Therapeutic compositions and methods for treating cancer

Abstract

Disclosed herein are enucleated cells comprising an exogenous nucleic acid molecule encoding a. CD40L polypeptide or engineered to express an exogenous CD40L polypeptide. Disclosed are enucleated cells comprising exogenous CD40L polypeptide. Also disclosed are methods of treating subjects comprising administering to the subject one or more enucleated cells disclosed herein.

Description

ATTORNEY DOCKET NO.38394.0028P1 THERAPEUTIC COMPOSITIONS AND METHODS FOR TREATING CANCER CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 677,991, filed on July 31, 2024, U.S. Provisional Patent Application No.63 / 678,023, filed on July 31, 2024, and U.S. Provisional Patent Application No.63 / 741,295, filed on January 2, 2025, each of which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing submitted July 31, 2025 as a text file named “38394.0028P1.xml,” created on July 31, 2025, and having a size of 12,020 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND

[0003] Cancers that arise from ovary, colorectal, liver or bladder tissue are difficult to identify and to treat due to their tendency to form adhesions on, and penetrate into, visceral organs, the peritoneal lining and the omentum. Further complicating treatment, systemic delivery of chemotherapeutic agents or biologicals to treat peritoneal carcinomatosis is challenging based upon their poor penetration into peritoneal cavity. The diffuse spread of metastatic cells within the peritoneal cavity highlights the need for improved locoregional therapeutic delivery options. The advent of immunotherapies that stimulate host immune cells and reverse the immunosuppressive tumor microenvironment (TME) has improved patient survival in many solid malignancies yet has shown mixed efficacy in treating peritoneal carcinomatoses. Indeed, in some clinical trials, immune checkpoint blockade (ICB) monotherapy has resulted in worse survival outcomes. Resistance to ICB in treating some peritoneal carcinomatoses may be a result of the unique immunological environment in the peritoneal cavity, as ICB-sensitive tumors can turn resistant in this unique metastatic niche. The TME in peritoneal carcinomatosis can consist of solid tumors growing on abdominal and peritoneal lining, and the omentum, the fatty tissue that filters peritoneal fluid and is a common site of ovarian cancer metastasis. In addition, malignant ascites, a hallmark of many peritoneal carcinomatoses, presents as a unique fluid TME. The localization and interplay between these forms of TME present intriguing opportunities and challenges when considering immunotherapeutic treatment of metastatic disease in the peritoneal cavity.

[0004] CD40-targeting therapies are designed to jump start anti-cancer immunity by servingATTORNEY DOCKET NO.38394.0028P1 as a molecular adjuvant that activates antigen presenting cells (APCs), thus priming tumor- specific T cells, and reprogramming immunosuppressive macrophages and other CD40- expressing immune cells in the TME. Recruitment of T cells through the use of CD40 immunoadjuvants in combination with ICB has gained traction as a strategy for boosting and prolonging antitumor immunity. While CD40 agonist antibodies (Abs) plus ICB has yielded promising clinical results, their use in treating ovarian cancer and other peritoneal carcinomatoses, such as colorectal, bladder or liver cancer metastases is hindered by their poor PK / PD profile and tendency to elicit peripheral toxicity. Thus, there is a need to develop better delivery systems for CD40 agonists that can effectively induce antitumor immunity and improve patient outcome. BRIEF SUMMARY

[0005] While immune checkpoint blockade (ICB) therapies have expanded the therapeutic arsenal in treating many cancers, many patients do not respond or achieve durable benefits from these therapies. CD40 ligand (CD40L) immunotherapy is an approach to increase the presence of tumor-specific T cells within the tumors, increasing the responsiveness to ICB therapies.

[0006] Disclosed are enucleated cells comprising an exogenous nucleic acid molecule encoding a CD40L polypeptide or engineered to express an exogenous CD40L polypeptide.

[0007] Disclosed are methods of increasing a subject’s responsiveness to immune checkpoint inhibitors comprising administering to the subject one or more enucleated cells disclosed herein.

[0008] Disclosed are methods of increasing T cells at a tumor site in a subject comprising administering to the subject one or more enucleated cells disclosed herein.

[0009] Disclosed are methods of treating a subject having cancer comprising administering to the subject one or more enucleated cells disclosed herein.

[0010] Disclosed are methods of recruiting one or more T cells to a tumor site comprising administering to a subject one or more enucleated cells disclosed herein.

[0011] Disclosed are methods of killing or inducing apoptosis of a cancer cell comprising administering one or more enucleated cells disclosed herein to the cancer cell or a subject comprising the cancer cell.

[0012] Disclosed are methods of inducing B cell proliferation comprising administering one or more enucleated cells disclosed herein to the B cell or a subject comprising the B cell, wherein the B cell proliferates upon exposure to the CD40L expressed by the one or more enucleated cells disclosed herein.ATTORNEY DOCKET NO.38394.0028P1

[0013] Disclosed are methods of activating NF-κB in a cell comprising administering one or more enucleated cells disclosed herein to the cell or a subject comprising the cell, wherein NF- κB is activated upon binding of the CD40L expressed by the one or more enucleated cells disclosed herein to CD40 on the cell.

[0014] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0016] FIGS.1A and 1B shows the interaction between CD40 on APCs and (FIG.1A) CA- CD40L or (FIG.1B) CD40 agonist antibody. CA-CD40L recapitulates native binding promoting CD40 trimerization and recruitment of the CD40 signaling complex (SC). In contrast, CD40 agonist antibodies require Fc binding by bystander cells to assemble the CD40 SC.

[0017] FIGS.2A-2D show FIG 2A) MSCs or CAs, labeled red with LifeAct RFP, were placed in a microfluidic device and timelapse images were acquired as they moved towards an FBS gradient through pores of 1 and 15 μm. Images are 10 hours after the start of migration. Nuclei are labeled blue. FIG.2B) Lung tumors were established in mice following i.v. injection of RFP-labeled EO771 cells. DiD-labeled Cargocytes were then injected and 24 hours later lungs were collected and sectioned for confocal imaging. FIG.2C) MSCs were transduced with a lentivirus expressing mouse CD40L and then enucleated to generate Cargocytes expressing CD40L (CA-CD40L). The level of expression was quantified using a CD40L-specific Ab via flow cytometry. FIG.2D) CD40L-HEK Blue cells, with an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter, were incubated with different numbers of MSC, MSC- CD40L or CA-CD40L overnight. SEAP substrate was added, and absorbance was quantified on a plate reader.

[0018] FIGS.3A-3B show FIG.3A) MSC and MSC-CD40L were plated in 24-well plates and allowed to attach. C57BL / 6 splenocytes were freshly isolated and added to wells containingATTORNEY DOCKET NO.38394.0028P1 MSCs or media only, at a ratio of 16:1 splenocytes:MSCs. Following a 24 hour incubation cells were collected and subjected to immune phenotyping by flow cytometry. DCs were defined as Live, CD45+, F4 / 80-, CD11b-, CD11c+. FIG.3B) MSC and MSC-CD40L were plated in 24-well plates with or without a CD40L blocking Ab. Negatively selected B cells from C57BL / 6 splenocytes were stained with the proliferation dye CellTrace FarRed then added to the cultures at a ratio of 20:1 B cells:MSCs. Four days later cells were collected for flow cytometric analysis to determine the division index: the average number of cell divisions that a cell in the original population has undergone. Data is presented as mean ± SD of triplicates. ****p<0.0001 by one- way ANOVA with Šídák’s multiple comparison test.

[0019] FIGS.4A-4B show FIG.4A) shows that i.p. injected Cargocytes are retained within the peritoneal cavity and dLNs (including mediastinal LNs in the thoracic cavity). Albino C57BL / 6 were injected i.p. with 1e6fLuc labeled Cargocytes (CA-fLuc). Mice were subjected to bioluminescent imaging at multiple time points following injection. FIG.4B) 5e6ID8-fLuc were injected i.p. into albino C57BL / 6 mice and monitored for tumor growth over time by bioluminescent imaging.20 days post injection, some mice were euthanized for immune phenotyping. Peritoneal cavity cells were collected from a peritoneal gavage and stained with specific Abs for flow cytometry. Cell populations were defined as live, CD45+; DCs: CD19-, Ly6G-, CD11cHi, MHCIIHi; Large peritoneal macrophages: CD19-, Ly6G-, CD11b+, F4 / 80Hi, Effector CD8 T cells: CD3+, CD8+, CD62L-, CD44+.

[0020] FIG. 5 shows stable expression of mouse CD40L using lentivirus transduction. Medinno MSCs were transduced with a Lentivirus expressing hTert and a CD4K mutant and placed under drug selection, generating TC-Medinno MSCs. TC-Medinno MSCs were then transduced with a mouse CD40L expressing Lentivirus and placed under drug selection, generating TC-Medinno-CD40L MSCs. These MSCs were enucleated, generating TC-Medinno- CD40L Cargocytes (CAs). MSCs or CAs were stained (or not) with an anti-mouse CD40L Ab and analyzed by flow cytometry.

[0021] FIG. 6 shows human MSC-CD40L induces B cell proliferation.

[0022] FIG. 7 shows both MSCs and cargocytes expressing CD40L have the same functional activity to activate CD40 and downstream NF-κB.

[0023] FIGS.8A and 8B show CD40L expressing D1-MSCs reduce mouse ovarian cancer tumor burden 24 hours post injection.

[0024] FIGS.9A-9C shows the ID8 mouse ovarian cancer model responds to CD40 activation.

[0025] FIG. 10 shows CD40L expressed on TC-Medinno MSCs is functional in activatingATTORNEY DOCKET NO.38394.0028P1 Human B cells.

[0026] FIGS.11A-11E show Cargocytes expressing CD40L (CA-CD40L) activate local but not systemic antigen-presenting cell populations. Serum cytokines were analyzed via a bead- based multiplex ELISA (FIG.11A) and Alanine aminotransferase (ALT) was quantified via ELISA (FIG.11B). Cells were stained with cocktails of fluorescently labeled antibodies in the presence of an Fc block and analyzed on a FACSCelesta. The expression of MCHI or CD86 was quantified as the geometric mean fluorescent intensity on cell populations in the peritoneal cavity (PerC; FIG.11C), omentum (FIG.11D) and spleen (FIG.11E). Cell populations were analyzed in FlowJo and defined in the live, CD45+population as follows. PerC Eosinphils: CD19-, Ly6G-, CD11c-, F4 / 80-, SSCHi. PerC B2 B cells: CD19+, CD11b-. PerC large peritoneal macrophages (LPMs) CD19-, Ly6G-, CD11c-, F4 / 80+, CD11b+, FSCHi. Omentume neutrophils: CD19-, CD11b+, Ly6G+. Omentum B2 B cells: CD19+, CD11b-. PerC conventional dendritic cells 2 (cDC2): CD19-, Ly6G-, CD11c+, CD11b+. Splenic neutrophils: CD19-, CD11b+, Ly6G+. Splenic B cells: CD19+. Splenic cDCs: CD19-, Ly6G-, SSCLo, CD11c+, CD11b+. Data is mean ± SEM of 4 per group. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s Multiple comparison test. *, p<0.05; **, p<0.01.

[0027] FIGS.12A-12C shows that CA-CD40L can synergize with other cell therapies (including CA-IL12) and induce superior immune cell activation. DETAILED DESCRIPTION

[0028] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0029] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0030] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids areATTORNEY DOCKET NO.38394.0028P1 discussed, each and every combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. A. Definitions

[0031] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a CD40L polypeptide" includes a plurality of such polypeptides, reference to "the CD40L polypeptide” is a reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth.

[0033] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0034] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting survival, growth, and / or spread of cancer cells. Treatment may be administered to a subject who does not exhibit signs of aATTORNEY DOCKET NO.38394.0028P1 disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0035] As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.

[0036] “Peptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A peptide is comprised of consecutive amino acids. The term “peptide” encompasses naturally occurring or synthetic molecules. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0037] As used herein, “subject” refers to the target of administration, e.g. an animal. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex. Subject can be used interchangeably with “individual” or “patient”.

[0038] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be consideredATTORNEY DOCKET NO.38394.0028P1 disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0040] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step. B. Enucleated Cells

[0041] In some embodiments, the disclosed enucleated cells can be referred to as cargocytes or cytoplasts.

[0042] Disclosed are enucleated cells engineered to express an exogenous CD40L polypeptide. In some embodiments, the cells can be engineered prior to enucleation. For example, exogenous DNA encoding a CD40L polypeptide can be introduced into the genome of the cell. In some embodiments the introduction of the exogenous DNA encoding CD40LATTORNEY DOCKET NO.38394.0028P1 polypeptide can be performed using a viral vector (e.g., lentivirus, adeno-associated virus) and / or Crispr / Cas9. In some embodiments, once the exogenous DNA has been introduced to the cell, the cells can then express or secrete the CD40L polypeptide, which is maintained after enucleation. Thus, the enucleated cells comprise the CD40L polypeptide.

[0043] In some embodiments, the cells can be engineered after enucleation. Thus, in some embodiments, the exogenous CD40L polypeptide can be introduced to an enucleated cell. For example, the exogenous RNA molecule that encodes a CD40L polypeptide can be transfected into an enucleated cell.

[0044] In some embodiments, the enucleated cells of the present disclosure are obtained or derived from a corresponding nucleated cell (referred to herein as a “parent cell”). The parent cell may be derived from a variety of different cell types, including eukaryotic cells. For example, an enucleated cell may be derived from any nucleated cell, an adult stem cell, a mesenchymal stromal cell (MSC), a natural killer (NK) cell, a macrophage, a myoblast, a neutrophil, endothelial cell, endothelial precursor cell, and / or a fibroblast. In some embodiments, an enucleated cell is derived from a mesenchymal stromal cell. For example, MSCs may be obtained from a number of sources known to one of skill in the art, including ATCC D1 ORL UVA cell line. In some embodiments, the enucleated cell is derived from an inducible pluripotent stem cell (iPSC). In some embodiments, the parent cell is derived from a cell is immortalized using suitable methods. In some embodiments, the enucleated cell includes or retains one or more structural features of the parent cell, including intracellular organelles, one or more tunneling nanotubes, or a combination thereof. In some embodiments, the enucleated cell comprises one or more intracellular organelles for synthesis or secretion of an exogenous polypeptide (e.g., therapeutic agent) in absence of the nucleus. In some embodiments, the one or more intracellular organelles comprise a Golgi apparatus, an endoplasmic reticulum, vesicular transport machinery, or a combination thereof. In some embodiments, the enucleated cell comprises or expresses any one of the therapeutic agents described herein. The enucleated cells described herein can also be purified. The enucleated cells may be purified methods described herein, thus yielding purified enucleated cells.

[0045] In some embodiments, the parent cell can originate from any organism having one or more cells. Non-limiting examples of cells include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell,ATTORNEY DOCKET NO.38394.0028P1 (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g., kelp), a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. In some embodiments, the parent cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell). In some embodiments, the parent cell is a somatic cell. In some embodiments, the parent cell is a stem cell or a progenitor cell. In some embodiments, the parent cell is a mesenchymal stem or progenitor cell. In some embodiments, the parent cell is a hematopoietic stem or progenitor cell. In some embodiments, the parent cell is a muscle cell, a skin cell, a blood cell, or an immune cell. Other non-limiting example of parent cells include, but are not limited to lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell), Natural killer cell, cytokine induced killer (CIK) cells; myeloid cells, such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte / Hypersegmented neutrophil), Monocyte / Macrophage, Red blood cell (Reticulocyte), Mast cell, Thrombocyte / Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell), parathyroid (Parathyroid chief cell, Oxyphil cell), adrenal (Chromaffin cell), pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia), Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph ); cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, Type II pneumocyte), Clara cell, Goblet cell, Dust cell; cells of the circulatory system, including Myocardiocyte, Pericyte; cells of the digestive system, including stomach (Gastric chief cell, Parietal cell), Goblet cell, Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (Hepatocyte, Kupffer cell), Cartilage / bone / muscle; bone cells, including Osteoblast, Osteocyte, Osteoclast, teeth (Cementoblast, Ameloblast); cartilage cells, including Chondroblast, Chondrocyte; skin cells, including Trichocyte, Keratinocyte, Melanocyte (Nevus cell); muscle cells, including Myocyte; urinary system cells, including Podocyte, Juxtaglomerular cell, Intraglomerular mesangial cell / Extraglomerular mesangial cell, Kidney proximal tubule brush border cell, Macula densa cell; reproductive system cells, including Spermatozoon, Sertoli cell, Leydig cell, Ovum; and other cells, including Adipocyte, Fibroblast, Tendon cell, Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte ofATTORNEY DOCKET NO.38394.0028P1 fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell), Wet stratified barrier epithelial cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts), Exocrine secretory epithelial cells, Salivary gland mucous cell (polysaccharide-rich secretion), Salivary gland serous cell (glycoprotein enzyme -rich secretion), Von Ebner's gland cell in tongue (washes taste buds), Mammary gland cell (milk secretion), Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear (wax secretion), Eccrine sweat gland dark cell (glycoprotein secretion), Eccrine sweat gland clear cell (small molecule secretion). Apocrine sweat gland cell (odoriferous secretion, sex -hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland), Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), Prostate gland cell (secretes seminal fluid components), Bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), Gland of Littre cell (mucus secretion), Uterus endometrium cell (carbohydrate secretion), Isolated goblet cell of respiratory and digestive tracts (mucus secretion), Stomach lining mucous cell (mucus secretion), Gastric gland zymogenic cell (pepsinogen secretion), Gastric gland oxyntic cell (hydrochloric acid secretion), Pancreatic acinar cell (bicarbonate and digestive enzyme secretion), Paneth cell of small intestine (lysozyme secretion), Type II pneumocyte of lung (surfactant secretion), Clara cell of lung, Hormone secreting cells, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, Magnocellular neurosecretory cells, Gut and respiratory tract cells, Thyroid gland cells, thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley dig cell of testes, Theca interna cell of ovarian follicle, Corpus luteum cell of ruptured ovarian follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cell (renin secretion), Macula densa cell of kidney, Metabolism and storage cells, Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract), Kidney, Type I pneumocyte (lining air space of lung), Pancreatic duct cell (centroacinar cell), Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.), Duct cell (of seminal vesicle, prostate gland, etc.), Epithelial cells lining closed internal body cavities,ATTORNEY DOCKET NO.38394.0028P1 Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell), Megakaryocyte (platelet precursor), Monocyte, Connective tissue macrophage (various types), Epidermal Langerhans cell, Osteoclast (in bone), Dendritic cell (in lymphoid tissues), Microglial cell (in central nervous system), Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types), Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germ cells, Oogonium / Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte), Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell (in testis), Thymus epithelial cell, Interstitial cells, and Interstitial kidney cells.

