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, and where such administration occurs one or more times over a given period of time. The exact dose depends on the purpose of the treatment and can be ascertained by one of skill in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0052] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide derived from various polypeptides that includes an antigen-binding portion (e.g., a polypeptide having at least an antigen-binding domain or an antigen-binding fragment thereof) fused to a primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner and may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or "ITAM." Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from CD3zeta (zeta), FcRgamma (gamma), FcRbeta (beta), CD3gamma, CD3delta (delta), CD3epsilon (epsilon), CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), and CD66d. CARs typically provide engineered immune cells, such as T lymphocytes, with antibody- or TCR-type specificity, to activate some or all of the functions of effector cells, including the production of IL-2 and lysis of target cells following signal transduction in the T cell.

[0053] The antigen-binding domain or antigen-binding fragment of the CAR described herein may exist in various forms, for example, the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single domain antibody fragment (sdAb) or heavy chain antibody (HCAb), single chain Fv antibody (scFv), either naturally derived or synthetic, and binds to the antigen. The antigen-binding domain or antigen-binding fragment of the CAR described herein may include any of the antibody or antibody fragment formats described herein. The antigen-binding domain or antigen-binding fragment of the CAR described herein may include sequences that are not derived from antibodies, including, but not limited to, chimeric or artificial T cell receptors (TCRs). These chimeric / artificial TCRs may include polypeptide sequences that recognize a target antigen, and the recognition sequence may be, for example, but is not limited to, a recognition sequence derived from a TCR or scFv. The intracellular domain polypeptide acts to activate the T cell. Chimeric / artificial TCRs are described, for example, in Gross, G., and Eshhar, Z., FASEB Journal 6:3370-3378(1992), and Zhang, Y., et al., PLOS Pathogens 6:1-13(2010).

[0054] A "CAR-T cell" is a T cell that has been transduced according to the methods disclosed herein and expresses a CAR gene, e.g., randomly integrated into the genome or purposefully integrated into the CCR5 and AAVS1 loci, or the T cell receptor alpha constant (TRAC) locus. In some embodiments, the T cell is a CD4 + T cells, CD8 + T cells, or CD4 + / CD8 + In some embodiments, the T cells are regulatory T cells. In some embodiments, the T cells are autologous, allogeneic, or xenogeneic with respect to the subject.

[0055] As used herein, the term "subject" refers to a mammalian subject. The term "subject" is intended to include a living organism (e.g., a mammal, a human) in which an immune response can be elicited. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. A "patient" is a subject suffering from or at risk of developing a disease, disorder, or condition, or in need of the compositions and methods provided herein.

[0056] As used herein, "preventing" refers to the prevention of a disease or condition, e.g., tumor formation, in a patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated by the methods of the invention and does not subsequently develop a tumor or other form of cancer, the disease is prevented in that individual, at least for a period of time.

[0057] As used herein, the term "CD19" refers to B lymphocyte antigen CD19, CD19 molecule (cluster of differentiation 19), B lymphocyte surface antigen B4, T cell surface antigen Leu-12, and CVID3, a transmembrane protein encoded by the gene CD19 in humans. In humans, CD19 is expressed in all B lineage cells except plasma cells, and in follicular dendritic cells. CD19 plays two major roles in human B cells. It acts as an adaptor protein that recruits cytoplasmic signaling proteins to the membrane, and acts within the CD19 / CD21 complex to reduce the threshold of the B cell receptor signaling pathway. Because it is present on all B cells, it is a biomarker for B lymphocyte development, lymphoma diagnosis, and can be used as a target for immunotherapy of leukemia and lymphoma.

[0058] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic or diagnostic product (e.g., a kit) that contain information regarding directions, usage, dosage, administration, combination therapy, contraindications, and / or warnings regarding the use of such therapeutic or diagnostic product.

[0059] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0060] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapeutics," which act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0061] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells or tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein. The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematological malignancy (blood tumor).

[0062] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient and / or maintains or improves the viability of a biological entity (e.g., a cell) contained therein, such that it is effective to treat a subject, and that does not contain additional ingredients that are unacceptably toxic to a subject in the amounts provided in the pharmaceutical composition.

[0063] The term "pharmaceutically acceptable carrier" includes those compatible with pharmaceutical administration, such as saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. In some embodiments, the pharmaceutically acceptable carrier is phosphate buffered saline, saline, Krebs buffer, Tyrode's solution, a contrast agent, or Omnipaque, or a mixture thereof. The term "pharmaceutical acceptable carrier" also includes sterile mitochondrial buffer (300 mM sucrose, 10 mM K+-HEPES (potassium buffered (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), pH 7.2), 1 mM K+-EGTA (potassium buffered ethylene glycol tetraacetic acid, pH 8.0). The term further includes respiration buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCh, 20 mM K-15 HEPES buffer, pH 7.2, and 0.5 mM K-EGTA, pH 8.0). The term further includes T cell media, such as RPMI 1640 medium GlutaMAX™ Supplement 500 ml (ThermoFisher, 61870010).

[0064] The terms "modulate" and "modulation" refer to either decreasing or inhibiting, or activating or increasing, the recited variable.

[0065] The terms "increase," "activate," and "enhance" mean to increase or decrease the activity of a listed variable by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 98%, 99%, 10 ...5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 98%, 99%, 100%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 95%, 98%, 99%, 100%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, This refers to an increase of .5x, 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 55x, 60x, 65x, 70x, 75x, 80x, 85x, 90x, 95x, 100x, or more.

[0066] The terms "reduce" and "inhibit" refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or greater decrease in the recited variable.

[0067] The term "agonize" refers to the activation of receptor signaling to induce a biological response associated with receptor activation. An "agonist" is an entity that binds to and agonizes a receptor.

[0068] The term "antagonize" refers to the inhibition of receptor signaling to inhibit a biological response associated with receptor activation. An "antagonist" is an entity that binds to and antagonizes a receptor.

[0069] The term "immune cells" refers to cells that belong to the immune system to protect the organism from infection or disease such as cancer. "Immune cells" are classified between the innate and adaptive immune responses.

[0070] The term "population of immune cells" or "population of adaptive immune cells" refers to a heterogenous group of immune cells or adaptive immune cells.

[0071] "Effector T cells" and "memory T cells" are T helper (i.e., CD4 + ) cells and cytotoxicity (i.e., CD8 + ) T cells. CD4 + Effector T cells typically contribute to the development of several immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. + Effector T cells typically kill virus-infected cells and tumor cells. CD8 + Memory T cells typically provide long-term protection against reinfection or cancer recurrence through enhanced recall capacity. For more information on effector T cells, see Seder and Ahmed, 2003, "Similarities and differences in CD4+ and CD8+ effector and memory T cell generation", Nat Immunol 4:835-42, which is incorporated by reference in its entirety. CD8 effector memory cells express surface markers CD62L-, CD45RA+, CD45RO-.

[0072] The term "central memory T cells" ("Tcm cells") refers to cells expressing the surface markers CD62L+, CD45RA-, CD45RO+. Human Tcm cells that constitutively express CD62L are required for cell extravasation through high endothelial venules (HEV) and migration to T cell areas of secondary lymphoid organs. Tcm cells efficiently mediate recall responses upon a second encounter with the same antigen.

[0073] The term "effector memory cells", such as CD8 effector memory cells, refers to cells that express the surface markers CD62L-, CD45RA-, CD45RO+ and are capable of migrating to inflamed peripheral tissues and exhibiting effector function.

[0074] The term "memory-like cells", such as "memory-like T cells", refers to cells that exhibit the characteristics and properties of memory cells. They may mimic one or more surface marker expression and exhibit at least one or more characteristics of memory cells, such as enhanced recall capacity, survival, and self-renewal. For example, in the presence of acute infection, T cells can be classified as memory depending on their surface expression and behavior. Some T cells exhibit memory-like properties during anti-tumor responses (Shiki Takamura, International Immunology, Volume 32, Issue 9, 1 September 2020, Pages 571-581).

[0075] The term "Tscm cells" or "stem cell-like memory cells" refers to memory cells that are at their earliest and longest lasting developmental stage, exhibit stem cell-like properties, and display a genetic profile between naive and central memory T cells. Stem cell-like memory cells express the surface markers CD62L+, CD45RA+, and CD45RO+.

[0076] "Trm cells" or "tissue-resident T cells" refer to a subset of long-term memory T cells that occupy epithelial and mucosal tissues (skin, mucosa, lung, brain, pancreas, gastrointestinal tract) without recirculation. Trm cells are a type of central memory T cell (T cell) that recirculates between blood, T cell zones of secondary lymphoid organs, lymphoid tissues, and non-lymphoid tissues. CM ) and effector memory T cells (T EM ) are transcriptionally, phenotypically, and functionally distinct. Trm cells themselves represent a diverse population due to the specialization of resident tissues.

[0077] The term "naive cells" refers to resting cells and cells that have not been activated. For example, naive T cells have not encountered antigens and circulate in vivo to screen peptides presented by APCs. Naive T cells express surface markers CD62L+, CD45RA+, and CD45RO-.

[0078] The term "adaptive immune cells" refers to cells that belong to the "adaptive immune system" or "acquired immune system" and play a key role in adaptive immunity. Adaptive immune cells are a subset of immune cells, which are highly specialized systemic cells. Specifically, lymphocyte B cells and T cells are adaptive immune cells. Adaptive immune cells can function to eliminate pathogens or prevent their growth. Adaptive immunity can create immune memory (e.g., memory B cells or memory T cells) after an initial response to a particular pathogen, resulting in an enhanced response to future encounters with that pathogen. The terms "adaptive immune cells" and "adaptive cells" can be used interchangeably.

[0079] The terms "CD4 T cell" and "CD8 T cell" refer to CD4 positive T cells and CD8 positive T cells, respectively. The terms "CD4 T cell", "CD4 immune T cell", and "CD4 immune cell" can be used interchangeably. The terms "CD8 T cell", "CD8 immune T cell", and "CD8 immune cell" can be used interchangeably.

[0080] The term "regulatory T cells" or "Treg" includes cells that regulate immunological tolerance, for example by suppressing effector T cells. In some embodiments, regulatory T cells are CD4 + CD25 + Foxp3 + In some embodiments, the regulatory T cells have a CD8 + CD25 + Regulatory T cells have a phenotype. For further information on regulatory T cells, see Nocentini et al., Br. J. Pharmacol., 2012, 165:2089-2099, which is incorporated by reference in its entirety. As used herein, the term "regulatory T cells (Treg)" preferably refers to a subset of CD4+ T cells that are important for immune homeostasis. Tregs are defined by the expression of the transcription factor forkhead box protein P3 (Foxp3), which is essential for their development and suppressive function. Loss of Foxp3 function leads to severe lymphoproliferative disease and autoimmunity. In addition to preventing autoimmune and inflammatory diseases, Tregs ensure a controlled immune response when pathogens are encountered, thereby preventing immune pathology. Conversely, excessive suppression by Tregs can interfere with pathogen clearance and promote chronic infection. In addition, Tregs can also suppress anti-tumor immune responses, thus promoting tumor progression.

[0081] The term "dendritic cell" refers to professional antigen-presenting cells that can activate naive T cells and stimulate the proliferation and differentiation of B cells.

[0082] The phrase "disease associated with expression of [target]" includes, but is not limited to, a disease associated with expression of [target] or a disease associated with a cell expressing [target], e.g., a proliferative disease such as a cancer or malignant tumor, or a precancerous disease such as a solid tumor or a hematological tumor. Non-cancer related indications associated with expression of [target] include, but are not limited to, for example, autoimmune diseases (e.g., lupus, rheumatoid arthritis, colitis), inflammatory disorders (allergies and asthma), and transplantation.

[0083] The term "stimulation" refers to a primary response induced by the binding of a stimulatory domain or molecule (e.g., CAR or TCR / CD3 complex) with its cognate ligand or antigen-independent CD3 / CD28 bead in vitro, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules and / or rearrangements of cytoskeletal structures.

[0084] The term "stimulatory molecule" or "stimulatory domain" refers to a molecule or portion thereof expressed by a T cell or engineered immune cell (e.g., an immune cell engineered to express a CAR) that provides a primary cytoplasmic signaling sequence that regulates primary activation of the TCR / CAR complex in a stimulatory way for at least some aspects of a signaling pathway, such as the T cell signaling pathway. In one embodiment, the primary signal is initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. In one embodiment, the primary signal is initiated, for example, by binding of the CAR (e.g., an antibody fragment or chimeric TCR) to its cognate antigen or epitope.

[0085] The term "antigen presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., dendritic cells, macrophages, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs typically process antigens and present them to T cells, but can be "loaded" with preprocessed antigenic peptides.

[0086] "Intracellular signaling domain", as the term is used herein, refers to the intracellular portion of a molecule involved in the generation of a signal that promotes immune effector functions, such as effector functions of TCR or CAR-expressing T cells. The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that may be required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137). A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83. The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment thereof.The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Acc. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and the "4-1BB costimulatory domain" is defined as amino acid residues 214 to 255 of GenBank Acc. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0087] The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, serving as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0088] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" can also include introns to the extent that a nucleotide sequence that encodes a protein may contain one or more introns in some versions.

[0089] The term "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

[0090] The term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system. In the case of a patient, the term "exogenous" can refer to material derived from the patient, donor, or cell culture. For example, mitochondria isolated from a patient's muscle tissue and then introduced into a population of immune cells, which may be autologous or autogenic, of the patient, are considered exogenous. The term "exogenous mitochondria" refers to any mitochondria isolated from autologous, allogeneic, and / or xenogeneic sources, and the nature of the source may be that of tissue, blood, or cultured cells.

[0091] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0092] As used herein, the term "expression vector" refers to a vector that contains a nucleic acid sequence that encodes at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0093] As used herein, an "expression construct" or "transgene" is defined as any type of genetic construct containing a nucleic acid that encodes a gene product, where part or all of the nucleic acid encoding sequence can be inserted into a vector.

[0094] As used herein with respect to a disease, disorder, or condition, the terms "treatment," "treat," "treated," or "treating" refer to prevention and / or treatment.

[0095] The term "lentivirus" refers to the genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and because they can deliver significant amounts of genetic information into the DNA of host cells, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0096] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009).

[0097] The term "homologous" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if each position in two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half of the positions in the two sequences (e.g., 5 positions in a polymer of length 10 subunits) are homologous, then the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) are matching or homologous, then the two sequences are 90% homologous.

[0098] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0099] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which results in expression of the latter.

[0100] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intranasal or intrasternal injection, intratumor, or infusion techniques.

[0101] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes, such as using conventional cloning techniques and polymerase chain reaction (PCR), and synthetic means. Additionally, polynucleotide includes mutations of polynucleotides, including, but not limited to, mutations of nucleotides or nucleosides by methods well known in the art. The nucleic acid can comprise one or more polynucleotides.

[0102] The term "promoter" refers to a DNA sequence recognized by the transcriptional or introduced synthetic machinery of a cell capable of initiating the specific transcription of a polynucleotide sequence.

[0103] The term "promoter / regulatory sequence" refers to a nucleic acid sequence that can be used to express a gene product operably linked to the promoter / regulatory sequence. In some instances, the sequence can be a core promoter sequence, and in other cases, the sequence can include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence can be, for example, one that expresses the gene product in a tissue-specific manner.

[0104] The term "constitutive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell under most or all physiological conditions of the cell.

[0105] The term "inducible promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0106] The term "tissue-specific promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0107] As used herein, "transient" refers to expression of a non-integrated transgene over a period of hours, days, or weeks, which period of expression is shorter than the period of expression of the gene when integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0108] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can receive and transmit a signal across a cell membrane.

[0109] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0110] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, amelioration, or eradication of a disease state.

[0111] The term "prophylaxis" as used herein means the prevention or prophylactic treatment of a disease or disease state.

[0112] In the context of the present invention, a "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, mesothelioma, renal cell carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, and gastric cancer.

[0113] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0114] "T cell exhaustion" and "exhausted T cells" refer to either hyporesponsive or "dysfunctional" T cells.

[0115] "Activity" or "activity of an immune cell" refers to cell effector functions such as cytotoxic activity against target cells expressing a particular antigen and detected by TCR or cytokine production specific for that antigen. It further refers to metabolic activity, proliferative potential and ability to grow and divide, ability to resist attrition, and suppressive activity.

[0116] The term "survival" of a cell or cell population refers to, but is not limited to, cell persistence, cell self-renewal, and cell durability. Cell viability can be defined as a process that encompasses the viability of a cell and its ability to sustain and maintain the integrity of cellular processes. Survival mechanisms ensure that cells are able to adapt and continue cellular activities such as replication, repair, and metabolism.

[0117] The term "enhancement of the survival" of immune cells, such as T cells, refers to, for example, the following properties of immune cells: (i) the ability to survive during resting periods and upon restimulation; (ii) responsiveness to interleukins, such as IL-7, IL-15 (e.g., cells may need to increase expression of receptors that respond to those interleukins so they may receive fewer signals to survive); (iii) resistance to activation-induced cell death (AICD); (iv) resistance to apoptosis by upregulating anti-apoptotic molecules, such as BCL-XL, BCL-2; (v) resistance to apoptosis by downregulating pro-apoptotic molecules, such as Fas and FasL expression; (vi) epigenetic modifications and transcriptional alterations that may affect the expression of certain genes, such as Bcl2, Bcl2l2, Mcl1, Bcl2a1d, Birc2, Birc3, Xiap, Cflar, etc. (viii) exhibiting one or more enhancements in telomere length.

[0118] The term "promoting the selection" refers to increasing the amount and / or enhancing some property of one or more subsets of immune cells relative to the majority of an immune cell or immune cell population.

[0119] The term "differentiation" or "cell / cellular differentiation" refers to the process of cells changing from one cell type to another, typically, but not exclusively, from a less specialized type (stem cell) to a more specialized type. Differentiation, particularly immune cell differentiation, can occur in response to antigen exposure. Differentiation can dramatically change a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals.

[0120] The term "immune cells treated with mitochondria" may refer to immune cells that have been exposed to, in close contact with, co-incubated with, or transplanted with mitochondria. The term "mitochondrial treatment" refers to the act of exposing a cell to mitochondria, or placing a cell in close contact / with mitochondria, or co-incubating a cell with mitochondria, or transplanting mitochondria into a cell.

