Mitochondrial transfer and methods related thereto

Abstract

The present disclosure is directed to the inhibition of tunneling nanotube-driven mitochondrial transfer that promotes tumor mutation, progression and drug resistance. In addition, method to "hi-jack" tunneling nanotube transfer mechanisms for the delivery of cancer therapies is also disclosed.

Description

[0001] DESCRIPTION

[0002] MITOCHONDRIAL TRANSFER AND METHODS RELATED THERETO PRIORITY CLAIM

[0003] This application claims benefit if priority to U.S. Provisional Application Serial No.

[0004] 63 / 755,798, filed February 7, 2025, the entire contents of which are hereby incorporated by reference.

[0005] BACKGROUND

[0006] 1. Field of the Disclosure

[0007] The present disclosure relates generally to the fields cell biology, immunology and oncology. More particularly, the disclosure relates to mitochondrial tunneling nanotubes and their use in treating cancer, including circumventing tumor resistance and T cell dysfunction.

[0008] 2. Background

[0009] The mitochondrion (MT) is a subcellular organelle that participates in critical physiological processes, such as energy production, calcium signaling, and programmed cell death. Mitochondria have their own 16.6Kb genome (16.3K for mouse) of circular DNA (mtDNA) with high mutation rate. Each somatic cell contains 102— 104copies of MT genomes, with a subset of mutations reaching high levels of heteroplasmy due to cytoplasmic inheritance or random drift. Recent studies used the single nucleotide variations (SNVs) in the mtDNA as the natural genetic barcodes to trace lineage relationship and clonal dynamics, providing insights about cancer cell evolution on the single cell level.

[0010] A growing body of literature has reported that mitochondria are not only inherited through lineage, but also laterally shared between different mammalian cells primarily through tunneling nanotubes (TNT). This phenomenon was first observed during coculture of mitochondria-deprived A549 lung cancer cell line with mesenchymal stem cells, where the latter transferred mitochondria to the former to restore their aerobic respiratory functions. Follow-up studies further demonstrated that this intercellular mitochondrial transfer occurs among diverse mammalian cell types in vitro and in vivo. Recent work examined the tunneling nanotube-based communication between cancer and immune cells, and reported unidirectional mitochondrial trafficking from T cells to cancer cells. As mitochondria provide both energy and essential metabolites for T cell activation, this raises issues relating mechanisms of T cell dysfunction and possible approaches to modulate this phenomenon.

[0011] 1

[0012] 4918-0430-5290, V. 1SUMMARY

[0013] Thus, in accordance with the present disclosure, a method of inhibiting mitochondrial transfer comprising between a cell, such as a T cell, and a cancer cell comprising contacting a cancer cell with an agent that inhibits CD38 expression. The agent may be an shRNA, an siRNA or an mRNA. The T cell may be a Treg cell, cytotoxic T cell, a helper T cell, an NK T cell, a yS T cell, a CAR-T cell, or a mucosal associated invariant T cell. The cancer cell may be a lung cancer cell, a gastric cancer cell, a skin cancer cell, a bladder cancer cell, a colorectal cancer cell, a breast cancer cell, a brain cancer cell, an ovarian cancer cell, a pancreatic cancer cell, or may be a primary cancer cell, a metastatic cancer cell or a drug -resistant cancer cell.

[0014] Also provided is a method of delivering an agent to a cancer cell comprising (a) providing a cell, such as a T cell, that contains an agent; and (b) culturing said cell / T cell in the presence of a cancer cell. The agent may be an RNA, a DNA, a protein, a small molecule or a metabolite. The T cell may be a Treg cell, cytotoxic T cell, a helper T cell, an NK T cell, a yS T cell, a CAR-T cell, or a mucosal associated invariant T cell. The cancer cell may be a lung cancer cell, a gastric cancer cell, a skin cancer cell, a bladder cancer cell, a colorectal cancer cell, a breast cancer cell, a brain cancer cell, an ovarian cancer cell, or a pancreatic cancer cell, and may be a primary cancer cell, a metastatic cancer cell or a drug-resistant cancer cell.

[0015] In another embodiment, there is provided a method of treating cancer in a subject comprising (a) providing a cell, such as a T cell, that contains an anti-cancer agent; and (b) delivering said cell / T cell to said subject. The T cell may be obtained from said subject prior to step (a). The anti-cancer agent may be an RNA, such as an shRNA or an siRNA or an mRNA encoding a viral or cancer antigen or an MHC molecule, a DNA, a protein, such as a viral or cancer antigen or an MIIC molecule, a small molecule, a metabolite, or an agent that targets an oncogene. The T cell may be a Treg cell, cytotoxic T cell, a helper T cell, an NK T cell, a yS T cell, a CAR-T cell, or a mucosal associated invariant T cell. The cancer cell may be a lung cancer cell, a gastric cancer cell, a skin cancer cell, a bladder cancer cell, a colorectal cancer cell, a breast cancer cell, a brain cancer cell, an ovarian cancer cell, or a pancreatic cancer cell, or may be a primary cancer cell, a metastatic cancer cell or a drug-resistant cancer cell.

