Production of cells and viral vectors

Abstract

Disclosed herein are methods of cell culture, methods of producing cells for adoptive cell therapy, methods of transducing immune cells with viral vectors, and uses of products produced by these methods, such as medical uses and therapeutic applications.

Description

[0001] This application claims priority to SG 10202250599M filed on July 27, 2022, the contents and elements of which are hereby incorporated by reference for all purposes. Field of the Invention

[0002] The present invention relates to molecular and cellular biology and particularly, but not exclusively, to methods of culturing cells and producing viral vectors.

[0003] background

[0004] Viral vectors, such as lentiviral vectors (LVs), are emerging as powerful tools for gene and cell therapy due to their ability to deliver long-term transgene expression to both dividing and non-dividing cells. Currently, viral vectors are produced using either adherent or suspension methods, each with advantages and disadvantages. Currently, 70% of viral vector products are manufactured using adherent culture methods, in which cells are cultured as a monolayer in a culture vessel rather than floating freely in culture medium (i.e., in suspension). Compared to three-dimensional platforms, adherent-based manufacturing methods can be easily purchased off-the-shelf and require less bioengineering expertise. However, most adherent production methods rely on animal-derived products, such as fetal bovine serum (FBS), which present safety, consistency, and regulatory challenges.

[0005] Poly-L-lysine is a nonspecific attachment molecule that allows cells to attach to the surface of the culture medium by increasing the number of positively charged cell binding sites on the surface of the polystyrene culture medium. This enhances the electrostatic interaction between the negatively charged cell membrane and the culture dish. Because HEK293 or derivative cells are easily detached from the cell culture vessel, some lentiviral production protocols recommend applying poly-L-lysine to the culture vessel to enhance cell adhesion. For example, Gandara and colleagues seeded HEK293T cells in DMEM culture dishes pre-coated with poly-L-lysine and containing 10% serum for the production of lentiviral vectors (Gandara et al., Hum Gene Ther Methods. (2018) 29(1): 1-15). However, the process still requires serum, and poly-L-lysine does not improve the transduction efficiency of peripheral blood mononuclear cells (PBMCs).

[0006] It is a recombinant human fibronectin fragment that is used as a gold standard transduction enhancer for retroviral and lentiviral gene transduction into hematopoietic cells. RetroNectin (recombinant human fibronectin fragment) has been widely used in a variety of clinical trials, including CAR-LV T cell transduction.

[0007] Vitronectin can bind to and stabilize plasminogen activator inhibitor-1 (PAI-1) (Zhou et al., Nature Structural Biology (2003) 10(7):541–4). Therefore, vitronectin can regulate proteolysis triggered by plasminogen activation. Studies have found that vitronectin helps the growth and differentiation of human pluripotent stem cells. SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention provides a method for cell culture, comprising attaching and culturing cells in a culture container, wherein the cells are cultured in a culture medium without fetal bovine serum, and the culture container contains a polypeptide having at least 70% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0010] The present invention also provides a method for cell culture, comprising attaching and culturing cells in a culture container, wherein the cells are cultured in a culture medium without fetal bovine serum, and the culture container contains a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or comprises an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0011] In another aspect, the present invention provides use of a polypeptide having at least 70% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 in a method for producing a viral vector, wherein the method comprises culturing cells in a culture container using a culture medium without FBS.

[0012] The present invention also provides the use of a polypeptide having at least 70% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 in a method for producing a viral vector, wherein the method comprises culturing cells in a culture container using a culture medium without FBS.

[0013] In some embodiments, the polypeptide used in the method enhances the attachment of cells to a culture container.

[0014] In some embodiments, the polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 comprises at least 70% sequence identity to one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23, preferably, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, comprises at least 70% sequence identity to one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23. One or more of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23 comprises at least 80%, 85%, 90%, 95% or 99% any sequence identity.In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2 or a fragment of SEQ ID NO:1, and an amino acid sequence having at least 70% sequence identity to one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23, preferably, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23. One or more of SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23 comprises at least 80%, 85%, 90%, 95% or 99% of any of the sequence identities.

[0015] In some embodiments, the polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 comprises at least 70% sequence identity to one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16 and / or SEQ ID NO:17, preferably, wherein the polypeptide comprises at least 80%, 85%, 90%, 95% or 99% any sequence identity to one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16 and / or SEQ ID NO:17.

[0016] In some embodiments, the polypeptide comprising an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, comprises at least 70% sequence identity with one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16 and / or SEQ ID NO:17, preferably, the polypeptide comprises at least 80%, 85%, 90%, 95% or 99% sequence identity with one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16 and / or SEQ ID NO:17.

[0017] In some embodiments, the polypeptide comprising an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:3 comprises at least 70% sequence identity with one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 and / or SEQ ID NO:12, preferably, wherein the polypeptide comprises at least 80%, 85%, 90%, 95% or 99% sequence identity with one or more of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 and / or SEQ ID NO:12.

[0018] In some embodiments, one or more of the cells express a viral vector.

[0019] In some embodiments, the viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector (AAV), or an adenoviral vector.

[0020] In some embodiments, the cell is a mammal or insect cell. In some embodiments, the mammal cell is a human cell. In some embodiments, the human cell is a human embryonic kidney cell, a HeLa cell, or a HT-1080 cell. In some embodiments, the human embryonic kidney cell is a HEK293 cell, a HEK293T cell, or a HEK293FT cell.

[0021] In some embodiments, the polypeptide has at least 75% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, preferably, the polypeptide has at least 80%, 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the polypeptide has the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, that is, it has 100% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0022] In some embodiments, the polypeptide is vitronectin or In some embodiments, the polypeptide is vitronectin or In some embodiments, the polypeptide is vitronectin or In some embodiments, the polypeptide is vitronectin or In certain embodiments, the polypeptide is vitronectin or a polypeptide that can enhance the adhesion of cells to culture containers, and / or enhance viral transduction ability, and / or reduce cell secretion of cytokines, and / or reduce cell secretion of cytokines while maintaining target cell killing, and / or enhance cell killing of target cells through adoptive cell transfer. Functional snippet.

[0023] In some embodiments, the polypeptide is vitronectin. In some embodiments, the polypeptide is a fragment of vitronectin. In some embodiments, the polypeptide is derived from vitronectin. In some embodiments, the polypeptide is a functional fragment of vitronectin. In some embodiments, the polypeptide is a fragment of vitronectin that can enhance the adhesion of cells to culture containers, enhance viral transduction ability, reduce cell secretion of cytokines, and / or reduce cell secretion of cytokines while maintaining killing of target cells, and / or enhance cell killing of target cells.

[0024] In some embodiments, the polypeptide is In some embodiments, the polypeptide is In some embodiments, the polypeptide is derived from In some embodiments, the polypeptide is In some embodiments, the polypeptide is a functional fragment that can enhance the adhesion of cells to culture containers, enhance viral transduction ability, reduce cell secretion of cytokines, and / or reduce cell secretion of cytokines while maintaining the killing of target cells, and / or enhance cell killing of target cells. Fragment of .

[0025] In some embodiments, the cytokine is one or more of IL-6, TNF-α, IFN-γ, and IL-8. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is TNF-α. In some embodiments, the cytokine is IFN-γ. In some embodiments, the cytokine is IL-8.

[0026] In some embodiments, the polypeptide has at least 75% sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and / or SEQ ID NO: 23, preferably, wherein the polypeptide has at least 75% sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and / or SEQ ID NO: NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and / or SEQ ID NO:23 have at least 80%, 85%, 90%, 95% or 99% sequence identity.

[0027] In some embodiments, the polypeptide increases cell attachment compared to cell attachment of control cells cultured in the same manner but without the polypeptide.

[0028] In some embodiments, the polypeptide increases cell attachment as compared to cell attachment of control cells cultured in the same manner but without the polypeptide, as assessed by microscopy, flow chamber pressure measurement, non-invasive label-free real-time impedance measurement, or measurement using an anti-cell attachment antibody.

[0029] In some embodiments, the polypeptide reduces cell aggregation as assessed by microscopic observation compared to cell aggregation of control cells cultured in the same manner but without the polypeptide.

[0030] In some embodiments, the polypeptide increases the distribution of cells in a culture vessel as assessed by microscopic observation compared to the distribution of cells of control cells cultured in the same manner but without the polypeptide.

[0031] In some embodiments, the polypeptide increases viral titer compared to viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0032] In some embodiments, when the polypeptide is used, the viral titer is increased by more than 1.5-fold compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0033] In some embodiments, when the polypeptide is used, the viral titer is increased by more than 2 times compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide, preferably, wherein, when the polypeptide is used, the viral titer is increased by more than 3 times, 4 times, 5 times, 6 times, or 7 times compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0034] In some embodiments, the polypeptide is provided in the form of a liquid solution. In some embodiments, the polypeptide is present in culture.

[0035] In some embodiments, the concentration of the polypeptide in the culture is at least 0.1 μg / mL of the total amount of the culture medium. In some embodiments, the concentration of the polypeptide in the culture is at least 0.1 μg / mL of the total amount of the culture medium, preferably, the added concentration of the polypeptide is at least 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL or 20 μg / mL.

[0036] In some embodiments, the polypeptide is added to the culture. In some embodiments, the polypeptide is added to the culture in the form of a liquid solution. In some embodiments, the polypeptide is added at a concentration of at least 0.1 μg / mL of the total volume of the culture. In some embodiments, the polypeptide is added at a concentration of at least 0.1 μg / mL of the total volume of the culture, preferably, wherein the polypeptide is added at a concentration of at least 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, or 20 μg / mL.

[0037] In some embodiments, the culture vessel comprises a coating. In some embodiments, the polypeptide is coated on the surface of the culture vessel. In some embodiments, the polypeptide is coated on the inner / inner surface of the culture vessel. In some embodiments, the culture surface comprises a coating.

[0038] In some embodiments, the culture vessel coating comprises at least 10% vitronectin and / or In some embodiments, the culture vessel coating comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of vitronectin and / or In some embodiments, the culture vessel coating is 100% vitronectin and / or In some embodiments, at least 10% of the culture vessel coating comprises a polypeptide having at least 70% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the culture vessel coating comprises a polypeptide having at least 70% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1.

[0039] In some embodiments, at least 10% of the culture vessel coating comprises a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or a fragment of SEQ ID NO: 1. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the culture vessel coating comprises a polypeptide having at least 70% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or a fragment of SEQ ID NO: 1.

[0040] In some embodiments, the culture vessel coating is composed of a polypeptide having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1.

[0041] In some embodiments, the culture vessel coating is composed of a polypeptide having at least 70% sequence identity with SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, or the polypeptide comprises an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.

[0042] In some embodiments, the polypeptide is applied to the culture container before the culture medium is added. In some embodiments, the culture container is coated by adding a solution containing the polypeptide to the culture container, incubating the culture container with the solution contained in the container, and discarding the remaining solution after the incubation. In some embodiments, the incubation is at least 1 hour. In some embodiments, the incubation is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the incubation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0043] In some embodiments, the incubation is performed in a refrigerator, at room temperature, or in an incubator at ≥30°C. In some embodiments, the solution comprising the polypeptide has a polypeptide concentration of at least 0.1 μg / mL. In some embodiments, the solution comprising the polypeptide has a polypeptide concentration of at least 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, or 20 μg / mL.

[0044] In some embodiments, the culture container is a culture plate, a culture dish, a culture bottle, a culture tank or a bioreactor. In some embodiments, the culture plate is a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate.

[0045] In some embodiments, the volume of the culture vessel is at least 0.2 mL. In some embodiments, the volume of the culture vessel is at least 0.5 mL, preferably, wherein the culture vessel has a volume of at least 0.6, 0.7, 0.8, 0.9, 1.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 125.0, 150.0, 175.0, or 200.0 mL.

[0046] In some embodiments, the polypeptide is provided as an additive to a commercially available FBS-free culture medium. In some embodiments, the culture medium does not contain animal serum. In some embodiments, the culture medium does not contain serum, xenobiotics, and / or animal products.

[0047] In some embodiments, a polypeptide having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1 is added to the protein-free culture medium.

[0048] In some embodiments, the polypeptide having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 is added to a protein-free culture medium.