[0121] The term "self-renewal capacity" or "cell self-renewal capacity" refers to the process of a cell to produce an unlimited number of cells of the same cell type. Self-renewal is the ability to divide and retain all the characteristics of the parent cell. Self-renewal keeps the number of cells roughly the same.

[0122] The term "recall capacity" refers to a secondary immune response elicited by memory cells that leads to rapid proliferation and differentiation into effector cells. This recall response is important in controlling the extent of infection and preventing disease.

[0123] The term "transplanted mitochondria" refers to exogenous mitochondria that are substantially integrated into a target cell (e.g., partially or completely integrated into the cell). "Mitochondrial transplantation," "transplantation of mitochondria," or "transfer of mitochondria" refers to the act of incorporating / transferring exogenous mitochondria into a host cell.

[0124] Ranges. Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, including subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the range.

[0125] Enhancement of immune cells and related compositions The present invention will be described in more detail below. Specifically, the present invention relates to the following items. 1. The present disclosure provides immune cells, e.g., human immune cells, treated with isolated viable mitochondria in an amount effective to enhance the viability and / or promote selection of adaptive immune cells, e.g., human adaptive immune cells, such as B cells or T cells, preferably CD8 immune cells or CD4 immune cells, compared to adaptive immune cells, e.g., human adaptive immune cells, that have not been treated with isolated viable mitochondria. 2. The present disclosure provides immune cells, e.g., human immune cells, treated with isolated viable mitochondria in an amount effective to promote memory cell differentiation and / or memory cell selection of adaptive immune cells, e.g., memory T cells, compared to immune cells that have not been treated with isolated viable mitochondria. 3. Specifically, the disclosure provides immune cells, e.g., human immune cells, such as human immune T cells, treated with isolated viable mitochondria in an amount effective to enhance viability of memory CD8 T cells and / or promote selection of memory CD8 T cells compared to immune cells, e.g., human adaptive immune cells, that are not treated with isolated viable mitochondria. In some specific embodiments, the isolated viable mitochondria is in an amount effective to enhance viability of central memory CD8 T cells and / or promote selection of central memory CD8 T cells compared to immune cells, e.g., human adaptive immune cells, that are not treated with isolated viable mitochondria. In some other specific embodiments, the isolated viable mitochondria is in an amount effective to enhance viability of effector memory CD8 T cells and / or promote selection of effector memory CD8 T cells compared to immune cells that are not treated with isolated viable mitochondria. 4. Specifically, the present disclosure provides immune cells, e.g., human immune cells, such as human T immune cells, treated with isolated viable mitochondria in an amount effective to enhance the viability of Treg cells and / or promote the selection of Treg cells compared to immune cells, e.g., human adaptive immune cells, such as CD4 immune cells, that are not treated with isolated viable mitochondria. 5. The present disclosure also provides immune cells, e.g., human immune cells, comprising exogenous isolated viable mitochondria (e.g., comprising mitochondria partially or fully integrated into the cell), in an amount effective to enhance the viability and / or facilitate the selection of adaptive immune cells, e.g., human adaptive immune cells, such as B cells or T cells, preferably T cells, such as CD8 or CD4 immune cells, compared to adaptive immune cells, e.g., human adaptive immune cells, that do not comprise exogenous isolated viable mitochondria. 6. The disclosure further provides immune cells, e.g., human immune T cells, comprising exogenous isolated viable mitochondria in an amount effective to promote memory cell differentiation and / or memory cell selection of adaptive immune cells, e.g., memory T cells, compared to immune cells, e.g., human adaptive immune cells, that do not comprise exogenous isolated viable mitochondria. 7. Specifically, the present disclosure provides immune cells, e.g., human T immune cells, comprising exogenous isolated viable mitochondria in an amount effective to enhance viability of memory CD8 T cells and / or promote selection of memory CD8 T cells, as compared to immune cells, e.g., human adaptive immune cells, that do not comprise isolated viable mitochondria. In some specific embodiments, the isolated viable mitochondria are in an amount effective to enhance viability of central memory CD8 T cells and / or promote selection of central memory CD8 T cells, as compared to immune cells that do not comprise exogenous isolated viable mitochondria. In some other specific embodiments, the isolated viable mitochondria are in an amount effective to enhance viability of effector memory CD8 T cells and / or promote selection of effector memory CD8 T cells, as compared to immune cells that do not comprise isolated viable mitochondria. 8. Specifically, the present disclosure provides immune cells, e.g., human immune cells, such as human T immune cells, comprising exogenous isolated viable mitochondria in an amount effective to enhance viability of Treg cells and / or promote selection of Treg cells compared to immune cells that do not comprise exogenous isolated viable mitochondria, e.g., human adaptive immune cells, such as CD4 immune cells. 9. Also provided herein is a population comprising an immune cell according to any one of the preceding paragraphs, such as a human immune cell, e.g., a human adaptive immune cell. 10. The present disclosure also provides a composition comprising a human immune cell, e.g., an immune cell, e.g., a human adaptive immune cell, or a composition of immune cells, treated with isolated viable mitochondria in an amount effective to enhance the viability and / or promote the selection of adaptive immune cells, e.g., human adaptive immune cells, such as B cells or T cells, preferably T cells, e.g., CD8 immune cells or CD4 immune cells, compared to adaptive immune cells, e.g., human adaptive immune cells, that have not been treated with isolated viable mitochondria. Specifically, the composition comprises immune cells, e.g., human immune cells, treated with isolated viable mitochondria in an amount effective to enhance the viability and / or promote the selection of memory CD8 T cells, such as central memory CD8 T, effector memory CD8 T cells, or a combination thereof, compared to immune cells, e.g., human adaptive immune cells, that have not been treated with isolated viable mitochondria. Specifically, the compositions comprise immune cells, such as human immune cells, e.g., human T cells, that have been treated with isolated viable mitochondria in an amount effective to enhance the viability and / or promote the selection of Treg cells compared to immune cells, such as CD4 immune cells, e.g., human adaptive immune cells, that have not been treated with isolated viable mitochondria. 11. The present disclosure provides a composition comprising immune cells, or a composition of immune cells, e.g., human immune cells, treated with isolated viable mitochondria in an amount effective to promote memory cell differentiation and / or memory cell selection of adaptive immune cells, e.g., memory T cells, compared to immune cells that have not been treated with isolated viable mitochondria. 12. The present disclosure provides compositions comprising immune cells or compositions of immune cells, e.g., human immune cells, where the immune cells comprise exogenous isolated viable mitochondria in an amount effective to enhance viability and / or facilitate selection of, e.g., human adaptive immune cells, e.g., B cells or T cells, preferably T cells, e.g., CD8 immune cells or CD4 immune cells, compared to adaptive immune cells, e.g., human adaptive immune cells, that do not comprise exogenous isolated viable mitochondria. Specifically, the compositions comprise immune cells, e.g., human immune cells, that comprise exogenous isolated viable mitochondria in an amount effective to enhance viability and / or facilitate selection of, memory CD8 T cells, e.g., central memory CD8 T cells, effector memory CD8 T cells, or a combination thereof, compared to immune cells, e.g., human adaptive immune cells, that do not comprise exogenous isolated viable mitochondria. Specifically, the compositions comprise immune cells, e.g., human immune cells, that comprise exogenous isolated viable mitochondria in an amount effective to enhance the viability of and / or promote the selection of Treg cells compared to immune cells, e.g., human immune cells, such as CD4 immune cells that do not comprise exogenous isolated viable mitochondria. 13. The disclosure provides a composition comprising or of immune cells, e.g., human immune cells, wherein the immune cells comprise exogenous isolated viable mitochondria in an amount effective to promote memory cell differentiation and / or memory cell selection of adaptive immune cells, e.g., memory T cells, compared to immune cells that do not comprise isolated viable mitochondria. 14. The present disclosure provides a composition comprising immune cells, e.g., human immune cells, or a composition of immune cells, e.g., human immune cells, treated with isolated viable mitochondria in an amount effective to enhance the viability and / or facilitate the selection of a population of adaptive immune cells, e.g., a population of human adaptive immune cells, such as B cells or T cells, preferably T cells, such as CD8 immune cells or CD4 immune cells, compared to a population of immune cells, e.g., human immune cells, that are not treated with mitochondria. 15. The present disclosure provides a composition comprising or of immune cells, e.g., human immune cells, treated with isolated viable mitochondria in an amount effective to enhance memory cell differentiation and / or promote memory cell selection of a population of adaptive immune cells, e.g., memory T cells, compared to a population of immune cells that have not been treated with isolated viable mitochondria. 16. The present disclosure provides compositions comprising immune cells, such as T cells, e.g., human T cells, treated with isolated viable mitochondria in an amount effective to enhance memory cell differentiation and / or promote selection of a population of memory CD8 T cells, such as central memory CD8 T cells, effector memory CD8 T cells, or a combination thereof, compared to immune cells, e.g., adaptive immune cells, that have not been treated with isolated viable mitochondria. In some embodiments, the mitochondria can enhance the percentage of memory adaptive cells, e.g., central memory CD8 T cells, or effector memory CD8 T cells, by at least 20%, preferably the last 30%, more preferably at least 50%. 17. The disclosure also provides compositions comprising immune cells, e.g., human immune cells, or compositions of immune cells, e.g., human immune cells, wherein the immune cells comprise exogenous isolated viable mitochondria in an amount effective to enhance viability and / or facilitate selection of a population of adaptive immune cells, e.g., human adaptive immune cells, such as B cells or T cells, preferably T cells, such as CD8 immune cells or CD4 immune cells, compared to a population of immune cells, e.g., human immune cells, that do not comprise exogenous isolated mitochondria. 18. The present disclosure provides a composition comprising immune cells, e.g., human immune cells, or a composition of immune cells, wherein the immune cells comprise exogenous isolated viable mitochondria in an amount effective to enhance memory cell differentiation and / or promote memory cell selection of adaptive immune cells within a population of adaptive immune cells, compared to a population of immune cells, e.g., memory T cells, that do not comprise exogenous mitochondria. 19. Specifically, the disclosure provides compositions comprising immune cells, e.g., human immune cells, e.g., human T cells, comprising exogenous isolated viable mitochondria in an amount effective to enhance the viability and / or facilitate the selection of a population of memory CD8 T cells, e.g., central memory CD8 T cells, effector memory CD8 T cells, or a combination thereof, compared to immune cells, e.g., adaptive immune cells, that do not comprise exogenous isolated viable mitochondria. In some embodiments, the mitochondria can enhance the percentage of memory adaptive cells, e.g., central memory CD8 T cells or effector memory CD8 T cells, by at least 20%, preferably at least 30%, more preferably at least 50%. 20. The composition according to any one of the preceding items may be formulated in solid or liquid form, preferably in liquid form. 21. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like cells described in any one of the preceding items, is the self-renewal capacity of the adaptive immune cells. In some other embodiments, the enhanced viability of adaptive immune cells selected by mitochondria described in any one of the preceding items is an improved self-renewal capacity. 22. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells, as described in any one of the preceding sections, is the viability of adaptive immune cells during resting and re-stimulation. In some other embodiments, the enhanced viability of adaptive immune cells selected by mitochondria as described in any one of the preceding sections is improved viability during resting and re-stimulation. 23. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells described in any one of the preceding sections, is in the ability of the adaptive immune cells to respond to interleukin signaling, such as IL-7 and / or IL-15 signaling. In some other embodiments, the enhanced viability of adaptive immune cells selected by mitochondria described in any one of the preceding sections is an improved ability to respond to interleukin signaling, such as IL-7 and / or IL-15 signaling. 24. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells described in any one of the preceding articles, resides in the ability of the adaptive immune cells to resist activation-induced cell death (AICD). In some other embodiments, the enhanced viability of adaptive immune cells results from an improved ability of adaptive immune cells with enhanced selection by mitochondria described in any one of the preceding articles to resist activation-induced cell death (AICD) of adaptive immunity. 25. In some embodiments, the survival rate of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells, described in any one of the preceding items, is due to the ability of the adaptive immune cells to resist apoptosis by upregulating anti-apoptotic molecules, such as BCL-XL, BCL-2, or by downregulating pro-apoptotic molecules, such as Fas and FasL expression. In some embodiments, the enhanced survival of adaptive immune cells selected by mitochondria described in any one of the preceding items is due to the improved ability to resist apoptosis by upregulating anti-apoptotic molecules, such as BCL-XL, BCL-2, or by downregulating pro-apoptotic molecules, such as Fas and FasL expression. 26. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells, described in any one of the preceding items, resides in the ability of the adaptive immune cells to express epigenetic and transcriptional modifications that affect the expression of genes selected from the group consisting of Bcl2, Bcl2l2, Mcl1, Bcl2a1d, Birc2, Birc3, Xiap, and Cflar. In some embodiments, the enhanced viability of adaptive immune cells selected by mitochondria as described in any one of the preceding items resides in an improved ability to produce epigenetic and transcriptional modifications that affect the expression of genes selected from the group consisting of Bcl2, Bcl2l2, Mcl1, Bcl2a1d, Birc2, Birc3, Xiap, and Cflar. 27. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells described in any one of the preceding items, resides in the ability of the adaptive immune cells to maintain their long telomeres. In some embodiments, the enhanced viability of adaptive immune cells whose selection of mitochondria has been promoted described in any one of the preceding items resides in an improved ability to maintain long telomeres compared to immune cells that have not been treated with isolated viable mitochondria or that do not contain exogenous isolated viable mitochondria. 28. In some embodiments, the viability of adaptive immune cells, such as memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells described in any one of the preceding items, resides in the ability of the adaptive immune cells to proliferate or show persistence, or a combination thereof, compared to immune cells that have not been treated with isolated viable mitochondria or that do not contain exogenous mitochondria. 29. In some embodiments, the viability of adaptive immune cells, e.g., memory immune cells (e.g., central memory CD8 T cells and effector memory CD8 T cells) or memory-like immune cells described in any one of the preceding paragraphs, is in reduced exhaustion of adaptive immune cells compared to immune cells that have not been treated with isolated viable mitochondria or that do not contain exogenous mitochondria. 30. In some embodiments, the composition according to any one of the preceding items is a pharmaceutical composition. In some other embodiments, the pharmaceutical composition further comprises at least one pharma- ceutically acceptable carrier. In some embodiments, the pharma-ceutically acceptable carrier is formulated for delivery to human immune cells. In some embodiments, the pharma-ceutically acceptable carrier is formulated for delivery to human tissues and / or organs. Pharmaceutically acceptable carriers include, but are not limited to, saline, dispersion media, isotonicity agents, etc., compatible with pharmaceutical administration. In some embodiments, the pharma-ceutically acceptable carrier is phosphate buffered saline, saline, Krebs buffer, Tyrode's solution, contrast agent, or Omnipaque, or mixtures thereof. In some other embodiments, the carrier is a buffer containing 300 mM sucrose, 10 mM K+-HEPES (potassium buffered (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), pH 7.2), 1 mM K+-EGTA, (potassium buffered ethylene glycol tetraacetic acid, pH 8.0), 250 mM sucrose, 2 mM KH2PO4, 10 mM MgCh, 20 mM K-15 HEPES buffer (pH 7.2), and 0.5 mM K-EGTA (pH 8.0), or a buffer containing RPMI1640 medium GlutaMAX™ Supplement 500 ml (ThermoFisher, 61870010). 31. In some embodiments, the immune cells according to any one of the preceding paragraphs, e.g., human immune cells such as adaptive immune cells, are derived from a biological sample selected from the group of blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived therefrom and their progeny, cells isolated from the biological sample. 32. In some embodiments, the immune cells of any one of the preceding paragraphs are produced from viable eukaryotic cells. In some other embodiments, the immune cells are produced in vitro or ex vivo. 33. In some embodiments, the immune cells according to any one of the preceding paragraphs, for example, human immune cells such as adaptive immune cells, are allogeneic or autologous immune cells. In some embodiments, the immune cells are xenogeneic. 34. In some embodiments, the immune cells of any one of the preceding paragraphs, e.g., human immune cells such as adaptive immune cells, are produced from stem cells, including mesenchymal stem cells or induced pluripotent stem cells (iPSCs). 35. In some embodiments, the immune cells according to any of the preceding paragraphs are preferably mammalian immune cells, more preferably human immune cells. 36. In some embodiments, immune cells may include, but are not limited to, natural immune cells that have been engineered or expanded in vitro. 37. In some embodiments, the immune cell according to any one of the preceding items is preferably an adaptive immune cell, such as a B or T lymphocyte, preferably a T lymphocyte. In some embodiments, the adaptive immune cell is an in vitro expanded B or T lymphocyte, preferably an in vitro expanded T lymphocyte. 38. In some aspects, the immune cells according to any one of the preceding embodiments are T lymphocytes, such as, but not limited to, alpha-beta T cells (αβ T cells), gamma-delta T cells (γδ T cells), CD4 immune cells, CD8 immune cells, or combinations thereof. In some aspects, the T lymphocytes are naive T cells, effector T cells, memory T cells (e.g., tissue-resident memory (Trm) cells, Tscm cells, central memory cells, and effector memory cells), memory-like T cells, or combinations thereof. In some other aspects, the T cells are preferably memory T cells. In some aspects, the immune cells, e.g., human immune cells, according to any one of the preceding items are pluripotent stem cell-derived immune cells. In some other aspects, the T lymphocytes are helper T cells (T H ), cytotoxic T cells (CTL), regulatory T (Treg) cells, memory T cells, or a combination thereof. In some other embodiments, the T cells are preferably regulatory T cells (Treg). In some other embodiments, the immune cells are mucosal-associated invariant T cells. In some other embodiments, the immune cells are T cells circulating in the blood or tumor-infiltrating lymphocytes (TIL). 39. In some embodiments, the immune cells of any of the preceding items are preferably CD8 T cells, such as but not limited to CD8 T cells circulating in the blood, tumor infiltrating lymphocytes (TILs), e.g., CD8 TILs, naive CD8 T cells, effector CD8 T cells, memory CD8 T cells (e.g., Trm CD8 T cells, Tscm CD8 T cells, central memory CD8 T cells and effector memory CD8 T cells), memory-like CD8 T cells, or combinations thereof. In some preferred embodiments, the CD8 T cells are TILs. In some more preferred embodiments, the immune cells are memory CD8 T cells, in particular effector memory CD8 T cells, central memory CD8 T cells, or combinations thereof. In some preferred embodiments, the immune cells are memory-like CD8 T cells. 40. In some embodiments, the T cells are CD4 T cells, such as naive CD4 T cells, effector CD4 T cells (e.g., Th1, Th2, or Th17), memory CD4 T cells, memory-like CD4 T cells, regulatory CD4 T cells (Treg), CD4 T cells circulating in the blood, tumor-infiltrating lymphocyte (TIL) CD4 T cells, or combinations thereof. Preferably, the CD4 T cells are Treg cells, such as human regulatory (Treg) CD4 T cells. 41. In some embodiments, the immune cells, e.g., human immune cells, described in any one of the preceding items include, but are not limited to, engineered immune cells, such as CAR-T cells, e.g., CD8 CAR-T cells, that express chimeric antigen receptors ("CARs") and / or artificial T cell receptor ("TCR") subunits. CAR T cells typically include an antigen-binding moiety (e.g., an antigen-binding domain or antigen-binding fragment thereof), a transmembrane component, and a primary cytoplasmic signaling sequence selected to activate the immune cell in response to the antigen-binding moiety binding to its cognate ligands. In some embodiments, the basic components of a chimeric antigen receptor (CAR) include: (1) a variable heavy chain (VH) of a tumor-specific monoclonal antibody; H ) and variable light chain (V L ) is fused in frame to the CD3 ζ chain from the T cell receptor complex. (2)V H and V L are usually linked together using a flexible glycine-serine linker, followed by a spacer (e.g., a CD8a stalk or C H 2-C H The scFv is linked to the transmembrane domain by a 3 constant domain) to extend the scFv from the cell surface so that it can readily interact with tumor antigens. In some embodiments, the engineered immune cells containing exogenous mitochondria are CAR-T cells. 