[0016] The subject may have had or may be receiving a distinct anti-cancer therapy, such as chemotherapy, radiation therapy, or immunotherapy. Steps (a) and (b) may be repeated 1, 2, 3, 4, 5 or more times. The method may comprise delivery of a checkpoint blockade reagent, such as shRNA against PD-L1, or delivery of an immune-boost reagent, such as an mRNA for an allogeneic MHC allele that alters cancer antigen presentation landscape.

[0017] 2

[0018] 4918-0430-5290, V. 1The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0019] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0020] 3

[0021] 4918-0430-5290, V. 1BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] FIG. 1. UMAP plot for the additional 34 single cell RNA-seq samples from cancer patients. For each sample, T cell mitochondrial fraction (Fr.T-Mito) was estimated using MERCI. Pearson’s correlation coefficient between this fraction and the UMAP coordinates was used to order the samples in the figure. Cancer cells within top 50% rank of Fr.T-Mito in the sample were labeled with red color. cor(.): Pearson’s correlation. Title of the sample indicated the cancer type and sample ID, with abbreviations listed at the end of the manuscript.

[0025] FIG.2. Flow sorting quantification of mitochondria transfer from primary T cells to A549 lung cancer cells after 24h coculture. MG labels cancer cell MT; MDR labels T cell MT.

[0026] FIG.3. Heatmap showing the relative fold change (y-axis) after treating A549 cells with shRNA targeting selected markers (x-axis). KIF1 A, CD38 and TRAK2 single KD saw the largest reductions.

[0027] FIG. 4. Confocal imaging showing. Figure shows reduction of nanotube formation after CD38 KD (left) and quantification of nanotubes in WT and KD A549 cells (right).

[0028] FIG. 5. Flow-sorting quantification (same setup as FIG. 2). Figure shows 10.9% of CD38 KD A549 cancer cells received MT from T cells after coculture.

[0029] FIG. 6. Diagram showing the experimental design to test shRNA transfer from T cells to cancer cells. This process permits MT transfer as a potential way to deliver reagents to cancer cells.

[0030] FIG. 7. shRNA sequencing confirmed the existence of shRNA transfer after coculturing of shRNA-transfected primary T cells with WT A549 cancer cells. Labeling: MC WT: monocultured wild-type A549 cells; CC WT: A549 cocultured with WT T cells; CC KD: A549 cocultured with shRNA transfected primary T cells, y-axis: anti-CD38 shRNA expression level.

[0031] 4

[0032] 4918-0430-5290, V. 1FIG.8. Flow sorting quantification of the percentage of MT acquisition. A549 cells were cocultured with shRNA transfected primary T cells, then after removal of these T cells they were cocultured again with WT T cells with fluorescently labeled MT (right group). These cells exhibited significantly reduced T cell MT hijack compared to WT cells (left group).

[0033] FIG. 9. Growth curves showing A549 cells under different treatment. WT, WT_KRAS, WT_Myc are A549 cells directly transfected with vehicle (empty) plasmid, KRAS-shRNA or Myc-shRNA. KRAS, Myc groups are A549 cells cocultured with T cells transfected with KRAS or Myc shRNAs. KRAS group is significantly lower than WT in Day 3.

[0034] FIG. 10. Transcript abundance measure in different groups of AAVS1 shRNA (typo in the figure). WT_WT: WT A549 cells treated with WT T cells; WT_AAVS: WT A549 cells treated with T cells with AAVS1 shRNA; CD38_WT: CD38 KD A549 cells treated with WT T cells; CD38_AAVS1: CD38 KD A549 cells treated with AAVS1 shRNA transfected T cells, y-axis measures the AAVS1 transcripts in the A549 cells in each group.

[0035] 5

[0036] 4918-0430-5290, V. 1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0037] The inventors have pioneered the genomic investigation of cancer-initiated T cell mitochondria hijack by developing a novel computational method, MERCI (Zhang et al., 2023). In brief, MERCI detects T cell specific mtDNA mutations in the cancer cells from single cell RNA-seq data to report a MT transfer event in human cancer samples. They observed MT transfer occurred in approximately 61% of the 61 solid tumor samples we analyzed, covering lung, gastric, skin, pancreatic, bladder, colorectal, and breast cancers (FIG. 1). Here, they describe further work exploring the nature of TNT for use both in preventing the interaction of T cells and cancer cells that result in MT transfer to cancer cell that further the oncogenic process and disease. In addition, they have tested their hypothesis that TNT can actually be converted into a therapeutic approach where T cells are “armed” with therapeutic agents that can be transferred via TNT to cancer cells, permitting new and highly effective treatments.

