Manufacturing of therapeutic cells

Abstract

A method for obtaining enucleated cells from nucleated cells is described herein. A cell processing method is also described herein, which includes providing a composition containing nucleated cells and enucleating at least a portion of the nucleated cells to produce an enucleated cell fraction.

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Application No. 63 / 495,697, filed on 12 April 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Current cell-based therapies tend to have undesirable side effects (such as carcinogenicity, limitations in manipulation capabilities, and adverse immune responses). Furthermore, producing a sufficient quantity of cell-based therapies remains an ongoing challenge. [Overview of the project]

[0003] Therefore, the need for large-scale production of cell-based therapies remains. In several embodiments, methods for obtaining a population of enucleated cells are described herein. The method comprises contacting a plurality of cells with a particle density gradient generated by fixed-angle centrifugation to produce a population of enucleated cells, the population of enucleated cells accumulating in a density range within the particle density gradient. In some embodiments, the method further comprises contacting the plurality of cells with a toxin, the toxin inhibiting cytoskeleton formation of the plurality of cells. In some embodiments, the method further comprises removing the population of enucleated cells from the particle density gradient. In some embodiments, the toxin comprises a mycotoxin. In some embodiments, the mycotoxin comprises a cytochalasin. In some embodiments, the cytochalasin comprises cytochalasin B. In some embodiments, the population of cells after enucleation comprises a reduced density compared to a plurality of cells with nuclei. In some embodiments, the particle density gradient before fixed-angle centrifugation comprises a plurality of particle solutions comprising various particle concentrations. In some embodiments, the fixed-angle centrifugation comprises centrifugation of the plurality of cells in the particle density gradient at an angle of 23 degrees, 24 degrees, 25 degrees, 45 degrees, 90 degrees, or 180 degrees. In some embodiments, fixed-angle centrifugation is performed at approximately 80,000 RCF, approximately 83,000 RCF, approximately 85,000 RCF, approximately 87,000 RCF, approximately 90,000 RCF, approximately 93,000 RCF, approximately 95,000 RCF, approximately 97,000 RCF, approximately 100,000 RCF, approximately 103,000 RCF, approximately 105,000 RCF, and approximately 107,000 RCF. This includes centrifugation at approximately 110,000 RCF, approximately 113,000 RCF, approximately 115,000 RCF, approximately 117,000 RCF, approximately 120,000 RCF, approximately 123,000 RCF, approximately 125,000 RCF, approximately 127,000 RCF, approximately 130,000 RCF, approximately 133,000 RCF, approximately 135,000 RCF, or approximately 137,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation at approximately 85,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation at approximately 127,000 RCF.In some embodiments, fixed-angle centrifugation includes centrifugation with an average R of approximately 85,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation with a maximum R of approximately 127,000 RCF. In some embodiments, fixed-angle centrifugation includes acceleration, which includes approximately 5.2 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes deceleration, which includes approximately 4.0 minutes to reach the stop of fixed-angle centrifugation. In some embodiments, fixed-angle centrifugation includes centrifugation for approximately 60 minutes. In some embodiments, the particle density gradient includes multiple particle solutions. In some embodiments, the multiple particle solutions include at least three particle solutions. In some embodiments, the particle density gradient includes a protein density gradient. In some embodiments, the protein density gradient includes a serum albumin gradient. In some embodiments, the particle density gradient includes a polysaccharide density gradient. In some embodiments, the polysaccharide density gradient includes multiple polysaccharide solutions. In some embodiments, the multiple polysaccharide solutions include 12.5% ​​polysaccharide solution, 15% polysaccharide solution, 16% polysaccharide solution, 17% polysaccharide solution, 25% polysaccharide solution, or any combination thereof. In some embodiments, the enucleated cell population accumulates in a density range of about 15% polysaccharide. In some embodiments, the polysaccharide density gradient includes a ficol density gradient. In some embodiments, the multiple cells include heterologous polynucleotides. In some embodiments, the method further includes cryopreserving the cell population. In some embodiments, fixed-angle centrifugation includes ultracentrifugation. In some embodiments, the multiple cells include stem cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, immortalized cells from cell lines, or any combination thereof. In some embodiments, the stem cells include mesenchymal stromal cells. In some embodiments, the multiple cells include immune cells. In some embodiments, the immune cells include lymphocytes or natural killer cells.In some embodiments, a population of cells comprises one or more intracellular organelles for synthesizing or secreting an exogenous polypeptide, the one or more organelles being retained within a population of enucleated cells, and the one or more organelles synthesizing or secreting the exogenous polypeptide in the absence of a nucleus. In some embodiments, a population of cells comprises heterologous polynucleotides encoding the exogenous polypeptide. In some embodiments, a population of enucleated cells comprises heterologous polynucleotides encoding the exogenous polypeptide, the heterologous polynucleotides being introduced into a population of enucleated cells, and one or more intracellular organelles synthesizing or secreting the exogenous polypeptide in the absence of a nucleus. In some embodiments, the exogenous polypeptide comprises a therapeutic agent. In some embodiments, a population of enucleated cells comprises at least one targeting moiety. In some embodiments, a population of enucleated cells comprises at least one fusionable moiety. In some embodiments, a population of enucleated cells comprises at least one immune-evading moiety. In some embodiments, the enucleated cells in a population of enucleated cells have diameters that include approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in a population of enucleated cells have diameters that include approximately 5 μm or more, approximately 10 μm or more, approximately 20 μm or more, approximately 30 μm or more, approximately 40 μm or more, approximately 50 μm or more, approximately 60 μm or more, approximately 70 μm or more, approximately 80 μm or more, or approximately 90 μm or more. In some embodiments, the diameter includes approximately 8 μm.

[0004] In some embodiments, pharmaceutical compositions comprising a population of enucleated cells produced by the method described herein and a pharmaceutically acceptable excipient, carrier, or diluent are described herein. In some embodiments, the pharmaceutical composition is in unit dose form. In some embodiments, the pharmaceutical composition is formulated for administration to a subject by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or any combination thereof. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition further comprises at least one additional activator. In some embodiments, the at least one additional activator comprises cytokines, growth factors, hormones, enzymes, small molecules, compounds, or any combination thereof.

[0005] In some embodiments, a kit comprising a population of cells produced by the method described herein, or a pharmaceutical composition described herein, and a container is described herein.

[0006] In some embodiments, instructions for enucleating multiple cells according to the method described herein to produce a population of enucleated cells, and a kit comprising one or more components of a polysaccharide density gradient are described herein.

[0007] In some embodiments, methods for treating a target disease or condition are described herein, which include administering a therapeutically effective amount of a population of enucleated cells produced by the method described herein, or of a pharmaceutical composition described herein.

[0008] In some embodiments, the present invention describes a method for treating cancer in a subject, comprising administering to a subject having cancer a population of enucleated cells produced by the method described herein, or a therapeutically effective amount of the pharmaceutical composition described herein.

[0009] In some embodiments, methods for treating a lung disease in a subject are described herein, comprising administering to the subject a population of enucleated cells produced by the method described herein, or a therapeutically effective amount of the pharmaceutical composition described herein.

[0010] In some embodiments, a method for obtaining a population of cells from a plurality of cells is described herein, comprising contacting the plurality of cells with a polysaccharide density gradient produced by fixed-angle centrifugation, wherein the population of cells accumulates within a density range in the polysaccharide density gradient. Also described herein, a method for enucleating a population of cells from a plurality of cells is described herein, comprising contacting the plurality of cells with a toxin that inhibits cytoskeleton formation of the plurality of cells, and contacting the plurality of cells with a polysaccharide density gradient produced by fixed-angle centrifugation, wherein the population of cells is enucleated by fixed-angle centrifugation and accumulates within a density range in the polysaccharide density gradient. In some embodiments, the method further comprises removing the population of cells from the polysaccharide density gradient. In some embodiments, the toxin comprises a mycotoxin. In some embodiments, the mycotoxin comprises a cytochalasin. In some embodiments, the cytochalasin comprises cytochalasin B. In some embodiments, the enucleated population of cells comprises a reduced density compared to the plurality of cells with nuclei. In some embodiments, the polysaccharide density gradient before fixed-angle centrifugation comprises a plurality of polysaccharide solutions comprising various polysaccharide concentrations. In some embodiments, fixed-angle centrifugation includes centrifugation of multiple cells in a polysaccharide density gradient at angles of 23 degrees, 24 degrees, 25 degrees, 45 degrees, 90 degrees, or 180 degrees. In some embodiments, fixed-angle centrifugation includes centrifugation at approximately 126,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation at an average R of approximately 85,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation at a maximum R of approximately 127,000 RCF. In some embodiments, fixed-angle centrifugation includes acceleration, which includes approximately 5.2 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes deceleration, which includes approximately 4.0 minutes to reach the stop of fixed-angle centrifugation. In some embodiments, fixed-angle centrifugation includes centrifugation for approximately 60 minutes. In some embodiments, the polysaccharide density gradient includes multiple polysaccharide solutions. In some embodiments, the multiple polysaccharide solutions include at least three polysaccharide solutions.In some embodiments, the polysaccharide solutions include 12.5% ​​polysaccharide solutions, 15% polysaccharide solutions, 16% polysaccharide solutions, 17% polysaccharide solutions, 25% polysaccharide solutions, or combinations thereof. In some embodiments, the cell population accumulates in a density range of approximately 15% polysaccharide. In some embodiments, the polysaccharide density gradient includes a Ficol density gradient. In some embodiments, the cells include heterologous polynucleotides. In some embodiments, the method further includes cryopreserving the cell population. In some embodiments, the method further includes thawing the cell population, after which the enucleated cells of the cell population are viable as otherwise equivalent, uncryopreserved enucleated cells. In some embodiments, the cell population includes stem cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells from cell lines, or combinations thereof. In some embodiments, the stem cells include mesenchymal stromal cells. In some embodiments, the cell population includes immune cells. In some embodiments, the immune cells include lymphocytes or natural killer cells. In some embodiments, the cell population includes one or more intracellular organelles for synthesizing or secreting exogenous polypeptides in the absence of a nucleus. In some embodiments, the exogenous polypeptide is encoded by heterologous polynucleotides. In some embodiments, the exogenous polypeptide includes a therapeutic agent. In some embodiments, the cell population includes at least one targeting moiety. In some embodiments, the cell population includes at least one fusion moiety. In some embodiments, the cell population includes at least one immune-evading moiety. In some embodiments, the enucleated cells of the cell population have a diameter including about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of a population of cells have diameters including approximately 5 μm or larger, approximately 10 μm or larger, approximately 20 μm or larger, approximately 30 μm or larger, approximately 40 μm or larger, approximately 50 μm or larger, approximately 60 μm or larger, approximately 70 μm or larger, approximately 80 μm or larger, or approximately 90 μm or larger.In some embodiments, the diameter includes approximately 8 μm.

[0011] In some embodiments, pharmaceutical compositions comprising a population of cells described herein and pharmaceutically acceptable excipients, carriers, or diluents are described herein. In some embodiments, the pharmaceutical composition is in unit dose form. In some embodiments, the pharmaceutical composition is formulated for administration to a subject by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or a combination thereof. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition further comprises at least one additional activator. In some embodiments, the at least one additional activator comprises cytokines, growth factors, hormones, enzymes, small molecules, compounds, or a combination thereof.

[0012] In some embodiments, a kit comprising a population of cells or a pharmaceutical composition described herein, and a container is described herein.

[0013] In some embodiments, methods for treating a target disease or condition are described herein, which include administering a therapeutically effective amount of a population of cells or a pharmaceutical composition described herein.

[0014] In some embodiments, methods for treating cancer in a subject are described herein, comprising administering a therapeutically effective amount of a population of cells described herein or a pharmaceutical composition described herein to a subject having cancer.

[0015] In some embodiments, methods for treating a lung disease in a subject are described herein, comprising administering a therapeutically effective amount of a population of cells described herein or a pharmaceutical composition described herein to a subject with a lung disease.

[0016] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict between a publication and / or patent or patent application incorporated by reference and the disclosure contained herein, the specification is intended to supersede and / or prevail over any such conflicting material.

[0017] Some novel features of the methods and compositions disclosed herein are described in this disclosure. A better understanding of the features and advantages of the methods and compositions disclosed herein can be obtained by reference to the following detailed description, which shows exemplary embodiments in which the principles of the disclosed compositions and methods are utilized, and to the accompanying drawings.

Brief Description of the Drawings

[0018] [Figure 1] A flowchart showing non-limiting steps for the treatment of a composition of enucleated cells or a pharmaceutical composition for the delivery of a therapeutic agent, according to one embodiment of the present disclosure. [Figure 2] A timeline of the production of enucleated cells for the delivery of single domain antibodies according to various embodiments, compared to the typical biological drug development timeline. [Figure 3A] Shows enucleation of cells in a fixed angle centrifuge with three Ficoll gradients. The three Ficoll gradients used were 12.5%, 15% and 17%, and after enucleation by fixed angle centrifugation, the enucleated cells are located at the 15% gradient. [Figure 3B] Shows an image of enucleated cells isolated from the 15% Ficoll gradient after enucleation in a fixed angle centrifuge. The central image shows enucleated cells imaged with a bright field microscope. The right image shows DAPI stained enucleated cells imaged with a fluorescence microscope. No DAPI staining was seen in the enucleated cells.

Modes for Carrying Out the Invention

[0019] Existing techniques that use centrifugal force to move nuclei in adherent cells (i.e., enucleation) have drawbacks and are not feasible for large-scale enucleation cell platforms for biomedical applications. These drawbacks include, but are not limited to, the small capacity of swing buckets, ranging from a few milliliters to several hundred milliliters, and the long overall pelletization time pathway, resulting in small-scale production and reduced efficiency. These drawbacks significantly limit the number of cells that can be processed at once, making it difficult to achieve optimal cell density within each centrifugation bucket in large-scale production.

[0020] To overcome these drawbacks, this application discloses a cell processing method comprising enucleating cells using fixed-angle centrifugation, including continuous-flow centrifugation. Fixed-angle rotors can hold a larger volume of tubes compared to their swing bucket counterparts due to their simple and efficient tube spacing. Furthermore, as a result of the rigid design of the metal alloy material, fixed rotors can withstand much higher gravitational forces, thus shortening the centrifugation time. Fixed-angle centrifugation, including continuous-flow centrifugation, allows for the centrifugation of large volumes of material at high centrifugal forces without the tedious task of filling and decanting numerous centrifuge tubes or frequently starting and stopping the rotor. The combination of high centrifugal force, short centrifugation time, and high throughput makes fixed-angle centrifugation processing useful for the large-scale production of enucleated cells for biomedical applications, ranging from bacterial and cell pelletization to isodensity separation of polymers.

[0021] Furthermore, quality control of enucleated cell platforms for biomedical applications is a challenge, and this challenge is exacerbated by large-scale manufacturing. Some of the many advantages of enucleated cells disclosed herein stem from the absence of a nucleus, such as the elimination of the need for in vivo gene transfer and the limitation of in vivo lifespan. However, existing large-scale manufacturing techniques result in the inclusion of nucleated parent cell portions in the resulting therapeutic compositions, thus undermining the advantages of the enucleated cell platform.

[0022] In addition to advancements in manufacturing scalability and quality control, the enucleated cell platform described herein offers certain advantages over existing cell-based therapeutic platforms and is unparalleled in suitability for large-scale use as a therapeutic composition. The enucleated cells described herein are described in U.S. Patent Application No. 10,927,349, which are incorporated herein by reference as a whole. Furthermore, the further utility and advantages of the enucleated cells disclosed herein are discussed in U.S. Patent Application No. 18 / 176,259, filed February 28, 2023, U.S. Patent Application No. 17 / 885,867, filed August 11, 2022, and U.S. Patent Application No. 18 / 190,838, filed March 27, 2023, each incorporated herein by reference as a whole. For example, cell delivery platforms have specific therapeutic applications (such as addressing pathogen outbreaks), and existing manufacturing timelines for such applications may limit the scalability and speed required to address public health emergencies resulting from pathogen outbreaks. Existing therapeutic cell therapies requiring extensive manipulation take at least 12 months to develop. In contrast, the enucleated cells disclosed herein can be extensively manipulated before and after enucleation (e.g., using targeting regions specific to target tissue, immune system evasion regions to reduce in vivo phagocytosis, etc.), and then stored for extended periods without sacrificing their restored viability by preferred means disclosed herein (e.g., lyophilization, cryopreservation, and cryopreservation). When a new pathogen or a new strain of a known pathogen is identified, the enucleated cells (already manipulated to express appropriate targeting regions, immune system evasion regions, immune activators, etc.) can be restored to their biological activity (e.g., rehydration, thawing, etc.) and further manipulated to express or deliver therapeutic agents for the prevention or treatment of infection by the recently discovered pathogen or strain. These advantages are illustrated in Figure 2, which shows that the manufacturing process of the enucleated cells of this disclosure takes approximately 2 months compared to a preferred timeline of 12 months or more.

[0023] Existing erythrocyte or platelet therapeutic platforms are enucleated by erythrocyte generation. In this erythrocyte generation, blood cells ultimately differentiate, and some intracellular organelles and ribosomes responsible for protein synthesis and secretion are eliminated. Therefore, the resulting erythrocytes or platelets lose cell-like functionality (e.g., protein expression, secretion, cell motility, chemokine sensing, homing ability, etc.) that may be important for therapeutic applications (e.g., production, delivery, or secretion of therapeutic agents in vivo) after enucleation by erythrocyte generation. In contrast, the enucleated cells described herein retain one or more intracellular organelles endogenous to the parent cell after enucleation. In some embodiments, all of the one or more intracellular organelles are retained. In some embodiments, fewer than all of the one or more intracellular organelles are retained. In some embodiments, the Golgi apparatus and / or endoplasmic reticulum, which are involved in protein synthesis and secretion, are retained. The retention of one or more intracellular organelles enables enucleated cells to synthesize or release biomolecules disclosed herein (e.g., single-domain antibodies, or portions thereof, targeting portions, immune-evading portions, etc.) in the absence of a nucleus. In some embodiments, a population of cells (e.g., enucleated cells) includes one or more intracellular organelles. In some embodiments, one or more intracellular organelles can synthesize and / or secrete exogenous polypeptides in the absence of a nucleus.

[0024] Enucleated cells disclosed herein may originate from substantially any nucleated cell (referred to herein as “parent” cell). In some embodiments, the parent cell is an immune cell. In some embodiments, the immune cell is a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, or lymphocyte (B cell and T cell). In some embodiments, the parent cell is a stem cell. In some embodiments, the parent cell is an adult stem cell. In some embodiments, the parent cell is a mesenchymal stromal cell (MSC). In some embodiments, the enucleated cells originate from inducible pluripotent stem cells (iPSCs). In some embodiments, the parent cell is not a erythrocyte. In some embodiments, the parent cell is not an erythrocyte progenitor cell. In some embodiments, the parent cell is not an endothelial cell. In some embodiments, the parent cell is not an endothelial progenitor cell.

[0025] Methods for producing enucleated cells in high quantities and with high purity are described herein. Herein, the produced enucleated cells can be formulated into compositions or pharmaceutical compositions for the treatment of a disease or pathological condition in a subject requiring treatment. Figure 1 shows a non-limiting example of the production of enucleated cells (100) as described herein. Nucleated cells (101) can be isolated from a subject and cultured in vitro for clonal proliferation. In some embodiments, nucleated cells (101) can also be immortalized or derived from a cell line. In some embodiments, nucleated cells can be manipulated to contain heterologous polynucleotides (103) (102). The nucleated cells can then be enucleated by fixed-angle centrifugation including continuous-flow centrifugation (104). Using fixed-angle centrifugation including continuous-flow centrifugation for cell enucleation is an improvement over currently available enucleation methods, and enucleation performed by fixed-angle centrifugation including continuous-flow centrifugation improves the quantity (yield, etc.) or purity of enucleated cells obtained from nucleated cells. In some embodiments, fixed-angle centrifugation increases the number of enucleated cells in the population. In some embodiments, fixed-angle centrifugation increases the number of enucleated cell populations by at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100%, 150%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 750%, at least 800%, at least 900%, or at least 1,000%. In some embodiments, fixed-angle centrifugation yields a higher yield of enucleated cell populations compared to swing-bucket centrifugation.In some embodiments, fixed-angle centrifugation yields a population of enucleated cells of at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 100%, 150%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 750%, at least 800%, at least 900%, at least 1,000%, at least 2,000%, at least 3,000%, at least 4,000%, at least 5,000%, at least 6,000%, at least 7,000%, at least 8,000%, or more compared to swing-bucket centrifugation. After obtaining a composition (105) of enucleated cells (which may have residual nucleated cells), the composition can be further purified with respect to enucleated cells by selecting markers for enucleated cells (106) to obtain a portion (107) of enucleated cells. The enucleated cells can be cryogenically dormant (108), cryopreserved (109), freeze-dried (110), or a combination thereof, and can be formulated into a composition or pharmaceutical composition for delivering a therapeutic agent to treat a target (111) disease or condition.

[0026] In some embodiments, the average enucleation yield is based on the total number of cells loaded per rotor. In some embodiments, the average enucleation yield is about 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the total number of cells loaded per rotor. In some embodiments, the average enucleation efficiency is based on the average enucleation yield. In some embodiments, the average enucleation efficiency is about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 98%, or 99%, based on the average enucleation yield. For example, after centrifugation of cells at a fixed angle, the fraction containing enucleated cells is the enucleation yield, and the actual number of enucleated cells in the fraction containing enucleated cells is the enucleation efficiency. In some embodiments, the enucleation efficiency is determined by contacting cells from the fraction containing enucleated cells with a stain that stains the nucleus. In some embodiments, the stain is Hoechst stain. In some embodiments, the stain is DAPI stain. For example, as shown in Figure 3B, cells collected from a fraction of a tube containing a 15% polysaccharide solution (left) were examined under bright-field (center) and fluorescence (right) microscopes. Many cells were visible under the bright-field microscope, but once stained with DAPI and analyzed under a fluorescence microscope, few cells were visible. In this experiment, DAPI stained the nuclei, and the cells that were not stained / not visible under the fluorescence microscope were successfully enucleated.

[0027] In some embodiments, this specification describes obtaining a population of enucleated cells from a plurality of cells, comprising contacting the plurality of cells with a particle density gradient generated by fixed-angle centrifugation. In some embodiments, the particle density gradient includes a polysaccharide density gradient. In some embodiments, the population of cells accumulates in a density range within the polysaccharide density gradient. In some embodiments, this method comprises contacting the plurality of cells with a toxin. In some embodiments, the toxin inhibits cytoskeleton formation in the plurality of cells. The toxin may be a mycotoxin. The mycotoxin may include a cytochalasin. The cytochalasin may include cytochalasin B. In some embodiments, this method comprises contacting the plurality of cells with a polysaccharide density gradient generated by fixed-angle centrifugation. In some embodiments, the population of cells is enucleated by fixed-angle centrifugation and accumulates in a density range within the polysaccharide density gradient. The population of cells may have a reduced density after enucleation compared to the plurality of cells with nuclei. In some embodiments, this method comprises removing the population of cells from the polysaccharide density gradient. In some embodiments, this method comprises contacting the population of cells with the polysaccharide density gradient. The polysaccharide density gradient may include multiple polysaccharide solutions containing various polysaccharide concentrations prior to fixed-angle centrifugation. In some embodiments, this method involves subjecting a population of cells within the polysaccharide density to fixed-angle centrifugation. In some embodiments, the fixed-angle centrifugation involves centrifugation of multiple cells in the polysaccharide density gradient at angles of approximately 20 degrees, approximately 21 degrees, approximately 22 degrees, approximately 23 degrees, approximately 24 degrees, approximately 25 degrees, approximately 26 degrees, approximately 27 degrees, approximately 28 degrees, approximately 29 degrees, approximately 30 degrees, approximately 35 degrees, approximately 40 degrees, approximately 45 degrees, approximately 50 degrees, approximately 60 degrees, approximately 70 degrees, approximately 80 degrees, approximately 90 degrees, approximately 100 degrees, approximately 120 degrees, approximately 150 degrees, or approximately 180 degrees. In some embodiments, fixed-angle centrifugation involves centrifugating a plurality of cells in a polysaccharide density gradient at angles of 23 degrees, 24 degrees, 25 degrees, 45 degrees, 90 degrees, or 180 degrees.In some embodiments, fixed-angle centrifugation includes centrifugation at approximately 120,000 RCF, approximately 122,000 RCF, approximately 124,000 RCF, approximately 126,000 RCF, approximately 127,000 RCF, approximately 128,000 RCF, approximately 130,000 RCF, approximately 132,000 RCF, approximately 134,000 RCF, or approximately 135,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation at approximately 127,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation at an average R of approximately 70,000 RCF, approximately 75,000 RCF, approximately 80,000 RCF, approximately 85,000 RCF, approximately 90,000 RCF, approximately 95,000 RCF, or approximately 100,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation with an average R of approximately 85,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation with a maximum R of approximately 120,000 RCF, approximately 122,000 RCF, approximately 124,000 RCF, approximately 126,000 RCF, approximately 127,000 RCF, approximately 128,000 RCF, approximately 130,000 RCF, approximately 132,000 RCF, approximately 134,000 RCF, or approximately 135,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation with a maximum R of approximately 127,000 RCF. In some embodiments, fixed-angle centrifugation includes acceleration of approximately 3.0 minutes (min), approximately 3.2 minutes, approximately 3.4 minutes, approximately 3.5 minutes, approximately 3.6 minutes, approximately 3.8 minutes, approximately 4.0 minutes, approximately 4.2 minutes, approximately 4.4 minutes, approximately 4.5 minutes, approximately 4.6 minutes, approximately 4.8 minutes, approximately 5.0 minutes, approximately 5.2 minutes, approximately 5.4 minutes, approximately 5.5 minutes, approximately 5.6 minutes, approximately 5.8 minutes, approximately 6.0 minutes, approximately 6.2 minutes, approximately 6.4 minutes, approximately 6.5 minutes, approximately 6.6 minutes, approximately 6.8 minutes, or approximately 7.0 minutes to reach RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration of approximately 5.2 minutes to reach RCF for centrifugation.In some embodiments, the fixed-angle centrifugation includes deceleration of approximately 2.0 minutes (min), approximately 2.2 minutes, approximately 2.4 minutes, approximately 2.5 minutes, approximately 2.6 minutes, approximately 2.8 minutes, approximately 3.0 minutes (min), approximately 3.2 minutes, approximately 3.4 minutes, approximately 3.5 minutes, approximately 3.6 minutes, approximately 3.8 minutes, approximately 4.0 minutes, approximately 4.2 minutes, approximately 4.4 minutes, approximately 4.5 minutes, approximately 4.6 minutes, approximately 4.8 minutes, approximately 5.0 minutes, approximately 5.2 minutes, approximately 5.4 minutes, approximately 5.5 minutes, approximately 5.6 minutes, approximately 5.8 minutes, or approximately 6.0 minutes before reaching the stop of the fixed-angle centrifugation. In some embodiments, the fixed-angle centrifugation includes deceleration of approximately 4.0 minutes before reaching the stop of the fixed-angle centrifugation. In some embodiments, fixed-angle centrifugation includes centrifugation for about 30 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, or about 90 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 60 minutes. In some embodiments, this method includes subjecting a group of cells to fixed-angle centrifugation for about 6 minutes. In some embodiments, this method includes subjecting a group of cells to fixed-angle centrifugation for about 5 minutes. In some embodiments, this method includes subjecting a group of cells to fixed-angle centrifugation for about 4 minutes.