42. In some embodiments, the CAR or artificial TCR subunit is introduced into the immune cell using a virus, such as a lentivirus or adenovirus or retrovirus, a nanoparticle, or a nanoparticle operably linked to a targeting moiety. In some embodiments, the exogenous polynucleotide encoding the CAR and / or artificial TCR subunit is introduced into the immune cell in vitro. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is derived from a retrovirus, lentivirus, adenovirus, adeno-associated virus, or hybrid vector. 43. In some embodiments, the immune cell or population of immune cells according to any one of the preceding items is a member selected from the group consisting of B cell maturation antigen (BCMA, also known as tumor necrosis factor receptor superfamily member 17, TNFRSF17), CD19, CD123, CD22, CD30, CD171, CS-1 (also known as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule-1 (CLL-1 or CLECL1), CD33, epidermal growth factor receptor variant III (EGFRvIII), ganglioside G2 (GD2), ganglioside GD3, Tn antigen (Tn Ag or GalNAca-Ser / Thr), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), B7H3 (CD276), KIT (CD117), interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), mesothelin, interleukin-11 receptor alpha (IL-11Ra), prostate stem cell antigen (PSCA), protease serine 21 (Testisin or PRSS21), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis Y antigen, CD24, platelet-derived growth factor receptor beta (PDGFR-beta), Stage-specific embryonic antigen-4 (SSEA-4), CD20, folate receptor alpha, receptor tyrosine-protein kinase ERBB2 (Her2 / neu), cell surface associated mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural cell adhesion molecule (NCAM), prostase, prostatic acid phosphatase (PAP), elongation factor 2 mutated (ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (macropain) subunit, beta, 9 (LMP2), glycoprotein 100 (gp100),Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (BCR-Abl), tyrosinase, ephrin type A receptor 2 (EphA2), fucosyl GM1, sialyl Lewis adhesion molecule (sLe), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl-GD2 ganglioside (OAcGD2), folate receptor beta, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), G protein-coupled receptor class C group 5, member D (GPRC5D), Chromosome X open reading frame 61 (CXORF61), CD97, CD179a, Anaplastic lymphoma kinase (ALK), Polysialic acid, Placenta specific 1 (PLAC1), Hexasaccharide moiety of GloboH glycoceramide (GloboH), Mammary differentiation antigen (NY-BR-1), Uroplakin 2 (UPK2), Hepatitis A virus cellular receptor 1 (HAVCR1), Adrenergic receptor beta 3 (ADRB3), Pannexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), Lymphocyte antigen 6 complex, locus K9 (LY6K), Olfactory receptor 51E2 (OR51E2), TCR gamma alternating open reading frame protein (TARP), Wilms tumor protein (WT1), Cancer / testis antigen 1 (NY-ESO-1), Cancer / testis antigen 2 (LAGE-1a), Melanoma-associated antigen 1 (MAGE-A1), ETS translocation mutant gene 6 located on chromosome 12p (ETV6-AML), sperm protein 17 (SPA17), X antigen family, member 1A (XAGE1), angiopoietin-binding cell surface receptor 2 (Tie2), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), Fos-related antigen 1, tumor protein p53 (p53), p53 mutant, prostein, survival, telomerase,Prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1), rat sarcoma tumor (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-acetylglucosamine transferase V (NA17), paired box protein PAX-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulatory of Imprinted Sites, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), Paired box protein Pax-5 (PAX5), Proacrosin-binding protein sp32 (OY-TES1), Lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), Synovial sarcoma X breakpoint 2 (SSX2), Receptor for Advanced Glycation Endproducts (RAGE-1), Renal ubiquitous 1 (RU1), Renal ubiquitous 2 (RU2), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), intestinal carboxyl esterase, heat shock protein 70-2 mutated (muthsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), and Immunoglobulin Lambda-Like Polypeptide 1 (IGLL1). In some embodiments, the immune cell or population of immune cells of any one of the preceding items comprises a first, second, third, or fourth generation CAR. 44. In some embodiments, the specific lymphocyte activation receptor agonist of the immune cells according to any one of the preceding items is conjugated to a cell mimicking cell-free support. In some embodiments, the cell mimicking support is a paramagnetic bead. 45. In some embodiments, the immune cells of any one of the preceding paragraphs are cells known in the art to have anti-tumor or immunosuppressive and activity, e.g., effector cells. In some other embodiments, the immune cells are cells with immunoregulating activity. 46. ​​The isolated viable mitochondria according to any one of the preceding items are preferably respiratory competent mitochondria. 47. An effective amount of isolated viable mitochondria according to any one of the preceding paragraphs is between 0.0001 ng and 2.5 ng of mitochondria per target cell, such as between 0.001 ng and 2.0 ng of mitochondria per target cell, such as between 0.01 ng and 1.5 ng or between 0.05 ng and 1.0 ng of mitochondria, such as between 0.1 ng and 0.5 ng of mitochondria. 48. The isolated viable mitochondria according to any one of the preceding items can be autologous or allogeneic mitochondria. In some other embodiments, they are heterologous mitochondria. 49. In some other embodiments, the isolated viable mitochondria described in any one of the preceding items can be freshly isolated or previously isolated and then stored until use, e.g., stored at a temperature below 0°C. 50. In some embodiments, the source of isolated viable mitochondria according to any one of the preceding items can be of different nature, for example, tissue, blood, more specifically cells circulating in the blood, or cultured cells. 51. In some embodiments, the isolated viable mitochondria are eukaryotic cell mitochondria. In some embodiments, the mitochondria are derived from a human cell line. 52. In some embodiments, the isolated viable mitochondria according to any one of the preceding items are derived from a healthy donor. In some embodiments, the isolated viable mitochondria are derived from a patient. In some embodiments, the patient is a cancer patient. In some other embodiments, the patient is a patient suffering from an autoimmune disease. In some other embodiments, the patient is a transplanted patient. In some other embodiments, the patient is a patient suffering from an infectious and / or inflammatory disease. 53. In some embodiments, the isolated viable mitochondria can be autologous or autologous viable mitochondria with genetic modifications. In some embodiments, the isolated viable mitochondria can be allogeneic viable mitochondria with genetic modifications. 54. In some embodiments, the isolated viable mitochondria disclosed in any one of the preceding items can be delivered to target cells, e.g., immune cells, both in vitro and in vivo. 55. In some embodiments, the isolated viable mitochondria disclosed in any one of the preceding items can be delivered to a target organ and / or tissue, e.g., to cells of a target organ and / or tissue, in vivo or ex vivo. 56. In some embodiments, viable mitochondria are isolated by using one of the isolation methods described below, each method comprising (i) isolating mitochondria from cultured cells, tissues, or organs by using an endopeptidase such as subtilisin A, or (ii) filtering the mitochondria through one or more filters, or (i) isolating mitochondria from cultured cells, tissues, or organs by using an endopeptidase such as subtilisin A, followed by (ii) filtering the mitochondria through one or more filters. 57. In some embodiments, the exogenous viable mitochondria are, but are not limited to, autologous or allogeneic mitochondria, genetically engineered mitochondria, or mitochondria encapsulated by liposomes or bound to specific drugs. 58. In some embodiments, the mitochondria according to any one of the preceding items, e.g., isolated viable mitochondria, can enhance the viability and / or promote the selection of an adaptive immune cell or population of adaptive immune cells, respectively, compared to an immune cell or population of immune cells that have not been treated with mitochondria, starting from day 3 after mitochondria treatment, e.g., day 3.5, or day 4 after mitochondria treatment, preferably from day 5 after mitochondria treatment, e.g., from day 6, day 7, or day 8 after mitochondria treatment, more preferably from day 9 after mitochondria treatment. 59. In some embodiments, the mitochondria described in any one of the preceding items, e.g., isolated viable mitochondria, can enhance the viability and / or facilitate the selection of an adaptive immune cell or population of adaptive immune cells, respectively, compared to an immune cell or population of immune cells that does not contain exogenous mitochondria, starting from day 3 after mitochondrial transplantation of the mitochondria into the immune cells, e.g., from day 3.5 or day 4 after mitochondrial transplantation, preferably from day 5 after mitochondrial transplantation, e.g., from day 6, day 7, or day 8 after mitochondrial transplantation, more preferably from day 9 after mitochondrial transplantation. 60. In some embodiments, the enhanced survival of an immune cell or population of immune cells, e.g., the enhanced viability of an adaptive immune cell or population of adaptive immune cells selected upon treatment with isolated viable mitochondria as described in any one of the preceding paragraphs, is at least 1.2-fold compared to immune cells not treated with mitochondria. In some embodiments, it is at least 1.3-fold, e.g., at least 1.5-fold or 2-fold compared to immune cells not treated with mitochondria. In some embodiments, it is in the range of 1.2-fold to 50-fold (expressed as a fold increase) compared to an immune cell, e.g., adaptive immune cell, or population of immune cells, that is not treated with mitochondria, e.g., 1.2-45, 1.2-40, 1.2-30, 1.2-20, 1.2-15, 1.2-10, 1.2-5, 1.2-2.5, 1.3-50, 1.3-40, 1.3-30, 1.3-20, 1.3-10, 1.3-5, 1.3-3.5, 1.5-30, 1.5-25, 1.5-20 , 1.5~15, 1.5~10, 1.5~5, 1.5~3, 1.5~2.5, 2~50, 2~40, 2~30, 2~20, 2~15, 2~10, 2~5, 2~4, 3~30, 3~20, 3~10, 3~5, 5~50, 5~40, 5~30, 5~20, 5~10, 5~8, 10~50, 10~40, 10~30, 10~20, 10~15, 15~50, 15~40, 15~30, 15~20, 20~50, 20~30, 20~25, 30~35, 30~40, 30~45, 40~50. 61. In some embodiments, the enhanced survival of an immune cell or population of immune cells, e.g., the enhanced survival of an adaptive immune cell or population of adaptive immune cells selected upon transplantation of exogenous mitochondria as described in any one of the preceding paragraphs, is at least 1.2-fold compared to immune cells not transplanted with exogenous mitochondria. In some embodiments, it is at least 1.3-fold, e.g., at least 1.5-fold or 2-fold compared to immune cells not transplanted with exogenous mitochondria. In some embodiments, this is in the range of 1.2-50 fold (expressed as fold) relative to immune cells, e.g., adaptive immune cells that do not include exogenous mitochondria (e.g., are not transplanted), e.g., 1.2-45, 1.2-40, 1.2-30, 1.2-20, 1.2-15, 1.2-10, 1.2-5, 1.2-2.5, 1.3-50, 1.3-40, 1.3-30, 1.3-20, 1.3-10, 1.3-5, 1.3-3.5, 1.5-30, 1.5-25, 1.5-20, 1. 5~15, 1.5~10, 1.5~5, 1.5~3, 1.5~2.5, 2~50, 2~40, 2~30, 2~20, 2~15, 2~10, 2~5, 2~4, 3~30, 3~20, 3~10, 3~5, 5~50, 5~40, 5~30, 5~20, 5~10, 5~8, 10~50, 10~40, 10~30, 10~20, 10~15, 15~50, 15~40, 15~30, 15~20, 20~50, 20~30, 20~25, 30~35, 30~40, 30~45, 40~50. 62. Also provided herein is a method for enhancing the viability and / or facilitating the selection of an immune cell or a population of immune cells described in any one of the preceding items, comprising: (a) activating the immune cells in vitro in a cell-free medium comprising a specific activating receptor agonist antibody capable of driving adaptive cell (such as T cell) activation; (b) exposing the immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days, for example at least 5 days. In some embodiments, the method for enhancing the viability and / or facilitating the selection of an immune cell or a population of immune cells described in any one of the preceding items alternatively comprises: (a) activating the immune cells in vitro in a cell-free medium with coated CD3 / CD28 beads, optionally in the presence of a recombinant interleukin such as IL-2; (b) exposing the immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days, for example at least 5 days. 63.Also provided herein is a method for promoting memory differentiation and / or memory selection of an immune cell or a population of immune cells according to any one of the preceding items, comprising: (a) activating the immune cells in vitro in a cell-free medium comprising a specific activating receptor agonist antibody capable of driving adaptive cell (such as T cell) activation; (b) exposing the immune cells to a pharmaceutical composition comprising isolated viable mitochondria according to any one of the preceding items for at least 3 days, e.g., at least 5 days. In some embodiments, the method for promoting memory differentiation and / or memory selection of an immune cell or a population of immune cells according to any one of the preceding items alternatively comprises: (a) activating the immune cells in vitro in a cell-free medium with coated CD3 / CD28 beads, optionally in the presence of a recombinant interleukin such as IL-2; (b) exposing the immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days, e.g., at least 5 days. 64. The pharmaceutical composition used in the method according to any one of the preceding items comprises isolated viable mitochondria, the mitochondria being disclosed in any one of the preceding items. The effective amount of isolated viable mitochondria contained in the pharmaceutical composition used in the method is 0.0001ng to 2.5ng of mitochondria per target cell, for example 0.001ng to 2.0ng, for example 0.01ng to 1.5ng or 0.05ng to 1.0ng, for example 0.1ng to 0.5ng of mitochondria per target cell. In some embodiments, the pharmaceutical composition used in the method according to any one of the preceding items further comprises one or more pharma- ceutically acceptable carriers. Carriers include saline, dispersion media, isotonic agents, etc., phosphate buffered saline, Krebs buffer, Tyrode's solution, contrast agents, Omnipac, a buffer containing 300 mM sucrose, a buffer containing 10 mM K+-HEPES (potassium buffered (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.2), 1 mM K+-EGTA (potassium buffered ethylene glycol tetraacetic acid, pH 8.0), 250 mM sucrose, 2 mM KH2PO4, 10 mM MgCh, 20 mM K-15 HEPES buffer (pH 7.2), and 0.5 mM K-EGTA (pH 8.0), or 500 ml of RPMI 1640 medium GlutaMAX™ Supplement. (ThermoFisher, 61870010). The pharmaceutical compositions include one or more pharma- ceutical acceptable carriers and isolated viable mitochondria in an amount effective to enhance the proportion of adaptive memory or memory-like immune cells by at least 1.1-fold, e.g., 1.2-fold, 1.3-fold, 1.5-fold, or 2-fold, compared to immune cells not treated with or transplanted with mitochondria.In some embodiments, the enhancement of the proportion of memory or memory-like immune cells is in the range of 1.1-fold to 100-fold (expressed as a fold), e.g., 1.1-99, 1.1-90, 1.1-80, 1.1-70, 1.1-60, 1.1-50, 1.1-40, 1.1-30, 1.1-20, 1.1-10, 1.1-5, 1.1-2, 1.1-1.8, 1.1-1.5, 1.2-99, 1.2-90, 1.2-80, 1.2-70, 1.2-60, 1.2-50, 1.2-20, 1.2-1 0, 1.2~5, 1.2~2.5, 1.3~90, 1.3~80, 1.3~70, 1.3~50, 1.3~40, 1.3~30, 1.3~20, 1.3~10, 1.3~5, 1.3~1.5, 1.4~100, 1.4~95, 1.4~90, 1.4~80, 1.4~70, 1.4~60, 1.4~50, 1.4~30, 1.4~25, 1.4~20, 1.4~10, 1.4~5, 1.4~3, 1.4~2.5, 1.5~99, 1.5~95, 1.5~90, 1.5~80, 1.5~7 0, 1.5~60, 1.5~50, 1.5~50, 1.5~40, 1.5~30, 1.5~20, 1.5~10, 1.5~5, 1.5~2.5, 2~99, 2~90, 2~80, 2~70, 2~60, 2~50, 2~40, 2~35, 2~30, 2~20, 2~10, 2~5, 2~4, 2~2.5, 3~99, 3~90, 3~80, 3~70, 3~60, 3~50, 3~40, 3~30, 3~25, 3~20, 3~10, 4~99, 4~80, 4~70, 4~60, 4~50, 4~5 5, 4~25, 4~20, 4~15, 4~10, 5~100, 5~80, 5~50, 5~30, 5~20, 5~10, 5.5~9, 5.5~7, 10~90, 10~50, 10~20, 20~100, 20~50, 25~40, 20~35, 30~100, 30~50, 40~100, 40~70, 40~60, 40~50, 50~100, 50~90, 50~80, 50~70, 55~65, 60~80, 75~90, 75~100, 80~90, 80~85, 85~100. 65. Also provided herein is an immune cell, e.g., a human immune cell, such as a human T cell, treated with the isolated viable mitochondria or exogenous isolated viable mitochondria described in any one of the preceding articles, for use in a method of treating a subject in need thereof, the method comprising administering to the subject the immune cell, or population of immune cells, described in any one of the preceding articles. 66. The present disclosure further provides an immune cell, e.g. a human immune cell, such as a human T cell, treated with the isolated viable mitochondria or exogenous isolated viable mitochondria according to any one of the preceding items, for use in treating cancer, an infectious disease, an inflammatory disease, or an autoimmune disease. 67. Also provided herein is a pharmaceutical composition according to any one of the preceding paragraphs, in an amount effective for use in treating cancer, an infectious disease, an inflammatory disease, or an autoimmune disease, such as a composition comprising immune cells treated with isolated viable mitochondria or immune cells comprising exogenous viable mitochondria. 68. In some embodiments, the immune cells or population of immune cells treated with isolated viable mitochondria or comprising exogenous isolated viable mitochondria according to any one of the preceding items are formulated in a pharmaceutical composition in an amount effective for use in a method for treating cancer in a human subject in need thereof. 69. In some embodiments, the immune cells or population of immune cells treated with isolated viable mitochondria according to any one of the preceding items or containing exogenous viable mitochondria are formulated in a pharmaceutical composition in an amount effective for use in a method for treating autoimmune diseases in a human subject in need thereof. Autoimmune diseases include, but are not limited to, multiple sclerosis, diabetes, irritable bowel syndrome, celiac disease, Crohn's disease, lupus, psoriasis, and rheumatoid arthritis. 70. In some embodiments, the immune cell or population of immune cells treated with isolated viable mitochondria or containing exogenous mitochondria according to any one of the preceding items is formulated into a pharmaceutical composition in an amount effective for use in a method for treating an inflammatory disease in a human subject in need thereof. 71. In some embodiments, the immune cell or population of immune cells treated with isolated viable mitochondria or containing exogenous mitochondria according to any one of the preceding items is formulated into a pharmaceutical composition in an amount effective for use in a method for treating graft vs host disease (GvHD) in a human subject in need thereof. 72. In some embodiments, immune cells, e.g., anti-tumor cells, e.g., CAR-T cells, or populations of immune cells, treated with isolated viable mitochondria or comprising exogenous mitochondria as described in any one of the preceding paragraphs, are formulated in a pharmaceutical composition in an amount effective for use in killing tumor cells, such as killing tumor cells more effectively and / or for longer than comparable immune cells or comparable populations of immune cells that are not treated with isolated viable mitochondria or lack exogenous mitochondria. 73. In some embodiments, the immune cells or population of immune cells treated with or containing isolated viable mitochondria according to any one of the preceding items are formulated in a pharmaceutical composition in an amount effective for use in autoimmune disease, for example, in an amount effective for use in reducing or preventing abnormal immune responses. Specifically, the abnormal immune response of the immune cells or population of immune cells treated with isolated viable mitochondria according to any one of the preceding items or containing exogenous isolated mitochondria, such as in the case of autoimmune disease, is milder (less) and / or completely absent when compared to the response of the equivalent immune cells or population of equivalent immune cells that are not treated with isolated viable mitochondria or lack exogenous mitochondria. 74. In some embodiments, the immune cells or population of immune cells treated with isolated viable mitochondria or containing exogenous mitochondria according to any one of the preceding items are formulated into a pharmaceutical composition and transplanted into a subject in need of cancer treatment in an amount effective to treat cancer in a human subject in need of cancer treatment via autologous or allogeneic cell transplantation. In some embodiments, the allogeneic cell transplantation includes (a) obtaining a sample of viable blood from a donor, (b) isolating immune cells from the blood sample obtained in step (a), (c) transducing the immune cells with one or more exogenous polynucleotides encoding a CAR or an artificial TCR subunit, (d) optionally contacting the immune cells with a small molecule, and (e) administering the modified immune cells to a subject in need. 75. The present disclosure further provides an immune cell, e.g., a human immune cell, or a population of immune cells, for co-administration with a pharmaceutical composition comprising isolated viable mitochondria formulated in a pharma- ceutically acceptable carrier according to any one of the preceding items in an amount effective for use in treating cancer, an infectious disease, an inflammatory disease, or an autoimmune disease in a subject in need thereof. The co-administration of the pharmaceutical composition comprising isolated viable mitochondria may be prior to, simultaneous with, or subsequent to administration of the immune cells. In some embodiments, the pharmaceutical composition is co-administered with the immune cells by intravenous injection to a subject in need thereof. In some embodiments, the pharmaceutical composition is co-administered with the immune cells by intratumoral injection. In some embodiments, the pharmaceutical composition is co-administered with the immune cells by intraorgan injection or via an organ-specific vasculature. In some embodiments, the subject is afflicted with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), or other inflammatory diseases. carcinoma), bone cancer, brain cancer (e.g., glioblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal cancer tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumors , liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, B chronic lymphocytic leukemia, hairy cell leukemia, Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0126] T cells In one embodiment, the immune cells that contain or are enhanced by exogenous mitochondria are T cells (also called T lymphocytes), which belong to a group of white blood cells called lymphocytes. Lymphocytes are usually involved in cell-mediated immunity. The "T" in "T cell" refers to a cell that originates from or whose maturation is influenced by the thymus. T cells can be distinguished from other lymphocyte types, such as B cells and natural killer (NK) cells, by the presence of a cell surface protein known as the T cell receptor (TCR), which recognizes antigens presented on the surface of the cell. During a typical immune response, the binding of these antigens to the T cell receptor, in the context of MHC antigen presentation, initiates intracellular changes that result in T cell activation.