[0038] These and other aspects of the disclosure are described in detail below.

[0039] I. Mitochondria and Mitochondrial Transfer

[0040] A. Mitochondria

[0041] A mitochondrion (pl. mitochondria) is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is used throughout the cell as a source of chemical energy. They were discovered by Albert von Kolliker in 1857 in the voluntary muscles of insects. Meaning a thread-like granule, the term mitochondrion was coined by Carl Benda in 1898. The mitochondrion is popularly nicknamed the "powerhouse of the cell", a phrase popularized by Philip Siekevitz in a 1957 Scientific American article of the same name.

[0042] Some cells in some multicellular organisms lack mitochondria (for example, mature mammalian red blood cells). The multicellular animal Henneguya salminicola is known to have retained mitochondrion-related organelles despite a complete loss of their mitochondrial genome. A large number of unicellular organisms, such as microsporidia, parabasalids and diplomonads, have reduced or transformed their mitochondria into other structures, e.g., hydrogenosomes and mitosomes. The oxymonads Monocercomonoides, Streblomastix, and Blattamonas have completely lost their mitochondria.

[0043] Mitochondria are commonly between 0.75 and 3 pm2 in cross section but vary considerably in size and structure. Unless specifically stained, they are not visible. In addition

[0044] 6

[0045] 4918-0430-5290, V. 1to supplying cellular energy, mitochondria are involved in other tasks, such as signaling, cellular differentiation, and cell death, as well as maintaining control of the cell cycle and cell growth. Mitochondrial biogenesis is in turn temporally coordinated with these cellular processes. Mitochondria have been implicated in several human disorders and conditions, such as mitochondrial diseases, cardiac dysfunction, heart failure and autism.

[0046] The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. A mature red blood cell has no mitochondria, whereas a liver cell can have more than 2000. The mitochondrion is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, intermembrane space, inner membrane, cristae, and matrix.

[0047] Although most of a eukaryotic cell's DNA is contained in the cell nucleus, the mitochondrion has its own genome ("mitogenome") that is substantially similar to bacterial genomes. This finding has led to general acceptance of the endosymbiotic hypothesis, which is that free-living prokaryotic ancestors of modern mitochondria permanently fused with eukaryotic cells in the distant past, evolving such that modern animals, plants, fungi, and other eukaryotes are able to respire to generate cellular energy.

[0048] Mitochondria may have a number of different shapes. A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes have different properties. Because of this double-membraned organization, there are five distinct parts to a mitochondrion: the outer mitochondrial membrane; the intermembrane space (the space between the outer and inner membranes); the inner mitochondrial membrane; the cristae space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane), which is a fluid. Mitochondria have folding to increase surface area, which in turn increases ATP (adenosine triphosphate) production. Mitochondria stripped of their outer membrane are called mitoplasts.

[0049] The most prominent roles of mitochondria are to produce the energy currency of the cell, ATP (i.e., phosphorylation of ADP), through respiration and to regulate cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs cycle, and oxidative phosphorylation. However, the mitochondrion has many other functions in addition to the production of ATP.

[0050] B. Mitochondrial Tunneling Nanotubes

[0051] A tunneling nanotube (TNT) or membrane nanotube is a term that has been applied to cytoskeletal protrusions that extend from the plasma membrane which enable different animal 7

[0052] 4918-0430-5290, V. 1cells to connect over long distances, sometimes over 100 (im between certain types of cells. Tunneling nanotubes that are less than 0.7 micrometers in diameter, have an actin structure and carry portions of plasma membrane between cells in both directions. Larger TNTs (>0.7 pm) contain an actin structure with microtubules and / or intermediate filaments and can cany components such as vesicles and organelles between cells, including whole mitochondria. The diameter of TNTs ranges from 0.05 to 1.5 pm and they can reach lengths of several cell diameters. There have been two types of observed TNTs: open ended and closed ended. Open ended TNTs connect the cytoplasm of two cells. Closed ended TNTs do not have continuous cytoplasm as there is a gap junction cap that only allows small molecules and ions to flow between cells. These structures have shown involvement in cell-to-cell communication, transfer of nucleic acids such as mRNA and miRNA between cells in culture or in a tissue, and the spread of pathogens or toxins such as HIV and prions. TNTs have observed lifetimes ranging from a few minutes up to several hours, and several proteins have been implicated in their formation and inhibition, including many that interact with Arp2 / 3.

[0053] As mentioned above, tunneling nanotubes have been implicated as one mechanism by which whole mitochondria can be transferred from cell to cell. One recent study has reported that cancer cells can hijack the mitochondria from immune cells via physical tunneling nanotubes (Saha et al., 2022). Mitochondrial DNA damage appears to be the main trigger for the formation of TNTs in order to traffic entire mitochondria, though the exact threshold of damage necessary to induce TNT formation is yet unknown. The maximum speed of mitochondria traveling over TNTs was found to be about 80 nm / s, lower than the measured speed of 100-1400 nm / s of axonal transport of mitochondria; this could be due to the smaller diameter of TNTs inhibiting mitochondrial migration.