[0028] In some embodiments, the method involves subjecting a plurality of cells to fixed-angle centrifugation containing at least a plurality of polysaccharide solutions. In some embodiments, the method involves subjecting a plurality of cells to fixed-angle centrifugation containing at least two, at least three, at least four, at least five, and at least six polysaccharide solutions. In some embodiments, the method involves subjecting a plurality of cells to fixed-angle centrifugation containing at least three polysaccharide solutions. In some embodiments, the plurality of polysaccharide solutions include 10% polysaccharide solution, 10.5% polysaccharide solution, 11% polysaccharide solution, 11.5% polysaccharide solution, 12% polysaccharide solution, 12.5% ​​polysaccharide solution, 13% polysaccharide solution, 13.5% polysaccharide solution, 14% polysaccharide solution, 14.5% polysaccharide solution, 15% polysaccharide solution, 15.5% polysaccharide solution, 16% polysaccharide solution, 16.5% polysaccharide solution, and 17% polysaccharide solutions. The polysaccharide solutions include 17.5% polysaccharide solution, 18% polysaccharide solution, 18.5% polysaccharide solution, 19% polysaccharide solution, 19.5% polysaccharide solution, 20% polysaccharide solution, 21% polysaccharide solution, 22% polysaccharide solution, 23% polysaccharide solution, polysaccharide solution, 24% polysaccharide solution, 25% polysaccharide solution, 26% polysaccharide solution, 28% polysaccharide solution, 29% polysaccharide solution, 30% polysaccharide solution, or any combination thereof. In some embodiments, the polysaccharide solutions include 12.5% ​​polysaccharide solution, 15% polysaccharide solution, 16% polysaccharide solution, 17% polysaccharide solution, 25% polysaccharide solution, or any combination thereof. In some embodiments, the polysaccharide solutions include 12.5% ​​polysaccharide solution, 15% polysaccharide solution, and 17% polysaccharide solution, as shown in Figure 3A. In some embodiments, the cell population is accumulated in density ranges of approximately 14% polysaccharide solution, approximately 14.5% polysaccharide solution, approximately 15% polysaccharide solution, approximately 15.5% polysaccharide solution, or approximately 16% polysaccharide solution. In some embodiments, the cell population is accumulated in density range of approximately 15% polysaccharide. In some embodiments, the polysaccharide density gradient includes a Ficol density gradient. In some embodiments, the multiple cells contain heterologous polynucleotides. In some embodiments, the method includes cryopreserving the cell population.In some embodiments, the method involves thawing a population of cells, after which the enucleated cells of the population are viable as otherwise equivalent, uncryopreserved enucleated cells. In some embodiments, fixed-angle centrifugation includes ultracentrifugation. In some embodiments, the population of cells includes stem cells. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, immortalized cells from cell lines, or any combination thereof. In some embodiments, the stem cells include mesenchymal stromal cells. In some embodiments, the population of cells includes immune cells. In some embodiments, the immune cells include lymphocytes or natural killer cells. In some embodiments, the multiple cells include heterologous polynucleotides, and the exogenous polypeptide is encoded by heterologous polynucleotides. In some embodiments, the exogenous polypeptide includes a therapeutic agent. In some embodiments, the population of cells includes at least one targeting moiety, at least one fusion moiety, at least one immune-evading moiety, or a combination thereof.

[0029] In some embodiments, the enucleation methods disclosed herein yield a composition containing enucleated cells (also referred herein as the “enucleated cell fraction” of the composition). In some embodiments, the composition further comprises approximately equal to or less than about 1 percent (%) by volume of residual nucleated cells (also referred herein as the “nucleated cell fraction” of the composition). In some embodiments, the nucleated cell fraction comprises approximately equal to or less than 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9% by volume of the composition. In some embodiments, the nucleated cell fraction comprises, on a volume basis, 0.1% to about 0.2%, about 0.1% to about 0.3%, about 0.1% to about 0.4%, about 0.1% to about 0.5%, about 0.1% to about 0.6%, about 0.1% to about 0.7%, about 0.1% to about 0.8%, about 0.1% to about 0.9%, or about 0.1% to about 1.0% of the composition. In some embodiments, the nucleated cell fraction comprises, on a volume basis, about 0.2% to about 0.3%, about 0.2% to about 0.4%, about 0.2% to about 0.5%, about 0.2% to about 0.6%, about 0.2% to about 0.7%, about 0.2% to about 0.8%, about 0.2% to about 0.9%, or about 0.2% to about 1.0% of the composition. In some embodiments, the nucleated cell fraction comprises about 0.3% to about 0.4%, about 0.3% to about 0.5%, about 0.3% to about 0.6%, about 0.3% to about 0.7%, about 0.3% to about 0.8%, about 0.3% to about 0.9%, or about 0.3% to about 1.0% by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 0.4% to about 0.5%, about 0.4% to about 0.6%, about 0.4% to about 0.7%, about 0.4% to about 0.8%, about 0.4% to about 0.9%, or about 0.4% to about 1.0% by volume of the composition. In some embodiments, the nucleated cell fraction comprises about 0.5% to about 0.6%, about 0.5% to about 0.7%, about 0.5% to about 0.8%, about 0.5% to about 0.9%, or about 0.5% to about 1.0% of the composition by volume.In some embodiments, the nucleated cell fraction comprises about 0.7% to about 0.8%, about 0.7% to about 0.9%, or about 0.7% to about 1.0% of the composition by volume.

[0030] Furthermore, pharmaceutical compositions and formulations comprising the compositions described herein and pharmaceutically acceptable carriers, excipients, diluents, or spray inhalants are also described herein. The pharmaceutical compositions are provided in the form of pharmaceutical formulations. In some embodiments, the pharmaceutical formulations are formulated for administration to a subject as a combination therapy (e.g., a prodrug, adjuvant, additional therapeutic agent, or other therapy) or as a monotherapy. In some embodiments, the pharmaceutical formulations are formulated for systemic administration or site-of-action administration (e.g., intratumoral administration).

[0031] A kit comprising a composition disclosed herein and packaging material configured to deliver the composition to an individual is disclosed herein. The kit disclosed herein may comprise a composition comprising an enucleated cell fraction and a nucleated cell fraction of less than 0.1%. In some embodiments, the kit further comprises instructions for further manipulating the enucleated cells in the enucleated cell fraction, for example, to produce or secrete a therapeutic agent disclosed herein. In some cases, the instructions may further comprise instructions for a method of formulating the resulting composition into a pharmaceutical formulation for administration to an object disclosed herein.

[0032] composition This specification discloses compositions and formulations thereof comprising enucleated cells that can be extensively manipulated to express an activator or a portion thereof in the absence of a nucleus. Such enucleated cells are viable cell-like entities capable of synthesizing, releasing (e.g., secreting), or delivering an activator to target cells or tissues in the absence of a nucleus. The compositions disclosed herein can be stored in a suspension biological stage for any period of time by means of cryopreservation, cryopreservation, or lyophilization, and once biological activity is restored, this does not affect the viability of the enucleated cells. Furthermore, the compositions disclosed herein contain approximately equal to or less than 0.1% nucleated cells (e.g., parent cells that were not enucleated during the enucleation treatment), making the compositions disclosed herein optimal for therapeutic use. The enucleated cells (referred to herein as “cytoplasm”) may further contain native cell surface molecules retained from the parent cells. In some embodiments, the enucleated cells further contain exogenous molecules, examples of which include targeted portions, transmembrane portions, and additional therapeutic agents (e.g., other than the activator), examples of which are disclosed herein.

[0033] enucleated cells The enucleated cells of this disclosure are obtained from or derived from corresponding nucleated cells (hereinafter referred to as “parent cells”). The parent cells may be derived from a variety of different cell types, including eukaryotic cells. For example, 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, enucleated cells are derived from mesenchymal stromal cells. In some embodiments, enucleated cells are derived from inducible pluripotent stem cells (iPSCs). In some embodiments, the parent cells are derived from cells immortalized using a preferred method. In some embodiments, enucleated cells include or retain one or more structural features of the parent cell, including intracellular organelles, one or more tunnel nanotubes, or a combination thereof. In some embodiments, enucleated cells include one or more intracellular organelles for synthesizing or secreting exogenous polypeptides (e.g., therapeutic agents) in the absence of a nucleus. In some embodiments, one or more intracellular organelles include the Golgi apparatus, the endoplasmic reticulum, or a combination thereof. In some embodiments, enucleated cells contain or express any one of the therapeutic agents described herein.

[0034] In some embodiments, the cells may originate from any organism having one or more cells. Non-limiting examples of cells include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of single-cell eukaryotes, protist cells, plant-derived cells (e.g., plant crops, fruits, vegetables, grains, soybeans, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), and algal cells (e.g., Botryococcus braunii, Conamidrim). This includes cells such as *Cypripedium macranthos*, *Nannochloropsis gaditana*, *Chlorella pyrenoides*, and *Sargassum fuciformis*, seaweed (e.g., kelp), fungal cells (e.g., yeast cells, mushroom cells), animal cells, invertebrate cells (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), vertebrate cells (e.g., fish, amphibians, reptiles, birds, mammals), and mammalian cells (e.g., pigs, cattle, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Cells may not originate from naturally occurring organisms (e.g., cells may be synthesized and manufactured, and are sometimes called artificial cells). In some embodiments, cells are somatic cells. In some embodiments, cells are stem cells or progenitor cells. In some embodiments, cells are mesenchymal stem cells or mesenchymal progenitor cells. In some embodiments, cells are hematopoietic stem cells or hematopoietic progenitor cells. In some embodiments, cells are muscle cells, skin cells, blood cells, or immune cells. Other non-exclusive examples of cells include lymphoid cells (examples include B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells); myeloid cells (examples include granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, platelets / megakaryocytes, dendritic cells); endocrine cells (including thyroid (thyroid epithelial cells, parafollicular cells), parathyroid (parathyroid chief cells, eosinophilic cells), adrenal (chromaffin cells), pineal (pineal cells) cells); nervous system cells (including glial cells (astrocytes, microglia), giant neurosecretory cells, astrocytes,This includes Bettcher cells and pituitary gland cells (gonadotropins, corticotropes, thyroid-stimulating hormone-secreting cells, growth hormone-secreting cells, mammary gland-stimulating hormone-secreting cells); respiratory system cells (including pneumocytes (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, dust cells); circulatory system cells (including cardiomyocytes, pericytes); digestive system cells (including stomach cells (gastrocnemiocytes, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells); enteroendocrine cells (and These include enterochromophilic cells, APUD cells, liver cells (hepatocytes, Kupffer cells), cartilage / bone / muscle cells; osteocytes (including osteoblasts, osteocytes, osteoclasts, teeth (cementoblasts, ameloblasts)); chondrocytes (including chondrocytes, chondrocytes); skin cells (including hair follicle cells, keratinocytes, melanocytes (nevus cells)); muscle cells (including cardiomyocytes); urinary system cells (including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells, renal proximal urine); This includes tubular brush border cells and macula densa cells; germline cells (including sperm, Sertoli cells, Leydig cells, and oocytes); and other cells (including adipocytes, fibroblasts, tendinocytes, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, cuticle hair stem cells, cuticle root sheath cells, root sheath cells of the Huxley layer, root sheath cells of the Henle layer, outer root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, cornea, tongue, oral cavity, esophagus, anal canal, and periphery. The epithelial cells include surface epithelial cells of stratified squamous epithelium of the urethra and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urethral epithelial cells (lining the inside of the urethra, vagina, bladder, and ureters), exocrine epithelial cells, salivary gland mucus cells (secreting polysaccharide-rich secretions), salivary gland serous cells (secreting glycoprotein enzyme-rich secretions), von Ebner's gland cells of the tongue (washing the taste buds), mammary gland cells (secreting milk), lacrimal gland cells (secreting tears), ceruminous gland cells of the ear (secreting earwax), eccrine gland dark cells (secreting glycoproteins), and eccrine gland clear cells (secreting small molecules).Apocrine sweat gland cells (odorous secretions, sex hormone sensitive), Mohl's gland cells of the eyelids (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretions), Bowman's gland cells of the nose (cleansing the olfactory epithelium), Brunner's gland cells of the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal components containing fructose to help sperm swim), prostate cells (secreting seminal components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubrication secretion), Littley's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), isolation of the respiratory and digestive tracts. Goblet cells (mucus secretion), mucus cells lining the inside of the stomach (mucus secretion), gastric gland enzyme progenitor cells (pepsinogen secretion), gastric gland acid-secreting cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Paneth cells of the small intestine (lysozyme secretion), type II pneumocytes of the lungs (surfactant secretion), Clara cells of the lungs, hormone-secreting cells, anterior pituitary cells, growth hormone-secreting cells, mammogenic hormone-secreting cells, thyroid-stimulating hormone-secreting cells, gonadotropins, corticotropes, pituitary intermediate cells, giant cell neurosecreting cells, cells of the intestinal and respiratory tracts, thyroid cells, Thyroid epithelial cells, parafollicular cells, parathyroid cells, chief parathyroid cells, eosinophilic cells, adrenal cells, chromaffin cells, Leydig cells of the testis, endometrial cells of follicular cells, luteal cells of ruptured follicles, granulosa lutein cells, follicular membrane lutein cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lungs, intestines, exocrine glands and urogenital tract), kidney, type I pneumocytes (lining the inside of the air spaces in the lungs), pancreatic duct cells (acinate central cells), non-striatal duct cells (of sweat glands, salivary glands, mammary glands, etc.), ductal cells (of seminal vesicles, prostate, etc.), closed interior Epithelial cells lining the inside of body cavities, ciliated cells with propulsive function, extracellular matrix secretory cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes, megakaryocytes (platelet progenitor cells), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes,This includes stem cells and differentiation-determining progenitor cells of the blood and immune systems (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transduction cells, autonomic nerve cells, sensory organ and peripheral neuron supporting cells, neurons and glial cells of the central nervous system, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (spermatocyte stem cells), sperm, nurse cells, ovarian follicular cells, Sertoli cells (in the testes), thymic epithelial cells, stromal cells, and interstitial kidney cells.

[0035] In some embodiments, the cells are eukaryotic cells. Non-limiting examples of eukaryotic cells include mammals (e.g., rodents, non-human primates, or humans), non-mammalian animals (e.g., fish, birds, reptiles, or amphibians), invertebrates, insects, fungi, or plant cells. In some embodiments, the eukaryotic cells are yeast cells such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cells are higher eukaryotes such as mammals, birds, plants, or insect cells. In some embodiments, the nucleated cells are primary cells. In some embodiments, the nucleated cells are immune cells (e.g., lymphocytes (e.g., T cells, B cells), macrophages, natural killer cells, neutrophils, mast cells, basophils, dendritic cells, monocytes, bone marrow-derived suppressor cells, eosinophils). In some embodiments, the nucleated cells are phagocytes or leukocytes. In some embodiments, nucleated cells are stem cells (e.g., adult stem cells (e.g., hematopoietic stem cells, mammary gland 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, or inducible pluripotent stem cells (iPS cells)). In some embodiments, nucleated cells are progenitor cells. In some embodiments, nucleated cells are derived from a cell line. In some embodiments, nucleated cells are suspension cells. In some embodiments, nucleated cells are adherent cells. In some embodiments, nucleated cells are cells immortalized by the expression of oncogenes. In some embodiments, nucleated cells are immortalized by the expression of human telomerase reverse transcriptase (hTERT) or any oncogene. In some embodiments, nucleated cells are patient- or subject-derived cells (e.g., autologous patient-derived cells or allogeneic 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 virus vector (e.g., a vesicular stomatitis virus (VSV) vector), or a hybrid viral vector) or plasmid) before the nucleated cells are denucleated using any denucleation technique described herein or known in the art.

[0036] In some embodiments, the cytoplasm is derived from the subject's own cells. In some embodiments, the cytoplasm is derived from the subject's allogeneic cells.

[0037] In some embodiments, the cytoplasm is derived from immune cells. In some embodiments, the cytoplasm is derived from natural killer (NK) cells, neutrophils, macrophages, lymphocytes, fibroblasts, adult stem cells (e.g., hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, mesenchymal stem cells, mesenchymal stromal cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, skin stem cells, or testicular cells), mast cells, basophils, eosinophils, endothelial cells, endothelial cell progenitor cells, or inducible pluripotent stem cells.

[0038] In some embodiments, parent cells may be enucleated or manipulated for therapeutic purposes. In some embodiments, parent cells may be treated with cytochalasin to soften the cortical actin cytoskeleton. In some embodiments, the nuclei are then physically extracted from the cell bodies by high-speed centrifugation in a polysaccharide gradient to produce enucleated cells. In some embodiments, the polysaccharide is ficol to generate a ficol gradient to produce enucleated cells. Since enucleated cells and intact nucleated cells precipitate into different layers in the ficol gradient, enucleated cells may be isolated or prepared for therapeutic purposes or for fusion with other cells (nucleated or enucleated cells). By utilizing the methods described herein, the enucleation process can be made clinically scalable to process tens of millions of cells. In some embodiments, enucleated cells may be used as disease-homing vehicles for delivering clinically relevant cargo or payloads to treat the various diseases or conditions described herein.

[0039] In some embodiments, the enucleated cell contains at least one therapeutic agent. In some embodiments, the enucleated cell disclosed herein expresses the therapeutic agent in the absence of a nucleus along with one or more intracellular organelles. In some embodiments, the therapeutic agent is exogenous to the enucleated cell or its parent (nucleated) cell. In some embodiments, the enucleated cell expresses the therapeutic agent on its surface. In some embodiments, the therapeutic agent is secreted by the enucleated cell into the extracellular space (e.g., the microenvironment) of the target tissue. In some embodiments, the therapeutic agent is the enucleated cell's cargo (e.g., encapsulated by the enucleated cell).

[0040] In some embodiments, enucleated cells are obtained from a first subset of multiple nucleated cells. In some embodiments, enucleated cells are present in a composition further comprising a second subset of multiple nucleated cells. In some embodiments, the second subset of nucleated cells comprises less than about 0.1% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 0.5% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 1% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 5% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 10% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 15% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 20% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 25% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 30% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 40% of the composition by volume. In some embodiments, the second subset of nucleated cells comprises less than about 50% of the composition by volume.

[0041] In some embodiments, the enucleated cells described herein can be preserved by cryopreservation, cryopreservation, freeze-drying, or a combination thereof. In some embodiments, cryopreserved enucleated cells are viable after thawing as otherwise equivalent, uncryopreserved enucleated cells. In some embodiments, freeze-dried enucleated cells are viable as otherwise equivalent, unfreeze-dried enucleated cells. In some embodiments, cryopreserved enucleated cells are viable as otherwise equivalent, unfreeze-dried enucleated cells. In some embodiments, cryopreservation involves storing the enucleated cells at a temperature below room temperature but not freezing. In some embodiments, cryopreservation involves storing the enucleated cells at approximately 4°C. In some embodiments, cryopreservation involves storing the enucleated cells at a maximum of approximately 4°C. In some embodiments, cryo-hibernation involves storing enucleated cells for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 1 month, at least about 1 month, or at least about 1 year. In some embodiments, cryo-hibernation involves contacting enucleated cells with a culture medium (such as a cell culture medium) or storing them in a culture medium. In some embodiments, the cell culture medium includes a xeno-free medium. In some embodiments, the cell culture medium contains about 5% to about 20% serum.

[0042] In some embodiments, enucleated cells, or compositions containing enucleated cells, may be cryopreserved (e.g., stored at freezing temperature) or cryopreserved (e.g., stored at a temperature between ambient temperature and freezing temperature). The duration of cryopreservation or cryopreservation may be approximately equal to or longer than 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, enucleated cells exhibit viability after cryopreservation or cryopreservation, and this viability is approximately equal to, or more similar to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of equivalent cells (e.g., parent cells, or enucleated cells described herein that have not been cryopreserved or cryopreserved) after the same period of cryopreservation or cryopreservation. In some embodiments, the post-cryohibernation viability exhibited by enucleated cells, measured 24 hours after cryopreservation, is greater than or equal to the viability of equivalent enucleated cells that have not been cryopreserved. In some embodiments, the post-cryohibernation viability exhibited by enucleated cells, measured 24 hours after cryopreservation, is greater than or equal to the viability of equivalent enucleated cells that have not been cryopreserved. Viability in this context may be measured by the trypan blue dye exclusion method described herein. In some embodiments, trypan blue dye exclusion is carried out by (a) centrifuging aliquots of nucleateless cells in a suspension to create a cell pellet; (b) resuspending the cell pellet in serum-free medium to produce a serum-free cell suspension; (c) mixing one part trypan blue dye with one part serum-free cell suspension; and (d) counting the nucleateless cells from (c) within 3-5 minutes, so that at least a portion of the nucleateless cells are not stained with trypan blue dye. This indicates viability. In some embodiments, viability is measured using annexin V cell surface staining. In some embodiments, viability is measured by the expression of exogenous polypeptides. For example, the viability of enucleated cells can be determined by the expression of exogenous antibodies or single-domain antibodies expressed by the enucleated cells.In some embodiments, viability is measured by the expression of any one of the cell surface markers described herein, examples of which include CD105, CD90, CD45, CXCR4, PSGL-1, or CCR2. In some embodiments, viability is measured by the cellular activity of enucleated cells. In some embodiments, viability is measured by the homing ability of enucleated cells, determined by chemosensing or chemokine homing activity described herein.

[0043] In some embodiments, enucleated cells, or compositions containing enucleated cells, may be lyophilized. In some embodiments, the enucleated cells exhibit viability after reconstitution from lyophilization, which is approximately equal to or greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of equivalent cells (e.g., parent cells, or enucleated cells described herein that have not been lyophilized). In some embodiments, the enucleated cells exhibit viability after rehydration from lyophilization, which is approximately equal to or greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of equivalent cells (e.g., parent cells, or enucleated cells described herein that have not been dehydrated).

[0044] In some embodiments, enucleated cells, or compositions containing enucleated cells, may be dehydrated. In some embodiments, the enucleated cells exhibit a viability after rehydration that is approximately equal to, or more similar to, that of equivalent cells (e.g., parent cells, or undehydrated enucleated cells described herein) at 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0045] In some embodiments, enucleated cells, or compositions containing enucleated cells, are stable at 4°C for approximately 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 compositions are stable at room temperature for approximately 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 compositions are stable at 37°C for approximately 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, enucleated cells, or compositions comprising enucleated cells, may continue to survive after being administered to a subject requiring it to treat a disease or condition described herein. In some embodiments, enucleated cells, or compositions comprising enucleated cells, may continue to survive for approximately equal to or longer than 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 after being administered to a subject.

[0046] In some embodiments, enucleated cells may be obtained from the parent cells of the subject requiring treatment with enucleated cells as described herein. In some embodiments, enucleated cells may be obtained from allogeneic parent cells of the subject requiring treatment with enucleated cells as described herein.

[0047] Enucleated cells may be smaller than their nucleated counterparts (e.g., nucleated parent cells), and for this reason, enucleated cells may be better able to migrate through vascular structures and small openings in tissue parenchyma. Furthermore, removing large, dense nuclei reduces major physical barriers, allowing cells to move freely through small openings in blood vessels and tissue parenchyma. Thus, enucleated cells have improved in vivo distribution within the body and migration to target tissues. In some embodiments, enucleated cells have a diameter of at least 1 μm. In some embodiments, the diameter of enucleated cells is greater than 1 μm. In some embodiments, the diameter of enucleated cells is 1-100 μm (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- The diameters are 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, and 15-20 μm. In some embodiments, the diameter of enucleated cells is 10-30 μm. In some embodiments, the diameter of the enucleated cells is 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 diameter of the enucleated cells is about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm or more. In some embodiments, the diameter of the enucleated cells is about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, or about 12 μm or more. In some embodiments, the enucleated cells have a diameter of about 8 μm. In some embodiments, it may be advantageous for some enucleated cells to be small enough to allow for better homing or delivery to the target site.For example, the enucleated cells described herein may be able to pass through narrow passages in lung tissue or lung structures (such as alveolar ducts or microcapillaries) that most cells (such as parent cells) cannot pass through.

[0048] In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated parent cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 50% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 60% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 70% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 80% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells in the enucleated cell fraction have a diameter that includes about 90% or less of the average diameter of the nucleated cells.

[0049] In some embodiments, enucleated cells have significant therapeutic value because they may continue to survive, not differentiate into other cell types, secrete bioactive molecules, physically migrate / homing within approximately 5 days or less, be extensively enucleated ex vivo to perform specific therapeutic functions, and fuse with the same or other cell types to transfer desired innate or enucleated products. Thus, enucleated cells have broad utility as cell vehicles for delivering therapeutically important biomolecules and disease-targeting cargo (such as genes, viruses, bacteria, mRNA, shRNA, siRNA, polypeptides (including antibody and antigen-binding fragments), plasmids, gene-editing mechanisms, or nanoparticles). This disclosure enables the creation of safe (e.g., no unwanted DNA is transferred to the target) and controllable cell-based carriers that may be genetically enucleated to deliver cargo to humans that combat specific diseases and promote health. In some embodiments, enucleated cells continue to survive, migrate, or homing for approximately 12, 24, 36, 48, 60, 72, 84, 96, 108 hours, 5, 6, 7, 8, 9 days, or longer, or more, after being administered to a target requiring administration.

[0050] In some embodiments, enucleated cells are manipulated to express at least one of the following: 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 single-stranded DNA, double-stranded DNA, oligonucleotides, plasmids, bacterial DNA molecules, DNA viruses, linear DNA, or a combination thereof. In some embodiments, the exogenous RNA molecule is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), RNA viruses, or a combination thereof. In some embodiments, the exogenous protein is a cytokine, a growth factor, a hormone, an antibody or its antigen-binding fragment, an enzyme, or a combination thereof. In some embodiments, the antibody is a single-domain antibody or its antigen-binding fragment. In some embodiments, the parental cell (e.g., a nucleated cell) is genetically enucleated before enucleation (e.g., pre-enucleation). In some embodiments, the parental cell is genetically enucleated after enucleation (e.g., post-enucleation).

[0051] Transmembrane part In some embodiments, enucleated cells comprising at least one transmembrane portion, or compositions comprising enucleated cells, are described herein. In some embodiments, the enucleated cells comprise an exogenous polypeptide. The exogenous polypeptide may be covalently fused to the transmembrane portion. In some embodiments, the exogenous polypeptide is complexed with the transmembrane portion. In some embodiments, the transmembrane portion comprises a full-length protein or a variant thereof or a fragment thereof. In some embodiments, the transmembrane portion is endogenous to the parent cell to be enucleated in order to obtain the enucleated cell. In some embodiments, the transmembrane portion may be an exogenous transmembrane portion to the parent cell or the enucleated cell. In some embodiments, the transmembrane portion is selected from transmembrane proteins (bitopic transmembrane proteins) comprising a single transmembrane α-helix. The transmembrane portion comprises a polytopic transmembrane α-helix 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.

[0052] 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 it into the cell membrane of the enucleated cell described herein. Non-limiting examples of modifications made to the exogenous polypeptide to add a transmembrane portion include adding glycosylphosphatidylinositol, farnesyl, palmitic acid, myristic acid, or a combination thereof to the exogenous polypeptide.

[0053] In some embodiments, the transmembrane portion is genetically modified to fuse with or complex with at least one exogenous therapeutic agent described herein. In some embodiments, the enucleated cell includes an immune evasion portion. In some embodiments, the immune evasion includes a "don't eat me" signaling peptide, e.g., CD47 (e.g., NCBI gene ID 961), programmed cell death ligand 1 (PD-L1, e.g., NCBI gene ID 29126), major histocompatibility complex, class I, E (HLA-E, e.g., NCBI gene ID 3133), major histocompatibility complex, class I, G (HLA-I, e.g., NCBI gene ID 3135), fragments thereof, or combinations thereof.