[0046] In some embodiments, the parent cell is a eukaryotic or nucleated cell. Non-limiting examples of eukaryotic cells include mammalian (e.g., rodent, non-human primate, or human), non-mammalian animal (e.g., fish, bird, reptile, or amphibian), invertebrate, insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. In some embodiments, the nucleated cell is a primary cell. In some embodiments, the nucleated cell is an immune cell (e.g., a lymphocyte (e.g., a T cell, a B cell), a macrophage, a natural killer cell, a neutrophil, a mast cell, a basophil, a dendritic cell, a monocyte, a myeloid-derived suppressor cell, an eosinophil). In some embodiments, the nucleated cell is a phagocyte or a leukocyte. In some embodiments, the nucleated cell is a stem cell (e.g., an adult stem cell (e.g., a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, mesenchymal stem cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a testicular cell), an embryonic stem cell, an inducible pluripotent stem cell (iPS)). In some embodiments, the nucleated cell is a progenitor cell. In some embodiments, the nucleated cell is from a cell line. In some embodiments, the nucleated cell is a suspension cell. In some embodiments, the nucleated cell is an adherent cell. In some embodiments, the nucleated cell is a cell that has been immortalized by expression of an oncogene. In some embodiments, the nucleated cell is immortalized by the expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, the nucleated cell is a patient or subject derived cell (e.g., an autologous patient-derived cell, or an allogenicATTORNEY DOCKET NO.38394.0028P1 patient-derived cell). In some embodiments, the nucleated cell is transfected with a vector (e.g., a viral vector (e.g., a retrovirus vector (e.g., a lentivirus vector), an adeno-associated virus (AAV) vector, a vesicular virus vector (e.g., vesicular stomatitis virus (VSV) vector), or a hybrid virus vector), a plasmid) before the nucleated cell is enucleated using any of the enucleation techniques described herein and known in the art.

[0047] In some embodiments, the enucleated cell is derived from a cell autologous to the subject. In some embodiments, the enucleated cell is derived from a cell allogenic to the subject.

[0048] In some embodiments, the enucleated cell is derived from an immune cell. In some embodiments, the enucleated cell is derived from a natural killer (NK) cell, a neutrophil, a macrophage, a lymphocyte, a fibroblast, an adult stem cell (e.g., hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a mesenchymal stem cell, a mesenchymal stromal cell, an endothelial stem cell, a neural stem cell, an olfactory adult stem cell, a neural crest stem cell, a skin stem cell, or a testicular cell), a mast cell, a basophil, an eosinophil, an endothelial cell, an endothelial cell precursor cell, or an inducible pluripotent stem cell.

[0049] In some embodiments, the parent cell may be enucleated and engineered for therapeutic use. In some embodiments, a parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton. In some embodiments, the nucleus is then physically extracted from the cell body by high-speed centrifugation in gradients of polysaccharide to generate an enucleated cell. In some embodiments, the polysaccharide is Ficoll for generating Ficoll gradients to generate an enucleated cell. Because enucleate cells and intact nucleated cells sediment to different layers in the Ficoll gradient, enucleated cells may be isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated). The enucleation process can be clinically scalable to process tens of millions of cells by utilizing the methods described herein. In some embodiments, enucleated cells may be used as a disease-homing vehicle to deliver clinically relevant cargos or payloads to treat various diseases or conditions described herein.

[0050] In some embodiments, the enucleated cell can be obtained from a first subset of a plurality of nucleated cells. In some embodiments, the enucleated cells are in a composition, which further comprises a second subset of the plurality of the nucleated cells. In some embodiments, the second subset of the nucleated cells comprises less than about 0.1% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 0.5% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 1% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less thanATTORNEY DOCKET NO.38394.0028P1 about 5% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 10% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 15% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 20% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 25% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 30% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 40% by volume of the composition. In some embodiments, the second subset of the nucleated cells comprises less than about 50% by volume of the composition.

[0051] In some embodiments, the one or more enucleated cells described herein can be cryopreserved, cryohibernated, lyophilized, or a combination thereof. In some embodiments, the cryopreserved purified enucleated cell, following thawing, the one or more enucleated cells is as viable as an otherwise comparable one or more enucleated cells that is not cryopreserved. In some embodiments, the lyophilized one or more enucleated cells is as viable as an otherwise comparable one or more enucleated cells that is not lyophilized. In some embodiments, the cryohibernated one or more enucleated cells is as viable as an otherwise comparable purified enucleated cell that is not cryohibernated.

[0052] In some embodiments, the one or more enucleated cells or the composition comprising the one or more enucleated cells described herein may be cryopreserved (e.g., storing the purified enucleated cell or the composition comprising the one or more enucleated cells at freezing temperature) or cryohibernated (e.g., storing the one or more enucleated cells or the composition comprising the one or more enucleated cells at a temperature that is between the ambient temperature and freezing temperature). The duration of cryopreservation or cryohibernation may be greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the one or more enucleated cells described herein exhibit a viability after cryopreservation or cryohibernation that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been cryopreserved or cryohibernated) after the same period of time of cryopreservation or cryohibernation. In some embodiments, the one or more enucleated cells described herein exhibit the viability following the cryohibernation as measured at 24 hours following the cryohibernation that is equal to or greater than the viabilityATTORNEY DOCKET NO.38394.0028P1 of a comparable purified enucleated cell that is not cryohibernated. In some embodiments, the one or more enucleated cells described herein exhibit the viability following the cryopreservation as measured at 24 hours following the cryopreservation that is equal to or greater than the viability of a comparable one or more enucleated cells that is not cryopreserved. Viability in this context may be measured by Trypan blue dye exclusion as described herein. In some embodiments, the Trypan blue dye exclusion is performed by: (a) centrifuging an aliquot of a plurality of the cell without the nucleus in a suspension to create a cell pellet; (b) resuspending the cell pellet in serum-free medium to produce a serum-free cell suspension; (c) mixing 1 part Trypan blue dye and 1 part of the serum-free cell suspension; (d) counting the plurality of the cells without the nucleus within 3-5 minutes of (c), wherein at least some of the plurality of cells without the nucleus are unstained with the Trypan blue dye, which is indicative of viability. In some embodiments, the viability is measured using Annexin-V cell surface staining. In some embodiments, the viability is measured by expression of the exogenous polypeptide. For example, the viability of a purified enucleated cell can be determined by the expression of the exogenous antibody or single-domain antibody expressed by the purified enucleated cell. In some embodiments, the viability can be measured by expression of cell surface markers of any one of the cell surface markers described herein such as CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2. In some embodiments, the viability can be measured by the cell activity of the purified enucleated cell. In some embodiments, the viability can be measured by the homing capability of the purified enucleated cell as determined by the chemosensing or chemokine homing activity described herein.

[0053] In some embodiments, the one or more enucleated cells or the composition comprising the one or more enucleated cell described herein may be lyophilized. In some embodiments, the one or more enucleated cells described herein exhibit a viability after being reconstituted from lyophilization that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been lyophilized). In some embodiments, the one or more enucleated cells described herein exhibit a viability after being rehydrated from lyophilization that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been dehydrated).

[0054] In some embodiments, the one or more enucleated cells or the composition comprising the one or more enucleated cells described herein may be dehydrated. In some embodiments, the one or more enucleated cells described herein exhibit a viability after beingATTORNEY DOCKET NO.38394.0028P1 rehydrated that is greater than or equal to about 1%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been dehydrated).

[0055] In some embodiments, the one or more enucleated cells or the composition comprising the one or more enucleated cells described herein is stable at 4 °C for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the composition comprising the one or more enucleated cells described herein is stable at room temperature for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the composition comprising the one or more enucleated cells described herein is stable at 37°C for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time. In some embodiments, the one or more enucleated cells or the composition comprising the one or more enucleated cells described herein may remain viable after being administered to a subject in need thereof for treating the disease or condition described herein. In some embodiments, the one or more enucleated cells or the composition comprising the one or more enucleated cells described herein may remain viable after being administered to the subject for greater than or equal to about one hour, two hours, six hours, 12 hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months, three months, or longer period of time.

[0056] In some embodiments, the one or more enucleated cells described herein may be obtained from a parent cell that is autologous to the subject, who is in need of the treatment by the one or more enucleated cells described herein. In some embodiments, the one or more enucleated cells described herein may be obtained from a parent cell that is allogenic to the subject, who is in need of the treatment by the one or more enucleated cells described herein.

[0057] In some embodiments, the one or more enucleated cells described herein may be smaller than their nucleated counterparts (e.g., the nucleated parent cells), and for this reason the one or more enucleated cells may migrate better through small openings in the vasculature and tissue parenchyma. In addition, removing the large dense nucleus alleviates a major physical barrier allowing the cell to move freely through small openings in the vessels and tissueATTORNEY DOCKET NO.38394.0028P1 parenchyma. Therefore, one or more enucleated cells described herein have improved bio- distribution in the body and movement into target tissues. In some embodiments, a one or more enucleated cells described herein comprises at least 1 μm in diameter. In some embodiments, a one or more enucleated cells described herein is greater than 1 μm in diameter. In some embodiments, a one or more enucleated cells described herein is 1-100 μm in diameter (e.g., 1- 90 μm, 1-80 μm, 1-70 μm, 1-60 μm, 1-50 μm, 1-40 μm, 1-30 μm, 1-20 μm, 1-10 μm, 1-5 μm, 5- 90 μm, 5-80 μm, 5-70 μm, 5-60 μm, 5-50 μm, 5-40 μm, 5-30 μm, 5-20 μm, 5-10 μm, 10-90 μm, 10-80 μm, 10-70 μm, 10-60 μm, 10-50 μm, 10-40 μm, 10-30 μm, 10-20 μm, 10-15 μm 15-90 μm, 15-80 μm, 15-70 μm, 15-60 μm, 15-50 μm, 15-40 μm, 15-30 μm, 15-20 μm). In some embodiments, a one or more enucleated cell is 10-30 μm in diameter. In some embodiments, the diameter of a one or more enucleated cells described herein is between 5-25 μm (e.g., 5-20 μm, 5-15 μm, 5-10 μm, 10-25 μm, 10-20 μm, 10-15 μm, 15-25 μm, 15-20 μm, or 20-25 μm). In some embodiments, the one or more enucleated cells described herein has a diameter that is more than or equal to about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some embodiments, the one or more enucleated cells described herein has a diameter that is more than or equal to about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, or about 12 μm. In some embodiments, the one or more enucleated cells described herein has a diameter that is about 8 μm. In some embodiments, the one or more enucleated cells described herein may advantageously be small enough to allow for better homing or delivery to a target site. In some embodiments, the one or more enucleated cells described herein may pass through passages in narrow lung tissues or lung structures such as alveolar duct or microcapillary that most cells such as the parent cells may not pass through.

[0058] In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of an average diameter of a nucleated parent cell. In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of an average diameter of the nucleated cells. In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 50% of an averageATTORNEY DOCKET NO.38394.0028P1 diameter of the nucleated cells. In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 60% of an average diameter of the nucleated cells. In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 70% of an average diameter of the nucleated cells. In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 80% of an average diameter of the nucleated cells. In some embodiments, the one or more enucleated cells of the one or more enucleated cell fractions described herein has a diameter comprising less than or equal to about 90% of an average diameter of the nucleated cells.

[0059] In some embodiments, one or more enucleated cells described herein possess significant therapeutic value, because they remain viable, do not differentiate into other cell types, secrete bioactive molecules, and may physically migrate / home for fewer than or equal to about 5 or 10 days, may be extensively enucleated ex vivo to perform specific therapeutic functions, and may be fused to the same or other cell types to transfer desirable production, natural or enucleated. Therefore, one or more enucleated cells described herein have wide utility as a cellular vehicle to deliver therapeutically important biomolecules and disease-targeting cargos comprising genes, viruses, bacteria, mRNAs, shRNAs, siRNA, polypeptides (comprising antibodies and antigen binding fragments), plasmids, gene-editing machinery, or nanoparticles. The present disclosure enables the generation of safe (e.g., no unwanted DNA is transferred to the subject), and controllable cell-based carrier that may be genetically enucleated to deliver specific disease-fighting and health promoting cargos to humans. In some embodiments, the one or more enucleated cells described herein remains viable and retain the function to migrate or home for greater than or equal to about 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 5 days, 6 days, 7 days, 8 days, 9 days, or longer after being administered to the subject in need thereof.

[0060] In some embodiments, the one or more enucleated cells described herein is engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous protein, gene-editing machinery or combinations thereof. In some embodiments, the exogenous DNA molecule is a single-stranded DNA, a double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule, a DNA virus, linear DNA, or combinations thereof. In some embodiments, the exogenous RNA molecule is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), an RNA virus, or combinations thereof. In some embodiments, theATTORNEY DOCKET NO.38394.0028P1 exogenous protein is a cytokine, a growth factor, a hormone, an antibody, or the antigen-binding fragment thereof, an enzyme, or combinations thereof. In some embodiments, the antibody is a single-domain antibody or antigen-binding fragment thereof. In some embodiments, parental cells (e.g., nucleated cells) are genetically enucleated before enucleation (e.g., pre-enucleation). In some embodiments, the parent cell is genetically enucleated after enucleation (e.g., post- enucleation).

[0061] The one or more enucleated cells disclosed herein may be derived from virtually any nucleated cell (referred to herein as “parent” cell). In some embodiments, the parent cell is an immune cell. In some embodiments, the immune cell is a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, or lymphocyte (B cells and T cells). In some embodiments, the parent cell is a stem cell. In some embodiments, the parent cell is an adult stem cell. In some embodiments, the parent cell is a mesenchymal stromal cell (MSC). In some embodiments, the parent cell may be selected from the list including, but not limited to: embryonic stem cells, mesenchymal stem cells, pluripotent stem cells, hematopoietic stem cells, neural stem cells, cancer stem cells, stem cells, multipotent stem cells, pluripotent stem cells, epithelial stem cells, bone marrow, differentiated cells, germ cells, totipotent cells, allogenic cells, autologous cells, multipotent stem cells, for example. In some embodiments, the enucleated cell is derived from an inducible pluripotent stem cell (iPSC). In some embodiments, the parent cell is not an erythrocyte. In some embodiments, the parent cell is not an erythroid precursor cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial precursor cell. 1. CD40L

[0062] Described herein are enucleated cells comprising CD40L polypeptide or a modified CD40L polypeptide. In some embodiments, the CD40L polypeptide has the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the modified CD40L polypeptide has the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, theCD40L or CD40L polypeptide can be a modified CD40L or modified CD40L polypeptide.

[0063] In some embodiments, the CD40L can be a human CD40L comprising the amino acid sequence of SEQ ID NO 1: MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHE DFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVATTORNEY DOCKET NO.38394.0028P1 NVTDPSQVSHGTGFTSFGLLKL

[0064] In some embodiments, the amino acid sequence of the CD40L is greater than or equal to about 80% identical to SEQ ID NO: 1. In some embodiments, the amino acid sequence of CD40L comprises s an amino acid sequence that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1.

[0065] In some embodiments, the CD40L can be encoded by a human CD40L nucleic acid. In some embodiments, the CD40L nucleic acid can be a human CD40L nucleic acid comprising the nucleic acid sequence of SEQ ID NO 2: aatcctgagt aaggtggcca ctttgacagt cttctcatgc tgcctctgcc accttctctg ccagaagata ccatttcaac tttaacacag catgatcgaa acatacaacc aaacttctcc ccgatctgcg gccactggac tgcccatcag catgaaaatt tttatgtatt tacttactgt ttttcttatc acccagatga ttgggtcagc actttttgct gtgtatcttc atagaaggtt ggacaagata gaagatgaaa ggaatcttca tgaagatttt gtattcatga aaacgataca gagatgcaac acaggagaaa gatccttatc cttactgaac tgtgaggaga ttaaaagcca gtttgaaggc tttgtgaagg atataatgtt aaacaaagag gagacgaaga aagaaaacag ctttgaaatg caaaaaggtg atcagaatcc tcaaattgcg gcacatgtca taagtgaggc cagcagtaaa acaacatctg tgttacagtg ggctgaaaaa ggatactaca ccatgagcaa caacttggta accctggaaa atgggaaaca gctgaccgtt aaaagacaag gactctatta tatctatgcc caagtcacct tctgttccaa tcgggaagct tcgagtcaag ctccatttat agccagcctc tgcctaaagt cccccggtag attcgagaga atcttactca gagctgcaaa tacccacagt tccgccaaac cttgcgggca acaatccatt cacttgggag gagtatttga attgcaacca ggtgcttcgg tgtttgtcaa tgtgactgat ccaagccaag tgagccatgg cactggcttc acgtcctttg gcttactcaa actctgaaca gtgtcacctt gcaggctgtg gtggagctga cgctgggagt cttcataata cagcacagcg gttaagccca ccccctgtta actgcctatt tataacccta ggatcctcct tatggagaac tatttattat acactccaag gcatgtagaa ctgtaataag tgaattacag gtcacatgaa accaaaacgg gccctgctcc ataagagctt atatatctga agcagcaacc ccactgatgc agacatccag agagtcctat gaaaagacaa ggccattatg cacaggttga attctgagta aacagcagat aacttgccaa gttcagtttt gtttctttgc gtgcagtgtc tttccatgga taatgcattt gatttatcag tgaagatgca gaagggaaat ggggagcctc agctcacatt cagttatggt tgactctggg ttcctatggc cttgttggag ggggccaggc tctagaacgt ctaacacagt ggagaaccga aacccccccc ccccccccgc caccctctcg gacagttatt cattctcttt caatctctct ctctccatct ctctctttca gtctctctct ctcaacctct ttcttccaat ctctctttct caatctctct gtttcccttt gtcagtctct tccctccccc agtctctctt ctcaatcccc ctttctaaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca gagtcaggcc gttgctagtc agttctcttc tttccaccct gtccctatct ctaccactat agatgagggt gaggagtagg gagtgcagcc ctgagcctgc ccactcctca ttacgaaatg actgtattta aaggaaatct attgtatcta cctgcagtct ccattgtttc cagagtgaac ttgtaattat cttgttattt attttttgaa taataaagac ctcttaacat ta

[0066] In some embodiments, the CD40L is encoded from a nucleic acid sequence that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orATTORNEY DOCKET NO.38394.0028P1 99% identical to SEQ ID NO:2.