[0054] In another study, Ahmad er al., EMBO J. 33(9): 994-1010 (2014) used four lines of mesenchymal stem cells, each expressing a differing phenotype of the Rho-GTPase Mirol. A higher level of Mirol was associated with more efficient mitochondrial transfer via TNTs. Several studies have shown, through the selective blockage of TNT formation, that TNTs are a primary mechanism for the trafficking of whole mitochondria between heterogeneous cells. One use of this phenomenon is in recovery from heart attacks. When cardiac muscle cells are injured by oxygen deprivation, the damaged mitochondria release reactive oxygen species, which trigger nearby mesenchymal stem cells to produce and donate healthy mitochondria to the damaged muscle cells through TNTs.

[0055] 8

[0056] 4918-0430-5290, V. 1C. Cells Capable of Forming TNTs

[0057] Many cell types are able to form TNTs. As has been reported in literature, mesenchymal stem cells can donate mitochondria via TNT to cancer cells under respiratory stress (Liu et al., 2014). Studies have also observed that macrophages / microglia form TNT with neuron cells to allow exchange of cytoplasmic contents to maintain a healthy neuroactivity (Chakraborty et al., 2023). In multiple myeloma, previous studies observed that stromal bone marrow cells form TNTs with the cancer cells to transfer MT to facilitate cancer growth (Marlein et al., 2019). The full landscape of TNT formation in different cell types remain unknown, but the current understanding is that many cancer cells (lung, breast, bone, brain, colon, skin, kidney cancers and multiple myeloma) are capable of generating TNTs to connect with each other, and with other cells, including T cells, such as Treg cells, cytotoxic T cells, helper T cells, NK T cells, yS T cells, CAR-T cells, and mucosal associated invariant T cells. There has been no report that normal somatic cells can connect with T cells via TNT.

[0058] IL Inhibition of TNT Mitochondrial Transfer

[0059] In one embodiment, there is provided method to prevent mitochondrial transfer via tunneling nanotubes. As noted in the literature, mitochondrial transfer between cells, including cancer cells, can lead to tumor growth, metastasis and resistance to therapy. The inventors have confirmed transfer of mitochondria from T cells to cancer cells, but they also determined that knock down of CD38 expression in cancer cells can significantly reduce the density and length of tunneling nanotubes that drive mitochondrial transfer. Thus, the hypothesized that the same mechanism of transfer could also drive the movement of agents from T cells into cancer cells and attempted to repurpose that mechanism to limit the degree of mitochondrial transfer that occurs between T cells and cancer cells.

[0060] CD38 (cluster of differentiation 38), also known as cyclic ADP ribose hydrolase, is a glycoprotein found on the surface of many immune cells (white blood cells), including CD4+, CD8+, B lymphocytes and natural killer cells. CD38 also functions in cell adhesion, signal transduction and calcium signaling. In humans, the CD38 protein is encoded by the CD38 gene which is located on chromosome 4. CD38 is a paralog of CD157, which is also located on chromosome 4 (4pl5) in humans. A representative accession number for human CD38 mRNA is NM_001775.

[0061] CD38 was first identified in 1980 as a surface marker (cluster of differentiation) of thymus cell lymphocytes. In 1992 it was additionally described as a surface marker on B cells, monocytes, and natural killer cells (NK cells). About the same time, CD38 was 9

[0062] 4918-0430-5290, V. 1discovered to be not simply a marker of cell types, but an activator of B cells and T cells. In 1992 the enzymatic activity of CD38 was discovered, having the capacity to synthesize the calcium-releasing second messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP).

[0063] CD38 is most frequently found on plasma B cells, followed by natural killer cells, followed by B cells and T cells, and then followed by a variety of cell types. CD38 has also been used as a prognostic marker in leukemia. Daratumumab (Darzalex) which targets CD38 has been used in treating multiple myeloma. The use of Daratumumab can interfere with preblood transfusion tests, as CD38 is weakly expressed on the surface of erythrocytes. Thus, a screening assay for irregular antibodies against red blood cell antigens or a direct immunoglobulin test can produce false-positive results. This can be sidelined by either pretreatment of the erythrocytes with dithiothreitol (DTT) or by using an anti-CD38 antibody neutralizing agent, e.g. DaraEx.

[0064] The inventors have designed shRNAs targeting CD38 mRNA that significantly reduce CD38 gene expression and reduce MT transfer by over 70%. They were able to demonstrate that shRNA against CD38 can be transferred to cancer cells through T cells with similar effects. They also confirmed the existence of CD38 shRNA in the cancer cells as well significantly reduced MT transfer.