[0054] targeting part In some embodiments, enucleated cells containing a targeting moiety are described herein. The targeting moiety described herein is designed to guide the enucleated cells to target cells or a target environment (e.g., tissue) within a target after delivery to the target (e.g., systemic delivery). 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. In some embodiments, enucleated cells containing a targeting moiety localize to target cells or a target environment with a 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold increase compared to the localization of equivalent enucleated cells lacking the targeting moiety. In some embodiments, enucleated cells containing the targeting region localize to target cells or target environments with an increase of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to equivalent enucleated cells lacking the targeting region. In some embodiments, the target cells or target environment are in vivo. In some embodiments, the target cells or target environment are ex vivo.

[0055] In some embodiments, the targeting portion includes an exogenous antibody or exogenous antigen-binding fragment for targeting a biomarker described herein. In some embodiments, the targeting portion includes an exogenous antibody or exogenous antigen-binding fragment for targeting a chemokine receptor or chemokine ligand involved in chemokine signaling, or a portion thereof. In some embodiments, the exogenous antibody is an exogenous single-domain antibody or a fragment thereof.

[0056] In some embodiments, the targeted portion targets a biomarker, which is 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 pathological condition. In some embodiments, the biomarker is expressed by immune cells in response to the target cell or microenvironment associated with the disease or pathological condition. In some embodiments, the biomarker may be an epitope or an antigen. In some embodiments, the biomarker, including an epitope, may be conjugated by an antibody different from the antibody or its antigen-binding fragment (e.g., a therapeutic agent) that confers therapeutic properties.

[0057] In some embodiments, the targeting moiety targets biomarkers expressed or released by lung cells or lung cancer cells. Non-limiting examples of cancer cell biomarkers include carbonic anhydrase 9 (CA9, e.g., NCBI gene ID 768), carbonic anhydrase 12 (CA12, e.g., NCBI gene ID 771), cancer / testicular antigen 83 (CXorf61, e.g., NCBI gene ID 203413), desmoglein 3 (DSG3, e.g., NCBI gene ID 1830), FAT atypical cadherin 2 (FAT2, e.g., NCBI gene ID 2196), G protein-coupled receptor 87 (GPR87, e.g., NCBI gene ID 53836), KISS1 receptor (KISS1R, e.g., NCBI gene ID 84634), LY6 / PLAUR domain-containing 3 (LYPD3, e.g., NCBI gene ID 27076), and lysinus. Quality carrier family 7 members 11 (SLC7A11, e.g., NCBI gene ID 23657), TMPRSS4 (e.g., NCBI gene ID 56649), transmembrane serine protease 4 (TFPI, e.g., NCBI gene ID 7035), midkine (MDK, e.g., NCBI gene ID 4192), secretory phosphorylated protein 1 (OPN, e.g., NCBI gene ID 6696), matrix metallopeptidase 2 (MMP2, e.g., NCBI gene ID 4313), TIMP metallopeptidase inhibitor 1 (TIMP1, e.g., NCBI gene ID 7076), cell adhesion molecule 5 (CEA, e.g., NCBI gene ID 1048), cytokeratin 19 fragments (CYFRA 21-1, e.g., NCBI gene ID 3880), Serpin family B member 3 (SCC, e.g., NCBI gene ID 6317), Advanced Glycation End Product organism-specific receptor (AGER, e.g., NCBI gene ID 177), Adipogenesis regulator (C10orf116, e.g., NCBI gene ID 10974), Adusin 2 (ADD2, e.g., NCBI gene ID 119), Periaxin (PRX, e.g., NCBI gene ID 57716), Laminin subunit beta 3 (LAMB3, e.g., NCBI gene ID 3914), Synemin (SYNM, e.g., NCBI gene ID 23336), Spectrin alpha, Red blood cell 1 (SPTA1, e.g., NCBI gene ID 6708),Ankyrin 1 (ANK1, e.g., NCBI gene ID 286), hemoglobin subunit epsilon 1 (HBE1, e.g., NCBI gene ID 3046), hemoglobin subunit gamma 1 (HBG1, e.g., NCBI gene ID 3047), carbonic anhydrase 1 (CA1, e.g., NCBI gene ID 759), tenascin XB (TNXB, e.g., NCBI gene ID 7148), multimelin 2 (MMRN2, e.g., NCBI gene ID 7 9812), hemoglobin subunit alpha-1 (HBA1, e.g., NCBI gene ID 3039), caveolin-1 (CAV1, e.g., NCBI gene ID 857), hemoglobin subunit beta (HBB, e.g., NCBI gene ID 3043), collagen type VI alpha-6 chain (COL6A6, e.g., NCBI gene ID 131873), chromosome 1 open reading frame 198 (C1orf198, e.g., NCBI gene ID84886), Chloride intracellular channel 2 (CLIC2, e.g., NCBI gene ID1193), SdpC synthetic operon transcription regulator (ArsR family) (SDPR, e.g., NCBI gene ID8436), EH domain-containing 2 (EHD2, e.g., NCBI gene ID30846), Apolipoprotein A2 (APOA2, e.g., NCBI gene ID336), NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7, e.g., NCBI gene ID4713), Protein kinase C delta-binding protein (PRKCDBP, e.g., NCBI gene ID112464), Laminin subunit alpha 3 (LAMA3, e.g., NCBI gene ID), EvC ciliary complex subunit 2 (LBN, e.g., NCBI gene ID132884), Serpin family A member 3 (ACT, e.g., NCBI gene ID12), Insulin-like growth factor-binding protein 3 (3 IGFBP3 (e.g., NCBI gene ID 3486), prostaglandin D2 synthase (L-PGDS, e.g., NCBI gene ID 5730), retinoic acid receptor beta (HAP, e.g., NCBI gene ID 5915), hepatocyte growth factor (HGF, e.g., NCBI gene ID 3082), eukaryotic translation initiation factor 4 gamma-2 (AAG1 / 2, e.g., NCBI gene ID 1982),Clusterin (CLU, e.g., NCBI gene ID 1191), Streptococcal superantigen SSA (SSA, e.g., NCBI gene ID 6737), Tetanic (TTA, e.g., NCBI gene ID 100189453), Apolipoprotein A4 (APOA4, e.g., NCBI gene ID 337), Fibrinogen-like protein A (FIBA, e.g., NCBI gene ID 105209070), Serum amyloid A cluster (SAA, e.g., NCBI gene ID 6288), Ceruloplasmin (CP, e.g., NCBI gene ID 1356) ), haptoglobin (HP, e.g., NCBI gene ID 3240), transthyretin (TTR, e.g., NCBI gene ID 7276), keratin 2 (KRT2A, e.g., NCBI gene ID 3849), glutamate transporter (GLT1B, e.g., NCBI gene ID 6506), casein kinase 1 (CK1, e.g., NCBI gene ID 1452), AKT serine / threonine kinase 1 (AKT, e.g., NCBI gene ID 207), mannose-binding lectin 2 (MBL2, e.g., NCBI gene ID 4153), fibril Norgen alpha chain (FGA, e.g., NCBI gene ID 2243), gelsolin (GSN, e.g., NCBI gene ID 2934), haptoglobin (HP, e.g., NCBI gene ID 3240), phycolin 3 (FCN3, e.g., NCBI gene ID 8547), carnosine dipeptidase 1 (CNDP1, e.g., NCBI gene ID 84735), calcitonin-related polypeptide alpha (CALCA, e.g., NCBI gene ID 796), carbamoyl phosphate synthase 1 (CPS1, e.g., NCBI gene ID 1373), clo Mogranin B (CHGB, e.g., NCBI gene ID 1114), Involucrin (IVL, e.g., NCBI gene ID 3713), Forward Gradient 2 (AGR2, e.g., NCBI gene ID 10551), Nuclear Autoantigen Sperm Protein (NASP, e.g., NCBI gene ID 4678), Phosphofructokinase, Platelet (PFKP, e.g., NCBI gene ID 5214), Thrombospondin 2 (THBS2, e.g., NCBI gene ID 7058), Thioredoxin Domain-containing 17 (TXNDC17, e.g., NCBI gene ID 84817),Proprotein convertase subtilisin / kexin type 1 (PCSK1, e.g., NCBI gene ID 5122), cellular retinoic acid-binding protein 2 (CRABP2, e.g., NCBI gene ID 1382), acyl-CoA-binding domain-containing protein 3 (ACBD3, e.g., NCBI gene ID 64746), desmoglein 2 (DSG2, e.g., NCBI gene ID 1829), LPS-responsive beige-like anchor protein (LRBA, e.g., NCBI gene ID 987), serine / threonine kinase receptor-related protein (STRAP, e.g., NCBI gene ID 5122), Gene ID 11171), VGF nerve growth factor-inducible (VGF, e.g., NCBI gene ID 7425), NOP2 nucleolar protein (NOP2, e.g., NCBI gene ID 4839), lipocalin 2 (LCN2, e.g., NCBI gene ID 3934), creatine kinase, mitochondrial 1B (CKMT1B, e.g., NCBI gene ID 1159), aldo-keto reductase family 1 member B10 (AKR1B10, e.g., NCBI gene ID 57016), carboxypeptidase D (CPD, e.g., NCBI gene ID 1362), p Roteasome activator subunit 3 (PSME3, e.g., NCBI gene ID 10197), virin 1 (VIL1, e.g., NCBI gene ID 7429), serpin family B member 5 (SERPINB5, e.g., NCBI gene ID 5268), ribosomal protein L5 (RPL5, e.g., NCBI gene ID 6125), placophyllin 1 (PKP1, e.g., NCBI gene ID 5317), ribosomal protein L10 (RPL10, e.g., NCBI gene ID 6134), aldo-keto reductase family 1 member B10 (AKR1B10, e.g., NCBI gene ID 57016), Aldo-keto reductase family member 1 C1 (AKR1C1, e.g., NCBI gene ID 1645), Proliferating cell nuclear antigen (PCNA, e.g., NCBI gene ID 5111), Ribosomal protein S2 (RPS2, e.g., NCBI gene ID 6187), Aldo-keto reductase family member 1 C3 (AKR1C3, e.g., NCBI gene ID 8644), Acyl-CoA binding domain-containing 3 (ACBD3, e.g., NCBI gene ID 64746), Bicinin-like 1 (VSNL1,For example, NCBI gene ID 7447), adenosyl homocysteine ​​(AHCY, e.g., NCBI gene ID 191), IMMP10, activated kinase 2 (PAK2, e.g., NCBI gene ID 5062), involucrin (IVL, e.g., NCBI gene ID 3713), isoleucine tRNA synthetase (IARS, e.g., NCBI gene ID 3376), proteasome 26S subunit ubiquitin receptor, non-ATPase 2 (PSMD2, e.g., NCBI gene ID 5708), guanylate-binding protein 5 (GBP5, e.g., NCBI gene ID 115362), minichromosome maintenance complex component 6 (MCM6, e.g., NCBI gene ID 4175), N-myc downstream regulation 1 (NDRG1, e.g., NCBI gene ID 10397), NOP58 ribonucleoprotein (NOP58, e.g., NCBI gene ID 51602), S100 calcium binding Protein A2 (S100A2, e.g., NCBI gene ID 6273), Neuregulin 1 (NRG1, e.g., NCBI gene ID 3084), Neuregulin 2 (NRG2, e.g., NCBI gene ID 9542), Carnosine dipeptidase 1 (CNDP1, e.g., NCBI gene ID 84735), Ubiquitin cross-reactive protein (UCRP, e.g., NCBI gene ID 9636), Kramer (CER, e.g., NCBI gene ID 9636) Gene ID 8110), plasminogen activator (UPA, e.g., NCBI gene ID 5328), matrix metallopeptidase 14 (MT1-MMP, e.g., NCBI gene ID 4323), stratifin (SFN, e.g., NCBI gene ID 2810), transferrin (TF, e.g., NCBI gene ID 7018), albumin (ALB, e.g., NCBI gene ID 213), S100 calcium-binding protein Protein A9 (S100A9, e.g., NCBI gene ID 6280), Stasmin 1 (STMN, e.g., NCBI gene ID 3925), Enolase (ENO), Plasminogen Activator (PLAU, e.g., NCBI gene ID 5328), Insulin-like Growth Factor Binding Protein 7 (IGFBP7, e.g., NCBI gene ID 3490), Matrix Metallopeptidase 14 (MMP14, e.g., NCBI gene ID 4323),It contains thrombospongin 1 (THBS1, e.g., NCBI gene ID 7057) or thrombospongin 2 (THBS2, e.g., NCBI gene ID 7058).

[0058] In some embodiments, the targeting portion targets biomarkers expressed or released by metastatic cancer cells. For example, cancer cells may originate in one tissue and then metastasize to another location. In some embodiments, metastatic cancer cells express a non-limiting example of cancer biomarkers described herein. In some embodiments, metastatic cancer cells express cancer biomarkers, including melanoma-associated antigen (MAGE family member A3 (MAGE-A3, e.g., NCBI gene ID 4102)), membrane-associated glycoprotein (MUC-1, e.g., NCBI gene ID 4582), glycoprotein epithelial cell adhesion molecule (EpCAM, e.g., NCBI gene ID 4072), KRAS proto-oncogene (KRAS, e.g., NCBI gene ID 3845), anaplastic lymphoma kinase (ALK, e.g., NCBI gene ID 238), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, e.g., NCBI gene ID 1493), programmed cell death protein 1 (PD-1, e.g., NCBI gene ID 513). 3) Epidermal growth factor (EGF, e.g., NCBI gene ID 1950), serine protease ester (EA, e.g., NCBI gene ID 5328), telomerase reverse transcriptase (TERT, e.g., NCBI gene ID 7015), PRAME nuclear receptor transcription regulator (PRAME, e.g., NCBI gene ID 23532), receptor tyrosine protein kinase erbB-2 (HER, e.g., NCBI gene ID 2064), or vascular endothelial growth factor (VEGF, e.g., NCBI gene ID 7422), carcinoembryonic antigen (CEA, e.g., NCBI gene ID 1048), MAGE family member A1 (MAGE-A1, e.g., NCBI gene ID 4100), MAGE family member A1 This includes MAGE-A4 (e.g., NCBI gene ID 4103), Survivin, six-transmembrane epithelial antigen of the prostate gland 1 (STEAP1, e.g., NCBI gene ID 26872), SRY (sex-determining region Y) box 2 (SOX2, e.g., NCBI gene ID 6657), or cancer / testis / antigen 1 (CTAG1B, e.g., NCBI gene ID 1485).

[0059] In some embodiments, the targeting portion targets a biomarker expressed or released by endothelial cells. In some embodiments, the endothelial cells are vascular cells. In some embodiments, the endothelial cells are lymphatic cells. In some embodiments, the biomarker is expressed or released by vascular cells. In some embodiments, the biomarker is expressed or released by lymphatic cells. Non-limiting examples of endothelial cell biomarkers include angiotensin I-converting enzyme (ACE / CD143, e.g., NCBI gene ID 1636), CD93 molecule (C1qR1 / CD93, e.g., NCBI gene ID 22918), cadherin 5 (VE-cadherin, e.g., NCBI gene ID 1003), D6 protein (CC chemokine receptor D6, e.g., NCBI gene ID 1238), platelet and endothelial cell adhesion molecule 1 (CD31 / PECAM-1, e.g., NCBI gene ID 5175), CD34 molecule (CD34, e.g., NCBI gene ID 947), CD36 molecule (CD36 / SR-B3, e.g., NCBI gene ID 948), CD151 molecule (CD151, e.g., NCBI gene ID 977), CD160 molecule (CD160, e.g., NCBI gene ID 11126),

[0060] CD300 molecule-like family member g (CD300g / nepmucin, e.g., NCBI gene ID 146894), CDC-like kinase 1 (CL-K1 / COLEC11, e.g., NCBI gene ID 78989), cleavage factor polyribonucleotide kinase subunit 1 (CL-P1 / COLEC12, e.g., NCBI gene ID 81035), coagulation factor III / tissue factor (e.g., NCBI gene ID 2152), C-type lectin domain family 4 member M (DC-SIGNR / CD299, e.g., NCBI gene ID 10332), discoidin, CUB and LCCL domain-containing 2 (DCBLD2 / ESDN, e.g., NCBI gene ID 131566), endothelial cell surface expression chemotaxis and apoptosis regulator (ECSCR, e.g., NCBI gene ID 641700), basidine (Ok blood type) (EMMPRIN / CD147, e.g., (NCBI gene ID 682), endoglin / CD105 (e.g., NCBI gene ID 5077), endomucin (e.g., NCBI gene ID 2022), endosialin / CD248 (e.g., NCBI gene ID 57124), protein C receptor (EPCR, e.g., NCBI gene ID 10544), erythropoietin R (e.g., NCBI gene ID 2056), endothelial cell adhesion molecule (ESAM, e.g., NCBI gene ID 90952), fatty acid binding protein 5 (FABP5 / E-FABP, e.g., NCBI gene ID 2171), fatty acid binding protein 6 (FABP6, e.g., NCBI gene ID 2172), intercellular adhesion molecule 1 (ICAM-1 / CD54, e.g., NCBI gene ID 3383), intercellular adhesion molecule 2 (ICAM-2 / CD102, e.g., NCBI gene ID 3384), interleukin 1 receptor (IL-1 RI (e.g., NCBI gene ID 3553), interleukin-13 receptor alpha-1 (IL-13 R alpha-1, e.g., NCBI gene ID 3597), integrin alpha-4 / CD49d (e.g., NCBI gene ID 3676), integrin α4β-1 (e.g., NCBI gene ID 3688), integrin α4β-7 / LPAM-1 (e.g., NCBI gene ID 3676), integrin β2 / CD18 (e.g., NCBI gene ID 3689), KLF transcription factor 4 (KLF4, e.g., NCBI gene ID 9314), lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1, e.g., NCBI gene ID 10894), melanoma cell adhesion molecule (MCAM / CD146, e.g., NCBI gene ID 4162), nectin cell adhesion molecule 2 (nectin-2 / CD112, e.g., NCBI gene ID 5819), PD-ECGF / thymidine phosphorylase (e.g., NCBI gene ID 1890), podocalyxin (e.g., NCBI Gene ID 5420), podoplanin (e.g., NCBI gene ID 10630), sphingosine-1-phosphate receptor 1 (S1P1 / EDG-1, e.g., NCBI gene ID 1901), sphingosine-1-phosphate receptor 2 (S1P2 / EDG-5, e.g., NCBI gene ID 9294), sphingosine-1-phosphate receptor 3 (S1P3 / EDG-3, e.g., NCBI gene ID 1903), sphingosine-1-phosphate receptor 4 (S1P4 / EDG-6, e.g., NCBI gene ID 8698), sphingosine-1-phosphate Receptor 5 (S1P5 / EDG-8, e.g., NCBI gene ID 53637), E-selectin / CD62E (e.g., NCBI gene ID 6401), P-selectin / CD62P (e.g., NCBI gene ID 6403), Molasses-like slow (SLAM / CD150, e.g., NCBI gene ID 6504), Stabilin-1 (e.g., NCBI gene ID 23166), Stabilin-2 (e.g., NCBI gene ID 55576), Plexin domain-containing 1 (TEM7 / PLXDC1, e.g., NCBI gene ID 57125), A NTXR cell adhesion molecule 1 (TEM8 / ANTXR1, e.g., NCBI gene ID 84168), thrombomodulin / BDCA-3 (e.g., NCBI gene ID thrombomodulin), thrombospondin type 1 domain-containing 1 (THSD1, e.g., NCBI gene ID 55901), thrombospondin type 1 domain-containing 7A (THSD7A, e.g., NCBI gene ID 221981), TEK receptor tyrosine kinase (Tie-2, e.g., NCBI gene ID 7010), TNF receptor superfamily member 1A (TNFRI / TNFRSF1A (e.g., NCBI gene ID 7132), TNF receptor superfamily member 1B (TNF RII / TNFRSF1B, e.g., NCBI gene ID 7133), basidine (Ok blood type) (TRA-1-85 / CD147, e.g., NCBI gene ID 682), TNF receptor superfamily member 10b (TRAIL R2 / TNFRSF10B, e.g., NCBI gene ID 8795), TNF receptor superfamily member 10a (TRAIL R1 / TNFRSF10A, e.g., NCBI gene ID 8797), vascular cell adhesion molecule 1 (VCAM-1 / CD106, e.g., NCBI gene ID 7412), EGF-like domain multiple 7 (VE-Statin, e.g., NCBI gene ID: 51162), fms-related receptor tyrosine kinase 1 (VEGFR1 / Flt-1, e.g., It includes, NCBI gene ID 2321), kinase insertion domain receptor (VEGFR2 / KDR / Flk-1, e.g., NCBI gene ID 3791), fms-related receptor tyrosine kinase 4 (VEGFR3 / Flt-4, e.g., NCBI gene ID 2324), angiogenic factor with G patch and FHA domain 1 (VG5Q, e.g., NCBI gene ID 55109), or von Willebrand factor domain 2 (vWF-A2, e.g., NCBI gene ID 7450).

[0061] In some embodiments, the targeting moiety includes a chemokine receptor or chemokine ligand involved in chemokine signaling, or a moiety thereof (e.g., SDF-1α / CXCR4, CCL2 / CCR2, etc.), or an adhesion molecule (e.g., PSGL-1, etc.). As shown herein, enucleated cells may be enucleated to express functional CXCR4, CCR2, or even glycosylated PSGL-1, which may significantly enhance the specific targeting of enucleated cells. In some embodiments, the targeting moiety (e.g., CXCR4, CCR2, or PSGL-1) may be expressed on the surface of enucleated cells. Non-limiting examples of cell surface proteins that can be expressed on the cell surface of enucleated cells as the targeting moiety include chemokines, such as CXCR4, CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. In some embodiments, enucleated cells can be enucleated to secrete targeted moieties or tethered to the extracellular matrix (e.g., SDF1α or CCL2). Non-limiting examples of targeted moieties that may be secreted by enucleated cells include SDF1α, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, CXCL10, CCL11, and CXCL12. In some embodiments, enucleated cells include cell matrix receptors, and intercellular adhesion molecules include integrins, cadherins, glycoproteins, and heparin sulfate proteoglycans.

[0062] In some embodiments, enucleated cells may further include surface markers that help evade the target immune system (e.g., by manipulation or from cells from which enucleated cells were obtained). For example, in some embodiments, enucleated cells may include CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof. Without being bound by any particular theory, CD47, PD-L1, HLA-E, HLA-G, fragments thereof, or combinations thereof are thought to help prevent enucleated cells from being phagocytosed by macrophages. Non-limiting examples of cell matrix receptors and cell adhesion molecules include integrins, cadherins, glycoproteins, or heparin sulfate proteoglycans. In some embodiments, the cell matrix receptor or cell adhesion molecule includes 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 can induce an immune response. For example, the therapeutic molecule may be either 4-1BB or one of the cytokines described herein for inducing an immune response.

[0063] Therapeutic drugs In some embodiments, the enucleated cells of the Disclosure comprise at least one therapeutic agent. In some embodiments, the enucleated cells of the Disclosure comprise at least two, three, four, five, six, seven, eight, nine, ten, or more therapeutic agents. In some embodiments, the therapeutic agent comprises an activator. In some embodiments, the therapeutic agent is exogenous to the enucleated cell or its parent cell. The activator comprises at least one of the following: DNA molecules, RNA molecules, proteins (e.g., enzymes, antibodies, antigens, toxins, cytokines, protein hormones, growth factors, cell surface receptors, or vaccines), peptides (e.g., peptide hormones or antigens), small molecules (e.g., steroids, polyketides, alkaloids, toxins, antibiotics, antivirals, colchicine, taxol, mitomycin, or emtansine), gene editing factors, nanoparticles, or other activators (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g., oncolytic viruses), exosomes, lipids, or ions). In some embodiments, the activator is a cytokine, growth factor, hormone, enzyme, small molecule, compound, or any combination thereof. In some embodiments, enucleated cells are manipulated to produce the therapeutic agent (e.g., expression, and possibly release or secretion). In some embodiments, the parent cells may be manipulated to produce the therapeutic agent before enucleation for the production of enucleated cells. Non-limiting examples of oncolytic viruses include tarimodine laherparepbec, ONYX-015, GL-ONC1, CV706, Voyager-V1, and HSV-1716. Some wild-type viruses also exhibit oncolytic behavior, examples of which include vaccinia virus, vesicular stomatitis virus, poliovirus, reovirus, seneca virus, ECHO-7, and Semryki forest virus.

[0064] The therapeutic agent may be or may contain the targeted moiety described herein. Non-limiting examples of targeted moieties that may be produced by or contained in enucleated cells include chemokine receptors, adhesion molecules, and antigens. In some embodiments, the therapeutic agent may be or may contain the transmembrane moiety described herein.

[0065] In some embodiments, the therapeutic agent is recombinantly expressed by enucleated cells or their parent cells. In some embodiments, the parent cells from which the enucleated cells are derived or obtained are manipulated to produce or express the therapeutic agent. In some embodiments, the expression of the therapeutic agent is stable (e.g., persistent). In some embodiments, the expression of the therapeutic agent by the parent cells is transient (e.g., non-persistent). In some embodiments, the parent cells are enucleated before manipulating the enucleated cells to recombinantly express the therapeutic agent.

[0066] In some embodiments, the therapeutic agent is not spontaneously expressed in the cells from which the enucleated cells originate or are obtained (e.g., without manipulation) (e.g., the therapeutic agent is exogenous to the parent cells). In some embodiments, the therapeutic agent is not spontaneously expressed in the target (e.g., the therapeutic agent is exogenous to the target). In some embodiments, the therapeutic agent is not spontaneously expressed in the target site of treatment (e.g., a tumor, or a specific tissue, such as the brain, intestines, lungs, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is exogenous to the target site). In some embodiments, levels of the therapeutic agent are not naturally present in the enucleated cells of the parent cells.

[0067] In some embodiments, the therapeutic agent is spontaneously expressed (e.g., without manipulation) in the cells from which enucleated cells originate or are obtained (e.g., the therapeutic agent is endogenous to enucleated cells). In some embodiments, the therapeutic agent is spontaneously expressed in the target (e.g., the therapeutic agent is endogenous to the target). In some embodiments, the therapeutic agent is spontaneously expressed in the target site of treatment (e.g., a tumor, or a specific tissue, such as the brain, intestines, lungs, heart, liver, spleen, pancreas, muscle, eye, etc.) (e.g., the therapeutic agent is endogenous to the target site of treatment).

[0068] In some embodiments, the therapeutic agent is derived from synthetic cells and loaded into enucleated cells. For example, the therapeutic agent may be taken up into cells. Alternatively, the therapeutic agent may be synthesized by cells and then delivered to target cells.

[0069] In some embodiments, compared to the original cells from which enucleated cells are derived or obtained, the therapeutic agent includes modified, cleaved, or non-mutated versions and / or copies of DNA molecules, RNA molecules, proteins, peptides, small molecule activators, and / or gene editing factors. For example, the therapeutic agent can modify mutated p53 or EGFR in target cells as part of the treatment of lung cancer.

[0070] In some embodiments, the therapeutic agent comprises any combination of at least two (e.g., at least two, three, four, five, or more) different therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule activators, or therapeutic gene editing factors. For example, in some embodiments, the therapeutic agent comprises a therapeutic DNA molecule and a small molecule activator. For example, in some embodiments, the therapeutic agent comprises two different small molecule activators. For example, in some embodiments, the therapeutic agent comprises a chemokine receptor (e.g., for targeting) and a small molecule activator.

