Compositions and methods for therapeutic delivery

Abstract

Described herein are compositions for delivering a single domain antibody or antigen-binding fragment thereof to a subject. The single domain antibody can be a therapeutic agent for treating a disease or condition in a subject, such as a disease or condition affecting the lungs of the subject. The composition comprises enucleated cells extensively engineered to produce the single domain antibody or antigen-binding fragment thereof, and optionally contains additional components such as a targeting moiety, an immune system evasion moiety, or an additional therapeutic agent or adjuvant. Methods of producing the compositions described herein are provided, including methods of enucleating parent cells to obtain enucleated cells comprising the single domain antibody or antigen-binding fragment thereof. Also provided are kits and methods for using the compositions described herein to treat a disease or condition by administering one or more compositions to a subject.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 154,591, filed February 26, 2021, and U.S. Provisional Patent Application No. 63 / 193,949, filed May 27, 2021, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on February 4, 2022 is named 53712-710_601_SL.txt and is 959,536 bytes in size. Summary of the Invention

[0003] In some aspects herein, an enucleating cell is described, the enucleating cell comprising a single domain antibody or fragment thereof that binds an immune checkpoint molecule, and one or more intracellular organelles configured to translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof. In some embodiments, the single domain antibody or fragment thereof is contained in the enucleating cell. In some embodiments, the single domain antibody or fragment thereof is released by the enucleating cell. In some embodiments, the enucleating cell further comprises a cell membrane, and the single domain antibody or fragment thereof is expressed on the luminal side of the cell membrane. In some embodiments, the enucleating cell further comprises a cell membrane, and the cell membrane comprises a transmembrane moiety attached to the single domain antibody or fragment thereof. In some embodiments, the transmembrane moiety comprises a transmembrane polypeptide. In some embodiments, the single domain antibody or fragment thereof is attached to the N-terminus or C-terminus of the transmembrane polypeptide. In some embodiments, the single domain antibody or fragment is conjugated to an anchor molecule that is attached to the cell surface of the enucleated cell, the anchor molecule comprising glycosylphosphatidylinositol, farnesyl, palmitate, myristate, or any combination thereof. In some embodiments, the enucleated cell further comprises a fusion protein configured to transfer the single domain antibody or fragment thereof from the enucleated cell to another cell. In some embodiments, the single domain antibody or fragment thereof is conjugated to a cytotoxic drug.In some embodiments, the immune checkpoint molecule is programmed cell death protein 1 (PD-1 or PDCD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (also known as cluster of differentiation 152 or CD152, CTLA-4), V domain Ig inhibitor of T cell activation (VISTA), programmed cell death 1 ligand 2 (also known as cluster of differentiation 273 or CD273, PDCD1LG2), B7 homolog 3 (cluster of differentiation 276 or CD277, PDCD1LG2), B7 homolog 3 (cluster of differentiation 277 or CD277, PDCD1LG2), B7 homolog 3 (cluster of differentiation 278 or CD278, PDCD1LG2), B7 homolog 3 (cluster of differentiation 279 or CD279 ... B7-H3), adenosine A2A receptor (A2AR), cluster of differentiation 27 (CD27), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and mucin domain containing-3 (also known as hepatitis A virus cell receptor 2 or HAVCR2, TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cluster of differentiation 73 (CD73), CD94 / NK group 2 member A (also known as cluster of differentiation 159 or CD159, NKG2A), poliovirus receptor-related immunoglobulin domain-containing (PVRIG), poliovirus receptor-related 2 (PVRL2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), focal adhesion kinase (FAK), CC chemokine receptor type 2 (CCR-2), chemokine (CC motif) ligand 2 (CCL-2), leukemia inhibitory factor (LIF), cluster of differentiation 47 (CD47), signaling In some embodiments, the immune checkpoint molecule comprises PD-L1, IL-1 regulatory protein alpha (SIRPα), macrophage colony-stimulating factor (M-CSF), colony-stimulating factor 1 receptor (CSF-1R), interleukin 3 (IL-3), interleukin-1 receptor accessory protein (IL-1RAP), interleukin 8 (IL-8), semaphorin-4D (SEMA4D), angiopoietin-2, CLEVER-1, tyrosine-protein kinase receptor UFO (Axl), phosphatidylserine, or fragments thereof. In some embodiments, the immune checkpoint molecule comprises PD-L1. In some embodiments, the immune checkpoint molecule comprises CTLA-4.In some embodiments, the immune checkpoint molecule comprises an amino acid sequence that is about 80% or more identical to any one of SEQ ID NOs: 155-164, 203, 204, 315-322, 511, 531-535, 551-554, 571, 594, 611-619, or 711. In some embodiments, the single domain antibody or fragment thereof is encoded by a deoxyribonucleic acid (DNA) sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 801. In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 851. In some embodiments, the single domain antibody or fragment thereof is encoded from a DNA sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 901. In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 951. In some embodiments, the enucleated cell further comprises a targeting moiety. In some embodiments, the targeting moiety comprises a homing receptor specific for a ligand expressed by cells in the lung tissue. In some embodiments, the targeting moiety comprises an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is different from the single domain antibody or fragment thereof. In some embodiments, the targeting moiety comprises a chemokine receptor. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cells are cells of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), adenocarcinoma, squamous cell carcinoma, large cell (anaplastic) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma. In some embodiments, the cancer cells are cells of a benign lung tumor. In some embodiments, the cancer cells are cells of a hamartoma. In some embodiments, the enucleated cells further comprise a therapeutic agent.In some embodiments, the therapeutic agent comprises interleukin 12 (IL-12). In some embodiments, the enucleated cells further comprise an immune evasion moiety comprising cluster of differentiation (CD47), PD-L1, major histocompatibility complex, class I, E (HLA-E), major histocompatibility complex, class I, G (HLA-G), fragments thereof, or combinations thereof. In some embodiments, the enucleated cells have a diameter of about 1 micrometer (μm) to about 100 μm. In some embodiments, the diameter is about 5 μm to 25 μm. In some embodiments, the diameter is about 8 μm to 12 μm. In some embodiments, the enucleated cells are reduced in diameter compared to an otherwise identical nucleated cell, the diameter being reduced by about 50% or more. In some embodiments, the enucleated cells further comprise an exogenous tumor necrosis factor (TNF) superfamily member polypeptide or catalytically active fragment thereof. In some embodiments, the exogenous TNF superfamily member polypeptide or catalytically active fragment thereof is soluble under aqueous conditions. In some embodiments, the exogenous TNF superfamily member polypeptide comprises tumor necrosis factor superfamily member 14 (LIGHT) or a catalytically active fragment thereof. In some embodiments, the enucleated cells are obtained from a parent cell, the parent cell comprising a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC), an adult stem cell, a mesenchymal stromal cell, an embryonic stem cell, or a fibroblast cell. In some embodiments, the enucleated cells are purified. In some embodiments, the enucleated cells are lyophilized.

[0004] In some embodiments herein, a plurality of cells are described, the cells comprising a plurality of enucleated cells, the enucleated cells comprising a single domain antibody or fragment thereof that binds an immune checkpoint molecule, and one or more intracellular organelles configured to translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof.

[0005] In some aspects herein, pharmaceutical formulations are described, the pharmaceutical formulations comprising an enucleated cell comprising a single domain antibody or fragment thereof that binds an immune checkpoint molecule, one or more intracellular organelles configured to translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, and a pharmaceutically acceptable excipient, carrier or diluent.

[0006] In some aspects herein, methods of delivering enucleated cells are described, the enucleated cells comprising a single domain antibody or fragment thereof that binds an immune checkpoint molecule and one or more intracellular organelles configured to translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, the methods comprising delivering the enucleated cells described herein to a subject. In some embodiments, the enucleated cells are autologous cells. In some embodiments, the enucleated cells are allogeneic cells. In some embodiments, the administering step is performed by systemic administration. In some embodiments, after the administering step, the enucleated cells are viable in the subject for 5 days or less.

[0007] In some aspects herein, a method of treating cancer in a subject is described, comprising administering to the subject a therapeutically effective amount of an enucleated cell, the enucleated cell comprising a single domain antibody or fragment thereof that binds an immune checkpoint molecule, and one or more organelles configured to translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, or a pharmaceutical formulation as described herein, thereby treating the cancer in the subject. In some embodiments, the enucleated cell is an autologous cell. In some embodiments, the enucleated cell is an allogeneic cell. In some embodiments, the administering step is performed by systemic administration. In some embodiments, after the administering step, the enucleated cell is viable in the subject for 5 days or less.

[0008] In some aspects herein, an enucleated cell is described, the enucleated cell comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF) and one or more intracellular organelles configured to (i) translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cell. In some embodiments, the single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 1701. In some embodiments, the single domain antibody or fragment thereof binds to an amino acid sequence of CTGF, the amino acid sequence of CTGF comprising SEQ ID NO: 1601 or SEQ ID NO: 1602. In some embodiments, the enucleated cell further comprises a targeting moiety specific for a ligand expressed by cells in the lung tissue. In some embodiments, the targeting moiety comprises a homing receptor specific for a ligand expressed by cells in the lung tissue. In some embodiments, the cell is an alveolar epithelial cell (AEC). In some embodiments, the cell is a bronchial cell. In some embodiments, the enucleated cell further comprises an immune evasion moiety comprising CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or any combination thereof. In some embodiments, the targeting moiety comprises a chemokine receptor. In some embodiments, the targeting moiety comprises an adhesion molecule. In some embodiments, the targeting moiety comprises an antibody or an antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is different from a single domain antibody or a fragment thereof. In some embodiments, the enucleated cell has a diameter of about 1 micrometer (μm) to about 100 μm. In some embodiments, the diameter is about 5 μm to 25 μm. In some embodiments, the diameter is about 8 μm to 12 μm. In some embodiments, the enucleated cell is reduced in diameter compared to an otherwise identical nucleated cell, the diameter being reduced by about 50% or more. In some embodiments, the enucleated cell further comprises an exogenous tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof.In some embodiments, the exogenous TNF superfamily member polypeptide or catalytically active fragment thereof is soluble under aqueous conditions. In some embodiments, the exogenous TNF superfamily member polypeptide comprises LIGHT or a catalytically active fragment thereof. In some embodiments, the enucleated cells are obtained from a parent cell, the parent cell comprising a stem cell. In some embodiments, the stem cell comprises an induced pluripotent stem cell (iPSC), an adult stem cell, a mesenchymal stromal cell, an embryonic stem cell, or a fibroblast cell. In some embodiments, the enucleated cells are purified. In some embodiments, the enucleated cells are lyophilized.

[0009] In some aspects herein, a plurality of cells are described, the cells comprising a plurality of enucleated cells, the enucleated cells comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF), and one or more intracellular organelles configured to (i) translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cells.

[0010] In some aspects herein, pharmaceutical formulations are described, the pharmaceutical formulations comprising an enucleated cell comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF), one or more intracellular organelles configured to (i) translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cell, and a pharmaceutically acceptable excipient, carrier or diluent.

[0011] In some aspects herein, methods of delivering enucleated cells to a subject are described, the methods comprising delivering to a subject an enucleated cell comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF) and one or more intracellular organelles configured to (i) translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cell. In some embodiments, the enucleated cell is an autologous cell. In some embodiments, the enucleated cell is an allogeneic cell. In some embodiments, the administering step is performed by systemic administration. In some embodiments, after the administering step, the enucleated cell is viable in the subject for 5 days or less.

[0012] In some aspects herein, methods of treating idiopathic pulmonary fibrosis (IPF) in a subject in need thereof are described, comprising administering to the subject a therapeutically effective amount of an enucleated cell, the enucleated cell comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF) and one or more intracellular organelles configured to (i) translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cell. In some embodiments, the enucleated cell is an autologous cell. In some embodiments, the enucleated cell is an allogeneic cell. In some embodiments, the administering step is performed by systemic administration. In some embodiments, after the administering step, the enucleated cell is viable in the subject for 5 days or less.

[0013] In some aspects herein, methods are described for treating a disease or condition in a subject in need of treatment, the methods comprising administering a therapeutically effective amount of enucleated cells to a subject having a disease or condition associated with a target cell of the subject, the enucleated cells comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF) and one or more intracellular organelles configured to (i) translate an exogenous mRNA molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cells, wherein the exogenous TNF superfamily member polypeptide or catalytically active fragment thereof normalizes vasculature associated with the disease or condition, and wherein normalizing the vasculature increases the therapeutic efficacy of treating the disease or condition compared to the therapeutic efficacy of a comparable method without normalizing the vasculature.

[0014] In some aspects herein, methods are described for treating a disease or condition characterized at least in part by abnormal vasculature in a subject, the methods comprising administering to a subject having the disease or condition an enucleated cell comprising a single domain antibody or fragment thereof that binds connective tissue growth factor (CTGF) and one or more intracellular organelles configured to (i) translate an exogenous mRNA molecule encoding the single domain antibody or fragment thereof, and (ii) release the single domain antibody or fragment thereof from the enucleated cell, wherein an exogenous tumor necrosis factor (TNF) superfamily member polypeptide or catalytically active fragment thereof synthesized or released by the enucleated cell is therapeutically effective in normalizing the abnormal vasculature of the subject.

[0015] In some embodiments herein, enucleated cells comprising antibodies, such as single domain antibodies, are provided. Also provided are methods of delivering the enucleated cells described herein to a subject, for example, to treat a disease or disorder in the subject. The enucleated cells and pharmaceutical compositions containing such enucleated cells, as well as methods of using them, offer several advantages over conventional cell-based therapeutics, including safety, defined lifespan, no risk of nuclear-encoded gene transfer to the host, and effective delivery of therapeutic cargo to target cells or tissues, even when administered systemically.

[0016] Aspects disclosed herein provide an enucleated cell obtained from a parent cell having a nucleus, the enucleated cell comprising one or more organelles for synthesizing an exogenous single domain antibody or fragment thereof in the absence of a nucleus. In some embodiments, the exogenous single domain antibody or fragment thereof is packaged in the enucleated cell. In some embodiments, the exogenous single domain antibody or fragment thereof is expressed on the side of the cell membrane of the enucleated cell by one or more organelles. In some embodiments, the exogenous single domain antibody or fragment thereof is expressed on the cytoplasmic side of the cell membrane of the enucleated cell by one or more organelles. In some embodiments, the exogenous single domain antibody or fragment thereof is complexed with a transmembrane moiety. In some embodiments, the transmembrane moiety comprises a transmembrane polypeptide. In some embodiments, the exogenous single domain antibody or fragment thereof is complexed with the N-terminus of the transmembrane polypeptide. In some embodiments, the exogenous single domain antibody or fragment thereof is complexed with the C-terminus of the transmembrane polypeptide. In some embodiments, the exogenous single domain antibody or fragment thereof comprises a modification to an otherwise identical reference single domain antibody or fragment thereof that anchors the exogenous single domain antibody or fragment thereof to the luminal or cytoplasmic side of the cell membrane of the enucleated cell. In some embodiments, the modification comprises complexing the exogenous single domain antibody or fragment thereof with glycosylphosphatidylinositol, farnesyl, palmitate, myristate, or a combination thereof. In some embodiments, the exogenous single domain antibody or fragment thereof is released by the enucleated cell by secreting the exogenous single domain antibody or fragment thereof from the enucleated cell. In some embodiments, the exogenous single domain antibody or fragment thereof is released upon death of the enucleated cell. In some embodiments, the exogenous single domain antibody or fragment thereof is released upon rupture of the enucleated cell. In some embodiments, the exogenous single domain antibody or fragment thereof is transferred from the enucleated cell to another cell by fusing the enucleated cell with another cell. In some embodiments, the exogenous single domain antibody or fragment thereof is conjugated to a cytotoxic drug.In some embodiments, the enucleated cell comprises an exogenous nucleotide having a polypeptide sequence encoding an exogenous single domain antibody or fragment thereof. In some embodiments, the polypeptide sequence comprises a sequence provided in SEQ ID NOs: 1-36, 101-111, 121-123, 165-192, 195, 205, 206, 211-213, 221-231, 241-245, 325-331, and 401-404. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen encoded by at least one nucleic acid of SEQ ID NOs: 131-134, 142-152, 201-202, 301-312, 501, 521-526, 541-545, 561, 584, 591-601, and 701-705. In some embodiments, the exogenous single domain antibody or fragment thereof is specific to an antigen comprising a peptide sequence encoding PD-L1. In some embodiments, the exogenous single domain antibody or fragment thereof is specific to an antigen comprising at least one peptide sequence of SEQ ID NOs: 155-164, 203, 204, 315-322, 511, 531-535, 551-554, 571, 594, 611-619, and 711. In some embodiments, the exogenous single domain antibody or fragment thereof is specific to an antigen associated with at least one pathogen in Table 1. In some embodiments, the exogenous single domain antibody or fragment thereof is specific to an antigen comprising a peptide sequence encoding connective tissue growth factor (CTGF), also known as Cellular Communication network factor 2 (CCN2). In some embodiments, the exogenous single domain antibody or fragment thereof is specific to an antigen comprising at least one peptide sequence of SEQ ID NOs: 1601 and 1602.

[0017] In some embodiments, the exogenous single domain antibody or fragment thereof is encoded from a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 801. In some embodiments, the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 851. In some embodiments, the exogenous single domain antibody or fragment thereof is encoded from a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 901. In some embodiments, the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 951. In some embodiments, the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 1701. In some embodiments, the exogenous single domain antibody or fragment thereof is specific to an antigen expressed by cancer cells in lung tissue. In some embodiments, the antigen expressed by cancer cells in lung tissue is PD-L1. In some embodiments, the antigen expressed by cancer cells in lung tissue is CTGF. In some embodiments, the cancer cells are non-small cell lung cancer (NSCLC) cells. In some embodiments, the cancer cells are cells of an adenocarcinoma, squamous cell carcinoma, large cell (anaplastic) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma. In some embodiments, the cancer cells are cells of a small cell lung cancer (SCLC). In some embodiments, the cancer cells are cells of a lung carcinoid tumor. In some embodiments, the cancer cells are cells of an adenoid cystic carcinoma. In some embodiments, the cancer cells are cells of a lymphoma. In some embodiments, the cancer cells are cells of a sarcoma. In some embodiments, the cancer cells are cells of a benign lung tumor.In some embodiments, the cancer cell is a hamartoma cell. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by a cell associated with idiopathic pulmonary fibrosis. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is a lung cell. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is an immune cell. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is an alveolar cell. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is an alveolar epithelial cell (AEC). In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is a bronchial cell. In some embodiments, the enucleated cell further comprises at least one additional exogenous therapeutic agent. In some embodiments, the enucleated cell further comprises a fusogenic moiety. In some embodiments, the fusogenic moiety comprises a viral fusogenic moiety. In some embodiments, the fusogenic moiety comprises a eukaryotic fusogenic moiety. In some embodiments, the enucleated cell further comprises an immune evasion moiety. In some embodiments, the immune evasion moiety comprises CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof. In some embodiments, the enucleated cell further comprises a targeting moiety. In some embodiments, the targeting moiety targets a biomarker of the cancer cell. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen expressed by the cancer cell, and the biomarker is a separate and distinct entity from the antigen targeted by the exogenous single domain antibody or fragment thereof. In some embodiments, the targeting moiety targets a biomarker of an immune cell within the tumor microenvironment. In some embodiments, the biomarker is expressed on the surface of the immune cell. In some embodiments, the biomarker is released by the immune cell.In some embodiments, the targeting moiety comprises a chemokine. In some embodiments, the targeting moiety comprises a chemokine receptor. In some embodiments, the targeting moiety comprises an adhesion molecule. In some embodiments, the targeting moiety comprises an antigen. In some embodiments, the targeting moiety comprises an antigen that is a separate and distinct entity from the antigen expressed by the cancer cell. In some embodiments, the targeting moiety comprises an antibody that is not expressed by the cancer cell. In some embodiments, the targeting moiety comprises a membrane-bound antibody. In some embodiments, the membrane-bound antibody is a membrane-bound single domain antibody. In some embodiments, the enucleated cells have a diameter of about 1 micrometer (μm) to about 100 μm. In some embodiments, the diameter is about 1 μm to about 10 μm. In some embodiments, the diameter is about 10 μm to about 100 μm. In some embodiments, the diameter is at least or about 1 μm, 5 μm, 8 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some embodiments, the diameter is about 8 μm. In some embodiments, the enucleated cells exhibit a diameter that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% smaller than the nucleated parent cell. In some embodiments, the parent cell is selected from the group consisting of stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, and cells from a cell line. In some embodiments, the parent cell is a mesenchymal stromal cell. In some embodiments, the enucleated cells exhibit viability after cryohibernation. In some embodiments, the enucleated cells exhibit a post-cryohibernation viability measured 24 hours after cryohibernation that is equal to or greater than the viability of comparable enucleated cells that are not cryohiberinated. In some embodiments, the enucleated cells exhibit a post-cryopreservation viability measured 24 hours after cryohibernation that is equal to or greater than the viability of comparable enucleated cells that are not cryohiberinated. In some embodiments, the enucleated cells exhibit a post-cryopreservation viability measured 24 hours after cryohibernation that is equal to or greater than the viability of comparable enucleated cells that are not cryopreserved.In some embodiments, the enucleated cells are isolated. In some embodiments, the enucleated cells are purified. In some embodiments, the enucleated cells are lyophilized. In some embodiments, the enucleated cells comprise an exogenous single domain antibody or fragment thereof, including a neutralizing antibody. In some embodiments, the exogenous single domain antibody or fragment thereof binds VEGF. In some embodiments, the exogenous single domain antibody or fragment thereof binds VEGF-A. In some embodiments, the enucleated cells comprise a targeting moiety that targets an endothelial cell biomarker. In some embodiments, the endothelial cell biomarker is expressed by vasculature cells. In some embodiments, the endothelial cell biomarker is expressed by vascular cells. In some embodiments, the endothelial cell biomarker is expressed by lymphatic cells. In some embodiments, the enucleated cells comprise at least one additional exogenous agent, including a polypeptide comprising a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof. In some embodiments, the TNF superfamily member polypeptide or catalytically active fragment thereof is soluble under aqueous conditions, as measured in vitro by turbidity solubility assay or thermodynamic solubility assay. In some embodiments, the TNF superfamily member polypeptide comprises tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT). In some embodiments, the TNF superfamily member polypeptide comprises soluble LIGHT. In some embodiments, the at least one additional exogenous agent comprises an immune checkpoint molecule. In some embodiments, the at least one additional exogenous agent comprises an immune checkpoint inhibitor molecule. In some embodiments, the at least one additional exogenous agent comprises an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor comprises a VEGF / VEGFR inhibitor. In some embodiments, the VEGF / VEGFR inhibitor comprises a VEGF-A inhibitor.

[0018] Aspects disclosed herein provide a cell line comprising the enucleated cells described herein.

[0019] Aspects disclosed herein provide a plurality of cells comprising the enucleated cells described herein.

[0020] Aspects disclosed herein provide a pharmaceutical composition comprising the enucleated cells described herein and a pharma- ceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition comprises a unit dosage form. In some embodiments, the pharmaceutical composition is formulated for administration to a subject in need thereof intrathecally, intraocularly, intravitreally, retina- ally, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intraventricularly, intraperitoneally, subcutaneously, intratumorally, pulmonary, intratracheally, intraperitoneally, intravesically, intravaginally, rectally, orally, sublingually, transdermally, by inhalation, by inhalation spray form, by intraluminal-GI route, or by combinations thereof. In some embodiments, the pharmaceutical composition is formulated for administration intravenously. In some embodiments, the pharmaceutical composition is formulated for administration intratumorally. In some embodiments, the pharmaceutical composition is formulated for administration pulmonary. In some embodiments, the medicament is formulated for administration intratracheally. In some embodiments, the pharmaceutical composition is formulated for administration by inhalation spray form. In some embodiments, the pharmaceutical composition comprises at least one additional active agent. In some embodiments, the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an antibody, an enzyme, a small molecule, a chemical compound, or a combination thereof.

[0021] Aspects disclosed herein provide a kit, the kit comprising an enucleated cell described herein, a cell line described herein, a plurality of cells described herein, or a pharmaceutical composition described herein, and a container.