[0049] In some embodiments, the cells are transfected with a packaging helper plasmid. In some embodiments, cells are transfected with 3 packaging helper plasmids. In some embodiments, the cells are transfected with a packaging helper plasmid carrying HIV1 Rev protein, a packaging helper plasmid carrying envelope protein, and / or a packaging helper plasmid carrying HIV1 gag / pol protein. In some embodiments, the envelope protein is vesicular stomatitis virus G (VSV-G) protein, baboon protein (e.g., BaEV), RD114 protein, measles virus protein (e.g., H / F), cocal virus protein (e.g., COCV), Nipah virus protein, Ebola protein, Mokola protein, LCMV protein, and / or MuLV glycoprotein. In some embodiments, the cells are transfected with a plasmid encoding a gene of interest.

[0050] In some embodiments, the method further comprises the step of transducing cells with a packaging helper plasmid and a plasmid containing a target gene before culturing the cells. In some embodiments, the target gene is a chimeric antigen receptor (CAR) gene.

[0051] In another aspect, the present invention provides a method for producing a viral vector, the method comprising culturing cells according to the above-mentioned method for culturing cells, the method further comprising the step of isolating or collecting viruses, viral vectors, and / or viral particles from the culture.

[0052] In another aspect, the present invention provides a method for producing cells for adoptive cell therapy, comprising the following steps:

[0053] (i) producing a viral vector according to the above method; and

[0054] (ii) Transduction of immune cells with viral vectors to generate cells for adoptive cell therapy.

[0055] In some embodiments, the cells for adoptive cell therapy are immune cells expressing CAR. In some embodiments, the cells for adoptive cell therapy are CAR T cells or CAR natural killer (CAR-NK) cells. In some embodiments, the immune cells are T cells or NK cells. In some embodiments, the immune cells are mammalian T cells or NK cells. In some embodiments, the immune cells are human T cells or NK cells. In some embodiments, the T cells are γδT cells, cytotoxic T cells, or helper T cells. In some embodiments, the viral vector is a lentiviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, or an adenoviral vector.

[0056] In some embodiments, the method steps (i) and (ii) are performed without changing the culture medium. In some embodiments, a normal culture medium without FBS is used in steps (i) and (ii).

[0057] In some embodiments, step (ii) is performed in the presence of a polypeptide having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1.

[0058] In some embodiments, step (ii) is performed in the presence of a polypeptide having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.

[0059] In some embodiments, a polypeptide having at least 70% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1 is used in step (ii) to enhance transduction capacity.

[0060] In some embodiments, a polypeptide having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1 is used in step (ii) to enhance transduction ability.

[0061] In some embodiments, the polypeptide is also used in another method step to enhance transduction of human cells by viral vectors to generate cells for adoptive cell therapy.

[0062] On the other hand, the present invention provides a method for producing cells for adoptive cell therapy, the method comprising contacting the cells with a viral vector encoding a target gene and a polypeptide, the polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, or comprising an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.

[0063] In another aspect, the present invention provides a method for introducing nucleic acid into mammalian cells using virus-mediated gene transduction, wherein a polypeptide having 70% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 2 is used as an enhancer for virus-mediated gene transduction.

[0064] In another aspect, the present invention provides a method for introducing nucleic acid into mammalian cells using virus-mediated gene transduction, wherein a polypeptide having 70% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 2, or a polypeptide comprising an amino acid sequence having 70% sequence identity with a fragment of SEQ ID NO: 3 or SEQ ID NO: 2 is used as an enhancer for virus-mediated gene transduction.

[0065] In another aspect, the present invention provides the use of a polypeptide having at least 70% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 2 as an enhancer of virus-mediated gene transduction in mammalian cells.

[0066] In another aspect, the present invention provides a polypeptide having at least 70% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 2, or a polypeptide comprising an amino acid sequence having 70% sequence identity to a fragment of SEQ ID NO: 3 or SEQ ID NO: 2 as an enhancer of virus-mediated gene transduction in mammalian cells.

[0067] On the other hand, the present invention provides a method for transducing immune cells with a viral vector encoding a target gene, the method comprising exposing immune cells to a viral vector encoding the target gene and a polypeptide having at least 70% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 under conditions suitable for transducing cells with the viral vector, thereby producing transduced cells for adoptive cell therapy.

[0068] In some embodiments, the polypeptide has at least 75% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23, preferably, wherein the polypeptide has at least 75% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23. NO:22, and / or SEQ ID NO:23 have at least 80%, 85%, 90%, 95%, or 99% sequence identity.

[0069] In some embodiments, the polypeptide has at least 75% sequence identity with any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and / or SEQ ID NO:12, preferably, the polypeptide has at least 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and / or SEQ ID NO:12.

[0070] In some embodiments, the polypeptide has at least 75% sequence identity with any one of SEQ ID NO:3 and / or SEQ ID NO:2, preferably, the polypeptide has at least 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:3 and / or SEQ ID NO:2.

[0071] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:6, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:6.

[0072] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:7, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7.

[0073] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:8, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8.

[0074] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:9, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:9.

[0075] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:10, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:10.

[0076] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:11, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:11.

[0077] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:12, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12.

[0078] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:13, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:13.

[0079] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:14, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14.

[0080] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:15, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:15.

[0081] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:16, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:16.

[0082] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:17, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0083] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:18, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0084] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:19, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0085] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:20, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:20.

[0086] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:21, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:21.

[0087] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:22, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0088] In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:23, preferably, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity with SEQ ID NO:23.

[0089] In some embodiments, the polypeptide increases the transduction efficiency compared to the transduction efficiency without the polypeptide. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.1 times compared to the transduction efficiency without the polypeptide. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.2 times compared to the transduction efficiency without the polypeptide, preferably, wherein the polypeptide increases the transduction efficiency by at least 1.3, 1.4, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times compared to the transduction efficiency without the polypeptide.

[0090] In some embodiments, the polypeptide increases the transduction efficiency compared to the transduction efficiency without the use of a transduction enhancer. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.1 times compared to the transduction efficiency without the use of a transduction enhancer. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.2 times compared to the transduction efficiency without the use of a transduction enhancer, preferably, wherein the polypeptide increases the transduction efficiency by at least 1.3, 1.4, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times compared to the transduction efficiency without the use of a transduction enhancer.

[0091] In some embodiments, the polypeptide increases the transduction efficiency compared to the transduction efficiency without the transduction enhancer. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.5 times compared to the transduction efficiency without the transduction enhancer. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.75 times compared to the transduction efficiency without the transduction enhancer, preferably, wherein the polypeptide increases the transduction efficiency by at least 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times compared to the transduction efficiency without the transduction enhancer.

[0092] In some embodiments, the polypeptide has at least 75% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1, preferably, wherein the polypeptide has at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1. In some embodiments, the polypeptide is vitronectin.

[0093] In some embodiments, the polypeptide has at least 75% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1, or has at least 75% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or a fragment of SEQ ID NO: 1, preferably, wherein the polypeptide has at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1, or to a fragment of SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 1. In some embodiments, the polypeptide is vitronectin or a fragment of vitronectin.

[0094] In some embodiments, the immune cell is a mammalian immune cell. In some embodiments, the mammalian immune cell is a human immune cell. In some embodiments, the cell for adoptive cell therapy is a cell expressing CAR. In some embodiments, the cell expressing CAR is a CAR T cell or a CAR natural killer (CAR-NK) cell.

[0095] In some embodiments, the method further comprises the step of collecting one or more transduced cells. In some embodiments, the method further comprises the step of expanding the transduced cells. In some embodiments, the method further comprises the step of expanding the transduced cells after collecting the transduced cells. In some embodiments, the method further comprises the step of storing the transduced cells. In some embodiments, the method further comprises the step of storing CAR T cells or CAR natural killer (CAR-NK) cells.

[0096] In some embodiments, the method comprises stimulating and / or expanding immune cells. In some embodiments, the method comprises stimulating and / or expanding peripheral blood cells. In some embodiments, the method comprises expanding an immune cell population (e.g., a T cell population) from an immune cell population (e.g., PBMCs).

[0097] In some embodiments, a population of T cells is expanded from a population of tumor infiltrating lymphocytes by stimulating T cells in the population of tumor infiltrating lymphocytes.

[0098] In some embodiments, the method further comprises the step of formulating the transduced mammalian cells with a pharmaceutically acceptable excipient into a medicament.

[0099] In some embodiments, the method further comprises expanding the T cell population from the heterogeneous T cells obtained from the blood sample, the PBMCs population, or the tumor infiltrating lymphocyte population.

[0100] In another aspect, the present invention provides an immune cell expressing CAR produced by the above method. In some embodiments, the cell expressing CAR is a CAR T cell or a CAR-NK cell, optionally a mammal or a human.

[0101] In another aspect, the present invention provides a CAR-expressing immune cell produced by the above method for use in a method for treating or preventing a disease or disorder.

[0102] In another aspect, the present invention provides a method for treating or preventing a disease or condition using CAR-expressing immune cells produced by the above method.

[0103] In another aspect, the present invention provides the use of CAR-expressing immune cells produced by the above method in the manufacture of a medicament for treating or preventing a disease or disorder.

[0104] In some embodiments, the CAR-expressing immune cell is a CAR T cell or a CAR-NK cell, optionally a mammal or a human.

[0105] In some embodiments, the disease or disorder is cancer.

[0106] In some embodiments, the cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon and rectal cancer, colon cancer, endometrial cancer, endometrial stromal sarcoma, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, leukemia Disease, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, plasma cell tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, respiratory system cancer, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, gastric cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0107] In some embodiments, the CAR is a CD19-CAR.

[0108] The present invention includes any combination of the various aspects and preferred features described, unless such a combination is expressly impermissible or explicitly avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The embodiments and experiments to illustrate the principles of the present invention are now presented in the form of drawings and citations as follows:

[0110] Figure 1 .Under the culture conditions of serum-reduced medium, Improves 293T cell adhesion. The virus titers produced by HEK293T cells were evaluated. There was no difference in virus titers between HEK293T cells cultured in DMEM containing 10% FBS under different growth conditions. However, in DMEM reduced to 5% FBS, compared to untreated groups, human platelets (hPLs), and medium containing FBS, The cell adhesion was significantly improved. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using Student's t-test. ＊ p<0.05 was significant; ＊＊ p<0.01; ＊＊＊ p<0.001.

[0111] Figure 2 AD. It can be used to promote the production of attached viral vectors in different serum media and is dose-dependent. Figure 2 A shows a microscopic image of the culture and illustrates the differences between cell attachment, cell aggregation, and cell distribution. Figure 2 B shows the difference in the size of the wells coated with serum-free medium compared to a control well containing similar medium without any coating (i.e., the same volume of PBS) in the corresponding wells. The proliferation of 293T cells in 6-well plates with 5 μg / mL PBS was significantly increased within 3 days. All cells were seeded at 300K / well and cells were counted by trypan blue dye exclusion test. Data are presented as mean ± standard error of the mean. Statistical significance was analyzed by Student's t test. ＊ p<0.05 was significant; ＊＊ p<0.01. Figure 2 C shows the presence or absence of ImmunoCult TM - Virus titer measurements of cells grown in XF T Cell Expansion Medium (Stem Cell Technologies), 4Cell Nutri-T Medium (abbreviated as NutriT; Sartorius), and TexMacs Medium (Miltenyi Biotec GmbH). Figure 2 D shows Effect of concentration on viral vector titer. All data in this study are expressed as mean ± standard error of the mean (SEM). Statistical significance was analyzed using Student's t test. ＊ p<0.05 was significant; ＊＊ p<0.01; ＊＊＊ p<0.001.

[0112] Figure 3 AC. Viral vector production using an adherent culture process and FBS-free medium can be reproducibly performed in different culture vessels. Figure 3 A shows 9.5cm 3 Viral vector titers of cultures. Figure 3 B shows 75cm 3 Viral vector titers of cultures. Figure 3 C shows 175cm 3 Viral vector titers of cultures All data in this study are presented as mean ± standard error of the mean (SEM).

[0113] Figure 4 AB. Amplification and purification of adherent viral vectors without FBS. Figure 4 Ai shows the morphology of HEK293T cells in DMEM medium containing 10% FBS. Figure 4 Aii shows the morphology of HEK293T cells in NutriT medium without FBS.