[0127] T cells are divided into two groups by their T cell receptors (TCRs), αβT cells and γδT cells. αβT cells with TCR2 mainly mediate cellular immunity and immune regulation, while γδT cells with TCR1 play important functions in wound healing, removal of damaged or transformed epithelial cells, and alleviating excessive inflammation, in addition to maintaining immune homeostasis in the local microenvironment. αβT cells and γδT cells play different roles in autoimmune diseases, tumors, and vascular diseases. αβT cells comprise 65-75% of peripheral blood mononuclear cells (PBMCs), while γδT cells account for less than 10%. They express distinct surface markers, CD4 and CD8, e.g., 60% of αβT cells are CD4 positive, 30% are CD8 positive, and less than 1% of αβT cells are both positive.

[0128] As used herein, the term "activated T cells" refers to T cells that have been stimulated to generate an immune response (e.g., clonal expansion of activated T cells) by recognition of antigenic determinants, e.g., as presented in the context of class I or class II major histocompatibility (MHC) markers. T cells are activated by the presence of antigenic determinants, cytokines, and / or lymphokines and cluster of differentiation cell surface proteins (e.g., CD3, CD4, CD8, etc., and combinations thereof). Cells that express a cluster of differentiation protein are often said to be "positive" for expression of that protein on the surface of the T cell (e.g., cells positive for expression of CD3, CD4, or CD8 are said to be "positive" for expression of that protein on the surface of the T cell). + , CD4 + , or CD8 + The CD3 and CD4 proteins are cell surface receptors or co-receptors that can be directly and / or indirectly involved in signal transduction in T cells.

[0129] In some embodiments, the immune cells comprising and / or augmented with exogenous mitochondria comprise a CAR-T cell population. In some embodiments, the CAR-T cell population is selected or enriched or purified to comprise at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of a cell type that expresses a particular marker, receptor, or cell surface glycoprotein, such as, for example, CD8, CD4, CD3, CD34, etc.

[0130] In some embodiments, the CAR-T cell population is CD4 + and CD8 +In some embodiments, the CAR-T cell population comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% CD8 T cells. + In some embodiments, the CAR-T cell population is enriched to contain at least 80% CD8 T cells. + In some embodiments, the CAR-T cell population is enriched to contain at least 90% CD8 T cells. + Thus, in some embodiments, the composition is enriched to contain genetically modified CD4 + More genetically modified CD8 than T cells + T cells are present, i.e., CD4 + Cellular CD8 + The ratio to cells is less than 1, for example, less than 0.9, less than 0.8, less than 0.7, less than 0.6, or less than 0.5.

[0131] Enriched immune cell populations In some embodiments, an enriched cell population is provided that includes or is augmented with exogenous mitochondria, and the enriched cell population is selected to include a specific ratio or percentage of one or more cell types. "Cell population" or "modified cell population" refers to a group of cells, such as more than two cells. A cell population can be homogenous, with cells of the same type, or each containing the same marker, or the cell population can be non-homogeneous. In some examples, the cell population is derived from a sample obtained from a subject, including cells prepared from bone marrow, umbilical cord blood, peripheral blood, or any tissue. In some examples, the cell population is contacted with a nucleic acid, the nucleic acid including a heterologous polynucleotide, such as a polynucleotide encoding a chimeric antigen receptor, an inducible chimeric pro-apoptotic polypeptide, or a costimulatory polypeptide, such as a chimeric myeloid differentiation primary response 88 (MyD88) or a truncated MyD88 and CD40 polypeptide. In some examples, the cell populations and modified cell populations are progeny of the original cells contacted with the nucleic acid comprising the heterologous polynucleotide. The cell population can be selected, enriched, or purified to contain at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of a cell type that expresses a particular marker, receptor, or cell surface glycoprotein, such as, for example, CD8, CD4, CD3, CD34.

[0132] Harvesting of T lymphocytes from patients' resected tumors and enrichment of TIL cells T cells such as TILs enhanced by exogenous mitochondria can be derived from cancer patients. TILs are obtained from resected tumors and expanded in vitro. Depending on the method applied, isolation of TILs results in reinfusion of “selected” or “young” TILs. Briefly, resected tumors are treated, such as by enzymatic digestion, and TILs are expanded and cultured in high doses of IL-2. An appropriate number of cells needs to be obtained for reinfusion of autologous TILs. “Selected” TILs are tested for cytokine production upon tumor cell recognition, but tumor reactivity of “young” TILs is not evaluated. In general, “selected” TILs require up to 36 days from culture to tumor reactivity evaluation before being reintroduced into cancer patients. Notably, the expansion process of “young” TILs requires only 10-22 days, while showing comparable clinical responses compared to “selected” TILs. According to the present disclosure, TILs transplanted with exogenous mitochondria can be excised from any tumor and any protocol for expansion and reinfusion can be applied.

[0133] Selection, enrichment, or purification of cell types in the modified cell population can be accomplished by any suitable method. In some embodiments, CD8 + T cells and CD4 + The percentage of T cells can be determined by flow cytometry. In some examples, a MAC column can be used. In some examples, the modified cell population is frozen and thawed before administration to a subject, and viable cells are tested for the percentage or ratio of specific cell types before administration to a subject.

[0134] In some embodiments, the cell population is CD8 + T cells or CD4 + Selected or enriched or purified to contain at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95, 96, 97, 98, or 99% of T cells.

[0135] According to the present disclosure, mitochondrial preparations including, for example, autologous mitochondria, allogeneic mitochondria, xenogeneic mitochondria, encapsulated mitochondria, or autologous mitochondria with appropriate genetic modifications can be delivered to enriched T cells.

[0136] Collection of T lymphocytes from the patient's blood and enrichment of the T cells T cells, such as T cells enhanced by exogenous mitochondria and / or engineered to express CAR, can be derived from any healthy donor. Donors are generally adults (at least 18 years old), but children are also suitable as T cell donors (Styczynski, 2018, "Young child as a donor of cells for transplantation and lymphocyte based therapies", Transfus Apher Sci 57:323-30). An example of a suitable process for obtaining T cells from a donor is described in (Di Stasi et al., 2011, "Inducible apoptosis as a safety switch for adoptive cell therapy", N Engl J Med 365:1673-83). In general, T cells can be obtained from a donor, subjected to genetic modification and selection, and then administered to a recipient subject. A useful source of T cells is the peripheral blood of the donor. Peripheral blood samples are generally subjected to leukapheresis to provide a sample enriched in white blood cells. This enriched sample (also known as a "leukopak") may be composed of a variety of blood cells including monocytes, lymphocytes, platelets, plasma, and red blood cells. Exclusion of contaminants such as red blood cells, platelets, monocytes, and tumor cells requires a multifaceted approach that is generally required using methods known in the art. Leukopaks typically contain a higher concentration of cells compared to venipuncture or buffy coat products.

[0137] Patients with recurrent cancer may have low T cell numbers, thus making it difficult to harvest sufficient autologous T cells. This problem can be overcome by methods known in the art, such as by using allogeneic T lymphocytes harvested from healthy donors.

[0138] Selection, enrichment, or purification of cell types in the modified cell population can be accomplished by any suitable method. In some embodiments, CD8 + T cells and CD4 + The percentage of T cells may be determined by flow cytometry. In some examples, a MAC column may be used. In some examples, the modified cell population is frozen and thawed prior to administration to a subject, and the viable cells are tested for the percentage or ratio of a particular cell type prior to administration to a subject. In leukopacs, CD8 + CD4 on cells + The ratio of CD8 cells is typically greater than 2, but in some embodiments, the ratio of CD8 + CD4 on cells + In some embodiments, the ratio of CD4 + More CD8 than T cells + T cells are present, i.e., CD8 + CD4 on cells + The ratio of CD4 T cells is less than 1, e.g., less than 0.9, less than 0.8, less than 0.7, less than 0.6, or less than 0.5. Thus, starting from donor cells, the overall procedure for producing T cells is + CD8 compared with T cells + In some embodiments, the T cells are designed to enrich for CD8 T cells. + T cells, and in some embodiments, 65% or more of the T cells are CD8 + T cells. CD3 + Within the population of T cells, in some embodiments, CD8 +The percentage of T cells is between 55-75%, e.g., between 55%-65%, 55%-70%, 56-71%, 63-73%, 60-70%, 59%-74%, 65-71%, or 65-75%. In some embodiments, a cell population is provided that is selected or enriched or purified to include a ratio of a particular cell type to another cell type, e.g., CD8 + T cells and CD4 + In some embodiments, the ratio of CD8 to T cells is at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97, 98, or 99%. + In some embodiments, the cells are selected, enriched, or purified to contain CD8 T cells. + T Cells vs. CD4 + The T cell ratio may be 4:1, or 9:1, or even higher.

[0139] In some embodiments, a genetically modified CD3 + For T cell populations, CD8 + The percentage of T cells is 55-75%, e.g., 55-65%, 55-70%, 56-71%, 59-74%, 63-73%, 60-70%, 60-75%, 65-75%, or 65-71%. In some embodiments, the percentage of CD8 + T cells and CD4 + In some embodiments, the ratio of CD8 to T cells is 3:2, 7:3, 4:1, 9:1, 19:1, or 39:1 or more. In some embodiments, the engineered cell population comprising a costimulatory polypeptide is at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96, 97, 98, or 99% CD8 to T cells. + In some embodiments, the cells are selected, enriched, or purified to contain CD8 T cells. + T cells and CD4 +The ratio of T cells is 4 to 1, or 9 to 1, or more. The costimulatory polypeptide can include one or more costimulatory signaling regions, such as CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, DAP10, MyD88, or CD40. The costimulatory polypeptide can include one or more costimulatory signaling regions that activate the signaling pathway activated by CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, DAP10, MyD88, or CD40. The costimulatory polypeptide can be inducibly or constitutively activated.

[0140] In some embodiments, the present invention relates to a method for detecting HIV-1 infection in which at least 80%, 85%, 90%, 95, 96, 97, 98, or 99% of the HIV-1 infection is CD8 + Compositions and methods are provided that include a CAR-T cell population comprising an inducible pro-apoptotic polypeptide that is a T cell. In some embodiments, the engineered cell population comprising an inducible pro-apoptotic polypeptide is at least 80% CD8 + In some embodiments, the engineered cell population is an inducible pro-apoptotic polypeptide 90% CD8 T cell. + Contains at least T cells.

[0141] In some embodiments, the present invention relates to a method for detecting HIV-1 infection in which at least 80%, 85%, 90%, 95, 96, 97, 98, or 99% of the HIV-1 infection is CD8 + Compositions and methods are provided that include a CAR-T cell population that is a T cell and that includes a costimulatory polypeptide and an inducible pro-apoptotic polypeptide. In some embodiments, the engineered cell population that includes a costimulatory polypeptide and an inducible pro-apoptotic polypeptide is at least 80% CD8 + In some embodiments, the engineered cell population comprising a costimulatory polypeptide and an inducible pro-apoptotic polypeptide is at least 90% CD8 T cells. + T cells.

[0142] According to the present disclosure, for example, mitochondrial preparations including autologous mitochondria, allogeneic mitochondria, xenogeneic mitochondria, encapsulated mitochondria, or autologous mitochondria with appropriate genetic modifications can be delivered to enriched T cells before, simultaneously with, or after genetic modifications (e.g., introduction of a CAR gene) are made.

[0143] Mitochondria The present invention is based, at least in part, on the discovery that isolated mitochondria can be delivered (also called transplanted) to cultured cells or to a patient's tissue by adding them to cell culture or by injecting them into the patient's tissue or into the blood vessels leading to the tissue, respectively (Cowan et al., 2017, "Transit and integration of extracellular mitochondria in human heart cells", Sci Rep 7:17450; McCully et al., 2017, "Mitochondrial transplantation: From animal models to clinical use in humans", Mitochondrion 34:127-34).

[0144] Mitochondria can be delivered ex vivo to cells of interest. Cells of interest include, but are not limited to, any of the cultured cells described herein, pre-engineered immune cells (e.g., CAR T cells), or cells that are further engineered (e.g., to express CAR or artificial TCR) and / or cultured (e.g., differentiated, activated, treated, or incubated). Mitochondria can be delivered ex vivo by liposome-mediated transfer using synthetic liposomes such as Lipofectin® (Shi et al., 2008. "Mitochondria transfer into fibroblasts: liposome-mediated transfer of labeled Mitochondria into cultured cells", Ethn.Dis.18:S1-43). Mitochondria can be delivered ex vivo by co-incubation (i.e., co-culture) of cells, such as any of the immune cells described herein, with mitochondria for a period of 2-24 hours (Masuzawa et al., 2013, “Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury”, Am J Physiol Heart Circ Physiol 304:H966-82). Without wishing to be bound by theory, transplanted mitochondria are internalized by an actin-dependent pathway. Mitochondrial internalization, as previously shown in cardiomyocytes, can occur with a 1-hour co-incubation. (Pacak et al., 2015, “Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function”, Biol Open 4:622-6).