[0067] Disclosed are enucleated cells comprising an exogenous nucleic acid molecule encoding a CD40L polypeptide. In some embodiments, the exogenous nucleic acid encoding the CD40L polypeptide has the nucleic acid sequence set forth in SEQ ID NO:2. Disclosed are enucleated cells comprising an exogenous nucleic acid encoding a modified CD40L polypeptide. In some embodiments, the exogenous nucleic acid encoding the modified CD40L polypeptide has the nucleic acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the CD40L polypeptide is expressed by the enucleated cell. In some embodiments, the CD40L polypeptide can be a modified CD40L polypeptide.

[0068] Disclosed are enucleated cells engineered to express an exogenous CD40L polypeptide. In some embodiments, the CD40L polypeptide can be a modified CD40L polypeptide.

[0069] In some embodiments, the enucleated cell comprises CD40L or a modified CD40L. In some embodiments, the enucleated cell may comprise an exogenously derived nucleic acid molecule encoding CD40L or a modified CD40L. In some embodiments, an enucleated cell may comprise CD40L or a modified CD40L expressed on the surface of the enucleated cell. In some embodiments, the enucleated cell may comprise one or more intracellular organelles. In some embodiments, the one or more intracellular organelles are for expressing the CD40L polypeptide. In some embodiments, the enucleated cell may comprise one or more ribosomes. In some embodiments, the enucleated cell may be sufficient for in vivo protein synthesis in the absence of the nucleus. In some embodiments, the enucleated cell may be sufficient for in vivo synthesis of CD40L in the absence of the nucleus. In some embodiments, the expressed CD40L may be present on the membrane of the enucleated cell. In a further embodiment, the CD40L may be modified. For example, In some embodiments, the CD40L may be modified to prevent cleavage. In some embodiments, uncleavable CD40L is a modified CD40L in which the sequence ‘SFEMQKG’ (SEQ ID NO:7) is deleted from the human CD40L. In some embodiments, uncleavable CD40L is a modified CD40L in which the sequence ‘SFEMQRG’ (SEQ ID NO:8) is deleted from the mouse CD40L.

[0070] In some embodiments, the modified CD40L can be a human modified CD40L comprising the amino acid sequence of SEQ ID NO:3 MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLH EDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENDEDPQIAAHVV SEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPS SQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEATTORNEY DOCKET NO.38394.0028P1 ASQVIHRVGFSSFGLLKL

[0071] In some embodiments, the modified CD40L can be a mouse modified CD40L comprising the amino acid sequence of SEQ ID NO:4 MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLH EDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENDEDPQIAAHVV SEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPS SQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTE ASQVIHRVGFSSFGLLKL.

[0072] In some embodiments, the amino acid sequence of the modified CD40L is greater than or equal to about 80% identical to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the amino acid sequence of modified CD40L comprises s an amino acid sequence that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:3 or 4.

[0073] In some embodiments, the modified CD40L can be encoded by a human modified CD40L nucleic acid. In some embodiments, the modified CD40L nucleic acid can be a human modified CD40L nucleic acid comprising the nucleic acid sequence of SEQ ID NO:5 atgattgaaacctatagccagccgagcccgcgcagcgtggcgaccggcctgccggcgagcatgaaaatttttatgtatctgctgaccgtgtt tctgattacccagatgattggcagcgtgctgtttgcggtgtatctgcatcgccgcctggataaagtggaagaagaagtgaacctgcatgaag attttgtgtttattaaaaaactgaaacgctgcaacaaaggcgaaggcagcctgagcctgctgaactgcgaagaaatgcgccgccagtttgaa gatctggtgaaagatattaccctgaacaaagaagaaaaaaaagaaaacgatgaagatccgcagattgcggcgcatgtggtgagcgaagc gaacagcaacgcggcgagcgtgctgcagtgggcgaaaaaaggctattataccatgaaaagcaacctggtgatgctggaaaacggcaaa cagctgaccgtgaaacgcgaaggcctgtattatgtgtatacccaggtgaccttttgcagcaaccgcgaaccgagcagccagcgcccgttta ttgtgggcctgtggctgaaaccgagcagcggcagcgaacgcattctgctgaaagcggcgaacacccatagcagcagccagctgtgcga acagcagagcgtgcatctgggcggcgtgtttgaactgcaggcgggcgcgagcgtgtttgtgaacgtgaccgaagcgagccaggtgattc atcgcgtgggctttagcagctttggcctgctgaaactg

[0074] In some embodiments, the modified CD40L can be encoded by a mouse modified CD40L nucleic acid. In some embodiments, the modified CD40L nucleic acid can be a mouse modified CD40L nucleic acid comprising the nucleic acid sequence of SEQ ID NO:6 atgattgaaacctatagccagccgagcccgcgcagcgtggcgaccggcctgccggcgagcatgaaaatttttatgtatctgctgaccgtgtt tctgattacccagatgattggcagcgtgctgtttgcggtgtatctgcatcgccgcctggataaagtggaagaagaagtgaacctgcatgaag attttgtgtttattaaaaaactgaaacgctgcaacaaaggcgaaggcagcctgagcctgctgaactgcgaagaaatgcgccgccagtttgaa gatctggtgaaagatattaccctgaacaaagaagaaaaaaaagaaaacgatgaagatccgcagattgcggcgcatgtggtgagcgaagc gaacagcaacgcggcgagcgtgctgcagtgggcgaaaaaaggctattataccatgaaaagcaacctggtgatgctggaaaacggcaaa cagctgaccgtgaaacgcgaaggcctgtattatgtgtatacccaggtgaccttttgcagcaaccgcgaaccgagcagccagcgcccgtttaATTORNEY DOCKET NO.38394.0028P1 ttgtgggcctgtggctgaaaccgagcagcggcagcgaacgcattctgctgaaagcggcgaacacccatagcagcagccagctgtgcga acagcagagcgtgcatctgggcggcgtgtttgaactgcaggcgggcgcgagcgtgtttgtgaacgtgaccgaagcgagccaggtgattc atcgcgtgggctttagcagctttggcctgctgaaactg

[0075] In some embodiments, the modified CD40L is encoded from a nucleic acid sequence that is greater than or equal to about 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:5 or 6.

[0076] In some embodiments, the CD40L polypeptide or modified CD40L polypeptide is expressed on a cell surface of the enucleated cell for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours. In some embodiments, the enucleated cell expresses an abundance of the CD40L polypeptide that is at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 1.0 fold, at least 2.0 fold, or at least 5.0 fold higher than the enucleated cell expressing an abundance of a comparable naturally-occurring CD40L polypeptide. In some embodiments, the abundance of the CD40L polypeptide and the abundance of the comparable naturally-occurring CD40L polypeptide is determined by FACS. In some embodiments, the CD40L polypeptide or modified CD40L polypeptide is not secreted by the enucleated cell

[0077] In some embodiments, the CD40L or modified CD40L of the disclosed invention can be replaced with any antigen presenting cell (APC) activator. For example, instead of CD40L, the disclosed enucleated cells can comprise IFN-beta or IL-12. In some embodiments, disclosed are enucleated cells comprising an exogenous RNA encoding an APC activator. For example, disclosed are enucleated cells comprising an exogenous RNA encoding IFN-beta or IL-12. In some embodiments, disclosed are enucleated cells engineered to express an APC activator. In some embodiments, disclosed are enucleated cells engineered to express IFN-beta or IL-12. 2. Transmembrane moiety

[0078] Described herein, are one or more enucleated cells or compositions comprising the one or more enucleated cells described herein comprising at least one transmembrane moiety. In some embodiments, the one or more enucleated cells described herein comprises an exogenous polypeptide. The exogenous polypeptide may be covalently fused to a transmembrane moiety. In some embodiments, the exogenous polypeptide is complexed to the transmembrane moiety. In some embodiments, the transmembrane moiety comprises a full length protein or a variation thereof or a fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the parent cell that is being enucleated for obtaining the enucleated cell. In some embodiments, the transmembrane moiety may be an exogenous transmembrane moiety to the parent cell or to the enucleated cell. In some embodiments, the transmembrane moiety is selected from a transmembrane protein comprising a single transmembrane α-helix (bitopicATTORNEY DOCKET NO.38394.0028P1 membrane protein). The transmembrane moiety comprises a polytopic transmembrane α-helical protein. In some embodiments, the transmembrane moiety comprises a polytopic transmembrane β-sheet protein. In some embodiments, the transmembrane moiety comprises a Type I, II, III, or IV transmembrane protein. Non-limiting examples of transmembrane protein may include CD4, CD14, glycophorin a (GPA), or any combination of integrins.

[0079] In some embodiments, the transmembrane moiety is added to the exogenous polypeptide (e.g. CD40L or modified CD40L) by way of a modification. For example, a transmembrane moiety may be added to the N or C-terminus of the CD40L or modified CD40L polypeptide to insert the exogenous polypeptide into the cell membrane of the one or more enucleated cells described herein. Non-limiting examples of modifications that are made to the exogenous polypeptide (e.g. CD40L or modified CD40L) to add the transmembrane moiety may include adding glycosylphosphatidylinositol, farnesyl, palmitate, myristate, or a combination thereof to the exogenous polypeptide.

[0080] In some embodiments, the transmembrane moiety is genetically modified to be fused or complexed with the at least one exogenous therapeutic agent described herein. In some embodiments, the transmembrane moiety is genetically modified to fuse to the at least one exogenous therapeutic agent described herein. In some embodiments, the one or more enucleated cells comprises an immune-evading moiety. In some embodiments, the immune- evading comprises a “don’t eat me” signaling peptide, such as CD47 (e.g., NCBI Gene ID 961), programmed cell death 1 ligand 1 (PD-L1,e.g., NCBI Gene ID 29126), major histocompatibility complex, class I, E (HLA-E, e.g., NCBI Gene ID 3133), major histocompatibility complex, class I, G (HLA-I, e.g., NCBI Gene ID 3135), a fragment thereof, or a combination thereof. 3. Targeting moiety

[0081] Described herein, are one or more enucleated cells or compositions comprising the one or more enucleated cells described herein further comprising a targeting moiety. The targeting moiety described herein can be designed to guide the one or more enucleated cells to a target cell or target environment (e.g., tissue) in a subject following delivery (e.g., systemic delivery) of the one or more enucleated cells to the subject. In some embodiments, the targeting moiety is expressed on the surface of the one or more enucleated cells. In some embodiments, the targeting moiety is complexed with a transmembrane moiety described herein. In some embodiments, the targeting moiety is secreted by the one or more enucleated cells. In some embodiments, the one or more enucleated cells comprising the targeting moiety localizes at the target cell or target environment with a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500- fold, 1,000-fold, 5,000-fold, or 10,000-fold increase as compared to localization of a comparableATTORNEY DOCKET NO.38394.0028P1 enucleated cell lacking the targeting moiety. In some embodiments, the one or more enucleated cells comprising the targeting moiety localizes at the target cell or target environment with an increase of 1%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared with a comparable enucleated cell lacking the targeting moiety. In some embodiments, the target cell or target environment is in vivo. In some embodiments, the target cell or target environment is ex vivo. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell is a multiple myeloma cell, glioblastoma cell, lymphoma cell, leukemia cell, mesothelioma cell, sarcoma cell, breast cancer cell, prostate cancer cell, ovarian cancer cell, pancreatic cancer cell, colon cancer cell, lung cancer cell, or any combination thereof. In some aspects, the cancer cell can be from any of the cancers described herein or any known cancers.

[0082] In some embodiments, the targeting moiety comprises an exogenous antibody or an exogenous antigen-binding fragment for targeting a biomarker described herein. In some embodiments, the targeting moiety comprises an exogenous antibody or an exogenous antigen- binding fragment for targeting a chemokine receptor or a chemokine ligand, or portion thereof, involved in chemokine signaling. In some embodiments, the exogenous antibody is an exogenous single-domain antibody or fragment thereof.

[0083] In some embodiments, the targeting moiety targets the biomarker expressed by, or associated with, a target cell or with a microenvironment. In some embodiments, the biomarker may be released by the target cell. The biomarker may indicate the presence of the disease or the condition. In some embodiments, the biomarker is expressed by immune cells responding to the target cell or the microenvironment associated with the disease or the condition. In some embodiments, the biomarker may be an epitope or antigen. In some embodiments, the biomarker comprising the epitope may be bound by an antibody that is different from the antibody or the antigen-binding fragment thereof that confers therapeutic property (e.g., the therapeutic agent).

[0084] In some embodiments, the targeting moiety comprises a homing receptor specific to a ligand or receptor expressed by a target cell. In some embodiments, the targeting moiety comprises a homing receptor specific to a ligand expressed by a target cell in a specific tissue, such as lung tissue. In some embodiments, the target cell or tissue is a cancer cell or tissue. In the case of enucleated cells that target a cancer cell or tissue, the enucleated cells may be engineered to express targeting moieties that recognize an antigen produced by the cancer cell or tissue. In some embodiments, the enucleated cells may be engineered to express other targeting moieties to guide the enucleated cells to the target tissue, including chemokines, integrins, adhesion molecules, membrane-bound antibody, or membrane-bound single-domain antibody. In some embodiments, the targeting moieties can facilitate efficient homing to target cell orATTORNEY DOCKET NO.38394.0028P1 tissue, even when administered to a subject systemically.