[0114] Figure 5 AD. Addition of vitronectin as a solution in the absence of FBS allowed 293T cells to attach. Figure 5 A shows a microscopic image of the culture and illustrates the differences between cell attachment, cell aggregation, and cell distribution. Figure 5 B shows that in different types of serum-free media, the proliferation of 293T cells in 6-well plates coated with vitronectin (5 μg / mL PBS) was significantly increased over 3 days compared to a control group (i.e., the same volume of PBS) with similar media without any coating in the corresponding wells. All cells were seeded at 300K / well and cells were counted by trypan blue dye exclusion test. Data are presented as mean ± standard error of the mean. Statistical significance was tested using Student's t test. ＊ p<0.05 was significant; ＊＊ p<0.01. Figure 5 C shows the expression of ImmunoCult in the presence or absence of vitronectin TM - Virus titer measurements of cells grown in -XF T cell expansion medium (StemCells Technologies Inc.), 4Cell Nutri-T medium (abbreviated as NutriT; Sartorius), and TexMacs medium (Miltenyi Biotec GmbH). Figure 5 D shows the effect of vitronectin concentration on viral vector titer. All data in this study are expressed as mean ± standard error of the mean (SEM). Statistical significance was determined using Student's t test. ＊ p<0.05 was significant; ＊＊ p<0.01; ＊＊＊ p<0.001.

[0115] Figure 6 AD. vitronectin and Enhances transduction of PBMCs to express target genes. Figure 6 A shows the relationship between the TM Compared with 4CellNutri-T medium (StemCells Technologies Inc.), 4CellNutri-T medium (abbreviated as NutriT; Sartorius) was superior in supporting T cell expansion. Measurements were made on day 7 using the trypan blue dye exclusion test. Figure 6 B, 6C, and 6D show the expression of 100 μM sera in the absence of virus (control 1) or transduction enhancer (control 2), or in the presence of The levels of CD3+, CD4+, and CD8+ of CD19CAR+ transduced PBMCs when treated with either vitronectin or vitronectin.

[0116] Figure 7 AD. Efficacy of lentiviral vectors encoding the gene of interest. Figure 7 A shows the cytotoxicity of CD19-expressing CAR-T lymphocytes generated in different culture media and different additives against Raji cells (Burkitt's lymphoma). Figure 7 B shows the cytotoxicity of CD19-expressing CAR-T lymphocytes generated in different culture media and different additives against Nalm6 cells (acute lymphoblastic leukemia). Figure 7 C shows the secretion level of IFN-γ in the Raji cell model. Figure 7 D shows the secretion level of γ-interferon in Nalm6 cell model. All data in this study are expressed as mean ± standard error of the mean (SEM).

[0117] Figure 8 . Schematic representation of representative methods for producing cells for adoptive cell therapy.

[0118] Fig. 9 AB. Effect of long-term coating. Fig. 9 A shows the growth of 293T cells in the presence of long-term The 293T cells formed a monolayer on the surface of the plate, while the control cells (293T cells grown in DMEM or NutriT medium supplemented with 10% FBS) showed obvious aggregation at the same time point. Fig. 9 B shows the long-term coating Cells seeded in the wells for 3 hours produced virus with the same efficiency as cells seeded 72 hours before transfection using standard techniques. All data in this study are presented as mean ± standard error of the mean (SEM).

[0119] Fig.10A-10C. Derived from vitronectin and Design of peptides. Fig.10 A shows a schematic diagram of the protein structure of the combination RV#1 and RV#2 and the structure of vitronectin. Figure 1 The location of the polypeptide in C in vitronectin. Fig.10 B shows the sequences of the proteins of compositions RV#1 and RV#2. Fig.10 C shows the proteins derived from vitronectin (P1–7) or (PR1, PR7.1 and PR7.2) polypeptide sequences.

[0120] Fig.11 A-11C. Derived from vitronectin and Effect of peptides on transduction efficiency. Fig.11 The bar graph shown in A shows the Comparison of the percentage of CD19 CAR+ cells in transduced cultures in the presence of vitronectin and predicted transduction enhancing proteins and peptides. As a control for the standard transduction enhancer. Fig.11 The bar graph shown in B shows a comparison of the percentage of CD19 CAR+ cells in transduced cultures in the presence of increasing concentrations of vitronectin. Fig.11 Bar graph shown in C compares the percentage of CD19 CAR+ cells in transduced cultures in the presence of increasing concentrations of peptide P1.

[0121] Fig.12 A-12I. Effects of transduction enhancers on IL-6 secretion after transduction. Fig.12 A shows the IL-6 concentrations secreted by hCD19-CAR-transduced T cells after 4 days of expansion in the presence of vitronectin or in the absence of any transduction enhancer. Fig.12 B shows the IL-6 concentrations secreted by hCD19-CAR-transduced T cells after 14 days of expansion in the presence of vitronectin or in the absence of any transduction enhancer. Fig.12 C shows the concentration of IL-6 secreted by T cells transduced with hCD19-CAR in the presence of increasing amounts of vitronectin. Fig.12 D shows the The proportion of specific cell killing mediated by T cells transduced with hCD19-CAR under transduction conditions in the presence or absence of vitronectin or any added reagents. Fig.12 E shows the detection of vitronectin in transduced CAR T cell cultures, with vitronectin detected in CAR T cells collected on day 4 (~20 ng / mL) and no vitronectin detected on day 14. Fig.12 F shows the concentration of TNF-α secreted by transduced T cells after expansion 4 days after transduction. Fig.12 G shows the concentration of IFN-γ secreted by transduced T cells after expansion 4 days after transduction. Fig.12 H shows the concentration of IL-8 secreted by transduced T cells after expansion for 14 days after transduction. Fig.12 I shows the levels of granzymes at a 5:1 effector to target ratio. DETAILED DESCRIPTION OF THE INVENTION

[0123] Various aspects and embodiments of the present invention will now be discussed with reference to the accompanying drawings. For those skilled in the art, more aspects and embodiments will be apparent. All documents mentioned herein are incorporated herein by reference.

[0124] In the process of optimizing the lentiviral vector production process, the inventors discovered that lentiviral vectors were very stable in a T cell culture medium without FBS, called TexMacs medium. The inventors then considered whether it was possible to use a serum-free / FBS-free culture medium to support T cell proliferation and cell expansion to produce lentiviral vectors. Using a universal culture medium to support the key processes from producing LVs to modifying T cells with LVs expressing chimeric antigen receptors (CARs) could improve the consistency of the final T cell products, which would otherwise exist in a variety of culture medium formulations.

[0125] The present invention is designed based on the above considerations.

[0126] The inventors have developed a novel serum-free attachment process (NSAP) for the production of viral vectors. ) for identification, characterization and reuse, the present method is realized.

[0127] Methods for culturing cells

[0128] Any suitable cell culture method, such as those listed in the Examples, can be used in the methods and compositions described herein.

[0129] Cell culture generally refers to the removal of cells from an organism and then growing them in a favorable artificial environment. Cells can be taken directly from tissues and dissociated enzymatically or mechanically before culture, or they can be extracted from established cell lines or cell strains. These cells can then be cultured under strictly controlled conditions.

[0130] In some embodiments, the methods described herein are related to the cultivation of a cell line. In some embodiments, the cell line is known. In some embodiments, the cell line is an insect or mammalian cell line. Preferably, the mammalian cell line is a human cell line. The human cell line can be a human embryonic kidney cell line, a HeLa cell line, or a HT-1080 cell line. In some embodiments, the human embryonic kidney cell line is a HEK293 cell line, a HEK293T cell line, or a HEK293FT cell line.

[0131] Cells can be cultured under a range of different conditions. However, in general, there are conditions that enhance the growth rate and yield of cells. These conditions vary depending on the cell type, but generally include providing a suitable culture vessel containing a matrix or culture medium that provides the necessary nutrients (e.g., amino acids, carbohydrates, vitamins and / or minerals), growth factors, hormones and / or gases (carbon dioxide, oxygen) for cell growth.

[0132] Many cells require a surface or artificial matrix to form a monolayer in adherent culture, while others can grow in free-floating culture in suspension culture.

[0133] Adherent cell culture Currently, 70% of viral vector products are produced using an adherent culture process, in which cells are cultured as a monolayer in a culture vessel rather than floating freely in a culture medium (i.e., suspension culture). Compared to 3D platforms, adherent-based manufacturing methods can be easily purchased off-the-shelf and require less bioengineering expertise. However, most adherent production methods rely on animal-derived products, such as fetal bovine serum (FBS), which presents challenges in safety, consistency, and regulation.

[0134] Various aspects of the invention provide methods of culturing adherent cells, including culturing cells in a cell culture vessel. Methods of culturing cells are also referred to herein as methods of expanding a cell population, because the cultured cell population is expanded using the method. The cell culture is not a suspension culture, because the cells are attached to a / specific surface of the culture vessel and / or a coating on a / specific surface of the culture vessel.

[0135] The method of the present invention may include seeding cells at a liquid-liquid interface of a cell culture container and contacting the seeded cells with a cell culture medium. The choice of cell culture medium depends on the cells being cultured and which cell culture medium is suitable for which type of cells as is well known to the skilled person. In some embodiments of the present invention, the cells are cultured for at least about 1 day, or at least about 5 days, or at least about 7 days, or at least about 14 days. In a preferred embodiment, the cells are cultured for at least about 7 days. In some embodiments of the present invention, the cells are cultured for less than about 5 days, about 7 days, about 14 days, about 28 days, about 56 days, or about 100 days. In a preferred embodiment, the cells are cultured for at least about 7 days. Advantageously, the cell culture system of the present invention is suitable for long-term culture of such adherent cells.

[0136] By "attachment culture" or "culture under attachment conditions" is meant culture under conditions that promote attachment of cells to a culture vessel, such as the bottom of a tissue culture plate or flask (Jaccard et al., JALA Charlottesv Va. (2014) 19(5):437–443). In some instances, cells can be induced to attach to a vessel simply by keeping the culture quiescent. The degree of attachment of cells to a surface, such as a culture vessel, can be determined in a variety of ways. First, the degree of attachment of cells to a given surface can be visually assessed by microscopic observation. The degree of cell attachment can also be assessed by flow chamber pressure measurement, in which pressure is applied to cultured cells through a flow chamber in a microfluidic device, which can optionally be connected to a microscope and / or camera to assess the degree of attachment. The level of cell attachment can be determined by non-invasive, label-free, real-time impedance detection using an electronic board to record cell viability and survival with time resolution. In addition, cells can be stained with anti-cell attachment antibodies to determine the number of attached cells compared to unattached cells. In addition, the degree of cell aggregation is also a strong indicator of the degree of cell attachment, ie, the lower the degree of cell aggregation, the higher the degree of cell attachment. The skilled artisan will be aware that there are other methods that can be used to determine the degree of cell attachment.

[0137] Cells for adherent cell culture

[0138] Aspects of the invention provide methods of culturing adherent cells, comprising culturing the cells in a cell culture vessel.

[0139] Attached cells can grow and expand while attached to a surface, such as the surface of a culture vessel. In some cases, attached cells grow and expand as a monolayer on a surface, such as the surface of a culture vessel. Based on their morphology, attached cells can be divided into two main categories: fibroblast-like cells, which are elongated in shape and often migrate on the culture dish, or epithelial-like cells, which are polygonal in shape, are often stationary, and grow in sheets.

[0140] Attached cells are commonly used to produce viral vectors. As mentioned above, 70% of viral vector products are produced using attached cells and attached culture processes. Most cells extracted from vertebrates and insects are anchorage-dependent and can be cultured on a suitable matrix. Therefore, examples of attached cells include insect cells and mammalian cells.

[0141] Mammalian cells are commonly used cells in biotechnology. Mammalian cells are excellent hosts for the production of recombinant proteins, viral vectors, and other products of interest. In addition, mammalian cells themselves can be used for therapy, such as adoptive cell transfer and stem cell therapy. Human cells are often used for human therapy. Mammalian cells, such as human cells, can be grown in adherent cell culture. For example, human cells in adherent culture can be human embryonic kidney cells, HeLa cells, or HT-1080 cells.

[0142] Mammalian cells can be extracted from any mammal or human, such as a non-human animal, such as a rabbit, guinea pig, rat, mouse or other rodent (including any animal from the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows, or any animal from the genus Bos), horse (including any animal from the family Equidae), donkey, and non-human primate or other non-human vertebrate; and / or non-human mammal; and / or human.

[0143] Human embryonic kidney (HEK) cells have been widely used in cell biology research for many years because of their reliable growth and ease of transfection. Examples of HEK cell lines include HEK293 cells, or cell lines derived from the HEK293 cell line, such as HEK293T, HEK293FT, HEK293S, HEK29FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEKEBNA1-6E, HEK293MSR and HEK293A cells. HEK293 cells were generated in 1973 by transfecting normal human embryonic kidney cell cultures with sheared adenovirus 5 DNA in the laboratory of Alex van der Eb in Leiden, the Netherlands (Graham et al., (1977) 36(1):59-74). HEK 293 cells are easy to culture and transfect.