[0071] In some embodiments, the therapeutic agent comprises RNA molecules, including messenger RNA (mRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA, long non-coding RNA (lncRNA), or RNA viruses. In some embodiments, the therapeutic agent comprises DNA molecules, such as single-stranded DNA, double-stranded DNA, oligonucleotides, plasmids, bacterial DNA molecules, or DNA viruses. In some embodiments, the therapeutic agent comprises a protein or a portion thereof. In some embodiments, the protein is a cytokine, growth factor, hormone, antibody or its antigen-binding fragment, small peptide-based drug, or enzyme. In some embodiments, enucleated cells transiently express the therapeutic agent. In some embodiments, the expression of the therapeutic agent is inducible. In some embodiments, the expression of the therapeutic agent is persistent.

[0072] In some embodiments, the therapeutic agent comprises an exogenous factor. In some embodiments, the exogenous factor is an exogenous polypeptide. In some embodiments, the exogenous polypeptide is encoded by an exogenous polynucleotide delivered to a parent cell or an enucleated cell. In some embodiments, the exogenous polypeptide is synthesized or released by at least one intracellular organelle of the enucleated cell. In some embodiments, the exogenous polypeptide is released by the enucleated cell. In some embodiments, the exogenous polypeptide is expressed on the cell surface or on the enucleated cell. In some embodiments, the enucleated cell delivers the exogenous polypeptide to a target cell. In some embodiments, the target cell is a cancer cell expressing any cancer biomarker described herein. In some embodiments, the target cell is a pneumocyte. In some embodiments, the target cell is an epithelial cell. In some embodiments, the epithelial cell is found on, within, or in lung tissue. In some embodiments, the target cell is an endothelial cell expressing an endothelial biomarker described herein. In some embodiments, the endothelial cell is an angiocyte. In some embodiments, the endothelial cell is a lymphatic cell.

[0073] In some embodiments, the exogenous polypeptide comprises one cytokine from among the cytokines described herein. In some embodiments, the exogenous polypeptide comprises a soluble cytokine. For example, the exogenous polypeptide may contain the extracellular domain or fragment of a cytokine. In some embodiments, the exogenous polypeptide comprises solubility determined by a turbidimetric solubility assay or a thermodynamic solubility assay by dissolving the exogenous polypeptide in a solvent, examples of which solvents include organic solvents (including dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, etc.) or inorganic solvents (including water or phosphate-buffered saline (PBS)). In some embodiments, the exogenous polypeptide contains 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.

[0074] In some embodiments, the exogenous polypeptide comprises a member of the tumor necrosis factor (TNF) superfamily or a catalytically active fragment thereof. Non-limiting examples of TNF superfamily members include lymphotoxin α (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 (4-1 This includes BBL, TNF-related apoptosis-inducing ligands (CD253 or APO-2L), nuclear factor-κB receptor activator ligands (CD254, OPGL, TRANCE or ODF), TNF-related weak apoptosis-inducing factors (APO-3L or DR3L), proliferation-inducing ligands (CD256, tumor marker genes (TALL-2 or TRDL1), B cell activators (CD257, BlyS, TALL-1 or TNFSF20), LIGHT (CD258 or HVEML), vascular endothelial growth inhibitors (TL1 or TL-1A), TNF superfamily member 18 (GITRL, AITRL or TL-6), or ectodysprasin A (ED1-A1 or ED1-A2).

[0075] In some embodiments, the therapeutic agent comprises any one of the immune checkpoint proteins described herein, or an immune checkpoint inhibitor for inhibiting any one of the immune checkpoint proteins described herein. Non-limiting examples of immune checkpoint proteins include PD-1, PD-L1, CTLA-4, VISTA, B7-H3 (also known as CD276), A2AR, CD27, LAG3, TIM-3, Ig and T cell immune receptors (TIGIT) containing the ITIM domain, 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 fragments thereof.

[0076] In some embodiments, the enucleated cells include additional therapeutic agents, examples of which are disclosed herein. In some embodiments, the composition containing the enucleated cells is formulated for administration to a subject disclosed herein together with the additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered to the subject sequentially, simultaneously, substantially sequentially, or substantially simultaneously.

[0077] Pharmaceutical composition In some embodiments, pharmaceutical compositions are disclosed herein, comprising the compositions disclosed herein and pharmaceutically acceptable carriers, excipients, diluents, or inhalants. In some embodiments, the compositions disclosed herein comprise a population of cells disclosed herein. In some embodiments, the compositions disclosed herein comprise one or more activators or therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein comprise at least one additional activator. In some embodiments, the pharmaceutical compositions disclosed herein comprise two or more activators or therapeutic agents.

[0078] In some embodiments, the pharmaceutical composition is in unit dose form. In some embodiments, the composition comprises two or more activators or two or more therapeutic agents disclosed herein. In some embodiments, two or more activators are contained in a single dosing unit, for example, when enucleated cells contain two or more therapeutic agents. In embodiments, two or more activators are contained in separate dosing units, for example, when enucleated cells are administered separately from additional therapeutic agents or adjuvants. In some embodiments, the pharmaceutical composition described herein comprises at least one additional activator other than the enucleated cells described herein. In some embodiments, the at least one additional activator is a cytokine, growth factor, hormone, enzyme, small molecule, compound, or any combination thereof. In some embodiments, the at least one additional activator is a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitor, antihormone agent, anti-angiogenic agent, cardioprotective agent and / or checkpoint inhibitor.Non-restrictive checkpoint inhibitors include IMP321 / eftilagimod alfa (Imtech), relatrimab BMS-986016, ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), semiplimab (Ributayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), LAG525, MK-4280, irinotecan, oxaliplatin, REGN3767, TSR-033, and BI. 754111, Sym022, FS118 (bispecific anti-LAG3 / PD-L1 antagonist mAb), MGD013 (bispecific anti-LAG3 / PD-1 antagonist mAb), TSR-022, niraparib, bevacizumab, MBG453, decitabine, spartalizumab, Sym023, INCAGN2390, LY3321367, ramucirumab, abemaciclib, merestinib, BMS-986258, SHR-1702, camrelizumab, MK-7684, etigirimab / OMP-313 M32, Tiragolumab / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA-170d, Enobrituzumab / MGA271, MGD009, I-8H9 / Ombrutamab, Trastuzumab, MGD013 (Anti-PD-1, Anti-LAG-3 Dual Checkpoint Inhibitor), BGB-A1217, CM-24 (MK-6018), BMS This includes 986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, utomirimab (PF-05082566), urerumab (BMS-663513)ES101, BMS-986156, TRX-518, AMG 228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or valrirumab (CDX-1127). Such compounds or drugs may be present in combination in amounts effective for the intended purpose.Further non-limiting examples of additional therapeutic agents include CPI-006 (to inhibit CD73 and enable T cell and APC activation); monalizumab (to inhibit NKG2A); COM701 (to inhibit PVRIG / PVRL2 and enable T cell activation); CM24 (to inhibit CEACAM1 and enable T cell and NK cell activation); NEO-201 (to inhibit CEACAM5 and CEACAM6, enabling T cell activation while inhibiting tumor cell growth); and defactinib (FAK (To inhibit and prevent tumor growth); PF-04136309 (To inhibit CCR-2 and CCL-2, enabling T cell recruitment and activation); MSC-1 (To inhibit LIF, enabling T cell and APC activation while preventing cancer growth); Hu5F9-G4 (5F9), ALX148, TTI-662 and RRx-001 (To inhibit CD47 or SIRPα, enabling T cell and APC activation); Lacunotuzumab (MCS-110), LY3022855, SNDX-6 352, 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 hindering tumor growth); pepinemab (VX15 / 2503) (to inhibit SEMA4D and hinder tumor growth) This includes: reducing the immunosuppressive tumor microenvironment; trevananib (to inhibit angiopoietin-2 and enable APC activation while hindering cancer growth); FP-1305 (to inhibit CLEVER-1 and enable APC activation); enapotamab vedotin (EnaV) (to inhibit Axl and enable APC activation while hindering cancer growth); or bavituximab (to inhibit phosphatidylserine and enable T cell and APC activation while hindering cancer growth).

[0079] The composition may contain at least an exogenous therapeutic agent as an active ingredient in the form of a free acid or free base, or a pharmaceutically acceptable salt. Furthermore, the methods and compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds having the same type of activity. In some embodiments, the therapeutic agent exists in a non-solvated form or in a solvated form with a pharmaceutically acceptable solvent (such as water or ethanol). The solvated form of the therapeutic agent is also considered to be disclosed herein.

[0080] In certain embodiments, the compositions provided herein include one or more preservatives for inhibiting microbial activity. Suitable preservatives include mercury-containing substances (such as melfen and thimerosal), stabilized chlorine dioxide, and quaternary ammonium compounds (such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride).

[0081] In some embodiments, the compositions described herein benefit from antioxidants, metal chelating agents, thiol-containing compounds, and other generally stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) glycerol at approximately 0.5% to 2% w / v, (b) methionine at approximately 0.1% to 1% w / v, (c) monothioglycerol at approximately 0.1% to 2% w / v, (d) EDTA at approximately 1 mM to 10 mM, (e) ascorbic acid at approximately 0.01% to 2% w / v, (f) polysorbate 80 at approximately 0.003% to 0.02% w / v, (g) polysorbate 20 at approximately 0.001% to 0.05% w / v, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparin analogs, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0082] Preparation for administration The compositions described herein may be formulated into any suitable dosage form, including, but are not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled-release formulations, rapid-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated tablets, capsules, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations, and mixed formulations of immediate-release and controlled-release formulations. In some embodiments, the compositions (e.g., the pharmaceutical compositions disclosed herein) may be formulated for administration to a target by intrathecal, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or any combination thereof. In some embodiments, the composition (e.g., the pharmaceutical composition disclosed herein) is formulated for intravenous administration. In one embodiment, the therapeutic agent (e.g., the therapeutic agent) discussed herein is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one embodiment, a formulation suitable for intramuscular, subcutaneous, or intravenous injection comprises a physiologically acceptable sterile aqueous or non-aqueous solution, a dispersion, a suspension or emulsion, and a sterile powder for rehydration into a sterile injectable solution or dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Adequate fluidity may be maintained, for example, by the use of a coating such as lecithin, maintaining the required particle size in the case of a dispersion, or by the use of a surfactant. In some embodiments, formulations suitable for subcutaneous injection also contain additives, such as preservatives, humectants, emulsifiers, and distributors. Microbial growth may be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. In some cases, it is desirable to include isotonic agents such as sugars and sodium chloride.Absorption may be prolonged by using agents that delay the absorption of injectable drug forms (such as aluminum monostearate and gelatin).

[0083] For intravenous injection, infusion, or infusion, the compositions described herein are formulated in aqueous solutions, preferably physiologically compatible buffers (such as Hanks' solution, Ringer's solution, or saline buffer). For transmucosal administration, appropriate penetrating agents are used in the formulation to penetrate the barrier. Such penetrating agents are generally known in the art. For other parenteral injections, suitable formulations preferably comprise aqueous or non-aqueous solutions containing physiologically compatible buffers or excipients. Such excipients are known.

[0084] Parenteral injection may include bolus injection or continuous infusion. The injectable composition may be supplied in unit dosage forms, such as ampoules or multi-dose containers, with added preservatives. The compositions described herein may be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation agents (such as suspending agents, stabilizers and / or dispersants). In one embodiment, the active ingredient is in powder form for preparation with a suitable vehicle (such as sterile pyrogen-free water) before use.

[0085] For inhalation administration, the therapeutic agent is formulated for use as an aerosol, mist, or powder. The pharmaceutical compositions described herein are conveniently delivered in the form of aerosol spray dispensing from a pressurized pack or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In the case of pressurized aerosols, the dosage unit may be determined by providing a valve for dispensing a measured amount. Capsules and cartridges (e.g., gelatin, to be an example) for use in inhalers or blowers may be formulated containing a powder mixture of the therapeutic agent described herein and a suitable powder base (e.g., lactose or starch). Formulations containing the compositions are prepared as a solution in saline by employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizers or dispersants known in the art. Preferably, these compositions and formulations are prepared using suitable non-toxic and pharmaceutically acceptable ingredients. The selection of a suitable carrier depends on the exact properties of the desired nasal dosage form (e.g., solution, suspension, ointment, or gel). Generally, nasal dosage forms contain a large amount of water in addition to the active ingredient. Other components (such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffers, as well as other stabilizers and solubilizers) may be present in trace amounts, at their discretion. Preferably, the nasal dosage form needs to be isotonic with nasal secretions.

[0086] Oral pharmaceutical preparations are obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally grinding the resulting mixture, adding a suitable adjuvant if desired, and then processing the granular mixture to obtain tablets or sugar-coated tablet cores. Suitable excipients include, for example, fillers (such as sugars, including lactose, sucrose, mannitol, or sorbitol); cellulose preparations (e.g., corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.); or other materials (such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate). Optionally, disintegrants may be added, examples of which include cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts (such as sodium alginate). In some embodiments, dyes or pigments are added to the tablet or sugar-coated tablet coating to identify or characterize different combinations of active therapeutic agent doses.

[0087] In some embodiments, the composition of the exogenous therapeutic agent is in the form of a capsule, which includes a push-in capsule made of gelatin, and a soft, sealed capsule made of gelatin and a plasticizer (such as glycerol or sorbitol). The push-in capsule contains the active ingredient mixed 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 the soft capsule, the active therapeutic agent is dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, a stabilizer is added. For example, the capsule may be prepared by placing a bulk blend of the therapeutic formulation inside the capsule. In some embodiments, the formulation (non-aqueous suspension and solution) 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 dispensing capsule, which is either swallowed whole or the capsule is opened and the contents are dispensed onto food before consumption.

[0088] Compositions for oral administration are in a state of dosage suitable for such administration. In one embodiment, a solid oral dosage form is prepared by mixing the composition with one or more of the following: antioxidants, flavoring agents, and carrier materials (such as binders, suspensions, 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 suspension tablets, fast-dissolving tablets, chewable disintegrating tablets, rapid disintegrating tablets, effervescent tablets, or caplets), pills, powders, capsules, solid dispersions, solid solutions, biodegradable dosage forms, controlled-release formulations, pulse-release formulations, multi-particle dosage forms, beads, pellets, and granules. In other embodiments, the composition is in the form of a powder. Compressed tablets are solid dosage forms prepared by compressing a bulk blend of the above formulations. In various embodiments, the tablets contain one or more flavoring agents. In other embodiments, the tablets contain a film surrounding the finally compressed tablet. In some embodiments, the film coating may provide delayed release of the therapeutic agent from the formulation. In other embodiments, the film coating assists patient compliance. The film coating may range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms (e.g., tablets, effervescent tablets, and capsules) are prepared by mixing particles of the therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into unit dosage forms (tablets, pills, and capsules, etc.) with equivalent effects. In some embodiments, each unit dose includes the film coating.

[0089] In another embodiment, the dosage form includes microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulated material. Non-limiting examples of materials include pH adjusters, erosion accelerators, defoamers, antioxidants, flavoring agents, and carrier materials (such as binders, suspenders, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents).

[0090] The dosage form of an orally administered liquid formulation is optionally an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form optionally contains additives, examples of which include (a) disintegrants, (b) dispersants, (c) wetting agents, (d) at least one preservative, (e) viscosity enhancers, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion further contains a crystal formation inhibitor.

[0091] In some embodiments, the compositions described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of immiscible phases in another phase, usually existing in the form of droplets. Generally, emulsions are produced by strong mechanical dispersion. SEDDS, in contrast to emulsions or microemulsifies, 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 disperse the droplets throughout the solution. Furthermore, the stability of unstable or hydrophobic active ingredients is ensured by optionally adding water or an aqueous phase immediately before administration. Thus, SEDDS provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS result in improved bioavailability of hydrophobic active ingredients.

[0092] The compositions described herein (e.g., pharmaceutical compositions) may be formulated for administration to a subject via a route of administration, including, but not limited to, intravenous, intra-arterial, oral, parenteral, oral, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes of administration. The compositions described herein may include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations, and mixed formulations of immediate-release and controlled-release.

[0093] Oral formulations are administered using a variety of formulations known in the art. Furthermore, the oral dosage forms described herein may further include a biodegradable (hydrolyzable) polymer carrier that also serves to adhere the dosage form to the buccal mucosa. For oral or sublingual administration, the composition may take the form of a tablet, lozenge, or gel formulated in a preferred manner.

[0094] For intravenous injection, the composition is optionally formulated as an aqueous solution, preferably as a physiologically compatible buffer (such as Hanks' solution, Ringer's solution, or saline buffer). For transmucosal administration, an appropriate penetrating agent is used in the formulation to penetrate the barrier. For other parenteral injections, the appropriate formulation preferably comprises an aqueous or non-aqueous solution containing a physiologically compatible buffer or excipient.

[0095] Parenteral injection may optionally involve bolus injection or continuous infusion. The injectable formulation may optionally be supplied in unit dosage forms, such as ampoules or multi-dose containers, with the addition of preservatives. In some embodiments, the compositions described herein are suitable for parenteral injection as sterile suspensions, solutions, or emulsions in oily or aqueous vehicles and contain formulation agents (such as suspending agents, stabilizers, and / or dispersants). The parenteral administration composition comprises an aqueous solution of an agent that modulates carotid body activity in an aqueous form. Furthermore, a suspension of the agent that modulates carotid body activity may optionally be prepared as needed (e.g., an oily injection suspension).

[0096] Suitable formulation techniques include, for example, one or a combination of the following methods: (1) dry mixing, (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., Ulster coating), tangential coating, top spray, tableting, extrusion molding, etc.

[0097] In some embodiments, compositions are provided that include a therapeutic agent particle and at least one dispersant or suspending agent for oral administration to a subject. The formulation may be a powder and / or granules for suspension, which, when mixed with water, yields a substantially homogeneous suspension.

[0098] Furthermore, the composition optionally includes one or more pH adjusters or buffers (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 citrate / dextrose, sodium bicarbonate, and ammonium chloride). Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0099] Furthermore, the composition optionally includes one or more salts in amounts necessary to bring the osmotic pressure of the composition within an acceptable range. Such salts include those having a sodium, potassium, or ammonium cation and an anion of chloride, citric acid, ascorbic acid, boric acid, phosphate, bicarbonate, sulfuric acid, thiosulfate, or bisulfite, and preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0100] Other compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances (such as melfen and thimerosal), stabilized chlorine dioxide, and quaternary ammonium compounds (such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride).

[0101] In one embodiment, the aqueous suspensions and dispersions described herein remain homogeneous for at least 4 hours. In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical stirring for less than 1 minute. In yet another embodiment, stirring is not required to maintain a homogeneous aqueous dispersion.

[0102] Nasal aerosol formulations are generally aqueous solutions designed to be administered into the nasal cavity as drops or sprays. Nasal solutions are generally isotonic and may be similar to nasal secretions in that they are slightly buffered to maintain a pH of approximately 5.5 to 6.5, although additionally, pH values ​​outside this range may be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0103] Aerosol formulations for inhalation and inhalation agents may be designed so that the agent, or combination of agents, is delivered to the target respiratory system when administered via the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer. Inhalations or blown-inhalations containing fine powder or liquid drugs may be delivered to the respiratory system as a pharmaceutical aerosol of the agent, or a solution or suspension of a combination of agents in a propellant, for example, to assist in dispersal. The propellant may be a liquefied gas, which may include halocarbons, such as fluorocarbons (fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons and hydrochlorocarbons, etc.), as well as hydrocarbons and hydrocarbon ethers.

[0104] Aerosol formulations may also contain other components (e.g., ethanol, isopropanol, propylene glycol), and even surfactants or other components (such as oils and detergents). These components may help stabilize the formulation and / or lubricate valve components.

[0105] Aerosol formulations may be packaged under pressure, and may be formulated as aerosols using solutions, suspensions, emulsions, powders, and semi-solid preparations. For example, a solution aerosol formulation comprises a solution of an agent (such as a transporter, carrier, or ion channel inhibitor) in a (substantially) pure propellant, or as a mixture of a propellant and a solvent. The solvent may be used to dissolve the agent and / or to slow the evaporation of the propellant. The solvent may include, for example, water, ethanol, and glycol. Any combination of suitable solvents may be used in optional combinations with preservatives, antioxidants, and / or other aerosol components.

[0106] Aerosol formulations may be dispersions or suspensions. A suspension aerosol formulation comprises a suspension of an agent, or a combination of agents (e.g., a transporter, a carrier, or an ion channel inhibitor), and a dispersant. The dispersant may include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil. A suspension aerosol formulation may also contain lubricants, preservatives, antioxidants, and / or other aerosol components.

[0107] Aerosol formulations may also be formulated as emulsions. Emulsified aerosol formulations may include, for example, an alcohol such as ethanol, a surfactant, water, and a propellant, or a combination of agents (e.g., a transporter, carrier, or ion channel). The surfactant used may be nonionic, anionic, or cationic. An example of an emulsion aerosol formulation includes, for example, ethanol, a surfactant, water, and a propellant. Another example of an emulsion aerosol formulation includes, for example, vegetable oil, glyceryl monostearate, and propane.

[0108] method In some embodiments, methods for producing or using compositions disclosed herein are disclosed herein. In some embodiments, the methods include high-throughput techniques for enucleated cells for producing compositions containing enucleated cells for biomedical applications with minimal residual nucleated parent cells. The methods disclosed herein also provide methods for using enucleated cells as fusion partners (e.g., fusion with other cells in vivo or ex vivo), therapeutic agent delivery vehicles, or combinations thereof.

[0109] In some embodiments, methods for producing enucleated cells described herein are disclosed, comprising enucleating nucleated parent cells. In some embodiments, parent cells may be treated with exogenous molecules to soften the cytoskeleton of the parent cells. For example, parent cells may be treated with cytochalasin to soften the cortical actin cytoskeleton. In some embodiments, the nuclei are physically extracted from the cell bodies by centrifugation to produce enucleated cells. In some embodiments, centrifugation involves the use of a density gradient, in which enucleated cells and intact nucleated cells precipitate into different layers, so that the enucleated cells are at least isolated. In some embodiments, the density gradient may be constructed from materials such as alkali metal salts (e.g., cesium chloride, sodium iodide, sodium bromide, cesium sulfate, cesium acetate, or potassium tartrate), neutral and water-soluble molecules (e.g., sugars), hydrophilic polymers (e.g., dextran), or synthetic molecules (e.g., sodium or methylglucamine salts of triiodobenzoic acid and metrizoic acid). In some embodiments, the density gradient is not composed of colloids such as Percoll. In some embodiments, the density gradient includes polysaccharides such as Ficol.

[0110] In some embodiments, centrifugation includes fixed-angle centrifugation, including continuous-flow centrifugation. Example 1 shows an exemplary fixed-angle centrifugation experiment for obtaining enucleated cells from nucleated cells. In some embodiments, fixed-angle centrifugation includes ultracentrifugation. In some embodiments, fixed-angle centrifugation includes continuous-flow centrifugation (e.g., fixed-angle centrifugation by Sorvall CC40NX centrifugation). In some embodiments, the use of fixed-angle centrifugation increases the volume that can be centrifuged. For example, using fixed-angle centrifugation increases the volume that can be centrifuged compared to swing-bucket centrifugation (to produce an equivalent density gradient). In some embodiments, using a fixed-angle rotor allows for holding a larger volume of tubing compared to a swing-bucket counterpart. In some embodiments, a fixed-angle rotor can withstand much higher gravity, and therefore, the centrifugation time is shorter. In some embodiments, by combining high centrifugal force and short centrifugation time, fixed-angle centrifugation is useful for a variety of applications ranging from bacterial and cell pelletization to isodense separation of macromolecules. In some embodiments, nucleated cells can be manipulated to have heterologous polynucleotides encoding heterologous gene products as described herein.

[0111] In some embodiments, a composition containing tens of millions of enucleated cells ("enucleated cell fraction") is obtained by the methods disclosed herein. In some embodiments, the composition also contains residual nucleated cells ("nucleated cell fraction"). In some embodiments, the composition is further processed to purify the enucleated cell fraction from the nucleated cell fraction. In some embodiments, the enucleated cell fraction is formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier, excipient, or diluent.

[0112] In some embodiments, the method for producing enucleated cells does not consist of, and does not involve, the differentiation of parental cells. For example, enucleated cells are not obtained by differentiating nucleated erythrocyte progenitor cells into differentiated enucleated erythrocytes. In some embodiments, enucleated cells are not terminally differentiated cells. In some embodiments, enucleated cells are not platelets. In some embodiments, enucleated cells are not obtained from platelet-derived cells. In some embodiments, enucleated cells are not erythrocytes. In some embodiments, enucleated cells are not obtained from erythrocyte-derived cells.

[0113] In some embodiments, a nucleus-containing parental cell is manipulated to express at least one of the therapeutic agents, transmembrane portions, immune-evading portions, or targeting portions described herein, and then the nucleus of the parental cell is removed. In some embodiments, a nucleus-containing parental cell is enucleated, and the enucleated cell is manipulated to express the therapeutic agent, transmembrane portion, immune-evading portion, or targeting portion described herein. In some embodiments, the parental cell is manipulated to express one or more of the above biomolecules (e.g., immune-evading portions and / or targeting portions), and the resulting enucleated cell (e.g., already expressing immune-evading portions and / or targeting portions) is further manipulated to express a second of the above biomolecules (e.g., a therapeutic agent). In this form, the enucleated cells of the Disclosure can be extensively manipulated before enucleation, can be stored for extended periods as needed (e.g., by lyophilization, cryopreservation, or freezing), and can be rapidly manipulated to express the therapeutic agent when the time is approaching.

[0114] In some embodiments, the compositions disclosed herein are loaded into tubes for fixed-angle centrifugation.

[0115] In some embodiments, the fixed-angle centrifuge includes at least one tube. In some embodiments, the fixed-angle centrifuge includes at least two tubes. In some embodiments, the fixed-angle centrifuge includes at least three tubes. In some embodiments, the fixed-angle centrifuge includes at least four tubes. In some embodiments, the fixed-angle centrifuge includes at least five tubes. In some embodiments, the fixed-angle centrifuge includes at least six tubes. In some embodiments, the fixed-angle centrifuge includes at least seven tubes. In some embodiments, the fixed-angle centrifuge includes at least eight tubes. In some embodiments, the fixed-angle centrifuge includes at least nine tubes. In some embodiments, the fixed-angle centrifuge includes at least ten tubes.