[0065] Nucleic acids (e.g., mRNA, shRNA, DNA) may be introduced to cells using viral delivery systems, such as lentiviral delivery systems, or using lipid nanoparticle-based delivery, such as Lipofectamine 3000. Presence of RNAs acids may be confirmed using a customized protocol to capture non-poly-A RNAs. This is achieved by attaching poly-A to the tail of the target shRNA followed by a standard qPCR protocol. T cells are the sole carrier of shRNA in this context due to its potentially high specificity to target the cancer cells.

[0066] III. TNT Mitochondrial Transfer

[0067] As discussed above, mitochondrial transfer between cells, including cancer cells, can lead to tumor growth, metastasis and resistance to therapy. As such, inhibition of this phenomenon can prove useful in protecting a patient from these pro-oncogenic functions. The inventors, however, also hypothesized that mitochondrial transfer via TNA could be “hijacked” as part of a tailored cancer therapy. As shown herein, they were able to demonstrate the transfer of an shRNA from T cells to cancer cells that greatly impaired mitochondrial transfer. The inventors the recognized that this same operation could deliver agents that either directly

[0068] 10

[0069] 4918-0430-5290, V. 1impede cancer cells or that can indirectly invoke other cancers therapies or make cancer therapies more effective.

[0070] Thus, in one embodiment, the inventors envision employing anti-cancer agents such as tumor specific inhibitory RNAs. such as shRNAs or siRNAs target features in cancer cells, such as oncogenes, anti-apoptotic functions, or checkpoint inhibitors. Alternatively, a “new” feature can be introduced selectively into cancers cells creating a highly specific and readily targetable surface antigens, such as mRNAs or DNAs encoding a viral or cancer antigens, or MHC molecules. Other nucleic acids, proteins, small molecules, metabolites, or other agents that can be produced in a “donor” cell are also envisioned.

[0071] Besides transferring shRNA or siRNA that directly target a cancer-related gene, the inventors also consider the following opportunities permitted with this technology. Transfer of mRNAs for certain genes that will induce a gain-of-function event in the cancer cells is another approach. One example is intact tumor suppressor gene, such as WT TP53, that causes cell cycle arrest. Second, one may transfer an allogeneic MHC allele to the cancer cells. Such allele will present a different landscape of antigens in the tumor proteome that make cancer cells generally ‘foreign’ to the host T cells, potentially inducing massive antigen- specific killing. Third, this approach also permits transfer of oncolytic viral particles, common choices including adenovirus, herpes simplex virus, AAV, and poxviruses (see Lv et al., 2024). The transferred viruses can replicate inside cancer cells and induce strong immune responses against cancer cells (possibly along with transfer of anti-tumor genes like tumor suppressors or inhibitory RNAs) without harming the normal somatic cells.

[0072] IV. Cancer Therapy

[0073] In accordance with the present disclosure, there are provided methods of treating cancers, particularly solid cancers. The methods involve the administration of cells to a subject such at the cells are brought in proximity with a cancer cell / cancer cell environment, thus permitting tunneling nanotube transfer. The administration may be performed multiple times (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a subject as needed. The subject may be an infant, a pediatric patient, a juvenile, a young adult, and adult or a senior. The subject may be a human, male or female, or may be a non-human mammal.

[0074] In certain aspects the cancer is a renal, melanoma, prostate cancer, chronic lymphocytic leukemia, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following 11

[0075] 4918-0430-5290, V. 1histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadcnocarcinoma; papillary cystadcnocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic 12

[0076] 4918-0430-5290, V. 1fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma: fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0077] A. Pharmaceutical Formulations and Routes of Administration Pharmaceutical compositions provided herein comprise an effective amount of one or more therapeutic compositions and, optionally, an additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains a therapeutic nucleic acid construct or a therapeutic engineered cell and one or more excipients will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0078] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except 13

[0079] 4918-0430-5290, V. 1insofar as any conventional earner is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0080] In certain embodiments, the pharmaceutical composition may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. In certain embodiments, pharmaceutical compositions provided herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (sec, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0081] In certain embodiments, the actual dosage amount of a composition administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0082] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the aclivc compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 15 microgram / kg / body weight, about 20 microgram / kg / body weight, about 25 microgram / kg / body weight, about 30 microgram / kg / body weight, about 35 microgram / kg / body weight, about 0.04 milligram / kg / body weight, about 0.05 milligram / kg / body weight, about 0.06 milligram / kg / body weight, about 0.07 milligram / kg / body weight, about 0.08 milligram / kg / body weight, about 0.09 milligram / kg / body weight, about 0.1 milligram / kg / body weight, about 0.2 milligram / kg / body weight, to about 0.5 mg / kg / body weight or more per 14

[0083] 4918-0430-5290, V. 1administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 0.01 mg / kg / body weight to about 0.1 mg / kg / body weight, about 0.04 microgram / kg / body weight to about 0.08 milligram / kg / body weight, etc., can be administered, based on the numbers described above.