[0144] An important variant of the HEK293 cell line is the HEK293T cell line. It contains the SV40 large T antibody, which allows exon replication of transfected plasmids containing the SV40 replication origin. This allows the transfected plasmid to be amplified and the expression of the desired gene product to be prolonged. HEK293 and HEK293T cells are commonly used to produce various viral vectors.

[0145] HeLa cells are an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The cell line was named after Henrietta Lacks' cervical cancer cells obtained on February 8, 1951. HeLa cells can be engineered to express viral vectors.

[0146] HT-1080 is a fibrosarcoma cell line that has been widely used in biomedical research. The cell line was derived from a fibrosarcoma biopsy from a 35-year-old male. HT-1080 cells can be engineered to express viral vectors.

[0147] Insect cells can efficiently express recombinant proteins and can be used to develop viral vectors, virus-like particles, and vaccines. Insect cell lines can be derived from Lepidoptera (moths and butterflies) and Diptera (flies). Insect cell lines can be extracted from Bombyx mori, Spodoptera frugiperda, Spodoptera frugiperda, Trichoplusia ni, and Drosophila melanogaster. Examples of insect cell lines are Sf9, IPLB-Sf21AE (Sf21), Sf900+, Schneider 2 (S2), TN-368, BTI-TN-5B1-4 (High Five), Tni PRO, and Bm-N. Insect cells can be engineered to express viral vectors.

[0148] Type of culture medium

[0149] Growth medium or culture medium is a liquid, semisolid or solid that is used to support the growth and expansion of cells. Culture medium is well known to scientists in the field of cell culture. Types and methods of cell culture media are comprehensively reviewed in "Cell Culture Media: A Review" (Meenakshi Arora, 2013, MATER METHODS 3:175), the contents of which are incorporated herein by reference.

[0150] The culture medium usually includes an appropriate energy source and compounds that regulate the cell cycle. A typical culture medium consists of amino acids, vitamins, inorganic salts, glucose, and serum, which is a source of growth factors, hormones, and attachment factors. In addition to nutrients, the culture medium also helps maintain pH and osmotic pressure.

[0151] Culture media can be liquid (such as water), semi-solid (such as gelatinous), or completely solid. Semi-solid and solid culture media may contain agar, silica gel, acrylamide, gellan gum, or other coagulants. Liquid culture media generally do not contain coagulants.

[0152] The culture medium can be a natural culture medium, such as biological fluids, tissue extracts or clots. In addition, the culture medium can also be an artificial culture medium, such as a balanced salt solution (such as PBS, DPBS, HBSS, EBSS), a basal culture medium (such as MEM and DMEM) or a compound culture medium (such as RPMI-1640 and IMDM).

[0153] Natural culture media consist entirely of naturally occurring biological fluids. Natural culture media are very useful and convenient for culturing various animal cells. The main disadvantage of natural culture media is poor reproducibility due to the lack of knowledge about the exact composition of these natural culture media. Natural culture media generally do not contain serum.

[0154] Artificial or synthetic culture media are obtained by adding nutrients (organic and inorganic), vitamins, salts, 2 and CO 2 Different artificial culture media have one or more of the following uses: 1) immediate survival (balanced salt solution with a specific pH and osmotic pressure); 2) long-term survival (balanced salt solution supplemented with various formulations of organic compounds and / or serum); 3) unlimited growth; 4) special functions.

[0155] Artificial culture media are generally divided into four categories: serum-containing media, serum-free media, chemically defined media, and protein-free media.

[0156] Serum-containing media usually contain FBS. FBS is the most commonly used supplement in animal cell culture media. It is a low-cost supplement that provides an optimal culture medium. Serum binds and neutralizes toxic substances and provides a carrier or chelator for labile or water-insoluble nutrients, hormones and growth factors, and protease inhibitors.

[0157] Serum-free media can be used to support the culture of a single cell type, such as Knockout Serum Replacement and Knockout DMEM from Thermo Fisher Scientific, and mTESR Medium for stem cells from Stem Cell Technologies. Serum-free media contain a defined amount of purified growth factors, lipoproteins, and other proteins that are normally provided by serum. These media are also called "defined media" because the components in these media are known.

[0158] Chemically defined media contain ultrapure inorganic or organic components that are free of contaminants and may also contain pure protein additives such as growth factors. Their components are produced in bacteria or yeast through genetic engineering and supplemented with vitamins, cholesterol, specific amino acids, and fatty acids.

[0159] Protein-free media do not contain any protein and only contain non-protein components. Compared with serum-containing media, the use of protein-free media can promote good cell growth and protein expression, and facilitate downstream purification of expressed products. Formulations such as MEM and RPMI-1640 do not contain protein, and protein supplements can be provided when needed.

[0160] The methods and compositions described herein may include culturing cells in a medium that does not contain fetal bovine serum (FBS). The term "FBS-free medium" or "FBS-free medium" may include cell culture medium in which the presence of fetal bovine serum (FBS) cannot be identified and / or detected.

[0161] Commercially available FBS-free media, e.g., ImmunoCult TM -XF T Cell Expansion Medium (Stem Cell Technologies), ImmunoCult TM -XF B Cell Expansion Medium (Stem Cell Technologies), ImmunoCult TM NK cell expansion medium (Stem Cell Technologies), ClonaCell TM -CHO ACF medium (Stem Cell Technologies), ImmunoCult TM -SF macrophage culture medium (Stem Cell Technologies), 4Cell Nutri-T culture medium (Sartorius), BIOTARGET TM 、DCCM-1SFM (Sartorius), BIO-MPM-1SFM (Sartorius), MSCgo TM Osteoblast differentiation medium (Sartorius), MSCgo TM Rapid osteoblast differentiation medium (Sartorius), hPSC X medium (Sartorius), MSC XF medium (Sartorius), EndoGo TM XF medium (Sartorius), MSCgo TM Adipogenic differentiation medium (Sartorius), D10 cryopreservation medium (Sartorius), D10 phenol red-free cryopreservation medium (Sartorius), TexMacs medium (Miltenyi Biotec GmbH), Gibco-BRL (Invitrogen), Virus Production (VP) medium from Gibco / Thermo Fisher Scientific (Cat. No. A4817902), OptiPRO SFM from Gibco / Thermo Fisher Scientific (Cat. No. 12309019), OpTmizer T cell expansion SFM from Gibco / Thermo Fisher Scientific (Cat. No. A1048501), LymphoONE medium from Takara (Cat. No. WK552s), X-Vivo medium from Lonza (Cat. No. 04-744Q), Prime-XV medium from T cell CDM Irvine Scientific (Cat. No. 91154), and AIM-V serum-free medium from Gibco / Thermo Fisher Scientific (Cat. No. 12055083). Many more are well known to the skilled artisan.

[0162] Serum is the amber blood component remaining after the blood has naturally coagulated; it is usually further purified by centrifugation to remove remaining blood cells. FBS is serum extracted from bovine fetal blood using a closed collection system. FBS can be used as a growth supplement for in vitro cell culture of eukaryotic cells. Animal sera (bovine and non-bovine sera) can be used for cell culture, of which fetal bovine serum (FBS) is the most widely used.

[0163] Serum is a complex mixture of albumin, growth factors and growth inhibitors. Serum has always been one of the most important components in cell culture media. It is a source of amino acids, proteins, vitamins (especially fat-soluble vitamins such as A, D, E and K), carbohydrates, lipids, hormones, growth factors, minerals and trace elements. Fetal serum and calf serum are commonly used to support the growth of cultured cells (von Seefried and Macmorine, 1976, published in "Dev Biol Stand." 37:83-9). Fetal serum is rich in growth factors and is suitable for cell cloning and cell growth that requires complex nutrition. Many growth media typically contain 2-10% serum.

[0164] Most adherent cell culture processes rely on animal-derived products such as FBS, which present safety, consistency, and regulatory challenges.

[0165] The methods and compositions described herein may include culturing cells in a serum-free medium, a defined medium, or a chemically defined medium. However, in some embodiments, the medium may not contain FBS, but may also contain other serum components.

[0166] The term "serum-free medium" may include cell culture media that do not contain serum proteins, such as FBS. Serum-free media are well known in the art, such as described in U.S. Pat. Nos. 5,631,159 and 5,661,034. Commercially available FBS-free media, such as ImmunoCult TM -XF T Cell Expansion Medium (Stem Cell Technologies), ImmunoCult TM -XF B Cell Expansion Medium (Stem Cell Technologies), ImmunoCult TM NK cell expansion medium (Stem Cell Technologies), ClonaCell TM -CHO ACF medium (Stem Cell Technologies), ImmunoCult TM -SF macrophage culture medium (Stem Cell Technologies), 4Cell Nutri-T culture medium (Sartorius), BIOTARGET TM 、DCCM-1SFM (Sartorius), BIO-MPM-1SFM (Sartorius), MSCgo TM Osteoblast differentiation medium (Sartorius), MSCgo TM Rapid osteoblast differentiation medium (Sartorius), hPSC X medium (Sartorius), MSC XF medium (Sartorius), EndoGo TM XF medium (Sartorius), MSCgo TM Adipogenic differentiation medium (Sartorius), D10 cryopreservation medium (Sartorius), D10 phenol red-free cryopreservation medium (Sartorius), TexMacs medium (Miltenyi Biotec GmbH), Gibco-BRL (Invitrogen), Virus Production (VP) medium from Gibco / Thermo Fisher Scientific (Cat. No. A4817902), OptiPRO SFM from Gibco / Thermo Fisher Scientific (Cat. No. 12309019), OpTmizer T cell expansion SFM from Gibco / Thermo Fisher Scientific (Cat. No. A1048501), LymphoONE medium from Takara (Cat. No. WK552s), X-Vivo medium from Lonza (Cat. No. 04-744Q), Prime-XV medium from T cell CDM Irvine Scientific (Cat. No. 91154), and AIM-V serum-free medium from Gibco / Thermo Fisher Scientific (Cat. No. 12055083). Many more are well known to the skilled artisan.

[0167] The serum-free medium may be supplemented with one or more components, such as serum replacement medium, at a concentration of 5%, 10%, 15%, etc. The serum-free medium may be supplemented with 10% serum replacement medium produced by Invitrogen-Gibco (Grand Island, NY).

[0168] The methods and compositions described herein may include culturing cells in a xeno-free medium. However, in some embodiments, the culture medium may be free of FBS but not free of xeno-free substances.

[0169] The term "xeno-free medium" can include cell culture media that do not contain biological components from organisms other than the organism from which the cultured cells originated. Thus, when culturing human cells, such as HEK cells, "xeno-free medium" refers to a formulation that contains only components of human origin and does not include foreign species such as calf (e.g., FBS).

[0170] The methods and compositions described herein may include culturing cells in a medium that does not contain animal products. However, in some embodiments, the medium may not contain FBS, but may contain other animal product components.

[0171] The term "animal product-free medium" is defined in the art as a medium that does not contain components from non-human animals. An "animal product-free medium" may contain a recombinant polypeptide designed based on a non-human animal polypeptide that is different from the related non-human animal polypeptide.

[0172] The methods and compositions described herein may include culturing cells in a protein-free medium. However, in some embodiments, the medium may not contain FBS but may contain other proteins.

[0173] The term "protein-free medium" may include cell culture medium that does not contain any protein components. The skilled person is already aware of proteins. Proteins are large biological molecules and macromolecules comprising one or more long chains of amino acid residues. Vitronectin, derived from The polypeptides and / or polypeptides derived from vitronectin can be added as supplements to the protein-free medium.

[0174] Culture container

[0175] According to the methods and compositions described herein, any suitable container or culture vessel can be used to propagate cells. In some aspects, the container or culture vessel should include a surface on which attached cells can attach or adhere to help them grow. Many different types of culture vessels are known to the technician. Typical culture vessels include: plates, dishes, bottles, tanks, cell factories and bioreactors. Also referred to as culture plates, culture dishes, culture bottles, culture tanks, culture cell factories or culture bioreactors. Culture vessels can be made of a variety of materials, such as metal, glass, plastic and polymers (such as polystyrene and polyester). Culture plates are low, flat-bottomed laboratory containers for culturing cells on a thin layer of nutrient medium. Culture plates and culture dishes can be used for liquid and solid culture media. Culture dishes are similar to culture plates. The most common culture plates and culture dishes are petri dishes and multi-well plates. Multi-well plates are available in a variety of sizes, such as 2-well plates, 4-well plates, 6-well plates, 12-well plates, 24-well plates, 48-well plates, 96-well plates or 384-well plates. Many manufacturers, such as Thermo Fisher Scientific, offer petri dishes and multiwell plates in different sizes. Petri dishes come in different colors (such as clear, black, and white) and are usually made of polystyrene.