[0116] In some embodiments, the composition has a volume ranging from about 0.001 L to about 10 L. In some embodiments, the composition has volumes ranging from about 0.001 L to about 0.01 L, about 0.001 L to about 0.1 L, about 0.001 L to about 1 L, about 0.001 L to about 2 L, about 0.001 L to about 3 L, about 0.001 L to about 5 L, about 0.001 L to about 6 L, about 0.001 L to about 7 L, about 0.001 L to about 8 L, about 0.001 L to about 9 L, about 0.001 L to about 10 L, and about 0.01 L to about 0.1 L. , about 0.01L to about 1L, about 0.01L to about 2L, about 0.01L to about 3L, about 0.01L to about 5L, about 0.01L to about 6L, about 0.01L to about 7L, about 0.01L to about 8L, about 0.01L ~9L, 0.01L~10L, 0.1L~1L, 0.1L~2L, 0.1L~3L, 0.1L~5L, 0.1L~6L, 0.1L~7L, 0.1L~7L 8L, approximately 0.1L to approximately 9L, approximately 0.1L to approximately 10L, approximately 1L to approximately 2L, approximately 1L to approximately 3L, approximately 1L to approximately 5L, approximately 1L to approximately 6L, approximately 1L to approximately 7L, approximately 1L to approximately 8L, approximately 1L to approximately 9L, approximately 1L ~10L, 2L~3L, 2L~5L, 2L~6L, 2L~7L, 2L~8L, 2L~9L, 2L~10L, 3L~5L, 3L~6L, 3L~6L The composition has a volume ranging from 7L, approximately 3L to 8L, approximately 3L to 9L, approximately 3L to 10L, approximately 5L to 6L, approximately 5L to 7L, approximately 5L to 8L, approximately 5L to 9L, approximately 5L to 10L, approximately 6L to 7L, approximately 6L to 8L, approximately 6L to 9L, approximately 6L to 10L, approximately 7L to 8L, approximately 7L to 9L, approximately 7L to 10L, approximately 8L to 9L, approximately 8L to 10L, or approximately 9L to 10L. In some embodiments, the composition has a volume ranging from approximately 0.001L, approximately 0.01L, approximately 0.1L, approximately 1L, approximately 2L, approximately 3L, approximately 5L, approximately 6L, approximately 7L, approximately 8L, approximately 9L, or approximately 10L. In some embodiments, the composition has a volume ranging from at least about 0.001 L, about 0.01 L, about 0.1 L, about 1 L, about 2 L, about 3 L, about 5 L, about 6 L, about 7 L, about 8 L, or about 9 L. In some embodiments, the composition has a volume ranging from up to about 0.01 L, about 0.1 L, about 1 L, about 2 L, about 3 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, or about 10 L.

[0117] In some embodiments, cell processing methods are described herein that involve using fixed-angle centrifugation, including continuous-flow centrifugation, to denucleate a portion of nucleated cells (parent cells) to produce an enucleated cell fraction.

[0118] In some embodiments, the resulting composition includes an enucleated cell fraction, which may constitute 100% of the composition. In other embodiments, there may be a nucleated cell fraction of the composition consisting of nucleated parent cells that were not enucleated. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 30% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 35% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 40% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 45% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 50% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 55% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 60% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 65% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 70% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 75% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 80% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 85% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 90% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 95% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 96% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 97% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 98% of the composition. In some embodiments, the enucleated cell fraction is approximately equal to or greater than 99% of the composition.

[0119] In some embodiments, cell isolation, cell segregation, or cell sorting is a process of isolating one or more specific cell populations from a heterogeneous mixture of cells. In some embodiments, the cell enucleation methods disclosed herein are performed on an isolated population of homogeneous cells. In some embodiments, the cell enucleation methods disclosed herein are performed on a heterogeneous mixture of cells. In some embodiments, the methods disclosed herein involve isolating a homogeneous population of cells from a heterogeneous mixture of cells using a preferred cell separation technique, which includes, but is not limited to, immunomagnetic cell separation, fluorescence-activated cell sorting, density gradient centrifugation, immunodensity cell isolation, microfluidic cell sorting, buoyancy-activated cell sorting, aptamer-based cell isolation, complement depletion, or any combination thereof.

[0120] During centrifugation, as the sample rotates, denser particles can move to the outer edges of the mixture, while less dense objects accumulate closer to the center. Biological samples can be centrifuged until cell types are isolated into layers. During centrifugation, each cell type can settle to its isodensity point, which is the point in the medium gradient where the density of the cells and the medium are equal. Examples of particle density gradient media include Lymphoprep®, Lympholyte®, Ficoll-Paque®, Percoll®, OptiPrep®, Cell Separation with Accuspin®, Aystem-Histopaque® medium, Histopaque® medium, Histopaque® iodation gradient medium, inorganic salts, nonionic iodation density gradient media, polyhydric alcohols, and polysaccharides. For example, Lymphoprep®, Lympholyte®, and Ficoll-Paque® are composed of sugars and sodium diatrizoate and can be used to isolate mononuclear cells from peripheral blood, umbilical cord blood, and bone marrow. Percoll® consists of colloidal silica particles coated with polyvinylpyrrolidone and is widely used to isolate cells, organelles, viruses, and other intracellular particles. OptiPrep® is a culture medium consisting of iodixanol in water and is used to isolate viruses, organelles, macromolecules, and cells. In some embodiments, a particle density gradient can be established by layering polyhydric (sugar) alcohols (e.g., sucrose, glycerol, or sorbitol), polysaccharides (e.g., ficol, polysucrose, or dextran), inorganic salts (CsCl, Cs2SO4, or KBr), iodinated compounds (diatrizoic acid, nycodenz, histodenz, or iodixanol), colloidal silica (e.g., Percoll), or combinations thereof. In some embodiments, a particle density gradient can be established by layering peptide or protein solutions of various concentrations.For example, a particle density gradient can be established using a serum albumin solution.

[0121] In some embodiments, lower-density particles can move to the outer edge of the mixture, while higher-density particles aggregate further inward. For example, using continuous flow centrifugation (e.g., fixed-angle centrifugation with a Sorvall CC40NX centrifuge), such a density gradient can be established, with the particle gradient loading proceeding from the bottom of the centrifuge to the lowest density and then to the highest density. In some embodiments, a particle density gradient can be established by loading a lower-density particle solution into a fixed-angle centrifuge, followed by a higher-density particle solution. In some embodiments, a particle density gradient can be established by loading a higher-density particle solution into a fixed-angle centrifuge, followed by a lower-density particle solution.

[0122] In some embodiments, cell processing methods are disclosed herein for producing an enucleated cell fraction by enucleating a portion of nucleated cells using fixed-angle centrifugation, including continuous-flow centrifugation. In some embodiments, cell processing methods are disclosed herein for producing an enucleated cell fraction by enucleating a portion of nucleated cells using zone centrifugation. In some embodiments, the fixed-angle centrifugation is continuous-flow centrifugation. In some embodiments, the fixed-angle centrifugation generates a density gradient. In some embodiments, the density gradient separates the enucleated cell fraction from nucleated cells in the composition. In some embodiments, the density gradient includes a polysaccharide density gradient. In some embodiments, the polysaccharide density gradient includes a ficol density gradient. In some embodiments, the method further includes producing a ficol gradient by polymerizing sucrose molecules with epichlorohydrin to obtain osmotically inert polysaccharides.

[0123] In some embodiments, the gradient includes a range of 2 to 20 of the density gradient. In some embodiments, the gradient is a density gradient of ranges 2-3, 2-4, 2-5, 2-6, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 3-4, 3-5, 3-6, 3-8, 3-10, 3-12, 3-14, 3-16, 3-18, 3-20, 4-5, 4-6, 4-8, 4-10, 4-12, 4-14, 4-16, 4-18, 4-20, 5-6, 5-8, 5-10, 5-12 Includes ranges, ranges 5-14, ranges 5-16, ranges 5-18, ranges 5-20, ranges 6-8, ranges 6-10, ranges 6-12, ranges 6-14, ranges 6-16, ranges 6-18, ranges 6-20, ranges 8-10, ranges 8-12, ranges 8-14, ranges 8-16, ranges 8-18, ranges 8-20, ranges 10-12, ranges 10-14, ranges 10-16, ranges 10-18, ranges 10-20, ranges 12-14, ranges 12-16, ranges 12-18, ranges 12-20, ranges 14-16, ranges 14-18, ranges 14-20, ranges 16-18, ranges 16-20, or ranges 18-20. In some embodiments, the gradient includes 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 ranges of density gradient. In some embodiments, the gradient includes at least 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, or 18 ranges of density gradient. In some embodiments, the gradient includes up to 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 ranges of density gradient.In some embodiments, the gradient includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 ranges of the density gradient. In some embodiments, the gradient includes at least 7 ranges of the density gradient. In some embodiments, the gradient includes at least 5 ranges of the density gradient. In some embodiments, the gradient includes at least 3 ranges of the density gradient. In some embodiments, the gradient includes 7 ranges of the density gradient. In some embodiments, the gradient includes 5 ranges of the density gradient. In some embodiments, the gradient includes 3 ranges of the density gradient.

[0124] In some embodiments, the gradient is approximately 7.5% to 30% density gradient, 7.5% to 10% density gradient, 7.5% to 12.5% ​​density gradient, 7.5% to 15% density gradient, 7.5% to 16% density gradient, 7.5% to 17% density gradient, 7.5% to 18% density gradient, 7.5% to 19% density gradient, and 7.5% to 20% density gradient. Medium, approximately 7.5% density gradient medium to approximately 25% density gradient medium, approximately 7.5% density gradient medium to approximately 27.5% density gradient medium, approximately 7.5% density gradient medium to approximately 30% density gradient medium, approximately 10% density gradient medium to approximately 12.5% ​​density gradient medium, approximately 10% density gradient medium to approximately 15% density gradient medium, approximately 10% density gradient medium to approximately 16% density gradient medium, approximately 10% density gradient medium to approximately 17% density gradient medium, approximately 10% density gradient medium to approximately 18% density gradient medium, approximately 10% density gradient medium to approximately 19% density gradient medium, approximately 10% density gradient medium to approximately 2 0% density gradient medium, approximately 10% density gradient medium to approximately 25% density gradient medium, approximately 10% density gradient medium to approximately 27.5% density gradient medium, approximately 10% density gradient medium to approximately 30% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 15% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 16% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 17% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 18% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 19% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 20% density gradient medium, Approximately 12.5% ​​density gradient medium to approximately 25% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 27.5% density gradient medium, approximately 12.5% ​​density gradient medium to approximately 30% density gradient medium, approximately 15% density gradient medium to approximately 16% density gradient medium, approximately 15% density gradient medium to approximately 17% density gradient medium, approximately 15% density gradient medium to approximately 18% density gradient medium, approximately 15% density gradient medium to approximately 19% density gradient medium, approximately 15% density gradient medium to approximately 20% density gradient medium, approximately 15% density gradient medium to approximately 25% density gradient medium, approximately 15% density gradient medium to approximately 27%.5% density gradient medium, approximately 15% density gradient medium ~ approximately 30% density gradient medium, approximately 16% density gradient medium ~ approximately 17% density gradient medium, approximately 16% density gradient medium ~ approximately 18% density gradient medium, approximately 16% density gradient medium ~ approximately 19% density gradient medium, approximately 16% density gradient medium ~ approximately 20% density gradient medium, approximately 16% density gradient medium ~ approximately 25% density gradient medium, approximately 16% density gradient medium ~ approximately 27.5% density gradient medium, approximately 16% density gradient medium ~ approximately 30% density gradient medium, approximately 17% density gradient medium ~ approximately 18% density gradient medium, approximately 17% density gradient medium ~ approximately 19% density gradient medium, approximately 17% density gradient medium ~ approximately 20% density gradient medium, approximately 17% density gradient medium ~ approximately 25% density gradient medium, approximately 17% density gradient medium ~ approximately 27.5% density gradient medium, approximately 17% density gradient medium ~ approximately 30% density gradient medium, approximately 18% density gradient medium ~ approximately Includes 19% density gradient media, approximately 18% to approximately 20% density gradient media, approximately 18% to approximately 25% density gradient media, approximately 18% to approximately 27.5% density gradient media, approximately 18% to approximately 30% density gradient media, approximately 19% to approximately 20% density gradient media, approximately 19% to approximately 25% density gradient media, approximately 19% to approximately 27.5% density gradient media, approximately 19% to approximately 30% density gradient media, approximately 20% to approximately 25% density gradient media, approximately 20% to approximately 27.5% density gradient media, approximately 20% to approximately 30% density gradient media, approximately 25% to approximately 27.5% density gradient media, approximately 25% to approximately 30% density gradient media, or approximately 27.5% to approximately 30% density gradient media. .

[0125] In some embodiments, the gradient is a ficol gradient. In some embodiments, the ficol gradient includes a range of 2 to 20 of the density ficol gradient. In some embodiments, the Ficol gradient is a density Ficol gradient of ranges 2-3, 2-4, 2-5, 2-6, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 3-4, 3-5, 3-6, 3-8, 3-10, 3-12, 3-14, 3-16, 3-18, 3-20, 4-5, 4-6, 4-8, 4-10, 4-12, 4-14, 4-16, 4-18, 4-20, 5-6, 5-8, 5-10, 5 Range ~12 range, Range 5 ~14 range, Range 5 ~16 range, Range 5 ~18 range, Range 5 ~20 range, Range 6 ~8 range, Range 6 ~10 range, Range 6 ~12 range, Range 6 ~14 range, Range 6 ~16 range, Range 6 ~18 range, Range 6 ~20 range, Range 8 ~10 range, Range 8 ~12 range, Range 8 ~14 range, Range 8 ~16 range, Range 8 ~18 range, Range 8 ~20 range The range includes ranges 10-12, 10-14, 10-16, 10-18, 10-20, 12-14, 12-16, 12-18, 12-20, 14-16, 14-18, 14-20, 16-18, 16-20, or 18-20. In some embodiments, the Ficol gradient includes ranges 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20. In some embodiments, the Ficol gradient includes at least ranges 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, or 18 of the density Ficol gradient. In some embodiments, the Ficol gradient includes up to 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 ranges of density Ficol gradient.In some embodiments, the ficol gradient includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes at least 7 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes at least 5 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes at least 3 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes 7 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes 5 ranges of the density ficol gradient. In some embodiments, the ficol gradient includes 3 ranges of the density ficol gradient.

[0126] In some embodiments, the Ficol density gradient is approximately 7.5% to 10% Ficol, approximately 7.5% to 12.5% ​​Ficol, approximately 7.5% to 15% Ficol, approximately 7.5% to 16% Ficol, approximately 7.5% to 17% Ficol, approximately 7.5% to 18% Ficol, approximately 7.5% to 19% Ficol, approximately 7.5% to 20% Ficol, approximately 7.5% to 25% Ficol, and approximately 7.5% to 27.5% Ficol. , approximately 7.5% Ficol to approximately 30% Ficol, approximately 10% Ficol to approximately 12.5% ​​Ficol, approximately 10% Ficol to approximately 15% Ficol, approximately 10% Ficol to approximately 16% Ficol, approximately 10% Ficol to approximately 17% Ficol, approximately 10% Ficol to approximately 18% Ficol, approximately 10% Ficol to approximately 19% Ficol, approximately 10% Ficol to approximately 20% Ficol, approximately 10% Ficol to approximately 25% Ficol, approximately 10% Ficol to approximately 27.5% Ficol, approximately 10% Ficol to approximately 30% Ficol, approximately 12.5% ​​Ficol Fikol ~ approx. 15% Fikol, approx. 12.5% ​​Fikol ~ approx. 16% Fikol, approx. 12.5% ​​Fikol ~ approx. 17% Fikol, approx. 12.5% ​​Fikol ~ approx. 18% Fikol, approx. 12.5% ​​Fikol ~ approx. 19% Fikol, approx. 12.5% ​​Fikol ~ approx. 20% Fikol, approx. 12.5% ​​Fikol ~ approx. 25% Fikol, approx. 12.5% ​​Fikol ~ approx. 27.5% Fikol, approx. 12.5% ​​Fikol ~ approx. 30% Fikol, approx. 15% Fikol ~ approx. 16% Fikol, approx. 15% Fikol ~ approx. 17% Fikol, approx. 15% Ficol to approximately 18% Ficol, approximately 15% Ficol to approximately 19% Ficol, approximately 15% Ficol to approximately 20% Ficol, approximately 15% Ficol to approximately 25% Ficol, approximately 15% Ficol to approximately 27.5% Ficol, approximately 15% Ficol to approximately 30% Ficol, approximately 16% Ficol to approximately 17% Ficol, approximately 16% Ficol to approximately 18% Ficol, approximately 16% Ficol to approximately 19% Ficol, approximately 16% Ficol to approximately 20% Ficol, approximately 16% Ficol to approximately 25% Ficol, approximately 16% Ficol to approximately 27% Ficol.5% Ficol, approximately 16% to approximately 30% Ficol, approximately 17% to approximately 18% Ficol, approximately 17% to approximately 19% Ficol, approximately 17% to approximately 20% Ficol, approximately 17% to approximately 25% Ficol, approximately 17% to approximately 27.5% Ficol, approximately 17% to approximately 30% Ficol, approximately 18% to approximately 19% Ficol, approximately 18% to approximately 20% Ficol, approximately 18% to approximately 25% Ficol, approximately 18% to approximately 27.5% Ficol, approximately Includes 18% to approximately 30% Ficol, approximately 19% to approximately 20% Ficol, approximately 19% to approximately 25% Ficol, approximately 19% to approximately 27.5% Ficol, approximately 19% to approximately 30% Ficol, approximately 20% to approximately 25% Ficol, approximately 20% to approximately 27.5% Ficol, approximately 20% to approximately 30% Ficol, approximately 25% to approximately 27.5% Ficol, or approximately 27.5% to approximately 30% Ficol.

[0127] In some embodiments, the ficol density gradient includes about 7.5% ficol, about 10% ficol, about 12.5% ​​ficol, about 15% ficol, about 16% ficol, about 17% ficol, about 18% ficol, about 19% ficol, about 20% ficol, about 25% ficol, about 27.5% ficol, or about 30% ficol. In some embodiments, the ficol density gradient includes at least about 7.5% ficol, about 10% ficol, about 12.5% ​​ficol, about 15% ficol, about 16% ficol, about 17% ficol, about 18% ficol, about 19% ficol, about 20% ficol, about 25% ficol, or about 27.5% ficol. In some embodiments, the Ficol density gradient includes up to about 10% Ficol, about 12.5% ​​Ficol, about 15% Ficol, about 16% Ficol, about 17% Ficol, about 18% Ficol, about 19% Ficol, about 20% Ficol, about 25% Ficol, about 27.5% Ficol, or about 30% Ficol. In some embodiments, the Ficol density gradient includes about 25% Ficol, about 17% Ficol, about 16% Ficol, about 15% Ficol, or about 12.5% ​​Ficol. In some embodiments, the Ficol density gradient includes about 25% Ficol. In some embodiments, the Ficol density gradient includes about 17% Ficol. In some embodiments, the Ficol density gradient includes about 16% Ficol. In some embodiments, the Ficol density gradient includes about 15% Ficol. In some embodiments, the Ficol density gradient includes about 12.5% ​​Ficol. In some embodiments, the proportion of Ficol is synonymous with the polysaccharide solution.

[0128] In some embodiments, fixed-angle centrifugation includes centrifugation at angles of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 degrees relative to the axis of rotation. It also includes centrifugation of multiple cells in a polysaccharide density gradient at angles of 43, 44, 45, 46, 47, 48, 49, 50, 90, or 180 degrees. In some embodiments, fixed-angle centrifugation includes centrifugation of multiple cells in a polysaccharide density gradient at angles of 23, 24, 25, or 45 degrees relative to the axis of rotation. In some embodiments, fixed-angle centrifugation includes centrifuging a plurality of cells in a polysaccharide density gradient at an angle of 23 degrees. In some embodiments, fixed-angle centrifugation includes centrifuging a plurality of cells in a polysaccharide density gradient at an angle of 24 degrees. In some embodiments, fixed-angle centrifugation includes centrifuging a plurality of cells in a polysaccharide density gradient at an angle of 25 degrees. In some embodiments, fixed-angle centrifugation includes centrifuging a plurality of cells in a polysaccharide density gradient at an angle of 45 degrees. In some embodiments, fixed-angle centrifugation includes centrifuging a plurality of cells in a polysaccharide density gradient at an angle of 90 degrees. In some embodiments, fixed-angle centrifugation includes centrifuging a plurality of cells in a polysaccharide density gradient at an angle of 180 degrees.

[0129] In some embodiments, fixed-angle centrifugation is achieved by a fixed-angle rotor. In some embodiments, fixed-angle centrifugation is achieved by a combination of a fixed-angle rotor and a swing bucket rotor. For example, cells and density gradients can be loaded into a vertical position (0 degrees) and then rotated while being swung to a fixed angle (e.g., 25 or 45 degrees) to spread them out. In some embodiments, fixed-angle centrifugation is not achieved by swing bucket centrifugation.

[0130] In some embodiments, a method for producing an enucleated cell fraction by enucleating a portion of nucleated cells using fixed-angle centrifugation for cell processing further includes generating a density gradient by centrifugation of polysaccharides.

[0131] In some embodiments, fixed-angle centrifugation includes centrifugation between approximately 10,000 relative centrifugal force (RCF) and approximately 250,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation between approximately 10,000 RCF and approximately 20,000 RCF, approximately 10,000 RCF and approximately 30,000 RCF, approximately 10,000 RCF and approximately 40,000 RCF, approximately 10,000 RCF and approximately 50,000 RCF, approximately 10,000 RCF and approximately 60,000 RCF, approximately 10,000 RCF and approximately 70,000 RCF, approximately 10,000 RCF and approximately 80,000 RCF, approximately 10,000 RCF and approximately 100,000 RCF, approximately 10,000 RCF and approximately 150,000 RCF, and approximately 10,000 RCF and approximately 200,000RCF, about 10,000RCF to about 250,000RCF, about 20,000RCF to about 30,000RCF, about 20,000RCF to about 40,000RCF, about 20,000RCF to about 50,000RCF, about 20,000RCF to about 60,0 00RCF, about 20,000RCF to about 70,000RCF, about 20,000RCF to about 80,000RCF, about 20,000RCF to about 100,000RCF, about 20,000RCF to about 150,000RCF, about 20,000RCF to about 200,000RCF F, about 20,000RCF to about 250,000RCF, about 30,000RCF to about 40,000RCF, about 30,000RCF to about 50,000RCF, about 30,000RCF to about 60,000RCF, about 30,000RCF to about 70,000RCF, about 30 ,000RCF~Approx. 80,000RCF, Approx. 30,000RCF~Approx. 100,000RCF, Approx. 30,000RCF~Approx. 150,000RCF, Approx. 30,000RCF~Approx. 200,000RCF, Approx. 30,000RCF~Approx. 250,000RCF, Approx. 40,00 0RCF~Approx. 50,000RCF, Approx. 40,000RCF~Approx. 60,000RCF, Approx. 40,000RCF~Approx. 70,000RCF, Approx. 40,000RCF~Approx. 80,000RCF, Approx. 40,000RCF~Approx. 100,000RCF, Approx. 40,000RCF~ Approx. 150,000RCF, Approx. 40,000RCF~Approx. 200,000RCF, Approx. 40,000RCF~Approx. 250,000RCF, Approx. 50,000RCF~Approx. 60,000RCF, Approx. 50,000RCF~Approx. 70,000RCF, Approx.000RCF, about 50,000RCF~about 100,000RCF, about 50,000RCF~about 150,000RCF, about 50,000RCF~about 200,000RCF F, approx. 50,000RCF ~ approx. 250,000RCF, approx. 60,000RCF ~ approx. 70,000RCF, approx. 60,000RCF ~ approx. 80,000RCF, approx. 60,00 0RCF~Approx. 100,000RCF, Approx. 60,000RCF~Approx. 150,000RCF, Approx. 60,000RCF~Approx. 200,000RCF, Approx. 60,000RCF~Approx. 250,000RCF, about 70,000RCF~about 80,000RCF, about 70,000RCF~about 100,000RCF, about 70,000RCF~about 150,000 RCF, approx. 70,000RCF ~ approx. 200,000RCF, approx. 70,000RCF ~ approx. 250,000RCF, approx. 80,000RCF ~ approx. 100,000RCF, approx. 80,000RCF~Approx. 150,000RCF, Approx. 80,000RCF~Approx. 200,000RCF, Approx. 80,000RCF~Approx. 250,000RCF, Approx. 100,00 This includes centrifugation between 0 RCF and approximately 150,000 RCF, approximately 100,000 RCF and approximately 200,000 RCF, approximately 100,000 RCF and approximately 250,000 RCF, approximately 150,000 RCF and approximately 200,000 RCF, approximately 150,000 RCF and approximately 250,000 RCF, or between approximately 200,000 RCF and approximately 250,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation between approximately 10,000 RCF, approximately 20,000 RCF, approximately 30,000 RCF, approximately 40,000 RCF, approximately 50,000 RCF, approximately 60,000 RCF, approximately 70,000 RCF, approximately 80,000 RCF, approximately 100,000 RCF, approximately 150,000 RCF, approximately 200,000 RCF, or approximately 250,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation between at least about 10,000 RCF, about 20,000 RCF, about 30,000 RCF, about 40,000 RCF, about 50,000 RCF, about 60,000 RCF, about 70,000 RCF, about 80,000 RCF, about 100,000 RCF, about 150,000 RCF, or about 200,000 RCF. In some embodiments, fixed-angle centrifugation includes centrifugation up to about 20,000 RCF, about 30,000 RCF, about 40,000 RCF, about 50,000 RCF, about 60,This includes centrifugation between 000 RCF, approximately 70,000 RCF, approximately 80,000 RCF, approximately 100,000 RCF, approximately 150,000 RCF, approximately 200,000 RCF, or approximately 250,000 RCF. In some embodiments, fixed-angle centrifugation includes approximately 124,000 RCF. In some embodiments, fixed-angle centrifugation includes approximately 125,000 RCF. In some embodiments, fixed-angle centrifugation includes approximately 126,000 RCF. In some embodiments, fixed-angle centrifugation includes approximately 127,000 RCF. In some embodiments, fixed-angle centrifugation includes approximately 128,000 RCF.