[0085] In some embodiments, the targeting moiety targets a biomarker expressed or released by a lung cell or a lung cancer cell. Non-limiting example of cancer cell biomarkers includes carbonic anhydrase 9 (CA9, e.g., NCBI Gene ID 768), carbonic anhydrase 12 (CA12, e.g., NCBI Gene ID 771), cancer / testis antigen 83 (CXorf61; e.g., NCBI Gene ID203413), desmoglein 3 (DSG3 (e.g., NCBI Gene ID 1830), FAT atypical cadherin 2 (FAT2 (e.g., NCBI Gene ID 2196), G protein-coupled receptor 87 (GPR87, e.g., NCBI Gene ID 53836), KISS1 receptor (KISS1R, e.g., NCBI Gene ID 84634), LY6 / PLAUR domain containing 3 (LYPD3. e.g., NCBI Gene ID 27076), solute carrier family 7 member 11 (SLC7A11, e.g., NCBI Gene ID 23657), TMPRSS4 (e.g., NCBI Gene ID 56649), transmembrane serine protease 4 (TFPI, e.g., NCBI Gene ID 7035), midkine (MDK, e.g., NCBI Gene ID 4192), secreted phosphoprotein 1 (OPN, e.g., NCBI Gene ID 6696), matrix metallopeptidase 2 (MMP2, e.g., NCBI Gene ID 4313), TIMP metallopeptidase inhibitor 1 (TIMP1, e.g., NCBI Gene ID 7076), cell adhesion molecule 5 (CEA, e.g., NCBI Gene ID 1048), cytokeratin 19 fragment (CYFRA 21–1, e.g., NCBI Gene ID 3880), serpin family B member 3 (SCC, e.g., NCBI Gene ID 6317), advanced glycosylation end-product specific receptor (AGER, e.g., NCBI Gene ID 177), adipogenesis regulatory factor (C10orf116, e.g., NCBI Gene ID 10974), adducin 2 (ADD2, e.g., NCBI Gene ID 119), periaxin (PRX, e.g., NCBI Gene ID 57716), laminin subunit beta 3 (LAMB3, e.g., NCBI Gene ID 3914), synemin (SYNM, e.g., NCBI Gene ID 23336), spectrin alpha, erythrocytic 1 (SPTA1, e.g., NCBI Gene ID 6708), ankyrin 1 (ANK1, e.g., NCBI Gene ID 286), hemoglobin subunit epsilon 1 (HBE1, e.g., NCBI Gene ID 3046), hemoglobin subunit gamma 1 (HBG1, e.g., NCBI Gene ID 3047), carbonic anhydrase 1 (CA1, e.g., NCBI Gene ID 759), tenascin XB (TNXB, e.g., NCBI Gene ID 7148), multimerin 2 (MMRN2, e.g., NCBI Gene ID 79812), hemoglobin subunit alpha 1 (HBA1, e.g., NCBI Gene ID 3039), caveolin 1 (CAV1, e.g., NCBI Gene ID 857), hemoglobin subunit beta (HBB, e.g., NCBI Gene ID 3043), collagen type VI alpha 6 chain (COL6A6, e.g., NCBI Gene ID 131873), chromosome 1 open reading frame 198 (C1orf198, e.g., NCBI Gene ID 84886), chloride intracellular channel 2 (CLIC2, e.g., NCBI Gene ID 1193), transcriptional regulator of SdpC synthesis operon (ArsR family) (SDPR, e.g., NCBI Gene ID 8436), EH domain containing 2 (EHD2, e.g., NCBI Gene ID 30846), apolipoprotein A2 (APOA2, e.g., NCBI Gene ID 336), NADH: ubiquinone oxidoreductase subunit B7 (NDUFB7, e.g., NCBI Gene ID 4713), protein kinase C delta binding protein (PRKCDBP, e.g., NCBI Gene ID 112464), laminin subunit alpha 3 (LAMA3, e.g., NCBI Gene ID ), EvC ciliary complex subunit 2 (LBN, e.g., NCBI Gene ID 132884), serpin family A member 3 (ACT, e.g., NCBI Gene ID 12), insulin like growth factor binding protein 3 (3ATTORNEY DOCKET NO.38394.0028P1 IGFBP3, e.g., NCBI Gene ID 3486), prostaglandin D2 synthase (L-PGDS, e.g., NCBI Gene ID 5730), retinoic acid receptor beta (HAP, e.g., NCBI Gene ID 5915), hepatocyte growth factor (HGF, e.g., NCBI Gene ID 3082), eukaryotic translation initiation factor 4 gamma 2 (AAG1 / 2, e.g., NCBI Gene ID 1982), clusterin (CLU, e.g., NCBI Gene ID 1191), streptococcal superantigen SSA (SSA, e.g., NCBI Gene ID 6737), tetanic (TTA, e.g., NCBI Gene ID 100189453), apolipoprotein A4 (APOA4, e.g., NCBI Gene ID 337), fibrinogen-like protein A (FIBA, e.g., NCBI Gene ID 105209070), serum amyloid A cluster (SAA, e.g., NCBI Gene ID 6288), ceruloplasmin (CP, e.g., NCBI Gene ID 1356), haptoglobin (HP, e.g., NCBI Gene ID 3240), transthyretin (TTR, e.g., NCBI Gene ID 7276), keratin 2 (KRT2A, e.g., NCBI Gene ID 3849), glutamate transporter (GLT1B, e.g., NCBI Gene ID 6506), casein kinase 1 (CK1, e.g., NCBI Gene ID 1452), AKT serine / threonine kinase 1 (AKT, e.g., NCBI Gene ID 207), mannose binding lectin 2 (MBL2, e.g., NCBI Gene ID 4153), fibrinogen alpha chain (FGA, e.g., NCBI Gene ID 2243), gelsolin (GSN, e.g., NCBI Gene ID 2934), haptoglobin (HP, e.g., NCBI Gene ID 3240), ficolin 3 (FCN3, e.g., NCBI Gene ID 8547), carnosine dipeptidase 1 (CNDP1, e.g., NCBI Gene ID 84735), calcitonin related polypeptide alpha (CALCA, e.g., NCBI Gene ID 796), carbamoyl-phosphate synthase 1 (CPS1, e.g., NCBI Gene ID 1373), chromogranin B (CHGB, e.g., NCBI Gene ID 1114), involucrin (IVL, e.g., NCBI Gene ID 3713), anterior gradient 2 (AGR2, e.g., NCBI Gene ID 10551), nuclear autoantigenic sperm protein (NASP, e.g., NCBI Gene ID 4678), phosphofructokinase, platelet (PFKP, e.g., NCBI Gene ID 5214), thrombospondin 2 (THBS2, e.g., NCBI Gene ID 7058), thioredoxin domain containing 17 (TXNDC17, e.g., NCBI Gene ID 84817), proprotein convertase subtilisin / kexin type 1 (PCSK1, e.g., NCBI Gene ID 5122), cellular retinoic acid binding protein 2 (CRABP2, e.g., NCBI Gene ID 1382), acyl-CoA binding domain containing 3 (ACBD3, e.g., NCBI Gene ID 64746), desmoglein 2 (DSG2, e.g., NCBI Gene ID 1829), LPS responsive beige-like anchor protein (LRBA, e.g., NCBI Gene ID 987), serine / threonine kinase receptor associated protein (STRAP, e.g., NCBI Gene ID 11171), VGF nerve growth factor inducible (VGF, e.g., NCBI Gene ID 7425), NOP2 nucleolar protein (NOP2, e.g., NCBI Gene ID 4839), lipocalin 2 (LCN2, e.g., NCBI Gene ID 3934), creatine kinase, mitochondrial 1B (CKMT1B, e.g., NCBI Gene ID 1159), aldo-keto reductase family 1 member B10 (AKR1B10, e.g., NCBI Gene ID 57016), carboxypeptidase D (CPD, e.g., NCBI Gene ID 1362), proteasome activator subunit 3 (PSME3, e.g., NCBI Gene ID 10197), villin 1 (VIL1, e.g., NCBI Gene ID 7429), serpin family B member 5 (SERPINB5, e.g., NCBI Gene ID 5268), ribosomal protein L5 (RPL5, e.g., NCBI Gene ID 6125), plakophilin 1 (PKP1, e.g., NCBI Gene ID 5317), ribosomal protein L10 (RPL10, e.g., NCBI Gene ID 6134), aldo-keto reductase family 1 member B10 (AKR1B10, e.g., NCBI GeneATTORNEY DOCKET NO.38394.0028P1 ID 57016), aldo-keto reductase family 1 member C1 (AKR1C1, e.g., NCBI Gene ID 1645), proliferating cell nuclear antigen (PCNA, e.g., NCBI Gene ID 5111), ribosomal protein S2 (RPS2, e.g., NCBI Gene ID 6187), aldo-keto reductase family 1 member C3 (AKR1C3, e.g., NCBI Gene ID 8644), acyl-CoA binding domain containing 3 (ACBD3, e.g., NCBI Gene ID 64746), visinin like 1 (VSNL1, e.g., NCBI Gene ID 7447), adenosylhomocysteinase (AHCY, e.g., NCBI Gene ID 191), IMMP10, activated kinase 2 (PAK2, e.g., NCBI Gene ID 5062), involucrin (IVL, e.g., NCBI Gene ID 3713), isoleucine-tRNA synthetase (IARS, e.g., NCBI Gene ID 3376), proteasome 26S subunit ubiquitin receptor, non-ATPase 2 (PSMD2, e.g., NCBI Gene ID 5708), guanylate binding protein 5 (GBP5, e.g., NCBI Gene ID 115362), minichromosome maintenance complex component 6 (MCM6, e.g., NCBI Gene ID 4175), N- myc downstream regulated 1 (NDRG1, e.g., NCBI Gene ID 10397), NOP58 ribonucleoprotein (NOP58, e.g., NCBI Gene ID 51602), S100 calcium binding protein A2 (S100A2, e.g., NCBI Gene ID 6273), neuregulin 1 (NRG1, e.g., NCBI Gene ID 3084), neuregulin 2 (NRG2, e.g., NCBI Gene ID 9542), carnosine dipeptidase 1 (CNDP1, e.g., NCBI Gene ID 84735), ubiquitin cross-reactive protein (UCRP, e.g., NCBI Gene ID 9636), crammer (CER, e.g., NCBI Gene ID 8110), plasminogen activator (UPA, e.g., NCBI Gene ID 5328), matrix metallopeptidase 14 (MT1-MMP, e.g., NCBI Gene ID 4323), stratifin (SFN, e.g., NCBI Gene ID 2810), transferrin (TF, e.g., NCBI Gene ID 7018), albumin (ALB, e.g., NCBI Gene ID 213), S100 calcium binding protein A9 (S100A9, e.g., NCBI Gene ID 6280), stathmin 1 (STMN, e.g., NCBI Gene ID 3925), Enolase (ENO), plasminogen activator (PLAU, e.g., NCBI Gene ID 5328), insulin like growth factor binding protein 7 (IGFBP7, e.g., NCBI Gene ID 3490), matrix metallopeptidase 14 (MMP14, e.g., NCBI Gene ID 4323), thrombospondin 1 (THBS1, e.g., NCBI Gene ID 7057), or thrombospondin 2 (THBS2, e.g., NCBI Gene ID 7058).

[0086] In some embodiments, the targeting moiety targets a biomarker expressed or released by a cancer cell that has metastasized. For example, the cancer cell may arise from one tissue and subsequently metastasizes to a different location. In some embodiments, the metastasized cancer cell expresses the non-limiting example of cancer biomarker described herein. In some embodiments, the metastasized cancer cell expresses cancer biomarker comprises Melanoma Associated Antigen (MAGE family member A3 (MAGE-A3, e.g., NCBI Gene ID 4102)), Membrane associated glycoprotein (MUC-1, e.g., NCBI Gene ID 4582), glycoproteine- epithelial cell adhesion molecule (EpCAM, e.g., NCBI Gene ID 4072), KRAS Proto-Oncogene (KRAS, e.g., NCBI Gene ID 3845), Anaplastic lymphoma kinase (ALK, e.g., NCBI Gene ID 238), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4, e.g., NCBI Gene ID 1493), Programmed cell death protein 1 (PD-1, e.g., NCBI Gene ID 5133), Epidermal growth factorATTORNEY DOCKET NO.38394.0028P1 (EGF, e.g., NCBI Gene ID 1950), Serine protease ester (EA, e.g., NCBI Gene ID 5328), Telomerase reverse transcriptase (TERT, e.g., NCBI Gene ID 7015), PRAME Nuclear Receptor Transcriptional Regulator (PRAME, e.g., NCBI Gene ID 23532), Receptor tyrosine-protein kinase erbB-2 (HER, e.g., NCBI Gene ID 2064), or Vascular endothelial growth factor (VEGF, e.g., NCBI Gene ID 7422), Carcinoembryonic antigen (CEA, e.g., NCBI Gene ID 1048), MAGE family member A1 (MAGE-A1, e.g., NCBI Gene ID 4100), MAGE family member A1 MAGE-A4, e.g., NCBI Gene ID 4103), Survivin, Six Transmembrane Epithelial Antigene of the Prostate 1 (STEAP1, e.g., NCBI Gene ID 26872), SRY (sex determining region Y)-box 2 (SOX2, e.g., NCBI Gene ID 6657), or Cancer / testis antigen 1 (CTAG1B, e.g., NCBI Gene ID 1485).

[0087] In some embodiments, the targeting moiety targets a biomarker expressed or released by an endothelial cell. In some embodiments, the endothelial cell is a blood vessel cell. In some embodiments, the endothelial cell is a lymphatic vessel cell. In some embodiments, the biomarker is expressed or released by a blood vessel cell. In some embodiments, the biomarker is expressed or released by a lymphatic vessel cell. Non-limiting examples of the endothelial cell biomarker include angiotensin I converting enzyme (ACE / CD143, e.g., NCBI Gene ID 1636), CD93 molecule (C1qR1 / CD93, e.g., NCBI Gene ID 22918), cadherin 5 (VE-Cadherin, e.g., NCBI Gene ID 1003), D6 protein (CC Chemokine Receptor D6, e.g., NCBI Gene ID 1238), platelet and endothelial cell adhesion molecule 1 (CD31 / PECAM-1, e.g., NCBI Gene ID 5175), CD34 molecule (CD34, e.g., NCBI Gene ID 947), CD36 molecule (CD36 / SR-B3, e.g., NCBI Gene ID 948), CD151 molecule (CD151, e.g., NCBI Gene ID 977), CD160 molecule (CD160, e.g., NCBI Gene ID 11126), CD300 molecule like family member g (CD300g / Nepmucin, e.g., NCBI Gene ID 146894), CDC like kinase 1 (CL-K1 / COLEC11, e.g., NCBI Gene ID 78989), cleavage factor polyribonucleotide kinase subunit 1 (CL-P1 / COLEC12, e.g., NCBI Gene ID 81035), Coagulation Factor III / Tissue Factor (e.g., NCBI Gene ID 2152), C-type lectin domain family 4 member M (DC-SIGNR / CD299, e.g., NCBI Gene ID 10332), discoidin, CUB and LCCL domain containing 2 (DCBLD2 / ESDN, e.g.,NCBI Gene ID 131566), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR, e.g., NCBI Gene ID 641700), basigin (Ok blood group) (EMMPRIN / CD147, e.g., NCBI Gene ID 682), Endoglin / CD105 (e.g., NCBI Gene ID 5077), Endomucin (e.g., NCBI Gene ID 2022), Endosialin / CD248 (e.g., NCBI Gene ID 57124), protein C receptor (EPCR, e.g., NCBI Gene ID 10544), Erythropoietin R (e.g., NCBI Gene ID 2056), endothelial cell adhesion molecule (ESAM, e.g., NCBI Gene ID 90952), fatty acid binding protein 5 (FABP5 / E-FABP, e.g., NCBI Gene ID 2171), fatty acid binding protein 6 (FABP6, e.g., NCBI Gene ID 2172), intercellular adhesion molecule 1 (ICAM-ATTORNEY DOCKET NO.38394.0028P1 1 / CD54, e.g., NCBI Gene ID 3383), intercellular adhesion molecule 2 (ICAM-2 / CD102, e.g., NCBI Gene ID 3384), interleukin 1 receptor (IL-1 RI, e.g., NCBI Gene ID 3553), Interleukin 13 receptor, alpha 1 (IL-13 R alpha 1, e.g., NCBI Gene ID 3597), Integrin alpha 4 / CD49d (e.g., NCBI Gene ID 3676), Integrin alpha 4 beta 1 (e.g., NCBI Gene ID 3688), Integrin alpha 4 beta 7 / LPAM-1 (e.g., NCBI Gene ID 3676), Integrin beta 2 / CD18 (e.g., NCBI Gene ID 3689), KLF transcription factor 4 (KLF4, e.g., NCBI Gene ID 9314), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1, e.g., NCBI Gene ID 10894), melanoma cell adhesion molecule (MCAM / CD146, e.g., NCBI Gene ID 4162), nectin cell adhesion molecule 2 (Nectin-2 / CD112, e.g., NCBI Gene ID 5819), PD-ECGF / Thymidine Phosphorylase (e.g., NCBI Gene ID 1890), Podocalyxin (e.g., NCBI Gene ID 5420), Podoplanin (e.g., NCBI Gene ID 10630), sphingosine- 1-phosphate receptor 1 (S1P1 / EDG-1, e.g., NCBI Gene ID 1901), sphingosine-1-phosphate receptor 2 (S1P2 / EDG-5, e.g., NCBI Gene ID 9294), sphingosine-1-phosphate receptor 3 (S1P3 / EDG-3, e.g., NCBI Gene ID 1903), sphingosine-1-phosphate receptor 4 (S1P4 / EDG-6, e.g., NCBI Gene ID 8698), sphingosine-1-phosphate receptor 5 (S1P5 / EDG-8, e.g., NCBI Gene ID 53637), E-Selectin / CD62E (e.g., NCBI Gene ID 6401), P-Selectin / CD62P (e.g., NCBI Gene ID 6403), slow as molasses (SLAM / CD150, e.g., NCBI Gene ID 6504), Stabilin-1 (e.g., NCBI Gene ID 23166), Stabilin-2 (e.g., NCBI Gene ID 55576), plexin domain containing 1 (TEM7 / PLXDC1, e.g., NCBI Gene ID 57125), ANTXR cell adhesion molecule 1 (TEM8 / ANTXR1, e.g., NCBI Gene ID 84168), Thrombomodulin / BDCA-3 (e.g., NCBI Gene ID Thrombomodulin), thrombospondin type 1 domain containing 1 (THSD1, e.g., NCBI Gene ID 55901), thrombospondin type 1 domain containing 7A (THSD7A, e.g., NCBI Gene ID 221981), TEK receptor tyrosine kinase (Tie-2, e.g., NCBI Gene ID 7010), TNF receptor superfamily member 1A (TNF RI / TNFRSF1A, e.g., NCBI Gene ID 7132), TNF receptor superfamily member 1B (TNF RII / TNFRSF1B, e.g., NCBI Gene ID 7133), basigin (Ok blood group) (TRA- 1-85 / CD147, e.g., NCBI Gene ID 682), TNF receptor superfamily member 10b (TRAIL R2 / TNFRSF10B, e.g., NCBI Gene ID 8795), TNF receptor superfamily member 10a (TRAILR1 / TNFRSF10A, e.g., NCBI Gene ID 8797), vascular cell adhesion molecule 1 (VCAM-1 / CD106, e.g., NCBI Gene ID 7412), EGF like domain multiple 7 (VE-Statin, e.g., NCBI Gene ID: 51162), fms related receptor tyrosine kinase 1 (VEGFR1 / Flt-1, e.g., NCBI Gene ID 2321), kinase insert domain receptor (VEGFR2 / KDR / Flk-1, e.g., NCBI Gene ID 3791), fms related receptor tyrosine kinase 4 (VEGFR3 / Flt-4, e.g., NCBI Gene ID 2324), angiogenic factor with G-patch and FHA domains 1 (VG5Q, e.g., NCBI Gene ID 55109), or von Willebrand Factor domain 2 (vWF-A2, e.g., NCBI Gene ID 7450).

[0088] In some embodiments, the targeting moiety comprises a chemokine receptor or aATTORNEY DOCKET NO.38394.0028P1 chemokine ligand, or portion thereof, involved in chemokine signaling, such as for example, SDF-1α / CXCR4, CCL2 / CCR2, or adhesion molecules, such as for example, PSGL-1. As shown herein, the one or more enucleated cells may be enucleated to express functional CXCR4, CCR2 as well as glycosylated PSGL-1, which may greatly promote the specific targeting of the enucleated cell. In some embodiments, the targeting moiety, such as CXCR4, CCR2 or PSGL-1 may be expressed on the surface of the enucleated cell. Non-limiting examples of cell surface proteins that may be expressed on the cell surface of the enucleated cell as the targeting moiety include chemokines such as CXCR4, CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. In some embodiments, the enucleated cell may be enucleated to secrete the targeting moiety or is tethered to the extracellular matrix, e.g., SDF1α or CCL2. Non-limiting examples of targeting moiety that may be secreted by the enucleated cell include SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11 and CXCL12. In some embodiments, the enucleated cell comprises cell-matrix receptors and cell-cell adhesion molecules comprise integrins, cadherins, glycoproteins, and heparin sulfate proteoglycans. 4. Immune Evasion

[0089] In some embodiments, the one or more enucleated cells may further comprise (e.g., by engineering or from the cell from which they were obtained) a surface marker that aids in their evasion of the subject immune system. For example, in some embodiments, the one or more enucleated cells may comprise a CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof. Without being bound by any particular theory, it is believed that a CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof helps to prevent the one or more enucleated cells from being phagocytosed by macrophages. Non-limiting examples of cell-matrix receptors and cell-cell adhesion molecules include integrins, cadherins, glycoproteins, or heparin sulfate proteoglycans. In some embodiments, the cell-matrix receptors or cell-cell adhesion molecules comprise PD-L1, HLA-E, or HLA-G. Non-limiting examples of therapeutic molecules include tumor antigens and immunomodulatory peptides, polyamines, and ATP. In some embodiments, the therapeutic molecules can be recognized by immune cells and can induce immune response. For example, the therapeutic molecules can be 4-1BB or any one of the cytokines described herein to induce immune response

[0090] In some embodiments, other examples of immune evasion molecules can be, but are not limited to, IL-1, IL-4, IL-6, IL-8, IL-10, TGF-b, IGF-2, VEGF, TNF-alpha, CD47, HLA-E, HLA-G, HLA-E / G, PD-1, PD-L1, TIGIT, CD112R, CTLA-4, a chemokine, chemokine ligand 1, C-C motif chemokine receptor 7, an NK inhibitor receptor, HLA-class I-specific inhibitory receptor, killer cell immunoglobulin-like receptor (KIR), NKG2A, lymphocyte activation gene-3ATTORNEY DOCKET NO.38394.0028P1 (LAG-3), or combinations thereof.