[0176] Culture flasks are generally used for liquid cultures and are usually similar to bottles. Culture flasks are generally made of glass or polystyrene. Glass culture flasks can be reused and sterilized, while disposable polystyrene culture flasks are widely available (such as Nunc EasYFlasks from Thermo Fisher Scientific). Culture flasks come in a variety of sizes, such as T25, T75, T175, T225, and T300. Different sized culture flasks have different volumes and surface areas for attached cell growth.

[0177] In some embodiments, the volume of the culture vessel is at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 125.0, 150.0, 175.0, or 200.0 mL.

[0178] Cell factories, or cell factory systems, are multi-layer systems for culturing cells. Cell factories can include multi-layer culture vessels (such as multi-layer plates, multi-layer bottles or multi-layer trays). Cell factories are available in various sizes and can have a variety of different plies. Various types of cell factories are known to technicians, and these cell factories are widely used (such as the Nunc EasyFill cell factory system of Thermo Fisher Scientific, or the high-yield performance bottle (HYPERFlask) of Corning).

[0179] A bioreactor can include a regulated bioreactor in which one or more conditions, such as oxygen partial pressure, can be controlled or monitored. Equipment for measuring and regulating these conditions is known in the art. For example, an oxygen electrode can be used to measure oxygen partial pressure. The oxygen partial pressure can be regulated by the amount and composition of a selected mixed gas (such as air or a mixture of air and / or oxygen and / or nitrogen and / or carbon dioxide). Suitable devices for measuring and regulating oxygen partial pressure are described in Bailey, J E. (Bailey, J E., author "Biochemical Engineering", McGraw-Hill, Inc., ISBN 0-07-003212-2 Higher Education, (1986) or Jackson A T., author "Verfahrenstechnik in der Biotechnologie", Springer, ISBN 3540561900 (1993)).

[0180] Other suitable vessels include rotators. Rotators are a type of bioreactor that can be stirred using a variety of stirring mechanisms, such as glass ball stirrers, impeller stirrers, and other suitable stirrers. The culture capacity of rotators is usually between 20mL and 500mL. Roller bottles are round cell culture bottles made of plastic or glass with a culture area of ​​400 to 2000cm 2 The cells are cultured along the entire inner surface of these bottles; the cells are coated with the medium by a "rolling" motion, i.e., rotation of the bottle around its own axis.

[0181] In addition, the culture can be static, i.e., the culture / substratum is not actively stirred. By reducing the stirring of the culture fluid, the cells / microcarriers can be made to form aggregates. Although some stirring can be used to promote the distribution and flow of the substratum on the cultured cells, the formation of the aggregates should be avoided as much as possible during use. For example, low speed stirring can be adopted, such as less than 30rpm or less than 20rpm.

[0182] In some embodiments, the culture container is coated with Vitronectin, derived from For example, one or more surfaces of the culture container may be coated with Vitronectin, derived from The coating may be a polypeptide derived from vitronectin, and / or a polypeptide derived from vitronectin, which may be used in combination with other nutrients or culture medium. One or more layers of coating may be applied to the culture container. The coating serves as a matrix for cell growth and can increase the adhesion of cells to the culture dish.

[0183] The culture vessel may be coated with a matrix, which in this context refers to a layer of material (such as a thin layer or film) attached to the culture vessel (such as a surface). The matrix may include RetroNectin (a recombinant human fibronectin fragment), vitronectin, The matrix may include other compatible or physiologically relevant matrices that can support cell growth.

[0184] Viral vectors

[0185] Viral vectors are well known in the art and are reviewed in, for example, Lundstrom, 2018, Disease, 6(2):42 and Morgan and Boyerinas, 2016, both of which are incorporated herein by reference in their entirety.

[0186] Viral vectors usually use viral genomes as carriers of target nucleic acids. This means that the target nucleic acid is inserted into the viral genome, and thus when the viral genome is introduced into the target cell, the target nucleic acid is also introduced into the target cell.

[0187] In some embodiments, the genome of the viral vector of the present invention is derived from an adenovirus (such as Ad5), an adeno-associated virus (such as AAV2, 3, 5, 6, 8 or 9), herpes simplex virus (such as HSV, HSV1), a retrovirus (such as MMSV, MSCV), a lentivirus (such as SIV, HIV-1, HIV-2) genome, an alphavirus (such as SFV, SIN, VEE, M1), a flavivirus (such as Kunjin, West Nile, dengue), a rhabdovirus (such as rabies, VSV), a measles virus (such as MV-Edm), a Newcastle disease virus, a poxvirus (such as VV) or a small RNA virus (such as a coxsackievirus) genome.

[0188] In some embodiments, the genome of the viral vector is derived from the genome of a retrovirus (i.e., the viral vector is a retroviral vector). In some embodiments, the genome of the viral vector is derived from the genome of a lentivirus (i.e., the viral vector is a lentiviral vector). In some embodiments, the genome of the viral vector is derived from the genome of HIV. In some embodiments, the genome of the viral vector is derived from the genome of HIV-1.

[0189] The ability of a viral vector to deliver a nucleic acid of interest into a cell depends on its ability to enter the cell and, therefore, on the ability of the viral vector to bind to and be internalized by the cell.

[0190] Viral vectors typically use an envelope glycoprotein that enables them to bind to and enter a variety of cell types.

[0191] Lentivirus is a subclass of retrovirus. Lentivirus consists of an envelope, a capsid, and an RNA genome. Lentivirus enters the target cell with extremely high efficiency. Once inside the cell, the RNA genome is reverse transcribed into DNA and integrated into the cell genome by the viral integrase. This wild-type biological property can be modified to make lentivirus an efficient gene transfer tool. To do this, the genes required for viral replication and induction of pathogenicity are replaced by the target gene, such as the CAR gene.

[0192] Because the lentiviral genome can efficiently and stably introduce nucleic acids into target cells, etc., it is highly favored in the preparation of viral vectors. However, the envelope glycoprotein of the lentivirus binds to receptors, which are only expressed in a few cell types. Therefore, viral vectors usually use a lentiviral genome with another viral envelope glycoprotein, which is called a pseudovirus. For a review of pseudoviral vectors, see Joglekar and Sandoval, Human Gene Therapy Approaches (2017) 28(6): 291-301, which is hereby incorporated by reference in its entirety.

[0193] Lentiviral vectors derived from human immunodeficiency virus have been extensively studied and used over the past two decades. Third-generation self-inactivating lentiviral vectors have recently been used in multiple clinical trials to introduce genes into hematopoietic stem cells to repair primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes into mature T cells to generate immunity against tumors by delivering chimeric antigen receptors (CARs) or cloned T cell receptors (Milone and O'Doherty, Leukemia, Vol. 32, pp. 1529–1541, 2018).

[0194] The first generation of lentiviral vectors contained a significant portion of the HIV genome, including the gag and pol genes, as well as several additional viral proteins (Vannucci L et al., New Microbiol. (2013) 36:1-22). The first generation of lentiviral vectors were designed to include an envelope protein from another virus, most commonly VSV-G. VSV-G recognizes a ubiquitously expressed receptor that was recently identified as the low-density lipoprotein (LDL) receptor, enabling lentiviral vectors to transduce a variety of cells (Burns et al., Proc Natl Acad Sci USA, (1993) 90:8033-7). The VSV-G gene is encoded on a plasmid separate from other lentiviral genes.

[0195] Subsequently, a safer second-generation lentiviral vector was developed, which lacks the auxiliary virulence factors vif, vpr, vpu and nef. The third-generation lentiviral vector divides the viral genome into independent plasmids, making it even less likely to produce recombinant viruses, thereby further improving safety.

[0196] Examples of envelope glycoproteins commonly used in viral vectors include vesicular stomatitis virus glycoprotein (VSV-G), Ebola glycoprotein, Mokola glycoprotein, LCMV glycoprotein, and MuLV glycoprotein. VSV-G is commonly used in viral vectors, and since its homologous LDL receptor is ubiquitously expressed, viral vectors having viral envelope glycoproteins including VSV-G are able to infect a variety of cell types.

[0197] In some embodiments, the envelope protein is a vesicular stomatitis virus G (VSV-G) protein, a baboon protein (such as BaEV), an RD114 protein, a measles virus protein (such as H / F), a cocal virus protein (such as COCV), a Nipah virus protein, an Ebola protein, a Mokola protein, a LCMV protein, or a MuLV glycoprotein.

[0198] In some embodiments, according to the present invention, the viral envelope glycoprotein is or is derived from VSV-G, Ebola glycoprotein, Mokola glycoprotein, LCMV glycoprotein, baboon glycoprotein, RD114 glycoprotein, measles glycoprotein, cocal glycoprotein, Nipah glycoprotein, or MuLV glycoprotein. In some embodiments, the viral envelope glycoprotein is or is derived from VSV-G.

[0199] As used herein, a viral envelope glycoprotein "derived from" a reference viral envelope glycoprotein may have a higher sequence identity to the amino acid sequence of the viral envelope glycoprotein from which it is derived. For example, a viral envelope glycoprotein "derived from" a reference viral envelope glycoprotein may have at least 60%, such as 65%, 70%, 75%, 80%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the viral envelope glycoprotein from which it is derived.

[0200] In some embodiments, the viral envelope glycoprotein comprises, or consists of, an amino acid sequence having at least 60%, such as 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1.

[0201] Methods for producing viruses, viral vectors and viral particles

[0202] Methods for producing viruses, viral vectors and viral particles are well known in the art and are reviewed by Merten et al. in Pharm. Bioprocess (2014) 2: 183-203 and van der Loo and Wright in Human Molecular Genetics (2015): 1-11, both of which are incorporated herein by reference in their entirety.

[0203] The methods generally involve introducing nucleic acid encoding components of the viral vector into cells, such as by transfecting or infecting the cells.

[0204] The most common method for producing retroviral, lentiviral and adeno-associated viral vectors and viral particles involves co-transfection of HEK 293 cells with a plasmid encoding the nucleic acid of interest to be delivered, packaging elements encoding the structural proteins required for viral assembly, and envelope proteins.

[0205] The co-transfected cells are cultured under conditions suitable for expressing the nucleic acid of interest and the viral proteins from the plasmid, and the components of the viral vector are assembled into viral particles, which are then released by the cultured cells into the cell culture supernatant. The cell culture supernatant containing the viral particles can be separated and / or collected, selectively treated to remove cells and cell debris, and selectively concentrated. Concentration can include centrifugation or precipitation of viral particles using calcium phosphate (see, e.g., Pham et al., J Gene Med, (2001) 3(2): 188-94).

[0206] The plasmid encoding the target nucleic acid generally also includes a promoter for initiating expression of the target nucleic acid. In the lentiviral system, viral long terminal repeat (LTR) sequences are generally located on both sides of the target nucleic acid, so that the target nucleic acid can be integrated into the target cell genome; the second generation lentiviral system uses a viral promoter at the 5'LTR, so that the target nucleic acid is expressed in a tat-dependent manner. The third generation lentiviral system uses a chimeric 5'LTR containing a non-tat-dependent promoter, such as a CMV or RSV promoter. The LTR provided to the 3' end of the target nucleic acid may contain a deletion that causes the virus to self-inactivate after integration. The transport plasmid may include an additional promoter for driving the expression of the target nucleic acid, and may also include other elements, such as a central polypurine region (cPPT) for promoting expression of the target nucleic acid, a Psi for promoting nucleocapsid packaging, and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) for enhancing expression of the target nucleic acid by increasing nuclear export.

[0207] The packaging plasmid provides genes encoding viral proteins required for the assembly of viral particles. In the lentiviral system, the packaging plasmid provides gag, which encodes the matrix, capsid, and nucleocapsid; pol, which encodes reverse transcriptase and integrase; and rev, which encodes a protein that binds to the rev corresponding element (RRE) to achieve nuclear export. The second-generation lentiviral system uses a single packaging plasmid encoding gag, pol, rev, and tat, encoding a transcriptional activator protein to activate transcription through the promoter of the 5'LTR. In the third-generation lentiviral system, gag, pol, and rev are encoded by two separate packaging plasmids. This system usually does not use tat (in this system, the 5'LTR of the transduction plasmid includes a non-tat-dependent promoter).