[0132] In some embodiments, fixed-angle centrifugation includes an R-average (average RCF value at the center of the tube during centrifugation) between approximately 10,000 RCF and approximately 150,000 RCF. In some embodiments, fixed-angle centrifugation includes approximately 10,000 RCF to approximately 30,000 RCF, approximately 10,000 RCF to approximately 50,000 RCF, approximately 10,000 RCF to approximately 60,000 RCF, approximately 10,000 RCF to approximately 70,000 RCF, approximately 10,000 RCF to approximately 80,000 RCF, approximately 10,000 RCF to approximately 85,000 RCF, approximately 10,000 RCF to approximately 90,000 RCF, approximately 10,000 RCF to approximately 95,000 RCF, approximately 10,000 RCF to approximately 100,000 RCF, and approximately 10,000 RCF to approximately 120,000RCF, about 10,000RCF to about 150,000RCF, about 30,000RCF to about 50,000RCF, about 30,000RCF to about 60,000RCF, about 30,000RCF to about 70,000RCF, about 30,000RCF to about 80,0 00RCF, about 30,000RCF to about 85,000RCF, about 30,000RCF to about 90,000RCF, about 30,000RCF to about 95,000RCF, about 30,000RCF to about 100,000RCF, about 30,000RCF to about 120,000RCF F, about 30,000RCF to about 150,000RCF, about 50,000RCF to about 60,000RCF, about 50,000RCF to about 70,000RCF, about 50,000RCF to about 80,000RCF, about 50,000RCF to about 85,000RCF, about 50 ,000RCF~Approx. 90,000RCF, Approx. 50,000RCF~Approx. 95,000RCF, Approx. 50,000RCF~Approx. 100,000RCF, Approx. 50,000RCF~Approx. 120,000RCF, Approx. 50,000RCF~Approx. 150,000RCF, Approx. 60,00 0RCF~Approx. 70,000RCF, Approx. 60,000RCF~Approx. 80,000RCF, Approx. 60,000RCF~Approx. 85,000RCF, Approx. 60,000RCF~Approx. 90,000RCF, Approx. 60,000RCF~Approx. 100,000RCF, about 60,000RCF to about 120,000RCF, about 60,000RCF to about 150,000RCF, about 70,000RCF to about 80,000RCF, about 70,000RCF to about 85,000RCF, about 70,000RCF to about 90,000RCF, about 70,000RCF~about 95,000RCF, about 70,000RCF~about 100,000RCF, about 70,000RCF~about 120,000RCF F, approx. 70,000RCF ~ approx. 150,000RCF, approx. 80,000RCF ~ approx. 85,000RCF, approx. 80,000RCF ~ approx. 90,000RCF, approx. 80,0 00RCF~Approx. 95,000RCF, Approx. 80,000RCF~Approx. 100,000RCF, Approx. 80,000RCF~Approx. 120,000RCF, Approx. 80,000RCF~ Approx. 150,000RCF, Approx. 85,000RCF~Approx. 90,000RCF, Approx. 85,000RCF~Approx. 95,000RCF, Approx. 85,000RCF~Approx. 100,00 0RCF, approximately 85,000RCF~approximately 120,000RCF, approximately 85,000RCF~approximately 150,000RCF, approximately 90,000RCF~approximately 95,000RCF, Approx. 90,000RCF~Approx. 100,000RCF, Approx. 90,000RCF~Approx. 120,000RCF, Approx. 90,000RCF~Approx. 150,000RCF, Approx. 95,0 Includes the R-mean between 00RCF and approximately 100,000RCF, approximately 95,000RCF and approximately 120,000RCF, approximately 95,000RCF and approximately 150,000RCF, approximately 100,000RCF and approximately 120,000RCF, approximately 100,000RCF and approximately 150,000RCF, or approximately 120,000RCF and approximately 150,000RCF. In some embodiments, fixed-angle centrifugation includes an R-average between approximately 10,000 RCF, approximately 30,000 RCF, approximately 50,000 RCF, approximately 60,000 RCF, approximately 70,000 RCF, approximately 80,000 RCF, approximately 85,000 RCF, approximately 90,000 RCF, approximately 95,000 RCF, approximately 100,000 RCF, approximately 120,000 RCF, or approximately 150,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average of at least about 10,000 RCF, about 30,000 RCF, about 50,000 RCF, about 60,000 RCF, about 70,000 RCF, about 80,000 RCF, about 85,000 RCF, about 90,000 RCF, about 95,000 RCF, about 100,000 RCF, or about 120,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average of at least about 30,000 RCF, about 50,000 RCF, about 60,000 RCF, about 70,000 RCF, about 80,000 RCF, about 85,This includes R-average values ​​between 000 RCF, approximately 90,000 RCF, approximately 95,000 RCF, approximately 100,000 RCF, approximately 120,000 RCF, or approximately 150,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 70,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 75,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 80,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 85,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 90,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 95,000 RCF. In some embodiments, fixed-angle centrifugation includes an R-average value of approximately 100,000 RCF.

[0133] In some embodiments, fixed-angle centrifugation includes a maximum R value (average RCF value at the bottom of the tube during centrifugation) between approximately 10,000 RCF and approximately 200,000 RCF. In some embodiments, fixed-angle centrifugation includes values ​​between approximately 10,000 RCF and approximately 30,000 RCF, approximately 10,000 RCF and approximately 50,000 RCF, approximately 10,000 RCF and approximately 60,000 RCF, approximately 10,000 RCF and approximately 80,000 RCF, approximately 10,000 RCF and approximately 100,000 RCF, approximately 10,000 RCF and approximately 120,000 RCF, approximately 10,000 RCF and approximately 130,000 RCF, approximately 10,000 RCF and approximately 140,000 RCF, approximately 10,000 RCF and approximately 160,000 RCF, and approximately 10,000 RCF ~ approx. 180,000 RCF, approx. 10,000 RCF ~ approx. 200,000 RCF, approx. 30,000 RCF ~ approx. 50,000 RCF, approx. 30,000 RCF ~ approx. 60,000 RCF, approx. 30,000 RCF ~ approx. 80,000 RCF, approx. ~100,000RCF, 30,000RCF~120,000RCF, 30,000RCF~130,000RCF, 30,000RCF~140,000RCF, 30,000RCF~160,000RCF, 30,000RCF~ Approx. 180,000RCF, Approx. 30,000RCF~Approx. 200,000RCF, Approx. 50,000RCF~Approx. 60,000RCF, Approx. 50,000RCF~Approx. 80,000RCF, Approx. 50,000RCF~Approx. 100,000RCF, Approx. 50,000RCF~Approx. 1 20,000RCF, about 50,000RCF to about 130,000RCF, about 50,000RCF to about 140,000RCF, about 50,000RCF to about 160,000RCF, about 50,000RCF to about 180,000RCF, about 50,000RCF to about 20 0,000RCF, about 60,000RCF to about 80,000RCF, about 60,000RCF to about 100,000RCF, about 60,000RCF to about 120,000RCF, about 60,000RCF to about 130,000RCF, about 60,000RCF to about 140 ,000RCF, about 60,000RCF to about 160,000RCF, about 60,000RCF to about 180,000RCF, about 60,000RCF to about 200,000RCF, about 80,000RCF to about 100,000RCF, about 80,000RCF to about 120,000RCF, about 80,000RCF to about 130,000RCF, about 80,000RCF to about 140,000RCF, about 80,000RCF to about 160,000RCF, about 80 ,000RCF~Approx. 180,000RCF, Approx. 80,000RCF~Approx. 200,000RCF, Approx. 100,000RCF~Approx. 120,000RCF, Approx. 100,000RCF~Approx. 130,000RCF, about 100,000RCF~about 140,000RCF, about 100,000RCF~about 160,000RCF, about 100,000RCF~about 180,000R CF, about 100,000RCF~about 200,000RCF, about 120,000RCF~about 130,000RCF, about 120,000RCF~about 140,000RCF, about 120, 000RCF~Approx. 160,000RCF, Approx. 120,000RCF~Approx. 180,000RCF, Approx. 120,000RCF~Approx. 200,000RCF, Approx. 130,000RCF~Approx. 140,000RCF, about 130,000RCF~about 160,000RCF, about 130,000RCF~about 180,000RCF, about 130,000RCF~about 200,000R CF includes the maximum R value between approximately 140,000 RCF and 160,000 RCF, 140,000 RCF and 180,000 RCF, 140,000 RCF and 200,000 RCF, 160,000 RCF and 180,000 RCF, 160,000 RCF and 200,000 RCF, or between approximately 180,000 RCF and 200,000 RCF. In some embodiments, the fixed-angle centrifugation includes R maximum values ​​between approximately 10,000 RCF, approximately 30,000 RCF, approximately 50,000 RCF, approximately 60,000 RCF, approximately 80,000 RCF, approximately 100,000 RCF, approximately 120,000 RCF, approximately 130,000 RCF, approximately 140,000 RCF, approximately 160,000 RCF, approximately 180,000 RCF, or approximately 200,000 RCF. In some embodiments, fixed-angle centrifugation is performed at a minimum of about 10,000 RCF, about 30,000 RCF, about 50,000 RCF, about 60,000 RCF, about 80,000 RCF, about 100,000 RCF, about 120,000 RCF, about 130,000 RCF, about 140,000 RCF, about 160,000 RCF, or about 180,The R maximum value includes a maximum of 000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of a maximum of about 30,000 RCF, about 50,000 RCF, about 60,000 RCF, about 80,000 RCF, about 100,000 RCF, about 120,000 RCF, about 130,000 RCF, about 140,000 RCF, about 160,000 RCF, about 180,000 RCF, or about 200,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of about 100,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of about 105,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of about 110,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of about 115,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 120,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 121,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 122,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 123,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 124,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 125,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 126,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 127,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 128,000 RCF. In some embodiments, fixed-angle centrifugation includes an R maximum value of approximately 129,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of approximately 130,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of approximately 135,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of approximately 140,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of approximately 145,000 RCF. In some embodiments, fixed-angle centrifugation includes a maximum R value of approximately 150,000 RCF.

[0134] In some embodiments, the cell processing method further includes generating a density gradient by centrifuging a density gradient medium at an acceleration for at least about 1 minute (min), at least about 5 minutes, at least about 9 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging polysaccharides at an acceleration for at least about 1 minute, at least about 5 minutes, at least about 9 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging the polysaccharides at an accelerated rate for at least about 9 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 50 minutes.

[0135] In some embodiments, the cell processing method further includes generating a density gradient by centrifuging a density gradient medium with a deceleration period of at least about 1 minute (min), at least about 5 minutes, at least about 9.5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging polysaccharides with a deceleration period of at least about 1 minute, at least about 5 minutes, at least about 9.5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes. In some embodiments, the cell processing method further includes generating a density gradient by centrifuging the polysaccharides at a slowdown for at least about 9.5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, or at least about 50 minutes.

[0136] In some embodiments, the fixed-angle centrifugation includes centrifugation for about 10 minutes to about 120 minutes. In some embodiments, the fixed-angle centrifugation includes centrifugation for about 20 minutes to about 110 minutes. In some embodiments, the fixed-angle centrifugation includes centrifugation for about 30 minutes to about 100 minutes. In some embodiments, the fixed-angle centrifugation includes centrifugation for about 40 minutes to about 90 minutes. In some embodiments, the fixed-angle centrifugation includes centrifugation for about 50 minutes to about 80 minutes. In some embodiments, the fixed-angle centrifugation includes centrifugation for about 50 minutes to about 70 minutes. In some embodiments, the fixed-angle centrifugation includes centrifugation for about 50 minutes to about 60 minutes.

[0137] In some embodiments, fixed-angle centrifugation includes centrifugation for about 10 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 20 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 30 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 40 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 50 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 60 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 70 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 80 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 90 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 100 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 110 minutes. In some embodiments, fixed-angle centrifugation includes centrifugation for about 120 minutes.

[0138] In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 1.0 minute to about 10.0 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 1.0 minute to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 1.5 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 2.0 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 2.5 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 3.0 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 3.5 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 4.0 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 4.5 minutes to reach the RCF for centrifugation. In some embodiments, fixed-angle centrifugation includes acceleration, which includes about 5.0 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 5.2 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 5.5 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 6.0 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 6.5 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 7.0 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 7.5 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 8.0 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which takes about 8.5 minutes to reach the RCF for centrifugation.In some embodiments, the fixed-angle centrifugation includes acceleration, which includes about 9.0 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which includes about 9.5 minutes to reach the RCF for centrifugation. In some embodiments, the fixed-angle centrifugation includes acceleration, which includes about 10.0 minutes to reach the RCF for centrifugation.

[0139] In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 1.0 to 10.0 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 1.0 minute to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 1.5 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 2.0 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 2.5 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 3.0 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 3.5 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 4.0 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which takes about 4.5 minutes to reach a stop. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 5.0 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 5.5 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 6.0 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 6.5 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 7.0 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 7.5 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 8.0 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 8.5 minutes until the fixed-angle centrifugation stops. In some embodiments, the fixed-angle centrifugation includes deceleration, which includes about 9.0 minutes until the fixed-angle centrifugation stops.In some embodiments, the fixed-angle centrifugal separation includes deceleration, which takes about 9.5 minutes to reach the stop of the fixed-angle centrifugal separation. In some embodiments, the fixed-angle centrifugal separation includes deceleration, which takes about 10.0 minutes to reach the stop of the fixed-angle centrifugal separation.

[0140] In some embodiments, the cell processing methods disclosed herein include denucleating a portion of nucleated cells to produce an enucleated cell fraction using fixed-angle centrifugation, including continuous-flow centrifugation, wherein the portion of nucleated cells to be enucleated is approximately equal to or greater than 10% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 20% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 25% of the nucleated cells. In some embodiments, the proportion of nucleated cells is approximately equal to or greater than 30% of the nucleated cells. In some embodiments, the proportion of nucleated cells is approximately equal to or greater than 35% of the nucleated cells. In some embodiments, the proportion of nucleated cells is approximately equal to or greater than 40% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 45% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 50% of the nucleated cells. In some embodiments, the portion of nucleated cells is approximately equal to or greater than 55% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 60% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 65% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 70% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 75% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 80% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 85% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 90% of the nucleated cells. In some embodiments, the nucleated cell portion is approximately equal to or greater than 95% of the nucleated cells.

[0141] In some embodiments, the enucleated cell fraction produced by the method disclosed herein is approximately 1 × 10⁻⁶ 5 Enucleated cells, 1 x 10 6Enucleated cells, about 1×10 7 Enucleated cells, 3×10 5 Enucleated cells, 5×10 5 Enucleated cells, 7×10 7 of enucleated cells, 8×10 7 of enucleated cells, 9×10 7 of enucleated cells, 10×10 7 of enucleated cells, 15×10 7 of enucleated cells, 20×10 7 of enucleated cells, 50×10 7 of enucleated cells, 70×10 7 Enucleated cells, 90×10 7 Enucleated cells, 100×10 7 of enucleated cells, 150×10 7 of enucleated cells, 200×10 7 of enucleated cells, 250×10 7 Enucleated cells, 300×10 7 Enucleated cells or 500×10 7 includes enucleated cells of or more.

[0142] In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 50% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 60% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 70% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 80% or less of the average diameter of the nucleated cells. In some embodiments, the enucleated cells of the enucleated cell fraction have a diameter that includes about 90% or less of the average diameter of the nucleated cells.

[0143] In some embodiments, the diameter of enucleated cells in the enucleated cell fraction includes approximately 5 μm or more, approximately 10 μm or more, approximately 20 μm or more, approximately 30 μm or more, approximately 40 μm or more, approximately 50 μm or more, approximately 60 μm or more, approximately 70 μm or more, approximately 80 μm or more, or approximately 90 μm or more. In some embodiments, the diameter of enucleated cells in the enucleated cell fraction is in the range of approximately 1 μm to approximately 10 μm. In some embodiments, the diameter of enucleated cells in the enucleated cell fraction is approximately 1 μm to 2 μm, 1 μm to 3 μm, 1 μm to 4 μm, 1 μm to 5 μm, 1 μm to 6 μm, 1 μm to 7 μm, 1 μm to 8 μm, 1 μm to 9 μm, 1 μm to 10 μm, 2 μm to 3 μm, 2 μm to 4 μm, 2 μm to 5 μm, 2 μm to 6 μm, 2 μm to 7 μm, 2 μm to 8 μm, 2 μm to 9 μm, 2 μm to 10 μm, 3 μm to 4 μm, 3 μm to 5 μm, 3 μm to 6 μm, 3 μm to 7 μm, 3 μm to 8 μm m is in the range of approximately 3μm to 9μm, approximately 3μm to 10μm, approximately 4μm to 5μm, approximately 4μm to 6μm, approximately 4μm to 7μm, approximately 4μm to 8μm, approximately 4μm to 9μm, approximately 4μm to 10μm, approximately 5μm to 6μm, approximately 5μm to 7μm, approximately 5μm to 8μm, approximately 5μm to 9μm, approximately 5μm to 10μm, approximately 6μm to 7μm, approximately 6μm to 8μm, approximately 6μm to 9μm, approximately 6μm to 10μm, approximately 7μm to 8μm, approximately 7μm to 9μm, approximately 7μm to 10μm, approximately 8μm to 9μm, approximately 8μm to 10μm, or approximately 9μm to 10μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is in the range of approximately 1 μm, approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, approximately 9 μm, or approximately 10 μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is in the range of at least approximately 1 μm, approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, or approximately 9 μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is in the range of at most approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, approximately 9 μm, or approximately 10 μm. In some embodiments, the diameter of the enucleated cells in the enucleated cell fraction is approximately 8 μm.

[0144] Disclosed cell modifications As shown in Figure 1, nucleated ("parent") cells may be manipulated to express one or more exogenous factors before or after enucleation, or in combination thereof. In some embodiments, one or more exogenous biomolecules include a targeting moiety, a transmembrane moiety, or a therapeutic agent, or a combination thereof. In some embodiments, the targeting moiety includes an adhesion molecule, a chemokine, or a retention receptor, or both. In some embodiments, the targeting moiety is manipulated to target a target tissue, cell, or environment (e.g., lymphoid tissue of the subject) as disclosed herein. Additionally or alternatively, the resulting enucleated cells are manipulated to express the therapeutic agent and, optionally, to secrete the therapeutic agent. In some embodiments, the therapeutic agent includes an antibody or its antigen-binding fragment (e.g., a single-domain antibody). In some embodiments, the enucleated cells may be administered to a subject in need to treat a disease or condition in the subject.

[0145] Various methods may be used to introduce biomolecules (e.g., therapeutic agents, transmembrane portions, immune-evading portions, and / or targeting portions) into parental or enucleated cells as described herein. Non-limiting examples of methods that may be used to introduce biomolecules into parental or enucleated cells include liposome-mediated transfer, adenovirus, adeno-associated virus, herpesvirus, retrovirus-based vector, lentiviral vector, electroporation, microinjection, lipofection, transfection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalement, hydrodynamic delivery, magnetofection, nanoparticle transfection, or combinations thereof. In some embodiments of any of the compositions and methods provided herein, therapeutic agents, viruses, antibodies, or nanoparticles may be introduced into enucleated cells.

[0146] In some embodiments, enucleated cells are preserved by cryopreservation, cryohibernation, or freeze-drying. Cryopreservation involves freezing the enucleated cells, while cryohibernation involves storing the enucleated cells at a temperature below room temperature but not freezing. In some embodiments, enucleated cells are freeze-dried. In some embodiments, freeze-dried enucleated cells can be reconstituted, and the reconstituted enucleated cells exhibit viability equivalent to that of non-freeze-dried enucleated cells. In some embodiments, freeze-drying comprises the components of freezing the cells and drying them under very low pressure (e.g., less than 3000 mTorr) using a vacuum. The drying component can induce sublimation, allowing the cells to be dehydrated while maintaining their 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 crystallization, and the rate of reconstitution. The triple point of a substance is the temperature and pressure at which the sublimation, melting, and evaporation curves intersect. Reaching the triple point, which varies for each substance, ensures that sublimation, rather than melting, occurs in the subsequent drying process. Larger ice crystals are preferable to promote faster and more efficient freeze-drying because they form a network within the product, which facilitates the faster removal of water vapor during sublimation. Producing larger crystals may require slow freezing of the product or a process called annealing, in which the temperature is periodically raised and lowered. Fresh or frozen biological tissues or cells do not have a single uniform melting point (eutectic point), and as a result, the freezing stage of the substance (cell or tissue) is cooled below the triple point (representing the temperature and pressure at which the solid, liquid, and gas phases of a substance can coexist). Living cells have a critical point on the phase diagram, at which point both the liquid and gas phases of the object or substance have the same density and are therefore indistinguishable. Maintaining the critical point temperature of the product is necessary to prevent meltback or cake collapse from occurring due to incomplete sublimation during primary and secondary drying.For substances that require structural preservation, such as living cells, large ice crystals can be harmful, potentially damaging cell walls, worsening texture, and leading to loss of nutrients. In this case, rapid freezing is necessary to quickly lower the temperature of the substance below its critical point, thereby avoiding the formation of large ice crystals. The freezing temperature of cells or tissues can vary, but is generally in the range of -50°C (-58°F) to -80°C (-112°F).

[0147] In the drying phase, the ambient pressure is reduced to a range of a few millibars, and as a result, heat is supplied to the material by conduction or radiation so that the ice sublimes. The amount of heat required can be calculated using the latent heat of sublimation of the molecules to be sublimated. In this initial drying phase, about 95% of the water in the material or substance sublimes. Often, this phase proceeds slowly and can last for several days depending on the material and technique employed. However, if too much heat is applied too quickly, the structure of the material may change. In this phase, the pressure is controlled by applying a partial vacuum. The vacuum accelerates sublimation and is useful as an intentional drying treatment. A cold condenser chamber and / or condenser plate is used as the surface(s) on which the water vapor reliquefies and solidifies. It is important to note that in this pressure range, the low air density prevents heat from being supplied by convection. The drying phase also aims to remove any remaining unfrozen water molecules, as the ice induced by freezing needs to be removed in the primary drying phase. This part of the freeze-drying process is governed by the adsorption isotherms of the material. At this stage, the temperature is raised higher than in the primary drying stage to break any physicochemical interactions formed between water molecules and the frozen material, sometimes exceeding 0°C (32°F). Typically, the pressure at this stage is also reduced to promote desorption. However, some products can similarly benefit from increased pressure. After the freeze-drying process is complete, the vacuum is usually broken using an inert gas (such as nitrogen) before sealing the material. At the end of the operation, the residual water content in the product should be very low, ranging from less than 1% to 4% of the original concentration.

[0148] In some embodiments, lyophilization of enucleated cells involves the use of cryoprotective agents to preserve cell viability and biological function. Cryoprotective agents include the addition of reagents, salts, or additives that protect cells during the drying process. Common cryoprotective agents include trehalose, DMSO, methylcellulose, sucrose, antioxidants, human or animal serum proteins, and cellular stress proteins. Furthermore, methods to increase the transport of cryoprotective agents within cells in suspension can be utilized as methods 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 may partially disrupt the integrity of the cell membrane and promote intracellular transport of cryoprotective agents.

[0149] In some embodiments, the nucleated cells described herein can be modified to express a targeting moiety (e.g., an antibody or its antigen-binding fragment), a therapeutic agent, a transmembrane moiety, a heterogeneous gene product, or a combination thereof. In some embodiments, the nucleated cells can be modified to express at least one heterogeneous polynucleotide, where at least one heterogeneous polynucleotide encodes a targeting moiety, a therapeutic agent, a transmembrane moiety, a heterogeneous gene product, or a combination thereof.

[0150] In one embodiment, a method for modifying cells by introducing at least one heterologous polynucleotide into the cells is disclosed herein. In some embodiments, the heterologous polynucleotide encodes a promoter, a heterologous gene product, or a combination thereof. In some embodiments, the method includes providing a composition comprising a first subset of nucleated cells and enucleated cells derived from a second subset of nucleated cells. In some embodiments, the first subset of nucleated cells comprises a heterologous polynucleotide encoding a heterologous gene product. Heterologous polynucleotides can be introduced into any type of enucleated cell by transfection, such as plasmids, transposons, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-Cas technology, or viral transduction, including, but not limited to, hTERT-immobilized mesenchymal stem cells.

[0151] In some embodiments, heterologous polynucleotides include a promoter. In some embodiments, the promoter includes an inducible promoter. In some embodiments, the promoter needs to be compatible with mammalian gene expression and need to provide rapid and potent gene expression only in the presence of an inducible stimulus. In some embodiments, the inducible promoter is hypothermic. In some embodiments, the inducible promoter is induced by exposing nucleated cells to a temperature below approximately 40°C. In some embodiments, the inducible promoter is induced by exposing nucleated cells to a temperature below approximately 39°C. In some embodiments, the inducible promoter is induced by exposing nucleated cells to a temperature below approximately 38°C. In some embodiments, the inducible promoter is induced by exposing nucleated cells to a temperature below approximately 37°C. In some embodiments, the inducible promoter is induced by exposing nucleated cells to a temperature below approximately 36°C. In some embodiments, the inducible promoter is induced by exposing nucleated cells to a temperature below approximately 35°C. In some embodiments, examples of inducible promoters include, but are not limited to, dsrA or CIRP.

[0152] In some embodiments, the inducible promoter is thermogenic. In some embodiments, the inducible promoter is induced by contacting nucleated cells at temperatures above 35°C. In some embodiments, the inducible promoter is induced by contacting nucleated cells at temperatures above 36°C. In some embodiments, the inducible promoter is induced by contacting nucleated cells at temperatures above 37°C. In some embodiments, the inducible promoter is induced by contacting nucleated cells at temperatures above 38°C. In some embodiments, the inducible promoter is induced by contacting nucleated cells at temperatures above 39°C. In some embodiments, the inducible promoter is induced by contacting nucleated cells at temperatures above 40°C. In some embodiments, examples of inducible promoters include, but are not limited to, heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), growth arrest and DNA damage-inducible gene 153 (GADD153), multidrug resistance mutation 1 (MDR1), or cytomegalovirus (HSE-CMV).

[0153] In some embodiments, the inducible promoter is induced by contacting nucleated cells with a molecule. In some embodiments, examples of molecules include, but are not limited to, rtTA, TRE, TetR, Kumat, rapamycin, abscisic acid, IPTG, or metallothionein.

[0154] In some embodiments, the inducible promoter is induced by exposing nucleated cells to light. In some embodiments, examples of inducible promoters include, but are not limited to, CIB1-CRY2 or GAL4-VVD.

[0155] In some embodiments, the inducible promoter is induced by contacting nucleated cells with the hormone. In some embodiments, an example of an inducible promoter includes, but is not limited to, estradiol-Gal4.

[0156] In some embodiments, the promoter comprises a constitutively active promoter. The promoter is continuously active, but suicide is induced under certain circumstances. In some embodiments, the constitutively active promoter is configured to activate the transcription of a heterologous polynucleotide under conditions sufficient to express a heterologous gene product. In some embodiments, the enucleated cells described herein can be preserved by cryopreservation, cryopreservation, lyophilization, or a combination thereof. In some embodiments, cryopreserved enucleated cells are as viable after thawing as otherwise equivalent, non-cryopreserved enucleated cells. In some embodiments, lyophilized enucleated cells are as viable as otherwise equivalent, non-lyophilized enucleated cells. In some embodiments, cryopreserved enucleated cells are as viable as otherwise equivalent, non-cryopreserved enucleated cells.

[0157] Treatment method In some embodiments, methods for using the enucleated cells, compositions, or pharmaceutical compositions described herein to treat a disease or condition are disclosed herein. In some embodiments, the method includes treating a target disease or condition by administering a composition described herein (for example, a pharmaceutical composition containing enucleated cells manipulated to express a therapeutic agent) to a target.

[0158] This disclosure also provides methods for using enucleated cells (natural or enucleated) as fusion partners with other cells (therapeutic or natural) to enhance and / or transport biomolecules described herein (e.g., therapeutic agents). In some embodiments, biomolecules include DNA / genes, RNA (mRNA, shRNA, siRNA, miRNA), nanoparticles, peptides, proteins, plasmids, bacteria, viruses, small molecule drugs, ions, cytokines, growth factors, and hormones. In some embodiments, enucleated cells are engineered to express a fusionable moiety. The fusionable moiety can be any biomolecule (e.g., sugars, lipids, or proteins) that facilitates membrane fusion. In some embodiments, the fusionable moiety is a fusionable protein. The fusionable protein enables enucleated cells expressing the fusionable protein to fuse with target cells. In some embodiments, the fusionable protein facilitates the integration of enucleated cells expressing the fusionable protein with target cells, allowing the contents of the enucleated cell to enter the target cell. In some embodiments, the fusionable protein is heteromorphic (e.g., viral classes I-III or HAP2 / GCS1 or SNARE). In some embodiments, the fusion protein is homoreptic, such as EFF-1 / AFF-1. Other non-limiting examples of the fusion protein 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-F+MV-Ed-H (hemagglutinin). Unlike nucleated cells, fusion of enucleated cells with another cell type of the same or similar or different origin produces a unique cell hybrid that lacks the problematic nuclear transfer while maintaining the desired therapeutic attributes. These desired therapeutic attributes include, but are not limited to, cell surface proteins, signaling molecules, secreted proteins, and epigenetic changes.

[0159] subject When performing the treatment or use methods provided herein, a therapeutically effective dose of the pharmaceutical composition described herein is administered to a mammal having the disease, disorder, or condition to be treated (e.g., cancer). In some embodiments, the mammal is a human. The therapeutically effective dose may vary significantly depending on the severity of the disease, the age and relative health status of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents, and optionally the compositions described herein, may be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0160] Disease or condition Methods for treating a disease or condition in a subject by administering a composition described herein (e.g., a pharmaceutical composition containing enucleated cells engineered to express a therapeutic agent) to the subject are provided herein. Methods for treating cancer in a subject by administering a composition described herein (e.g., a pharmaceutical composition containing enucleated cells engineered to express a therapeutic agent) to the subject are provided herein. Methods for treating lung disease in a subject by administering a composition described herein (e.g., a pharmaceutical composition containing enucleated cells engineered to express a therapeutic agent) to the subject are provided herein. In some embodiments, administration is carried out by any preferred mode of administration, including systemic administration (e.g., intravenous, inhalation, etc.). In some embodiments, the subject is human.