[0145] Mitochondria can also be delivered to an organ or tissue by direct injection into the target site or by delivery via organ- or tissue-specific vasculature, such as the subject's coronary artery, the subject's pulmonary artery, the subject's hepatic portal vein, the subject's large pancreatic artery, the subject's renal artery, or the subject's prostatic artery. In the latter case, mitochondria are retained in the downstream organ or tissue. For example, when administered via the coronary artery, mitochondria are delivered almost exclusively to the heart (Shin et al., 2019, "Myocardial Protection by Intracoronary Delivery of mitochondria: Safety and Efficacy in the Ischemic Myocardium", JACC: Basic to Translational Science Vol. 4, No. 8, 2019), whereas mitochondria can be delivered to the lungs via the pulmonary artery or to the kidneys by delivery through the renal artery. Direct injection of mitochondria allows for localized enrichment of the injected mitochondria. The number of mitochondria used for injection can vary depending on the size of the target organ or tissue, as well as the intended use. Mitochondria can be suspended in homogenization buffer and injected into various sites using, for example, a tuberculin syringe with a 28-32 gauge needle (Emani et al., 2017, “Autologous Mitochondrial Transplantation for Dysfunction after Ischemia-Reperfusion Injury”, J Thorac Cardiovasc Surg 154:286-9; McCully et al., 2017, “Mitochondrial Transplantation: From Animal Models to Clinical Use in Humans”, Mitochondrion 34:127-34).

[0146] In vivo mitochondrial transplantation can be performed using single or continuous injections of autologous or heterologous mitochondria without direct or indirect, acute or chronic alloreactivity, allorecognition, or damage-associated molecular pattern molecules (Ramirez-Barbieri et al., 2019, “Alloreactivity and allorecognition of syngeneic and allogeneic mitochondria”, Mitochondrion 46:103-15).

[0147] Without wishing to be bound by theory, viable, respiration competent mitochondria are taken up by endocytosis into both ischemic and non-ischemic tissues (Cowan et al., 2016, “Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection”, PLoS One 11:e0160889; Kesner et al., 2016, “Characteristics of Mitochondrial Transformation into Human Cells”, Sci Rep 6:26057; Cowan et al., 2017, “Transit and integration of cellular mitocellular mitochondondria in Human heart cells”, Sci Rep 7:17450).

[0148] Using relatively simple medical procedures, skilled physicians can distribute mitochondria locally and / or systemically to patient tissues and / or cells for various purposes.It is further noted that, compared to some traditional therapeutic regimens, including nanoparticles, mitochondria are non-toxic and do not induce any substantial adverse immune or autoimmune responses.

[0149] Without intending to be bound by any theory, it is believed that the injected mitochondria extravasate through the capillary wall by first attaching to the endothelium. After they are injected or infused into the artery, the mitochondria can cross the endothelium of the blood vessel and be taken up by tissue cells through an endosomal actin-dependent internalization process.

[0150] In vivo mitochondrial transplantation can include co-administration of any of the cells of interest described herein with exogenous mitochondria (e.g., exogenous isolated viable mitochondria) provided herein. In some embodiments, the exogenous mitochondria and the cells of interest are co-administered to promote or enhance the desired therapeutic effect of the cells of interest to treat a disease in a patient. The cells of interest include, but are not limited to, any of the immune cells described herein, cultured cells, pre-engineered immune cells (e.g., CAR T cells), or cells that are further engineered (e.g., to express a CAR or artificial TCR). In embodiments in which the exogenous mitochondria and the cells of interest are included in different pharmaceutical compositions, administration of the exogenous mitochondria can occur before, simultaneously with, or after administration of the cells of interest. In some aspects, administration of the exogenous mitochondria and the cells of interest occurs within about one month of each other. In some aspects, administration of the exogenous mitochondria and the cells of interest occurs within about one week of each other. In some aspects, administration of the exogenous mitochondria and the cells of interest occurs within about 5, 4, 3, or 2 days of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about one day of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 12 hours of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 6 hours of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 3 hours of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 2 hours of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 1 hour of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 30 minutes of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within about 15 minutes of each other. In some embodiments, administration of the exogenous mitochondria and the cells of interest occurs within a few minutes of each other.In some embodiments, co-administration of exogenous mitochondria and cells of interest comprises repeated administration of exogenous mitochondria and / or cells of interest.

[0151] Isolation of mitochondria Mitochondria for use in the methods described herein can be isolated or provided from any source, for example, isolated from cultured cells or tissue. Exemplary cells include, but are not limited to, muscle tissue cells, cardiac fibroblasts, HeLa cells, prostate cancer cells, yeast, and any mixture thereof, among others. Exemplary tissues include, but are not limited to, liver tissue, skeletal muscle, heart, brain, and adipose tissue. Mitochondria can be isolated from autologous, allogeneic, and / or xenogeneic cells or tissues (e.g., biopsy material). In some examples, mitochondria are isolated from cells with genetic modifications, for example, cells with modified mtDNA or modified nuclear DNA.

[0152] Mitochondria can be isolated from cells or tissues by any means known to those skilled in the art. In one example, tissue or cell samples are harvested and then homogenized. Following homogenization, mitochondria are isolated by repeated centrifugation (Kesner et al., 2016, "Characteristics of Mitochondrial Transformation into Human Cells", Sci Rep 6:26057). Alternatively, cell homogenates can be filtered through nylon mesh filters. Exemplary methods for isolating mitochondria are described, for example, in McCully JD, Cowan DB, Pacak CA, Toumpoulis IK, Dayalan H and Levitsky S, "Injection of isolated mitochondria during early reperfusion for cardioprotection", Am J Physiol 296, H94-H105 PMC2637784 (2009); Frezza, C., Cipolat, S., & Scorrano, L, "Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts", Nature protocols, 2(2), 287-295 (2007), and in PCT applications entitled "Products and Methods to Isolate Mitochondria" (PCT / US2015 / 035584; WO 2015192020), each of which is incorporated by reference.

[0153] The mitochondria used in treatment or contained in pharmaceutical compositions can be isolated from cells or tissues of autologous, allogeneic, or xenogeneic origin.In some cases, mitochondria are collected from the cultured cells or tissues of a subject, and these mitochondria are administered back to the same subject (autologous).In some other cases, mitochondria are collected from the cultured cells (e.g., human cardiac fibroblasts) or tissues of a second subject, and these mitochondria are administered to the first subject (allogeneic).In some cases, mitochondria are collected from the cultured cells or tissues of different species (e.g., mouse, pig, and yeast) (xenogeneic).

[0154] In certain embodiments of the methods described herein, mitochondria may have different sources, for example, exogenous mitochondria may be autologous, autologous, allogeneic, or xenogeneic. In certain embodiments, mitochondria are freshly isolated (within 120 minutes, preferably within 60 minutes, more preferably within 30 minutes after taking tissue biopsy sample). In some embodiments, mitochondria are isolated and then stored until use. In certain embodiments, autologous mitochondria may have exogenous mtDNA. In some embodiments, mitochondria are derived from the subject's first-degree relative. In some embodiments, mitochondria are encapsulated.

[0155] In some embodiments, the methods described include harvesting isolated mitochondria from cells prior to administration. The isolated mitochondria can be transplanted into a cell of interest, such as any of the immune effector cells described herein, or can be administered to a subject in conjunction with treatment with a cell of interest.

[0156] Engineering Expression Constructs In some embodiments, immune cells that contain exogenous mitochondria or are enhanced by exogenous mitochondria are engineered to express CAR, and the term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as a template, as used herein. The advantage of using cDNA, as opposed to DNA polymerized from genomic DNA or genomic, unprocessed or partially processed RNA templates, is that cDNA mainly contains the coding sequence of the corresponding protein. Complete or partial genomic sequences may be used, such as when non-coding regions are required for optimal expression or when non-coding regions such as introns are targeted in antisense strategies.

[0157] In some embodiments, the nucleic acid construct, for example any of the chimeric antigen receptors described herein, is contained within a viral vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is an adenoviral vector or a lentiviral vector. It should be understood that in some embodiments, the cell is contacted with the viral vector ex vivo, and in some embodiments, the cell is contacted with the viral vector in vivo. Thus, the expression construct can be inserted into a vector, for example, a viral vector or a plasmid. The steps of the provided methods can be performed using any suitable method, including, but not limited to, the methods of transducing, transforming, or otherwise providing a nucleic acid to a cell described herein.

[0158] As used herein, the term "gene" is defined as a functional protein, polypeptide, or peptide coding unit. As will be understood, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or are adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and / or mutants.

[0159] Promoters and other regulatory elements are selected such that they are functional in the desired cells or tissues. Furthermore, this list of promoters should not be construed as exhaustive or limiting, and other promoters that may be used in conjunction with the promoters and methods disclosed herein.

[0160] Expression constructs such as CAR genes can be randomly integrated into the genome, such as through viral-mediated integration, or can be purposefully integrated into specific sites in immune cell genomes, such as the T cell genome, including but not limited to the CCR5 and AAVS1 loci, or the T cell receptor alpha constant (TRAC) locus. Targeted integration can use gene editing tools such as nuclease-mediated genome editing systems, including the clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) system, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) (Liu et al., 2019, "Building Potent Chimeric Antigen Receptor T Cells With CRISPR Genome Editing", Front Immunol 10:456).

[0161] co-stimulation In some embodiments, the immune cells that include exogenous mitochondria or are enhanced with exogenous mitochondria are immune cells engineered to express a CAR, such as CAR-T cells, that include a costimulatory polypeptide. In some embodiments, the immune cells that include exogenous mitochondria or are enhanced with exogenous mitochondria are CAR-T cells that include a costimulatory polypeptide. CARs can be engineered to contain costimulatory domains, such as those derived from the cytoplasmic portions of T cell costimulatory molecules, including, but not limited to, CD28, 4-1BB, OX40, ICOS, DAP10 (see, e.g., Carpenito et al. (2009) Proc Natl Acad Sci USA 106:3360-3365; Finney et al. (1998) J Immunol 161:2791-2797; Hombach et al. J Immunol 167:6123-6131; Maher et al. (2002) Nat Biotechnol 20:70-75; Imai et al. (2004) Leukemia 18:676-684; Wang et al. (2007) Hum Gene Ther 18:712-725; Zhao et al. (2009) J Immunol 183:5563-5574; Milone et al. (2009) Mol Ther 17:1453-1464; Yvon et al. (2009) Clin Cancer Res 15:5852-5860), thereby enabling CAR-T cells to receive appropriate costimulation upon engagement of the target antigen.

[0162] The costimulatory polypeptide of the present invention can be inducibly or constitutively activated. The costimulatory polypeptide can include one or more costimulatory signaling regions, such as CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, DAP10, MyD88, or CD40, or, for example, their cytoplasmic regions. The costimulatory polypeptide can include one or more suitable costimulatory signaling regions that activate the signaling pathways activated by CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, DAP10, MyD88, or CD40. The costimulatory polypeptide includes any molecule or polypeptide that activates the NF-κB pathway, Akt pathway, and / or p38 pathway of the tumor necrosis factor receptor (TNFR) family (i.e., CD40, RANK / TRANCE-R, OX40, 4-1BB) and CD28 family members (CD28, ICOS). Multiple costimulatory polypeptides or costimulatory polypeptide cytoplasmic regions can be expressed in the engineered T cells discussed herein.

[0163] In some embodiments, an inducible chimeric signaling polypeptide comprises two costimulatory polypeptide cytoplasmic signaling regions, such as, for example, 4-1BB and CD28, or one or more costimulatory polypeptide cytoplasmic signaling regions selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, DAP10.

[0164] vector In some embodiments, the population of immune cells that includes or is enhanced by exogenous mitochondria (e.g., as autologous, allogeneic, xenogeneic, encapsulated, or autologous with appropriate genetic modification) includes a CAR or artificial TCR subunit produced from a DNA, double-stranded RNA, single-stranded mRNA, or circular RNA vector. It is understood that the vectors provided herein can be modified using methods known in the art to change the location or order of regions or replace one region with another, and the vector can encode an antigen binding domain specific for one or more target antigens, such as, for example, BCMA, CD123, CD20, CD22, CD30, CD33, EGFR, EGFRvIII, GD2, Her2, Mesothelin, MUC1, MUC16, NKG2D, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, for example, as part of a CAR construct. The vector can also be modified with appropriate substitutions of each polypeptide region, as discussed herein.

[0165] The vector can encode a costimulatory polypeptide cytoplasmic signaling region, for example, as part of a CAR construct, including one or more costimulatory polypeptide cytoplasmic signaling regions, such as those selected from the group consisting of CD27, CD28, 4-1BB, OX40, ICOS, RANK, TRANCE, and DAP10. The vector can encode a linker, for example, as part of a CAR construct, such as a linker between a CAR polypeptide and a costimulatory polypeptide. Engineered immune cells, such as T cells (e.g., CAR T cells) of the present invention, can express a safety switch, also known as an inducible suicide gene or suicide switch, which can be used to eradicate the engineered immune cells in vivo if desired, for example, when graft-versus-host disease (GVHD) develops. In some examples, engineered immune cells expressing chimeric antigen receptors are provided to patients that induce adverse events, such as on-target off-tumor toxicity. In some therapeutic cases, patients may experience some negative symptoms during treatment using CAR modified cells. In some cases, these therapies have resulted in adverse events, in part, due to non-specific attacks on healthy tissues. In some instances, the therapeutic engineered immune cells may no longer be needed or treatment is intended for a specified amount of time, e.g., the therapeutic engineered immune cells may act to reduce tumor cells or tumor size and may no longer be needed. Thus, in some embodiments, nucleic acids, cells, and methods are provided in which the engineered immune cells also express a safety switch, such as an inducible caspase-9 polypeptide.Other suicide switch systems known in the art include, but are not limited to, (a) herpes simplex virus (HSV)-tk, which converts the non-toxic prodrug ganciclovir (GCV) to GCV-triphosphate, resulting in cell death by halting DNA replication; (b) iCasp9 binds to the small molecule AP1903, resulting in dimerization that activates the intrinsic apoptosis pathway; and (c) targetable surface antigens (e.g., CD20 and truncated EGFR) expressed on transduced iNKT cells allow for efficient elimination of modified cells via complement / antibody-dependent cytotoxicity (CDC / ADCC) after administration of the relevant monoclonal antibody. For example, when the number of engineered immune cells needs to be reduced, an inducible ligand can be administered to the patient, thereby inducing apoptosis of the engineered immune cells. These switches are supplied when it is desired to eradicate the engineered immune cells, and respond to triggers such as drugs that result in cell death (e.g., by inducing necrosis or apoptosis). These drugs can result in the expression of toxic gene products, but a more rapid response can be obtained if the engineered immune cells already express proteins that are switched to a toxic form in response to the drug.

[0166] Selection marker In certain embodiments, the expression construct comprises a nucleic acid construct whose expression is identified in vitro or in vivo by including a marker in the expression construct. Such a marker confers an identifiable change to the cell, allowing easy identification of cells containing the expression construct. Usually, the inclusion of a drug selection marker aids in cloning and selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) are used. Immunological surface markers that contain an extracellular, non-signaling domain, or various proteins (e.g., CD34, CD19, LNGFR) can also be used, allowing for simple methods for magnetic or fluorescent antibody-mediated selection. The selectable marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable markers include reporters such as, for example, GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT).

[0167] Linker Polypeptides Linker polypeptides include, for example, cleavable and non-cleavable linker polypeptides. Non-cleavable polypeptides can include, for example, any polypeptide that can be operatively linked between a cytoplasmic signaling region of a costimulatory polypeptide and an ITAM portion of a chimeric antigen receptor (e.g., CD3ζ). Linker polypeptides include, for example, those consisting of about 2 to about 30 amino acids (e.g., (GGGGS) nor a furin cleavage site, or a glycine-serine linker). In some embodiments, the linker polypeptide consists of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the linker polypeptide consists of about 18-22 amino acids. In some embodiments, the linker polypeptide consists of 20 amino acids. In some embodiments, the cleavable linker comprises a linker that is cleaved by an enzyme exogenous to the engineered cells in the population, e.g., an enzyme encoded by a polynucleotide introduced into the cells by transfection or transduction, either at the same time or at a different time as the polynucleotide encoding the linker. In some embodiments, cleavable linkers include linkers that are cleaved by enzymes endogenous to the engineered cells in the population, including, for example, enzymes naturally expressed in the cells and enzymes encoded by polynucleotides native to the cells, such as, for example, lysozyme.

[0168] Therapeutic Applications Exogenous mitochondria provided herein, for example immune cells augmented with exogenous isolated viable mitochondria (such as immune cells transplanted with autologous, allogeneic, xenogeneic, encapsulated or genetically modified mitochondria), may be useful for treating any disease or condition involving a target. Where the present application discloses a general application of immune cells (not binding agent specific), a "tumor associated antigen" ("TAA") may be used as the target cell molecule. In some embodiments, the disease or condition is a disease or condition that may benefit from treatment with adoptive cell therapy. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is a viral infection. In some embodiments, the disease or condition is an autoimmune disease.

[0169] In some embodiments, provided herein is a method of treating a disease or condition in a subject in need of such treatment, by administering to the subject an effective amount of immune cells augmented with exogenous mitochondria provided herein, e.g., immune cells previously transplanted with exogenous mitochondria ex vivo. In some embodiments, provided herein is a method of treating a disease or condition in a subject in need of such treatment, by co-administering to the subject an effective amount of immune cells with exogenous mitochondria provided herein. In some aspects, the disease or condition is cancer. In some aspects, the disease or condition is a viral infection. In some embodiments, the disease or condition is an autoimmune disease.

[0170] Any suitable cancer may be treated with the exogenous mitochondria-enhanced immune cells provided herein. Exemplary suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, tracheal tumor, carcinoma of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, fibrous histiocytoma, Ewing's lymphoma, and leukemia. Sarcoma, Eye Cancer, Germ Cell Tumor, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gestational Trophoblastic Disease, Glioma, Head and Neck Cancer, Hepatocellular Carcinoma, Histiocytosis, Hodgkin's Lymphoma (HL), Subpharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumor, Kaposi's Sarcoma Kidney Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lobular Carcinoma in Epithelial Cell, Lung Cancer, Macroglobulinemia, Malignant Fibrous Histiocytoma, Melanoma, Merkel Cell Carcinoma , mesothelioma, metastatic squamous cell carcinoma with occult primary, midline duct carcinoma with NUT gene, oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma (NHL), non-small cell lung cancer (NSCLC), oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, brown cell carcinoma, alveolar cyst, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0171] Combination therapy In some embodiments, immune cells such as T cells or CAR T cells enhanced with exogenous mitochondria provided herein are administered with at least one additional therapeutic agent. The immune cells enhanced with exogenous mitochondria may include immune cells that have been previously transplanted with exogenous mitochondria ex vivo, or immune cells that have been co-administered with exogenous mitochondria so that exogenous mitochondria are transplanted into immune cells in vivo. Any suitable additional therapeutic agent may be administered with the immune cells enhanced with exogenous mitochondria provided herein. In some aspects, the additional therapeutic agent is selected from radiation, cytotoxic agents, chemotherapeutic agents, cytostatic agents, antihormonal agents, EGFR inhibitors, immunostimulants, antiangiogenic agents, checkpoint blockers, and combinations thereof.