[0208] The envelope plasmid provides nucleic acid encoding the viral envelope glycoproteins.

[0209] The viral vector used in the method of the present invention can be prepared by co-transfection and expression in cells of a plasmid encoding a target nucleic acid, a plasmid encoding gag and pol, a plasmid encoding rev, and a plasmid encoding the composite viral envelope glycoprotein described herein.

[0210] In some embodiments, the viral vector used in the methods of the present invention can be prepared from a viral vector comprising a composite viral envelope glycoprotein and a viral envelope glycoprotein.

[0211] In some embodiments, the viral vector is prepared by a method comprising transfecting cells with nucleic acids encoding the composite viral envelope glycoprotein and the viral envelope glycoprotein at a specific ratio / ratio.

[0212] In some embodiments, greater than 5%, such as greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of any nucleic acid encoding a viral envelope protein encodes a composite viral envelope glycoprotein.

[0213] In some embodiments, less than 100% of the viral vectors used in the production methods of the invention, such as less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of any nucleic acid encoding a viral envelope protein encodes a composite viral envelope glycoprotein.

[0214] In some embodiments, greater than 5% and less than 100% of the viral vectors used in the methods of the invention are produced, such as greater than 5% and less than 90%, greater than 5% and less than 85%, greater than 10% and less than 80%, greater than 10% and less than 75%, greater than 10% and less than 70%, greater than 15% and less than 65%, greater than 15% and less than 60%, greater than 15% and less than 55%, greater than 20% and less than 50%, greater than 25% and less than 45%, greater than 25% and less than 40%, or greater than 30% and less than 35% of any nucleic acid encoding a viral envelope glycoprotein encodes a composite viral envelope glycoprotein. In some embodiments, about 33% of the nucleic acid encoding a viral envelope glycoprotein of the viral vectors used in the methods of the invention are produced, and encode a composite viral envelope glycoprotein.

[0215] In some embodiments, the ratio of nucleic acid encoding a composite viral envelope glycoprotein to nucleic acid encoding a viral envelope glycoprotein (e.g., nucleic acid encoding anti-CD3 scFv-VSV-G: nucleic acid encoding VSV-G) for producing the viral vector used in the method of the invention is greater than 1:19 and less than 1:0, such as greater than 1:19 and less than 9:1, greater than 1:19 and less than 17:3, greater than 1:9 and less than 4:1, greater than 1:9 and less than 3:1, greater than 1:9 and less than 7:3, greater than 3:17 and less than 13:7, greater than 3:17 and less than 6:4, greater than 3:17 and less than 11:9, greater than 1:4 and less than 1:1, greater than 1:3 and less than 9:11, greater than 1:3 and less than 4:6, or greater than 3:7 and less than 7:13. In some embodiments, the ratio of nucleic acid encoding composite viral envelope glycoprotein to viral envelope glycoprotein nucleic acid (such as nucleic acid encoding anti-CD3scFv-VSV-G: nucleic acid encoding VSV-G) in producing the viral vector used in the method of the present invention is about 1:2.

[0216] After transfection, viral vectors and viral particles are produced and released into the culture supernatant of the cells. The viral vectors and viral particles can then be collected and used in subsequent downstream processes, such as transduction of immune cells.

[0217] A representative simplified viral vector production scheme is as follows:

[0218] 1. Inoculate the culture medium in the culture container with an appropriate density of packaging cells;

[0219] 2. Incubate the cells for about 20 hours;

[0220] 3. Prepare transfection plasmids (such as target gene plasmids and packaging plasmids);

[0221] 4. Transfect packaging cells with plasmid;

[0222] 5. Incubate the cells to grow and expand;

[0223] 6. Collect the virus / viral vector / particles from the supernatant;

[0224] 7. Optionally store the virus / viral vector / particles (store at 4°C for several hours but freeze in liquid nitrogen at -80°C as soon as possible).

[0225] Cells used to produce viral vectors

[0226] Part of the viral vector production process is to grow cells that express the viral vector. These cells are often called packaging cells. Effective packaging cells should be easy to transfect and support high levels of viral protein expression.

[0227] Packaging cells are usually transfected with at least one plasmid. Cells can be transfected with a variety of plasmids, such as a packaging helper plasmid and a plasmid containing a target gene. One or more of these plasmids contain a viral genome, for example, a target gene plasmid can contain a target gene and a viral plasmid.

[0228] The skilled artisan is aware of a variety of cell types suitable for transfection with viral plasmids (eg, packaging viruses). Mammalian cells and insect cells are excellent hosts for the production of viral vectors and are suitable for transfection with viral plasmids.

[0229] Human cells are examples of mammalian cells that are used to produce viral vectors and are suitable for transfection with viral plasmids. The human cells can be human embryonic kidney cells, HeLa cells, or HT-1080 cells.

[0230] Human embryonic kidney (HEK) cells have been widely used in cell biology research for many years because of their stable growth and ease of transfection. Examples of HEK cell lines are HEK293 cells, or cell lines derived from the HEK293 cell line such as HEK293T, HEK293FT, HEK293S, HEK29FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEKEBNA1-6E, HEK293MSR, and HEK293A cells.

[0231] An important variant of the HEK293 cell line is the HEK293T cell line. It contains the SV40 giant T antigen, allowing exon replication of transfected plasmids containing the SV40 replication origin. This allows purification of the transfected plasmid and amplification of the desired gene product expressed in real time. HEK293 and HEK293T cells are commonly used for the production of a variety of viral vectors.

[0232] HeLa cells are an immortal cell line used in scientific research. They are the oldest and most commonly used human cell line. HeLa cells can be engineered to express viral vectors.

[0233] HT-1080 is a fibrosarcoma cell line that has been widely used in biomedical research. HT-1080 cells can be engineered to express viral vectors.

[0234] Insect cells can be used to produce viral vectors. Insect cell lines can be from the orders Lepidoptera (moths and butterflies) and Diptera (flies). Insect cell lines can be extracted from Bombyx mori, Spodoptera frugiperda, Spodoptera frugiperda, Trichoplusia ni, and Drosophila melanogaster. Examples of insect cell lines are Sf9, IPLB-Sf21AE (Sf21), Sf900+, Schneider 2 (S2), TN-368, BTI-TN-5B1-4 (High Five), Tni PRO, and Bm-N. Insect cells can be engineered to express viral vectors.

[0235] and vitronectin

[0236] Vitronectin is a glycoprotein found in the extracellular matrix.

[0237] The length of the amino acid sequence of Homo sapiens vitronectin (including the signal peptide) is 478 amino acids (SEQ ID NO: 3). The length of the human vitronectin dephosphorylation signal peptide (P04004) is 459 amino acids (SEQ ID NO: 2).

[0238] The vitronectin sequence available from Genbank is sequence no. AAH05046.1 GI:13477169.

[0239] In the present specification, "vitronectin" includes an amino acid sequence having any sequence identity of 70%, preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with one or more of the above-mentioned vitronectin amino acid sequences.

[0240] Vitronectin can be from or derived from any animal or human, such as a non-human animal, such as a rabbit, guinea pig, rat, mouse or other rodent (including any animal from the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows, or any animal from the genus Bos), horse (including any animal from the family Equidae), donkey, and non-human primate or other non-human vertebrate; and / or non-human mammal; and / or human.

[0241] It is a recombinant human fibronectin (CH-296) containing three functional domains: the human fibronectin cell binding domain (C-domain), the heparin binding domain (H-domain), and the CS-1 sequence domain. The amino acid sequence length is 574 (SEQ ID NO: 1).

[0242] In this manual, Including one or more of the above Amino acid sequences have any one of 70%, preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0243] It is a recombinant human fibronectin fragment containing three functional domains: (1) an N-terminal cell-binding domain that binds to cells through the VLA-5 integrin (this domain contains an RGDS motif and includes three fibronectin type III domains 8, 9 and 10), (2) a heparin-binding domain that includes three fibronectin type III domains 12, 13 and 14, and (3) a C-terminal CS-1 that binds to cells through the VLA-4 integrin.

[0244] Vitronectin is a 54 kDa glycoprotein composed of three major domains: (1) an N-terminal somatostatin B domain (residues 1-39) that binds to various proteins such as the urokinase-type plasminogen activator receptor, (2) a central domain (residues 131-342) that is a hemopexin homologue, and (3) a C-terminal domain (residues 347-459) that contains a heparin-binding domain that interacts with heparin and other glycosaminoglycans and may help bind vitronectin to cell surfaces and extracellular matrix components.

[0245] Vitronectin also contains an RGD sequence located at residues 45-47. This sequence serves as a recognition site for integrin receptors, particularly the αvβ3 and αvβ5 integrins on the cell surface. Vitronectin binds to these integrins to enhance a variety of cellular processes such as cell adhesion, migration, and signaling.

[0246] There is also a plasminogen binding site in the first 45 amino acids. Plasminogen is the precursor of the proteolytic enzyme fibrinolytic. Binding of plasminogen to vitronectin promotes the activation of plasminogen to plasmin, which is responsible for the degradation of fibrin clots and other extracellular matrix proteins. Therefore, its function is mainly to regulate fibrinolysis and extracellular matrix remodeling. The N-terminal region of vitronectin includes a series of repetitive alpha helical segments that are intertwined. This region is responsible for the dimerization of vitronectin. It forms a rod-like structure and contributes to the stability of the protein.

[0247] Derived from and vitronectin peptides

[0248] In some aspects, the polypeptides of the present disclosure are or a fragment of vitronectin, a protein derived from or vitronectin polypeptides, or polypeptides derived from or a polypeptide of a vitronectin fragment.

[0249] It is understood that a polypeptide derived from an amino acid sequence of interest includes a peptide / polypeptide that comprises or consists of: (i) an amino acid sequence of a protein of interest, or (ii) an amino acid sequence similar to the amino acid sequence of a protein of interest (i.e., an amino acid sequence similar to the amino acid sequence of (i)).

[0250] In some embodiments, the first polypeptide includes the target amino acid sequence and includes other additional amino acids. In some embodiments, the first peptide / polypeptide includes the target amino acid sequence and any number of additional amino acids 1-3, 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40 or 1-50 located at one or both ends (i.e., N or C terminus) of the target amino acid sequence. In some embodiments, the additional amino acids are related to the target amino acid sequence relative to the amino acids provided at these positions, and the target amino acid sequence is from a protein. For example, if the target amino acid sequence is relative to the 20th to 30th amino acids of the target protein amino acid sequence, and the first peptide / polypeptide includes the target amino acid sequence and another 5 amino acids located at the N-terminus of the target amino acid sequence, then these other 5 amino acids may correspond to the 15th to 19th positions of the target protein amino acid sequence.

[0251] The polypeptide may include more than one peptide / polypeptide chain. For example, the first peptide / polypeptide may be a complex of peptides / polypeptides, such as comprising 2, 3, 4, 5 or 6 or more peptides / polypeptides. The peptide / polypeptide may be conjugated with other peptides / polypeptides, or may form a composition (such as a mixture) with two or more unconjugated peptides / polypeptides.

[0252] As used herein, a polypeptide derived from a reference polypeptide, or an isomer, fragment, variant or homolog of a given reference polypeptide, can be characterized as having at least 70% sequence identity, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity to the amino acid sequence of the reference polypeptide.

[0253] A polypeptide derived from a reference polypeptide generally refers to a polypeptide that includes an amino acid sequence in which one or more amino acids are substituted, inserted, deleted or otherwise modified relative to the amino acid sequence of the reference protein, but still retains a considerable degree of sequence identity (e.g., at least 70%) with the amino acid sequence of the reference protein.

[0254] A "fragment" generally refers to a portion of a reference protein from which the fragment is derived.

[0255] Fragments of vitronectin can include a minimum length of at least, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% relative to the full-length sequence. Fragments can have a maximum length, such as no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% relative to the full-length sequence. Fragments can include at least, such as having 5 amino acids, or any number of 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids. Fragments can have a maximum length, such as no more than 10 amino acids, or less than any number of 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids. Fragments can have any of the minimum and maximum lengths described.

[0256] A fragment may include at least, such as having a minimum length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% relative to the full-length sequence. A fragment may have a maximum length, such as having no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or 99% relative to the full-length sequence. Fragments can include at least, such as having 5 amino acids, or any number of 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids. Fragments can have a maximum length, such as no more than 10 amino acids, or less than any number of 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids. Fragments can have any of the minimum and maximum lengths described.