[0091] In some embodiments, the immune evasion molecule is expressed on the surface of the enucleated cell.

[0092] In some embodiments, the enucleated cells can provide immune evasion by expression an immune evasion molecule on its surface, by depleting one or more immune recognition molecules, or a combination thereof.

[0093] In some embodiments, the one or more immune recognition molecules can be, but are not limited to, a human leukocyte antigen (HLA), a proteoglycan, a sugar moiety, an embryonic antigen, or any combination thereof.

[0094] Provided herein are methods of governing immune recognition in a subject, the method comprising: administering to the subject an enucleated cell, wherein the enucleated cell is genetically engineered to evade recognition by the immune system. In some embodiments, the enucleated cell is genetically engineered to deplete the enucleated cell of immune recognition molecules. 5. Therapeutic Agent

[0095] In some embodiments, the disclosed enucleated cell comprises at least one therapeutic agent. In some embodiments, the one or more enucleated cells disclosed herein express the therapeutic agent with one or more intracellular organelles in the absence of the nucleus. In some embodiments, the therapeutic agent is exogenous to the enucleated cell or parent (nucleated) cell thereof. In some embodiments, the one or more enucleated cells expresses the therapeutic agent at the surface of the enucleated cell. In some embodiments, the therapeutic agent is secreted by the one or more enucleated cells. For example, the therapeutic agent can be secreted into extracellular space at a target tissue (e.g., a microenvironment). In some embodiments, the therapeutic agent is cargo (e.g., encapsulated by the enucleated cell) of the one or more enucleated cells.

[0096] In some embodiments, the one or more enucleated cells of the present disclosure comprises at least one therapeutic agent. In some embodiments, the one or more enucleated cells of the present disclosure comprises at least two, three, four, five, six, seven, eight, nine, ten, or more therapeutic agents. In some embodiments, the therapeutic agent comprises an active agent. In some embodiments, the therapeutic agent is exogenous to the one or more enucleated cells or parent cell thereof. An active agent comprises at least one of a DNA molecule, a RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, cytokine, a protein hormone, a growth factor, a cell surface receptor, or a vaccine), a peptide (e.g., a peptide hormone or an antigen), a small molecule (e.g., a steroid, a polyketide, an alkaloid, a toxin, an antibiotic, anATTORNEY DOCKET NO.38394.0028P1 antiviral, a colchicine, a taxol, a mitomycin, or emtansine), a gene editing factor, a nanoparticle, or another active agent (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g., oncolytic viruses), exosomes, lipids, or ions). In some embodiments, the active agent is a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or any combination thereof. In some embodiments, a enucleated cell is engineered to produce (e.g., express, and in some cases, release or secrete) the therapeutic agent. In some embodiments, the parent may be engineered to produce the therapeutic agent prior to enucleation to produce the enucleated cell. Non-limiting examples of oncolytic viruses include Talimogene laherparepvec, Onyx-015, GL-ONC1, CV706, Voyager-V1, and HSV-1716. Some wild-type viruses also show oncolytic behavior, such as Vaccinia virus, Vesicular stomatitis virus, Poliovirus, Reovirus, Senecavirus, ECHO-7, and Semliki Forest virus.

[0097] The therapeutic agent may be, or comprise, a targeting moiety described herein. Non- limiting example of the targeting moieties that may be produced by or contained in a enucleated cell includes chemokine receptors, adhesion molecules, and antigens. In some embodiments, the therapeutic agent may be, or comprise, a transmembrane moiety described herein.

[0098] In some embodiments, the therapeutic agent is recombinantly expressed by the enucleated cell or parent cell thereof. In some embodiments, the parent cell from which the enucleated cell is derived or obtained is engineered to produce or express the therapeutic agent. In some embodiments, expression of the therapeutic agent is stable (e.g., permanent). In some embodiments, the expression of the therapeutic agent by the parent cell is transient (e.g., non- permanent). In some embodiments, the parent cell is enucleated prior to engineering the enucleated cell to recombinantly express the therapeutic agent.

[0099] In some embodiments, the therapeutic agent is not naturally expressed (e.g., in the absence of engineering) in the cell from which the enucleated cell was derived or obtained (e.g., the therapeutic agent is exogenous to the parent cell). In some embodiments, the therapeutic agent is not naturally expressed in the subject (e.g., the therapeutic agent is exogenous to the subject). In some embodiments, the therapeutic agent is not naturally expressed in the subject at the intended site of therapy (e.g., a tumor, or a particular tissue, such as the brain, the intestine, the lungs, the heart, the liver, the spleen, the pancreas, muscles, eyes, and the like) (e.g., the therapeutic agent is exogenous to the intended site of therapy). In some embodiments, the level of the therapeutic agent is not naturally occurring in the enucleated cell of the parent cell.

[0100] In some embodiments, the therapeutic agent is naturally expressed (e.g., in the absence of engineering) in the cell from which the enucleated cell was derived or obtained (e.g., the therapeutic agent is endogenous to the enucleated cell). In some embodiments, theATTORNEY DOCKET NO.38394.0028P1 therapeutic agent is naturally expressed in the subject (e.g., the therapeutic agent is endogenous to the subject). In some embodiments, therapeutic agent is naturally expressed in the subject at the intended site of therapy (e.g., a tumor, or a particular tissue, such as the brain, the intestine, the lungs, the heart, the liver, the spleen, the pancreas, muscles, eyes, and the like) (e.g., the therapeutic agent is endogenous to the intended site of therapy).

[0101] In some embodiments, the therapeutic agent is derived from a synthetic cell and loaded into the enucleated cell. For example, the therapeutic agent may be endocytosed into the cell. Alternatively, the therapeutic agent may be synthesized by the cell and subsequently delivered to a target cell.

[0102] In some embodiments, the therapeutic agent comprises a corrected, a truncated, or a non-mutated version and / or copy of the DNA molecule, the RNA molecule, the protein, the peptide, the small molecule active agent, and / or the gene-editing factor as compared to the cell from which the enucleated cell was derived or obtained. For example, the therapeutic agent can correct a mutated p53 or EGFR in the target cell as part of the treatment for lung cancer.

[0103] In some embodiments, therapeutic agent comprises at least 2 (e.g., at least 2, 3, 4, 5, or more) different therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule active agents, or therapeutic gene-editing factors, in any combination. For example, in some embodiments, a therapeutic agent comprises a therapeutic DNA molecule and a small molecule active agent. For example, in some embodiments, the therapeutic agent comprises two different small molecule active agents. For example, in some embodiments, the therapeutic agent comprises a chemokine receptor (e.g., for targeting) and a small molecule active agent.

[0104] In some embodiments, the therapeutic agent comprises an RNA molecule comprising messenger RNA (mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA (lncRNA) or an RNA virus. In some embodiments, the therapeutic agent comprises a DNA molecule that is single-stranded DNA, double-stranded DNA, an oligonucleotide, a plasmid, a bacterial DNA molecule or a DNA virus. In some embodiments, the therapeutic agent comprises a protein, or a portion thereof. In some embodiments, the protein is a cytokine, a growth factor, a hormone, an antibody or an antigen- binding fragment thereof, a small-peptide based drug, or an enzyme. In some embodiments, the enucleated cell transiently expresses the therapeutic agent. In some embodiments, the expression of the therapeutic agent is inducible. In some embodiments, the expression of the therapeutic agent permanent.

[0105] In some embodiments, the therapeutic agent comprises an exogenous agent. In someATTORNEY DOCKET NO.38394.0028P1 embodiments, the exogenous agent is an exogenous polypeptide. In some embodiments, the exogenous polypeptide is encoded by an exogenous polynucleotide delivered into the parent cell or the enucleated cell. In some embodiments, the exogenous polypeptide is synthesized or released by at least one intracellular organelle of the enucleated cell. In some embodiments, the exogenous polypeptide is released by the enucleated cell. In some embodiments, the exogenous polypeptide is expressed on the cell surface or the enucleated cell. In some embodiments, the enucleated cell delivers the exogenous polypeptide to a target cell. In some embodiments, the target cell is a cancer cell expressing the cancer biomarker of any cancer described herein. In some embodiments, the target cell is a pneumocyte. In some embodiments, the target cell is an epithelial cell. In some embodiments, the epithelial cells are located on, in, or from lung tissue. In some embodiments, the target cell is an endothelial cell expressing an endothelial biomarker described herein. In some embodiments, the endothelial cell is a blood vessel cell. In some embodiments, the endothelial cell is a lymphatic vessel cell.

[0106] In some embodiments, the exogenous polypeptide comprises a cytokine of any one of the cytokine described herein. In some embodiments, the exogenous polypeptide comprises a soluble cytokine. For example, the exogenous polypeptide can comprise an extracellular domain or fragment of the cytokine. In some embodiments, the exogenous polypeptide comprises a solubility as determined by turbidimetric solubility assay or thermodynamic solubility assay by dissolving the exogenous polypeptide in solvent such as organic solvent, comprising dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc., or inorganic solvent, comprising water or phosphate-buffered saline (PBS). In some embodiments, the exogenous polypeptide comprises a solubility that is at least 0.0001 mg / ml, 0.0005 mg / ml, 0.001 mg / ml, 0.005 mg / ml, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 5.0 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 500 mg / ml 1,000 mg / ml 5,000 mg / ml, 10,000 mg / ml, 50,000 mg / ml, or 100,000 mg / ml.

[0107] In some embodiments, the exogenous polypeptide comprises a tumor necrosis factor (TNF) superfamily member or a catalytically active fragment thereof. Non-limiting examples of the TNF superfamily member include Lymphotoxin alpha (TNFβ), Tumor necrosis factor (TNFα), Lymphotoxin beta (TNFγ), OX40 ligand (CD252, Gp34, or CD134L), CD40 ligand (CD154, TRAP, Gp39, or T-BAM), Fas ligand (CD178, APTL, or CD95L), CD27 ligand (CD70), CD30 ligand (CD153), CD137 ligand (4-1 BBL), TNF-related apoptosis-inducing ligand (CD253 or APO-2L), Receptor activator of nuclear factor kappa-Β ligand (CD254, OPGL, TRANCE, or ODF), TNF-related weak inducer of apoptosis (APO-3L or DR3L), a proliferation-inducing ligand (CD256, TALL-2, or TRDL1), B-cell activating factor (CD257,ATTORNEY DOCKET NO.38394.0028P1 BLyS, TALL-1, or TNFSF20), LIGHT (CD258 or HVEML), Vascular endothelial growth inhibitor (TL1 or TL-1A), TNF superfamily member 18 (GITRL, AITRL, or TL-6), or Ectodysplasin A (ED1-A1 or ED1-A2).

[0108] In some embodiments, the therapeutic agent comprises any one of the immune checkpoint proteins described herein or an immune checkpoint inhibitor (immune checkpoint blockade) for inhibiting any one of the immune checkpoint protein described herein. Non- limiting examples of the immune checkpoint protein include PD-1, PD-L1, CTLA-4, VISTA, B7-H3 (also called CD276), A2AR, CD27, LAG3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CD73, NKG2A, PVRIG, PVRL2, CEACAM1, CEACAM5, CEACAM6, FAK, CCR-2, CCL-2, LIF, CD47, SIRPα, M-CSF, CSF-1R, IL-3, IL-1RAP, IL-8, SEMA4D, Angiopoietin-2, CLEVER-1, Axl, phosphatidylserine or a fragment thereof.

[0109] In some embodiments, the immune checkpoint inhibitor can include a PD-1 inhibitor including, but not limited to, Pembrolizumab, Nivolumab, or Cemiplimab. In some embodiments, the immune checkpoint inhibitor can include a PD-L1 inhibitor including, but not limited to, Atezolizumab, Avelumab, or Durvalumab. In some embodiments, the immune checkpoint inhibitor can include a LAG-3 inhibitor including, but not limited to, relatlimab. In some embodiments, the immune checkpoint inhibitor can include a CTLA-4 inhibitor including, but not limited to, Ipilimumab. In some embodiments, the immune checkpoint inhibitors are administered. In some embodiments, the immune checkpoint inhibitors can include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, CD47 inhibitors, B7 inhibitors, CD 137 inhibitors, or CTLA-4 inhibitors.

[0110] In some embodiments, the composition including the enucleated cell is administered simultaneously with the one or more additional therapies. In some embodiments, the composition including the enucleated cell is administered separately from the one or more additional therapies.

[0111] In some embodiments, the enucleated cells comprise an additional therapeutic agent, such as those disclosed herein. In some embodiments, the composition comprising the enucleated cells is formulated for administration to a subject disclosed herein with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject sequentially, simultaneously, substantially sequentially, or substantially simultaneously.

[0112] In some embodiments of any of the compositions provided herein, the composition further includes one or more additional therapies (e.g., chemotherapy (e.g., a chemotherapeutic agent (e.g., doxorubicin, paclitaxel, cyclophosphamide), cell-based therapy, radiation therapy, immunotherapy, a small molecule, an inhibitory nucleic acid (e.g., antisense RNA, antisenseATTORNEY DOCKET NO.38394.0028P1 DNA, miRNA, siRNA, IncRNA) or surgery). C. Compositions

[0113] Disclosed herein are compositions and formulations thereof comprising one or more enucleated cells described herein capable of being extensively engineered to express an active agent, or portion thereof, in the absence of a nucleus. Such enucleated cells are viable cell-like entities capable of synthesizing, releasing (e.g., secreting), or delivering the active agent to a target cell or tissue in the absence of the nucleus. The compositions disclosed herein can be stored in a suspended biological stage by means such as cryohibernation, cryopreservation, or lyophilization for any period of time without impacting the viability of the enucleated cell once the biological activity is revived. Moreover, the compositions disclosed herein comprise less than or equal to about 0.05%, or less than or equal to about 1% of nucleated cells (e.g., parent cells that were not enucleated during the enucleation process), rendering the compositions disclosed herein optimal for therapeutic applications. The enucleated cells (as referred to here as “cytoplasts”) may further comprise naturally occurring cell-surface molecules retained from the parent cell. In some embodiments, the enucleated cells further comprise exogenous molecules, such as a targeting moiety, a transmembrane moiety, an additional therapeutic agent (e.g., other than the active agent) such as those disclosed herein 1. Pharmaceutical Compositions

[0114] In some instances, the compositions can further comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0115] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising the compositions disclosed herein and a pharmaceutically acceptable: carrier, excipient, diluent, or nebulized inhalant. In some embodiments, the compositions disclosed herein comprise one or more of the enucleated cells disclosed herein. In some embodiments, the compositions disclosed herein comprise one or more active agents or therapeutic agents. In some embodiments, the pharmaceutical composition disclosed herein comprise at least one additional active agent. In some embodiments, the pharmaceutical compositions disclosed herein comprise two or more active or therapeutic agents.

[0116] In some embodiments, the pharmaceutical composition is in a unit dose form. In some embodiments, the compositions comprise two or more active agents, or two or more therapeutic agents as disclosed herein. In some embodiments, the two or more active agents are contained in a single dosage unit, such as for example, when the enucleated cell comprises twoATTORNEY DOCKET NO.38394.0028P1 or more therapeutic agents. In embodiments, the two or more active agents are contained in separate dosage units, such as when the enucleated cell is administered separately from an additional therapeutic agent or adjuvant. In some embodiments, the pharmaceutical composition described herein comprises at least one additional active agent other than the enucleated cell described herein. In some embodiments, the at least one additional active agent is a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or any combination thereof. In some embodiments, the at least one additional active agent is a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, cardio protectant, and / or checkpoint inhibitor. Non-limiting checkpoint inhibitor includes IMP321 / Eftilagimod alpha (Immutep), Relatlimab BMS-986016, Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy), LAG525, MK- 4280, Irinotecan, Oxaliplatin, REGN3767, TSR-033, BI754111, Sym022, FS118 (a bi-specific anti-LAG3 / PD-L1 antagonistic mAb), MGD013 (a bi-specific anti-LAG3 / PD-1 antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym023, INCAGN2390, LY3321367, Ramucirumab, Abemaciclib, Merestinib, BMS-986258, SHR-1702, Camrelizumab, MK-7684, Etigilimab / OMP-313 M32, Tiragolumab / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA-170d, Enoblituzumab / MGA271, MGD009, I-8H9 / omburtamab,Trastuzumab, MGD013 (Anti-PD-1, anti-LAG-3 dual checkpoint inhibitor), BGB-A1217, CM-24 (MK-6018), BMS 986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, Utomilimab (PF-05082566), Urelumab (BMS-663513) ES101, BMS-986156, TRX-518, AMG 228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or Varlilumab (CDX-1127). Such compounds or drugs may be present in combination in amounts that are effective for the purpose intended. Additional non-limiting examples of the additional therapeutic agent comprise CPI-006 (for inhibiting CD73 and allowing T cell and APC activation); Monalizumab (for inhibiting NKG2A); COM701 (for inhibiting PVRIG / PVRL2 and activating T cell); CM24 (for inhibiting CEACAM1 and allowing T and NK cells activation); NEO-201 (for inhibiting CEACAM5 and CEACAM6 which allows T cell activation while interfering with tumor cell growth); Defactinib (for inhibiting FAK and interfering with tumor growth); PF-04136309 (for inhibiting CCR-2 and CCL-2 and allowing T cell recruitment and activation); MSC-1 (for inhibiting LIF and allowing T cell and APC activation while interfering with cancer growth); Hu5F9-G4 (5F9), ALX148, TTI-662, and RRx-001 (for inhibiting CD47 or SIRPα and allowing T cell and APC activation); Lacnotuzumab (MCS-110), LY3022855, SNDX-6352, Emactuzumab (RG7155), and Pexidartinib (PLX3397) (for inhibiting M-CSF orATTORNEY DOCKET NO.38394.0028P1 CSF-1R and allowing APC activation); CAN04 and Canakinumab (ACZ885) (for inhibiting IL- 3 or IL-1RAP and allowing T cell and APC activation); BMS-986253 (for inhibiting IL-8 and decreasing immunosuppressive tumor microenvironment while interfering with tumor growth); Pepinemab (VX15 / 2503) (for inhibiting SEMA4D and decreasing immunosuppressive tumor microenvironment while interfering with tumor growth); Trebananib (for inhibiting Angiopoietin-2 and allowing APC activation while interfering with cancer growth); FP-1305 (for inhibiting CLEVER-1 and allowing APC activation); Enapotamab vedotin (EnaV) (for inhibiting Axl and allowing APC activation while interfering with cancer growth); or Bavituximab (for inhibiting phosphatidylserine and allowing T cell and APC activation while interfering with cancer growth).