[0022] Aspects disclosed herein provide a method of treating a disease or condition in a subject in need of such treatment, comprising administering a therapeutically effective amount of a cell disclosed herein or a pharmaceutical composition disclosed herein to a subject having a disease or condition associated with a target cell in the subject, wherein the exogenous single domain antibody or fragment thereof binds to an antigen expressed by the target cell in the subject, thereby treating the disease or condition in the subject. In some embodiments, the enucleated cell is an autologous cell. In some embodiments, the enucleated cell is an allogeneic cell. In some embodiments, the antigen comprises a tumor associated antigen (TAA). In some embodiments, the antigen comprises a tumor specific antigen (TSA). In some embodiments, the binding of the exogenous single domain antibody or fragment thereof to an antigen directly kills the cancer cell. In some embodiments, the binding of the exogenous single domain antibody or fragment thereof to an antigen disrupts cell cycle signaling of the cancer cell. In some embodiments, the binding of the exogenous single domain antibody or fragment thereof to an antigen disrupts angiogenesis signaling of the cancer cell. In some embodiments, binding of the exogenous single domain antibody or fragment thereof to the antigen recruits immune cells to the cancer cells. In some embodiments, the immune cells are T cells. In some embodiments, the enucleated cells or pharmaceutical compositions are administered to the subject intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intraventricularly, intraperitoneally, subcutaneously, intratumorally, pulmonary, intratracheally, intraperitoneally, intravesically, intravaginally, rectally, orally, sublingually, transdermally, by inhalation, inhalation spray form, intraluminal-GI route, or combinations thereof. In some embodiments, the enucleated cells or pharmaceutical compositions are administered intravenously. In some embodiments, the enucleated cells or pharmaceutical compositions are administered intratumorally. In some embodiments, the enucleated cells or pharmaceutical compositions are administered intrapulmonary. In some embodiments, the enucleated cells or pharmaceutical compositions are administered intratracheally. In some embodiments, the enucleated cells or pharmaceutical compositions are administered by inhalation spray form.In some embodiments, following administration of the enucleated cells or pharmaceutical composition to a subject, the enucleated cells are viable within the subject for 14 days or less. In some embodiments, following administration of the enucleated cells or pharmaceutical composition to a subject, the enucleated cells are viable within the subject for 4 days or less. In some embodiments, the target cells are cancer cells. In some embodiments, the disease or condition is cancer or neoplasm.

[0023] Aspects described herein provide an enucleated cell obtained from a parent cell having a nucleus, the enucleated cell comprising one or more organelles for synthesizing an exogenous polypeptide, including a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof, in the absence of a nucleus. In some embodiments, the enucleated cell comprises at least one exogenous targeting moiety. In some embodiments, the exogenous polypeptide comprises a solubility of 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 under aqueous conditions, where the solubility is measured by a turbidimetric solubility assay or a thermodynamic solubility assay. In some embodiments, the exogenous polypeptide is expressed on the luminal side of the cell membrane of the enucleated cell by one or more intracellular organelles. In some embodiments, the exogenous polypeptide is released by the enucleated cell. In some embodiments, the enucleated cell further comprises an exogenous polynucleotide encoding the exogenous polypeptide. In some embodiments, the exogenous polypeptide comprises a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1501-1511. In some embodiments, the exogenous polypeptide comprises a sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1511. In some embodiments, the TNF superfamily member polypeptide is LIGHT. In some embodiments, the enucleated cell further comprises a second exogenous polypeptide. In some embodiments, the second exogenous polypeptide comprises an antibody, an immune checkpoint molecule, or a fragment thereof. In some embodiments, the second exogenous polypeptide comprises an antibody or an antigen-binding fragment thereof, or a single domain antibody or an antigen-binding fragment thereof.In some embodiments, the antibody or antigen-binding fragment thereof, or single domain antibody or antigen-binding fragment thereof, is a neutralizing antibody or neutralizing antigen-binding fragment thereof. In some embodiments, the neutralizing antibody or neutralizing antigen-binding fragment thereof targets an immune checkpoint molecule. In some embodiments, the neutralizing antibody or neutralizing antigen-binding fragment thereof targets angiopoietin-1, angiopoietin-2, endostatin, FGF, MMP, DII4, class 3 semaphorins, FGF, VEGFR, NRP-1, PDGF (BB homodimer), PDGFR, TGF-β, endoglin, TGF-β receptor, CCL2, integrin αVβ3, αVβ5 or α5β1, VE-cadherin, CD31, ephrin, plasminogen activator, plasminogen activator inhibitor-1, eNOS, COX-2, AC133, ID1 / ID3, class 3 semaphorins, or Nogo-A. In some embodiments, the neutralizing antibody or neutralizing antigen-binding fragment thereof targets VEGF. In some embodiments, the neutralizing antibody or neutralizing antigen-binding fragment thereof targets VEGF-A. In some embodiments, the immune checkpoint molecule comprises PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), B7-H3 (also referred to as 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. In some embodiments, at least one exogenous targeting moiety comprises an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof.In some embodiments, the antibody or antigen-binding fragment thereof, or the single domain antibody or antigen-binding fragment thereof, comprises an exogenous single domain antibody or fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof, or the single domain antibody or antigen-binding domain thereof, targets a cancer cell marker. In some embodiments, the antibody or antigen-binding fragment thereof, or the single domain antibody or antigen-binding fragment thereof, targets an endothelial cell biomarker. In some embodiments, the endothelial cell biomarker is expressed by vasculature cells. In some embodiments, the endothelial cell biomarker is expressed by blood vessel cells. In some embodiments, the endothelial cell biomarker is expressed by lymphatic vessel cells.

[0024] Aspects provided herein are methods of treating a disease or condition characterized at least in part by abnormal vasculature in a subject, the method comprising administering to a subject having the disease or condition an enucleated cell comprising one or more organelles that synthesize or release an exogenous polypeptide comprising a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof in the absence of a nucleus, wherein the exogenous polypeptide synthesized or released by the cell is therapeutically effective in normalizing the abnormal vasculature of the subject. In some embodiments, the exogenous polypeptide comprises a soluble TNF superfamily member polypeptide. In some embodiments, the exogenous polypeptide is released by the enucleated cell. In some embodiments, the exogenous polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1501-1511. In some embodiments, the exogenous polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1508. In some embodiments, the exogenous polypeptide comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1511. In some embodiments, the TNF superfamily member is LIGHT. In some embodiments, the enucleated cell further comprises at least one exogenous targeting moiety comprising an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof, or the single domain antibody or antigen-binding fragment thereof comprises an exogenous single domain antibody or fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof, or the single domain antibody or antigen-binding fragment thereof targets a cancer cell marker. In some embodiments, the antibody or antigen-binding fragment thereof, or the single domain antibody or antigen-binding fragment thereof targets an endothelial cell biomarker. In some embodiments, the endothelial cell biomarker is expressed by vasculature cells. In some embodiments, the endothelial cell biomarker is expressed by vascular cells.In some embodiments, the endothelial cell biomarker is expressed by lymphatic cells. In some embodiments, the enucleated cells deliver exogenous polypeptides to cells in the abnormal vasculature of the subject. In some embodiments, the enucleated cells comprise at least one additional exogenous factor. In some embodiments, the at least one additional exogenous agent comprises an immune checkpoint molecule. In some embodiments, the at least one additional exogenous agent comprises an immune checkpoint molecule inhibitor. In some embodiments, the at least one additional exogenous agent comprises an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor comprises a VEGF / VEGFR inhibitor. In some embodiments, the VEGF / VEGFR inhibitor comprises a VEGF-A inhibitor. In some embodiments, the at least one exogenous agent kills cancer in the abnormal vasculature. In some embodiments, the at least one exogenous agent recruits endogenous immune cells to the abnormal vasculature to kill cancer in the abnormal vasculature. In some embodiments, the enucleated cells are administered to the subject intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intraventricularly, intraperitoneally, subcutaneously, intratumorally, intrapulmonary, intratracheally, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation spray form, by intraluminal-GI route, or combinations thereof. In some embodiments, following administration of the enucleated cells to the subject, the enucleated cells are viable within the subject for up to 14 days. In some embodiments, following administration of the enucleated cells to the subject, the enucleated cells are viable within the subject for up to 4 days. In some embodiments, the disease or condition is cancer or neoplasm. In some embodiments, the abnormal vasculature is in the lungs of the subject.In some embodiments, the method includes administering to the patient an effective amount of any of the following: CPI-006, Monalizumab, COM701, CM24, NEO-201, Defactinib, PF-04136309, MSC-1, Hu5F9-G4 (5F9), ALX148, TTI-662, RRx-001, Lacnotuzumab (MCS110), LY3022855, SNDX-6 352, Emactuzumab (RG7155), Pexidartinib (PLX3397), CAN04, Canakinumab (ACZ885), BMS-986253, Pepinemab (VX15 / 2503), Trebananib, FP-1305, Enapotamab vedotin (EnaV), Bavituximab, or a combination thereof, to the subject.

[0025] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, the specification is intended to supersede and / or take precedence over any such conflicting material. [Brief description of the drawings]

[0026] The novel features of the inventive concept are set forth with particularity in the appended claims. A better understanding of the features and advantages of the inventive concept will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concept are utilized, and the accompanying drawings. [Figure 1] 1 shows a process for generating enucleated cells for delivery of single domain antibodies according to various embodiments described herein. [Diagram 2]1 shows a timeline for the production of enucleated cells for delivery of single domain antibodies according to various embodiments compared to a typical biological drug development timeline. [Figure 3A] 1 is a representative graph showing relative fold changes in viable or enucleated cells ("cytoplasts") over time. [Figure 3B] 1 is a representative graph showing viable cells and cytoplasts following recovery from cryopreservation (cryopreservation). [Figure 3C] Representative graph showing relative viability of cytoplasts 24 hours after enucleation (fresh cytoplasts) or 24 hours after recovery from cryopreservation after enucleation (cryopreservation), comparing fresh and cryopreserved cytoplasts with viability of cytoplasts 4 hours after enucleation. Mean ± SEM, n=10. [Figure 4A] 1 is a representative line graph showing the viability of mesenchymal stem cells (MSCs) and MSC-derived cytoplasts immediately after recovery from cryohibernation at 4° C. for the indicated times. Viability was assessed by automated cell counting (Cell Countess) using trypan blue dye exclusion and expressed as a ratio of input cell number. [Figure 4B] Representative bar graph comparing migrated MSCs and MSC-derived cytoplasts in a Boyden chamber assay immediately after recovery from cryohibernation at 4° C. for the indicated times. Cells and cytoplasts were allowed to migrate for 3 hours in the bottom chamber using no serum (negative control) or 10% premium FBS (P-FBS) as a chemoattractant, and counts were normalized to a loading control. [Figure 5A] Representative flow cytometry graphs showing the number of events counted versus the signal intensity of cell surface C-X-C motif chemokine receptor 4 (CXCR4) expression by fluorescent antibody on engineered cytoplasts and engineered parental MSCs as analyzed by FlowJo. [Figure 5B]Representative bar graph showing the percentage of migratory cells or cytoplasts that migrated to the underside of the Boyden chamber assay compared to loading control. Mean ± SEM, n = 10. MSCs and MSC-derived cytoplasts, with or without engineered CXCR4 receptors, were allowed to migrate towards the indicated concentrations of stromal cell-derived factor 1α (SDF-1α) for 2 h in a Boyden chamber assay. [Figure 6A] Representative flow cytometry graphs showing the number of events counted versus the signal intensity of cell surface P-selectin glycoprotein ligand 1 (PSGL1) expression by fluorescent antibody on engineered cytoplasts and engineered parental MSCs as analyzed by FlowJo. [Figure 6B] 1 is a representative graph showing cell surface binding of P-selectin to engineered MSCs and MSC-derived cytoplasts as determined by flow cytometry. MSC control = parental MSCs. Engineered MSCs = PSGL1 / (Fucosyltransferase 7)Fut7 engineered MSCs. Engineered cytoplasts = PSGL1 / Fut7 engineered MSC-derived cytoplasts. [Figure 7A] 13 is a representative flow cytometry graph showing the number of events counted versus cell surface signal intensity of mCD47 expression on engineered cytoplasts and MSCs as analyzed by FlowJo. [Figure 7B] Representative bar graph showing the number of viable cytoplasmic bodies (DiD+) that were not phagocytosed by macrophages (F4 / 80- and CD11b-), indicating that the cytoplasmic bodies escaped macrophage phagocytosis in the lung. Mean ± SEM, n=3. DiD dye-labeled control cytoplasmic bodies or engineered cytoplasmic bodies (mCD47 cytoplasmic bodies) were injected retro-orbitally into the vasculature of mice. After 24 hours, tissues were harvested and stained with two different pan-macrophage markers (F4 / 80 and CD11b). [Figure 7C]Representative bar graph showing live cytoplasts (DiD+) that were not phagocytosed by macrophages (F4 / 80- and CD11b-), indicating that cytoplasts escaped macrophage phagocytosis in the liver. Mean ± SEM, n=3. DiD dye-labeled control cytoplasts or engineered cytoplasts (mCD47 cytoplasts) were injected retroorbitally into the vasculature of mice. After 24 hours, tissues were harvested and stained with two different pan-macrophage markers (F4 / 80 and CD11b). [Figure 8A] Representative scatter plots showing the number of DiD-labeled MSCs or cytoplasts detected in the lung. MSCs were cultured under standard adherent conditions (2D) or in suspension by the handling drop method (3D) to generate 3D cytoplasts. MSCs and cytoplasts were labeled with Vybrant® DiD dye and injected retro-orbitally into the vasculature of C57BL / 6 mice. Tissues were harvested 24 hours later and cell suspensions were analyzed by flow cytometry. Mean ± SEM, n=2. [Figure 8B] Representative scatter plots showing the number of DiD-labeled MSCs or cytoplasts detected in the liver. MSCs were cultured under standard adherent conditions (2D) or in suspension by handling drop method (3D) to generate 3D cytoplasts. MSCs and cytoplasts were labeled with Vybrant® DiD dye and injected retro-orbitally into the vasculature of C57BL / 6 mice. Tissues were harvested 24 hours later and cell suspensions were analyzed by flow cytometry. Mean ± SEM, n=2. [Figure 8C] Representative scatter plots showing the number of Vybrant® DiD-labeled MSCs or cytoplasts detected in the spleen. MSCs were cultured under standard adherent conditions (2D) or in suspension by handling drop method (3D) to generate 3D cytoplasts. MSCs and cytoplasts were labeled with DiD dye and injected retro-orbitally into the vasculature of C57BL / 6 mice. Tissues were harvested 24 hours later and cell suspensions were analyzed by flow cytometry. Mean ± SEM, n=2. [Figure 9]FIG. 1 shows cell surface staining of fluorescein isothiocyanate (FITC) labeled Annexin V on mesenchymal stromal cells (MSCs) or cytoplasts analyzed by flow cytometry for cell viability analysis. [Figure 10A] Representative graph showing the abundance of secreted single domain antibodies as measured by enzyme-linked immunosorbent assay (ELISA) in conditioned medium of non-transfected (hTERT) cells or cells transfected with vectors encoding single domain antibodies (scFv). Transfected cells were enucleated and plated in 6-well plates (0.5×106 / well), and conditioned medium was collected for ELISA detection 24 and 48 hours after enucleation. Mean ± SEM, n=3 biological replicates. [Figure 10B] Representative graph showing secreted anti-programmed death-ligand 1 (PD-L1) nanobody, single domain antibody, or scFv (NB) measured by ELISA in conditioned medium of non-transfected (hTERT-MSC only) or transfected (enucleated cells NBαPD-L1) cells. Cells were seeded in 6-well plates (0.5×106 / well) and conditioned medium was collected for ELISA detection 24 and 48 hours after enucleation. Mean ± SEM, n=3 biological replicates. [Figure 10C] Representative graph showing secreted anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) NB measured by ELISA in conditioned medium of non-transfected (hTERT-MSC only) and transfected (enucleated cells NBCTLA-4) cells. Cells were seeded in 6-well plates (0.5×106 / well) and conditioned medium was collected for ELISA detection 24 and 48 hours after enucleation. Mean ± SEM, n=3 biological replicates. Detailed Description of the Invention

[0027] Disclosed herein are enucleated cells that can be extensively engineered to express single domain antibodies or portions thereof in the absence of a nucleus. Such enucleated cells can be used to express and deliver single domain antibodies or portions thereof in vivo to target cells or tissues, even when administered systemically. The single domain antibodies or portions thereof or fragments thereof can exert a therapeutic effect. For example, the single domain antibodies or portions thereof or fragments thereof can target and bind to immune checkpoint molecules, thus reducing the expression of immune checkpoint molecules in a subject and treating a disease or condition in the subject. In some embodiments, the enucleated cells can be engineered to express an exogenous tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof. In some embodiments, the enucleated cells can be engineered to express both a single domain antibody and exogenous TNF. In some embodiments, the exogenous TNF normalizes the abnormal vasculature of the subject. In some embodiments, the normalization of the abnormal vasculature of the subject increases the therapeutic efficacy of the enucleated cells. For example, the normalized vasculature allows for the delivery of single domain antibodies to target sites associated with a disease or condition.

[0028] The enucleated cells described herein can be engineered to express one or more targeting moieties that, when expressed by the enucleated cells (e.g., on their surface), direct the enucleated cells to target cells or tissues in vivo. To avoid unintended clearance of the enucleated cells in vivo, the enucleated cells can also include immune system evasion moieties ("don't eat me" signaling polypeptides), such as CD47, PD-L1, HLA-E, or HLA-G. In some embodiments, the enucleated cells described herein include an additional active agent, such as a therapeutic agent.

[0029] This has two major problems that arise with the systemic administration of conventional therapeutic cells. First, a large portion of the cells may be trapped in small capillaries in the lungs or other tissues, which may adversely affect the biodistribution of the therapeutic cargo and cause serious side effects such as pulmonary thromboembolism. Second, conventional therapeutic cells that lack tissue-specific homing receptors and adhesion molecules (e.g., SDF-1α / CXCR4, CCL2 / CCR2, PSGL-1) to target intended cells or tissues cannot efficiently home to such intended cells or tissues.

[0030] The enucleated cells of the present disclosure, in some embodiments, solve such problems by being much smaller than conventional therapeutic cells and their parent cells (e.g., about 60% of the diameter and 1 / 8 of the volume of the parent cells) and not having a rigid nucleus. Thus, the enucleated cells may pass through small capillaries and blood vessels better than their conventional counterparts. In addition, the enucleated cells described herein may be engineered to express functional targeting moieties (e.g., homing receptors, adhesion molecules, or membrane-bound antibodies such as membrane-bound single domain antibodies) to facilitate efficient homing to target cells or tissues, even when administered systemically to a subject. In some embodiments, the target cells or tissues are cancer cells or tissues. In the case of enucleated cells targeting cancer cells or tissues, the enucleated cells may be engineered to express targeting moieties that recognize antigens produced by the cancer cells or tissues. 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 antibodies, or membrane-bound single domain antibodies.

[0031] In some embodiments, the single domain antibody or portion thereof is exogenous to the enucleated cell or its parent cell. The exogenous single domain antibody or portion thereof may confer a therapeutic effect, such as for treating a disease or disorder described herein. In some embodiments, the single domain antibody or portion thereof is fused to multiple antibodies or multiple single domain antibodies that may target a single epitope or multiple epitopes (e.g., bispecific). In some embodiments, the single domain antibody is conjugated to a small molecule, such as an antibody-drug conjugate (ADC). In some embodiments, the small molecule is a cytotoxic drug or a therapeutically effective portion thereof.

[0032] In some embodiments, the enucleated cell comprises a polynucleotide encoding a single domain antibody or a portion thereof. In some embodiments, the polynucleotide is exogenous to the enucleated cell or a parent cell thereof. In some embodiments, the polynucleotide encodes a single domain antibody fused to multiple antibodies or multiple single domain antibodies described herein.

[0033] Also disclosed herein are methods of using enucleated cells and compositions comprising the enucleated cells of the present disclosure. In some embodiments, methods are provided for treating a disease or condition by delivering enucleated cells or compositions containing enucleated cells to a subject. In some embodiments, the disease or condition is associated with a target cell or tissue that the enucleated cells are engineered to target. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is an epithelial cell. In some cases, the tissue is a tumor. In some embodiments, the tissue is a lung tissue. In some embodiments, the disease or condition comprises cancer. In some embodiments, the disease or condition comprises idiopathic pulmonary fibrosis. In some embodiments, the method comprises using the enucleated cells of the present disclosure to transfer a cargo (e.g., a single domain antibody, a therapeutic agent) into the target cell, e.g., using a fusogenic moiety or a tunneling nanotube, etc.

[0034] Disclosed herein are methods of producing enucleated cells as described herein, for example, by removing the nucleus from a nucleated parent cell without differentiation of the parent cell. For example, the parent cell containing the nucleus can be engineered to express a single domain antibody or portion thereof, a targeting moiety, or a "don't eat me" signaling peptide, and the nucleus of the parent cell can then be removed. In another example, the parent cell containing the nucleus is enucleated and the enucleated cell is engineered to express a domain antibody or portion thereof, a targeting moiety, or a "don't eat me" signaling peptide. In some embodiments, removing the nucleus comprises mechanically removing the nucleus.

[0035] Following enucleation of the parent cell, the enucleated cells described herein retain one or more organelles endogenous to the parent cell. In some embodiments, all of the one or more organelles are retained. In some embodiments, less than all of the one or more organelles are retained. In some embodiments, the Golgi apparatus and / or the endoplasmic reticulum are retained, which are involved in protein synthesis and secretion. Retention of one or more organelles at least partially allows the enucleated cells to synthesize or release a biomolecule disclosed herein (e.g., a single domain antibody or portion thereof, a targeting moiety, an immune evasion moiety, etc.) in the absence of a nucleus.

[0036] In some embodiments, the parent cell is any one of the nucleated cells described herein. 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 enucleated cell is derived from an induced pluripotent stem cell (iPSC). In some embodiments, the parent cell is not an erythrocyte or erythroid progenitor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial progenitor cell or erythroid progenitor cell. In some embodiments, the parent cell is not a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial progenitor cell. In some embodiments, the parent cell does not express complement receptor 1 (CR1). In some embodiments, the parent cell does not express CD44. In some embodiments, the parent cell does not express VLA-4. In some embodiments, the parent cell does not express BCAM. In some embodiments, the parent cell does not express ICAM. In some embodiments, the parent cell does not express a receptor for collagen. In some embodiments, the parent cell does not express a receptor for thrombopoietin. In some embodiments, the parent cell does not express a receptor for collagen. In some embodiments, the parent cell does not express a receptor for von Willebrand factor (VWF). In some embodiments, the parent cell does not express a receptor for fibrinogen. In some embodiments, the parent cell does not express a GP1b-IX-V receptor. In some embodiments, the parent cell does not express a GPIIb / IIIa receptor. In some embodiments, the parent cell does not express a prostanoid receptor. In some embodiments, the parent cell does not express a purinergic receptor. In some embodiments, the parent cell does not express a thromboxane receptor.

[0037] composition Provided herein are cells comprising a single domain antibody or portion thereof, and compositions comprising such enucleated cells. In some embodiments, the cells are enucleated. In some embodiments, the single domain antibody is a therapeutic agent. In some embodiments, the enucleated cells can express the single domain antibody (e.g., a therapeutic agent) in the absence of a nucleus, using one or more intracellular organelles retained by the enucleated cell from the parent cell. In some embodiments, the single domain antibody or portion thereof (e.g., a therapeutic agent) is exogenous to the enucleated cell or its parent cell. In some embodiments, the enucleated cell expresses the single domain antibody or portion thereof (e.g., a therapeutic agent) on the surface of the enucleated cell. In some embodiments, the single domain antibody or portion thereof is secreted by the enucleated cell into the extracellular space of a target tissue (e.g., a microenvironment). In some aspects, the composition comprises an enucleated cell as described herein. In some aspects, the enucleated cell is obtained or derived from an enucleated cell (e.g., a parent cell). In some aspects, the enucleated cell comprises a transmembrane moiety. In some embodiments, the enucleated cell comprises a targeting moiety. In some embodiments, the enucleated cells comprise a therapeutic agent. In some embodiments, the enucleated cells comprise an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof. In some aspects, the targeting moiety comprises an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof. In some embodiments, the therapeutic agent comprises an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof. In some embodiments, the enucleated cells are formulated in a pharmaceutical formulation as described herein.

[0038] enucleated cells The enucleated cells of the present disclosure are obtained or derived from a corresponding nucleated cell (herein referred to as a "parent cell"). The parent cell may be derived from a variety of different cell types, including eukaryotic cells. For example, the enucleated cells may be derived from adult stem cells, mesenchymal stromal cells (MSCs), natural killer (NK) cells, macrophages, myoblasts, neutrophils, endothelial cells, endothelial progenitor cells, and / or fibroblasts. In some embodiments, the enucleated cells are derived from mesenchymal stromal cells. In some embodiments, the enucleated cells are derived from induced pluripotent stem cells (iPSCs). In some embodiments, the parent cell is derived from a cell that is to be immortalized using a suitable method. For example, the parent cell is immortalized by expressing human telomerase reverse transcriptase (hTERT), an oncogene, or a viral gene such as Simian Virus 40 (SV40). In some embodiments, the cytoplasts are derived from the parent cell using suitable methods provided in U.S. Patent No. 10,927,349, which is incorporated herein by reference in its entirety. In some embodiments, the enucleated cell retains one or more organelles for synthesis of the exogenous single domain antibody or fragment thereof in the absence of a nucleus.