[0257] "Isoforms" generally refer to variants of a reference protein expressed by the same species as the reference protein. "Homologs" generally refer to variants of a reference protein produced by a species different from the reference protein. Homologs include orthologs.

[0258] An isomer, fragment, variant or homologue of a given reference protein can be selectively characterized as having at least 70%, preferably ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% or 100% amino acid sequence identity with the amino acid sequence of an immature or mature (i.e., signal peptide removed) form of a related polypeptide of a given species, such as human.

[0259] In some aspects, the polypeptide is derived from In some aspects, the polypeptide is Or a fragment of vitronectin. In some aspects, the polypeptide is derived from or fragments of vitronectin.

[0260] In some aspects, the polypeptide is derived from SEQ ID NO: 3, SEQ ID NO: 2, and / or SEQ ID NO: 1. In some aspects, the polypeptide is a fragment of SEQ ID NO: 3, SEQ ID NO: 2, and / or SEQ ID NO: 1. In some aspects, the polypeptide is derived from a fragment of SEQ ID NO: 3, SEQ ID NO: 2, and / or SEQ ID NO: 1. For example, P1 (SEQ ID NO: 6) is a fragment of SEQ ID NO: 3, and thus P1 (SEQ ID NO: 6) is derived from SEQ ID NO: 3.

[0261] By further exemplification, P6 (SEQ ID NO: 9) is derived from SEQ ID NO: 10, and SEQ ID NO: 10 is a fragment of SEQ ID NO: 3. Therefore, P6 is derived from a vitronectin fragment.

[0262] In some aspects, the polypeptide comprises at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2, or a fragment of SEQ ID NO:1.

[0263] For example, P6 (SEQ ID NO: 9) includes an amino acid sequence with more than 70% sequence identity to SEQ ID NO: 3. It can be seen that SEQ ID NO: 10 is a fragment of SEQ ID NO: 3, and P6 (SEQ ID NO: 9) includes an amino acid sequence with more than 70% sequence identity to SEQ ID NO: 10. Relative to SEQ ID NO: 10, P6 (SEQ ID NO: 9) includes more amino acids at the N-terminus and C-terminus and includes an insertion, but the amino acid sequence included in P6 (SEQ ID NO: 9) has more than 70% sequence identity to SEQ ID NO: 10. By completing the sequence alignment, it can be seen that the sequence identity percentage between SEQ ID NO: 9 and SEQ ID NO: 10 is 92.86%.

[0264] In some embodiments, the polypeptide derived from SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.

[0265] In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 6. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 7. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 8. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 9. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 10. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 11. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 12. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 13. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 14. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 15. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 16. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 17. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 18. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 19. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 20.In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 21. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 22. In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 23.

[0266] In some embodiments, the polypeptide derived from SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0267] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.

[0268] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 6. In some embodiments, the polypeptide derived from a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 7.

[0269] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:8.

[0270] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:9.

[0271] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:10.

[0272] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:11.

[0273] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:12.

[0274] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:13.

[0275] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:14.

[0276] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:15.

[0277] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:16.

[0278] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:17.

[0279] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:18.

[0280] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:19.

[0281] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:20.

[0282] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:21.

[0283] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:22.

[0284] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:23.

[0285] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0286] In some embodiments, the polypeptide derived from a fragment of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3 is SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:17.

[0287] In some embodiments, the vitronectin fragment comprises an amino acid sequence derived from the N-terminal growth factor B domain of vitronectin. In some embodiments, the vitronectin fragment comprises an amino acid sequence derived from the central domain of vitronectin. In some embodiments, the vitronectin fragment comprises an amino acid sequence derived from the C-terminal domain of vitronectin.

[0288] In some embodiments, the vitronectin fragment comprises an amino acid sequence having greater than 70% sequence identity with an amino acid sequence in the N-terminal somatostatin B domain of vitronectin. In some embodiments, the vitronectin fragment comprises an amino acid sequence having greater than 70% sequence identity with an amino acid sequence in the central domain of vitronectin. In some embodiments, the vitronectin fragment comprises an amino acid sequence having greater than 70% sequence identity with an amino acid sequence in the C-terminal domain of vitronectin.

[0289] In some embodiments, the vitronectin fragment comprises an amino acid sequence taken from the N-terminal growth factor B domain of vitronectin. In some embodiments, the vitronectin fragment comprises an amino acid sequence taken from the central domain of vitronectin. In some embodiments, the vitronectin fragment comprises an amino acid sequence taken from the C-terminal domain of vitronectin.

[0290] In some embodiments, the vitronectin fragment includes a vitronectin N-terminal somatomedin B domain. In some embodiments, the vitronectin fragment includes a vitronectin central domain. In some embodiments, the vitronectin fragment includes a vitronectin C-terminal domain.

[0291] In some embodiments, the fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include an amino acid sequence derived from the N-terminal growth factor B domain of vitronectin. In some embodiments, the fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include an amino acid sequence derived from the central domain of vitronectin. In some embodiments, the fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include an amino acid sequence derived from the C-terminal domain of vitronectin.

[0292] In some embodiments, a fragment of SEQ ID NO:3 and / or SEQ ID NO:2 comprises an amino acid sequence having greater than 70% sequence identity with the vitronectin N-terminal somatomedin B domain. In some embodiments, a fragment of SEQ ID NO:3 and / or SEQ ID NO:2 comprises an amino acid sequence having greater than 70% sequence identity with the vitronectin central domain. In some embodiments, a fragment of SEQ ID NO:3 and / or SEQ ID NO:2 comprises an amino acid sequence having greater than 70% sequence identity with the vitronectin C-terminal domain.

[0293] In some embodiments, fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include an amino acid sequence taken from the N-terminal growth factor B domain of vitronectin. In some embodiments, fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include an amino acid sequence taken from the central domain of vitronectin. In some embodiments, fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include an amino acid sequence taken from the C-terminal domain of vitronectin.

[0294] In some embodiments, the fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include the vitronectin N-terminal somatomedin B domain. In some embodiments, the fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include the vitronectin central domain. In some embodiments, the fragments of SEQ ID NO:3 and / or SEQ ID NO:2 include the vitronectin C-terminal domain.

[0295] In some embodiments, Fragments include those derived from In some embodiments, Fragments include those derived from In some embodiments, Fragments include those derived from Amino acid sequence of the C-terminal CS-1 sequence.

[0296] In some embodiments, The snippet includes The N-terminal cell binding domain has an amino acid sequence with greater than 70% sequence identity. In some embodiments, The snippet includes The heparin binding domain has an amino acid sequence with greater than 70% sequence identity. In some embodiments, The snippet includes The C-terminal CS-1 sequence has an amino acid sequence with more than 70% sequence identity.

[0297] In some embodiments, The excerpts include those taken from In some embodiments, The excerpts include those taken from In some embodiments, The excerpts include those taken from Amino acid sequence of the C-terminal CS-1 sequence.

[0298] In some embodiments, Clips include In some embodiments, Clips include In some embodiments, Clips include C-terminal CS-1 sequence.

[0299] In some embodiments, a fragment of SEQ ID NO: 1 comprises a fragment derived from In some embodiments, Fragments include those derived from In some embodiments, Fragments include those derived from Amino acid sequence of the C-terminal CS-1 sequence.

[0300] In some embodiments, the fragment of SEQ ID NO: 1 includes The N-terminal cell binding domain has an amino acid sequence with greater than 70% sequence identity. In some embodiments, the fragment of SEQ ID NO: 1 includes The heparin binding domain has an amino acid sequence with greater than 70% sequence identity. In some embodiments, the fragment of SEQ ID NO: 1 includes The C-terminal CS-1 sequence has an amino acid sequence with more than 70% sequence identity.

[0301] In some embodiments, a fragment of SEQ ID NO: 1 comprises a fragment taken from In some embodiments, the fragment of SEQ ID NO: 1 includes an amino acid sequence taken from In some embodiments, the fragment of SEQ ID NO: 1 comprises an amino acid sequence taken from Amino acid sequence of the C-terminal CS-1 sequence.

[0302] In some embodiments, the fragment of SEQ ID NO: 1 comprises N-terminal cell binding domain. In some embodiments, the fragment of SEQ ID NO: 1 includes Heparin binding domain. In some embodiments, the fragment of SEQ ID NO: 1 comprises C-terminal CS-1 sequence.

[0303] In some embodiments, the fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.

[0304] In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 6. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 8. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 10. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 12. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 13. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 14. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 15. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 19. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 20. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 21. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 22. In some embodiments, a fragment of SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3 is SEQ ID NO: 23.

[0305] In some embodiments, the polypeptide has at least 70% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23, preferably, wherein the polypeptide has at least 70% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23. NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23 have at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In some embodiments, the polypeptide has at least 70% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17, preferably, wherein the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17.

[0306] In some embodiments, the polypeptide has at least 70% sequence identity with any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17, preferably, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17.

[0307] In some embodiments, the polypeptide has at least 70% sequence identity with any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:12, preferably, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:12.

[0308] In some embodiments, the polypeptide has at least 70% sequence identity with any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:12, preferably, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and / or SEQ ID NO:12.

[0309] In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:3. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:2. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:1. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:4. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:5. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:6. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:7. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:8. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:9. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:10. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO:11. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 13. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 14. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 15. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 16. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 17. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 18. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 19. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 20. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 21. In some embodiments, the polypeptide has at least 70% sequence identity with SEQ ID NO: 22. In some embodiments, the polypeptide has at least 70% sequence identity to SEQ ID NO:23.

[0310] In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:3. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:2. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:1. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:4. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:5. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:6. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:7. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:8. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:9. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:10. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO:11. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 12. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 13. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 14. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 16. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 17. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 18. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 19. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 21. In some embodiments, the polypeptide has at least 90% sequence identity with SEQ ID NO: 22. In some embodiments, the polypeptide has at least 90% sequence identity to SEQ ID NO:23.

[0311] In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:3. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:1. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:4. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:5. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:16.In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 18. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 19. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 20. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 21. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 22. In some embodiments, the polypeptide has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 23.

[0312] In some embodiments, the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23, or comprises an amino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23. NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23, an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications (such as substitutions, insertions, or deletions).

[0313] In some embodiments, the polypeptide includes an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17, or an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17.

[0314] In some embodiments, the polypeptide includes an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17, or an amino acid sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17.

[0315] In some embodiments, the polypeptide includes an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17, or an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17.

[0316] In some embodiments, the polypeptide includes an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17, or an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and / or SEQ ID NO:17.

[0317] In some embodiments, the polypeptide includes an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17, or an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, and / or SEQ ID NO:17.

[0318] In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:1. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:2. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:3. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:4. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:5. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:6. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:7. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:8. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:9. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:10. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:11. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO:12. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 13. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 14. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 15.In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 16. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 17. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 18. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 19. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 20. In some embodiments, the polypeptide includes an amino acid sequence comprising up to 10 modifications (such as substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 21. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 10 modifications (e.g., substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 22. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 10 modifications (e.g., substitutions, insertions, or deletions) relative to the amino acid sequence as shown in SEQ ID NO: 23.

[0319] In some embodiments, the polypeptide includes an amino acid sequence comprising at most 1 modification (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide includes an amino acid sequence comprising at most 2 modifications (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide includes an amino acid sequence comprising at most 3 modifications (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide includes an amino acid sequence comprising at most 4 modifications (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide includes an amino acid sequence comprising at most 5 modifications (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide includes an amino acid sequence comprising at most 6 modifications (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide includes an amino acid sequence comprising at most 7 modifications (such as replacement, insertion, or deletion) relative to the reference polypeptide. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 8 modifications (e.g., substitutions, insertions, or deletions) relative to the reference polypeptide. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 9 modifications (e.g., substitutions, insertions, or deletions) relative to the reference polypeptide. In some embodiments, the polypeptide comprises an amino acid sequence comprising up to 10 modifications (e.g., substitutions, insertions, or deletions) relative to the reference polypeptide.

[0320] In some embodiments, the polypeptide is used as a conjugate of another polypeptide / protein (such as an immunoglobulin or a carrier protein). The polypeptide can be conjugated, connected, or fused to another component. Additional components can improve the functional properties of the polypeptide. For example, the polypeptide can be connected to another component to improve stability, reduce immunogenicity, and / or increase half-life. In some aspects, the polypeptide can be conjugated, connected, or fused to another polypeptide. The polypeptide can be conjugated, connected, or fused to an immunoglobulin.