[0084] In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0085] In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.

[0086] In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present embodiments. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in preferred embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

[0087] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the 15

[0088] 4918-0430-5290, V. 1preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[0089] The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng / mg protein.

[0090] In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

[0091] B. Combination Therapies

[0092] In order to increase the effectiveness of a cancer therapy of the present disclosure, it may be desirable to combine these agents with other agents effective in the treatment of the disease of interest. As a non-limiting example, the treatment of cancer may be implemented with a cell of the present disclosure along with other anti-cancer agents. An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the cell and the other agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or

[0093] 16

[0094] 4918-0430-5290, V. 1formulations, at the same time, wherein one composition includes the cell of the present disclosure and the other includes the second agent(s).

[0095] Treatment with the cell may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and the anti-cancer peptide or nanoparticle complex are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the anti-cancer peptide or nanoparticle complex would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly where several days (e.g., 2, 3, 4, 5. 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations.

[0096] Various combinations may be employed, where the cell therapy is “A” and the other agent is “B”:

[0097] A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / B / B / B B / A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A A / A / B / A In certain embodiments, administration of the cell therapy and / or other agent(s) to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy.

[0098] Chemotherapy. Cancer therapies also include a variety of combination therapies. In some aspects, a cell therapeutic of the embodiments is administered (or formulated) in conjunction with a chemotherapeutic agent. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor such as a EGFR, VEGFR, AKT, Erbl, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitors. Nonlimiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus.

[0099] 17

[0100] 4918-0430-5290, V. 1everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof.

[0101] Yet further combination chemotherapies include, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mcchlorcthaminc oxide hydrochloride, mclphalan, novcmbichin, phcncstcrinc, prcdnimustinc, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone;

[0102] 18

[0103] 4918-0430-5290, V. 1etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone: mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogeimanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, famcsyl-protcin tansfcrasc inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Gleevac (e.g., from about 400 to about 800 mg / day of Gleevac may be administered to a patient). In certain embodiments, one or more chemotherapeutic may be used in combination with the compositions provided herein.

[0104] Radiotherapy. Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as micro waves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[0105] The terms “contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with

[0106] 19

[0107] 4918-0430-5290, V. 1the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.

[0108] Immunotherapy. Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0109] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with a cell therapy of the present embodiments. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p 155.

[0110] Checkpoint inhibitor therapy is another powerful form of cancer immunotherapy. The therapy targets immune checkpoints, which are key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function. The first anti-cancer drug targeting an immune checkpoint was ipilimumab, a CTLA4 blocker approved in the United States in 2011. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD-1 on an immune cell surface, which inhibits immune cell activity. Among PD-L1 functions is a key regulatory role on T cell activities. It appears that (cancer-mediated) upregulation of PD-L1 on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to either PD-1 orPD-Ll and therefore block the interaction may allow the T-cells to attack the tumor.

[0111] 20

[0112] 4918-0430-5290, V. 1Gene Therapy. In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the therapeutic composition. Viral vectors for the expression of a gene product are well known in the art, and include such eukaryotic expression systems as adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, lentiviruses, poxviruses including vaccinia viruses, and papiloma viruses, including SV40. Alternatively, the administration of expression constructs can be accomplished with lipid-based vectors such as liposomes or DOTAPicholesterol vesicles. All of these methods are well known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).

[0113] Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on target tissues. A variety of proteins are encompassed within the present embodiments, some of which are described below.

[0114] As noted above, the tumor suppressor oncogenes inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation.

[0115] Genes that may be employed as secondary treatment in accordance with the present embodiments include p53, pl 6, Rb, APC, DOC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27 / pl6 fusions, p21 / p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb,fms, trk, ret, gsp, list, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors), MCC and other genes listed in Table IV.

[0116] Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family of proteins and ICE- like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems. The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, Proc. Nat’l. Acad. Sci. USA, 82(21):7439-43, 1985; Cleary el al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.

[0117] Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These 21

[0118] 4918-0430-5290, V. 1different family members have been shown to either possess similar functions to Bcl-2 (e.g., BCIXL, Bclw, Bcls, Mcl-1, Al, Bfl-1 ) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).

[0119] Surgery. Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0120] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs’ surgery). It is further contemplated that the present embodiments may be used in conjunction with removal of superficial cancers, prccanccrs, or incidental amounts of normal tissue.

[0121] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.

[0122] Other agents. It is contemplated that other agents may be used in combination with the compositions provided herein to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abililties of the compositions provided herein by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperprolileralive effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the compositions provided herein to improve the anti-hyerproliferative efficacy of the treatments.

[0123] 22

[0124] 4918-0430-5290, V. 1Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hypeiproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the compositions provided herein to improve the treatment efficacy.