[0172] In some embodiments, the additional therapeutic agent comprises an immunostimulant.

[0173] In some embodiments, the immunostimulant is an agent or its ligand that blocks the signal transduction of inhibitory receptors of immune cells. In some aspects, the inhibitory receptor or ligand is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), programmed cell death protein 1 (also known as PD-1 or CD279), programmed death-ligand 1 (also known as PD-L1 or CD274), transforming growth factor beta (TGFβ), lymphocyte activation gene 3 (LAG-3, also known as CD223), Tim-3 (Hepatitis A virus cell receptor 2 or HAVCR2 or CD366), neuritin, B and T lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptor (KIR), and combinations thereof. In some embodiments, the agent is selected from an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), and an anti-PD-L1 antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-TIM3 antibody, carcinoembryonic antigen-related cell adhesion molecule 1 (CECAM-1, also CD66a), and 5 (CEACAM-5, also CD66e), a vset immunoregulatory receptor (also VISR or VISTA), a leukocyte-associated immunoglobulin-like receptor 1 (also LAIR1 or CD305), CD160, a natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof. In some embodiments, the agent is pembrolizumab. In some embodiments, the agent is nivolumab. In some embodiments, the agent is atezolizumab.

[0174] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from an antibody, a peptidomimetic, and a small molecule. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from pembrolizumab (Keytruda™), nivolumab (Opdivo™), atezolizumab (Tecentriq™), avelumab (Bavencio™), pidilizumab, durvalumab, BMS-936559, sulfamonomethoxine 1, and sulfamethizole 2. In some embodiments, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is any therapeutic agent known in the art to have such activity, e.g., as described in Weinmann et al. (Weinmann, 2016, "Corrigendum: Cancer Immunotherapy: Selected Targets and Small-Molecule Modulators", ChemMedChem 11:1576), which is incorporated by reference in its entirety. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition as an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition than an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered prior to administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered after administration of an antibody provided herein. In some embodiments, an agent that inhibits the interaction between PD-1 and PD-L1 is administered simultaneously with an antibody provided herein, although the agent and the antibody are administered in separate pharmaceutical compositions.

[0175] In some embodiments, the immunostimulatory agent is an agonist of a costimulatory receptor of an immune cell. In some aspects, the costimulatory receptor is selected from GITR, OX40, ICOS, LAG-2, CD27, CD28, 4-1BB, CD40, STING, toll-like receptor, RIG-1, and NOD-like receptor. In some embodiments, the agonist is an antibody.

[0176] In some embodiments, the immunostimulatory agent modulates the activity of arginase, indoleamine-2 3-dioxygenase, or adenosine A2A receptor.

[0177] In some embodiments, the immune stimulatory agent is a cytokine. In some aspects, the cytokine is selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof. In some aspects, the cytokine is IL-2.

[0178] In some embodiments, the immunostimulatory agent is an oncolytic virus. In some aspects, the oncolytic virus is selected from herpes simplex virus, vesicular stomatitis virus, adenovirus, Newcastle disease virus (NDV), vaccinia virus, and Maraba virus.

[0179] Further examples of additional therapeutic agents include taxanes (e.g., paclitaxel or docetaxel), platinum agents (e.g., carboplatin, oxaliplatin, and / or cisplatin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, and / or mitoxantrone), folinic acid (e.g., leucovorin), or nucleoside metabolic inhibitors (e.g., fluorouracil, capecitabine, and / or gemcitabine). In some embodiments, the additional therapeutic agent is folinic acid, 5-fluorouracil, and / or oxaliplatin. In some embodiments, the additional therapeutic agent is 5-fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexed. In some embodiments, the additional therapeutic agent is a targeted therapeutic agent, such as an EGFR, RAF, or MEK targeting agent.

[0180] The additional therapeutic agent can be administered by any suitable means. In some embodiments, the medicament provided herein and the additional therapeutic agent are contained in the same pharmaceutical composition. In some embodiments, the antibody provided herein and the additional therapeutic agent are contained in different pharmaceutical compositions.

[0181] In embodiments in which the antibody provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the antibody can occur prior to, concurrently with, and / or after administration of the additional therapeutic agent. In some aspects, administration of the antibody provided herein and the additional therapeutic agent occurs within about one month of each other. In some aspects, administration of the antibody provided herein and the additional therapeutic agent occurs within about one week of each other. In some aspects, administration of the antibody provided herein and the additional therapeutic agent occurs within about one day of each other. In some aspects, administration of the antibody provided herein and the additional therapeutic agent occurs within about 12 hours of each other. In some aspects, administration of the antibody provided herein and the additional therapeutic agent occurs within about one hour of each other.

[0182] How to use Provided herein are methods for delivering isolated mitochondria or pharmaceutical compositions of isolated mitochondria to cells of a patient or allogeneic donor ex vivo and / or to tissues of a patient in vivo. Without wishing to be bound by theory, the mitochondria are taken up by tissue cells or cultured cells via actin-dependent endocytosis, thereby providing methods for delivering pharmaceutical compositions directly to cells. In a non-limiting illustrative example, mitochondria are cultured in culture medium for, e.g., 2-24 hour periods, for 10-24 hours, for ... 6Mitochondria (100 μg / well) are implanted into target immune cells by co-incubation with mitochondria (100 μg / well) (100 μg / well). One skilled in the art will recognize that the dosage of mitochondria administered to immune cells ex vivo or administered to patient tissues in vivo can vary based on the intended outcome of enhancing the target immune cell(s), such as optimizing viability, survival, durability, self-renewal capacity, and / or selection. For example, in delivery of mitochondria to immune cells ex vivo by co-incubation, the dosage of mitochondria can be from 0.0001 ng of mitochondria per target cell to 2.5 ng of mitochondria per target cell. In delivery of mitochondria in vivo to patient tissues, the dosage of mitochondria can be from 1 mitochondrion per mL to 10 mitochondrion per mL. 7 Mitochondria may be delivered.

[0183] The present disclosure contemplates compositions comprising enhanced immune cells (such as αβT cells, γδT cells, memory immune cells (e.g., central memory CD8 T cells, effector memory CD8 T cells, or memory-like T cells), Treg cells (e.g., Treg CD4 T cells), CAR-T cells, etc.), which comprise or are enhanced with exogenous mitochondria, which may be autologous, allogeneic, xenogeneic, encapsulated, or autologous with genetic modification. These cells may be any effector cell known in the art with anti-tumor activity or immunosuppressive immune cells capable of preventing autoimmunity. Thus, the present disclosure provides methods for delivering immune cells comprising or enhanced with exogenous mitochondria, or pharmaceutical compositions of immune cells comprising or enhanced with exogenous mitochondria, to cells and / or tissues of a patient, or to cells derived from an allogeneic donor. Immune cells containing or augmented with exogenous mitochondria can be used to treat a variety of diseases, including, but not limited to, various forms of cancer, tumors, and autoimmune diseases.

[0184] In some embodiments, the preparation of CAR T cells may comprise the following steps. 1. T lymphocytes are collected from the patient's blood through leukapheresis. 2. Enrichment of T cells by density gradient centrifugation, elutriation, and immunomagnetic bead selection. 3. Genetic modification using electroporation, retroviral / lentiviral transduction, or nuclease-mediated genome editing (e.g., introduction of a CAR gene into the genome of a target cell). 4. Activation and expansion of CAR-T cells by polyclonal activation with an artificial antigen-presenting system (anti-CD8 / anti-CD28 immunomagnetic beads / LV-APC) using methods known in the art. Consistency is generally achieved by standardization and validation of raw materials and protocols according to cGMP (Good Manufacturing Practice). 5. Quality Assurance - Testing for viability, phenotyping, Gram staining, endotoxin, and bacterial, fungal, and mycoplasma contaminants in accordance with FDA guidelines using methods known in the art. 6. Formulation and Administration - Test for clinically prescribed doses and routes of administration using methods known in the art. Storage, packaging, shipping, receipt, and administration of therapeutic cells generally require maintaining product stability and chain of custody.

[0185] In certain embodiments, the mitochondrial preparation is delivered to immune cells (1) before genetic modification (e.g., introduction of a CAR gene), (2) simultaneously with genetic modification, or (3) after genetic modification. In certain embodiments, the mitochondrial preparation is delivered to immune cells ex vivo before genetic modification (e.g., introduction of a CAR gene), (2) simultaneously with genetic modification, or (3) after genetic modification, such as in a method including ex vivo genetic modification. In certain embodiments, the mitochondrial preparation is delivered to immune cells ex vivo before genetic modification (e.g., introduction of a CAR gene) in vivo (e.g., in vivo virus-mediated genetic modification). Without wishing to be bound by theory, step (1) is typically important for regenerating autologous T cells (exhausted T cells or senescent T cells) taken from immunocompromised cancer patients. Mitochondria can be co-incubated with cells ex vivo at a ratio between 0.2:1 and 5000:1, for example, 0.2:1, 0.5:1, 1:1, 10:1, 50:1, 100:1, 200:1, 500:1, 1000:1, or 5000:1.

[0186] To boost immune cell activity, mitochondria, such as CAR-T cell activity, can also be delivered to patients in vivo together with immune cells (4). In certain embodiments, mitochondrial preparations are delivered to immune cells in vivo (1) before in vivo genetic modification (e.g., introduction of CAR gene), (2) simultaneously with in vivo genetic modification, or (3) after in vivo genetic modification (e.g., in vivo virus-mediated genetic modification). In certain embodiments, mitochondrial preparations are delivered to immune cells in vivo after ex vivo genetic modification (e.g., introduction of CAR gene). In certain embodiments of the present invention, CAR-T cells or other immune cells are delivered via systemic (intravenous) injection, while mitochondria are delivered via (5) intratumoral injection, (6) intraorgan injection, (7) intratissue injection, or (8) organ- or tissue-specific vasculature. EXAMPLES

[0187] The following are examples of methods and compositions of the present invention. It will be understood from the general description herein that various other embodiments may be practiced.

[0188] Example 1a. Isolation of mitochondria from tissue samples or cultured cells Experiments were performed to isolate mitochondria from tissue samples or cultured cells.

[0189] preparation The following solutions were prepared to isolate intact, viable, and respiration-capable mitochondria. To successfully isolate mitochondria using this method, solutions and tissue samples must be kept on ice to preserve mitochondrial viability. Even when kept on ice, isolated mitochondria show a decrease in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). The following solutions should be prepared in advance, if possible. -1M K-HEPES stock solution (pH adjusted to 7.2 with KOH). -0.5M K-EGTA stock solution (adjust pH to 8.0 with KOH). -1M KH2PO4 stock solution. -1M MgCl2 stock solution. - Homogenization buffer (pH 7.2), 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. Stored at 4°C. -1×PBS (ThermoFisher, 10010031) -1x PBS was prepared by pipetting 100mL of 10x PBS into 1L of double distilled H2O. Subtilisin A Stock was prepared by weighing 2mg of Subtilisin A into a 1.5mL microfuge tube. Store at -20°C until use. Prepared at 2mg / ml in Homogenizing Buffer.

[0190] Isolation of mitochondria from tissues A scheme outlining the procedural steps in the isolation of mitochondria using tissue dissociation and differential filtration is shown in Figure 2. Two 6 mm biopsy fresh sample punches taken from skeletal muscle were transferred to 5 mL of homogenization buffer in a gentleMACS C tube (Miltenyi Biotec, Somerville, MA) and the samples were homogenized using the 1-minute homogenization program of the gentleMACS™ Dissociator (Miltenyi Biotec). Subtilisin A stock solution (250 μL) was added to the homogenate in the gentleMACS C tube and incubated on ice for 10 minutes. The homogenate was centrifuged at 750×g for 4 minutes (as an optional step). The homogenate was then filtered through a pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was refiltered through a new pre-wet 40 μm mesh filter in a 50 mL conical centrifuge on ice. The filtrate was refiltered again through a new pre-wet 10 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was refiltered through a new pre-wet 6 μm mesh filter in a 50 mL conical centrifuge tube on ice. The resulting filtrate was either used immediately or concentrated by centrifugation. In the case of concentration, the filtrate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 9000 × g for 10 min at 4 °C. The supernatant was removed and the pellet containing the mitochondria was resuspended and mixed in 1 mL of homogenization buffer.

[0191] Isolation of mitochondria from cultured cells Mitochondria were also isolated from cultured cells, for example, from the human cardiac fibroblast (HCF) cell line (obtained from ScienCell Research Laboratories, Carlsbad, Calif.).

[0192] Culture of human cardiac fibroblast (HCF) cells Human cardiac fibroblasts (HCFs) were maintained in Fibroblast Medium-2 containing fetal bovine serum, fibroblast growth supplement-2, and antibiotic (penicillin / streptomycin) solution according to the supplier's instructions (ScienCell). Cells were maintained as monolayers at 37°C in a humidified atmosphere of 5% CO2 and passaged when they reached 90% confluence.

[0193] Preparation of human cardiac fibroblast (HCF) cells HCF cells from two flasks (T150) at 80% confluency were washed once with PBS. Trypsin was then used to detach the cells according to the supplier's instructions (ScienCell Research Laboratories, Carlsbad, CA). The reaction was stopped by adding trypsin neutralizing solution according to the supplier's instructions (ScienCell Research Laboratories, Carlsbad, CA). The cells were collected in a 50 ml centrifuge tube and centrifuged at 1000 rpm (190 x g) for 5 min. The supernatant was discarded and washed a total of three times with 1 x PBS.

[0194] The preparation of cultured cells different from HCFs needs to be performed according to the manufacturer's instructions. Of note, the cells used as a source of mitochondria can be adherent, semi-adherent, or in suspension.

[0195] The mitochondrial isolation procedure was essentially the same as that for isolating mitochondria from tissue samples, except that human fibroblasts were used rather than biopsy samples.

[0196] Alternatively, mitochondria can be isolated by repeated centrifugation (Kesner et al., 2016, “Characteristics of Mitochondrial Transformation into Human Cells”, Sci Rep 6:26057). Briefly, cells were harvested by trypsinization, suspended in PBS, and centrifuged (5 min, 250 × g) twice. The mitochondrial isolation procedure was performed at 4 °C or on ice. Centrifuged cells were resuspended in mitochondrial isolation buffer (320 mM sucrose, 5 mM Tris-HCl, pH 7.4, 2 mM EGTA) and homogenized in a Dounce homogenizer. Nuclei and cell debris were removed by two centrifugations at 3000 × g for 5 min, and the supernatant was collected (optional step). The supernatant was then centrifuged at 12,000 × g for 10 min, and the mitochondrial pellet was resuspended in mitochondrial isolation buffer. Mitochondrial concentrations were determined by Bradford assay.

[0197] Mitochondrial number Viable mitochondrial numbers were determined by labeling an aliquot (10 μL) of isolated mitochondria with MitoTracker Orange CMTMRos (5 μmol / L, Thermo Fisher Scientific). An aliquot of labeled mitochondria was spotted onto a slide and counted using a spinning disk confocal microscope with a 63× C-apochromat objective (1.2W Korr / 0.17NA, Zeiss). Mitochondria were counterstained with the mitochondrial-specific dye MitoFluor Green (Thermo Fisher Scientific). For the measurement of autofluorescence and background fluorescence, appropriate wavelengths were selected using unstained cells and tissues. Briefly, 1 μL of labeled mitochondria was placed on a microscope slide and covered. Mitochondria numbers were determined using MetaMorph Imaging Analysis software at low (×10) magnification covering the entire specimen area.

[0198] Example 1b. Isolation of mitochondria from cultured cells Experiments were carried out to isolate mitochondria from cultured cells.

[0199] preparation The following solutions were prepared to isolate intact, viable, and respiration-capable mitochondria. To successfully isolate mitochondria using this method, solutions and tissue samples must be kept on ice to preserve mitochondrial viability. Even when kept on ice, isolated mitochondria show a decrease in functional activity over time (Olson et al., J Biol Chem 242:325-332, 1967). The following solutions should be prepared in advance, if possible. -1M K-HEPES stock solution (pH adjusted to 7.2 with KOH). -0.5M K-EGTA stock solution (adjust pH to 8.0 with KOH). - Homogenization buffer (pH 7.2), 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. Stored at 4°C. -1×PBS(ThermoFisher,10010031) - Subtilisin A stock was prepared by weighing 2 mg of Subtilisin A into a 1.5 mL microcentrifuge tube. Store at 20° C. until use. Prepared at 2 mg / ml in homogenization buffer.

[0200] Culture of human cardiac fibroblast (HCF) cells Human cardiac fibroblasts (HCF) (obtained from ScienCell Research Laboratories, Carlsbad, Calif.) were cultured as described in Example 1a, and the cells were passaged when they reached 90% confluence.

[0201] The preparation of cultured cells different from HCFs needs to be performed according to the manufacturer's instructions. Of note, the cells used as a source of mitochondria can be adherent, semi-adherent, or in suspension.

[0202] Isolation of mitochondria from cultured cells Mitochondria were also isolated from cultured cells, such as human cardiac fibroblast (HCF) cell lines. Preparation of HCF cells was performed according to the method of Example 1a. HCF cells from each flask were then transferred to 5 mL of homogenization buffer in a gentleMACS C Tube (Miltenyi Biotec, Somerville, MA), and the samples were homogenized using the 1 minute homogenization program of the gentleMACS™ Dissociator (Miltenyi Biotec). Subtilisin A stock solution (250 μL) was added to the homogenate in the gentleMACS C tube and incubated on ice for 10 minutes. The homogenate was filtered through a pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge tube on ice. The filtrate was re-filtered through a new pre-wetted 40 μm mesh filter in a 50 mL conical centrifuge on ice. The filtrate was refiltered again through a new pre-wet 10 μm mesh filter in a 50 mL conical centrifuge tube on ice. Optionally, the filtrate was refiltered again through a new pre-wet 5 μm mesh filter in a 50 mL conical centrifuge tube on ice. The resulting filtrate was either used immediately or concentrated by centrifugation. In the case of concentration, the filtrate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 9500×g for 5 min at 4° C. Three washes were performed at the same centrifugation speed.