[0117] The compositions may comprise at least an exogenous therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and compositions described herein include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some embodiments, therapeutic agents exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents are also considered to be disclosed herein.

[0118] In certain embodiments, compositions provided herein comprise one or more preservatives to inhibit microbial activity. Suitable preservatives comprise mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0119] In some embodiments, compositions described herein benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof. 2. Formulations for Administration

[0120] The compositions described herein are formulated into any suitable dosage form,ATTORNEY DOCKET NO.38394.0028P1 including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations. In some embodiments, the compositions (e.g., pharmaceutical compositions disclosed herein) are formulated for administering intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, intratumorally, pulmonarily, endotracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhaled nebulized form, by intraluminal-GI route, or any combination thereof, to a subject. In some embodiments, the compositions (e.g., pharmaceutical compositions disclosed herein) are formulated for administering intravenously. In one embodiment, a therapeutic agent as discussed herein, e.g., therapeutic agent is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one embodiment, formulations suitable for intramuscular, subcutaneous, or intravenous injection comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms may be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0121] For intravenous injections or drips or infusions, a composition described herein is formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosalATTORNEY DOCKET NO.38394.0028P1 administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations comprise aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[0122] Parenteral injections may involve bolus injection or continuous infusion. Compositions for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0123] For administration by inhalation, a therapeutic agent is formulated for use as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base such as lactose or starch. Formulations that comprise a composition are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. The choice of suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0124] Pharmaceutical preparations for oral use are obtained by mixing one or more solid excipient with one or more of the compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, forATTORNEY DOCKET NO.38394.0028P1 example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents are added, such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic agent doses.

[0125] In some embodiments, the compositions of the exogenous therapeutic agents are in the form of a capsules, including push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push fit capsules contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active therapeutic agent is dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some embodiments, stabilizers are added. A capsule may be prepared, for example, by placing the bulk blend of the formulation of the therapeutic agent inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule is swallowed whole or the capsule is opened and the contents sprinkled on food prior to eating.

[0126] Compositions for oral administration are in dosages suitable for such administration. In one embodiment, solid oral dosage forms are prepared by mixing a composition with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some embodiments, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite- disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the composition is in the form of a powder. Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, tablets will include one or more flavoring agents. In other embodiments, the tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating may provide a delayed release of a therapeutic agent from theATTORNEY DOCKET NO.38394.0028P1 formulation. In other embodiments, the film coating aids in patient compliance. Film coatings may range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit dosages comprise film coatingsIn another embodiment, dosage forms comprise microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Non-limiting example of materials includes pH modifiers, erosion facilitators, anti- foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0127] Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to therapeutic agent the liquid dosage forms optionally comprise additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions further comprise a crystal-forming inhibitor.

[0128] In some embodiments, the compositions described herein are self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.

[0129] The compositions (e.g., pharmaceutical compositions) described herein may be formulated for administration to a subject by administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes.ATTORNEY DOCKET NO.38394.0028P1 The composition described herein may include, but not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended-release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0130] Buccal formulations are administered using a variety of formulations known in the art. In addition, the buccal dosage forms described herein may further comprise a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a suitable manner.

[0131] For intravenous injections, a composition is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For other parenteral injections, appropriate formulations comprise aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

[0132] Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some embodiments, a composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and / or dispersing agents. The compositions for parenteral administration comprise aqueous solutions of an agent that modulates the activity of a carotid body in water soluble form. Additionally, suspensions of an agent that modulates the activity of a carotid body are optionally prepared as appropriate, e.g., oily injection suspensions.

[0133] Suitable formulation techniques comprise, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods comprise, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Wurster coating), tangential coating, top spraying, tableting, extruding and the like.

[0134] In some embodiments, the compositions are provided that comprise particles of a therapeutic agent and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations may be a powder and / or granule for suspension, and upon admixtureATTORNEY DOCKET NO.38394.0028P1 with water, a substantially uniform suspension is obtained.

[0135] Furthermore, the compositions optionally comprise one or more pH adjusting agents or buffering agents, comprising acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0136] Additionally, the compositions optionally comprise one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0097] Other compositions optionally comprise one or more preservatives to inhibit microbial activity. Suitable preservatives comprise mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0137] In one embodiment, the aqueous suspensions and dispersions described herein remain in a homogenous state for at least 4 hours. In one embodiment, an aqueous suspension is re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

[0138] An aerosol formulation for nasal administration is generally an aqueous solution designed to be administered to the nasal passages in drops or sprays. Nasal solutions may be similar to nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values outside of this range may additionally be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0139] An aerosol formulation for inhalations and inhalants may be designed so that the agent or combination of agents is carried into the respiratory tree of the subject when administered by the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer. Inhalations or insufflations, comprising finely powdered or liquid drugs, may be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant, e.g., to aid in disbursement. Propellants may be liquefied gases, comprising halocarbons, for example, fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, asATTORNEY DOCKET NO.38394.0028P1 well as hydrocarbons and hydrocarbon ethers.

[0140] Aerosol formulations may also comprise other components, for example, ethanol, isopropanol, propylene glycol, as well as surfactants or other components such as oils and detergents. These components may serve to stabilize the formulation and / or lubricate valve components.

[0141] The aerosol formulation may be packaged under pressure and may be formulated as an aerosol using solutions, suspensions, emulsions, powders and semisolid preparations. For example, a solution aerosol formulation comprises a solution of an agent such as a transporter, carrier, or ion channel inhibitor in (substantially) pure propellant or as a mixture of propellant and solvent. The solvent may be used to dissolve the agent and / or retard the evaporation of the propellant. Solvents may comprise, for example, water, ethanol and glycols. Any combination of suitable solvents may be use, optionally combined with preservatives, antioxidants, and / or other aerosol components.

[0142] An aerosol formulation may be a dispersion or suspension. A suspension aerosol formulation comprises a suspension of an agent or combination of agents, e.g., a transporter, carrier, or ion channel inhibitor, and a dispersing agent. Dispersing agents may comprise, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil. A suspension aerosol formulation may also comprise lubricants, preservatives, antioxidant, and / or other aerosol components.

[0143] An aerosol formulation may similarly be formulated as an emulsion. An emulsion aerosol formulation may comprise, for example, an alcohol such as ethanol, a surfactant, water and a propellant, as well as an agent or combination of agents, e.g., a transporter, carrier, or ion channel. The surfactant used may be nonionic, anionic or cationic. One example of an emulsion aerosol formulation comprises, for example, ethanol, surfactant, water and propellant. Another example of an emulsion aerosol formulation comprises, for example, vegetable oil, glyceryl monostearate and propane. D. Methods

[0144] Disclosed herein are methods of treating cancer in a subject, methods of increasing a subject’s responsiveness to immune checkpoint inhibitors, methods of increasing a subject's responsiveness to immunotherapy, methods of increasing the amount of T cells at the site of a tumor, methods of activating and reactivating innate cells like macrophages, etc.

[0145] In some embodiments the methods described herein comprise administering one or more of the enucleated cells described herein.

[0146] In some embodiments, delivering CD40L to a cancer patient via the disclosedATTORNEY DOCKET NO.38394.0028P1 enucleated cells can activate CD40 expressing antigen presenting cells (e.g., macrophages, B cells). Activation of antigen presenting cells can facilitate and improve their antigen presentation function, allowing them to acquire antigen from their environment (i.e., the tumor microenvironment) and present antigen to and activate T cells. In some embodiments, this facilitates de novo T cell activation against one or multiple tumor antigens. In some embodiments, activation of antigen presenting cells from an enucleated cell expressing CD40L, as disclosed herein, can be beneficial in a tumor in which specific immunogenic antigens have not been identified, or where there is not a single immunodominant epitope. Thus, in some embodiments, the disclosed methods are directed to a more generic or global response rather than a single antigen specific response.

[0147] Disclosed are methods of increasing a subject’s responsiveness to immune checkpoint inhibitors comprising administering to the subject one or more enucleated cells disclosed herein. In some aspects, increasing a subject’s responsiveness to immune checkpoint inhibitors results in a reduction in the subject’s tumor burden.

[0148] In some embodiments, the subject has previously been administered an immune checkpoint inhibitor. In some embodiments, the subject has previously been administered an immune checkpoint inhibitor and has a decreased response to the immune checkpoint inhibitor or no longer is responsive to the immune checkpoint inhibitor.

[0149] In some embodiments, the subject has not previously been administered an immune checkpoint inhibitor. In some embodiments, the subject is simultaneously administered an immune checkpoint inhibitor. In some embodiments of a subject being simultaneously administered an immune checkpoint inhibitor and an enucleated cell, the enucleated cell can comprise the immune checkpoint inhibitor. In some embodiments of a subject being simultaneously administered an immune checkpoint inhibitor and an enucleated cell, they are each formulated separately.

[0150] In some embodiments, the subject is administered an immune checkpoint inhibitor at least 24hrs after receiving the one or more enucleated cells. In some embodiments, the subject is administered an immune checkpoint inhibitor at least 1, 2, 3, 4, 5, 6, or 7 days after receiving the one or more enucleated cells. In some embodiments, the subject is administered an immune checkpoint inhibitor at least 1, 2, 3, or 4 weeks after receiving the one or more enucleated cells

[0151] Disclosed are methods of increasing T cells at a tumor site in a subject comprising administering to the subject one or more of the disclosed enucleated cells. In some embodiments, the one or more enucleated cells activate antigen presenting cells capable of priming T cells causing an increase of T cells at the tumor site.ATTORNEY DOCKET NO.38394.0028P1

[0152] Disclosed are methods of treating a subject having cancer comprising administering to the subject one or more enucleated cells disclosed herein.

[0153] In some embodiments, the cancer is multiple myeloma, glioblastoma, lymphoma, leukemia, mesothelioma, sarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, lung cancer, or any combination thereof. In some embodiments, the cancer is Acute biphenotypic leukemia, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute Lymphocytic Leukemia, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Adamantinoma, Adenocarcinoma of the lung, Adrenal Cancer, Adrenocortical adenoma, Adrenocortical carcinoma, AIDS-related lymphoma, Anal cancer, Anaplastic large cell lymphoma, Angioimmunoblastic T-cell lymphoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal and Squamous Cell Skin Cancer, Basal cell carcinoma, Basaloid squamous cell lung carcinoma, B-cell prolymphocytic leukemia, Bile Duct Cancer, Bladder cancer, Blastoma, Blood Cancer, Bone Cancer, Brainstem glioma, Breast cancer, Bronchial adenomas / carcinoids, Burkitt's lymphoma, Carcinoid, Carcinoid tumor, Cerebellar astrocytoma, Cerebral astrocytoma, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus carcinoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia, Colon cancer, Colorectal Cancer, Conjunctival melanoma, Craniopharyngioma, Cutaneous T- cell lymphoma, Diffuse large B-cell lymphoma, Ductal carcinoma in situ, Duodenal cancer, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Esophagus Cancer, Ewing Family of Tumors, Ewing's sarcoma, Extragonadal germ cell tumor, Extrahepatic bile duct cancer, Fallopian tube cancer, Fibrosarcoma, Follicular lymphoma, Gallbladder cancer, Ganglioneuroma, Gastric cancer, Gastrinoma, gastrointestinal, Gastrointestinal carcinoid tumor, Gastrointestinal Neuroendocrine (Carcinoid) Tumors, Gastrointestinal stromal tumor (GIST), Gestational trophoblastic tumor, Giant-cell carcinoma of the lung, Glioblastoma, Glioma, Glucagonoma, Hairy cell leukemia, Head and neck cancer, Head and Neck Cancers, Hemangioblastoma, Hepatoblastoma, Hepatocellular cancer, Hepatosplenic T-cell lymphoma, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal cancer, Inflammatory breast cancer, Insulinoma, Intravascular large B-cell lymphoma, Invasive cribriform carcinoma), Invasive ductal carcinoma, Invasive lobular carcinoma, islet cell, Islet cell carcinoma, Kaposi Sarcoma, Keratoacanthoma, Kidney cancer, Large granular lymphocytic leukemia, Large-cell lung carcinoma, Laryngeal and Hypopharyngeal Cancer, Laryngeal cancer, Leiomyosarcoma, Leukemia, Leukemia in Children, Leydig cell tumor, Liposarcoma, Liver Cancer, Lung Cancer, Lung Carcinoid Tumor, Lymphoma, Lymphoma of the Skin,ATTORNEY DOCKET NO.38394.0028P1 Lymphomatoid granulomatosis, Lymphoplasmacytic lymphoma, Male breast cancer, Malignant fibrous histiocytoma of bone / osteosarcoma, Malignant Oncocytoma, Mammary secretory carcinoma, Mantle cell lymphoma, Marginal zone B-cell lymphoma, Mast cell leukemia, Mediastinal large B cell lymphoma, Medullary carcinoma, Medulloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesothelioma, Mucinous carcinoma of the breast, Mucosa-associated lymphoid tissue lymphoma, Multiple endocrine neoplasia syndrome, Multiple myeloma / plasma cell neoplasm, Mycosis fungoides, Myelodysplastic syndromes, Myxosarcoma, Nasal Cavity and Paranasal Sinuses Cancer, Nasopharyngeal carcinoma, Neuroblastoma, Neurofibroma, Nodal marginal zone B cell lymphoma, Non-Hodgkin lymphoma, Non-small cell lung cancer, Non-small cell lung carcinoma, Ocular Melanoma, Oligodendroglioma, Optic nerve glioma, Oral cancer, Orbital lymphoma, Oropharyngeal cancer, Osteosarcoma, Ovarian cancer, Pancreatic cancer, Papillary carcinomas of the breast, Paraganglioma, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Phyllodes tumor, Pilocytic astrocytoma, Pineal astrocytoma, Pineoblastoma, Pineocytoma, Pituitary Tumors, Plasmablastic lymphoma, Pleuropulmonary blastoma, Precursor B lymphoblastic leukemia, Primary central nervous system lymphoma, Primary cutaneous follicular lymphoma, Primary cutaneous immunocytoma, Primary effusion lymphoma, Primitive neuroectodermal tumor, Prostate cancer, Rectal cancer, Renal cell carcinoma, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcomatoid carcinoma of the lung, Schwannoma, Seminoma, Serous tumour, Sertoli cell tumour, Sézary syndrome, Skin adnexal tumors, Skin Cancer, Small cell lung cancer, Small intestine cancer, Soft Tissue Sarcoma, Somatostatinoma, Splenic marginal zone lymphoma, Squamous cell carcinoma, Squamous cell skin cancer, Squamous-cell carcinoma of the lung, Stomach Cancer, Synovial sarcoma, T-cell prolymphocytic leukemia, Teratoma, Testicular cancer, Thymus Cancer, Thyroid cancer, Tubular carcinoma, Ureter and renal pelvis, Urethral cancer, urothelial carcinoma, Uterine sarcoma, Uveal melanoma, Vaginal cancer, VIPoma, Vulvar cancer, Waldenstrom Macroglobulinemia, Wilms tumor, or any other cancer known in the art.

[0154] In some embodiments, the adaptive immune system is activated. In some embodiments, this can occur once the CD40L polypeptide on the enucleated cells helps activate antigen presenting cells which allows them to acquire antigen from the environment (e.g., acquire tumor antigens from the tumor microenvironment)

[0155] In some embodiments of the disclosed methods, tumor growth is slowed.

[0156] In some embodiments, the method of treating further comprise administering an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor can beATTORNEY DOCKET NO.38394.0028P1 formulated within the enucleated cell comprising the CD40L polypeptide or can be formulated separately. In some embodiments, when formulated separately, the immune checkpoint inhibitor can be still be formulated in an enucleated cell, just not the same one comprising the CD40L polypeptide. Thus, disclosed are methods of treating a subject comprising administering one or more of the enucleated cells that comprises CD40L or a modified CD40L, and administering one or more of the enucleated cells comprising an immune checkpoint inhibitor. In some embodiments the one or more enucleated cells that comprise CD40L or a modified CD40L and the one or more enucleated cells comprising an immune checkpoint inhibitor can be the same or different enucleated cell.

[0157] In some embodiments of the disclosed methods, particularly upon combination treatment with the disclosed enucleated cells and an immune checkpoint inhibitor, there is an improvement in tumor clearance, immunosuppressive state is reversed, and / or overall survival rates are increased.