[0125] In certain embodiments, hormonal therapy may also be used in conjunction with the present embodiments or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

[0126] V. Examples

[0127] The following examples arc included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0128] Example 1 - Inhibition of Mitochondrial Transfer

[0129] The inventors performed in vitro experiments by coculturing human primary T cells with human cancer cell lines and repeatedly confirmed mitochondrial transfer from T cells to the cancer cells. They observed that over 40% of the A549 lung cancer cells received T cell mitochondria after 24 hours of coculture (FIG. 2). Similar results were observed for another non-small cell lung cancer cell line, PC9, with over 45% recipients.

[0130] They next performed shRNA interference experiments to selectively knock down (KD) regulators of mitochondria transfer. Relevant to this disclosure, they chose CD38 to be the target. CD38 has been reported to promote mitochondria transfer from bone marrow stem cells to multiple myeloma cells (Marlein et al., 2019). They designed shRNA targeting CD38 mRNA and transfected the plasmid to A549 cancer cells. The inventors detected significantly reduced CD38 gene expression (FIG. 3). CD38 KD significantly reduced the density and length

[0131] 23

[0132] 4918-0430-5290, V. 1of tunneling nanotubes between cells (FIG. 4). Consistently, after coculture of primary T cells with A549 cells treated with anti-CD38 shRNA, they observed that MT transfer was reduced by over 70% (FIG. 5).

[0133] The inventors next examined if the mechanism that allowed T cell MT transfer also allows the targeted transfer of other cellular reagents, such as RNAs. Using this existing system, they tested if shRNA of CD38 can be transferred to the cancer cells through T cells with similar effects with the following experiment (FIG. 6): They transfected primary T cells with CD38-targeting shRNA DNA plasmid, and performed puromycin selection to keep the cells with successful transfection. They cocultured transfected T cells with WT A549 cells, expecting shRNA to be transferred along with mitochondria. Finally, they remove the transfected T cells from coculture; 4) WT primary T cells are added to the culture medium with A549 cells for 3 hours.

[0134] The inventors confirmed the existence of shRNA in the cancer cells after the first coculturc (FIG. 7). This is a critical result, as it conclusively supports the claim that the mechanism, mainly nanotube, that allows the transfer of mitochondria, also allows the transfer of RNA, and possibly other molecules, such as DNA, protein, metabolites or small molecule drugs, from T cells to the cancer cells. The consequence of this transfer can generate a cellular, tissue or physiological effect. In this experiment, the inventors observed significantly reduced MT transfer in the 2ndcoculture (FIG. 8), indicating that T-cell-transferred shRNA KD of CD38 generated a phenotype in recipient cancer cells.

[0135] This system has two unique advantages over traditional approaches. First, it is cancerspecific. Acquisition of T cell mitochondria has never been reported for normal tissue. The inventors have also conducted experiments to show that primary kidney epithelial cells cannot obtain mitochondria from T cells. Second, it is theoretically feasible to target many undruggable oncogenes, for example, c-Myc, TP53, KRAS, etc, with shRNA that do not perturb T cell migration and survival. The inventors have tested this hypothesis using T cells carrying shRNAs against c-Myc and KRAS to deliver to the A549 lung cancer cell line, and confirmed that after 3 -days of coculture, the cancer cells exhibited significantly reduced growth rate (FIG. 9).

[0136] 24

[0137] 4918-0430-5290, V. 1Example 2 - T cell Mitochondria “Hijack”

[0138] Further, to confirm that shRNA transfer from T cells to cancer cells occurs dominantly through TNTs, the inventors conducted the following experiment. In the control group, they used WT A549 cells cocultured with T cells transfected with shRNA targeting AAVS1, a putative non-functional region of the genome. In the treatment group, CD38 KD A549 cells were cocultured with the same T cells. The inventors measured the transfer of AAVS1 shRNA in the tumor cells and observed about 90% reduction in the CD38 KD group (FIG. 10), confirming that shRNA transfer is dominantly through nanotubes.

[0139] This finding opens up the opportunity of a new paradigm of adaptive T cell therapy, i.e., a new method of delivery therapeutic agents to cancer cells. First, one transfects certain reagents into ex vivo expanded T cells, and then infuses these modified cells back into the patients, thus allowing cancer-specific transfer of the reagents. The consequence of this transfer can result in direct tumor killing, or a beneficial modification of the tumor cells. For example, delivery of mRNA (instead of shRNA) can further ‘knock-in’ certain functionalities into the cancer cells that would be useful for anti-cancer killing, such as short mRNAs encoding cancer antigens or molecules that boost cancer antigen presentation and / or increase immune recognition. Feeding cancer cells with mRNAs of allogeneic MHC molecules via TNTs could force cancer cells to present completely different epitopes that could be recognized by specifically designed effector T cells. Alternatively, feeding cancer cells with viral epitopes from common viruses, such as SARS-CoV-2 spike protein, or influenza Ml protein, could significantly boost immune killing of tumor. This approach could be naturally combined with CAR-T therapy, or used as adjuvant for checkpoint blockade therapies. Similar to CAR-T therapy, it could also be repeated administered to increase cancer killing efficiency.