[0203] Quantification of isolated mitochondria The isolated mitochondria were suspended in homogenization buffer of Example 1b and kept on ice until use. The amount of mitochondria in the preparations for different dose administration was measured using Qubit™ Fluorometer (ThermoFisher Scientific / Invitrogen) with Qubit™ Protein Assay kit according to the manufacturer's instructions. For protein concentration measurement, mitochondria were resuspended in PBS (ThermoFisher, 10010031). Mitochondria dosage was estimated in terms of protein content expressed in μg.

[0204] Example 2 T cell isolation, activation and culture CD8 + T cells were isolated from buffy coats of healthy donors. Peripheral blood mononuclear cells (PBMCs) were collected by density gradient centrifugation using Ficoll Paque plus (Cytiva, 17144002) according to the manufacturer's instructions. EasySep™ Human CD8 + Human CD8 T Cell Isolation Kit (Stemcell, 17953) and the Big Easy EasySep™ Magnet (Stemcell, 18001) were used to isolate human CD8 T Cells from PBMCs. + T cells were harvested. Isolated CD8 + T cells were activated at a 1:1 ratio using Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher, 111.32D) in the presence of 100 U / ml recombinant human IL-2 (Peprotech, 200-02). + T cells were cultured in 500 mL of RPMI 1640 medium GlutaMAX™ Supplement (ThermoFisher, 61870010) supplemented with 1% L-glutamine (ThermoFisher, 25030024), 1% penicillin-streptomycin (10,000 U / mL, Gibco, 15140122), 1% non-essential amino acids (NEAA, ThermoFisher, 11140050), 1% sodium pyruvate (ThermoFisher, 11360070), 10% fetal bovine serum, and 0.1% 2β-mercaptoethanol (Gibco, 31350-010). + T cells were plated at 0.5 million cells / mL and split when the cells reached a confluency of 2 million cells / mL or when the medium turned yellow.

[0205] Example 3. T cell transplantation CD8 +T cells were plated at 0.5 million cells / mL in 24-well plates 24 hours prior to mitochondrial transfer. + The T cells were harvested and centrifuged at 1500 rpm (430×g) for 5 min. The supernatant was discarded and the cells were resuspended in fresh T cell medium at a concentration of 1 million cells / 100 μL. The T cell medium is described in Example 2. + T cells were cultured in 200 µL final volume of T cell medium in each well of a 24-well plate, with 10% CD8 + T cells were incubated with isolated mitochondria for 4 hours in a range of 10 μg to 100 μg of protein per million. + Four hours after T cell co-incubation, 1.8 ml of fresh T cell medium was added per well.

[0206] Example 4. Mitochondrial labeling and internalization Example 4.1 - T Cell Transplantation with Stained Isolated Mitochondria CD8 + T cells are seeded at 500,000 cells / mL in 24-well plates 24 hours prior to mitochondrial transfer. Mitochondria are isolated according to the procedure described in Example 1b. Mitochondria are then stained with 200 nM Mitotracker Red CMXRos (ThermoFisher, M7512) and Mitotracker Green FM (ThermoFisher, M7514) in homogenization buffer of Example 1b for 10-15 minutes at 37°C. Three washes of the stained mitochondria are performed with homogenization buffer of Example 1b at 9500×g for 5 minutes at 4°C, and the supernatant of the last wash is saved as a control. CD8 +T cells are harvested and centrifuged at 1500 rpm (430×g) for 5 min. The supernatant is removed and the cells are resuspended in fresh T cell medium at 1 million cells / 100 μL. T cell medium is described in Example 2. The stained mitochondria are added (immediately) to the T cells to give a final volume of 200 μL per well of a 24-well plate. The final wash of stained mitochondria is then transferred to control non-transplanted CD8 + The cells are added in equal volumes to the CD8 T cells. The incorporation of stained mitochondria is assessed 5 min to 24 h after transplantation by flow cytometry (data acquired on e.g. FACSLyric (BD Biosciences)) or by fluorescence microscopy (Keyence microscope, BZ-X810). + For T cell co-incubation, add 1.8 ml of fresh T cell medium per well.

[0207] Example 4.2 - Staining after mitochondrial transfer Transplanted CD8 + T cells were cultured in each well of a 24-well plate in a final volume of 200 µL of CD8 T cell medium. + Incubate for 4 h with isolated mitochondria ranging from 10 µg to 100 µg of protein per million of T cells. Exogenous mitochondria and CD8 + After 4 h of T cell co-incubation, 1.8 ml of fresh T cell medium is added per well. Mitochondrial respiration and mass are assessed in transplanted cells 24 h after co-incubation. The dyes Mitotracker Red CMXRos (ThermoFisher, M7512) and Mitotracker Green FM (ThermoFisher, M7514) are diluted to a final concentration of 100 nM in phenol red-free RPMI1640 medium (ThermoFisher, 11835030) supplemented with 1% penicillin-streptomycin (10,000 U / mL, Gibco, 15140122), 5% fetal bovine serum. One million CD8 +100 μl of stain per T cell is added and staining is carried out for 15 min at 37° C. The cells are then washed twice for 5 min at 1500 rpm (430×g) with FACS buffer (1×PBS (ThermoFisher, 10010031), 2% FBS, 1% EDTA 0.5M (Sigma-Aldrich, E6758)). The supernatant is discarded and CD8 + T cells are resuspended in 300 μL of FACS buffer and acquired on a FACS machine (FACSLyric, BD Biosciences).

[0208] Example 5. Memory CD8 in vitro during mitochondrial transplantation + Increased proportion of T cells The percentage of memory T cells was determined by measuring the percentage of CD8 + Exogenous mitochondria were assessed by flow cytometry 9 days after transfer into T cells.

[0209] procedure (i) T cell isolation, activation, and culture were performed as described in Example 2. (ii) Mitochondrial Isolation Mitochondria were isolated from human cardiac fibroblasts (HCF) as described above in Example 1b. The isolated mitochondria were suspended in homogenization buffer of Example 1b and kept on ice until use. The amount of mitochondria in the preparations for various dose administration was measured using a Qubit™ Protein Assay Kit using a Qubit™ Fluorometer (Thermo Fisher Scientific / Invitrogen) according to the manufacturer's instructions. Mitochondrial dosage is estimated in terms of protein content expressed in μg. (iii) Nine days after transplantation, staining was performed on ice according to the manufacturer's instructions using anti-human CD45RA APC (Biolegend, 304112), anti-human CD45RO PB (Biolegend, 304223), and anti-human CD62L FITC (Biolegend, 304804). Depending on the surface expression, CD8 +T cells were classified as naive (CD62L+, CD45RA+, CD45RO-), stem cell-like memory (CD62L+, CD45RA+, CD45RO+), central memory (CD62L+, CD45RA-, CD45RO+), effector memory (CD62L-, CD45RA-, CD45RO+) or effector (CD62L-, CD45RA+, CD45RO-). A portion of the different subsets were naive CD8 + T cells and CD8 + T cells are compared.

[0210] result Central memory CD8 cells 9 days after mitochondrial transplantation + T cells and effector memory CD8 + Increased proportion of T cells Transplanted mitochondria were found to be memory CD8 from a mixed population. + To test for their ability to favor T cell survival and / or differentiation and / or selection, mitochondria were transfected into 1 million CD8 T cells at day 12 post-activation. + CD8 at dose levels of 30 μg and 100 μg per T cell + On day 9 after transplantation, CD8 + T cells were stained and analyzed by flow cytometry using FACSLyric (BD Biosciences) and classified as naïve (CD62L+, CD45RA+, CD45RO-), stem cell-like memory (CD62L+, CD45RA+, CD45RO+), central memory (CD62L+, CD45RA-, CD45RO+), effector memory (CD62L-, CD45RA-, CD45RO+), or effector (CD62L-, CD45RA+, CD45RO-).

[0211] As shown in Figure 3, untreated CD8 + Compared with T cells, central memory CD8 + T cells and effector memory CD8 +The proportion of T cells is clearly increased. Central memory CD8 + T cells and effector memory CD8 + A significant enhancement of T cells was detected at doses of 30 μg and 100 μg of mitochondria.

[0212] Example 6. Memory CD8 in vivo during mitochondrial transplantation + Increased proportion of T cells The proportion of effector and memory T cells is evaluated over time in the immune response induced to acute infection. CD45.1 murine OT-I T cells bound to ovalbumin (OVA) peptide are activated and transplanted with exogenous mitochondria, followed by injection into CD45.2 C57 / B6 mice, which are then infected with Listeria-OVA. Treatment groups are compared to the induced immune response of OT-I T cells not transplanted with mitochondria.

[0213] means (i) Mouse CD8 + Isolation of T cells was performed using EasySep™ Mouse CD8 + The T cell isolation kit (StemCell, Cat. #19853) is followed. T cell activation and proliferation is performed using CD3 / CD28 Dynabeads (Gibco, Cat. #11456.D) in a 1:1 ratio with recombinant IL-2 (50 U / ml). CD8 + T cells are plated at 0.5 million cells / mL and split when the cells reach a confluency of 2 million cells / mL or when the medium turns yellow. (ii) Isolation of OT-I mouse mitochondria Mitochondria are isolated from skeletal muscle as described above in Example 1a. The isolated mitochondria are suspended in homogenization buffer of Example 1a and kept on ice until use. The amount of mitochondria is measured using Qubit™ Protein Assay Kit using Qubit™ Fluorometer (ThermoFisher Scientific / Invitrogen) according to the manufacturer's instructions in preparation for various dose administration. Mitochondrial dosage is estimated in terms of protein content expressed in μg. (iii) Mouse CD8 + T cells are transferred 7 days after activation as described above in Example 3. (iv) Mice (CD45.2) were transplanted with 20,000 OT-I CD8 + T cells (CD45.1) are injected and subsequently infected with 2,000 colony forming units (cfu) of Listeria-OVA. +T cell persistence and memory differentiation is assessed over time in the blood (days 7, 14, 21) and organs (spleen, lymph nodes (LN) on day 21) of the animals. Staining from blood or processed organs: LIVE / DEAD Fixable dye Aqua DEAD (ThermoFisher, L34957), anti-mouse CD8α PE / Texas Red (Abcam ab25294), anti-mouse CD45.1 BV650 (BD 563754), anti-mouse CD45.2 BV421 (BD 562895), anti-mouse KLRG1 PE-Cy7 (BioLegend 138415), anti-mouse CD127 PE (BioLegend 121111), anti-mouse CD44 APC-Cy7 (BD 560568), anti-mouse CD62L PerCP / Cyanine5.5 (BioLegend 104431). The elicited immune response of OT-I T cells against Listeria-OVA is classified as short-lived effector cells (SLEC) (KLRG1+ CD127- and / or CD44+ CD62L-) and memory progenitor cells (MPEC) (KLRG1-CD127+ and / or CD44+ CD62L+). (v) Cytokine production is assessed on the day of sacrifice, 4 hours after peptide restimulation (OVA peptide). Cells are harvested from homogenized spleens and LNs plated in 96-well plates. Cells are incubated with 10 μM SIINFEKL (OVA) peptide or PMA / ionomycin for 30 minutes, followed by restimulation for an additional 4 hours in the presence of Golgistop (BD) and Golgiplug (BD). Cells are harvested, fixed, and permeabilized for intracellular cytokine staining to assess anti-mouse IFNγ PerCP / Cyanine5.5 (BioLegend 505821), anti-mouse TNFα Pacific Blue (BioLegend 506318), anti-mouse IL-2 PE (BioLegend 503807), and anti-mouse Granzyme B FITC (BioLegend 515403) production.

[0214] result Exogenous mitochondrial transplantation promotes the formation and persistence of memory cells during a triggered immune response. Over time, the proportion of mouse short-lived effector cells (SLECs) (KLRG1+ CD127- and / or CD44+ CD62L-) decreased, and memory progenitor cells (MPECs) (KLRG1-CD127+ and / or CD44+ CD62L+) increased in proportion to the transplanted OT-1 CD8 + Increased in mice injected with T cells. Upon peptide restimulation, OT-I CD8 in the treatment group with exogenous mitochondria + Cytokine production of T cells is higher compared to the untreated group.

[0215] Example 7. Memory-like CD8 from TILs in vitro upon mitochondrial transplantation + Increased proportion of T cells After exogenous mitochondrial transfer into cultured human TILs, the percentage of memory-like T cells was assessed over time by flow cytometry.

[0216] means (i) Isolation and culture of TILs Surgically resected tumor masses are digested with enzymes such as collagenase type IV (Sigma Aldrich) and Pulmozyme (Roche) to generate single cell suspensions. TILs are expanded with high doses of IL-2 as previously described (van den Berg JH, et al. J Immunother Cancer 2020;8:e000848,doi:10.1136 / jitc-2020-000848). TIL CD8 + If the proportion of T cells is sufficient in the bulk TIL population, CD8 + T cell isolation is performed as described in Example 2. (ii) Mitochondrial Isolation Mitochondria are isolated from human cardiac fibroblasts (HCF) as described above in Example 1b. The isolated mitochondria are suspended in homogenizing buffer of Example 1b and kept on ice until use. The amount of mitochondria is measured using Qubit™ Protein Assay Kit using Qubit™ Fluorometer (ThermoFisher Scientific / Invitrogen) according to the manufacturer's instructions in preparation for various dose administration. Mitochondrial dosage is estimated in terms of protein content expressed in μg. (iii) Over time, from 4 hours to 2 weeks after transplantation, stain with anti-human CD45RA APC (Biolegend, 304112), anti-human CD45RO PB (Biolegend, 304223), and anti-human CD62L FITC (Biolegend, 304804) on ice according to the manufacturer's instructions. + T cells are classified as naive (CD62L+, CD45RA+, CD45RO+), stem cell-like memory (CD62L+, CD45RA+, CD45RO+), central memory (CD62L+, CD45RA-, CD45RO+), effector memory (CD62L-, CD45RA-, CD45RO+), or effector (CD62L-, CD45RA+, CD45RO-). Parts of the different subsets were analyzed in control and exogenously mitochondrially transplanted TIL CD8 + Compare with T cells.

[0217] result Memory-like TIL CD8 after mitochondrial transplantation + Increased proportion of T cells Transfer of exogenous mitochondria into TILs from bulk populations promotes survival and selection of memory-like TILs.

[0218] Example 8. Adoptive cell transfer of TILs rechallenged in tumor-bearing mice or engrafted during acute infection shows enhanced recall responses Exogenous mitochondria are transplanted into OT-I TILs extracted and isolated from OVA-expressing tumors. To evaluate the properties of selected memory-like TILs after in vitro culture, treated or untreated TILs are adoptively transferred and re-administered into tumor-bearing mice or during acute infection. In OVA-restricted tumor-bearing mice, the recall capacity of OT-I TILs is evaluated over time by measuring tumor growth, mouse survival, and persistence of transferred cells infiltrating the cancer mass and lymphoid organs. In the context of acute infection, OT-I TILs are adoptively transferred into animals and subsequently infected with OVA-expressing virus or bacteria. The evoked immune response is evaluated over time in blood and lymphoid organs between transplanted and untreated TILs.

[0219] means (i) Generation and extraction of mouse TILs. CD45.2 C57 / B6 mice are engrafted subcutaneously in one flank with 200,000 OVA-expressing tumor cells. Six days after engraftment, 100,000 CD45.1 OT-I T cells are adoptively transferred intravenously. 21 days after engraftment and / or when an appropriate tumor size is reached, tumors are harvested and dissociated using a Tumor Dissociation Kit (130-096-730, Miltenyi Biotec) according to the manufacturer's instructions. Mouse CD8 TILs are extracted from the tumors. + To select T cells, EasySep™ Mouse CD8 + Isolation is performed according to the T cell isolation kit (StemCell, Cat. #19853). To further select OT-I TILs, FACS-based cell sorting is performed according to LIVE / DEAD-, CD45.1+, CD8+. (ii) Isolation of mitochondria from mouse OT-I Mitochondria are isolated from skeletal muscle as described above in Example 1a. The isolated mitochondria are suspended in homogenization buffer of Example 1a and kept on ice until use. The amount of mitochondria is measured in preparation for various dose administration using Qubit™ Fluorometer (ThermoFisher Scientific / Invitrogen) and Qubit™ Protein Assay Kit according to the manufacturer's instructions. Mitochondrial dosage is estimated in terms of protein content expressed in μg. (iii) Mouse CD8 + T cells are transferred as described above in Example 3. (iv) Rechallenge in Tumor-Bearing Mice CD45.2 C57 / B6 mice are subcutaneously implanted with 200,000 OVA-expressing tumor cells in one flank. Five days after engraftment, 5 Gy total body irradiation is applied. Six days after engraftment, 10,000 transplanted CD45.1 OT-I TILs are adoptively transferred intravenously. Tumor growth is measured every 2-3 days using a caliper. 21 days after engraftment and / or when an appropriate tumor size is reached, tumors are harvested and dissociated using a Tumor Dissociation kit (130-096-730, Miltenyi Biotec) according to the manufacturer's instructions. Invasion in tumors and persistence in lymphoid organs are assessed by flow cytometry with the following staining: LIVE / DEAD Fixable dye Aqua DEAD (ThermoFisher, L34957), anti-mouse CD8α Pe / Texas Red (Abcam ab25294), anti-mouse CD45.1 BV650 (BD 563754), anti-mouse CD45.2 BV421 (BD 562895), anti-mouse KLRG1 PE-Cy7 (BioLegend 138415), anti-mouse CD127 PE (BioLegend 121111), anti-mouse CD44 APC-Cy7 (BD 560568), anti-mouse CD62L PerCP / Cyanine5.5 (BioLegend 104431). (v) Rechallenge for Acute Infection Mice (CD45.2) are injected with 10,000 OT-I TILs (CD45.1) one day after transplantation and subsequently infected with 2,000 cfu of Listeria-OVA. Persistence and memory differentiation of OT-I TILs is assessed over time in blood of animals (days 7, 14, 21) and organs (spleen, LN on day 21). Staining from blood or processed organs LIVE / DEAD fixed dye Aqua DEAD (ThermoFisher, L34957), anti-mouse CD8α Pe / Texas Red (Abcam ab25294), anti-mouse CD45.1 BV650 (BD 563754), anti-mouse CD45.2 BV421 (BD 562895), anti-mouse KLRG1 PE-Cy7 (BioLegend 138415), anti-mouse CD127 PE (BioLegend 121111), anti-mouse CD44 APC-Cy7 (BD 560568), anti-mouse CD62L PerCP / Cyanine 5.5 (BioLegend 104431). Recall immune responses of OT-I TILs against Listeria-OVA are classified as short-lived effector cells (SLECs) (KLRG1+ CD127- and / or CD44+ CD62L-) and memory progenitor cells (MPECs) (KLRG1-CD127+ and / or CD44+CD62L+).