[0158] In some embodiments, the methods described here further comprise an additional treatment. For example, the additional treatment may comprise a radiation therapy, tumor irradiation, or other tumor therapy. For example, the additional treatment may comprise administration of an immunotherapy. Disclosed are methods of treating a subject comprising: administering to the subject one or more enucleated cells described herein, and administering to the subject a radiation therapy.

[0159] In some embodiments, disclosed are methods of recruiting one or more T-cells to a tumor site comprising administering to a subject one or more enucleated cells described herein.

[0160] In some embodiments, the methods can relate to increasing the concentration of T cells at a tumor site, comprising the administration of an enucleated cell described herein.

[0161] In some embodiments, disclosed are methods of killing or inducing apoptosis of a cancer cell comprising administering one or more enucleated cells described herein. In some embodiments, administering can mean contacting a cancer cell with one or more enucleated cells described herein.

[0162] In some embodiments, disclosed are methods of inducing B cell proliferation comprising administering one or more enucleated cells described herein. In some embodiments, administering can mean contacting a B cell with one or more enucleated cells described herein. In some embodiments, the B cell proliferates upon exposure to the CD40L polypeptide of the enucleated cell. In some embodiments, CD40 on the surface of a B cell binds to CD40L polypeptide on the surface of the one or more enucleated cells, allowing for activation and proliferation.ATTORNEY DOCKET NO.38394.0028P1

[0163] In some embodiments, disclosed are methods of activating NF- κB comprising administering one or more enucleated cells described herein. In some embodiments, administering can mean contacting a cell (e.g., B cell) with one or more enucleated cells described herein. In some embodiments, NF-κB is activated upon binding of the CD40L polypeptide on the surface of the one or more enucleated cells to CD40 on the cell.

[0164] Also described herein are methods of administering compositions, such as pharmaceutical compositions, comprising one or more enucleated cells described herein in any of the disclosed methods. E. Kits

[0165] The compositions and materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits for producing the disclosed enucleated cells.

[0166] Disclosed herein are kits comprising the enucleated cells or compositions disclosed herein and packaging material configured to deliver the composition to an individual. The kits disclosed herein may comprise a composition comprising a enucleated cell fraction and less than 0.05% nucleated cell fraction. The kits disclosed herein may comprise a composition comprising a enucleated cell fraction and less than 0.1% nucleated cell fraction. The kits disclosed herein may comprise a composition comprising a enucleated cell fraction and less than 1% nucleated cell fraction. The kits disclosed herein may comprise a composition comprising an enucleated cell fraction and less than 5% nucleated cell fraction. In some embodiments, the kits further comprise instructions for further engineering the enucleated cells in the enucleated cell fraction, such as for example, to produce or secrete a therapeutic agent disclosed herein. In some cases, the instructions may further comprise instructions for how to formulate the resulting composition into a pharmaceutical formulation for administration to a subject disclosed herein. Examples A. Example 1 -Active delivery of native CD40 agonist to the tumor microenvironment

[0167] Cargocytes are an innovative approach to deliver native CD40 agonists into the diffuse and complex TME of peritoneal carcinomatosis. A major challenge in delivering CD40 agonist therapy lies in the physical events that must occur to initiate CD40 signaling in a target cell. CD40 is a member of the tumor necrosis factor receptor superfamily, and as such requires trimerization in order to recruit and engage its intracellular signaling complex partners. SolubleATTORNEY DOCKET NO.38394.0028P1 versions of CD40L, while having high affinity for CD40, are weak activators of CD40 signaling compared to membrane-bound CD40L. While CD40 agonist Abs have demonstrated robust immune activity in preclinical animal models, efficacy in clinical studies has been disappointing. This poor efficacy and some peripheral toxicity events can be explained by the interaction ofCD40 Abs with different Fc receptors (Fc R). The ability to exert robust agonist activityrequires binding of CD40 Abs to Fc Rs, but, depending on the Fc R subtype, has the potential totrigger Ab-dependent cellular phagocytosis or Ab-dependent cellular cytotoxicity. Cargocytes, by contrast, overcome these barriers as exemplified in FIG.1 by delivering membrane-boundCD40L and bypassing the need and potential complication of requiring Fc R binding.

[0168] CD40 is a powerful immunoadjuvant, and multiple clinical trials are in progress, or have been completed, to address its potential as a cancer immunotherapeutic. Clinical use products include CD40 agonist Abs with Fc domain modifications or soluble CD40L peptide fragments, commonly in context with ICB. The agonist potential of anti-CD40 Abs is a complex interplay of affinity, epitope location and interaction with native FcγRs. Thus, the translation of promising pre-clinical efficacy has been slow, with adverse effects resulting in the need to reduce to sub-optimal doses. Cell-based delivery of a native CD40 agonist on CAR-T and mesenchymal stromal cells (MSCs) has been achieved in nonclinical studies. While the addition of CD40L to CAR-T cells has improved therapy to liquid tumors, CAR-T treatment of solid tumors is still impaired by exclusion from the TME. In contrast, delivery of CD40L on MSCs capitalizes on their innate tumor trophic capacity. However, MSCs present safety concerns as they retain genomic DNA and produce a range of immunosuppressive cytokines at high concentration raising concerns regarding their potential to engraft and support a pro-tumor environment.

[0169] CA-CD40L has advantages of over other CD40-targeted immunotherapies. A drug delivery platform of enucleated MSCs called “CargocytesTM” are ideally suited for local delivery of native CD40 agonist, CD40L into the TME. These advantages include, but are not limited to the following: 1) Like MSCs, Cargocytes are migratory and tumor-trophic. Cargocytes lack a rigid nucleus yet retain the ability to reorganize their actin cytoskeleton and are therefore able to migrate towards chemokines released by inflamed tissue. Since Cargocytes are morphologically malleable, they can migrate through smaller spaces. Most importantly, when administered systemically, cargocytes penetrate deeply into the TME (See e.g. FIG.2B).2) Cargocytes are safe. Removal of the nucleus and genomic DNA eliminates primary safety concerns of unpredictable cellular differentiation, the potential to produce unwanted gene products and carcinogenicity in vivo.3) Cargocytes are biosynthetically active. Cargocytes retain allATTORNEY DOCKET NO.38394.0028P1 biosynthetic organelles, including endoplasmic reticulum, Golgi and vesicular transport machinery, enabling them to translate synthetic mRNAs encoding immunomodulatory proteins and biologics into functional proteins at the desired therapeutic target site. Thus, Cargocytes that are derived from lentivirus-transduced MSCs encoding CD40L retain steady-state levels of CD40L over their lifespan, largely because the parental MSC derivatives express thousands of copies of CD40L mRNA that can be translated into plasma membrane CD40L (See e.g. FIG. 2C).4) Cargocytes express plasma membrane CD40L in its native state in its natural environment. Unlike soluble CD40 agonists (CD40 Abs or peptide fragments of CD40L), expression of CD40L in its native state on the plasma membrane of a Cargocyte more closely mimics the natural presentation of CD40L to CD40 on the membrane of an opposing antigen presenting cell and activated T cells. The biological activity of CA-CD40L is shown in FIGS .2- 4.5) Cargocytes exhibit an improved pharmacokinetic (PK) profile relative to inert therapeutic delivery systems. Because Cargocytes are an active delivery platform that home towards and penetrate into target organs, their absorption, distribution, metabolism and elimination (ADME) characteristics are improved over passive delivery systems, like antibodies (and other targeting motifs), nanoparticles, exosomes, RBCs, and various combinations of encapsulations platforms. 6) Cargocytes can be cryopreserved, biobanked and stored in liquid nitrogen without loss of viability or therapeutic function. The enucleation process and proprietary post-enucleation purification is highly efficient (99.9999%) and is readily scalable for commercial manufacturing. Cytonus has the capacity to enucleate 8L of MSCs per manufacturing run per day, with collaboration partners Thermo Fisher. 1. Experimental Data i. Cargocytes deliver native CD40 agonist (CA-CD40L) to the TME.

[0170] Cargocytes retain the ability of their parental MSC to migrate towards chemoattractant gradients. Without a rigid nucleus they can traverse smaller constrictive areas than nucleated MSCs (FIG.2A). The combination of chemotaxis and mobility allows Cargocytes to migrate to and intercalate into tumor mass in vivo (FIG.2B). This allows for the delivery of CD40 agonist directly to the tumor and surrounding tissue. To generate Cargocytes that express native CD40L, MSCs were first stably engineered with a CD40L expressing lentivirus. Flow cytometry analysis of these cells demonstrates high expression of CD40L (FIG. 2C). Enucleation of the MSC-CD40L for the generation of Cargocytes expressing CD40L (CA- CD40L) does not diminish CD40L expression (FIG. 2C). CD40L expressed on both MSCs and Cargocytes is capable of activating NF-κB downstream of CD40 in a reporter system (FIG.2D), demonstrating that the ligand is bioactive.ATTORNEY DOCKET NO.38394.0028P1 ii. Membrane CD40L stimulates the activation of antigen-presenting cells.

[0171] CD40 ligation on immune cell subsets triggers different signaling pathways with cell- dependent outcomes. Dendritic cells (DCs) upregulate MHC and T cell co-stimulatory molecules, gearing up to present antigen to and activate cognate T cells. B cell CD40 activation readies antigen-presentation machinery but also induces robust proliferation and differentiation. These diverse outcomes of CD40 agonism were tested in vitro. Co-culture of mouse splenocytes with MSC-CD40L activated DC populations and induced the expression of CD86 (FIG.3A), a key T cell co-stimulatory molecule. CD40L also increased the expression of MHC-I on this population (FIG.3A), indicating the licensing of DCs by CD40L expressing cells to interact with CD8+T cells, the critical anti-tumor population. Next, MSCs were cultured with freshly isolated splenic B cells and observed robust B cell proliferation when the MSCs expressed CD40L. Furthermore, this proliferation was abrogated when a CD40L blocking Ab was added to the culture (FIG.3B), highlighting the specificity of the CD40-CD40L interaction. Together, these experiments demonstrate the predicted action of CD40 activation on mouse immune cells by the natively expressed CD40L. iii. Delivery of Cargocytes provides ideal biodistribution to safely deliver CD40L to peritoneal carcinomas.

[0172] Delivery of Cargocytes via intraperitoneal (i.p.) injection results in their distribution throughout the peritoneal cavity with strong retention for at least 24 hours (FIG.4A). Areas of strong Cargocyte retention mimic the areas that ID8 ovarian carcinoma cells gather and grow in (FIG.4B), indicating that even without chemotaxis to cancerous growth factors, Cargocytes are uniquely capable to accumulating in sites key to metastatic growth. The ID8 model is ideal for testing the efficacy and ability to improve upon immune checkpoint blockade. Tumor growth is slow and steady for the first 20 days after implantation (FIG.4B), giving the immune system time to respond to immune therapy before serious disease sets in. DCs are elevated in the peritoneal cavity in ID8-bearing mice (FIG. 4B), a key immunologic target population of CD40L therapy. Macrophages in the peritoneal cavity display a distinct pro-tumor M2-like phenotype with elevated CD206 expression (FIG. 4B). Furthermore, mice bearing ID8 tumors fail to produce CD8+effector T cells (FIG.3B, bottom right), highlighting a key reason that PD- 1 blockade has been shown to be ineffective in this model. Delivery of CA-mCD40L in this model can activate the accumulated DCs in the tumor microenvironment which will lead to activation and recruitment of tumor-specific effector CD8+T cells which can then respond to ICB treatment.ATTORNEY DOCKET NO.38394.0028P1 2. Compare the antitumor efficacy, immunity and toxicity of CA-CD40L versus CD40 agonistic antibodies for treatment of peritoneal carcinomatosis. 1. Rationale.

[0173] CD40-targeted therapy holds great promise as an immunoadjuvant therapy due to its ability to 1) stimulate maturation of professional APCs, i.e. DCs and B cells, leading to the licensing of tumor-specific CD8+ T cells, 2) promote macrophage-mediated destruction of the tumor stroma, releasing neoantigens, and 3) destroy the immunosuppressive TME by reducing the presence of T regulatory cells and M2-like macrophages. However, systemic administration of CD40 agonist therapies is linked with dose-limiting cytokine release syndrome and hepatotoxicity. In this aim, the hypothesis that Cargocytes expressing the native transmembrane CD40 agonist, CD40L, will actively penetrate the TME and ignite local and systemic adaptive immune response with little peripheral immunotoxicity is tested. Changes in the activation and function of key immune cell subsets are measured using established nonclinical murine models of peritoneal carcinomatosis. CA-CD40L can transform the TME from an immunologically “cold” to “hot” tumor based on key biomarkers of antitumor immunity. The other studies can address changes in anti-tumor efficacy of CA-CD40L in combination with ICB therapy. 2. Experimental Design.

[0174] Peritoneal metastases can be formed by inoculating ID8-fLuc or MC38-fLuc cells i.p. into Albino C57BL / 6 mice, and tumor growth can be monitored by whole body luminescence following administration of luciferin. Mice can be stratified into 4 treatment groups (n=6): 1) control (IgG), 2) empty Cargocytes (1e6) (CA), 3) CA-CD40L (1e6), or 4) αCD40 (clone IC10; 50 μg). Equivalent second and third doses can be administered in 3–5-day intervals. As necessary, in follow-on experiments, CA-CD40L dosing can be recalibrated based upon measures of CD40L-driven immune activation, including increased expression of MHC and costimulatory molecules CD86 / CD80 on APCs. Immune activity can be measured by leukocyte composition within local and systemic sites. Toxicity can be monitored by measuring serum concentrations of liver enzymes and cytokines. a) Analysis of tumor burden, toxicity and immune (re)activation

[0175] Animal health can be monitored after every treatment to identify signs of overt toxicity. Tumor burden can be quantified by bioluminescent imaging (BLI) 24 hours after each treatment. Between 1-7 days after the final treatment mice can be euthanized and immune cell phenotype and function can be assessed in local (peritoneal cavity / ascites, omentum and draining lymph node (dLN)) and systemic (blood, spleen and non-draining LNs) sites. The activation of DCs and B cells can be assessed by flow cytometry, quantifying the expression ofATTORNEY DOCKET NO.38394.0028P1 MHC-I, MHC-II, CD80 and CD86. The ability of DCs to stimulate naïve T cells, a function that is suppressed in the context of ovarian cancer, can be assessed using an in vitro suppression assay, where DCs from tumor bearing mice are cultured with naïve T cells of mixed allotypes. Frequencies of other immunosuppressive cells can be quantified by flow cytometry. CD206 expression on macrophages can be evaluated to distinguish anti-tumor M1-like (CD206-) from pro-tumor M2-like (CD206+). Myeloid-derived suppressor cells and T regulatory cells (TReg) are also quantified. The induction of effective anti-tumor T cell responses can be assessed by phenotyping CD8 T effector cells (TEff) and central memory cells. T cell function can be assessed by their ability to secrete Granzyme B, IFN-γ, or TNF-α. T cell exhaustion can be assessed by the expression of PD-1, CTLA-4, LAG-3 and Tim-3. Differences in immune cell phenotypes and functional status can be compared between groups using a Kruskal-Wallis test with Dunn’s multiple comparison test. b) Serum measures of toxicity.

[0176] Plasma concentrations of liver ALT / AST enzymes and cytokines (TNFα, IL-1β, IL-6 and IL-8) will be determined by ELISA and the specific expression of TNF-α in the liver will be taken as measures of hepatotoxicity and immunotoxicity. 3. Results a) Efficacy of CA-CD40L in activating antitumor immunity.

[0177] CA-CD40L can engage CD40 on B cells and DCs within the local environment of the peritoneal cavity, omentum and dLNs, resulting in their activation (increased expression of MHC and co-stimulatory molecules) and reversal of tumor-derived suppressive phenotype. Subsequently, a decrease in immunosuppressive cells (MDSCs, M2-like macrophages and TReg) in these tumor-adjacent tissues can occur, while observing an influx of functional TEff. The data shown in FIG.4A indicate that i.p. injected Cargocytes are retained within the peritoneal cavity and dLNs (including mediastinal LNs in the thoracic cavity). Thus, strong immune activation is not expected in systemic sites (spleen and ndLNs) when compared to the i.p. injected αCD40, nor associated signs of peripheral or hepatotoxicity. Nonetheless, effective immunotherapies, even when locally delivered, are tied with the generation of systemic immune memory. Thus, the emergence of T cell memory at systemic sites is not expected. Ultimately, CA-CD40L can induce adaptive and innate immune responses that can reprogram the immunologically “cold” TME into a “hot” TME without overt animal toxicity. b) Reduction in tumor burden.

[0178] The dosing regimen of CA-CD40L can slow tumor growth and metastatic progression. However, this response can be attenuated by immune cell exhaustion. Thus, localATTORNEY DOCKET NO.38394.0028P1 delivery of CA-CD40L in combination with PD-1 therapy can result in more robust and sustained antitumor activity. c) Measures of CA-CD40L toxicity.

[0179] 50 μg / dose CD40 agonist Ab can elicit anti-tumor immunity and efficacy but can result in increased ALT / AST serum levels and increased expression of IL-1β, IL-6, IL-8 and TNFα. In contrast, no increase in the serum concentrations of ALT / AST or cytokines can be measured in mice receiving CA-CD40L. 3. Determine if the addition of CA-CD40L to ICB improves tumor clearance and survival. 1. Rationale.

[0180] The advent of ICB has revolutionized cancer immunotherapy, however response rates in peritoneal carcinomatosis to ICB alone have been low, even if the primary tumor is responsive. Many factors contribute to this resistance, including the immunosuppressive state of the peritoneal cavity containing metastatic tumors and malignant ascites. Recent studies have demonstrated success in overcoming ICB resistance by combining ICB therapy with adjuvants that can reverse the immunosuppressive environment in the peritoneal cavity. The purpose of this study is to test if CA-CD40L therapy can sensitize peritoneal carcinomatosis to ICB therapy. T ability of CA-CD40L to improve tumor clearance and overall survival in combination with PD-1 blockade is evaluated. 2. Experimental Design.