[0140] * * * * * * * * * * * * * * * ^ ^

[0141] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or

[0142] 25

[0143] 4918-0430-5290, V. 1similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0144] 26

[0145] 4918-0430-5290, V. 1VI. References

[0146] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0147] Marlein et al., Cancer Res. 9(9): 2285-2297, 2019.

[0148] Saha el al., Nature Nanotechnology, 17(1): 98-106, 2022.

[0149] Zhang et al., Cancer Cell, 2023.

[0150] Liu et al., Microvasc. Res., 92:10-8, 2014.

[0151] Chakraborty et al., Cell Death Dis., 14(5):329, 2023.

[0152] Lv et al.. Front. Microbiol., 15:1356415, 2024.

[0153] 27

[0154] 4918-0430-5290, V. 1

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting mitochondrial transfer comprising between a T cell and a cancer cell comprising contacting a cancer cell with an agent that inhibits CD38 expression.

2. The method of claim 1, wherein the agent is an shRNA, an siRNA or an niRNA.

3. The method of claim 1 or claim 2, wherein the T cell is a Treg cell, cytotoxic T cell, a helper T cell, an NK T cell, a yS T cell, a CAR-T cell, or a mucosal associated invariant T cell.

4. The method of any one of claims 1-3, wherein the cancer cell is a lung cancer cell, a gastric cancer cell, a skin cancer cell, a bladder cancer cell, a colorectal cancer cell, a breast cancer cell, or a brain cancer cell, an ovarian cancer cell, a pancreatic cancer cell.

5. The method of any one of claims 1-4, wherein the cancer cell is a primary cancer cell, a metastatic cancer cell or a drug-resistant cancer cell.

6. A method of delivering an agent to a cancer cell comprising:(a) providing a T cell that contains an agent: and(b) culturing said T cell in the presence of a cancer cell.

7. The method of claim 6, wherein the agent is an RNA, a DNA, a protein, a small molecule or a metabolite.

8. The method of claim 6 or claim 7, wherein the T cell is a Treg cell, cytotoxic T cell, a helper T cell, an NK T cell, a yS T cell, a CAR-T cell, or a mucosal associated invariant T cell.

9. The method of any one of claims 6-8, wherein the cancer cell is a lung cancer cell, a gastric cancer cell, a skin cancer cell, a bladder cancer cell, a colorectal cancer cell, a breast cancer cell, a brain cancer cell, an ovarian cancer cell, or a pancreatic cancer cell.

10. The method of any one of claims 6-9, wherein the cancer cell is a primary cancer cell, a metastatic cancer cell or a drug-resistant cancer cell.284918-0430-5290, V.

111. A method of treating cancer in a subject comprising:(a) providing a T cell that contains an anti-cancer agent; and(b) delivering said T cell to said subject.

12. The method of claim 11, wherein said T cell is obtained from said subject prior to step (a).

13. The method of claim 11 or claim 12, wherein the anti-cancer agent is an RNA, such as an shRNA or an siRNA or an mRNA encoding a viral or cancer antigen or an MHC molecule, a DNA, a protein, such as a viral or cancer antigen or an MHC molecule, a small molecule, a metabolite, or an agent that targets an oncogene.

14. The method of any one of claims 11-13, wherein the T cell is a Trcg cell, cytotoxic T cell, a helper T cell, an NK T cell, a yS T cell, a CAR-T cell, or a mucosal associated invariant T cell.

15. The method of any one of claims 11-14, wherein the cancer cell is a lung cancer cell, a gastric cancer cell, a skin cancer cell, a bladder cancer cell, a colorectal cancer cell, a breast cancer cell, a brain cancer cell, an ovarian cancer cell, or a pancreatic cancer cell.

16. The method of any one of claims 11-15, wherein the cancer cell is a primary cancer cell, a metastatic cancer cell or a drug-resistant cancer cell.

17. The method of any one of claims 11-16, wherein said subject has had or is receiving a distinct anti-cancer therapy, such as chemotherapy, radiation therapy, or immunotherapy.

18. The method of any one of claims 11-17, wherein steps (a) and (b) are repeated 1, 2, 3, 4, 5 or more times.

19. The method of claim 11, wherein said method comprises delivery of a checkpoint blockade reagent, such as shRNA against PD-L1.294918-0430-5290, V.

120. The method of claim 11, wherein said method comprises delivery of an immune-boost reagent, such as an mRNA encoding an allogeneic MHC allele that alters cancer antigen presentation landscape.304918-0430-5290, V. 1

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