[0161] In some embodiments, the disease or condition includes infectious diseases (e.g., human immunodeficiency virus (HIV) infection, Chagas disease, tuberculosis), neurological diseases (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease), autoimmune diseases (e.g., diabetes mellitus, Crohn's disease, multiple sclerosis, sickle cell anemia), cardiovascular diseases (e.g., acute myocardial infarction, heart failure, refractory angina), eye diseases, skeletal diseases, metabolic diseases (e.g., phenylketonuria, glycogen storage deficiency type 1A, Gaucher disease), inflammatory diseases (e.g., cancer, inflammatory bowel disease), or diseases caused by external pathogens or toxins in the subject. In some embodiments, the disease or condition includes idiopathic pulmonary fibrosis. In some embodiments, the subject is in need of, or is determined to need, such enucleated cell treatment.

[0162] In some embodiments, the cancer may be lung cancer including non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), or any other type of lung cancer. For example, lung cancer may include adenocarcinoma, squamous cell carcinoma, large cell (undifferentiated) carcinoma, large cell neuroendocrine carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, pulmonary carcinoid 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, colorectal cancer, prostate cancer, sarcoma, bladder cancer, neuroblastoma, and Wilms' tumor.

[0163] In some embodiments, the enucleated cells described herein include a targeting moiety described herein for binding to an epitope expressed by cancer cells or an epitope associated with the tumor microenvironment. In some embodiments, the targeting moiety includes an antibody or an antigen-binding fragment described herein. In some embodiments, the antibody or antigen-binding fragment includes a single-domain antibody. In some embodiments, the antibody or antigen-binding fragment binds to an epitope expressed by cancer cells or an epitope associated with the tumor microenvironment. In some embodiments, the binding of the targeting moiety (e.g., an antibody or an antigen-binding fragment) to the epitope results in a therapeutic effect that treats cancer in the target. In some embodiments, the binding of the targeting moiety (e.g., an antibody or an antigen-binding fragment) to the epitope recruits immune cells and activates an immune response against cancer.

[0164] In some embodiments, enucleated cells and methods for treating diseases or conditions associated with abnormal vascular systems in a subject using these enucleated cells are described herein. Abnormal vascular systems may be associated with diseases or conditions such as inflammation and cancer (e.g., any one of the cancers described herein). In some embodiments, when the enucleated cells described herein come into contact with an abnormal vascular system, they enhance the normalization of the abnormal vascular system, improving endothelial cell adhesion and preventing leakage of endovascular factors from the vascular system. In some embodiments, the normalization of the abnormal vascular system includes a reduction in damage such as cell death of endothelial cells of the vascular system. In some embodiments, the normalization of the abnormal vascular system includes angiogenesis of immature or leaky blood vessels. In some embodiments, the normalization exerted by enucleated cells may include the normalization of blood vessels, lymphatic vessels, or a combination thereof.

[0165] In some embodiments, the disease or condition may be caused by a pathogen. In some embodiments, the enucleated cells described herein include an antibody or its antigen-binding fragment or a single-domain antibody that binds to an epitope expressed by the pathogen or to an epitope associated with the pathogen-related microenvironment. In some cases, the binding of the antibody or its antigen-binding fragment or a single-domain antibody to the epitope confers therapeutic properties against the pathogen. In some embodiments, the binding of the antibody or its antigen-binding fragment or a single-domain antibody to the epitope mobilizes immune cells and activates an immune response that confers therapeutic properties against the pathogen. For example, the disease or condition may be caused by a virus, bacteria, fungi, parasites, or molecules produced by detoxification. In some embodiments, the pathogen may be able to spread or be transmitted from person to person, have a high mortality rate and have a significant public health impact, may cause mass panic and social disruption and require special measures for public health preparedness. Examples of these pathogens may include anthrax (Bacillus anthracis), botulism (Clostridium botulinum toxin), plague (Bacillus yersinus), smallpox (Vaccinium pallidum), tularemia (Bacillus tularensis), or viral hemorrhagic fever (including filoviruses (Ebola, Marburg) and arenaviruses (Lassa, Machupo)).

[0166] In some embodiments, the pathogen may spread, resulting in moderate morbidity and low mortality rates, necessitating specific enhancements to diagnostic capabilities and improved disease surveillance. Examples of these pathogens may include brucellosis (Brucella species), epsilon toxin from Clostridium perfringens, threats to food safety (e.g., Salmonella species, Escherichia coli O157:H7, or Shigella), glanders (Glandworm), meridian (Meridian), psittacosis (Chlamydia psittaci), Q fever (Coxiella barneti), lysine toxin from castor bean, Staphylococcus enterotoxin B, typhus (Rickettsia prowazekii), viral encephalitis (alphaviruses, including eastern equine encephalitis, Venezuelan equine encephalitis, and western equine encephalitis), or threats to water safety (e.g., Vibrio cholerae, Cryptosporidium parvum).

[0167] In some embodiments, the pathogens may include emerging pathogens that are likely to cause death and morbidity, but whose extent is not fully understood. Non-limiting examples of these pathogens may include Nipah virus and Hantavirus, among others.

[0168] The enucleated cells described herein, or compositions containing such enucleated cells (hereinafter referred to as “Compositions” in this section), may be administered to subjects in a suitable dose, mode of administration, and frequency, depending on the intended effect.

[0169] In some embodiments, the composition is administered at least once over a period of time (e.g., every two days, twice a week, once a week, weekly, three times a month, twice a month, once a month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or once a year). In some embodiments, the composition is administered two or more times within a period of time (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times).

[0170] In some embodiments, the composition is administered in therapeutically effective doses by various forms and routes, including, for example, oral or topical administration. In some embodiments, the composition may be administered parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, subarachnoidally, intraocularly, intrasternally, ophthalmologically, endothelially, topically, intranasally, intrapulmonaryly, rectally, intraarterially, intrathecally, by inhalation, intrafocally, intradermally, epidurally, intracapsularly, subcardiacly, transtracheally, subepidermally, subarachnoidally, or intraspinally (e.g., by injection or infusion). In some embodiments, the composition may be administered by absorption through the epithelium or mucocutaneous surface (e.g., oral mucosa, rectal, and intestinal mucosa). In some embodiments, the composition is delivered via multiple routes of administration.

[0171] In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the composition is administered by slow, continuous infusion over a long period (e.g., more than 24 hours). In some embodiments, the composition is administered by intravenous injection or short-duration infusion.

[0172] The composition may be administered topically (for example, by direct injection of the agent into an organ), but may optionally be administered as a depot, 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 formulation may result in immediate release. The sustained-release formulation may result in controlled release or sustained delayed release. In some embodiments, a pump may be used for delivery of the composition. In some embodiments, a pen-type delivery device may be used, for example, for subcutaneous delivery of the composition of this disclosure. The composition provided herein may be administered in combination with other therapeutic agents, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents.

[0173] The composition (for example, the enucleated cells described herein, or a pharmaceutical composition containing enucleated cells) may be administered before, during, or after the onset of a disease or condition, and the timing of administration of the composition containing the therapeutic agent may vary. In some cases, the composition may be used as a prophylactic agent and may be administered continuously to subjects who are susceptible to coronavirus or have a tendency toward coronavirus-related conditions or diseases (for example, subjects receiving immunization or treatment). Prophylactic administration may reduce the likelihood of developing an infection, disease, or condition, or reduce the severity of an infection, disease, or condition.

[0174] The composition may be administered to a subject before the onset of symptoms. The composition may be administered to a subject (e.g., an immunization subject or a treatment subject) after a test result (e.g., as early as possible thereafter), examples of such test results include a test result that provides a diagnosis, a test that indicates the presence of coronavirus in the subject (e.g., an immunization subject or a treatment subject), or a test result that indicates the progression of the disease (e.g., a decrease in blood oxygen levels). The composition may be administered after the onset of the disease or condition has been detected or suspected (e.g., as early as possible thereafter). The composition may be administered after potential exposure to coronavirus (e.g., as early as possible thereafter), examples of which include administration after the subject (e.g., an immunization subject or a treatment subject) has come into contact with an infected subject or has come into contact with an infected subject that may be contagious by contact.

[0175] The actual dosage level of the agents of this disclosure (e.g., antibodies or their antigen-binding fragments, or therapeutic agents) may be modified to obtain an amount of the agent that is non-toxic to the subject (e.g., an immunization subject, or a treatment subject) and achieves the desired treatment response for a particular subject, composition, and mode of administration. The selected dosage level may depend on a variety of pharmacokinetic factors, including the activity of the particular composition employed herein, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors well known in the medical technology.

[0176] The drug regimen may be adjusted to obtain the optimal desired response (e.g., a therapeutic response and / or a prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over a long period, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions into unit dosage forms. As used herein, unit dosage forms refer to physically distinct units suitable as unit doses for a subject (e.g., an immunization subject or a treatment subject), each unit containing a predetermined amount of activator calculated to produce a desired therapeutic effect in conjunction with the necessary pharmaceutical carrier. The specifications of the unit dosage forms in this disclosure may be determined by, or directly depend on, (a) the inherent properties of the activator and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the art of formulating such activators for the treatment of susceptibility in an individual. The dose may be determined by reference to the plasma or local concentration of the cyclic polyribonucleotide or antibody or its antigen-binding fragment. The dose may be determined by referring to the plasma or local concentration of linear polyribonucleotides, antibodies, or their antigen-binding fragments.

[0177] The compositions described herein may be in unit dosage forms suitable for single-dose administration of precise dosages. In unit dosage forms, the formulation may be divided into unit doses containing appropriate amounts of the composition. In unit dosage forms, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies or antigen-binding fragments, and / or therapeutic agents. Unit doses may be in the form of packaging containing individual amounts of the formulation. Non-limiting examples include packaged injectable drugs, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in non-resealable single-dose containers. Resealable multi-dose containers may be used, for example, with or without preservatives. The injectable formulations disclosed herein may be present in unit dosage forms, examples of which include ampoules or multi-dose containers containing preservatives.

[0178] The dose may be based on the amount of the agent per kilogram of body weight of the subject (e.g., the subject to immunization or treatment). The dose of the agent (e.g., antibody) is in the range of 10 to 3000 mg / kg, for example, 100 to 2000 mg / kg (e.g., 300 to 500 mg / kg / day for 1 to 10 days or 1 to 5 days, e.g., 400 mg / kg / day for 3 to 6 days, e.g., 1 g / kg / day for 2 to 3 days). In some embodiments, the dose may be based on the number of enucleated cells per kilogram of body weight of the subject. In some embodiments, the dose may be administered in amounts ranging from approximately 1,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose is approximately 1,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000 cells / kg body weight to approximately 10,000 cells / kg body weight, approximately 1,000 cells / kg body weight to approximately 100,000 cells / kg body weight, approximately 1,000 cells / kg body weight to approximately 1,000,000 cells / kg body weight, and approximately 1,000 cells / kg body weight to approximately 10,000,000 cells / kg body weight. 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 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 ~ approx. 1,000, 000,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 100,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 1,000,000 cells / kg body weight, approx. 10 ,000 cells / kg body weight ~ approx. 10,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 100,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 1,000 ,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 10,000 cells / kg body weight ~ approximately 100,000,000,000 cells 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 1,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 10,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 100,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 10,000,000 cells / kg body weight 0,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 100,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 1,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 10,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 100,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight to approximately 1,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight to approximately 100,000,000 cells / kg body weight Cells / kg body weight, approximately 10,000,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight ~ approximately 100,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight 0,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight ~ approximately 100,000,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight ~ approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 10,000,The drug may be administered at dosages of approximately 1,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, approximately 10,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, or approximately 100,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose may be administered in amounts of approximately 1,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000 cells / kg body weight, approximately 10,000 cells / kg body weight, approximately 100,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight, approximately 10,000,000,000 cells / kg body weight, or approximately 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose may be administered in amounts ranging from about 1,000 cells / kg body weight to about 1,000,000,000,000 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 100,000,000,000 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 approximately 1,000 cells / kg body weight to approximately 1,000,000,000,000 cells / kg body weight, up to approximately 10,000 cells / kg body weight, approximately 100,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight, approximately 100,000,000 cells / kg body weight, approximately 1,000,000,000 cells / kg body weight, approximately 10,000,000,000 cells / kg body weight, approximately 100,000,000,000 cells / kg body weight or approximately 1,000,000,000,The drug may be administered at a dose of 000 cells / kg body weight. In some embodiments, the non-nucleated cells are administered to the subject twice within the following periods, which are 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.

[0179] Methods for repeatedly administering a composition or pharmaceutical composition to a subject in need thereof are disclosed herein. In some embodiments, an initial administration of the composition or pharmaceutical composition containing enucleated cells normalizes blood vessels or lymphatic vessels. In some embodiments, the same composition or pharmaceutical composition containing enucleated cells may be subsequently administered to the subject for the purpose of maintaining the normalization of blood vessels or lymphatic vessels and delivering exogenous factors for treating the disease or condition described herein.

[0180] kit In some embodiments, kits for using the compositions described herein are disclosed herein. In some embodiments, the kits disclosed herein may be used to treat a disease or condition in a subject. In some embodiments, the kit includes an assembly of materials or components in addition to the compositions. In some embodiments, the kit includes nucleated cells described herein (e.g., nucleated cells engineered to express a targeting portion (e.g., an antibody or its antigen-binding fragment), a therapeutic agent, a transmembrane portion, an immune-evading portion, a heterologous gene product, or a combination thereof). In some embodiments, the kit may include a population of cells disclosed herein. In some embodiments, the kit may include enucleated cells obtained from nucleated cells. In some embodiments, the kit may include a mixed population of nucleated cells and enucleated cells obtained from nucleated cells. In some embodiments, the kit may include a substantially pure population of enucleated cells. In some embodiments, the kit includes nucleated cells, enucleated cells, or a combination thereof suspended in at least one density gradient.

[0181] In some embodiments, the kit comprises a pharmaceutical formulation disclosed herein, which comprises enucleated cells, which are engineered to express (and possibly secrete) a targeting moiety (e.g., an antibody or its antigen-binding fragment), a therapeutic agent, a transmembrane moiety, an immune-evading moiety, a heterogeneous gene product, or a combination thereof. In some embodiments, the enucleated cells express or secrete a therapeutic agent, such as an immune checkpoint molecule or immune checkpoint inhibitor for treating a disease or condition in a subject. 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 its antigen-binding fragment, which, once administered, allows the enucleated cells to efficiently migrate to a target tissue within the subject. 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 a subject for treating a disease or condition (such as cancer). In some embodiments, cancer comprises cancer of lung tissue. In some embodiments, cancer is lung cancer.

[0182] In some embodiments, the kit includes components for purifying enucleated cells from nucleated cells or other cell debris. For example, the kit may include filter membranes with different pore sizes for isolating and purifying enucleated cells. In some embodiments, the kit includes components for staining and selecting enucleated cells. For example, the kit may include a fluorescent dye for staining the nucleus. Here, nucleated cells can be stained and selectively removed, leaving a population of enucleated cells.

[0183] In some embodiments, the kit described herein includes components for selecting a homogeneous population of enucleated cells. In some embodiments, the kit described herein includes components for selecting a heterogeneous population of enucleated cells. In some embodiments, the kit includes components for assaying the number of units of biomolecules (e.g., therapeutic agents) synthesized and / or released or expressed on the surface by enucleated cells. In some embodiments, the kit includes components for performing assays, examples of which include enzyme-linked immunosorbent assay (ELISA), single-molecule array (Simoa), PCR, and qPCR. The exact nature of the components comprising the kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating diseases or conditions disclosed herein (e.g., cancer) in a subject. In some embodiments, the kit is configured specifically for treating mammalian subjects. In some embodiments, the kit is configured specifically for treating human subjects.

[0184] A container may be included in the kit. Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the composition to a subject that requires it. 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 a composition that may have been frozen, freeze-dried, or cryopreserved during storage or transport, or otherwise restoring its biological activity. 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).

[0185] Optionally, the kit may also contain other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandage materials, or other useful instruments. The materials or components assembled in the kit may be provided to the practitioner in any convenient and suitable manner that maintains their applicability and usefulness. For example, components may be in a dissolved, dehydrated, or lyophilized form and may be provided at room temperature, refrigerated, or frozen. Components may be housed in suitable packaging materials.

[0186] definition The use of absolute or sequential terms, such as “to do,” “not to do,” “shall do,” “shall not do,” “must do,” “must not do,” “firstly,” “initially,” “nextly,” “following,” “before,” “after,” “finally,” and “ultimately,” is intended to be illustrative and not to limit the scope of the embodiments disclosed herein.

[0187] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context otherwise explicitly indicates. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” and “with,” or their variants, are used in any of the detailed descriptions and / or claims, such terms are intended to be inclusive in a similar manner to the term “equipped with.”

[0188] As used herein, the terms “at least one,” “one or more,” and “and / or” are open-ended expressions that have the function of both logical AND and logical OR in an operation. For example, 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” mean A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together, respectively.

[0189] As used herein, “or” may refer to “and,” “or,” or “and / or,” and may be used both exclusively and comprehensively. For example, the term “A or B” may refer to “A or B,” “A but not B,” “B but not A,” and “A and B.” In some cases, the specific meaning may be determined by the context.

[0190] Any systems, methods, software, platforms, compositions, pharmaceutical compositions, and kits described herein are modular. Therefore, terms such as “First” and “Second” do not necessarily imply priority, order of importance, or order of actions.

[0191] When referring to a number or range of numbers, the term "approximately" means that the number or range being referenced is an approximation within experimental variation (or within the statistical error margin), and that the number or range may vary, for example, by 1% to 15% of the stated number or range. In the example, "approximately" refers to ±10% of the stated number or value.

[0192] In this specification, the terms “increased,” “increasing,” or “growing” are generally used to mean an increase of a statically significant amount. In some embodiments, the terms “increased” or “growing” mean an increase of at least 10% compared to a reference level, examples of which include an increase of 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 an increase up to 100%, including 100%, or any increase between 10% and 100%. Other examples of “increase” include an increase of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times or more compared to a reference level.

[0193] In this specification, the terms “reduced,” “decreasing,” or “declining” are generally used to mean a statistically significant reduction. In some embodiments, “reduced” or “declining” means a decrease of at least 10% compared to a reference level, examples of which include a decrease of 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 a decrease up to 100% including 100% (e.g., a level that is absent or undetectable compared to a reference level), or any decrease between 10% and 100%. In relation to markers or symptoms, these terms mean a statistically significant decrease to such a level. The decrease may be, for example, at least 10%, at least 20%, at least 30%, or at least 40% or more, and preferably to a level that is acceptable as within the normal range for a given disease-free individual. Other examples of “decrease” include a decrease of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times or more compared to the reference level.

[0194] The terms “individual” and “subject” are used interchangeably and include mammals. Non-restrictive examples of mammals include any member of the mammalian class, namely humans, non-human primates (such as chimpanzees, other apes and monkey species); domesticated animals (such as cattle, horses, sheep, goats, and pigs); pet animals (such as rabbits, dogs, and cats); laboratory animals including rodents (such as rats, mice, and guinea pigs), and similar species. Mammals may also be humans. The term “animal” as used herein includes humans and non-human animals. In one embodiment, “non-human animal” is a mammal, and examples include rodents (such as rats or mice). The term “patient” as used herein means a subject who is suffering from or has been diagnosed with one of the diseases or conditions described herein.

[0195] As used herein, the term “immune evasion moiety” refers to a signaling peptide or a portion thereof that reduces the phagocytic activity of cells through interaction with signal receptor proteins expressed by phagocytic cells (such as macrophages and dendritic cells). In some embodiments, the immune evasion moiety blocks the recognition or activation of immune cells.

[0196] As used herein, the term “targeting portion” refers to an entity that guides cells, such as enucleated cells, to a target tissue or target cell. The targeting portion can be substantially any biomolecule, including proteins, polypeptides, sugars, nucleic acids, or small molecules, or portions thereof.

[0197] As used herein, the term “transmembrane portion” refers to an entity that crosses (at least partially) the cell membrane of a cell (e.g., an enucleated cell).

[0198] The terms “expression” or “to express” refer to one or more processes in which a polynucleotide is transcribed from a DNA template (such as mRNA or other RNA transcripts), and / or the process in which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides are sometimes collectively referred to as “gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. With respect to expression, “upregulation” generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the expression level in the wild-type state, and “downregulation” generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence compared to the expression level in the wild-type state.

[0199] As used herein, “cell” generally refers to a living cell.

[0200] As used herein, "enucleation" refers to the process of making a cell non-replicating, which includes, for example, the removal of the nucleus.

[0201] As used herein, the terms “cytoplasm,” “non-nucleated cell,” or “enucleated cell” are used interchangeably to refer to a non-nucleated cell obtained from a previously nucleated cell (e.g., any cell described herein). In some embodiments, the nucleated cell contains organelles, and the cytoplasm derived from the nucleated cell holds such organelles and, in some cases, enables cellular functions (such as cell movement, protein synthesis, and protein secretion). In some embodiments, “obtaining” does not involve differentiating a nucleated cell into an enucleated cell using natural processes or other methods.

[0202] As used herein, the term “gene” refers to a segment of nucleic acid (also called a “coding sequence” or “coding region”) that encodes an individual protein or RNA, along with optionally associated regulatory regions, such as promoters, operators, and terminators, which may be located upstream or downstream of the coding sequence. The term “gene” should be interpreted broadly and may encompass the mRNA, cDNA, genomic DNA, and other forms of genes.

[0203] In some usages, the term “gene” encompasses the transcribed sequence, including the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region may contain an “open reading frame” that encodes the polypeptide. In some uses of the term, “gene” includes only the coding sequence (e.g., “open reading frame” or “coding region”) necessary to encode the polypeptide. In some embodiments, a gene does not encode a polypeptide; for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some embodiments, the term “gene” includes not only the transcribed sequence but also the untranscribed region, including upstream and downstream regulatory regions, enhancers, and promoters. The term “gene” may encompass the mRNA, cDNA, and genomic form of a gene.

[0204] The term "packaging material" refers to one or more physical structures used to contain the contents of a kit (e.g., compositions). The packaging material is constructed in a well-known manner, preferably to provide a sterile environment free from contaminants. The packaging material employed in the kit is one that is conventionally used in gene expression assays and the administration of treatments.

[0205] As used herein, the term “packaging” refers to a suitable solid matrix or material capable of holding individual kit components, such as glass, plastic, paper, or foil. For example, packaging may be a glass vial or pre-filled syringe used to contain a suitable amount of pharmaceutical product. The packaging material may have an external label indicating the contents and / or purpose of the kit and its components.

[0206] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably to refer to a multimeric form of a nucleotide of any length, either deoxyribonucleotide or ribonucleotide, or an analogue thereof (which may be in single-stranded, double-stranded, or multi-stranded form). Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. A polynucleotide may contain one or more analogues (e.g., a modified skeleton, sugar, or nucleic acid base). Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small 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 may be interrupted by non-nucleotide components.

[0207] As used herein, the terms “polypeptide,” “peptide,” and “protein” may be used interchangeably with respect to polymers 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 into a mature form; however, polypeptides or peptides may still refer to a degraded or processed fragment of a protein that is uniquely or identifiablely mapped to a particular protein. A polypeptide may be a single linear polymer chain of amino acids linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Polypeptides may be modified, for example, by carbohydrate addition, phosphorylation, etc.

[0208] As used herein, the terms “fragment” or “part,” or equivalent terms, may refer to a portion of an entity having a length less than the full length of the entity and optionally retaining the function of the entity. In some embodiments, the entity is a protein.

[0209] As used herein, the terms “complement,” “multiple complements,” “complementary,” and “complementarity” generally refer to sequences that are fully complementary to and hybridizable to a given sequence. In some cases, a sequence hybridized with a given nucleic acid is called a “complementary sequence” or “reverse complementary sequence” of a particular molecule if its sequence of bases across a given region is capable of complementaryly binding the bases of its binding partner, resulting in the formation of, for example, AT, AU, GC, and GU base pairs. Generally, a first sequence hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, and as a result, hybridization with the second sequence, or a set of second sequences, is preferred over hybridization with a non-target sequence during the hybridization reaction (e.g., thermodynamically more stable under a given set of conditions (such as the strict conditions used in the relevant field)). Typically, hybridizable sequences share some degree of sequence complementarity (such as 25% to 100% complementarity) across their entire or partial lengths, including sequences with approximately equal or greater complementarity of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Sequence identity (for example, to assess the percentage of complementarity) may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Bunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, with optional use of default settings) and the BLAST algorithm. Optimal alignment may be evaluated using any suitable parameters (including default parameters) of the selected algorithm.

[0210] As used herein, the term “percent (%) identity” generally refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after the sequences have been aligned and gaps introduced as necessary to achieve maximum percentage identity (for example, gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Alignment for the purpose of determining percentage identity may be achieved by various methods known in the relevant art. Percent identity of two sequences may be calculated by aligning the test sequence and the comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides at the same position 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.

[0211] As used herein, the term "in vivo" may be used to describe events occurring within a living organism, such as the body of a subject.

[0212] As used herein, the term “ex vivo” may be used to describe events occurring outside the organism, such as in vitro. “Ex vivo” assays cannot be performed on the subject; rather, they may be performed on a sample other than the subject. “Ex vivo” may be used to describe events occurring in intact cells outside the subject.

[0213] As used herein, the term “in vitro” may be used to describe an event that occurs within a container for holding experimental reagents and, as a result, is isolated from a living biological source (from which the material is derived). In vitro assays may encompass cell-based assays in which viable or dead cells are employed. In vitro assays may also encompass cell-free assays in which intact cells are not employed.

[0214] As used herein, “to treat,” “treating,” or “treatment” means to reduce or eliminate a disorder, disease, or condition, or to reduce or eliminate one or more symptoms associated with a disorder, disease, or condition, or to reduce or eliminate the cause of the disorder, disease, or condition itself. The desired effects of treatment may include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving the condition or relieving pain, and achieving remission or improving the prognosis.

[0215] The terms “effective amount” and “therapeutic effective amount,” as used interchangeably herein, generally refer to an amount of a composition, such as a composition containing immune cells, such as lymphocytes (e.g., T lymphocytes and / or NK cells) comprising the system of the Disclosure, such amount is sufficient to produce the desired activity when administered to a target requiring administration. In the context of the Disclosure, the term “therapeutic effective” refers to an amount of a composition sufficient to delay, halt the progression of, or alleviate or reduce at least one symptom of a disorder treated by the method of the Disclosure.

[0216] The terms “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refer to a pharmaceutically acceptable material, composition, or vehicle (such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material). A component may be “pharmaceutically acceptable” in the sense that it is compatible with other components of a pharmaceutical formulation. It may also be suitable for use in contact with human and animal tissues or organs, provided that the benefit-risk ratio is reasonable and without causing excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications.

[0217] As used herein, the terms “administer,” “administer,” and their variants mean introducing a composition or agent to a subject, including simultaneous and sequential introduction of the composition or agent. Introduction of a composition or agent to a subject is carried out by any preferred route, including oral, intrapulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, lymphatic, or topical. Administration includes self-administration and administration by another person. The preferred route of administration allows the composition or agent to exert its intended function. For example, if the preferred route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. Administration may be carried out by any preferred route. In some embodiments, administration is intravenous. In some embodiments, administration is pulmonary. In some embodiments, administration is inhalation.