[0039] In some embodiments, the cells may be from any organism that has one or more cells. Non-limiting examples of cells include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, cereals, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, mosses, plants from mosses), algae cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, pyrenoidosa, Sargassumpatens C.Agardh, etc.), seaweed (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes the cells are not from a natural organism (e.g., the cells are artificially created and sometimes referred to as artificial cells). In some embodiments, the cells are somatic cells. In some embodiments, the cells are stem or progenitor cells. In some embodiments, the cells are mesenchymal stem or progenitor cells. In some embodiments, the cells are hematopoietic stem or progenitor cells. In some embodiments, the cell is a muscle cell, a skin cell, a blood cell, or an immune cell. Other non-limiting examples of cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, and cytokine-induced killer (CIK) cells; granulocytes (basophil granulocytes, eosinophil granulocytes, and eosinophil granulocytes);myeloid cells such as granulocytes, neutrophilic granulocytes / hypersegmented neutrophils, monocytes / macrophages, red blood cells (reticulocytes), mast cells, platelets / megakaryocytes, and dendritic cells; thyroid (thyroid epithelial cells, parafollicular cells) Cells from the endocrine system, including the parathyroid glands (Parathyroid Chief Cells, Oxyphil cells), adrenal glands (Chromaffin Cells), pineal cells (Pinealocytes), glial cells (astrocytes, microglia), Magnocellular Neurosecretory cells, stellate cells. cell), Boettcher cell Cells of the nervous system, including the pituitary gland (gonadotrope, corticotrope, thyrotrope, somatotrope, lactotrope), cells of the respiratory system, including lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, and dust cells, cells of the circulatory system, including myocarduocytes and pericytes, cells of the digestive system, including stomach (gastric chief cell, parietal cell, goblet cell, Paneth cell, G cell, D cell, ECL cell, I cell, K cell, S cell), enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (hepatocyte, Kupffer cell), and cartilage / bone / muscle Bone cells, including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts),Cartilage cells, including chondroblasts and chondrocytes; Skin cells, including trichocytes, keratinocytes, and melanocytes (nevus cells); Muscle cells, including mycotes; Urinary tract cells, including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, kidney proximal tubule brush border cells, and macula densa cells; Spermatozoon, Sertoli cells, and Leydig cells. Reproductive lineage cells, including ovum, adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, cuticular hair root sheath cells, Huxley's layer hair root sheath cells, Henle's layer hair root sheath cells, outer root sheath cells, hair matrix cells (stem cells), wet stratified barrier epithelial cells, cornea, tongue, oral cavity, esophagus, anal canal, distal urethra urethra, and stratified squamous epithelium of the vaginaSurface epithelial cells of the epithelium, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, urothelial cells (lining the bladder and ureters), Exocrine secretory epithelial cells, Salivary gland mucous cells (secreting polysaccharides), Salivary gland serous cells (secreting glycoprotein enzymes), Von Ebner's gland cells in the tongue (washes Taste buds), Mammary gland cells (secreting milk), Lacrimal gland cells (secreting tears), Ceruminous gland cells in the ear (secreting wax), Eccrine sweat gland dark cells (secreting glycoproteins), Eccrine sweat gland clear cells (secreting wax), Other cells in the gland include Apocrine sweat gland cells (smell secretion, sex hormone sensitive), Gland of Moll cells in the eyelids (specialized sweat glands), Sebaceous gland cells (secrete lipid-rich sebum), Bowman's gland cells in the nose (cleanses the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), Seminal vesicle cells (secrete seminal fluid components including fructose for swimming), Prostate gland cells (secrete seminal fluid components), Bulbourethral gland cells (secrete mucus), Bartholin's gland cells (secrete vaginal lubricant), Liter cells glands (mucus secretion), endometrial cells (Uterus endometriumcell (secretes carbohydrates), isolated goblet cells of the respiratory and digestive tract (secretes mucus), stomach lining mucous cells (secretes mucus), gastric gland zymogenic cells (secretes pepsinogen), gastric gland oxyntic cells (secretes hydrochloric acid), pancreatic gland cells (secretes bicarbonate and digestive enzymes), Paneth cells of the small intestine (secretes lysozyme), type II pneumocytes of the lungs (secretes surfactant), Clara cells of the lungs, hormone-secreting cells, anterior pituitary gland cells, somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, intermediate pituitary cells, magnocellular neurosecretory cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells cell, parathyroid cell, parathyroid chief cell, oxyphil cell, adrenal cell, chromaffin cells, Leydig cell of the testis, Theca interna cell, luteal cell of ruptured follicle, granulosa lutein cell, theca lutein cell, Juxtaglomerular cell (secreting renin), Macula densa cell of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands, and urogenital tract), kidney, type I pneumocyte (lining the air spaces of the lung), pancreatic duct cell (central acinar cell), Nonstriated duct cell (sweat gland, salivary gland, mammary gland, etc.) cells, duct cells (such as those in the seminal vesicles and prostate), epithelial cells lining closed body cavities, ciliated cells that function as propulsion cells, extracellular matrix secretion cells, contractile cells,Skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglial cells (in the central nervous system), neutrophil granulocyte, eosinophil granulocyte, basophil granulocyte, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitors of the blood and immune system (various types), multipotent Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, Adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sensory organs and peripheral nerve supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocytes, Retinal pigment epithelial cells, Germ cells, Oogonium / Oocytes, Spermatids, Spermatocytes, Spermatogonium cells (stem cells of spermatocytes), Sperm, Nurse cells, Ovarian follicle cells, Sertoli cells (in the testis), Thymus epithelial cells, Interstitial cells, and Interstitial kidney cells.

[0040] In some embodiments, the cell is a eukaryotic cell. Non-limiting examples of eukaryotic cells include mammalian (e.g., rodent, non-human primate, or human), non-mammalian (e.g., fish, avian, 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 a mammalian, avian, plant, or insect cell. In some embodiments, the nucleated cell is a primary cell. In some embodiments, the nucleated cell is an immune cell (e.g., lymphocyte (e.g., T cell, B cell), macrophage, natural killer cell, neutrophil, mast cell, basophil, dendritic cell, monocyte, myeloid-derived suppressor cell, eosinophil). In some embodiments, the nucleated cell is a phagocyte or a leukocyte. In some embodiments, the nucleated cells are stem cells (e.g., adult stem cells (e.g., hematopoietic stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells), embryonic stem cells, induced pluripotent stem cells (iPS)). In some embodiments, the nucleated cells are progenitor cells. In some embodiments, the nucleated cells are derived from a cell line. In some embodiments, the nucleated cells are suspension cells. In some embodiments, the nucleated cells are adherent cells. In some embodiments, the nucleated cells are cells immortalized by expression of an oncogene. In some embodiments, the nucleated cells are immortalized by expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, the nucleated cells are immortalized by expression of a viral gene, such as Simian Virus 40 (SV40). In some embodiments, the nucleated cells are cells derived from a patient or subject (e.g., autologous patient-derived cells or allogenic patient-derived cells).In some embodiments, nucleated cells are transfected with a vector (e.g., a viral vector (e.g., a retroviral vector (e.g., a lentiviral vector), an adeno-associated virus (AAV) vector, a vesicular viral vector (e.g., a vesicular stomatitis virus (VSV) vector), or a hybrid viral vector), a plasmid) before the nucleated cells are enucleated using any of the enucleation techniques described herein.

[0041] In some embodiments, the cytoplasts are derived from cells that are autologous to the subject. In some embodiments, the cytoplasts are derived from cells that are allogeneic to the subject.

[0042] In some embodiments, the cytoplast is derived from an immune cell, hi some embodiments, the cytoplast is derived from a natural killer (NK) cell, a neutrophil, a macrophage, a lymphocyte, a fibroblast, an adult stem cell (e.g., a hematopoietic stem cell, a breast 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 progenitor cell, or an induced pluripotent stem cell.

[0043] In some embodiments, the parent cell is not an erythrocyte or erythroid progenitor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial progenitor cell. In some embodiments, the enucleated cell is not an erythrocyte or erythroid progenitor cell. In some embodiments, the enucleated cell is not a platelet cell. In some embodiments, the enucleated cell is not an endothelial cell. In some embodiments, the enucleated cell is not an endothelial progenitor cell. In some embodiments, the enucleated cell does not express complement receptor 1 (CR1). In some embodiments, the enucleated cell does not express CD44. In some embodiments, the enucleated cell does not express VLA-4. In some embodiments, the enucleated cell does not express BCAM. In some embodiments, the enucleated cell does not express ICAM. In some embodiments, the enucleated cell does not express a receptor for collagen. In some embodiments, the enucleated cell does not express a receptor for thrombopoietin. In some embodiments, the enucleated cells do not express a receptor for collagen. In some embodiments, the enucleated cells do not express a receptor for von Willebrand factor (VWF). In some embodiments, the enucleated cells do not express a receptor for fibrinogen. In some embodiments, the enucleated cells do not express a GP1b-IX-V receptor. In some embodiments, the enucleated cells do not express a GPIIb / IIIa receptor. In some embodiments, the enucleated cells do not express a prostanoid receptor. In some embodiments, the enucleated cells do not express a purinergic receptor. In some embodiments, the enucleated cells do not express a thromboxane receptor.

[0044] In some embodiments, the parent cell may be enucleated and manipulated for therapeutic use. In some embodiments, the parent cell is any of the nucleated cells described herein. 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 enucleated cell is derived from an induced pluripotent stem cell (iPSC). In some embodiments, the parent cell is not an erythroid or erythroid progenitor cell. In some embodiments, the parent cell is a platelet cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial progenitor cell. The parent cell may be treated with cytochalasin to soften the cortical actin cytoskeleton. The nucleus may then be physically extracted from the cell body by high speed centrifugation on a Ficoll gradient to generate an enucleated cell. Because the enucleated cells and intact nucleated cells settle into different layers in the Ficoll gradient, the enucleated cells may be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated). The enucleation process is clinically scalable to process tens of millions of cells. In some embodiments, the enucleated cells can be used as disease-homing vehicles to deliver clinically relevant cargo / payloads to treat a variety of diseases.

[0045] In some embodiments, the enucleated cells contain one or more organelles or cytoskeleton and form tunneling nanotubes (TNTs) or membrane nanotubes. The inventors of the present disclosure have discovered that enucleating a cell can, in some cases, increase TNTs in the cell, which may provide a therapeutic advantage. In some embodiments, the increase in TNT of the enucleated cells is determined by the enucleated cells exhibiting an increased number of TNT formations compared to TNT formations by otherwise identical cells that have not been enucleated. In some embodiments, the increase in TNT of the enucleated cells is determined by the enucleated cells exhibiting an increased length of TNT formations compared to TNT formations by otherwise identical cells that have not been enucleated. In some embodiments, the increase in TNT of the enucleated cells is determined by the enucleated cells exhibiting an increased diameter of TNT formations compared to TNT diameters by otherwise identical cells that have not been enucleated. In some embodiments, the tunneling nanotubes deliver at least one exogenous agent as described herein to the target cell. In some embodiments, the increased formation of TNTs, the increased length of TNTs, or the increased diameter of TNTs increases the effectiveness of the delivery of at least one exogenous agent to a target cell by the enucleated cell. In some embodiments, the exogenous agent is a therapeutic agent. In some embodiments, the exogenous agent can normalize the vasculature. In some embodiments, the tunneling nanotube is a protrusion extending from the plasma membrane of the enucleated cell, and the tunneling nanotube can be about 1 μm to about 1000 μm in length. In some embodiments, the tunneling nanotube is about 0.1 nm to about 10.0 μm in diameter. In some embodiments, the tunneling nanotube comprises actin. In some embodiments, the tunneling nanotube comprises both actin and microtubules. In some embodiments, the tunneling nanotube comprises the cytoplast content of the enucleated cell. For example, the tunneling nanotube can contain vesicles, organelles, or at least one exogenous agent described herein. In some embodiments, the tunneling nanotube can deliver at least one exogenous agent by contacting the target cell.In some embodiments, the tunneling nanotubes can deliver at least one exogenous agent to the target cell by forming fluid communication between the enucleated cell and the target cell. In some embodiments, upon delivery of at least one exogenous agent from the enucleated cell to the target cell, the target cell can form additional tunneling nanotubes with other non-target cells. In such a scenario (similar to the bystander effect), the target cell can communicate at least one exogenous agent and its effect to other non-target cells, thus propagating the therapeutic effect of the enucleated cell to other non-target cells.

[0046] After enucleation of the parent cell, the enucleated cells described herein retain one or more organelles endogenous to the parent cell. In some embodiments, all of the one or more organelles are retained. In some embodiments, less than all of the one or more organelles are retained. In some embodiments, the Golgi apparatus and / or the endoplasmic reticulum are retained, which are involved in protein synthesis and secretion. Retention of one or more organelles at least partially allows the enucleated cells to synthesize or release biomolecules disclosed herein (e.g., single domain antibodies or portions thereof, targeting moieties, immune evasion moieties, etc.) in the absence of a nucleus. In some embodiments, the enucleated cells retain a cytoskeleton, such as a filament or tubule, for the formation of TNTs.

[0047] Enucleated cells can be smaller than their nucleated counterparts (e.g., nucleated parent cells) and therefore can better migrate through small openings in the vasculature and tissue parenchyma. In addition, removing the large density nucleus alleviates a major physical barrier, allowing the cells to freely migrate through small openings in blood vessels and tissue parenchyma. Thus, the enucleated cells have improved biodistribution in the body and migration to target tissues. In some embodiments, the enucleated cells are at least 1 μm in diameter. In some embodiments, the enucleated cells are greater than 1 μm in diameter. In some embodiments, the enucleated cells are 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 10-20 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, the enucleated cells are 10-30 μm in diameter. In some embodiments, the enucleated cells have a diameter of 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 enucleated cells have a diameter of about 8 μm. In some embodiments, some enucleated cells may advantageously be small enough to allow better homing or delivery to a target site. For example, the enucleated cells described herein may pass through passages in narrow lung tissue or structures, such as alveolar ducts or microcapillaries, that most cells, such as parent cells, cannot pass through.

[0048] In some embodiments, enucleated cells have significant therapeutic value because they remain viable, do not differentiate into other cell types, do not secrete bioactive molecules, can physically migrate / home within about 5 days, can be extensively enucleated ex vivo to perform specific therapeutic functions, and can be fused to the same or other cell types to transfer the desired production, either natural or enucleated. Thus, enucleated cells have broad utility as cellular vehicles for delivering therapeutically significant biomolecules and disease-targeting cargoes, including genes, viruses, bacteria, mRNA, shRNA, siRNA, polypeptides (including antibodies and antigen-binding fragments), plasmids, gene editing mechanisms, or nanoparticles. The present disclosure enables the generation of safe (e.g., no unwanted DNA is transferred to the subject) and controllable (e.g., cell death occurs within 3-4 days) cell-based carriers that can be genetically enucleated to deliver specific disease-therapeutic and health-promoting cargoes to humans. In some embodiments, the enucleated cells remain viable and retain the ability to migrate or home for about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 5 days or more after administration to a subject in need thereof.

[0049] In some embodiments, the enucleated cells are engineered to express at least one of an exogenous DNA molecule, an exogenous RNA molecule, an exogenous protein, or an exogenous protein, a gene editing mechanism, or a combination 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, or a combination thereof. In some embodiments, the exogenous RNA molecule is a messenger RNA (mRNA), a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an RNA virus, or a combination thereof. In some embodiments, the exogenous protein is a cytokine, a growth factor, a hormone, an antibody or an antigen-binding fragment thereof, an enzyme, or a combination thereof. In some embodiments, the antibody is a single domain antibody or an antigen-binding fragment thereof. In some embodiments, the parent cell (e.g., a nucleated cell) is genetically enucleated prior to enucleation (e.g., before enucleating). In some embodiments, the parent cell is genetically enucleated after enucleation (e.g., after enucleation).

[0050] In some embodiments, the enucleated cells described herein comprise a transmembrane moiety. In some embodiments, the transmembrane moiety is genetically modified to fuse or complex with a single domain antibody or antigen-binding fragment thereof, or a therapeutic agent described herein, or combinations thereof. In some embodiments, the transmembrane moiety is genetically modified to fuse with a single domain antibody or antigen-binding fragment thereof, or a therapeutic agent described herein, or combinations thereof. In some embodiments, the enucleated cells comprise the immune-evading moiety. In some embodiments, the immune-evading comprises a "don't eat me" signaling peptide, such as CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof.

[0051] In some embodiments, the enucleated cells or compositions comprising the enucleated cells may be cryopreserved (e.g., the enucleated cells or compositions comprising the enucleated cells are stored at freezing temperatures) or cryohibernated (e.g., the enucleated cells or compositions comprising the enucleated cells are stored at temperatures between ambient and freezing temperatures). The duration of cryopreservation or cryohibernation may be for a period of about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or longer. In some embodiments, the enucleated cells exhibit a viability following cryopreservation or freeze-freeze hibernation that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more similar to a comparable cell (e.g., a parent cell or an enucleated cell described herein that has not been cryopreserved or freeze-freeze hibernated) following the same period of cryopreservation or freeze-freeze hibernation. For example, in some embodiments, the viability is reduced by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a comparable cell. In another example, in some embodiments, the viability is increased by 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to a comparable cell. Viability in this context may be measured by Trypan blue dye exclusion as described herein. In some embodiments, Trypan blue dye exclusion is performed by (a) centrifuging an aliquot of the nucleated cells in suspension to produce a cell pellet, (b) resuspending the cell pellet in serum-free medium to generate a serum-free cell suspension, (c) mixing a portion of the Trypan blue dye with a portion of the serum-free cell suspension, and (d) counting the nucleated cells within 3-5 minutes of (c), where at least some of the nucleated cells do not stain with Trypan blue dye, indicating viability. In some embodiments, viability is measured using Annexin-V cell surface staining. In some embodiments, viability is measured by expression of an exogenous polypeptide.For example, the viability of the enucleated cells can be determined by the expression of an exogenous antibody or a single domain antibody expressed by the enucleated cells. In some embodiments, the viability is measured by the expression 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 is measured by the cellular activity of the enucleated cells. In some embodiments, the viability is measured by the homing ability of the enucleated cells, as determined by chemosensing or chemokine homing activity as described herein.

[0052] In some embodiments, the enucleated cells or compositions comprising the enucleated cells may be lyophilized. In some embodiments, the enucleated cells, after reconstitution from lyophilization, exhibit a viability similar to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more relative to a comparable cell (e.g., a parent cell or a non-lyophilized enucleated cell described herein).

[0053] In some embodiments, the enucleated cells or compositions comprising enucleated cells may be dehydrated. In some embodiments, the enucleated cells, after being rehydrated from lyophilization, exhibit a viability similar to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more relative to comparable cells (e.g., parent cells or enucleated cells described herein that have not been dehydrated).

[0054] In some embodiments, the enucleated cells, or compositions comprising the enucleated cells, are stable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more at 4° C. In some embodiments, the compositions are stable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more at room temperature. In some embodiments, the compositions are stable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more at 37° C. In some embodiments, the enucleated cells, or compositions comprising the enucleated cells, may remain viable after being administered to a subject in need thereof to treat a disease or condition described herein. In some embodiments, the enucleated cells or compositions comprising the enucleated cells may remain viable for about 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, or more after administration to a subject.

[0055] In some embodiments, the enucleated cells may be obtained from parent cells that are autologous to a subject in need of treatment with the enucleated cells described herein. In some embodiments, the enucleated cells may be obtained from parent cells that are allogeneic to a subject in need of treatment with the enucleated cells described herein.

[0056] Transmembrane part In some embodiments herein, a cell or composition comprising an enucleated cell comprising at least one transmembrane moiety is described. In some embodiments, the cell is enucleated using, such as, the enucleation method disclosed herein. In some embodiments, the transmembrane moiety is linked to a polypeptide, such as a single domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed herein, or a combination thereof. In some embodiments, the transmembrane moiety is linked by a covalent bond. In some embodiments, the transmembrane moiety is a fusion protein comprising a single domain antibody or antigen-binding fragment thereof, a therapeutic agent disclosed herein, or a combination thereof. In some embodiments, an exogenous polypeptide is complexed to the transmembrane moiety. In some embodiments, the transmembrane moiety comprises a full-length protein or a variant thereof or a fragment thereof. In some embodiments, the transmembrane moiety is endogenous to the parent cell that is enucleated to obtain the enucleated cell. In some embodiments, the transmembrane moiety can be an exogenous transmembrane moiety to the parent cell or the enucleated cell. In some embodiments, the transmembrane moiety comprises a single transmembrane α-helix (bitopic membrane protein). The transmembrane portion comprises a polymorphic transmembrane α-helical protein. In some embodiments, the transmembrane portion comprises a polytopic transmembrane β-sheet protein. In some embodiments, the transmembrane portion comprises a type I, type II, type III, or type IV transmembrane protein. Non-limiting examples of transmembrane proteins may include any combination of CD4, CD14, glycophorin a (GPA), or integrins.

[0057] In some embodiments, the transmembrane portion is added to the exogenous polypeptide by modification. For example, the transmembrane portion may be added to the N-terminus or C-terminus of the exogenous polypeptide to insert the exogenous polypeptide into the cell membrane of the enucleated cell described herein. A non-limiting example of a modification made to the exogenous polypeptide to add a transmembrane portion may include the addition of an anchor molecule. The anchor molecule may be any molecule (e.g., a glycolipid) that can be inserted into and remain in the cell membrane. In some embodiments, the anchor molecule comprises glycosylphosphatidylinositol, farnesyl, palmitate, myristate, or a combination thereof.

[0058] targeting part In some embodiments herein, cells are described that include a targeting moiety. In some embodiments, the cells are enucleated using, for example, the enucleation methods disclosed herein. The targeting moiety described herein is designed to direct the enucleated cell to a target cell or target environment (e.g., tissue) in the subject following delivery (e.g., systemic delivery) of the enucleated cell to a subject. In some embodiments, the targeting moiety is expressed on the surface of the enucleated cell. In some embodiments, the targeting moiety is complexed with a transmembrane moiety described herein. In some embodiments, the targeting moiety is secreted by the enucleated cell. An enucleated cell that includes a targeting moiety localizes to a target cell or target environment in a subject 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 over the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 2-fold increase over the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 5-fold increase over the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 10-fold increase over the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 20-fold increase over the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 50-fold increase over the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 5% increase over the localization of a comparable enucleated cell lacking the targeting moiety, hi some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject with at least a 10% increase over the localization of a comparable enucleated cell lacking the targeting moiety.In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 20% increased relative to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 30% increased relative to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 40% increased relative to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 50% increased relative to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 60% increased relative to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 70% increased compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 80% increased compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 90% increased compared to the localization of a comparable enucleated cell lacking the targeting moiety. In some embodiments, enucleated cells comprising a targeting moiety localize to a target cell or target environment in a subject at least 100% increased compared to the localization of a comparable enucleated cell lacking the targeting moiety.

[0059] 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 a portion thereof, involved in chemokine signaling. In some embodiments, the exogenous antibody is an exogenous single domain antibody or a fragment thereof.

[0060] In some embodiments, the targeting moiety targets a biomarker expressed by or associated with the target cell or microenvironment. In some embodiments, the biomarker may be released by the target cell. The biomarker may indicate the presence of a disease or condition. In some embodiments, the biomarker is expressed by an immune cell responsive to the target cell or a disease or condition-associated microenvironment. In some embodiments, the biomarker may be an epitope or antigen. In some embodiments, a biomarker that includes an epitope may be bound by an antibody that is distinct from the antibody or antigen-binding fragment thereof that confers the therapeutic property (e.g., a therapeutic agent).