[0181] PD-1 Ab therapy can be added to the CA-CD40L in the two peritoneal metastasis models. Tumor burden can be monitored by BLI and mice can be stratified into 4 treatment groups (n= 12): 1) CA + control hamster IgG (hIgG; 200 μg), 2) CA + PD-1 (clone J43; hIgG; 200 μg) 3) CA-CD40L + hIgG, and 4) CA-CD40L+ PD-1. Mice can receive the CA-CD40L optimal dose as determined in the earlier study described above. The effectiveness of CD40 agonism can be altered depending on the timing of ICB therapy. The kinetics of T cell exhaustion can be used to predict the dosing schedule of PD-1. Studies can be performed to test initiating ICB treatment before, concomitant with, or after CA-CD40L treatment in inducing the most robust tumor clearance. Tumor burden can be quantified by bioluminescent imaging (BLI) 24 hours after each treatment and then regularly throughout the course of disease. Survival rates can be determined from Kaplan Meier plots and the log-rank test. The durability and locality of the antitumor response can be evaluated by rechallenging surviving mice with tumor cells. Mice with no measurable primary tumors 60 days after treatment can be re-injected with tumor cells either i.p. (testing local immune memory) or s.c. in the flank (testing systemicATTORNEY DOCKET NO.38394.0028P1 immune memory) and tumor growth can again be monitored by BLI and measurement of flank tumor growth. 3. Results

[0182] I.p. delivery of CA-CD40L + PD-1 can reduce tumor burden, and prolong overall survival compared to CA-CD40L or PD-1 only animals. CA-CD40L + ICB treated mice that have cleared their tumor burden can have developed durable anti-tumor immune memory and can be resistant to tumor re-challenge, regardless of the location of tumor re-challenge. B. Example 2 – Stable Expression of Mouse CD40L

[0183] As shown in FIG.5, stable expression of mouse CD40L was achieved using lentivirus transduction and antibiotic selection. CD40L expression was uniform and high without additional enrichment or sorting procedures. The generation of Cargocytes by enucleation of CD40L expressing MSCs did not alter the expression of CD40L.

[0184] MSCs were transduced with a Lentivirus expressing hTert and a CD4K mutant and placed under drug selection, generating TC-MSCs. TC-MSCs were then transduced with a mouse CD40L expressing Lentivirus and placed under drug selection, generating TC-CD40L MSCs. These MSCs were enucleated using methods described in US Patent No.10,927,349, which is hereby incorporated herein by reference, generating TC-CD40L Cargocytes (CAs). MSCs or CAs were stained (or not) with an anti-mouse CD40L Ab and analyzed by flow cytometry. C. Example 3 - CD40L expressing MSCs activate mouse immune cells

[0185] shown in FIG.6, CD40L expressed on MSCs is functional in activating mouse immune cells. Mouse B cells proliferate when exposed to CD40L MSCs. This proliferation is inhibited when an antibody binds to CD40L and blocks interaction with CD40 in B cells.

[0186] MSCs or CD40L-MSCs were plated in triplicate. In some wells an anti-CD40L blocking Ab was added at 1μg / mL. MSCs were incubated for 1-2 hours to allow them to attach and spread. Mouse B cells were isolated from freshly prepared mouse splenocytes using magnetic bead negative selection. B cells were then stained with CellTrace Far red and plated over adherent MSCs.4 days later, B cells were collected from wells and analyzed for evidence of proliferation by dye dilution using flow cytometry. The division index, the average number of cell divisions that a cell in the original population has undergone, was determined using FlowJo analysis software. Data is mean ± SD. Statistical analysis between MSC-CD40L with and without anti-CD40L was calculated using a students-unpaired t-test. ***, p<0.001.ATTORNEY DOCKET NO.38394.0028P1 D. Example 4 - MSCs and cargocytes activate CD40 and downstream NF-κB in a reporter cell line

[0187] As shown in FIG.7, both MSCs and Cargocytes expressing CD40L have the same ability, per cell, to activate CD40 and downstream NF-κB in a reporter cell line. Thus, the CD40L expressed by both MSCs and Cargocytes is bioactive.

[0188] TC-MSCs, TC-CD40L MSCs and TC-CD40L Cargocytes (CAs) were serially diluted in a 96-well plate. HEK-Blue CD40L cells, HEK 293 cells expressing human CD40 and an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter, were added to the wells. Cultures were left to incubate overnight. After incubation, supernatant from cultures was added to SEAP detection reagent, QUANTI-Blue, and O.D. measurements were acquired using a TECAN reader. E. Example 5 - CD40L expressing D1-MSCs reduce mouse ovarian cancer tumor burden 24 hours post injection

[0189] As shown in FIGS.8A and 8B , CD40L expressing D1-MSCs reduce mouse ovarian cancer tumor burden 24 hours post injection. Post 24 hours, the increase in tumor burden is likely due to the immunosuppressive nature of fully nucleated MSCs, a response that has not been observed in the presence of Cargocytes.

[0190] Female, 8 week old Albino C57BL / 6 mice were injected with 5x106ID8-KMF-fLuc cells by intraperitoneal (i.p.) route. Average radiance of tumor cells was quantified 10 minutes post i.p. administration of fLuc substrate D-Luciferin by IVIS (PerkinElmer). Mice were stratified into two groups; Untreated and D1-mCD40L treated. D1 MSCs were transfected with a plasmid expressing mouse CD40L. Treated mice received 1x106D1-mCD40L MSCs i.p. on days 10, 15 and 21 post tumor cell implantation. BLI was quantified prior to MSC injection and 24 hours post injection. n=5 per group. F. Example 6 - ID8 mouse ovarian cancer model responds to CD40 activation

[0191] As shown in FIG.9A, The ID8 mouse ovarian cancer model responds to CD40 activation. Anti-CD40 Ab treatment reduces tumor burden in mice 24 hours post treatment.. The same treatment recruits Effector CD8+ T cells to the peritoneal space, which the tumor alone failed to do (FIG.9B), making this treatment an ideal model to test the theory that CD40L therapy would be effective at rendering checkpoint-blockade resistant tumors into sensitive tumors. Activation of CD40 in mice also reduced the expression of CD206 on peritoneal macrophages (FIG.9C), restoring an anti-tumor “M1-like” phenotype that is lost in the presence of the tumor alone.

[0192] Female, 8 week old Albino C57BL / 6 mice were injected with 5x106 ID8-KMF-fLucATTORNEY DOCKET NO.38394.0028P1 cells by intraperitoneal (i.p.) route. Average radiance of tumor cells was quantified 10 minutes post i.p. administration of fLuc substrate D-Luciferin by IVIS (PerkinElmer). Mice were stratified on day 7 and divided into groups. Some mice received 100μg of anti-CD40 Ab (Rat IgG, Clone 1C10) on days 7, 12 and 17. (FIG.9A) BLI was monitored on the day of injection and 24 hours post injection. On day 20, mice were euthanized and a peritoneal lavage was performed to isolated cells in the peritoneal cavity. Cells were stained with cocktails of fluorescently labeled antibodies in the presence of an Fc block and analyzed on a FACSCelesta. Cell populations were analyzed in FlowJo. (FIG.9B) Effector CD8+ T cells were defined as Live, CD45+, CD3+, CD8+, CD44+, CD62L-. (FIG.8C) CD206 expression on large peritoneal macrophages, defined as Live, CD45+, CD19-, Ly6G-, CD11b+, F4 / 80Hi. Data is mean ± SEM of 10 (FIG.9A) or 5 (FIG.9B, FIG.9C) per group. Statistical analysis (FIG.9B, FIG.9C) was performed by Kruskal-Wallis test with Dunn’s Multiple comparison test. *, p<0.05. G. Example 7

[0193] As shown in FIG.10, CD40L expressed on TC-Medinno MSCs is functional in activating Human B cells. Human B cells proliferate when exposed to MSCs or Cargocytes (CAs) expressing CD40L (mouse).

[0194] TC-Medinno or TC-Medinno-CD40L MSCs or CAs were plated in triplicate and incubated for 1-2 hours to allow them to attach and spread. Human PBMCs from 4 individual donors were thawed and stained with CellTrace Far red and plated over adherent MSCs / CAs.4 days later, PBMCs were collected from wells and analyzed for evidence of proliferation by dye dilution using flow cytometry. The division index, the average number of cell divisions that a cell in the original population has undergone, was determined using FlowJo analysis software. Data is mean ± SD, combine from 4 independent experiments using 4 unique donors. Statistical analysis was calculated via a repeated measures one-way ANOVA with the Holm-Šídák’s multiple comparison test. *, p<0.05; **, p<0.01. H. Example 8

[0195] Cargocytes expressing CD40L (CA-CD40L) activate local but not systemic antigen- presenting cell populations. A conventional method of activating CD40 by the intra-peritonealinjection of an agonist antibody ( CD40) leads to systemic immune activation includingcirculating inflammatory cytokines (A) and the liver enzyme ALT (B), and activation of both local (peritoneal cavity), draining (omentum) and systemic (spleen) localized immune cells. By contrast, the expression of CD40L on carogocytes restricts immune activation to key antigen presenting cell populations (B cells and dendritic cells (DCs)) only in local and draining sites and does not result in off-target cell activation (neutrophils and eosinophils) or systemicATTORNEY DOCKET NO.38394.0028P1 inflammation.

[0196] Female, 8 week old C57BL / 6 mice were injected with saline, 50μg CD40 agonist antibody (clone 1C10), 1x106naked cargocytes (CA) or 1x106naked cargocytes expressing mouse CD40L (CA-CD40L).24 hours later, mice were euthanized, blood was collected via cardiac puncture and a peritoneal lavage was performed to isolated cells in the peritoneal cavity, and spleen and the omentum were collected. Single cell suspensions were prepared from the omentum and spleen by mechanical separation (following digestion in Collagenase Type 1A and DNAse I for the omentum). Serum was isolated from blood. Serum cytokines were analyzed via a bead-based multiplex ELISA (FIG.11A) and Alanine aminotransferase (ALT) was quantified via ELISA (FIG.11B). Cells were stained with cocktails of fluorescently labeled antibodies in the presence of an Fc block and analyzed on a FACSCelesta. The expression of MCHI or CD86 was quantified as the geometric mean fluorescent intensity on cell populations in the peritoneal cavity (PerC; FIG.11C), omentum (FIG.11D) and spleen (FIG.11E). Cell populations were analyzed in FlowJo and defined in the live, CD45+population as follows. PerC Eosinphils: CD19-, Ly6G-, CD11c-, F4 / 80-, SSCHi. PerC B2 B cells: CD19+, CD11b-. PerC large peritoneal macrophages (LPMs) CD19-, Ly6G-, CD11c-, F4 / 80+, CD11b+, FSCHi. Omentume neutrophils: CD19-, CD11b+, Ly6G+. Omentum B2 B cells: CD19+, CD11b-. PerC conventional dendritic cells 2 (cDC2): CD19-, Ly6G-, CD11c+, CD11b+. Splenic neutrophils: CD19-, CD11b+, Ly6G+. Splenic B cells: CD19+. Splenic cDCs: CD19-, Ly6G-, SSCLo, CD11c+, CD11b+. Data is mean ± SEM of 4 per group. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s Multiple comparison test. *, p<0.05; **, p<0.01. I. Example 9

[0197] CA-CD40L can synergize with other cell therapies delivering immune activation signals (here CA-IL12) and induce superior immune cell activation.

[0198] Cargocytes (CA) expressing CD40L, secreting IL12, or empty controls (CA group) were seeded in 24-well plates and allowed to attach (100K per well). C57BL / 6 splenocytes were isolated by mechanical separation and red blood cells were lysed. Splenocytes (1M per well) were seeded over added to the wells and the cells were incubated for 24 hours at 37°C. Cells were collected and stained with a cocktail of antibodies in the presence of Tc block. Cells were analyzed on a FACSCelesta and phenotypes were identified using FlowJo. As shown in FIGS. 12A- 12C, CA-CD40L can synergize with other cell therapies (here CA-IL12) and induce superior immune cell activation.

[0199] The following phenotypes were identified in live, CD45+cells; FIG.12A shows B cells: CD19+; FIG.12B shows dendritic cells: CD19-, CD11cHi, MHCIIHi; FIG 12C showsATTORNEY DOCKET NO.38394.0028P1 macrophages: CD19-, F4 / 80+, CD11b+.

[0200] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

ATTORNEY DOCKET NO.38394.0028P1 CLAIMS We claim:

1. An enucleated cell comprising an exogenous nucleic acid molecule encoding a CD40L polypeptide or engineered to express a CD40L polypeptide.

2. The enucleated cell of claim 1, wherein the CD40L polypeptide is expressed by the enucleated cell.

3. The enucleated cell of any one of claims 1-2, wherein the CD40L polypeptide is expressed on the surface of the enucleated cell.

4. The enucleated cell of any one of claims 1-3, further comprising one or more organelles for expressing the CD40L polypeptide, 5. The enucleated cell of any one of claims 1-4, wherein the one or more organelles are endoplasmic reticulum, golgi apparatus, or vesicular transport machinery.

6. The enucleated cell of any one of claims 1-5, wherein the enucleated cell is an enucleated stem cell.

7. The enucleated cell of any one of claims 1-6, further comprising a targeting moiety.

8. The enucleated cell of claim 7, wherein the targeting moiety comprises a homing receptor specific to a ligand or receptor expressed by a target cell.

9. The enucleated cell of claim 8, wherein the target cell is a cancer cell.

10. The enucleated cell of claim 9, wherein the cancer cell is a multiple myeloma cell, glioblastoma cell, lymphoma cell, leukemia cell, mesothelioma cell, sarcoma cell, breast cancer cell, prostate cancer cell, ovarian cancer cell, pancreatic cancer cell, colon cancer cell, lung cancer cell, or any combination thereof.

11. The enucleated cell of any one of claims 1-10, further comprising an immune evasion moiety.

12. The enucleated cell of claim 11, wherein the immune evasion moiety comprises CD47, PD-L1, HLA-E, HLA-G, or a combination thereof.ATTORNEY DOCKET NO.38394.0028P1 13. The enucleated cell of any one of claims 1-12, wherein the enucleated cell is depleted of one or more immune recognition molecules.

14. The enucleated cell of claim 13, wherein the one or more immune recognition molecules are a human leukocyte antigen (HLA), a proteoglycan, a sugar moiety, an embryonic antigen, or any combination thereof.

15. The enucleated cell of any one of claims 1-14, further comprising an immune checkpoint inhibitor.

16. The enucleated cell of claim 15, wherein the immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a B7 inhibitor, a CD137 inhibitor, a CTLA-4 inhibitor, or any combination thereof.

17. The enucleated cell of any one of claims 1-16, wherein the CD40L polypeptide is a modified CD40L polypeptide.

18. The enucleated cell of claim 17, wherein the modified CD40L polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

19. The enucleated cell of any one of claims 1-18, wherein the CD40L polypeptide is expressed on the enucleated cell for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours.

20. The enucleated cell of any one of claims 1-19, wherein the enucleated cell expresses an abundance of the CD40L polypeptide that is at least 0.1 fold, at least 0.2 fold, at least 0.5 fold, at least 1.0 fold, at least 2.0 fold, or at least 5.0 fold higher than the enucleated cell expressing an abundance of a comparable naturally-occurring CD40L polypeptide.

21. The enucleated cell of claim 20, wherein the abundance of the CD40L polypeptide and the abundance of the comparable naturally-occurring CD40L polypeptide is determined by FACS.

22. The enucleated cell of any one of claims 1-21, wherein the exogenous nucleic acid molecule is an exogenous RNA molecule or an exogenous DNA molecule.ATTORNEY DOCKET NO.38394.0028P1 23. A method of increasing a subject’s responsiveness to immune checkpoint inhibitors comprising administering to the subject one or more enucleated cells of any one of claims 1-22.

24. The method of claim 23, wherein the subject has previously been administered an immune checkpoint inhibitor.

25. The method of claim 23, wherein the subject is simultaneously administered an immune checkpoint inhibitor.

26. The method of claim 23, wherein the subject is administered an immune checkpoint inhibitor at least 24hrs after receiving the one or more enucleated cells.

27. A method of increasing T cells at a tumor site in a subject comprising administering to the subject one or more enucleated cells of any one of claims 1-22.

28. The method of claim 27, wherein the one or more enucleated cells activate antigen presenting cells capable of priming T cells causing an increase of T cells at the tumor site.

29. A method of treating a subject having cancer comprising administering to the subject one or more enucleated cells of any one of claims 1-22.

30. The method of claim 29, wherein the cancer is multiple myeloma, glioblastoma, lymphoma, leukemia, mesothelioma, sarcoma, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, lung cancer, or any combination thereof.

31. The method of any one of claims 29-30, wherein the adaptive immune system is activated.

32. The method of any one of claims 29-31, wherein tumor growth is slowed.

33. The method of any one of claims 29-32, further comprising administering an immune checkpoint inhibitor.

34. The method of claim 33, wherein there is an improvement in tumor clearance.

33. The method of any one of claims 33-34, wherein immunosuppressive state is reversed.ATTORNEY DOCKET NO.38394.0028P1 34. The method of any one of claims 29-33, wherein overall survival rates are increased.

35. A method of treating a subject comprising: administering to the subject one or more enucleated cells of any one of claims 1-22, and administering to the subject a radiation therapy.

36. A method of recruiting one or more T cells to a tumor site comprising administering to a subject one or more enucleated cells of any one of claims 1-22.

37. A method of killing or inducing apoptosis of a cancer cell comprising administering one or more enucleated cells of any one of claims 1-22 to the cancer cell or a subject comprising the cancer cell.

38. A method of inducing B cell proliferation comprising administering one or more enucleated cells of any one of claims 1-22 to the B cell or a subject comprising the B cell, wherein one or more enucleated cells expresses CD40L, and wherein the B cell proliferates upon exposure to the CD40L expressed by the one or more enucleated cells.

39. A method of activating NF-κB in a cell comprising administering one or more enucleated cells of any one of claims 1-22, wherein one or more enucleated cells express CD40L, to the cell or a subject comprising the cell, wherein NF-κB is activated upon binding of the CD40L expressed by the one or more enucleated cells to CD40 on the cell.

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