[0218] The term "pharmaceutical composition" refers to a mixture of the compositions disclosed herein with other chemical components, examples of which include diluents or carriers (e.g., pharmaceutically acceptable inert components), and examples of which include carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, wetting agents, defoamers, antioxidants, preservatives, or one or more of these. Pharmaceutical compositions may facilitate the administration of compositions to living organisms. In the art, there are several techniques for administering compounds, including, but not limited to, oral, injectable, aerosol, parenteral, and topical administration.

[0219] As used herein, the term “fusion protein” refers to polypeptides that, when expressed on the surface of cells such as enucleated cells, promote intercellular membrane fusion of cells expressing fusion proteins and target cells.

[0220] While preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. The present invention is not limited by the specific examples shown herein. Although the present invention is described with reference to the preceding specification, the descriptions and illustrations of embodiments herein should not be interpreted restrictively. Thus, those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions that depend on various conditions and variables as described herein. When practicing the present invention, it should be understood that various alternative forms to the embodiments of the present invention described herein may be adopted. Therefore, it is assumed that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention and are intended to encompass the methods and structures within these claims, as well as their equivalents.

[0221] Embodiment Embodiment 1. A method for obtaining a population of cells from a plurality of cells, comprising contacting the plurality of cells with a polysaccharide density gradient generated by fixed-angle centrifugation, wherein the population of cells accumulates within a density range in the polysaccharide density gradient.

[0222] Embodiment 2. A method for denucleating a population of cells from a plurality of cells, comprising: contacting the plurality of cells with a toxin that inhibits cytoskeleton formation of the plurality of cells; and contacting the plurality of cells with a polysaccharide density gradient generated by fixed-angle centrifugation, wherein the population of cells is denucleated by fixed-angle centrifugation and accumulates within a density range in the polysaccharide density gradient.

[0223] Embodiment 3. The method according to Embodiment 1 or Embodiment 2, further comprising removing the population of cells from the polysaccharide density gradient.

[0224] Embodiment 4. The method according to Embodiment 2, wherein the toxin includes a mycotoxin.

[0225] Embodiment 5. The method according to Embodiment 4, wherein the mycotoxin includes cytochalasin.

[0226] Embodiment 6. The method according to Embodiment 5, wherein the cytochalasin comprises cytochalasin B.

[0227] Embodiment 7. The method according to Embodiment 2, wherein the population of cells after enucleation includes a reduced density compared to the plurality of cells having nuclei.

[0228] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the polysaccharide density gradient before fixed-angle centrifugation comprises a plurality of polysaccharide solutions containing various polysaccharide concentrations.

[0229] Embodiment 9. The method according to any one of Embodiments 1 to 8, wherein the fixed-angle centrifugation comprises centrifugation of the plurality of cells in the polysaccharide density gradient at an angle of 23 degrees, 24 degrees, 25 degrees, 45 degrees, 90 degrees, or 180 degrees.

[0230] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the fixed-angle centrifugal separation includes centrifugal separation at approximately 126,000 RCF.

[0231] Embodiment 11. The method according to any one of Embodiments 1 to 9, wherein the fixed-angle centrifugation includes centrifugation at an average R of approximately 85,000 RCF.

[0232] Embodiment 12. The method according to any one of Embodiments 1 to 9, wherein the fixed-angle centrifugation includes centrifugation at a maximum R value of approximately 127,000 RCF.

[0233] Embodiment 13. The method according to any one of Embodiments 1 to 12, wherein the fixed-angle centrifugal separation includes acceleration, which takes about 5.2 minutes to reach the RCF for centrifugal separation.

[0234] Embodiment 14. The method according to any one of Embodiments 1 to 13, wherein the fixed-angle centrifugal separation includes deceleration, which takes about 4.0 minutes to reach the cessation of the fixed-angle centrifugal separation.

[0235] Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the fixed-angle centrifugal separation includes centrifugal separation for approximately 60 minutes.

[0236] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the polysaccharide density gradient comprises a plurality of polysaccharide solutions.

[0237] Embodiment 17. The method according to Embodiment 16, wherein the plurality of polysaccharide solutions comprises at least three polysaccharide solutions.

[0238] Embodiment 18. The method according to Embodiment 17, wherein the plurality of polysaccharide solutions include a 12.5% ​​polysaccharide solution, a 15% polysaccharide solution, a 16% polysaccharide solution, a 17% polysaccharide solution, a 25% polysaccharide solution, or a combination thereof.

[0239] Embodiment 19. The method according to any one of Embodiments 1 to 18, wherein the population of cells accumulates in the aforementioned density range of approximately 15% polysaccharides.

[0240] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the polysaccharide density gradient includes a Ficol density gradient.

[0241] Embodiment 21. The method according to any one of Embodiments 1 to 20, wherein the plurality of cells contain heterogeneous polynucleotides.

[0242] Embodiment 22. The method according to any one of the prior embodiments, further comprising cryopreserving the population of cells.

[0243] Embodiment 23. The method of Embodiment 22, further comprising thawing the population of cells, wherein, after thawing, the enucleated cells of the population of cells are viable in the same way as otherwise equivalent, uncryopreserved enucleated cells.

[0244] Embodiment 24. The method according to any one of the prior embodiments, wherein the population of cells includes stem cells.

[0245] Embodiment 25. The method according to Embodiment 24, wherein the stem cells include induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, or immortalized cells from a cell line, or a combination thereof.

[0246] Embodiment 26. The method according to Embodiment 25, wherein the stem cells include the mesenchymal stromal cells.

[0247] Embodiment 27. The method according to any one of Embodiments 1 to 23, wherein the population of cells includes immune cells.

[0248] Embodiment 28. The method according to Embodiment 25, wherein the immune cells include lymphocytes or natural killer cells.

[0249] Embodiment 29. The method according to any one of Embodiments 1 to 26, wherein the population of cells comprises one or more intracellular organelles for synthesizing or secreting exogenous polypeptides in the absence of a nucleus.

[0250] Embodiment 30. The method according to Embodiment 29, wherein the exogenous polypeptide is encoded by the heterologous polynucleotide.

[0251] Embodiment 31. The method according to Embodiment 29, wherein the exogenous polypeptide comprises a therapeutic agent.

[0252] Embodiment 32. The method according to any one of Embodiments 1 to 31, wherein the population of cells comprises at least one targeted region.

[0253] Embodiment 33. The method according to any one of Embodiments 1 to 32, wherein the population of cells includes at least one fusionable portion.

[0254] Embodiment 34. The method according to any one of Embodiments 1 to 33, wherein the population of cells includes at least one immune-evading portion.

[0255] Embodiment 35. The method according to any one of Embodiments 1 to 34, wherein the enucleated cells of the cell population have a diameter including about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated cells.

[0256] Embodiment 36. The method according to any one of Embodiments 1 to 34, wherein the enucleated cells of the cell population have diameters including approximately 5 μm or more, approximately 10 μm or more, approximately 20 μm or more, approximately 30 μm or more, approximately 40 μm or more, approximately 50 μm or more, approximately 60 μm or more, approximately 70 μm or more, approximately 80 μm or more, or approximately 90 μm or more.

[0257] Embodiment 37. The method according to Embodiment 36, wherein the diameter includes approximately 8 μm.

[0258] Embodiment 38. A pharmaceutical composition comprising a population of cells described in any one of Embodiments 1 to 37 and a pharmaceutically acceptable excipient, carrier, or diluent.

[0259] Embodiment 39. The pharmaceutical composition of Embodiment 38, wherein the pharmaceutical composition is in unit dose form.

[0260] Embodiment 40. The pharmaceutical composition according to Embodiment 38 or Embodiment 39, which is formulated for administration to a target by intrathecal cavity, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or a combination thereof.

[0261] Embodiment 41. The pharmaceutical composition according to Embodiment 40, wherein the pharmaceutical composition is formulated for intravenous administration.

[0262] Embodiment 42. The pharmaceutical composition according to any one of Embodiments 38 to 41, further comprising at least one additional activator.

[0263] Embodiment 43. The pharmaceutical composition according to Embodiment 42, wherein the at least one additional activator comprises a cytokine, growth factor, hormone, enzyme, small molecule, compound, or combination thereof.

[0264] Embodiment 44. A kit comprising a population of cells according to any one of Embodiments 1 to 37, or a pharmaceutical composition according to any one of Embodiments 38 to 43, and a container.

[0265] Embodiment 45. A method for treating a target disease or condition, comprising administering to the target a therapeutically effective amount of a population of cells described in any one of Embodiments 1 to 37, or a pharmaceutical composition described in any one of Embodiments 38 to 43.

[0266] Embodiment 46. A method for treating cancer in a subject, comprising administering a therapeutically effective amount of a population of cells described in any one of Embodiments 1 to 37, or a pharmaceutical composition described in any one of Embodiments 38 to 43, to the subject having cancer.

[0267] Embodiment 47. A method for treating a lung disease in a subject, comprising administering a therapeutically effective amount of a population of cells described in any one of Embodiments 1 to 37, or a pharmaceutical composition described in any one of Embodiments 38 to 43, to the subject having the lung disease. [Examples]

[0268] The following exemplary examples are representative of, and not intended to limit, embodiments of the stimuli, systems, compositions, and methods described herein.

[0269] Example 1. Production of enucleated cells by fixed-angle centrifugation. material: aMSCs hTERT CCP cells; Ficol PM400; Ultrapure water (Invitrogen; 10977-015); Refractometer (Reichert; 13940000); 10x MEM (Gibco; 11430-030); 7.5% sodium bicarbonate (Gibco; 25080-094); 100x Penstrep (Gibco; 15140-122); 0.22 μm film filter (Olympus; 25-227); Alpha MEM (Gibco; 12561-056); Premium FBS (Atlanta Biologics; S1150); 1M HEPES (Gibco; 15630-80); 100x anti-inflammatory (Gibco; 15240-062); 100x GlutaMAX (Gibco; 35050-061); Cytochalasin B (Cyto B) derived from Drechslera dematioidea; 94ml ultra-clear tube (Beckman); PBS (Gibco; 14190-144); 40μm cell strainer (Falcon; 352340); Type 45Ti rotor; Beckman L8M ultracentrifuge; Stemful low cell adhesion tube (Sbio;MS90150Z); 0.4% trypan blue (Invitrogen; T10282); and Hoechst 33342 (ThermoFisher; H3570) [10 mg / mL stock; 2 μg / mL final; 1:5,000 final] protocol: Generate a stock of double the amount of MEM 1. To prepare 50 mL, 10 mL of 10x MEM, exactly 2.94 mL of NaHCO3, 1 mL of 100x pen / strep, and 36 mL of ultrapure water were added to each 50 mL tube. 2. Filtered through a 0.22 μm filter and then 50 mL aliquots were made and stored at 4 °C for up to 1 month or until precipitation. Preparation of cells for enucleation 1. Seed into 12 - 14 roller bottles so that it can be prepared by the day of enucleation. Day 0 - Prepare enucleation standard solution / gradient 1. 100 μL of [10 mg / mL] Cyto B was added to 400 μL of DMSO to generate a [2 mg / mL] standard solution (residual solution can be stored at 4 °C for up to 2 weeks) 2. 10 μL of 2 mg / mL Cyto B was added per 1 mL of 2x MEM [Final: 20 μg / mL Cyto B] One tube: 500 μL of 2 mg / mL Cyto B into 50 mL of 2x MEM (Table 1)

Table 1

Table 2

[0270] Centrifugation was performed using a Type 45 Ti fixed-angle rotor. After centrifugation, the enucleated cells were located in the 15% ficoll layer of the ficoll gradient (Figure 3A). The number of cells isolated from the 15% ficoll layer was 1.82×10 6 cells, and a 90% yield was obtained for the enucleated cells after enucleation. The enucleated cells were imaged after fixed-angle centrifugation. To show the cell yield after enucleation, the enucleated cells were imaged with a bright-field microscope (Figure 3B, central image). Then, the cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) that stains the nuclei of the cells. The fluorescence microscope image of the DAPI-stained enucleated cells showed that more than 95% of the cells isolated from the 15% ficoll layer after centrifugation did not have nuclei (Figure 3B, right image).

[0271] Example 2. Comparison between fixed-angle centrifugation and swing-bucket centrifugation An example of swing-bucket centrifugation enucleation is described in U.S. Patent Application No. 16 / 636,249, filed on August 7, 2018, which is hereby incorporated by reference in its entirety. For fixed-angle centrifugation enucleation, the cells were grown to a density of 80 - 90% based on the cell type. Then, a ficoll gradient was prepared from 50% ficoll as shown in Table 4. The frozen or cryopreserved 50% ficoll was thawed in a water bath heated to 25 °C for at least 10 minutes until the 50% ficoll was completely thawed. [Table 4]

[0272] The double-layer MEM was removed from the refrigerator at 4°C and placed in a 25°C water bath for at least 15 minutes. Cytochalasin B aliquots (10 mg / mL) were also thawed at room temperature. 120 μl of cytochalasin B (10 mg / mL) was transferred to a new Eppendorf container containing 480 μl of DMSO. The cytochalasin B solution was added to the double-layer MEM to a final concentration of 20 μg / mL (100 μl of 2 mg / mL solution per 9.9 mL of double-layer MEM).

[0273] To prepare 1x MEM, an equal volume of ultrapure water was mixed with 2x MEM. To prepare 25% Ficol, an equal volume of 2x MEM containing cytochalasin B and 50% Ficol were vortexed at 3000 rpm for 30 seconds. To prepare an additional Ficol gradient, 2x MEM and 50% Ficol were mixed to generate a gradient as shown in Table 4. The amount of the Ficol gradient was evaluated by refractive index, and the correction concentration of the Ficol gradient was determined. Subsequently, the Ficol gradient could be superimposed on cells for enucleation. Table 5 shows the number of tubes (e.g., ultraclear tubes) subjected to centrifugation and examples of each Ficol gradient. The first layer of the Ficol gradient loaded into the tube was 25% Ficol at 14.25 mL. The second layer of the Ficol gradient in the tube was 17% Ficol at 14.25 mL. The third layer of the Ficol gradient in the tube was 16% Ficol at 3.56 mL. The fourth layer of the Ficol gradient in the tube was 15% Ficol at 3.56 mL. The fifth layer of the Ficol gradient in the tube was 12.5% ​​Ficol at 14.25 mL. [Table 5]

[0274] After loading the Ficol layers, the top of each ultraclear tube was covered with Parafilm. Each Ficol layer was marked with a marker and incubated overnight at 37°C in a CO2 incubator with 1x MEM, 12.5% ​​Ficol, any excess 25% Ficol, and 2x MEM with the cap loosened. A centrifuge rotor (e.g., Type 45Ti rotor and bucket) was placed in a 37°C incubator without CO2.

[0275] Cells (84-177 × 10⁶ per Ficol gradient tube) 6 Collect MSCs and resuspend them in PBS to increase the cell concentration to 1-5 × 10⁻⁶. 6 The cells were diluted to ml, and the number and viability were determined by trypan blue assay. Approximately 84-177 × 10⁴ 6 Cells were loaded into 50 mL conical tubes. The cells were washed and resuspended in 12.5% ​​Ficol. The cells were then filtered through a strainer and added dropwise to a new 50 mL conical tube. The number of cells that passed through the filter and their viability were determined again by trypan blue assay.

[0276] Using a 3.2 mL syringe with a 21 gauge needle, 3 mL of 12.5% ​​Ficol containing cells was collected and placed on top of the Ficol in an ultraclear tube, with the needle beveled against the side of the tube so that it was approximately 1 cm above the liquid surface. The 12.5% ​​Ficol containing cells was then slowly released at a rate of 1 mL per 15 seconds. A total of 6 mL of 12.5% ​​Ficol containing cells was added to each tube. After loading the cells, 10.35 mL to 16.35 mL of 1x MEM was added to each tube to balance the weight of each tube to within ±0.05 g for centrifugation. The tubes containing cells were then centrifuged in an ultracentrifuge at 33,000 rpm (or 85,000 RCF) for 60 minutes. Accelerated to 7, decelerated to 7.

[0277] Once the ultracentrifuge operation was complete, each tube was divided into four layers: the upper 12.5% ​​with some cells and a large amount of cell debris; the 12.5 / 15% interface with a large number of enucleated cells; 17% (a large or small number of enucleated cells); and the lower 25% with nucleated cells and free nuclei. The layer with enucleated cells was collected, washed, and pelleted in a conical tube. The enucleated cells were then resuspended for additional assays and the Hoechst assay to determine the enucleation efficiency. The enucleated cells were also cultured and attached to cell culture dishes or plates for additional assays.

[0278] Next, the ability of enucleated cells to secrete IL-12 to exert therapeutic efficacy was either immediately evaluated, or they were first cryopreserved, thawed, and then evaluated for IL-12 secretion to exert therapeutic efficacy. In the in vitro IL-12 secretion assay, nucleated and enucleated MSCs (human Wharton's jelly cells or human umbilical cord cells) were placed in antibiotic-free culture medium at a rate of 1 × 10⁶ 6 The cells were resuspended to the desired cell / ml level. Then, following the manufacturer's protocol, a total of 1 ug of mouse IL-12 mRNA was mixed with MessengerMAX in OptiMEM and incubated at room temperature for 5 minutes. The mRNA-MessengerMAX mixture was added to the cells and incubated for a further 30 minutes in a 37°C, 5% CO2 incubator. The cells were washed once and seeded into 24 wells at a rate of 25,000 cells per well. The acclimatization medium was collected and analyzed every 24 hours.

[0279] Table 6 shows a comparison of enucleation of human adipose-derived mesenchymal stem cells (MSCs) using two different rotors (swing bucket (SW41) and fixed-angle (Type 45Ti)). Average data were collected from at least three runs. As shown in Table 6, fixed-angle centrifugation yielded more cells per run (852 × 10⁶). 6 Although it was only a single cell, the swing bucket centrifugation used for comparison was 120 × 10⁶ 6Enucleation of the cells (which were [number] cells) was possible. The mean RCF value was 85,000 for both fixed-angle centrifugation and swing-bucket centrifugation. The maximum RCF was 126,847 for fixed-angle centrifugation. The maximum RCF was 115,839 for swing-bucket centrifugation. Fixed-angle centrifugation also increased the percentage of enucleated cells per run (65%, compared to 50% for the comparative swing-bucket centrifugation). Similar enucleation efficiencies were obtained with both fixed-angle centrifugation and swing-bucket centrifugation. [Table 6]

[0280] While the foregoing disclosure is described in some detail for clarity and comprehensibility, it will be apparent to those skilled in the art that various modifications may be made to the form and details without departing from the true scope of this disclosure. For example, all the techniques and apparatus described above may be used in various combinations. All publications, patents, patent applications, and / or other documents referenced in this application are incorporated by reference as a whole for all purposes, to the same extent as if each of the publications, patents, patent applications, and / or other documents were individually and separately indicated to be incorporated by reference for all purposes.

Claims

1. A method for obtaining a population of enucleated cells, This method involves bringing multiple cells into contact with a particle density gradient generated by fixed-angle centrifugation to produce a population of enucleated cells. The method wherein the enucleated population of cells accumulates in a density range within the particle density gradient.

2. The method according to claim 1, further comprising contacting the plurality of cells with a toxin that inhibits the formation of the cytoskeleton of the plurality of cells.

3. The method according to claim 1 or claim 2, further comprising removing the population of enucleated cells from the particle density gradient.

4. The method according to claim 2, wherein the toxin includes a mycotoxin.

5. The method according to claim 4, wherein the mycotoxin comprises a cytochalasin.

6. The method according to claim 5, wherein the cytochalasin includes cytochalasin B.

7. The method according to claim 2, wherein the population of cells after enucleation includes a reduced density compared to the plurality of cells having nuclei.

8. The method according to any one of claims 1 to 7, wherein the particle density gradient before fixed-angle centrifugation comprises a plurality of particle solutions containing various particle concentrations.

9. The method according to any one of claims 1 to 8, wherein the fixed-angle centrifugation comprises centrifugation of the plurality of cells in the particle density gradient at an angle of 23 degrees, 24 degrees, 25 degrees, 45 degrees, 90 degrees, or 180 degrees.

10. The aforementioned fixed-angle centrifugation is performed at approximately 80,000 RCF, approximately 83,000 RCF, approximately 85,000 RCF, approximately 87,000 RCF, approximately 90,000 RCF, approximately 93,000 RCF, approximately 95,000 RCF, approximately 97,000 RCF, approximately 100,000 RCF, approximately 103,000 RCF, approximately 105,000 RCF, approximately 107,000 RCF, and approximately 110,000 RCF. The method according to any one of claims 1 to 9, comprising centrifugation at approximately 113,000 RCF, approximately 115,000 RCF, approximately 117,000 RCF, approximately 120,000 RCF, approximately 123,000 RCF, approximately 125,000 RCF, approximately 127,000 RCF, approximately 130,000 RCF, approximately 133,000 RCF, approximately 135,000 RCF, or approximately 137,000 RCF.

11. The method according to claim 10, wherein the fixed-angle centrifugal separation includes centrifugal separation at approximately 85,000 RCF.

12. The method according to claim 10, wherein the fixed-angle centrifugal separation includes centrifugal separation at approximately 127,000 RCF.

13. The method according to any one of claims 1 to 12, wherein the fixed-angle centrifugal separation includes centrifugal separation at an average R of approximately 85,000 RCF.

14. The method according to any one of claims 1 to 13, wherein the fixed-angle centrifugal separation includes centrifugal separation at a maximum R value of approximately 127,000 RCF.

15. The method according to any one of claims 1 to 14, wherein the fixed-angle centrifugal separation includes acceleration, which includes about 5.2 minutes to reach the RCF for centrifugal separation.

16. The method according to any one of claims 1 to 15, wherein the fixed-angle centrifugal separation includes deceleration, which takes about 4.0 minutes to reach the cessation of the fixed-angle centrifugal separation.

17. The method according to any one of claims 1 to 16, wherein the fixed-angle centrifugal separation includes centrifugal separation for approximately 60 minutes.

18. The method according to any one of claims 1 to 17, wherein the particle density gradient comprises a plurality of particle solutions.

19. The method according to claim 18, wherein the plurality of particle solutions comprises at least three particle solutions.

20. The method according to any one of claims 1 to 19, wherein the particle density gradient includes a protein density gradient.

21. The method according to claim 20, wherein the protein density gradient includes a serum albumin gradient.

22. The method according to any one of claims 1 to 19, wherein the particle density gradient includes a polysaccharide density gradient.

23. The method according to claim 22, wherein the polysaccharide density gradient comprises a plurality of polysaccharide solutions.

24. The method according to claim 23, wherein the plurality of polysaccharide solutions include a 12.5% ​​polysaccharide solution, a 15% polysaccharide solution, a 16% polysaccharide solution, a 17% polysaccharide solution, a 25% polysaccharide solution, or any combination thereof.

25. The method according to claim 24, wherein the enucleated cell population accumulates in a density range of approximately 15% polysaccharides.

26. The method according to claim 22, wherein the polysaccharide density gradient includes a ficol density gradient.

27. The method according to any one of claims 1 to 26, wherein the plurality of cells contain heterologous polynucleotides.

28. The method according to any one of claims 1 to 27, further comprising cryopreserving the population of cells.

29. The method according to any one of claims 1 to 28, wherein the fixed-angle centrifugal separation includes ultracentrifugation.

30. The method according to any one of claims 1 to 29, wherein the plurality of cells include stem cells.

31. The method according to claim 30, wherein the stem cells include induced pluripotent stem cells (iPSCs), adult stem cells, mesenchymal stromal cells, embryonic stem cells, fibroblasts, immortalized cells from cell lines, or any combination thereof.

32. The method according to claim 31, wherein the stem cells include the mesenchymal stromal cells.

33. The method according to any one of claims 1 to 32, wherein the plurality of cells include immune cells.

34. The method according to claim 33, wherein the immune cells include lymphocytes or natural killer cells.

35. The method according to any one of claims 1 to 34, wherein the plurality of cells comprises one or more intracellular organelles for synthesizing or secreting exogenous polypeptides, the one or more intracellular organelles are maintained within the population of enucleated cells, and the one or more intracellular organelles synthesize or secrete the exogenous polypeptides in the absence of a nucleus.

36. The method according to claim 35, wherein the plurality of cells contain heterologous polynucleotides encoding the exogenous polypeptide.

37. The method according to claim 35, wherein the enucleated cell population comprises heterologous polynucleotides encoding the exogenous polypeptide, the heterologous polynucleotides are introduced into the enucleated cell population, and one or more intracellular organelles synthesize or secrete the exogenous polypeptide in the absence of a nucleus.

38. The method according to any one of claims 35 to 37, wherein the exogenous polypeptide comprises a therapeutic agent.

39. The method according to any one of claims 1 to 38, wherein the population of enucleated cells includes at least one targeted region.

40. The method according to any one of claims 1 to 39, wherein the population of enucleated cells includes at least one fusionable portion.

41. The method according to any one of claims 1 to 40, wherein the population of enucleated cells includes at least one immune-evading portion.

42. The method according to any one of claims 1 to 41, wherein the enucleated cells of the population of enucleated cells have a diameter that includes about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or less of the average diameter of the nucleated cells.

43. The method according to any one of claims 1 to 42, wherein the enucleated cells of the population of enucleated cells have a diameter including approximately 5 μm or more, approximately 10 μm or more, approximately 20 μm or more, approximately 30 μm or more, approximately 40 μm or more, approximately 50 μm or more, approximately 60 μm or more, approximately 70 μm or more, approximately 80 μm or more, or approximately 90 μm or more.

44. The method according to claim 43, wherein the diameter includes approximately 8 μm.

45. (a) A population of enucleated cells produced by the method of any one of claims 1 to 40, (b) A pharmaceutical composition comprising a pharmaceutically acceptable excipient, carrier, or diluent.

46. The pharmaceutical composition according to claim 45, wherein the pharmaceutical composition is in the form of a unit dose.

47. The pharmaceutical composition according to claim 45 or 46, which is formulated for administration to a target by intrathecal cavity, intraocular, intravitreous, intraretinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, intratumoral, intrapulmonary, intratracheal, intraperitoneal, intrabladder, vaginal, intrarectal, oral, sublingual, transdermal, inhalation, inhalation spray form, intracavitary GI route, or any combination thereof.

48. The pharmaceutical composition according to claim 47, wherein the pharmaceutical composition is formulated for intravenous administration.

49. The pharmaceutical composition according to any one of claims 45 to 48, further comprising at least one additional activator.

50. The pharmaceutical composition according to claim 49, wherein the at least one additional activator comprises a cytokine, growth factor, hormone, enzyme, small molecule, compound, or any combination thereof.

51. a) A population of cells produced by the method described in any one of claims 1 to 44, or a pharmaceutical composition described in any one of claims 45 to 50, b) A kit including a container.

52. a) Instructions for enucleating a plurality of cells according to any one of claims 1 to 44 to produce a population of enucleated cells, b) A kit comprising one or more components of a polysaccharide density gradient.

53. A method for treating a target disease or condition, comprising administering to the target a therapeutically effective amount of a population of enucleated cells produced by the method of any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 45 to 50.

54. A method for treating cancer in a subject, comprising administering to the subject having cancer a therapeutically effective amount of a population of enucleated cells produced by the method of any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 45 to 50.

55. A method for treating a lung disease in a subject, comprising administering to the subject having the lung disease a therapeutically effective amount of a population of enucleated cells produced by the method of any one of claims 1 to 44, or a pharmaceutical composition according to any one of claims 45 to 50.

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