[0061] In some embodiments, the targeting moiety targets a biomarker expressed or released by lung cells or lung cancer cells. Non-limiting examples of cancer cell biomarkers include carbonic anhydrase 9 (CA9), carbonic anhydrase 12 (CA12), Kita-Kyushu lung cancer antigen 1 (CXorf61), desmoglein 3 (DSG3), FAT atypical cadherin 2 (FAT2), G protein-coupled receptor 87 (GPR87), kisspeptin receptor (KISS1R), LY6 / PLAUR domain containing 3 (LYPD3), solute carrier family 7 member kinase 1 (SEQ ID NO: 1), and IL-11. bar 11 (SLC7A11), transmembrane serine protease 4 (TMPRSS4), tissue factor pathway inhibitor (TFPI), midkine (MDK), secreted phosphoprotein 1 (OPN), matrix metallopeptidase 2 (MMP2), TIMP metallopeptidase inhibitor 1 (TIMP1), carcinoembryonic antigen (CEA), keratin 19 (CYFRA21-1), SCC, advanced glycosylation end-product specific receptor (AGER), adipogenesis regulatory factor (C10orf116), adducin 2 (ADD2), periaxin (PRX), laminin subunit β3 (LAMB3), (synemin (SYNM), spectrin α, erythrocytic 1 (SPTA1), ankyrin 1 (ANK1), hemoglobin subunit epsilon 1 (HBE1), hemoglobin subunit gamma 1 (HBG1), carbonic anhydrase 1 (CA1), tenascin XB (TNXB), multimerin 2 (MMRN2), hemoglobin subunit alpha 1 (HBA1), caveolin 1 (CAV1), hemoglobin subunit beta (HBB), type VI collagen alpha 6 chain (COL6A6), chromosome 1 open reading frame 198 (C1orf198), chloride intracellular channel 2 (CLIC2), serum deprivation response protein (SDPR), EH domain containing 2 (EHD2), apolipoprotein A2 (APOA2), NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), protein kinase C delta binding protein (PRKCDBP),Laminin subunit α3 (LAMA3), EvC ciliary complex subunit 2 (LBN), actin-like protein (ACT), insulin-like growth factor binding protein 3 (IGFBP3), prostaglandin D2 synthase (L-PGDS), haples (HAP), hepatocyte growth factor (HGF), eukaryotic translation initiation factor 4 gamma 2 (AAG1 / 2), clusterin (CLU), calreticulin (SSA), tRNA suppressor anticodon 2-1 (TTA), apolipoprotein A4 (APOA4), fibrinogen α chain (FIBA), serum amylo id cluster A (SAA), ceruloplasmin (CP), haptoglobin (HP), transthyretin (TTR), keratin 2 (KRT2A), solute carrier family 1 (GLT1B), casein kinase 1 (CK1), serine / threonine kinase 1 (AKT), MBL2 (mannose-binding lectin 2), fibrinogen α chain (FGA), gelsolin (GSN), ficolin 3 (FCN3), carnosin dipeptidase 1 (CNDP1), calcitonin-related polypeptide α (CALCA), carbamoyl phosphate synthase 1 (CPS1), chromoglobulin Ranin B (CHGB), involucrin (IVL), anterior gradient 2, protein disulfide isomerase family member (AGR2), nuclear autoantigenic sperm protein (NASP), phosphofructokinase, platelet (PFKP), thrombospondin 2 (THBS2), thioredoxin domain-containing 17 (TXNDC17), proprotein convertase subtilisin / kexin type 1 (PCSK1), cellular retinoic acid-binding protein 2 (CRABP2), acyl-CoA binding domain-containing 3 (ACBD3), desmoglein 2 (DSG2), LPS response beige-like anchor protein (LRBA), serine / threonine kinase receptor-associated protein (STRAP), VGF nerve growth factor-inducible (VGF), NOP2 nucleolar protein (NOP2), lipocalin 2 (LCN2), creatine kinase, mitochondrial 1B (CKMT1B), aldo-keto reductase family 1 member B10 (AKR1B10), carboxypeptidase D (CPD), proteasome activator subunit 3 (PSME3), villin 1 (VIL1), serpin family B member 5 (SERPINB5),Ribosomal protein L5 (RPL5), plakophilin 1 (PKP1), ribosomal protein L10 (RPL10), aldo-keto reductase family 1 member B10 (AKR1B10), aldo-keto reductase family 1 member C1 (AKR1C1), proliferating cell nuclear antigen (PCNA), ribosomal protein S2 (RPS2), aldo-keto reductase family 1 member C3 (AKR1C3), acyl-CoA-linked dodecyl sulfate main-containing 3 (ACBD3), visinin-like 1 (VSNL1), adenosylhomocysteinase (AHCY), stromal interaction molecule 1 (STIM1 or IMMP10), p21(RAC1)-activated kinase 2 (PAK2), involucrin (IVL), isoleucine-tRNA synthetase (IARS), proteasome 26S subunit ubiquitin receptor, non-ATPase 2 (PSMD2), guanylate-binding protein 5 (GBP5), minichromosome Maintenance complex component 6 (MCM6), N-myc downstream regulated 1 (NDRG1), NOP58 ribonucleoprotein (NOP58), S100 calcium-binding protein A2 (S100A2), neuregulin 1 (NRG1-2), carnosine dipeptidase 1 (CNDP1), ubiquitin cross-reactive protein (UCRP1), cerberus (CER), plasminogen activator, urokinase (UPA or PLAU), matrix metallopeptide These include thrombopoietinase 14 (MT1-MMP), stratifin (SFN), transferrin (TF), albumin (ALB), S100 calcium-binding protein A9 (S100A9), stathmin 1 (STMN), enolase (ENO), insulin-like growth factor binding protein 7 (IGFBP7), matrix metallopeptidase 14 (MMP14), thrombospondin 1 (THBS1), and thrombospondin 2 (THBS2).

[0062] In some embodiments, the targeting moiety targets a biomarker expressed or released by metastatic cancer cells. For example, cancer cells may originate from one tissue and then metastasize to a different location. In some embodiments, the metastatic cancer cells express non-limiting examples of cancer biomarkers described herein. In some embodiments, the metastatic cancer cells express Melanoma Associated Antigen (MAGE-A3), Membrane associated glycoprotein (MUC-1), glycoprotein-epithelial cell adhesion molecule (EpCAM), KRAS proto-oncogene (KRAS), Anaplastic lymphoma kinase (ALK), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), Programmed cell death protein 1 (PD-1), Epidermal growth factor receptor 2 (EGFR-2), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-39, IL-40, IL-41, IL-42, IL-53, IL-54, IL-55, IL-61, IL-72, IL-83, IL-84, IL-85, IL-95, IL-196, IL-197, IL-198, IL-199, IL-210, IL-2211, IL-231, IL-242, IL-251, IL-252, IL-253, IL-254, IL-255, IL-255, IL express cancer biomarkers including epidermal growth factor (EGF), serine protease easter (EA), telomerase reverse transcriptase (TERT), PRAME nuclear receptor transcription factor (PRAME), receptor tyrosine-protein kinase erbB-2 (HER), or vascular endothelial growth factor (VEGF), carcinoembryonic antigen (CEA), MAGE-A1, MAGE-A4, survivin, six transmembrane epithelial antigen of prostate 1 (STEAP1), sex determining region Y-box 2 (SOX2), or cancer / testis antigen 1 (CTAG1B).

[0063] In some embodiments, the targeting moiety targets a biomarker expressed or released by an endothelial cell. In some embodiments, the endothelial cell is a vascular cell. In some embodiments, the endothelial cell is a lymphatic cell. In some embodiments, the biomarker is expressed or released by a vascular cell. In some embodiments, the biomarker is expressed or released by a lymphatic cell. Non-limiting examples of endothelial cell biomarkers include angiotensin I converting enzyme (ACE or CD143), C1qR1 / CD93, VE-cadherin, CC chemokine receptor D6, CD31 / PECAM-1, CD34, CD36 / SR-B3, CD151, CD160, CD300g / nepmucin, CL-K1 / COLEC11, CL-P1 / COLEC12, coagulation factor III / tissue factor, DC-SIGNR / CD299, DCBLD2 / E SDN, ECSCR, EMMPRIN / CD147, endoglin / CD105, endomucin, endosialin / CD248, EPCR, erythropoietin R, endothelial cell adhesion molecule (ESAM), fatty acid binding protein 5 (FABP5 or E-FABP), fatty acid binding protein 6 (FABP6), ICAM-1 / CD54, ICAM-2 / CD102, IL-1RI, IL-13Rα1, integrin α4 / CD49d, integrin α4 β1, integrin α4β7 / LPAM-1, integrin β2 / CD18, Kruppel-like factor 4 (KLF4), lymphatic endothelial hyaluronan receptor 1 (LYVE-1), MCAM / CD146, nectin-2 / CD112, PD-ECGF / thymidine phosphorylase, podocalyxin, podoplanin, sphingosine-1-phosphate receptor 1 (S1P1 or EDG-1), sphingosine-1-phosphate receptor 2 (S1P2 or is EDG-5), sphingosine-1-phosphate receptor 3 (S1P3 or EDG-3), sphingosine-1-phosphate receptor 4 (S1P4 or EDG-6), sphingosine-1-phosphate receptor 5 (S1P5 or EDG-8), E-selectin / CD62E, P-selectin / CD62P, SLAM / CD150, stabilin-1, stabilin-2, plexin domain containing 1 (TEM7 or PLXDC1),These include ANTXR cell adhesion molecule 1 (TEM8 or ANTXR1), thrombomodulin / BDCA-3, thrombospondin type 1 domain containing (THSD1), thrombospondin type 1 domain containing 7A (THSD7A), TEK receptor tyrosine kinase (Tie-2), TNFRI / TNFRSF1A, TNFRII / TNFRSF1B, TRA-1-85 / CD147, TRAILR2 / TNFRSF10B, TRAILR1 / TNFRSF10A, VCAM-1 / CD106, VE-statin, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGFR3 / Flt-4, angiogenic factor with G patch and FHA domain 1 (VG5Q), or von Willebrand factor (vWF-A2).

[0064] In some embodiments, the targeting moiety comprises a chemokine receptor or chemokine ligand, or a portion thereof, including chemokine signaling, such as SDF-1α / CXCR4, CCL2 / CCR2, or adhesion molecules, such as PSGL-1. As shown herein, enucleated cells can be enucleated to express functional CXCR4, CCR2, and glycosylated PSGL-1, which can greatly facilitate specific targeting of the enucleated cells. In some embodiments, targeting moieties, such as CXCR4, CCR2, or PSGL-1, can be expressed on the surface of the enucleated cells. Non-limiting examples of cell surface proteins that can be expressed on the cell surface of the enucleated cells as targeting moieties include chemokines, such as CXCR4, CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. In some embodiments, the enucleated cells can be enucleated to secrete a targeting moiety or tethered to the extracellular matrix, such as SDF1α or CCL2. Non-limiting examples of targeting moieties that may be secreted by enucleated cells include SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, and CXCL12. In some embodiments, the enucleated cells comprise cell-matrix receptors and cell-cell adhesion molecules including integrins, cadherins, glycoproteins, and heparin sulfate proteoglycans.

[0065] In some embodiments, the enucleated cells may further comprise surface markers (e.g., by genetically engineering the cells from which they are obtained or derived therefrom) that aid in evading the subject's immune system. For example, in some embodiments, the enucleated cells may comprise CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof. Without being bound to any particular theory, it is believed that CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof help prevent the 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 include 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 molecule can be recognized by immune cells and induce an immune response. For example, the therapeutic molecule can be 4-1BB or any one of the cytokines described herein for inducing an immune response.

[0066] Therapeutic Agents In some embodiments, the cells of the present disclosure include at least one therapeutic agent. In some embodiments, the cells are enucleated using the enucleation methods disclosed herein or the like. In some embodiments, the active agent includes at least one of a DNA molecule, an RNA molecule, a protein (e.g., an enzyme, an antibody, an antigen, a toxin, a 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, an antiviral drug, colchicine, taxol, mitomycin, or emtansine), a gene editor, a nanoparticle, or another active agent (e.g., a bacterium, a bacterial spore, a bacteriophage, a bacterial component, a virus (e.g., an oncolytic virus), an exosome, a lipid, or an ion). Non-limiting examples of RNA molecules include messenger RNA (mRNA), short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA, long non-coding RNA (lncRNA), and RNA viruses. Non-limiting examples of DNA molecules include single-stranded DNA, double-stranded DNA, oligonucleotides, plasmids, bacterial DNA molecules, and DNA viruses. Non-limiting examples of proteins include cytokines, growth factors, hormones, antibodies or antigen-binding fragments thereof, single domain antibodies or antigen-binding fragments thereof, small peptide-based drugs, and enzymes. Non-limiting examples of oncolytic viruses include Talimogene laherparepvec, Onyx-015, GL-ONC1, CV706, Voyager-V1, and HSV-1716. Some wild-type viruses, such as vaccinia virus, vesicular stomatitis virus, poliovirus, reovirus, Senecavirus, ECHO-7, or Semliki forest virus, also exhibit oncolytic behavior.

[0067] In some embodiments, the enucleated cell is engineered to produce (e.g., express, and in some cases release or secrete) a therapeutic agent. In some embodiments, the parent cell from which the enucleated cell is derived may be engineered to produce a therapeutic agent prior to enucleation to produce the enucleated cell. In some embodiments, the enucleated cell is engineered to produce a therapeutic agent after enucleation (in the absence of a nucleus). In some embodiments, the therapeutic agent is exogenous to the enucleated cell or its parent cell. In some embodiments, the therapeutic agent is endogenous to the enucleated cell or its parent cell. In some embodiments, the enucleated cell of the present disclosure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more therapeutic agents.

[0068] In some embodiments, the therapeutic agent comprises a modified form of the DNA molecule, RNA molecule, protein, peptide, small molecule active agent, and / or gene editing factor compared to the naturally occurring version. In some embodiments, the therapeutic agent is a modified, truncated, or non-mutated version and / or copy of the DNA molecule, RNA molecule, protein, peptide, small molecule active agent, and / or gene editing factor. For example, the therapeutic agent can modify mutated tumor protein p53 (p53) or epidermal growth factor receptor (EGFR) in target cells as part of the treatment of lung cancer.

[0069] The therapeutic agent may be or may include a targeting moiety as described herein. Non-limiting examples of targeting moieties that may be produced by or contained in enucleated cells include chemokine receptors, adhesion molecules, and antigen-binding polypeptides (e.g., single domain antibodies and antigen-binding fragments thereof), or portions thereof. In some embodiments, the therapeutic agent may be or may include a transmembrane moiety as described herein.

[0070] In some embodiments, the therapeutic agent is recombinantly expressed by the enucleated cell or a 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, the 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. In some embodiments, the parent cell is engineered to recombinantly express the therapeutic agent prior to enucleation.

[0071] In some embodiments, the therapeutic agent is not naturally expressed (e.g., in the absence of manipulation) in the cell from which the enucleated cell is 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 treatment (e.g., a tumor or a particular tissue such as the brain, intestine, lung, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is exogenous to the intended site of treatment). In some embodiments, the level of therapeutic agent does not naturally occur in the enucleated cell of the parent cell, such as overexpression or underexpression of the therapeutic agent.

[0072] 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 taken up by the cell before or after enucleation of the cell. Alternatively, the therapeutic agent may be synthesized by the cell and then delivered to the target cell.

[0073] In some embodiments, the therapeutic agent comprises at least two (e.g., at least 2, 3, 4, 5, or more) different DNA molecules, RNA molecules, proteins, peptides, small molecule active agents, or gene editing factors in any combination. For example, in some embodiments, the therapeutic agent comprises a 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.

[0074] In some embodiments, the therapeutic agent comprises a polypeptide. In some embodiments, the polypeptide is exogenous. In some embodiments, the polypeptide is encoded by an exogenous polynucleotide delivered to the parent cell or the enucleated cell. In some embodiments, the polypeptide is synthesized or released by at least one intracellular organelle of the enucleated cell. In some embodiments, the polypeptide is released by the enucleated cell. In some embodiments, the polypeptide is expressed on the cell surface or on the enucleated cell. In some embodiments, the enucleated cell delivers the polypeptide to a target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell expresses a cancer biomarker for any cancer described herein. In some embodiments, the target cell is an endothelial cell. In some embodiments, the endothelial cell expresses an endothelial biomarker described herein. In some embodiments, the endothelial cell is a vascular cell. In some embodiments, the endothelial cell is a lymphatic cell.

[0075] In some embodiments, the exogenous polypeptide comprises any one of the cytokines 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 a cytokine. In some embodiments, the exogenous polypeptide comprises a solubility determined by a turbidimetric or thermodynamic solubility assay by dissolving the exogenous polypeptide in a solvent such as an organic solvent including dimethylsulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc., or an inorganic solvent including 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.

[0076] In some embodiments, the exogenous polypeptide comprises a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof. Non-limiting examples of TNF superfamily member polypeptides include lymphotoxin alpha (TNFβ), tumor necrosis factor (TNFα), lymphotoxin β (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 (4-1BBL), TNF-related apoptosis-inducing ligand (CD253 or APO-2L), receptor activator of nuclear factor kappa B ligand (CD254, Exogenous polypeptides include OPGL, TRANCE, or ODF), weak inducer of TNF-related apoptosis (APO-3L or DR3L), proliferation-inducing ligand (CD256, TALL-2, or TRDL1), B-cell activating factor (CD257, 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). In some embodiments, the exogenous polypeptide comprises a peptide sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 1501, 1504, 1505, or 1508. In some embodiments, the exogenous polypeptide comprises LIGHT or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 1508. In some embodiments, the exogenous polypeptide comprises LIGHT or a catalytically active fragment thereof. In some embodiments, the exogenous polypeptide comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 1511.

[0077] In some embodiments, the exogenous polypeptide comprises a soluble member of the TNF superfamily or a soluble catalytically active fragment thereof. In some embodiments, the soluble TNF superfamily member polypeptide or a catalytically active fragment thereof comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NOs: 1501-1511. In some embodiments, the soluble TNF superfamily member polypeptide or a catalytically active fragment thereof comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NOs: 1508-1510. In some embodiments, the soluble TNF superfamily member polypeptide or a catalytically active fragment thereof comprises a peptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to SEQ ID NO: 1511.

[0078] In some embodiments, the therapeutic agent comprises any one of the immune checkpoint molecules described herein, or an immune checkpoint molecule inhibitor for inhibiting any one of the immune checkpoint molecules described herein. Non-limiting examples of immune checkpoint molecules include PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), 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.

[0079] In some embodiments, the therapeutic agent comprises an antibody, such as a single domain antibody described herein. In some embodiments, the antibody or single domain antibody binds to an immune checkpoint molecule. In some embodiments, the single domain antibody binds to an immune checkpoint molecule and modulates its expression or activity. In some embodiments, the single domain antibody is an inhibitor of immune checkpoint molecule activity or expression. In some embodiments, the single domain antibody is an activator of immune checkpoint molecule activity or expression. In some embodiments, the antibody or single domain antibody binds to PD-1, PD-L1, CTLA-4, VISTA, PDCD1LG2 (CD273), 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. In some embodiments, the therapeutic agent comprising the antibody or single domain antibody binds to PD-L1. In some embodiments, the therapeutic agent comprising the antibody or single domain antibody binds to CTLA-4. In some embodiments, the immune checkpoint molecule comprises an amino acid sequence that is about 80% or more identical to any one of SEQ ID NOs: 155-164, 203, 204, 315-322, 511, 531-535, 551-554, 571, 594, 611-619, or 711. In some embodiments, the single domain antibody or fragment thereof is encoded by a deoxyribonucleic acid (DNA) sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 801. In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 851.In some embodiments, the single domain antibody or fragment thereof is encoded from a DNA sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 901. In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO:951.

[0080] In some embodiments, the therapeutic agent comprises an antibody, such as a single domain antibody, that binds connective tissue growth factor (CTGF). In some embodiments, the single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 1701. In some embodiments, the single domain antibody or fragment thereof binds to an amino acid sequence of CTGF, wherein the amino acid sequence of CTGF comprises SEQ ID NO: 1601 or SEQ ID NO: 1602.

[0081] In some embodiments, the therapeutic agent comprises an exogenous agent comprising an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitor comprises an inhibitor that inhibits a vascular endothelial growth factor (VEGF) receptor (VEGFR), or a combination thereof (VEGF / VEGFR). In some embodiments, the VEGF / VEGFR inhibitor comprises a compound, a small molecule, a peptide, an antibody, or a combination thereof. In some embodiments, the VEGF inhibitor inhibits VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), or a combination thereof. In some embodiments, the VEGF inhibitor is a VEGF-A inhibitor. In some embodiments, the VEGF inhibitor comprises an antibody or a single domain antibody for targeting and inhibiting VEGF-A. In some embodiments, the VEGF inhibitor comprises a peptide, such as a KDR or FLT1 peptide. In some embodiments, the VEGF inhibitor comprises an antibody or a single domain antibody for targeting and inhibiting the VEGF-A receptor.

[0082] Antibodies and Single Domain Antibodies In some embodiments, the enucleated cell comprises an antibody or an antigen-binding fragment. In some embodiments, the enucleated cell comprises a polynucleotide encoding the antibody or an antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment of the present disclosure may be a neutralizing antibody, a non-neutralizing antibody, or a combination thereof. In some embodiments, the antibody or antigen-binding fragment is a single domain antibody. The usefulness and advantages of single domain antibodies (sdAbs) include, but are not limited to, their smaller size, more epitopes available, relatively low production costs, and improved robustness compared to their full-length antibodies.

[0083] In some embodiments, the antibody or antigen-binding fragment thereof described herein is a humanized antibody, variant, or derivative thereof, which can be formulated, for example, for administration to humans. In some embodiments, the humanized antibody is a chimeric humanized antibody or a fully human antibody, for example, comprising an amino acid sequence derived from or similar to a human antibody amino acid sequence, and a non-human amino acid sequence. For example, a portion of the heavy and / or light chain of the chimeric humanized antibody can be identical or similar to the corresponding sequence in a human antibody, while the remainder of the chain can be identical or similar to the corresponding sequence in a non-human, for example, antibody derived from another species or belonging to another antibody class or subclass. The non-human sequence can be humanized to reduce the possibility of immunogenicity while preserving target specificity, for example, by incorporating human DNA into the gene sequence of the gene that produces the antibody in the non-human animal. The humanized antibody can be, for example, a fully human antibody containing an amino acid sequence that is a human antibody amino acid sequence.

[0084] The antibody or antigen-binding fragment thereof of the present disclosure may comprise a basic 4-chain antibody unit. The basic 4-chain antibody unit comprises two heavy chain (H) polypeptide sequences and two light chain (L) polypeptide sequences. Each of the heavy chains comprises one N-terminal variable (VH) region and three or four C-terminal constant (CH1, CH2, CH3, and CH4) regions. Each light chain comprises one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chain variable region is aligned with the heavy chain variable region, and the light chain constant region is aligned with the first heavy chain constant region CH1. The pairing of the heavy chain variable region and the light chain variable region together forms a single antigen-binding site. Each light chain is linked to the heavy chain by one covalent disulfide bond. The two heavy chains are linked to each other by one or more disulfide bonds depending on the heavy chain isotype. Each heavy and light chain may also contain regularly-spaced intrachain disulfide bridges. The C-terminal constant region of the heavy chain contains the Fc region of an antibody, which may mediate effector functions, for example, by interacting with Fc receptors or complement proteins.

[0085] The light chain may be called kappa or lambda based on the amino acid sequence of the constant region. The heavy chain may be named alpha, delta, epsilon, gamma, or mu based on the amino acid sequence of the constant region. Antibodies are classified into five immunoglobulin classes or isotypes based on the heavy chain. IgA contains an alpha heavy chain, IgD contains a delta heavy chain, IgE contains an epsilon heavy chain, IgG contains a gamma heavy chain, and IgM contains a mu heavy chain. The IgG, IgD, and IgE class antibodies contain monomers of the above four chain units (two heavy chains and two light chains), while the IgM and IgA classes contain multimers of four chain units. The alpha and gamma classes may be further divided into subclasses based on differences in the sequence and function of the heavy chain constant region. The subclasses of IgA and IgG expressed by humans include IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The antibodies of the present disclosure comprise a human light chain constant domain sequence, e.g., a kappa (IgK) chain or a lambda (IgL) chain. In some embodiments, the antibodies comprise a human IgK constant domain, a variant, derivative, or fragment thereof. In some embodiments, the antibodies comprise a human IgL constant domain, a variant, derivative, or fragment thereof.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof, or single domain antibody or antigen-binding fragment thereof described herein comprises a signal peptidase. The signal peptide may result in higher protein expression and / or secretion by cells. The signal peptidase may cleave the signal peptide from the antibody or antigen-binding fragment thereof, or single domain antibody or antigen-binding fragment thereof, for example, during the secretion process, to generate a mature antibody that does not contain the signal peptide sequence.

[0087] The constant region of an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof, may mediate various effector functions and may be minimally involved in antigen binding. Different IgG isotypes or subclasses may be associated with different effector functions or therapeutic properties, for example, by interacting with different Fc receptors and / or complement proteins. Antibodies that include an Fc region that engages an activating Fc receptor may be involved in, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), induction of signal transduction via immunoreceptor tyrosine-based activation motifs (ITAMs), and induction of cytokine secretion. Antibodies that include an Fc region that binds an inhibitory Fc receptor may be involved in, for example, signal transduction via immunoreceptor tyrosine-based inhibitory motifs (ITIMs).

[0088] Different antibody subclasses include different abilities to induce immune effector functions. For example, IgG1 and IgG3 can effectively recruit complement and activate CDC, while IgG2 induces minimal ADCC. IgG4 has a low ability to induce immune effector functions. Modifications to the constant region can also affect antibody properties, such as enhanced or reduced Fc receptor ligation, enhanced or reduced ADCC, enhanced or reduced ADCP, enhanced or reduced CDC, enhanced or reduced signaling through ITAMs, enhanced or reduced cytokine induction, enhanced or reduced signaling through ITIMs, enhanced or reduced half-life, or enhanced or reduced co-binding of antigens to Fc receptors. Modifications can include, for example, amino acid mutations, altered post-translational modifications (e.g., glycosylation), combinations of domains from different isotypes or subclasses, or combinations thereof.

[0089] The antibodies or antigen-binding fragments thereof of the present disclosure comprise constant regions or Fc regions that are selected or modified to provide suitable antibody properties, e.g., properties suitable for treating a disease or condition disclosed herein. In some embodiments, IgG1 may be used to promote immune effector functions, e.g., for the treatment of inflammation, immune activation, and infection. In some embodiments, IgG4 may be used when the antagonistic properties of an antibody with reduced immune effector function are desired (e.g., to neutralize coronavirus antigens and inhibit viral entry into cells without promoting inflammation and immune activation).

[0090] The variable (V) region mediates antigen binding and may define the specificity of a particular antibody for an antigen. The variable region contains relatively invariant sequences called framework regions and hypervariable regions whose sequences vary considerably among antibodies of different binding specificities. The variable region of each antibody heavy or light chain contains four framework regions separated by three hypervariable regions. The variable regions of the heavy and light chains fold together to bring the hypervariable regions into close proximity to create the antigen-binding site. The four framework regions primarily adopt an f3 sheet structure, while the three hypervariable regions form loops that connect and in some cases form part of the f3 sheet structure.