[0220] result Engrafted TILs show improved recall capacity during acute infection in tumor-bearing mice. Re-administered engrafted TILs in tumor-bearing or infected mice exhibit hallmark characteristics of memory cells, as demonstrated by enhanced persistence, improved recall capacity, and improved tumor control in tumor-bearing animals.

[0221] Example 9. Transplanted CD8 + T cells have an enhanced ability to compete for survival signals To evaluate the ability of transplanted T cells to compete efficiently for survival signals, co-transplantation of treated and untreated cells is performed in the same host. CD45.1 mouse OT-I T cells restricted to ovalbumin (OVA) peptide are activated and transplanted with exogenous mitochondria, but CD45.1.2 OT-I T cells are not transplanted. CD45.1 treated OT-I and CD45.1.2 untreated OT-I are co-transferred into CD45.2 C57 / B6 mice, followed by infection with Listeria-OVA. The treatment groups are compared to the induced immune response of OT-I T cells not transplanted with mitochondria and competing for limited survival signals in the same host.

[0222] means (i) Mouse CD8 + T cell isolation was performed using EasySep™ Mouse CD8 + The T cell isolation kit (StemCell, Cat. #19853) is followed. T cell activation and proliferation is performed by using CD3 / CD28 Dynabeads (Gibco, Cat. #11456.D) in a 1:1 ratio and recombinant IL-2 (50 U / ml). CD8 + T cells are plated at 0.5 million cells / mL and split when the cells reach a confluency of 2 million cells / mL or when the medium turns yellow. (ii) Mitochondrial isolation from OT-I mice Mitochondria are isolated from skeletal muscle as described above in Example 1a. The isolated mitochondria are suspended in homogenization buffer of Example 1a and kept on ice until use. The amount of mitochondria is measured using Qubit™ Protein Assay Kit using Qubit™ Fluorometer (ThermoFisher Scientific / Invitrogen) according to the manufacturer's instructions in preparation for various dose administration. Mitochondrial dosage is estimated in terms of protein content expressed in μg. (iii) Mouse CD8 + T cells are transferred 7 days after activation as described above in Example 3. (iv) Mice (CD45.2) were injected with 10,000 OT-I CD8 + T cells (CD45.1), and 10,000 naïve OT-I CD8 + T cells (CD45.1.2). Mice are then infected with 2,000 cfu of Listeria-OVA. CD8 + T cell persistence and memory differentiation will be assessed over time in the blood (days 7, 14, 21) and organs (spleen, LN on day 21) of the animals. Staining from blood or processed organs: LIVE / DEAD Fixable dye Aqua Dead (ThermoFisher, L34957), anti-mouse CD8α Pe / Texas Red (Abcam ab25294), anti-mouse CD45.1 BV650 (BD 563754), anti-mouse CD45.2 BV421 (BD 562895), anti-mouse KLRG1 PE-Cy7 (BioLegend 138415), anti-mouse CD127 Pe (BioLegend 121111), anti-mouse CD44 APC-Cy7 (BD 560568), anti-mouse CD62L PerCP / Cyanine5.5 (BioLegend 104431). The elicited immune response of OT-I T cells against Listeria-OVA is classified as short-lived effector cells (SLEC) (KLRG1+ CD127- and / or CD44+ CD62L-) and memory progenitor cells (MPEC) (KLRG1- CD127+ and / or CD44+ CD62L+).

[0223] result Enhancement of the ability of transplanted cells to compete for limited survival signals OT-I T cells transplanted with exogenous mitochondria are better able to compete for limited survival signals following acute infection, resulting in an enhanced proportion of treated T cells circulating in the blood and lymphoid organs compared to untreated T cells.

[0224] Example 10. Mitochondrial import increases the persistence of CAR-T cells in vivo Bulk CD8 T cells from healthy donors are transferred with exogenous mitochondria and cultured over time to select for central and effector memory T cells. CD8 T cells from healthy donors are transduced to express an anti-CD19 CAR-T construct (anti-CD19scFv-FLAG-CD28-CD3ζ, Promab). The induced immune response of CAR-T cells treated or not with mitochondria is evaluated in a mouse xenograft model of B cell lymphoma.

[0225] means (i) CAR-T cell culture is performed as described in Example 2. (ii) Mitochondria Isolation Mitochondria are isolated from human cardiac fibroblasts (HCF) according to the procedure described in Example 1b. (iii) Quantification of Isolated Mitochondria Mitochondrial dosage is estimated in terms of protein content expressed in μg according to the procedure described in Example 1b. (iv) CAR-T cell transplantation according to the procedure of Example 3. Mitochondria are transplanted into the CAR-T cells in an amount of 30 μg or 100 μg.

[0226] Lymphoma Models Nine-week-old female NOD / SCID mice (non-obese diabetic, T-cell, macrophage and NK cell deficient, Taconic, Denmark) were transfected with human Burkitt lymphoma CD19 + Raji cells (2.5×10 6 100 cells / mouse) were injected subcutaneously (sc). 3 Once tumors reach a size of 10 mm, animals are randomized into treatment groups, with 5-8 mice per group with equal tumor size selected for treatment. Animals are cultured at 10 7 Mock-transduced T cells, or anti-CD19 CAR-T cells, or mitochondrial-enhanced anti-CD19 + They received IV injections of CAR-T cells. Tumor size is measured in two dimensions using a caliper-like device. Individual tumor volume (V) is calculated according to the formula V = 0.56 x (length + width).2 If the tumor volume is 1,500 mm 3 Once the human end point of 10 days has been reached, animals are sacrificed by cervical dislocation. Kaplan-Meier survival plots are generated using the software program PRISM (GraphPad) and survival curves are compared using the log-rank (Mantel-Cox) test.

[0227] Leukemia model Eight-week-old male NSG (NOD / SCID gamma mice, deficient in T, B, and NK cells) mice purchased from Jackson Laboratories are housed in a vivarium in a sterile cage. Raji / Luc-GFP cells (10 6 ) is injected intravenously via the lateral tail vein using an insulin syringe (designated as day 0). On day 6, luciferase activity is measured by bioluminescence imaging to assess tumor burden. On day 7, 10 7 Mock-transduced T cells, anti-CD19 CAR-T cells, or mitochondrial-enhanced anti-CD19 CAR-T cells are prepared in 100 µL of PBS and injected intravenously using an insulin syringe. Tumor progression is monitored by bioluminescence imaging using an IVIS imaging system. On day 60, surviving mice are euthanized and spleen and bone marrow cells are harvested and resuspended in a total volume of 2 mL of flow cytometry (FACS) buffer (PBS supplemented with 2% FCS). Two hundred microliters of the cell suspension are then labeled with anti-human CD3 PE and anti-human CD45 APC antibodies and analyzed by flow cytometry to determine the percentage of human T cells.

[0228] Compared to non-enhanced or control CAR-T cells, CAR-T cells enhanced with exogenous mitochondria exhibit greater anti-tumor activity (longer median survival) in treated mice.

[0229] Example 11. Effect of mitochondrial transfer on Treg survival and selection in vitro CD4 isolated from healthy donors and subsequently cultured + The proportion of Tregs will be assessed by flow cytometry over time following exogenous mitochondria transfer into mixed populations of T cells.

[0230] means (i) CD4 + Isolation, activation, and culture of CD4 T cells + T cells are isolated from buffy coats of healthy donors. Peripheral blood mononuclear cells (PBMCs) are harvested by density gradient centrifugation using Ficoll Paque plus (cytiva, 17144002) according to the manufacturer's instructions. EasySep™ Human CD4 + Human CD4 T Cell Isolation Kit (Stemcell, 117952) and “The Big Easy” EasySep™ Magnet (Stemcell, 18001) were used to isolate human CD4 T Cells from PBMCs. + Harvest T cells. Isolated CD4 + T cells are activated with Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher, 111.32D) in a 1:1 ratio in the presence of 100 U / ml of recombinant human IL-2 (Peprotech, 200-02). + T cells are cultured in RPMI 1640 medium GlutaMAX™ Supplement (ThermoFisher, 61870010) supplemented with 1% L-glutamine (ThermomFisher, 25030024), 1% penicillin-streptomycin (10,000 U / ml, Gibco, 15140122), 1% non-essential amino acids (NEAA, ThermoFisher, 11140050), 1% sodium pyruvate (ThermoFisher, 11360070), 10% fetal bovine serum, and 0.1% 2β-mercaptoethanol (Gibco, 31350-010). + T cells are plated at 0.5 million cells / ml and split when the cells reach a confluency of 2 million cells / ml or when the medium turns yellow. (ii) CD4 + T cell transplantation CD4 + T cells were treated with CD4 + T cells are co-transplanted with various doses of mitochondria. (iii) CD4 + T cell staining after transplantation. From 1 to 20 days, CD4 + T cells are stained and analyzed by flow cytometry at various time points. Staining is performed on ice with anti-human CD45RA APC (BioLegend, 304112), anti-human CD45RO PB (BioLegend, 304223), anti-human CD25 FITC (BioLegend, 302604), and anti-human CD127Pe (BioLegend, 351304) according to the manufacturer's instructions. Depending on the surface expression of the mentioned markers, CD4 + T cells are classified as naïve (CD25-, CD127+, CD45RA+, CD45RO-), Treg (CD25+, CD127-, CD45RA+, CD45RO-), central memory (CD25+, CD127+, CD45RA-, CD45RO+), effector memory (CD25-, CD127+, CD45RA-, CD45RO+), or effector (CD25+, CD127-, CD45RA+ / -, CD45RO+ / -). The different subsets were analyzed in control and exogenously mitochondrially transplanted CD4 + In addition, the levels of FOXP3 in the Treg population will be assessed after mitochondrial transfer using the True-Nuclear Human Treg Flow Kit (BioLegend, 320027) according to the manufacturer's instructions.

[0231] result Transplantation of exogenous mitochondria is a novel method to transfect CD4 + Facilitates Treg selection from a mixed population of T cells Upon mitochondrial transplantation, Tregs were expressed as CD4 T cells that were not treated with exogenous mitochondria. +This selection method can be used to select CD4+ T cells for adoptive cell therapy to treat autoimmune diseases. + The proportion of Tregs from a mixed population of T cells can be increased.

[0232] Incorporation by Reference The entire disclosures of all patent and non-patent publications cited herein are each incorporated by reference in their entirety for all purposes.

[0233] Other embodiments The above disclosure may encompass multiple separate inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments as disclosed and illustrated herein are not to be considered in a limiting sense, as numerous variations are possible. The subject matter of the invention includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically point out the particular combinations and subcombinations that are considered novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and / or properties may be claimed in this application, in an application claiming priority from this application, or in a related application. Such claims are also considered to be within the subject matter of the invention of the present disclosure, whether directed to different inventions or to the same invention, and whether broader, narrower, equal, or different in scope as compared to the original claims.

Claims

1. Use of exogenous mitochondria, A. (i) (a) producing modified human immune cells from human immune cells, including memory immune cells or memory-like cells, and (ii) enhancing the survival rate of said memory immune cells or said memory-like cells and / or facilitating the selection of said memory immune cells or said memory-like cells, said use comprising: exposing said human immune cells to said exogenous mitochondria such that said exogenous mitochondria are delivered to said human immune cells, thereby generating said modified human immune cells, including modified memory immune cells or modified memory-like cells; the modified memory immune cells or the modified memory-like cells have increased viability compared to the memory immune cells or the memory-like cells before exposure, and / or the modified memory immune cells or the modified memory-like cells are selectively enhanced compared to the memory immune cells or the memory-like cells prior to exposure; (b) producing modified human immune cells from human immune cells, including engineered immune cells that express chimeric antigen receptor ("CAR") and / or artificial T-cell receptor ("TCR") subunits; and (ii) enhancing the survival rate of said engineered immune cells that express chimeric antigen receptor ("CAR") and / or artificial T-cell receptor ("TCR") subunits and / or facilitating the selection of said engineered immune cells that express chimeric antigen receptor ("CAR") and / or artificial T-cell receptor ("TCR") subunits, wherein said use comprises: exposing said human immune cells to said exogenous mitochondria such that said exogenous mitochondria are delivered to said human immune cells, thereby generating said modified human immune cells, including modified and engineered immune cells; the modified engineered immune cells have increased viability compared to the engineered immune cells before exposure, and / or the modified engineered immune cells are selectively enhanced compared to the engineered immune cells prior to exposure; (c) producing modified human immune cells from human immune cells, including tumor infiltrating lymphocytes (TILs); and (ii) enhancing the survival rate of said tumor infiltrating lymphocytes (TILs) and / or promoting the selection of said tumor infiltrating lymphocytes (TILs), said use comprising: exposing the human immune cells to the exogenous mitochondria such that the exogenous mitochondria are delivered to the human immune cells, thereby generating the modified human immune cells, including modified tumor-infiltrating lymphocytes (TILs); the modified tumor infiltrating lymphocytes (TILs) have increased survival compared to the tumor infiltrating lymphocytes (TILs) before exposure, and / or the modified tumor infiltrating lymphocytes (TILs) are selectively enhanced compared to the tumor infiltrating lymphocytes (TILs) prior to exposure; or (d) producing modified human immune cells from human immune cells, including memory immune cells; and (ii) promoting differentiation of said human immune cells, including memory immune cells, and / or promoting selection of said memory immune cells, wherein said use comprises: exposing the human immune cells to the exogenous mitochondria such that the exogenous mitochondria are delivered to the human immune cells, thereby generating the modified human immune cells, including modified memory immune cells; the modified memory immune cells have a greater degree of differentiation compared to the memory immune cells prior to exposure; and the modified memory immune cells are selectively enhanced compared to the memory immune cells prior to exposure; or B. (i) producing modified tumor infiltrating lymphocytes (TILs), including memory-like tumor infiltrating lymphocytes; and (ii) enhancing the survival rate of said tumor infiltrating lymphocytes (TILs), including memory-like tumor infiltrating lymphocytes, and / or promoting the selection of said tumor infiltrating lymphocytes (TILs), including memory-like tumor infiltrating lymphocytes, wherein said use comprises: exposing the tumor infiltrating lymphocytes (TILs) to the exogenous mitochondria such that the exogenous mitochondria are delivered to the tumor infiltrating lymphocytes (TILs), thereby generating modified tumor infiltrating lymphocytes (TILs); the modified memory-like tumor-infiltrating lymphocytes have increased survival compared to said memory-like tumor-infiltrating lymphocytes prior to exposure, and / or the modified memory-like tumor-infiltrating lymphocytes are selectively enhanced compared to the memory-like tumor-infiltrating lymphocytes before exposure; The use, wherein the modified human immune cells, the human immune cells, the memory immune cells or memory-like cells, the engineered immune cells, the TILs, the modified TILs, and the memory-like TILs are CD8 positive.

2. The use of claim 1, wherein (i) the memory immune cells are effector memory CD8 T cells, central memory CD8 T cells, or a combination thereof, or (ii) the memory immune cells are tissue-resident memory cells (Trm cells), stem cell-like memory cells, CD8 memory-like cells, or persistent or memory-like tumor-infiltrating lymphocytes (TILs).

3. The use described in claim 1, wherein the human immune cells are memory T cells.

4. The use described in claim 1, wherein the human immune cells are naive T cells, effector T cells, memory T cells, or memory-like T cells, or a combination of two or more thereof.

5. The use of claim 1, wherein the human immune cells are circulating CD8 T cells, tumor-infiltrating lymphocytes (TILs), naive CD8 T cells, effector CD8 T cells, memory CD8 T cells, or memory-like CD8 T cells, or a combination of two or more thereof.

6. The use described in claim 1, wherein the human immune cells are gamma-delta T cells (γδ T cells), alpha-beta T cells (αβ T cells), or a combination of two or more thereof.

7. The use described in claim 1, wherein the human immune cells are T cells circulating in the blood and / or tumor infiltrating lymphocytes (TILs).

8. The use described in claim 1, wherein the human immune cells are tumor-infiltrating lymphocytes (TILs) and the memory cells are memory-like TILs.

9. Activating said human immune cells in vitro in a cell-free medium comprising an activating receptor agonist antibody capable of driving adaptive cell activation, comprising: activating, wherein the exposing comprises exposing the human immune cells to a pharmaceutical composition comprising the exogenous mitochondria for at least 3 days, and the exogenous mitochondria are isolated viable mitochondria. The use according to claim 1, comprising:

10. The use described in claim 9, wherein the human immune cells are exposed to the pharmaceutical composition for at least 5 days.

11. Activating said human immune cells in vitro in a cell-free medium containing coated CD3 / CD28 beads in the presence of a recombinant interleukin, comprising: activating, wherein said exposing comprises exposing said human immune cells to a pharmaceutical composition comprising said exogenous mitochondria for at least 3 days, wherein said exogenous mitochondria are isolated viable mitochondria, and optionally said recombinant interleukin comprises IL-2, and optionally wherein exposing said human immune cells to said pharmaceutical composition is for at least 5 days. The use according to claim 1, comprising:

12. The use of claim 1, wherein (i) the exogenous mitochondria are genetically engineered mitochondria, mitochondria encapsulated in liposomes, or mitochondria bound to a specific drug, or a combination of two or more thereof, or (ii) exposing the human immune cells to exogenous mitochondria comprises exposing the human immune cells to 0.0001 ng to 2.5 ng of exogenous mitochondria.

13. A method for promoting (i) memory differentiation of human immune cells, (ii) memory selection of said human immune cells, or (iii) a combination thereof, comprising: (a) activating said human immune cells in vitro in a cell-free medium containing a specific activating receptor agonist antibody capable of driving adaptive cell activation; (b) exposing said human immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days; or (a) activating said human immune cells in vitro in a cell-free medium containing coated CD3 / CD28 beads in the presence of recombinant interleukin; (b) exposing the human immune cells to a pharmaceutical composition comprising isolated viable mitochondria for at least 3 days; Optionally, said recombinant interleukin comprises IL-2; The method, wherein the human immune cells are CD8 positive.

14. A method for differentiating naive T cells, comprising a step of treating the naive T cells with isolated viable mitochondria, wherein the naive T cells are CD8 positive.

15. Modified human immune cells or modified tumor infiltrating lymphocytes (TILs) produced by the use described in claim 1.

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