[0091] Within the hypervariable region are the amino acid residues that primarily determine the binding specificity of the antibody. The sequence that contains these residues is known as the complementarity determining region (CDR). One antigen-binding site of an antibody contains six CDRs, three in the hypervariable region of the light chain and three in the hypervariable region of the heavy chain. The CDRs in the light chain can be called LCDR1, LCDR2, and LCDR3, while the CDRs in the heavy chain can be called HCDR1, HCDR2, and HCDR3.

[0092] In some embodiments, the antibody or antigen-binding fragment of the present disclosure includes variants or derivatives thereof. For example, non-human animals can be genetically modified to produce antibody variants or derivatives. In some embodiments, the antibody can be a single domain antibody (sdAb), such as a heavy chain only antibody (HCAb), VHH, or nanobody. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, FabIL-6R dimers and trimers, Fv, scFv, minibodies, diabodies, triabodies, and tetrabodies, and linear antibodies. Fab and Fab' are antigen-binding fragments that include the VH and CH1 domains of the heavy chain linked to the VL and CL domains of the light chain via disulfide bonds. F(ab')2 includes two Fabs or Fab's linked by disulfide bonds. Fv includes the VH and VL domains held together by non-covalent interactions. scFv (single chain variable fragment) is a fusion protein that contains VH and VL domains linked by a peptide linker. Manipulation of the orientation of VH and VL domains and linker length can be used to create different forms of the molecule that can be monomers, dimers (diabodies), trimers (triabodies), or tetramers (tetrabodies). Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers.

[0093] In other embodiments, the antibody is a binding fragment thereof. In some cases, the antibody is a humanized antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a multispecific antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a single domain antibody (e.g., a nanobody®) thereof. In some cases, the antibody is a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an IgNAR, a camelid antibody or a binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody may be a multispecific antibody. In some cases, a multispecific antibody comprises two or more target binding moieties, each of which specifically binds to an antigen, and the two or more antigens are different. In some cases, a multispecific antibody comprises target binding moieties that specifically bind to three or more different antigens, four or more different antigens, or five or more different antigens. In some embodiments, an antibody may be a bispecific antibody.In some cases, the bispecific antibody or binding fragment is referred to as Knobs-into-Holes (KiH), Asymmetric Re-engineering Technology-immunoglobulin (ART-Ig), Triomabquadroma, bispecific monoclonal antibody (BiMAb, BsmAb, BsAb, BsmAb, BS-MAb, or Bi-MAb), FcΔAdp, XmAb, Azymetric, Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT), Bispecific T-cell Engager (BiTE), Biclonics, Fab-scFv-Fc, Two-in-one / Dual Action Fab (DAF), FinomAb, scFv-Fc-(Fab)-fusion, Dock-aNd-Lock (DNL), Adaptir (formerly SCORPION), Tandem The bispecific antibody may be a diAbody (TandAb), a Dual-affinity-ReTargeting (DART), or a nanobody. In some embodiments, the bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, the bispecific antibody is a trifunctional antibody. A trifunctional antibody may be a full-length monoclonal antibody that contains binding sites for two different antigens.

[0094] In some cases, the bispecific antibody is a bispecific miniantibody. In some cases, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE).

[0095] In some embodiments, the bispecific T cell engager is a fusion protein containing two single chain variable fragments (scFv), where the two scFvs target epitopes of two different antigens. In some embodiments, the antibodies described herein comprise an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some cases, the antibodies comprise an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the antibodies comprise an IgG1 framework. In some cases, the antibodies comprise an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the antibodies comprise an IgG2a framework. In some cases, the antibodies comprise an IgG2b framework. In some cases, the antibodies comprise an IgG3 framework. In some cases, the antibodies comprise an IgG4 framework.

[0096] In some cases, the antibody described herein comprises one or more mutations in the framework region, such as the CH1 domain, the CH2 domain, the CH3 domain, the hinge region, or a combination thereof. In some cases, the one or more mutations are for stabilizing the antibody and / or increasing half-life. In some cases, the one or more mutations are for modulating Fc receptor interaction and reducing or eliminating Fc effector functions such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In further cases, the one or more mutations are for modulating glycosylation.

[0097] In some embodiments, the antibody comprises a humanized antibody or a binding fragment thereof, or a chimeric antibody or a binding fragment thereof. In some embodiments, the antibody comprises a multispecific antibody or a binding fragment thereof. In some embodiments, the antibody comprises a bispecific antibody or a binding fragment thereof. In some embodiments, the antibody can be an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, a scDiabody, a scDiabody-CH3, a triple body, a miniantibody, a minibody, a TriBi minibody, a scFv-CH3KIH, a Fab-scFv-FcKIH, a Fab-scFv, a scFv-CH-CL-scFv, a F(ab')2, a F(ab')2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a diabody-Fc, a tandem scFv-Fc, or an intrabody. In some cases, the antibody is a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), a single domain antibody (e.g., scFv), an IgNAR, a camelid antibody, or a binding fragment or chemically modified derivative thereof.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof, or single domain antibody or antigen-binding fragment thereof, binds to an epitope expressed by a target cell associated with a disease or disorder described herein. In some embodiments, the antibody or antigen-binding fragment thereof, or single domain antibody or antigen-binding fragment thereof, binds to an epitope associated with a microenvironment described herein. Non-limiting examples of epitopes include peptide fragments of cytokines, immune checkpoint molecules, or any other protein associated with a disease or disorder. Non-limiting examples of cytokines include 4-1BBL, acylated stimulatory protein, adipokines, albiinterferon, APRIL, Arh, BAFF, Bcl-6, CCL1, CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2, CCL2 / MCP-1, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL69, CL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CC L7, CCL8, CCL9, CCR10, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CD153, CD154, CD178, CD40LG, CD70, CD95L / CD178, Kel Cerberus (protein), chemokine, CLCF1, CNTF, colony-stimulating factor, common b chain (CD131), common g chain (CD132), CX3CL1, CX3CR1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXC L6, CXCL7, CXCL9, CXCR3, CXCR4, CXCR5, EDA-A1, Epo, erythropoietin, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Flt-3L, FMS-like tyrosine kinase 3 ligand, Foxp3, GATA-3, GcMAFG-CSF, GITRL, GM-CSF, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor,Hepatocyte growth factor, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5 / IFNaG, IFNA7, IFNA8, IFNB1, IFNE, IFNG, IFNZ, IFN-α, IFN-β, IFN-γ, IFNω / IFNW1, IL-1 , IL-10, IL-10 family, IL-10-like, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17 family, IL-17A-F, IL-18, IL-18BP, IL-19, IL-1A, IL-1B, IL-1F1 0, IL-1F3 / IL-1RA, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1-like, IL-1RA, IL-1RL2, IL-1α, IL-1β, IL-2, IL-20, IL-21, IL-22, IL-23, IL-24, IL-28A , IL-28B, IL-29, IL-3, IL-31, IL-33, IL-35, IL-4, IL-5, IL-6, IL-6-like, IL-7, IL-8 / CXCL8, IL-9, inflammasome, interferome, interferon, interferon β-1a, interferon interferon beta-1b, interferon gamma, interferon type I, interferon type II, interferon type III, interferon, interleukin, interleukin 1 receptor antagonist, interleukin 8, IRF4, leptin, leukemia inhibitory factor (LIF), leukocyte promoting factor, LIGHT, LTA / TNFB, LT-beta, lymphokine, lymphotoxin, lymphotoxin alpha, lymphotoxin beta, macrophage colony-stimulating factor, macrophage inflammatory protein, macrophage activating factor, M-CSF, MHC class III, miscellaneous Blood agents, monokines, MSP, myokines, myonectin, nicotinamide phosphoribosyltransferase, oncostatin M (OSM), oprelvekin, OX40L, platelet factor 4, promegapoietin, RANKL, SCF, STAT3, STAT4, STAT6, stromal cell-derived factor 1, TALL-1, TBX21, TGF-α, TGF-β, TGF-β1, TGF-β2, TGF-β3, TNF, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF4, TNFSF8, TNF-α,Examples of immune checkpoint molecules include TNF-β, Tpo, TRAIL, TRANCE, TWEAK, vascular endothelial growth inhibitor, XCL1, or XCL2. Examples of immune checkpoint molecules include VISTA, PDCD1LG2 (CD273), PD-L1, CTLA-4, PD-L2, B7-1 (CD80), B7-2 (CD86), B7-H3 (CD276), B7-H2, B7-H4 (VTCN1), HVEM (CD270, TNFRSF14), galectin 9, galectin 3, CEACAM1 (CD66a), OX-2 (CD200), PVR (CD155), PVRL2 (nectin-2, CD112), FGL-1, PECAM-1, TSG-6, C The epitope may include D47, stabilin-1 (Clever-1), neuropilin 1, neuropilin 2, CD158 (family), IGSF2 (CD101), CD155, GITRL, CD137L, OX40L, LIGHT, CD70, PD-1, RGMB, CTLA-4 (CD152), BTLA, CD160, Tim-3, CD200R, TIGIT, CD112R (PVRIG), LAG-3 (CD223), PECAM-1, CD44, SIRPα (CD172a), or IGSF11. In some embodiments, the epitope is a peptide sequence selected from LAG-3, P2X7, or albumin, or any combination or portion thereof. In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to an epitope expressed by a target cell associated with idiopathic pulmonary fibrosis. In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to an epitope comprising a peptide sequence encoding CTGF. In some embodiments, the exogenous single domain antibody or fragment thereof is specific for an antigen comprising at least one peptide sequence in SEQ ID NOs: 1601-1602. In some embodiments, the exogenous single domain antibody or fragment comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 1701.

[0099] In some embodiments, the exogenous antibody or single domain antibody functions as an agonist or antagonist, and upon binding to any one of the epitopes described herein, the binding of the antibody or single domain antibody induces an agonist or antagonist effect. For example, upon binding to an immune checkpoint inhibitor such as PD-L1 / PD-1 or CTLA-4, the exogenous antibody or single domain antibody exerts an agonist or antagonist effect on the immune checkpoint signaling pathway (SEQ ID NOs: 801, 851, 901, and 951). In some embodiments, the exogenous antibody or single domain antibody expressed by the enucleated cell is encoded from a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 801. In some embodiments, the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 851. In some embodiments, the exogenous single domain antibody or fragment thereof is encoded from a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 901. In some embodiments, the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 951.

[0100] In some cases, the epitope may include a peptide sequence of a pathogen protein. For example, the epitope may be a viral protein or a fragment thereof. In some embodiments, the epitope may be a viral protein of a coronavirus. In some embodiments, the coronavirus may be a severe acute respiratory syndrome-related virus (SARS-CoV). In some embodiments, the SARS-CoV is SARS-CoV-2. In some embodiments, the epitope may be a viral protein selected from orf1a, orf1ab, spike protein (S protein), 3a, 3b, envelope protein (E protein), matrix protein (M protein), p6, 7a, 7b, 8b, 9b, nucleocapsid protein (N protein), orf14, nsp1 (leader protein), nsp2, nsp3, nsp4, nsp5 (3C-like proteinase), nsp6, nsp7, nsp8, nsp9, nsp10 (growth factor-like protein), nsp12 (RNA-dependent RNA polymerase, or RdRp), nsp13 (RNA 5'-triphosphatase), nsp14 (3'-to-5' exonuclease), nsp15 (endoRNAse), and nsp16 (2'-O-ribose methyltransferase), portions thereof, or combinations thereof.

[0101] Another example of an epitope includes a peptide sequence of a viral protein of an influenza virus. In some embodiments, the influenza virus is selected from the genus consisting of influenza virus A, influenza virus B, influenza virus C, and influenza virus D. In further embodiments, the influenza A virus is of subtype H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, or H6N1. In further embodiments, the influenza B virus is of influenza B virus of B / Yamagata / 16 / 88-like lineage, or B / Victoria / 2 / 87-like lineage. In some embodiments, the influenza can be any strain of influenza virus or any serotype within a strain of influenza virus. In some cases, the influenza virus includes any combination of the viral surface glycoproteins hemagglutinin (H or HA) and neuraminidase (N or NA).

[0102] In some embodiments, the epitopes are encoded by the nucleic acid sequences provided in SEQ ID NOs: 131-134, 142-152, 201, 202, 301-312, 501, 521-526, 541-545, 561, 584, 591-601, and 701-705. In some embodiments, the epitope is encoded by a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleic acid sequence found in SEQ ID NOs: 131-134, 142-152, 201, 202, 301-312, 501, 521-526, 541-545, 561, 584, 591-601, and 701-705.

[0103] In some embodiments, the epitope comprises a peptide sequence provided in SEQ ID NOs: 155-164, 203, 204, 315-322, 511, 531-535, 551-554, 571, 594, 611-619, and 711. In some embodiments, the epitope comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the peptide sequence found in SEQ ID NOs: 155-164, 203, 204, 315-322, 511, 531-535, 551-554, 571, 594, 611-619, and 711.

[0104] In some cases, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is encoded from a polynucleotide sequence described herein. In some embodiments, the polynucleotide sequence is exogenous to the enucleated cell or parent cell. In some embodiments, the polynucleotide comprises a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an mRNA or cDNA sequence encoding an antibody or antigen-binding fragment thereof described herein.

[0105] In some embodiments, the enucleated cell comprises a polynucleotide comprising a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof (e.g., a single domain antibody). Non-limiting examples of antibodies or antigen-binding fragments thereof encoded by the polynucleotides herein can be found in SEQ ID NOs: 1-36, 101-111, 121-123, 165-192, 195, 205, 206, 211-213, 221-231, 241-245, 325-331, and 401-404.

[0106] In some embodiments, the enucleated cell comprises an antibody or antigen-binding fragment (e.g., a single domain antibody) comprising an amino acid sequence provided in SEQ ID NOs: 1-36, 101-111, 121-123, 165-192, 195, 205, 206, 211-213, 221-231, 241-245, 325-331, and 401-404. In some embodiments, the single domain antibody comprises an amino acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences listed in SEQ ID NOs: 1-36, 101-111, 121-123, 165-192, 195, 205, 206, 211-213, 221-231, 241-245, 325-331, and 401-404.

[0107] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to tumor necrosis factor (TNF). Examples of peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies against TNF can be found in SEQ ID NOs: 1-36.

[0108] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to albumin. Non-limiting examples of peptide sequences of antibodies or single domain antibodies to albumin may be found in SEQ ID NOs: 101-111. In some embodiments, the exogenous single domain antibody or fragment comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 101-111.

[0109] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to the purinergic receptor P2X7 (P2X7). Examples of peptide sequences for antibodies or antigen-binding fragments thereof, or single domain antibodies to P2x7 may be found in SEQ ID NOs: 121-123. In some embodiments, the exogenous single domain antibody or fragment comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 121-123.

[0110] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human PD-L1 encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 131-152. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human PD-L1 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 155-164. Examples of peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies to human PD-L1 can be found in SEQ ID NOs: 165-192. In some embodiments, the exogenous single domain antibody or fragment comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 165-192.

[0111] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to the mouse CD274 antigen (PD-L1). Exemplary peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies to mouse PD-L1 are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:195.

[0112] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human CTLA-4 encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 201 or SEQ ID NO: 202. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human CTLA-4 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 203 or SEQ ID NO: 204. Examples of peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies against human CTLA-4 may be found in SEQ ID NOs: 205 and 206. In some embodiments, the exogenous single domain antibody or fragment comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NO: 205 or 206.

[0113] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to mouse CTLA-4. Exemplary peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies to mouse CTLA-4 can be found in SEQ ID NOs: 211-213. In some embodiments, the exogenous single domain antibodies or fragments comprise peptide sequences that are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 211-213.

[0114] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to the interleukin-6 receptor (IL-6R). Examples of peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies to IL-6R can be found in SEQ ID NOs: 221-231 and 241-245. In some embodiments, the exogenous single domain antibodies or fragments comprise peptide sequences that are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 221-231 and 241-245.

[0115] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human lymphocyte activation gene 3 (LAG-3) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 301-312. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human LAG-3 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 315-321. Examples of peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies to human LAG-3 may be found in SEQ ID NOs: 325-331. In some embodiments, the exogenous single domain antibody or fragment comprises a peptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 325-331.

[0116] In some embodiments, the antibodies or antigen-binding fragments thereof or antigen-binding fragments thereof or single domain antibodies described herein may bind to spike proteins, such as, for example, the spike glycoprotein of coronaviruses. Examples of peptide sequences of antibodies or antigen-binding fragments thereof, or single domain antibodies or antigen-binding fragments thereof, to spike proteins can be found in SEQ ID NOs: 401-404. In some embodiments, the exogenous single domain antibodies or fragments comprise peptide sequences that are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more identical to SEQ ID NOs: 401-404.

[0117] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies or antigen-binding fragments thereof described herein can bind to an epitope of any of the pathogens described herein. Non-limiting examples of pathogens can be found in Table 1.

[0118] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies or antigen-binding fragments thereof described herein may bind to a programmed cell death 1 ligand 2 (PDCD1LG2) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 501. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to a PDCD1LG2 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:511.

[0119] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to programmed cell death protein 1 (PDCD-1 or PD-1) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 521-526. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to PDCD-1 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 531-535.

[0120] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to PD-1 encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 541-545. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to PD-1 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 551-554. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to PD-1 encoded from a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 561.

[0121] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to a LAG3-related protein (LAG3P) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 561. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to a LAG3P comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 571.

[0122] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to a T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 581. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to a TIM3 comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO:594.

[0123] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind a T cell immunoreceptor having an Ig and ITIM domain (TIGIT) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 591-601. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind a TIGIT comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 611-619.

[0124] In some embodiments, an antibody or antigen-binding fragment thereof, or single domain antibody described herein may bind a V-domain Ig inhibitor of T cell activation (VISTA) encoded by a nucleic acid that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID NOs: 701-705. In some embodiments, an antibody or antigen-binding fragment thereof, or single domain antibody described herein may bind a VISTA comprising a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 711.

[0125] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human brain-derived neurotrophic factor (BDNF) and exert an antagonistic effect on human BDNF. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to mouse BDNF and exert an antagonistic effect on mouse BDNF. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human tropomyosin receptor kinase B (TrkB) and exert an antagonistic effect on human TrkB. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to mouse TrkB and exert an antagonistic effect on mouse TrkB. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to human TrkB and exert an agonistic effect on human TrkB. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein can bind to mouse TrkB and exert an agonistic effect on mouse TrkB. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein can bind to human PD-1 and exert an agonistic effect on human PD-1. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein can bind to mouse PD-1 and exert an agonistic effect on mouse PD-1. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein can bind to human PD-L1 and exert an antagonistic effect on human PD-L1. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein can bind to mouse PD-L1 and exert an antagonistic effect on mouse PD-L1.

[0126] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to CTLA-4, such as Yervoy (Ipilimumab). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to PD-1, such as Nivolumab (Opdivo), Pembrolizumab (Keytruda), or Cemiplimab (Libtayo). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to PD-L1, such as Avelumab (Bavencio), Durvalumab (Imfinzi), or Atezolizumab (Tecentriq). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to HER2, such as Herceptin (Trastuzumab), Margetuximab-cmkb (MARGENZA), or Pertuzumab (Perjeta). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to TROP-2, such as Sacituzumab (Trodelvy). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to IL-6, such as Siltuximab (Sylvant). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to IL-6R, such as Tocilizumab (Actemra). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to CD20, such as Rituximab (MabThera).

[0127] In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies or antigen-binding fragments thereof described herein may be conjugated with a drug to form an antibody-drug conjugate (ADC). Examples of drugs or compounds that may be part of an ADC may include chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitors, antihormonal agents, antiangiogenic agents, cardio protectants, and / or checkpoint inhibitors. Non-limiting checkpoint inhibitors include IMP321 / Eftilagimod alpha (Immutep), Relatlimab,BMS-986016, ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), semilimumab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), LAG525, MK-4280, irinotecan, oxaliplatin, REGN3767, TSR-033, BI754111, Sym022, FS118 (bispecific anti-LAG3 / P D-L1 antagonistic mAb), MGD013 (bispecific anti-LAG3 / PD-1 antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym023, INCAGN2390, LY3321367, Ramucirumab, Abemaciclib, Merestinib, BMS-986258, SHR-1702, Cammaleri Camrelizumab, MK-7684, Etigilimab / OMP-313M32, 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 inhibitors) These include: BGB-A1217, CM-24 (MK-6018), BMS986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, Utomilimab (PF-05082566), Urelumab (BMS-663513) ES101, BMS-986156, TRX-518, AMG228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or Varlilumab (CDX-1127).

[0128] Such compounds or drugs may be present in the combination in amounts effective for the intended purpose. In some embodiments, the ADC comprises a molecule for treating idiopathic pulmonary fibrosis. In some embodiments, the ADC comprises nintedanib or pirfenidone. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to VEGF, VEGFR, or a combination thereof (VEGF / VEGFR). In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to VEGF-A, VEGF-B, VEGF-C, VEGF-D, placenta growth factor (PIGF), angiopoietin-1, angiopoietin-2, endostatin, FGF, MMP, DII4, class 3 semaphorin, FGF, VEGFR, NRP-1, PDGF (BB homodimer), PDGFR, TGF-β, endoglin, TGF-β receptor, CCL2, integrin αVβ3, αVβ5, or α5β1, VE-cadherin, CD31, ephrin, plasminogen activator, plasminogen activator inhibitor-1, eNOS, COX-2, AC133, ID1 / ID3, class 3 semaphorin, or Nogo-A, or a combination thereof. In some embodiments, the antibodies or antigen-binding fragments thereof, or single domain antibodies described herein may bind to a VEGF-A receptor, a VEGF-B receptor, a VEGF-C receptor, a VEGF-D receptor, a placenta growth factor receptor (PIGF), or a combination thereof.

[0129] Pharmaceutical preparations Described herein are pharmaceutical formulations comprising the enucleated cells or compositions described herein. In some embodiments, the pharmaceutical formulations further comprise a pharma- ceutically acceptable carrier, excipient, diluent, or nebulizer.

[0130] In some embodiments, the pharmaceutical formulation comprises two or more active agents, or two or more therapeutic agents, as disclosed herein. In some embodiments, the two or more active agents are included in a single dosage unit, such as, for example, when the enucleated cells comprise two or more therapeutic agents. In embodiments, the two or more active agents are included in separate dosage units, such as when the enucleated cells are administered separately from the additional therapeutic agent or adjuvant. In some embodiments, the active agent, which in some embodiments may be an additional therapeutic agent, comprises a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an antihormonal agent, an antiangiogenic agent, a cardioprotectant, and / or a checkpoint inhibitor.Non-limiting checkpoint inhibitors include IMP321 / eftilagimod alfa (Immutep), lilatolimab BMS-986016, ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), cemipimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), LAG525, MK-4280, irinotecan, oxaliplatin, REGN3767, TSR-033, BI754111, and Sym022. , FS118 (bispecific anti-LAG3 / PD-L1 antagonistic mAb), MGD013 (bispecific anti-LAG3 / PD-1 antagonistic mAb), TSR-022, niraparib, bevacizumab, MBG453, decitabine, spartalizumab, Sym023, INCAGN2390, LY3321367, ramucirumab, abemaciclib, meletinib, BMS-986258, SHR-1702, camalelizumab, MK-7684, Tizilimab / OMP-313M32, tiragolumab / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA-170d, enoblitutumab / MGA271, MGD009, I-8H9 / omburtamab, trastuzumab, MGD013 (anti-PD-1, anti-LAG-3 dual checkpoint inhibitor), BGB-A1217, CM-24 (MK- 6018), BMS986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, utomirimab (PF-05082566), urelumab (BMS-663513) ES101, BMS-986156, TRX-518, AMG228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or varlilumab (CDX-1127). Such compounds or drugs may be present in the combination in amounts effective for the intended purpose.In some embodiments, non-limiting examples of active agents that may be additional therapeutic agents include CPI-006 (to inhibit CD73 and allow activation of T cells and APCs), monalizumab (to inhibit NKG2A), COM701 (to inhibit PVRIG / PVRL2 and activate T cells), CM24 (to inhibit CEACAM1 and allow T and NK cell activation), NEO-201 (to inhibit CEACAM5 and CEACAM6 to allow T cell activation while interfering with tumor cell proliferation), and defibrillation. Actinib (to inhibit FAK and disrupt tumor growth), PF-04136309 (to inhibit CCR-2 and CCL-2 and enable T cell recruitment and activation), MSC-1 (to inhibit LIF and enable T cell and APC activation while disrupting cancer growth), Hu5F9-G4 (5F9), ALX148, TTI-662, and RRx-001 (to inhibit CD47 or SIRPα and enable T cell and APC activation), lacnotuzumab (MCS-110), LY3022855 , SNDX-6352, emactuzumab (RG7155), and pexidartinib (PLX3397) (to inhibit M-CSF or CSF-1R and enable APC activation), CAN04 and canakinumab (ACZ885) (to inhibit IL-3 or IL-1RAP and enable T cell and APC activation), BMS-986253 (to inhibit IL-8 and reduce the immunosuppressive tumor microenvironment while interfering with tumor growth), and pepinemab (VX15 / 2503) (to inhibit SEMA4D and reduce tumor proliferation). These include trebananib (to reduce the immunosuppressive tumor microenvironment while interfering with cancer growth), trebananib (to inhibit angiopoietin-2, to enable APC activation while interfering with cancer growth), FP-1305 (to inhibit CLEVER-1, to enable APC activation), enapotamab vedotin (EnaV) (to inhibit Axl, to enable APC activation while interfering with cancer growth), or bavituximab (to inhibit phosphatidylserine, to enable T cell and APC activation while interfering with cancer growth).

[0131] In carrying out the methods of treatment or use provided herein, the pharmaceutical preparations described herein are administered in a therapeutically effective amount to a mammal having a disease, disorder, or illness, such as cancer, to be treated. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents, and in some cases, the pharmaceutical preparations described herein, can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0132] The pharmaceutical preparations described herein can be administered to a subject by suitable administration route, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation or intraperitoneal.The compositions described herein can include but are 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 dissolving formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and immediate and controlled release mixed formulations.

[0133] Pharmaceutical preparations containing a therapeutic agent may be manufactured in a conventional manner, such as, by way of example only, conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or compressing processes.

[0134] Pharmaceutical preparations may contain at least exogenous therapeutic agents as active ingredients in free acid or free base form, or in pharma- ceutically acceptable salt form.In addition, the methods and compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds with the same type of activity.In some embodiments, therapeutic agents are present in unsolvated form or in solvated form with pharma-ceutically acceptable solvents such as water, ethanol, etc.Solvated forms of therapeutic agents are also considered to be disclosed herein.

[0135] In some embodiments, the pharmaceutical formulations provided herein contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0136] In some embodiments, the pharmaceutical formulations described herein benefit from antioxidants, metal chelators, thiol-containing compounds, and other general stabilizing agents. Examples of such stabilizers 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, 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.

[0137] The pharmaceutical preparations described herein are formulated into any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.In one aspect, the therapeutic agent discussed herein, for example, the therapeutic agent, is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection.In one aspect, the formulation suitable for intramuscular, subcutaneous, or intravenous injection comprises 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 (propylene glycol, polyethylene-glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity can 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 preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. In some cases, it is desirable to include isotonic agents such as sugars, sodium chloride, etc. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

[0138] For intravenous injection or drip or infusion, the pharmaceutical preparations described herein are formulated in aqueous solution, preferably in physiologically compatible buffer such as Hanks' solution, Ringer's solution or physiological saline buffer.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffer or excipient.Such excipients are well known.

[0139] Parenteral injection may include bolus injection or continuous infusion. Pharmaceutical preparations for injection may be provided in unit dosage form, for example, in ampoules or multi-dose containers, with the addition of preservatives. The compositions described herein may be in a form suitable for parenteral injection as sterile suspension, solution or emulsion in oily or aqueous vehicles, and may contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0140] For administration by inhalation, the therapeutic agent is formulated for use as an aerosol, mist or powder. The pharmaceutical formulations described herein are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. By way of example only, capsules and cartridges of gelatin or the like for use in an inhaler or insufflator can be formulated to contain a powder mix of the therapeutic agent described herein and a suitable powder base, such as lactose or starch. The formulations containing the composition are prepared as a solution in saline using 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 using suitable non-toxic pharmaceutically acceptable ingredients. The selection of suitable carrier depends on the exact nature of the desired nasal dosage form, for example, solution, suspension, ointment or gel.Nasal dosage forms generally contain a large amount of water in addition to active ingredient.Other components such as pH adjusters, emulsifiers, or dispersants, preservatives, surfactants, gelling agents, or buffers and other stabilizers and solubilizers are optionally present in small amounts.Preferably, nasal dosage forms should be isotonic with nasal secretions.

[0141] Pharmaceutical preparations for oral use are obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules to obtain tablets or dragee cores, after adding suitable auxiliaries, if desired. Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, or others such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrants are added, such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or salts such as alginic acid or sodium alginate. In some embodiments, dyes or pigments are added to tablets or dragee coatings for identification or to characterize different combinations of active therapeutic doses.

[0142] In some embodiments, the pharmaceutical formulation of the exogenous therapeutic agent is in the form of a capsule, including push-fit capsules made of gelatin and soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules contain the active ingredient in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active therapeutic agent is dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In some embodiments, a stabilizer is added. The capsules may be prepared, for example, by placing a bulk blend of the formulation of the therapeutic agent inside the capsule. In some embodiments, the formulation (non-aqueous suspensions and solutions) is placed in a soft gelatin capsule. In other embodiments, the formulation is placed in a standard gelatin capsule or a non-gelatin capsule, such as a capsule containing HPMC. In other embodiments, the formulation is placed in a sprinkle capsule and the capsule is swallowed whole, or the capsule is opened and the contents sprinkled on food before eating.

[0143] Pharmaceutical formulations for oral administration are in dosages suitable for such administration. In one aspect, solid oral dosage forms are prepared by mixing the composition with one or more of antioxidants, flavorings, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some embodiments, the solid dosage forms disclosed herein are in the form of tablets (including suspensions, fast-dissolving tablets, bite-disintegrating tablets, fast-disintegrating tablets, effervescent tablets, or caplets), pills, powders, capsules, solid dispersions, solid solutions, biodegradable dosage forms, controlled release formulations, pulsed 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 compressing bulk blends of the above formulations. In various embodiments, the tablets will include one or more flavorings. In other embodiments, the tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating can provide a delayed release of the therapeutic agent from the formulation. In other embodiments, the film coating aids in patient compliance. Film coatings generally range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms, such as 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 easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit doses include a film coating. These formulations are manufactured by conventional formulation techniques.

[0144] In another aspect, the dosage form comprises microencapsulated formulation.In some embodiments, one or more other compatible materials are present in the microencapsulated material.Non-limiting examples of materials include pH adjusters, erosion promoters, antifoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0145] The liquid pharmaceutical dosage form for oral administration is optionally an aqueous suspension selected from the group including, but not limited to, pharma- ceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form optionally includes additives such as (a) a disintegrant, (b) a dispersant, (c) a wetting agent, (d) at least one preservative, (e) a viscosity enhancer, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion further includes a crystal formation inhibitor.

[0146] In some embodiments, the pharmaceutical formulations described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase into another immiscible phase, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Furthermore, water or an aqueous phase is optionally added immediately prior to administration, which ensures the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides improved bioavailability of hydrophobic active ingredients.

[0147] Oral preparations are administered using various formulations well known in the art.In addition, the oral dosage forms described herein can further comprise a biodegradable (hydrolyzable) polymer carrier, which also helps the dosage form to adhere to oral mucosa.For buccal or sublingual administration, the composition can take the form of tablets, lozenges, or gels that are formulated in conventional manner.

[0148] For intravenous injection, pharmaceutical preparations are optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' 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, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.

[0149] Parenteral injections optionally include bolus injections or continuous infusions. Preparations for injection are optionally presented in unit dosage form, for example in ampoules or multi-dose containers, with the addition of preservatives. In some embodiments, the compositions described herein are in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and contain formulation agents such as suspending agents, stabilizing agents and / or dispersing agents. Compositions for parenteral administration include aqueous solutions of the agent that modulates the activity of the carotid body in water-soluble form. In addition, suspensions of the agent that modulates the activity of the carotid body, for example oily injection suspensions, are optionally prepared as needed.

[0150] Conventional formulation techniques include, for example, one or a combination of the following methods: (1) dry blending, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Wurster coating), tangential coating, top spray, tableting, extrusion, and the like.

[0151] In some embodiments, a composition is provided comprising particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, which upon mixing with water results in a substantially uniform suspension.

[0152] In addition, the pharmaceutical formulations optionally contain one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane, and buffers such as citric acid / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0153] In addition, the pharmaceutical formulations optionally contain one or more salts in an amount necessary to bring the 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.

[0154] Other pharmaceutical formulations optionally contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0155] In one embodiment, the aqueous suspension and dispersion described herein remain homogeneous for at least 4 hours.In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical stirring lasting less than 1 minute.In yet another embodiment, no stirring is required to maintain a homogeneous aqueous dispersion.

[0156] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal cavity in drops or sprays. Nasal solutions may resemble 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 this range may also be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0157] Aerosol formulations for inhalation and inhalation can be designed to deliver the drug or drug combination to the subject's respiratory tree when administered via nasal or oral respiratory routes. Inhalation solutions can be administered, for example, by nebulizers. Inhalation or insufflation with fine powder or liquid drugs can be delivered to the respiratory system as pharmaceutical aerosols of solutions or suspensions of the drug or drug combination in propellants, for example, to aid distribution. Propellants can be liquefied gases, including halocarbons, e.g., fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

[0158] Halocarbon propellants include fluorocarbon propellants in which all hydrogens are replaced by fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced by chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Useful hydrocarbon propellants include, for example, propane, isobutane, n-butane, pentane, isopentane, and neopentane. Blends of hydrocarbons can also be used as propellants. Ether propellants include, for example, dimethyl ether and ether. Aerosol formulations can also include one or more propellants. For example, aerosol formulations include one or more propellants from the same class, such as two or more fluorocarbons, or one or more, two or more, three or more propellants from different classes, such as fluorohydrocarbons and hydrocarbons. The compositions of the present disclosure can also be dispensed with compressed gas, for example, an inert gas, such as carbon dioxide, nitrous oxide, or nitrogen.

[0159] The aerosol formulation may also contain other components, such as ethanol, isopropanol, propylene glycol, as well as surfactants or other components, such as oils and detergents, which may help stabilize the formulation and / or lubricate the valve components.

[0160] Aerosol formulations can be packaged under pressure and can be formulated as aerosols using solutions, suspensions, emulsions, powders and semi-solid preparations.For example, solution aerosol formulations include solutions of agents such as transporters, carriers, or ion channel inhibitors in (substantially) pure propellants or as a mixture of propellants and solvents.Solvents can be used to dissolve agents and / or slow the evaporation of propellants.Solvents can include, for example, water, ethanol and glycol.Any combination of suitable solvents can be used, optionally in combination with preservatives, antioxidants, and / or other aerosol components.

[0161] Aerosol formulations can be dispersions or suspensions.Suspension aerosol formulations include suspensions of drug or drug combinations, such as transporters, carriers, or ion channel inhibitors, and dispersants.Dispersants include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil.Suspension aerosol formulations can also include lubricants, preservatives, antioxidants, and / or other aerosol components.

[0162] Aerosol formulations can also be formulated as emulsions.Emulsion aerosol formulations can include, for example, alcohol, such as ethanol, surfactant, water, and propellant, and a drug or drug combination, such as a transporter, carrier, or ion channel.The surfactant used can be non-ionic, anionic, or cationic.One example of emulsion aerosol formulation includes, for example, ethanol, surfactant, water, and propellant.Another example of emulsion aerosol formulation includes, for example, vegetable oil, glyceryl monostearate, and propane.

[0163] methodIn some embodiments herein, methods of making and using the cells of the present disclosure are disclosed. Methods disclosed herein include methods of generating enucleated cells from nucleated cells (parent cells) using high speed centrifugation. In some embodiments, the methods of generating enucleated cells of the present disclosure do not include differentiation of the nucleated cells. In some embodiments, the enucleated cells are stored under conditions that slow or suspend the biological activity of the cells, such as cryopreservation, freeze-drying or lyophilization. In some embodiments, the biological activity of enucleated cells stored under such conditions can be restored at a time of need and further manipulated as needed. Methods of delivering enucleated cells to a subject as disclosed herein are also provided. In some embodiments, the delivering step includes administering the enucleated cells or a composition comprising the enucleated cells to the subject, which can, for example, treat a disease or disorder or subject as disclosed herein. In some embodiments, the methods described herein include methods for treating a disease or condition characterized at least in part by abnormal vasculature in a subject, the method comprising administering to a subject having the disease or condition an enucleated cell comprising one or more organelles that synthesize or release an exogenous polypeptide comprising a tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof in the absence of a nucleus, wherein the exogenous polypeptide synthesized or released by the cell is therapeutically effective in normalizing the abnormal vasculature of the subject. In some embodiments, the methods described herein include treating the disease or condition by administering to a subject an enucleated cell expressing a single domain antibody or antigen-binding fragment thereof, wherein the single domain antibody or antigen-binding fragment thereof targets an immune checkpoint molecule. In some embodiments, the methods described herein include treating the disease or condition by administering to a subject an enucleated cell expressing a single domain antibody or antigen-binding fragment thereof, wherein the single domain antibody or antigen-binding fragment thereof targets an immune checkpoint molecule as described herein.In some embodiments, the methods described herein comprise treating a disease or condition by administering to a subject enucleated cells expressing a single domain antibody, or antigen-binding fragment thereof, wherein the single domain antibody, or antigen-binding fragment thereof, targets CTGF.

[0164] Methods for generating enucleated cells Disclosed herein are methods of generating the nucleated cells described herein by removing the nucleus of a nucleated cell (parent cell). In some embodiments, the method of generating an enucleated cell does not require differentiation of the parent cell. In some embodiments, the parent cell containing the nucleus is engineered to express a single domain antibody or antigen binding fragment thereof, a therapeutic agent, a transmembrane moiety, an immune evasion moiety, and / or a targeting moiety described herein, and then the nucleus of the parent cell is removed. In some embodiments, the parent cell containing the nucleus is enucleated and the enucleated cell is engineered to express a single domain antibody or antigen binding fragment thereof, a therapeutic agent, a transmembrane moiety, an immune evasion moiety, and / or a targeting moiety described herein. In some embodiments, the parent cell is engineered to express one or more of the above biomolecules (e.g., immune evasion moiety and / or targeting moiety), and the resulting enucleated cell (e.g., already expressing an immune evasion moiety and / or targeting moiety) is further engineered to express a second of the above biomolecules (e.g., a therapeutic agent). In this manner, the enucleated cells of the present disclosure can be extensively manipulated prior to enucleation, stored for long periods of time if necessary (e.g., via lyophilization, freeze-drying, cryopreservation), and rapidly engineered to express therapeutic agents when they are needed.

[0165] As shown in Figure 1, the parent cells can be engineered to express adhesion molecules, chemokines or retention receptors, or both, that target lymphoid tissues (e.g., lymph nodes) of the subject prior to enucleation. Additionally or alternatively, the resulting enucleated cells are engineered to express and optionally secrete a therapeutic agent, such as, for example, an antibody or antigen-binding fragment thereof (e.g., a single domain antibody). In some embodiments, the enucleated cells can be administered to a subject in need thereof to treat a disease or disorder of the subject.

[0166] The ability to extensively engineer the enucleated cells described herein before and after enucleation significantly simplifies the manufacturing process compared to comparable biological drug development timelines. The process to manufacture the enucleated cells of the present disclosure is approximately 2 months compared to traditional biological drug development timelines of 12 months or more. Referring now to FIG. 2, the enucleated cells of the present disclosure may be prepared in advance and cryopreserved for a period of time. This means that the enucleated cells of the present disclosure (e.g., engineered to express homing receptors, immune activators, etc.) can be rapidly deployed. Such technical aspects are particularly important when the enucleated cells are used to treat diseases or illnesses resulting from pathogen exposure or infection outbreaks.

[0167] In some embodiments, removal of the nucleus involves mechanically removing the nucleus. The parent cells can be treated with cytochalasin to soften the cortical actin cytoskeleton. The nucleus is then physically extracted from the cell body by high-speed centrifugation on a Ficoll gradient to generate enucleated cells. Because the enucleated cells and intact nucleated cells sediment in different layers in the Ficoll gradient, the enucleated cells can be easily isolated and prepared for therapeutic purposes or fusion to other cells (nucleated or enucleated). The enucleation process is clinically scalable to process tens of thousands of cells. In some embodiments, the enucleated cells can be used as disease-homing vehicles to deliver clinically relevant cargo / payloads to treat various diseases.

[0168] Various methods can be used to introduce biomolecules (therapeutic agents, transmembrane moieties, immune evasion moieties, and / or targeting moieties described herein) into parent or enucleated cells as described herein. Non-limiting examples of methods that can be used to introduce biomolecules into parent or enucleated cells include liposome mediated transfer, adenovirus, adeno-associated virus, herpes virus, retrovirus-based vectors, lentivirus vectors, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetic transfection, nanoparticle transfection, or combinations thereof. In some embodiments of any of the compositions and methods provided herein, therapeutic agents, viruses, antibodies, or nanoparticles can be introduced into enucleated cells.

[0169] In some embodiments, the enucleated cells are preserved by cryopreservation, freeze-freeze hibernation, or freeze-drying. Cryopreservation involves freezing the enucleated cells, while freeze-hibernation involves storing the enucleated cells at temperatures below room temperature without freezing the enucleated cells. In some embodiments, the enucleated cells are freeze-dried. In some embodiments, the freeze-dried enucleated cells can be reconstituted, and the reconstituted enucleated cells exhibit viability comparable to non-freeze-dried enucleated cells. In some embodiments, freeze-drying involves a component that freezes the cells and a component that uses a vacuum to dry the cells under very low pressure (e.g., <3000 mTorr). The drying component can result in sublimation and dehydrate the cells while maintaining cell viability and biological function. In some embodiments, the freezing step involves balancing the duration and temperature of freezing to maintain cell viability and stability, proper crystal formation, and rate of reconstitution. The triple point of a substance is the temperature and pressure at which the sublimation curve, melting curve, and evaporation curve meet. Achieving a triple point, which varies for different substances, ensures that sublimation, rather than melting, occurs in the subsequent drying step. To facilitate faster and more efficient freeze-drying, larger ice crystals are preferred because they form a network within the product that facilitates faster removal of water vapor during sublimation. To produce larger crystals, the product must be frozen slowly or the temperature can be raised and lowered in a process called annealing. Fresh or frozen biological tissue or living cells do not have a single homogenous melting point (eutectic point), and as a result, the freezing stage of the material (cells or tissue) is cooled below its triple point, which represents the temperature and pressure at which the solid, liquid and gas phases of the material can coexist. Living cells have a critical point on the phase diagram, which is one where both the liquid and gas phases of an object or substance have the same density and are therefore indistinguishable. The product critical point temperature must be maintained to prevent meltback or cake collapse that occurs during primary and secondary drying, which reflects incomplete sublimation.For materials where preservation of structure is required, such as living cells, large ice crystals can be harmful and can destroy cell walls, which can result in poorer texture and loss of nutritional content. In this case, freezing needs to be done quickly to quickly bring the material below its critical point and thus avoid the formation of large ice crystals. The freezing temperature for cells or tissues can vary, but generally ranges between -50°C (-58°F) and -80°C (-112°F).

[0170] During the drying stage, the ambient pressure is reduced to the range of a few millibars, and then heat is supplied by conduction or radiation to the material for sublimation of the ice. The amount of heat required may be calculated using the latent heat of sublimation of the sublimating molecules. In this initial drying stage, about 95% of the water in the material or substance is sublimated. This phase is often slow and may even last for days depending on the substance and technique used, but the application of excess heat may quickly change the structure of the material. In this stage, the pressure is controlled through the application of a partial vacuum. The vacuum accelerates the sublimation, making it useful as a planned drying process. A low-temperature condenser chamber and / or condenser plates are used as a surface for the water vapor to re-liquefy and solidify. It is important to note that in this pressure range, heat cannot be provided by the convection effect due to the low air density. The drying stage also aims to remove the remaining unfrozen water molecules, since the ice induced by freezing needs to be removed during the primary drying stage. This part of the freeze-drying process is governed by the adsorption isotherm of the material. In this stage, the temperature is raised higher than in the primary drying stage, and may even go above 0 °C (32 °F), to break any physicochemical interactions formed between the water molecules and the frozen material. Usually, in this stage, the pressure is also reduced to facilitate desorption; however, there are some outputs that benefit from increased pressure. After the freeze-drying process is completed, the vacuum is usually broken with an inert gas such as nitrogen before the material is sealed. At the end of the operation, the residual water content in the output should be extremely low, in the range of <1%-4% of the original concentration.

[0171] In some embodiments, lyophilization of enucleated cells involves the use of lyoprotectants to preserve cell viability and biological function. Lyoprotectants include the addition of reagents, salts, or additives that protect cells during the drying process. Common lyoprotectants include trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Additionally, methods to increase the transport of cryoprotectants inside cells in suspension can be utilized as a way to improve cell viability and function after lyophilization. These methods include electroporation, the addition of reagents that enhance intracellular transport, genetic modification of cells to upregulate the expression of pores on the cell membrane, and mechanical microfluidic devices that partially disrupt the integrity of the cell membrane and potentially facilitate the intracellular transport of lyoprotectants.

[0172] How to use In some embodiments herein, methods of using the compositions described herein are disclosed. In some embodiments, the methods include treating a disease or condition in a subject by administering to the subject a composition described herein (e.g., a pharmaceutical composition containing enucleated cells engineered to express a therapeutic agent). In some embodiments, the methods utilize the enucleated cells described herein to generate tissues or organs ex vivo (e.g., by 3D bioprinting for whole tissue and organ development for transplantation, skin grafts, development of synthetic meat sources, bioscaffolds, etc.). In some embodiments, the methods utilize the enucleated cells described herein to grow and manufacture cytoplasmic replicating viruses such as VSV, rabies, etc. In some embodiments, the methods utilize the enucleated cells described herein as a diagnostic tool to detect disease or disease locus in the body. In some embodiments, the methods utilize the enucleated cells described herein for cosmetic applications. In some embodiments, the methods utilize the enucleated cells described herein as a source for purifying any cellular components that need to be free of proteins, membranes, lipids, various RNAs, organelles, or nuclear DNA. In some embodiments, the methods utilize the enucleated cells described herein as a fusogen, which can be an ex vivo source for transferring gene editing modalities into various cell types in vitro, in some embodiments, the methods utilize the enucleated cells described herein to promote wound healing or as regenerative medicine.

[0173] The present disclosure also provides methods for the use of enucleated cells (natural or enucleated) as fusion partners to other cells (therapeutic or natural) to enhance and / or transfer biomolecules described herein, such as, for example, single domain antibodies or antigen-binding fragments thereof and / or therapeutic agents. In some embodiments, biomolecules include DNA / genes, RNA (mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, and plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones. In some embodiments, the enucleated cells are engineered to express a fusogenic moiety. The fusogenic moiety may be any biomolecule (e.g., sugar, lipid, or protein) that promotes membrane fusion. In some embodiments, the fusogenic moiety is a fusogenic protein. The fusion protein allows the enucleated cell expressing the fusion protein to fuse with a target cell. In some embodiments, the fusion protein promotes fusion between the enucleated cell expressing the fusion protein and the target cell, allowing the contents of the enucleated cell to enter the target cell. In some embodiments, the fusion protein is heterotypic, such as viral class I-III or Hapless2 precursor HAP2 or SNARE. In some embodiments, the fusion protein is homoleptic, such as EFF-1 / AFF-1. Other non-limiting examples of fusion proteins are Izumo1 or Syncytin. In some embodiments, the fusion protein is a viral protein. In some embodiments, the viral-derived fusion protein is VSV-g, hERV-W-ENV (Syncytin), or MV-Ed-FMV-Ed-H (Hemagglutinin). Unlike nucleated cells, fusion of enucleated cells to the same or another cell type of similar or different origin produces unique cellular hybrids that lack problematic nuclear transfer while maintaining desirable therapeutic attributes, including but not limited to cell surface proteins, signaling molecules, secreted proteins, and epigenetic changes.In some embodiments, the fusion protein may be expressed on the TNT, thereby promoting fusion between the enucleated cell and the target cell when the TNT is contacted with the target cell.

[0174] In some embodiments, the fusion protein is encoded by a polynucleotide that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to any one of the nucleic acid sequences in SEQ ID NOs: 1001-1106. In some embodiments, the fusion protein comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to any one of the peptide sequences in SEQ ID NOs: 1201-1205, 1207, 1209-1211, 1213-1229, 1231-1233, 1237-1247, 1249, 1251-1257, and 1259-1305. In some embodiments, the fusion protein is encoded by a polynucleotide that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to any one of SEQ ID NOs: 1001-1106. In some embodiments, the fusion protein comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to any one of SEQ ID NOs: 1201-1305.

[0175] In some embodiments, immortalized cells such as human telomerase reverse transcriptase (hTERT) cells or cancer cells, when enucleated, can be administered to a subject in need thereof to vaccinate the subject against a disease or disorder disclosed herein. Non-limiting examples of human immortalized cells include HeLa (human epithelial) cells, 293 / 293T / HEK-293T (human embryonic kidney) cells, SH-SY5Y (human neuroblastoma, cloned from bone marrow) cells, and the like. Non-limiting examples of mammalian immortalized cells include 3T3 (mouse embryonic fibroblast) cells, COS (monkey kidney) cells, MDCK (canine kidney epithelial) cells, CHO (Chinese hamster ovary) cells, PC12 (rat pheochromaffin) cells, and Neuro-2a / N2a (mouse neuroblastoma) cells. For example, once enucleated, cancer cells obtained from a subject can be administered to the subject to induce immunity against the tumor without the risk of metastasis. Also, the enucleated cancer cells may be allogeneic and therefore do not induce other deleterious immune responses to the subject. In some embodiments, the enucleated cells in this context may not have a therapeutic payload (e.g., a single domain antibody or a therapeutic agent). In some embodiments, the enucleated cells may include one or more of a targeting moiety, an immune system evasion moiety, a therapeutic agent (e.g., an mRNA encoding an immune checkpoint molecule, an immune checkpoint molecule inhibitor, a single domain antibody or an antigen-binding fragment thereof, or an oncolytic virus), or a combination thereof. In some embodiments, the enucleated cells may activate the innate or adaptive immunity of a subject by immunizing the subject with a pathogen or antigen. In some embodiments, the enucleated cells may activate the innate or adaptive immunity of a subject by delivering a therapeutic agent that can activate the immune response of the subject.

[0176] Treatment method Provided herein are methods of treating a disease or condition in a subject by administering a composition described herein to the subject. In some embodiments, administration is by any suitable mode of administration, including systemic administration (e.g., intravenous, inhalation, etc.). In some embodiments, the subject is a human. In some embodiments, the disease or condition comprises a disease or condition of the lung tissue. In some embodiments, the disease or condition comprises cancer. In some embodiments, the disease or condition comprises idiopathic pulmonary fibrosis. In some embodiments, the composition comprises an enucleated cell disclosed herein that has been engineered to express, and in some cases secrete, a therapeutic agent comprising a single domain antibody or antigen binding fragment that binds to an immune checkpoint molecule, such as PD-L1. In some embodiments, the composition comprises an enucleated cell disclosed herein that has been engineered to express, and in some cases secrete, a therapeutic agent comprising a single domain antibody or antigen binding fragment that binds to an epithelial biomarker, such as CTGF.

[0177] In some embodiments, the disease or condition comprises an infection (e.g., human immunodeficiency virus (HIV) infection, Chagas disease, tuberculosis), a neurological disease (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease), an autoimmune disease (e.g., diabetes, Crohn's disease, multiple sclerosis, sickle cell anemia), a cardiovascular disease (e.g., acute myocardial infarction, heart failure, refractory angina), an ophthalmological disease, a skeletal disease, a metabolic disease (e.g., phenylketonuria, glycogen storage disease type 1A, Gaucher disease), an inflammatory disease (e.g., cancer, inflammatory bowel disease), or a disease caused by an external pathogen or toxin in the subject. In some embodiments, the disease or condition comprises idiopathic pulmonary fibrosis. In some embodiments, the subject is in need of, or has been determined to be in need of, such an enucleation cell treatment.

[0178] In some embodiments, the enucleated cells described herein comprise an antibody or antigen-binding fragment thereof (e.g., a single domain antibody) that binds to an epitope expressed by a cancer cell or an epitope associated with the tumor microenvironment. In some embodiments, the binding of the antibody or antigen-binding fragment thereof to the epitope provides a therapeutic effect for treating cancer in a subject. In some embodiments, the binding of the antibody or antigen-binding fragment thereof to the epitope recruits immune cells to activate an immune response against the cancer. In some embodiments, the cancer comprises cancer of lung tissue. In some embodiments, the cancer is lung cancer. Non-limiting examples of cancer include carcinoma, carcinomatous cell carcinoma, acoustic neuronoma, ventral substantia nigra melanoma, acrospirorroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatous adenocarcinoma, adrenal cortical carcinoma, Adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, osteoblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, berry duct cancer, biliary tract cancer, bladder cancer , blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt's lymphoma, carcinoma of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary site, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, brain astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, tendon tumor, choriocarcinoma , choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, bile cell neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small cell neoplasm, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endothelial sinus tumor, endometrial cancer, endometrial ovarian cancer,Endometrioid tumor, Bowel disease associated T-cell lymphoma, Pituitary blastoma, Ependymoma, Epithelial sarcoma, Erythroleukemia, Esophageal cancer, Infectious neuroblastoma, Ewing family tumors, Ewing family sarcoma, Ewing sarcoma, Cranial ectodermal cell tumor, Gonadal ectodermal cell tumor, Extrahepatic cholestatic carcinoma, Extramammary Paget's disease, Fallopian tube carcinoma, Fetal fetus, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder cancer, Gallbladder cancer, Ganglioneuroma, Ganglioneuroma, Gastric cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal carcinoid tumor, Gastrointestinal stromal tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germ cell tumor, Germ cell tumor, Gestational choriocarcinoma, Gestational trophoblastic tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy cell leukemia, Hairy cell leukemia, Head and neck cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematologic malignancies, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, Hypopharyngeal cancer, Hypothalamic glioma, Inflammatory breast cancer, Intraocular melanoma, Pancreatic islet cell carcinoma, Pancreatic islet cell tumor, Juvenile myelomonocytic leukemia, Kaposi's sarcoma , Kaposi's sarcoma, kidney cancer, Klatzkin's tumor, Krukenberg's tumor, laryngeal cancer, laryngeal cancer, lentiginomatous melanoma, leukemia, leukemia, fat and oral cancer, liposarcoma, lung cancer, xanthomas, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mesodermal tumor of the breast, mesodermal tumor of the breast, thyroid medullary tumor cancer, medulloblastoma, medulloblastoma, meningoepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, obstructive primary metastatic squamous cell carcinoma, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, murine carcinoma, mucinous tumor, multiple endocrine neoplastic syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder, myxoma, nasal carcinoma, nasopharyngeal carcinoma, neoplasm, neuroma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma,Non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cavity cancer, oral cancer, pharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, pancreatic tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, senile cancer, perivascular epithelial cell tumor, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumor of intermediate differentiation, medulloblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell neoplasm, alveolar epithelial blastoma, multiple germinoma, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma , Primary hepatocellular carcinoma, Primary liver cancer, Primary peritoneal cancer, Primary neuroectodermal tumor, Prostate cancer, Pseudomyxomab peritoneal cancer, Rectal cancer, Renal cell carcinoma, NUT gene on chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Respiratory failure cancer with Richter transformation, Sacroiliac joint teratoma, Salivary gland cancer, Sarcoma, Schwannomatosis, Sebaceous gland cancer, Secondary neoplasms, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord stromal tumor, Sezary syndrome, Signature ring cell carcinoma, Skin cancer, Small blue round cell tumor, Small cell carcinoma, Small cell lung cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Hot wort wart), spinal cord tumor, spinal tumor, splenic marginal zone lymphoma, squamous cell carcinoma, stromal carcinoma, superficial spreading melanoma, epithelial primitive neuroectodermal tumor, superficial epithelial stromal tumor, sinoviral sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, peripheral lymphatic carcinoma, testicular cancer, sarcoma, throat cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and urinary tract, transitional cell carcinoma, tracheal cancer, urethral cancer, genitourinary neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Berner-Morrison syndrome, bullous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the target cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells.

[0179] In some embodiments, the cancer may be lung cancer, including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or any other lung cancer type. For example, lung cancer may include adenocarcinoma, squamous cell carcinoma, large cell (undifferentiated) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, lung cancer tumor, or adenoid cystic carcinoma. Other non-limiting examples of lung cancer include lymphoma, sarcoma, benign lung tumor, or hamartoma. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer has metastasized to the lung from a different tissue or source. For example, metastatic cancers that may be found in the lung may include breast cancer, colon cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma, and Wilms' tumor.

[0180] In some embodiments herein, enucleated cells and methods of using these enucleated cells to treat a lung disease or condition are described. In some embodiments, the lung disease or condition is asthma, partial or total collapse of the lung (pneumothorax or atelectasis), swelling and inflammation in the main passageways that carry air to the lungs (bronchi) (bronchitis), chronic obstructive pulmonary disease (COPD), lung cancer as described herein, lung infection (e.g., pneumonia), blockage of the pulmonary artery (pulmonary embolism) including abnormal accumulation of fluid in the lungs (pulmonary edema), or idiopathic pulmonary fibrosis (IPF).

[0181] In some embodiments herein, enucleated cells and methods of using these enucleated cells to treat a disease or condition associated with abnormal vasculature in a subject are described. Abnormal vasculature may be associated with a disease or condition such as inflammation (e.g., IPF) and cancer (e.g., any one of the cancers described herein). In some embodiments, the enucleated cells described herein, when contacted with abnormal vasculature, increase normalization of the abnormal vasculature, increasing adhesion between endothelial cells to prevent leakage of intravascular factors from the vasculature. In some embodiments, normalization of abnormal vasculature includes a reduction in damage, such as cell death of endothelial cells of the vasculature. In some embodiments, normalization of abnormal vasculature includes angiogenesis of immature or leaky blood vessels. In some embodiments, normalization provided by the enucleated cells may include normalization of blood vessels, lymphatic vessels, or a combination thereof.

[0182] In some embodiments, normalization of blood vessels or lymphatic vessels allows for the delivery of therapeutic agents to target cells. For example, the enucleated cells described herein can deliver an exogenous agent to first normalize abnormal blood vessels or lymphatic vessels that allow disease or illness-associated cells (e.g., cancer cells) to receive nutrients from the bloodstream, but are small or leaky enough to effectively prevent therapeutic delivery to the disease or illness-associated cells. Normalization of blood vessels or lymphatic vessels then allows for the delivery of therapeutic agents to treat the disease or illness-associated cells. In some embodiments, the enucleated cells described herein include an exogenous agent for normalizing abnormal blood vessels or lymphatic vessels. In some embodiments, the same enucleated cells that include an exogenous agent for normalizing abnormal blood vessels or lymphatic vessels can further include a second exogenous agent that is delivered to the target cells for the treatment of the disease or illness. In some embodiments, the disease or illness includes cancer or IPF. For example, the enucleated cells can first deliver a soluble form of a TNF member (e.g., a soluble form of LIGHT) to normalize abnormal blood or lymphatic vessels. After normalization, the same enucleated cells can deliver an anti-cancer therapeutic agent, such as an immune checkpoint molecule inhibitor, to treat cells associated with a disease or disorder.

[0183] In some embodiments, the disease or illness may be caused by a pathogen. In some embodiments, the enucleated cells described herein include an antibody or antigen-binding fragment thereof, or a single domain antibody that binds to an epitope expressed by a pathogen or an epitope associated with a microenvironment associated with the pathogen. In some cases, the binding of the antibody or antigen-binding fragment thereof, or the single domain antibody to the epitope confers a therapeutic property against the pathogen. In some embodiments, the binding of the antibody or antigen-binding fragment thereof, or the single domain antibody to the epitope recruits immune cells to activate an immune response, conferring a therapeutic property against the pathogen. For example, the disease or illness may be caused by a virus, a bacterium, a fungus, a parasite, or a molecule resulting from detoxification. In some embodiments, the pathogen may be easily spread or transmitted from person to person, may cause major mortality, pose a risk of significant impact on public health, may cause public panic and social unrest, and require special actions for public health preparedness. Examples of these pathogens may include Anthrax (Bacillus anthracis), Botulism (Clostridium botulinum toxin), Plague (Yersiniapestis), Smallpox (variolamajor), Tularemia (Francisella tularensis), or viral hemorrhagic fevers, including Filoviruses (Ebola, Marburg) and Arenaviruses (Lassa, Machupo).

[0184] In some embodiments, the pathogen may be reasonably easy to spread, causing moderate morbidity and low mortality, requiring specific enhanced diagnostic capabilities and enhanced disease surveillance. Examples of these pathogens include Brucellosis (Brucella species), Epsilon toxin of Clostridium perfringens, food safety threats (e.g., Salmonella species, Escherichia coli O157 H7, or Shigella), Glanders (Burkholderia mallei), Melioidosis (Burkholderia pseudomallei), Psittacosis (Chlamydia psittaci), Q fever (Coxiella burnetii), Ricin toxin from Ricus communis (castor bean), Staphylococcal enterotoxin B, typhoid fever (Rickettsia prowazekii), viral encephalitis (alpha viruses such as Australian equine encephalitis, Venezuelan equine encephalitis, and Western equine encephalitis), or water safety threats (e.g., Vibrio cholerae). cholerae) and Cryptosporidium parvum).

[0185] In some embodiments, the pathogen may include emerging pathogens that have a high potential for mortality and morbidity, but whose spread is not fully understood. Non-limiting examples of these pathogens may include Nipah virus and Hantavirus.

[0186] In some embodiments, the pathogen comprises a toxin. In some cases, the toxin may be secreted by any of the pathogens described herein. Non-limiting examples of pathogens and diseases or illnesses associated with these pathogens that can be treated with the compositions described herein can be found in Table 1.

[0187] [Table 1-1]

[0188] [Table 1-2]

[0189] Dosage and frequency of administration The enucleated cells described herein, or compositions containing such enucleated cells (referred to in this paragraph as "compositions"), can be administered to a subject at an appropriate dose, mode of administration, and frequency, depending on the intended effect.

[0190] In some embodiments, the composition is administered at least once during a period of time (e.g., every 2 days, twice a week, once a week, weekly, three times a month, twice a month, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is administered more than once during a period of time (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times).

[0191] In some embodiments, the composition is administered in a therapeutically effective amount by various forms and routes, including, for example, oral or topical administration. In some embodiments, the composition can be administered parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, intrathecally, intraocularly, intrasternally, ophthalmically, intradermally, topically, intranasally, intrapulmonary, rectally, intraarterially, intrathecally, by inhalation, intralesional, intradermal, epidurally, intracapsularly, subcapsularly, intracardially, transtracheally, subcutaneously, intrathecally, or intraspinally, for example, by injection or infusion. In some embodiments, the composition can be administered by absorption through epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa administration). In some embodiments, the composition is delivered via multiple administration routes.

[0192] In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the composition is administered by slow continuous infusion over an extended period of time, such as more than 24 hours. In some embodiments, the composition is administered as an intravenous injection or short-term infusion.

[0193] The composition may be administered in a localized manner, for example, by injecting the drug directly into an organ, optionally in a depot or sustained release formulation or implant. The composition may be provided in the form of a rapid release formulation, a sustained release formulation, or an intermediate release formulation. The rapid release form may provide immediate release. The sustained release formulation may provide controlled release or sustained delayed release. In some embodiments, a pump may be used to deliver the composition. In some embodiments, a pen delivery device may be used, for example, for subcutaneous delivery of the composition of the present disclosure.

[0194] The compositions provided herein may be administered in conjunction with other therapies disclosed herein, including, for example, antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. In some embodiments, the cyclic polyribonucleotides or antibodies or antigen-binding fragments thereof described herein may be used alone or in combination with one or more therapeutic agents as a component of a mixture. In some embodiments, the linear polyribonucleotides or antibodies or antigen-binding fragments thereof described herein may be used alone or in combination with one or more therapeutic agents as a component of a mixture.

[0195] The compositions (e.g., enucleated cells or pharmaceutical preparations comprising enucleated cells described herein) may be administered before, during, or after the onset of a disease or condition, and the timing of administering the composition containing the therapeutic agent may vary. In some cases, the compositions may be used as prophylactics and may be administered continuously to subjects (e.g., subjects for immunization or subjects for treatment) with a susceptibility to coronavirus or a predisposition to a disease or condition associated with coronavirus. Prophylactic administration may reduce the likelihood of the onset of an infection, disease, or condition, or may reduce the severity of an infection, disease, or condition.

[0196] The composition may be administered to a subject prior to the onset of symptoms. The composition may be administered to a subject (e.g., an immunized or treated subject) after a test result (e.g., as soon as possible), for example, a test result providing a diagnosis, a test showing the presence of coronavirus in a subject (e.g., an immunized or treated subject), or a test showing disease progression, e.g., a drop in blood oxygen levels. The therapeutic agent may be administered after a disease or disease onset is detected or suspected (e.g., as soon as practicable). The therapeutic agent may be administered after a potential exposure to coronavirus (e.g., as soon as practicable thereafter), for example, after a subject (e.g., a subject for immunization or a subject for treatment) comes into contact with an infected subject, or comes into contact with an infected subject who may be infectious and knows that he or she may be contagious.

[0197] Actual dosage levels of agents of the present disclosure (e.g., antibodies or antigen-binding fragments thereof, or therapeutic agents) may be varied to obtain an amount of agent to achieve a desired therapeutic response for a particular subject, composition, and mode of administration without toxicity to the subject (e.g., subject for immunization or subject for treatment). The dosage level selected may depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention used, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, disease, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0198] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. Unit dosage form as used herein refers to a physically discrete unit suitable as a unitary dosage for a subject (e.g., a subject for immunization or a subject for treatment), each unit containing a predetermined amount of active agent calculated to produce a desired therapeutic effect, associated with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present disclosure can be determined by and directly depend on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active agents for the treatment of sensitivities in individuals. The dose can be determined with reference to the plasma concentration or local concentration of the cyclic polyribonucleotide or antibody or antigen-binding fragment thereof. Dosage can be determined with reference to plasma or local concentrations of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.

[0199] The compositions described herein may be in unit dosage form suitable for single administration of precise dosage amounts. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of the composition. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosage form may be in the form of a package containing a discrete amount of the formulation. Non-limiting examples are packaged injections, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with or without preservatives. The injectable formulations disclosed herein may be in unit dosage form, for example, in ampoules, or in multi-dose containers containing preservatives.

[0200] Dosages may be based on the amount of agent per kilogram of subject body weight (e.g., immunized or treated subject). Doses of agent (e.g., antibody) may range from 10-3000 mg / kg, e.g., 100-2000 mg / kg, e.g., 300-500 mg / kg / day for 1-10 days or 1-5 days, e.g., 400 mg / kg / day for 3-6 days, e.g., 1 g / kg / d for 2-3 days. In some embodiments, dosages may be based on the number of enucleated cells per kg of subject body weight, in some embodiments, dosages may be administered at about 1000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 1,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose is from about 1,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 10,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 100,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 1,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 10,000 cells / kg body weight, 000 cells / kg body weight, approximately 1,000 cells / kg body weight ~ approximately 100,000,000 cells / kg body weight, approximately 1,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 1, 000 cells / kg body weight ~ approx. 10,000,000,000 cells / kg body weight, approx. 1,000 cells / kg body weight ~ approx. 100,000,000,000 cells / kg body weight, approx. 1,000 cells / kg body weight ~1,000,000,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight~approximately 100,000 cells / kg body weight, approximately 10,000 cells / kg body weight~approximately 1,000,000 cells cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 10,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 100,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approx. 1,000,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 10,000,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 10 0,000,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 1,000,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,0 cells / kg body weight, about 100,000 cells / kg body weight to about 10,000,000 cells / kg body weight, about 100,000 cells / kg body weight to about 100,000,000 cells / kg body weight, about 100,000 cells / kg body weight to about 1,000,000,000 cells / kg body weight, about 100,000 cells / kg body weight to about 10,000,000,000 cells / kg body weight, about 100,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 100,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 1,000,000 cells / kg body weight to about 10,000,000 cells / kg body weight, about 1,000,000 cells / kg body weight to about 100,000,000 cells / kg body weight, about 1,000,000 cells / kg body weight to about 1,000,000,000 cells / kg body weight, about 1,000,000 cells / kg body weight to about 10,000,000,000 cells / kg body weight, about 1,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 1,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight to about 100,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight to about 1,000,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight to about 10,000,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight to about 1,000,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight to about 10,000,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight to about 10,000,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, or about 100,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight. In some embodiments, doses may be administered at dosages of about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight, about 1,000 cells / kg body weight, about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, about 100,000,000,000 cells / kg body weight, or about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose may be administered at a dosage of about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight, at least about 1,000 cells / kg body weight, about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, or about 100,000,000,000 cells / kg body weight. In some embodiments, the dose is from about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight, up to about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, about 100,000,000,000 cells / kg body weight, or about 1,000,000,000 cells / kg body weight.In some embodiments, the nucleus-free cells are administered to a subject twice within at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, or 4 years.

[0201] In some embodiments, the same enucleated cell-containing composition or pharmaceutical composition can be repeatedly administered to a subject in need thereof. In some embodiments, a first administration of the enucleated cell-containing composition or pharmaceutical composition normalizes blood or lymphatic vessels. In some embodiments, the same enucleated cell-containing composition or pharmaceutical composition can be subsequently administered to the subject to maintain blood or lymphatic vessel normalization and for delivery of exogenous agents to treat a disease or condition described herein.

[0202] kit In some embodiments herein, kits are disclosed for using the compositions described herein. In some embodiments, the kits disclosed herein can be used to treat a disease or illness in a subject. In some embodiments, the kits include a collection of materials or components separate from the composition.

[0203] In some embodiments, the kits described herein comprise a pharmaceutical formulation disclosed herein, which comprises enucleated cells engineered to express (and in some cases secrete) a single domain antibody or antigen-binding fragment disclosed herein. In some embodiments, the single domain antibody or antigen-binding fragment is specific for an immune checkpoint molecule, such as PD-L1. In some embodiments, the enucleated cells are further engineered to express a targeting moiety, such as a chemokine receptor, an integrin signaling molecule, or an antibody or antigen-binding fragment thereof, which allows the enucleated cells, once administered, to efficiently migrate to the target tissue of the subject. In some embodiments, the target tissue comprises lung tissue. In some embodiments, the kit further comprises an additional therapeutic agent, such as those disclosed herein. In some embodiments, the kit further comprises instructions for administering the pharmaceutical formulation and / or the additional therapeutic agent to the subject to treat a disease or disorder of the subject, such as cancer. In some embodiments, the cancer comprises cancer of lung tissue. In some embodiments, the cancer is lung cancer.

[0204] In some embodiments, the kits described herein comprise a pharmaceutical formulation disclosed herein, which comprises enucleated cells engineered to express (and in some cases secrete) a single domain antibody or antigen-binding fragment specific for an epithelial marker, such as CTGF. In some embodiments, the enucleated cells are further engineered to express a targeting moiety, such as a chemokine receptor, an integrin signaling molecule, or an antibody or antigen-binding fragment thereof, which allows the enucleated cells, once administered, to efficiently migrate to the target tissue of the subject. In some embodiments, the target tissue comprises lung tissue. In some embodiments, the kit further comprises an additional therapeutic agent, such as those disclosed herein. In some embodiments, the kit further comprises instructions for administering the pharmaceutical formulation and / or additional therapeutic agent to the subject to treat a disease or condition of the subject, such as pulmonary fibrosis. In some embodiments, the pulmonary fibrosis comprises idiopathic pulmonary fibrosis,

[0205] In some embodiments, the kits described herein include components for selecting a homogenous population of enucleated cells. In some embodiments, the kits described herein include components for selecting a heterogeneous population of enucleated cells. In some embodiments, the kits include components for assaying the number of units of a biomolecule (e.g., a therapeutic agent) synthesized by the enucleated cells and / or released or expressed on a surface. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or disorder disclosed herein (e.g., cancer) in a subject. In some embodiments, the kits are configured specifically for the purpose of treating a mammalian subject. In some embodiments, the kits are configured specifically for the purpose of treating a human subject.

[0206] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the composition to a subject in need thereof. In some embodiments, the kit includes instructions for further manipulating the composition to express a biomolecule (e.g., a therapeutic agent). In some embodiments, the kit includes instructions for thawing or restoring biological activity of the composition, which may have been cryopreserved, lyophilized, or freeze-suspended during storage or transport. In some embodiments, the kit includes instructions for measuring the viability of the restored composition to ensure its effectiveness for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0207] The kit also optionally includes other useful components such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandages or other useful paraphernalia. The assembled materials or components in the kit can be provided to the practitioner and stored in any convenient and suitable manner that preserves their operability and usefulness. For example, the components can be in dissolved, dehydrated, or lyophilized form, and they can be provided at room temperature, refrigerated, or frozen temperatures. The components are generally contained in suitable packaging materials.

[0208] definition The use of absolute or sequential terms, such as "will," "willnot," "shall," "shallnot," "must," "mustnot," "first," "initially," "next," "subsequently," "before," "after," "lastly," and "finally," is exemplary and not limiting of the scope of the embodiments of the invention disclosed herein.

[0209] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising."

[0210] As used herein, "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions "at least one of A, B and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0211] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusive and inclusive. For example, the term "AorB" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0212] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0213] The term "about," when referring to a number or numerical range, means that the number or numerical range referred to is approximate within experimental variability (or within statistical experimental error), and that the number or numerical range may vary, for example, by 1% to 15% of the stated number or numerical range. For example, the term "about" refers to ±10% of the stated number or value.

[0214] "Increased", "increasing", or "increase" are used herein generally to mean an increase by a statistically significant amount. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a reference level, e.g., at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100%, or any increase between 10-100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0215] The terms "decreased", "decreasing" or "decrease" are used herein to generally mean a decrease by a statistically significant amount. In some embodiments, "decreased" or "decreasing" means a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, and up to 100% (e.g., non-existent or undetectable levels compared to a reference level), or any decrease between 10-100% compared to a reference level. In the context of a marker or condition, these terms mean a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to a level that is accepted as within the normal range for an individual without a given disease. Other examples of a "reduction" include at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 20-fold, at least a 50-fold, at least a 100-fold, at least a 1000-fold or more decrease compared to a reference level.

[0216] The terms "individual" or "subject" are used interchangeably and include mammals. Non-limiting examples of mammals include any member of the mammalian class, such as humans, non-human primates such as chimpanzees, and other ape and monkey species, farm animals such as cows, horses, sheep, goats, pigs, domestic animals such as rabbits, dogs, and cats, and laboratory animals including rodents such as rats, mice, and guinea pigs. A mammal may be a human. The term "animal" as used herein includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, e.g., a rodent such as a rat or mouse. A "patient" as used herein refers to a subject having or diagnosed with a disease or disorder as described herein.

[0217] The term "immune-evading moiety," as used herein, refers to a signaling peptide or portion thereof that reduces cell phagocytosis through its interaction with a signal receptor protein expressed by phagocytes, such as macrophages and dendritic cells. In some embodiments, the immune-evading moiety blocks immune cell recognition or immune cell activation.

[0218] The term "targeting moiety," as used herein, refers to an entity that directs a cell, such as an enucleated cell, to a target tissue or cell. A targeting moiety can be virtually any biomolecule, including a protein, polypeptide, sugar, nucleic acid, or small molecule, or portion thereof. In some embodiments, a targeting moiety disclosed herein comprises a chemokine receptor, a chemokine, an integrin signaling molecule, an antibody or antigen-binding fragment thereof, or a single domain antibody or antigen-binding fragment thereof.

[0219] The term "transmembrane moiety," as used herein, refers to an entity that spans (at least partially) the cell membrane of a cell (eg, an enucleated cell).

[0220] The term "expression" or "expressing" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. "Up-regulated" in the context of expression generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to the expression level in a control, e.g., wild-type state, whereas "down-regulated" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to the expression level in a control, e.g., wild-type state.

[0221] As used herein, a "cell" generally refers to a biological cell.

[0222] As used herein, "enucleation" refers to the rendering of a cell non-replicating, such as through removal of the nucleus.

[0223] As used herein, the terms "cytoplast," "cell without a nucleus," or "enucleated cell" are used interchangeably to refer to an anucleated cell obtained from a previously nucleated cell (e.g., any cell described herein). In some embodiments, a nucleated cell contains organelles, and a cytoplast derived from a nucleated cell retains such organelles, which in some cases allows for cellular functions such as cell motility, protein synthesis, protein secretion, formation of tunneling nanotubes, etc. In some embodiments, "obtaining" refers to a process that encompasses the enucleation of a nucleated cell. In some embodiments, enucleation does not involve differentiating a nucleated cell into an enucleated cell using natural processes or another method.

[0224] The term "gene" as used herein refers to a segment of nucleic acid that codes for an individual protein or RNA (also called a "coding sequence" or "coding region"), optionally together with associated regulatory regions such as promoters, operators, terminators, etc., that may be located upstream or downstream of the coding sequence. The term "gene" should be interpreted broadly and may include mRNA, cDNA, cRNA, and genomic DNA forms of a gene. In some uses, the term "gene" includes a transcribed sequence, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region may include an "open reading frame" that codes for a polypeptide. In some uses of the term, a "gene" includes only the coding sequences (e.g., "open reading frame" or "coding region") necessary to code for a polypeptide. In some embodiments, a gene does not code for a polypeptide, e.g., a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some embodiments, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. The term "gene" can encompass mRNA, cDNA, and genomic forms of a gene.

[0225] The term "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as compositions. The packaging material is preferably constructed by well-known methods to provide a sterile, contaminant-free environment. The packaging materials used in the kits are routinely utilized in gene expression assays and administration of treatments.

[0226] As used herein, the term "package" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, etc., that can hold individual kit components.For example, the package can be a glass vial or a pre-filled syringe that is used to contain an appropriate amount of pharmaceutical agent.The packaging material has an external label that indicates the contents and / or purpose of the kit and its components.

[0227] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogs, either single-stranded, double-stranded, or multi-stranded. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can be present in a cell-free environment. A polynucleotide can be a gene or a fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide contains one or more analogs (e.g., modified backbones, sugars, or nucleobases). Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (locuses) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0228] The terms "polypeptide", "peptide" and "protein", as used herein, may be used interchangeably herein in reference to a polymer of amino acid residues. A protein may refer to a full-length polypeptide translated from a coding open reading frame, or a full-length polypeptide processed to its mature form, while a polypeptide or peptide may refer to a degradation or processing fragment of a protein that still uniquely or identifiably maps to a particular protein. A polypeptide may be a single linear polymeric chain of amino acids bound together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. A polypeptide may be modified, for example, by the addition of carbohydrates, phosphorylation, and the like.

[0229] As used herein, the term "fragment" or "portion" or equivalent terms may refer to a portion of a protein having less than the full length of the protein and optionally maintaining a function of the protein.

[0230] The terms "complement," "complements," "complementary," and "complementarity," as used herein, generally refer to a sequence that is completely complementary to and can hybridize to a given sequence. In some cases, a sequence hybridized to a given nucleic acid is called the "complement" or "reverse complement" of a given molecule if its sequence of bases on a given region can bind complementarily to that of its binding partner, for example, so that AT, AU, GC, and GU base pairs are formed. Generally, a first sequence that can hybridize to a second sequence is specifically or selectively hybridizable to the second sequence, so that hybridization to a second sequence or set of second sequences is more favorable than hybridization to a non-target sequence during a hybridization reaction (e.g., more thermodynamically stable under a given set of conditions, such as stringent conditions commonly used in the art). Generally, hybridizable sequences will share a degree of sequence complementarity over all or a portion of their respective lengths, such as from 25% to 100% complementarity, including about 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and 100% sequence complementarity. Sequence identity for purposes of assessing percent complementarity can be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, for example, the EMBOSSNeedle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html), the BLAST algorithm, etc. Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including default parameters.

[0231] The term "percent (%) identity" as used herein generally refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to those of a reference sequence after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent identity (e.g., gaps can be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). Alignment can be achieved in a variety of commonly known ways for purposes of determining percent identity. The percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0232] As used herein, the term "in vivo" may be used to describe events that take place in a living organism, such as the body of a subject.

[0233] As used herein, the term "ex vivo" may be used to describe events that occur outside of an organism, such as the body of a subject. An "ex vivo" assay cannot be performed on a subject. Rather, it may be performed on a sample separate from the subject. Ex vivo may be used to describe events that occur in intact cells outside the subject's body.

[0234] The term "in vitro" as used herein may be used to describe an event that is contained within a container for holding laboratory reagents, such as being separated from the living biological source organism from which the material is obtained. In vitro assays may include cell-based assays in which live or dead cells are used. In vitro assays may also include cell-free assays in which no intact cells are used.

[0235] "Treat", "treating" or "treatment" as used herein refers to alleviating or inhibiting a disorder, disease or condition, or to alleviating or suppressing one or more of the symptoms associated with a disorder, disease or condition, or to alleviating or eradicating the cause of the disorder, disease or condition itself. Desirable effects of treatment can include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or ameliorating the disease state, and remission or improved prognosis.

[0236] The terms "effective amount" and "therapeutically effective amount", when used interchangeably herein, generally refer to an amount of a composition, e.g., a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells) comprising the system of the present disclosure, sufficient to produce a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay the onset, halt the progression, and reduce or alleviate at least one symptom of a disorder treated by the method of the present disclosure.

[0237] The terms "pharmacologically acceptable carrier", "pharmacologically acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. An ingredient may be "pharmacologically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It may also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0238] As used herein, the terms "administration", "administering" and variations thereof refer to the introduction of a composition or agent into a subject, including simultaneous and sequential introduction of compositions or agents. The introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, or topical administration. Administration includes self-administration and administration by another. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein. Administration can be performed by any suitable route. In some embodiments, the administration is intravenous administration. In some embodiments, the administration is pulmonary administration. In some embodiments, the administration is inhalation.

[0239] The term "pharmaceutical formulation" refers to a mixture of a composition disclosed herein with other chemical components, such as a diluent or carrier (e.g., a pharma- ceutically acceptable inactive ingredient), carrier, excipient, binder, filler, suspending agent, flavoring agent, sweetener, disintegrant, dispersant, surfactant, lubricant, colorant, diluent, solubilizer, wetting agent, plasticizer, stabilizer, permeation enhancer, wetting agent, antifoaming agent, antioxidant, preservative, or one or more combinations thereof. A pharmaceutical formulation may facilitate administration of a composition to an organism. Numerous techniques of administering a compound exist in the art, including, but not limited to, oral, injection, aerosol, parenteral, and topical administration.

[0240] The term "fusogenic protein," as used herein, refers to a polypeptide that, when expressed on the surface of a cell, such as an enucleated cell, promotes fusion of the cell containing the fusion protein with a target cell.

[0241] The term "anchor molecule" includes molecules that can be inserted into and remain in a cell membrane, for example, glycosylphosphatidylinositol (GPI) is an anchor molecule that can form a complex with a protein, where GPI can be inserted into and remain in a cell membrane, thereby anchoring the protein to the cell membrane.

[0242] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions described herein, which depend upon a variety of conditions and variables. It will be understood that various alternatives to the embodiments of the present invention described herein may be employed in carrying out the present invention. It is therefore contemplated that the present invention shall also encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0243] EMBODIMENT 1 An enucleated cell obtained from a parent cell having a nucleus, said enucleated cell comprising one or more organelles for the synthesis of an exogenous single domain antibody or fragment thereof in the absence of a nucleus. EMBODIMENT 2 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is encapsulated in said enucleated cell. EMBODIMENT 3 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is expressed on the luminal side of the plasma membrane of said enucleated cell by said one or more intracellular organelles. EMBODIMENT 4 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is expressed by said one or more intracellular organelles on the cytoplasmic side of the plasma membrane of said enucleated cell. EMBODIMENT 5 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is conjugated to a transmembrane moiety. EMBODIMENT 6 6. The enucleated cell of embodiment 5, wherein the transmembrane moiety comprises a transmembrane polypeptide. EMBODIMENT 7 The enucleated cell of embodiment 6, wherein said exogenous single domain antibody or fragment thereof is complexed to the N-terminus of said transmembrane polypeptide. EMBODIMENT 8 The enucleated cell of embodiment 6, wherein said exogenous single domain antibody or fragment thereof is complexed to the C-terminus of said transmembrane polypeptide. EMBODIMENT 9 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof comprises a modification relative to an otherwise identical reference single domain antibody or fragment thereof, said modification anchoring said exogenous single domain antibody or fragment thereof to the luminal or cytoplasmic side of the cell membrane of said enucleated cell. EMBODIMENT 10 10. The enucleated cell of embodiment 9, wherein said modification comprises conjugating said exogenous single domain antibody or fragment thereof to an anchor molecule comprising glycosylphosphatidylinositol, farnesyl, palmitate, myristate, or a combination thereof. EMBODIMENT 11 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody, or fragment thereof, is released by said enucleated cell by secreting said exogenous single domain antibody, or fragment thereof, from said enucleated cell. EMBODIMENT 12 The enucleated cell of embodiment 10, wherein said exogenous single domain antibody or fragment thereof is released upon death of said enucleated cell. EMBODIMENT 13 The enucleated cell of embodiment 11, wherein said exogenous single domain antibody or fragment thereof is released upon rupture of said enucleated cell. EMBODIMENT 14 The enucleated cell of embodiment 11, wherein said exogenous single domain antibody or fragment thereof is transferred from said enucleated cell to another cell by fusing said enucleated cell with said another cell. EMBODIMENT 15 The enucleated cell according to any one of embodiments 1 to 14, wherein said exogenous single domain antibody or fragment thereof is conjugated to a cytotoxic drug. EMBODIMENT 16 2. The enucleated cell of embodiment 1, wherein the enucleated cell comprises an exogenous polynucleotide encoding a polypeptide sequence corresponding to the exogenous single domain antibody or fragment thereof. EMBODIMENT 17 17. The enucleated cell of embodiment 16, wherein the polypeptide sequence comprises a sequence as provided in SEQ ID NOs: 1-36, 101-111, 121-123, 165-192, 195, 205, 206, 211-213, 221-231, 241-245, 325-331, and 401-404. EMBODIMENT 18 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof is specific to an antigen encoded by at least one nucleic acid in SEQ ID NOs: 131-134, 142-152, 201-202, 301-312, 501, 521-526, 541-545, 561, 584, 591-601, and 701-705. EMBODIMENT 19 2. The enucleated cell of embodiment 1, wherein the exogenous single domain antibody, or fragment thereof, is specific to an antigen comprising a peptide sequence encoding PD-L1. EMBODIMENT 20 2. The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof is specific for an antigen comprising at least one peptide sequence in SEQ ID NOs: 155-164, 203, 204, 315-322, 511, 531-535, 551-554, 571, 594, 611-619, and 711. EMBODIMENT 21 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen associated with at least one pathogen in Table 1. EMBODIMENT 22 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen comprising at least one peptide sequence encoding connective tissue growth factor (CTGF). EMBODIMENT 23 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof is specific for an antigen comprising at least one peptide sequence in SEQ ID NOs: 1601-1602. EMBODIMENT 24 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof is encoded from a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO:801. EMBODIMENT 25 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO:851. EMBODIMENT 26 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof is encoded from a nucleic acid sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO:901. EMBODIMENT 27 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO:951. EMBODIMENT 28 The enucleated cell of embodiment 1, wherein the exogenous single domain antibody or fragment thereof comprises a polypeptide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more identical to SEQ ID NO: 1701. EMBODIMENT 29 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by cancer cells in lung tissue. EMBODIMENT 30 The enucleated cell of embodiment 29, wherein the cancer cell is a non-small cell lung cancer (NSCLC) cell. EMBODIMENT 31 The enucleated cell of embodiment 29, wherein the cancer cell is an adenocarcinoma, squamous cell carcinoma, large cell (undifferentiated) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma cell. EMBODIMENT 32 The enucleated cell of embodiment 29, wherein the cancer cell is a small cell lung cancer (SCLC) cell. EMBODIMENT 33 The enucleated cell of embodiment 29, wherein the cancer cell is a cell of a lung carcinoid tumor. EMBODIMENT 34 The enucleated cell of embodiment 29, wherein the cancer cell is an adenoid cystic carcinoma cell. EMBODIMENT 35 The enucleated cell of embodiment 29, wherein the cancer cell is a lymphoma cell. EMBODIMENT 36 The enucleated cell of embodiment 29, wherein the cancer cell is a cell of a sarcoma. EMBODIMENT 37 The enucleated cell of embodiment 29, wherein the cancer cell is a cell of a benign lung tumor. EMBODIMENT 38 The enucleated cell of embodiment 37, wherein said cancer cell is a hamartoma cell. EMBODIMENT 39 2. The enucleated cell of embodiment 1, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by cells associated with idiopathic pulmonary fibrosis. EMBODIMENT 40 The enucleated cell of embodiment 39, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is a lung cell. EMBODIMENT 41 The enucleated cell of embodiment 39, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is an immune cell. EMBODIMENT 42 The enucleated cell of embodiment 39, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by cells associated with idiopathic pulmonary fibrosis that are alveolar cells. EMBODIMENT 43 The enucleated cell of embodiment 39, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by cells associated with idiopathic pulmonary fibrosis that are alveolar epithelial cells (AECs). EMBODIMENT 44 The enucleated cell of embodiment 39, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by a cell associated with idiopathic pulmonary fibrosis that is a bronchial cell. EMBODIMENT 45 The enucleated cell according to any one of embodiments 1 to 44, wherein the enucleated cell further comprises at least one additional exogenous agent. EMBODIMENT 46 2. The enucleated cell of embodiment 1, wherein the enucleated cell further comprises a fusogenic moiety. EMBODIMENT 47 The enucleated cell of embodiment 46, wherein the fusogenic moiety comprises a viral fusogenic moiety. EMBODIMENT 48 47. The enucleated cell of embodiment 46, wherein the fusogenic moiety comprises a eukaryotic fusogenic moiety. EMBODIMENT 49 The enucleated cell of embodiment 1, wherein the enucleated cell further comprises an immune evasion moiety. EMBODIMENT 50 The enucleated cell of embodiment 49, wherein the immune evasion portion comprises CD47, PD-L1, HLA-E, HLA-G, a fragment thereof, or a combination thereof. EMBODIMENT 51 2. The enucleated cell of embodiment 1, wherein the enucleated cell further comprises a targeting moiety. EMBODIMENT 52 The enucleated cell of embodiment 51, wherein the targeting moiety targets a biomarker of the cancer cell. EMBODIMENT 53 The enucleated cell of embodiment 52, wherein said exogenous single domain antibody or fragment thereof is specific to an antigen expressed by a cancer cell, and said biomarker is a separate and distinct entity from said antigen targeted by said exogenous single domain antibody or fragment thereof. EMBODIMENT 54 The enucleated cell of embodiment 51, wherein said targeting moiety targets a biomarker of an immune cell within the tumor microenvironment. EMBODIMENT 55 The enucleated cell of embodiment 54, wherein said biomarker is expressed on the surface of said immune cell. EMBODIMENT 56 The enucleated cell of embodiment 54, wherein said biomarker is released by said immune cell. EMBODIMENT 57 The enucleated cell of any one of embodiments 51-56, wherein the targeting moiety comprises a chemokine. EMBODIMENT 58 57. The enucleated cell of any one of embodiments 51-56, wherein the targeting moiety comprises a chemokine receptor. EMBODIMENT 59 57. The enucleated cell of any one of embodiments 41 to 56, wherein the targeting moiety comprises an adhesion molecule. EMBODIMENT 60 57. The enucleated cell of any one of embodiments 51 to 56, wherein the targeting moiety comprises an antigen. EMBODIMENT 61 The enucleated cell of any one of embodiments 51-56, wherein the targeting moiety comprises an antigen that is a separate and distinct entity from an antigen expressed by the cancer cell. EMBODIMENT 62 The enucleated cell of any one of embodiments 51 to 56, wherein the targeting moiety comprises an antibody that is not expressed by the cancer cell. EMBODIMENT 63 57. The enucleated cell of any one of embodiments 51 to 56, wherein the targeting moiety comprises a membrane-bound antibody. EMBODIMENT 64 64. The enucleated cell of embodiment 63, wherein said membrane-bound antibody is a membrane-bound single domain antibody. EMBODIMENT 65 2. The enucleated cell of embodiment 1, wherein the enucleated cell has a diameter of about 1 micrometer (μm) to about 100 μm. EMBODIMENT 66 The enucleated cell of embodiment 65, wherein the diameter is from about 1 μm to about 10 μm. EMBODIMENT 67 The enucleated cell of embodiment 65, wherein the diameter is from about 10 μm to about 100 μm. EMBODIMENT 68 66. The enucleated cell of embodiment 65, wherein the diameter is at least or about 1 μm, 5 μm, 8 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. EMBODIMENT 69 The enucleated cell of embodiment 68, wherein said diameter is about 8 μm. EMBODIMENT 70 The enucleated cell of embodiment 1, wherein the enucleated cell exhibits a diameter that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% smaller than a nucleated parent cell. EMBODIMENT 71 The enucleated cell of embodiment 1, wherein the parent cell is selected from the group consisting of stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, and cells from a cell line. EMBODIMENT 72 The enucleated cell of embodiment 71, wherein said parent cell is a mesenchymal stromal cell. EMBODIMENT 73 2. The enucleated cell of embodiment 1, wherein said enucleated cell exhibits viability after cryohibernation. EMBODIMENT 74 The enucleated cell of embodiment 73, wherein the enucleated cell exhibits a post-freeze hibernation viability measured 24 hours after freeze hibernation that is equal to or greater than the viability of a comparable enucleated cell that has not been freeze hibernated. EMBODIMENT 75 2. The enucleated cell of embodiment 1, wherein said enucleated cell exhibits viability after cryopreservation. EMBODIMENT 76 The enucleated cell of embodiment 75, wherein said enucleated cells exhibit a post-cryopreservation viability measured 24 hours after cryopreservation that is equal to or greater than the viability of comparable enucleated cells that have not been cryopreserved. EMBODIMENT 77 The enucleated cell of any one of embodiments 1 to 76, wherein the enucleated cell is isolated. EMBODIMENT 78 The enucleated cell of any one of embodiments 1 to 77, wherein the enucleated cell is purified. EMBODIMENT 79 The enucleated cell of any one of embodiments 1 to 77, wherein the enucleated cell is lyophilized. EMBODIMENT 80 A cell line comprising an enucleated cell according to any one of embodiments 1 to 79. Embodiment 81 A cell comprising a plurality of the enucleated cells according to any one of embodiments 1 to 80. EMBODIMENT 82 A pharmaceutical composition comprising the enucleated cells of any one of embodiments 1 to 80 and a pharma- ceutically acceptable excip...

Claims

1. a single domain antibody or fragment thereof that binds an immune checkpoint molecule; one or more intracellular organelles configured to translate an exogenous messenger ribonucleic acid (mRNA) molecule encoding said single domain antibody or fragment thereof; Enucleated cells containing

2. The enucleated cell of claim 1 , wherein the single domain antibody or fragment thereof is contained in the enucleated cell.

3. The enucleated cell of claim 1 , wherein the single domain antibody or fragment thereof is released by the enucleated cell.

4. The enucleated cell of claim 1, further comprising a cell membrane, wherein said single domain antibody or fragment thereof is expressed on the luminal side of said cell membrane.

5. The enucleated cell of claim 1 , further comprising a cell membrane, said cell membrane comprising a transmembrane portion attached to said single domain antibody or fragment thereof.

6. The enucleated cell of claim 5 , wherein the transmembrane portion comprises a transmembrane polypeptide.

7. The enucleated cell of claim 6 , wherein the single domain antibody or fragment thereof is linked to the N-terminus or C-terminus of the transmembrane polypeptide.

8. The enucleated cell of claim 1, wherein the single domain antibody or fragment thereof is linked to an anchor molecule attached to the cell surface of the enucleated cell, the anchor molecule comprising glycosylphosphatidylinositol, farnesyl, palmitate, myristate, or any combination thereof.

9. The enucleated cell of claim 1, further comprising a fusion protein configured to import said single domain antibody or fragment thereof from said enucleated cell into another cell.

10. The enucleated cell of any one of claims 1 to 9, wherein said single domain antibody or fragment thereof is linked to a cytotoxic drug.

11. The immune checkpoint molecule is selected from the group consisting of programmed cell death protein 1 (PD-1 or PDCD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (also known as cluster of differentiation 152 or CD152, CTLA-4), V domain Ig inhibitor of T cell activation (VISTA), programmed cell death 1 ligand 2 (also known as cluster of differentiation 273 or CD273, PDCD1LG2), B7 homolog 3 (also known as cluster of differentiation 276 or CD276), and IL-11 (IL-11). known as B7-H3), adenosine A2A receptor (A2AR), cluster of differentiation 27 (CD27), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and mucin domain containing-3 (also known as hepatitis A virus cell receptor 2 or HAVCR2, TIM-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cluster of differentiation 73 (CD73), CD94 / NK group 2 member A (also known as cluster of differentiation 159 or CD159, NKG2A ), poliovirus receptor-related immunoglobulin domain-containing (PVRIG), poliovirus receptor-related 2 (PVRL2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), focal adhesion kinase (FAK), C-C chemokine receptor type 2 (CCR-2), chemokine (C-C motif) ligand 2 (CCL-2), leukemia inhibitory factor (LIF), cluster of differentiation 47 (CD47), signaling 2. The enucleated cell of claim 1, comprising a IL-regulatory protein alpha (SIRPα), macrophage colony-stimulating factor (M-CSF), colony-stimulating factor 1 receptor (CSF-1R), interleukin 3 (IL-3), interleukin-1 receptor accessory protein (IL-1RAP), interleukin 8 (IL-8), semaphorin-4D (SEMA4D), angiopoietin-2, CLEVER-1, tyrosine-protein kinase receptor UFO (Axl), phosphatidylserine, or a fragment thereof.

12. The enucleated cell of claim 1, further comprising a targeting moiety.

13. The enucleated cell described in claim 12, wherein the targeting moiety includes a homing receptor specific to a ligand expressed by cells in pulmonary tissue.

14. The enucleated cell described in claim 12, wherein the targeting moiety comprises a chemokine receptor.

15. The enucleated cell described in claim 13, wherein the cell is a cancer cell.

16. The enucleated cell of claim 15, wherein the cancer cell is a cell of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), adenocarcinoma, squamous cell carcinoma, large cell (undifferentiated) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, or sarcomatoid carcinoma.

17. An enucleated cell described in any one of claims 1 to 11, 12 to 13, or 14 to 16, wherein the enucleated cell further contains a therapeutic agent.

18. The enucleated cell of claim 1, further comprising an immune evasion portion comprising cluster of differentiation (CD47), PD-L1, major histocompatibility complex, class I, E (HLA-E), major histocompatibility complex, class I, G (HLA-G), fragments thereof, or combinations thereof.

19. The enucleated cell of claim 1, wherein the enucleated cell is reduced in diameter compared to an otherwise identical nucleated cell, the diameter being reduced by about 50% or more.

20. The enucleated cell of claim 1, further comprising an exogenous tumor necrosis factor (TNF) superfamily member polypeptide or a catalytically active fragment thereof.

21. The enucleated cell of claim 1, wherein the enucleated cell is obtained from a parent cell, the parent cell comprising a stem cell.

22. The enucleated cell of any one of claims 1 to 21, wherein the enucleated cell is freeze-dried.

23. a) an enucleated cell according to any one of claims 1 to 22; b) a pharma- ceutically acceptable excipient, carrier, or diluent; 13. A pharmaceutical formulation comprising:

24. A method for delivering an enucleated cell to a subject, the method comprising a step of delivering an enucleated cell described in any one of claims 1 to 23 to the subject.

25. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an enucleated cell described in any one of claims 1 to 22 or a pharmaceutical preparation described in claim 23, thereby treating the cancer in the subject.

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