[0321] Immunoglobulins and their structures are described in Schroeder and Cavacini in J Allergy Clin Immunol. (2010) 125 (202): S41-S52, etc., the entire contents of which are incorporated herein by reference. G-type immunoglobulins (i.e., IgG) are glycoproteins of about 150 kDa, including two heavy chains and two light chains. From the N-terminus to the C-terminus, the immunoglobulin heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region includes three constant regions (CH1, CH2, and CH3, with a CH1-CH2 hinge region between CH1 and CH2). The immunoglobulin light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Depending on the heavy chain, immunoglobulins can be divided into IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE or IgM. Light chains can be kappa (κ) or lambda (λ).

[0322] In some embodiments, the polypeptide is conjugated, linked, or fused to another component (eg, an immunoglobulin) via a linker.

[0323] The skilled person is aware of linker sequences, such as Chen et al., in Adv Drug Deliv Rev. (2013) 65 (10): 1357-1369, etc., the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence can be a flexible linker sequence. The flexible linker sequence allows the amino acid sequence connected to the linker sequence to move relative to each other. The skilled person is aware of flexible linkers, and Chen et al., in Adv Drug Deliv Rev. (2013) 65 (10): 1357-1369, point out several flexible linkers. Flexible linker sequences generally include a high proportion of glycine and / or serine residues. In some embodiments, the linker sequence includes at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence includes or consists of glycine and serine residues. In some embodiments, the polypeptide is conjugated, linked or fused to another component (such as an immunoglobulin) through a linker having the amino acid sequence EPKSA (SEQ ID NO:27) or an amino acid sequence comprising up to three modifications (such as substitutions, insertions or deletions) relative to SEQ ID NO:27.

[0324] In some embodiments, derived from (SEQ ID NO: 1) or vitronectin (SEQ ID NO: 2 or SEQ ID NO: 3) have the same function as the polypeptide from which it is derived. In some embodiments, the polypeptide derived from (SEQ ID NO: 1) or vitronectin (SEQ ID NO: 2 or SEQ ID NO: 3) polypeptides have improved functions compared to the polypeptides from which they are derived.

[0325] In some embodiments, derived from (SEQ ID NO: 1) or vitronectin (SEQ ID NO: 2 or SEQ ID NO: 3) has one or more of the following properties:

[0326] Improve cell adhesion

[0327] Improves cell attachment to surfaces

[0328] Improve cell attachment to culture vessels

[0329] Improves cell attachment to the inner surface of culture vessels.

[0330] Cell attachment is enhanced as compared to cell attachment of control cells cultured in the same manner but without the polypeptide, as assessed by microscopy, flow chamber pressure measurement, non-invasive label-free real-time impedance measurement, or measurement using anti-cell attachment antibodies.

[0331] Improves cell attachment by 1.5-fold compared to control cells cultured in the same manner but without the peptide

[0332] Compared to the cell adhesion of control cells cultured in the same manner but without the polypeptide, the cell adhesion is increased by 2-fold, 3-fold, 4-fold, or 5-fold

[0333] Improves cell attachment in the absence of fetal bovine serum (FBS)

[0334] Improved cell attachment in FBS-free medium

[0335] Improve cell attachment in the absence of serum

[0336] Improved cell attachment in serum-free medium

[0337] Improve cell attachment without animal products

[0338] Improved cell attachment in animal product-free media

[0339] Improved cell attachment in xeno-free media

[0340] Reduce cell aggregation

[0341] By microscopic observation, cell aggregation is reduced compared to aggregation of control cells cultured in the same manner but without the polypeptide.

[0342] Cell aggregation was reduced 1.5-fold compared to aggregation of control cells cultured in the same manner but without the polypeptide.

[0343] Cell aggregation was reduced 2-fold compared to aggregation of control cells cultured in the same manner but without the polypeptide.

[0344] Increase virus titer

[0345] Increase the virus titer of culture supernatant

[0346] Increase the viral titer of the culture supernatant after transfection

[0347] Increase the viral titer of culture supernatant after transfection with a plasmid containing the gene of interest

[0348] Increase the viral titer of the culture supernatant 6 hours after transfection incubation

[0349] Increase the viral titer of the culture supernatant 12 hours after transfection incubation

[0350] Increase the viral titer of the culture supernatant 24 hours after transfection incubation

[0351] Increase the viral titer of the culture supernatant 48 hours after transfection incubation

[0352] Increase the viral titer of the culture supernatant 72 hours after transfection incubation

[0353] The increase in viral titer when the polypeptide is used is compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide

[0354] The viral titer was increased more than 1.5-fold when the polypeptide was used compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0355] The use of the polypeptide increases viral titer by more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0356] Improve / enhance virus-mediated gene transfer

[0357] Improve / enhance virus-mediated gene transduction in mammalian cells

[0358] Improve transduction efficiency compared to transduction without the polypeptide

[0359] Improves transduction efficiency compared to transduction without transduction enhancers

[0360] Compared to transduction without the use of a transduction enhancer, the transduction efficiency is increased by at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times

[0361] Compared to transduction without a transduction enhancer, the transduction efficiency is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times. Compared to transduction without a transduction enhancer, the transduction efficiency is increased by at least 1.1 times.

[0362] Compared to transduction without the polypeptide, the transduction efficiency is increased by at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times

[0363] Compared to transduction without the polypeptide, the transduction efficiency is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times; Compared to transduction without the polypeptide, the transduction efficiency is increased by 1.1 times

[0364] Reduced cytokine secretion from cells transduced with chimeric antigen receptor (CAR)

[0365] Reduced cytokine secretion by cells used for adoptive cell transfer

[0366] Reduce CAR-T cell secretion of cytokines

[0367] Improved cell killing ability of CAR-transduced cells

[0368] Improving the cell killing ability of cells used for adoptive cell transfer

[0369] Improving the cell-killing ability of CAR T cells

[0370] and vitronectin as an attachment enhancer

[0371] The inventors have demonstrated that when cells are grown in an adherent medium, and vitronectin can enhance cell adhesion to the culture vessel. In addition, studies have shown that in serum-free culture medium, and vitronectin can improve the yield of viral vectors without adding FBS. As shown in the examples, the improvement of adhesion leads to an increase in viral vector yield and an improvement in viral vector yield.

[0372] It has been demonstrated that this increased adhesion and improved viral vector production can be achieved in a variety of ways.

[0373] Vitronectin, derived from The polypeptides, and / or polypeptides derived from vitronectin can be present in the culture medium, added to the culture medium, and / or as a coating on a culture vessel.

[0374] In the culture medium and vitronectin

[0375] Most adherence processes rely on animal-derived products, such as fetal bovine serum (FBS), which presents challenges for safety, consistency, and regulation. In fact, most media used to culture adherent cells contain FBS and / or other serum components. The inventors have demonstrated that And vitronectin can be used in culture media to replace serum components such as FBS, allowing cells to effectively attach to the culture container.

[0376] Vitronectin, derived from The polypeptides, and / or polypeptides derived from vitronectin may be present in the culture medium at the beginning of the process, or may be added to the culture medium during the culture process, e.g. Vitronectin, derived from The polypeptides and / or polypeptides derived from vitronectin can be added to the culture medium during cell inoculation. Vitronectin, derived from The polypeptides described herein and / or polypeptides derived from vitronectin may be considered as components of the culture medium or culture medium additives.

[0377] Vitronectin, derived from The polypeptides and / or polypeptides derived from vitronectin can be added to the culture medium in various forms. For example, Vitronectin, derived from The polypeptides, and / or polypeptides derived from vitronectin can be added to the culture medium as a liquid / aqueous solution, or as a powder or gel.

[0378] Vitronectin, derived from The polypeptides, and / or polypeptides derived from vitronectin can be added to the culture medium in the form provided by the manufacturer, for example, a 1 mg / mL sterile solution containing 12.5 mM sodium citrate (pH 6.2) and 1.25% sucrose.

[0379] Vitronectin, derived from The polypeptides and / or polypeptides derived from vitronectin can be added to the culture medium via diluted stock solutions. Vitronectin, derived from The polypeptide, and / or polypeptide derived from vitronectin can be added to the culture medium at any concentration of 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg / mL.

[0380] Vitronectin, derived from The polypeptide and / or the polypeptide derived from vitronectin can be added to the culture medium at a concentration of 20 μg / mL. Vitronectin, derived from The polypeptide, and / or polypeptide derived from vitronectin can be added to the culture medium at a concentration of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg / mL of culture medium.

[0381] Vitronectin, derived from The polypeptide, and / or the polypeptide derived from vitronectin can be added to the culture medium at a concentration exceeding 20 μg / mL. Vitronectin, derived from The polypeptide, and / or polypeptide derived from vitronectin can be added to the culture medium at a concentration greater than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg / mL of the culture medium.

[0382] Vitronectin, derived from The polypeptide, and / or polypeptide derived from vitronectin can be added to the culture medium at a concentration of less than 20 μg / mL. Vitronectin, derived from The polypeptide, and / or polypeptide derived from vitronectin can be added to the culture medium at a concentration of less than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μg / mL of culture medium.

[0383] and vitronectin coating

[0384] As mentioned above, most adhesion processes rely on animal-derived products, such as fetal bovine serum (FBS), which presents challenges for safety, consistency, and regulation. The inventors have demonstrated that and vitronectin can be coated on the culture vessel to avoid the use of serum components such as FBS, thereby allowing cells to effectively attach to the culture vessel.

[0385] As shown in the examples, despite the absence of serum components such as FBS, and vitronectin to coat culture vessels to increase adhesion, virus yield, and virus productivity.

[0386] Technicians already know Vitronectin, derived from Various methods for applying polypeptides derived from vitronectin to culture containers. Vitronectin, derived from The polypeptide and / or the polypeptide derived from vitronectin may be applied only to the inner surface of the culture vessel, or the entire culture vessel may be coated. Vitronectin, derived from and / or polypeptides derived from vitronectin.

[0387] The examples illustrate the use of An exemplary method of coating a culture vessel with vitronectin and / or vitronectin. Other methods are also possible.

[0388] and / or vitronectin, or derived from Peptides of vitronectin and / or vitronectin can be diluted in PBS to a range of concentrations (e.g., 0.1, 1, 4, 8, and 16 μg / mL in a solvent such as PBS). The appropriate amount of solution can be added to the culture vessel and then incubated overnight at 4°C. Studies have also shown that placing it at room temperature or in an incubator for 2-3 hours can also promote smearing. The technician can adjust the appropriate concentration, solution volume, incubation time and incubation temperature.

[0389] In some embodiments, the incubation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the incubation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0390] In some embodiments, incubation is performed in a refrigerator, at room temperature, or in an incubator at ≥ 30°C.

[0391] In some embodiments, the solution comprising the polypeptide comprises a polypeptide concentration of at least 0.1 μg / mL. In some embodiments, the solution comprising the polypeptide comprises a polypeptide concentration of at least 5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, or 20 μg / mL.

[0392] In some embodiments, the culture container is a culture plate, a culture dish, a culture bottle, a culture tank or a bioreactor. In some embodiments, the culture plate is a 2-well plate, a 4-well plate, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate.

[0393] In some embodiments, the volume of the culture container is at least 0.2 mL. In some embodiments, the volume of the culture container is at least 0.5 mL, preferably, the volume of the culture container is at least 0.6, 0.7, 0.8, 0.9, 1.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 125.0, 150.0, 175.0, or 200.0 mL.

[0394] In some embodiments, the culture container is coated with and / or vitronectin, or derived from and / or vitronectin polypeptides. One or more layers of coating can be applied to the culture vessel. The coating acts as a matrix for cell growth and can increase the adhesion of cells to the culture vessel.

[0395] The culture container may be coated with a matrix, in this context, a matrix refers to a layer of material (such as a thin layer or film) attached to the culture container (such as a surface). The matrix may include RetroNectin (a recombinant human fibronectin fragment) and / or vitronectin, and / or a protein derived from The matrix may include other compatible or physiologically relevant matrices that can support cell growth.

[0396] The matrix may include other components of an extracellular matrix (ECM). Any known component of the ECM may be used, such as components that support cell growth. The components of the extracellular matrix are known in the art, such as those described in Chapter 4 of Molecular Biology of Cells (2002) by Alberts et al. and the references cited therein.

[0397] In some embodiments, the matrix is ​​free of fetal bovine serum, serum-free, xenobiotic-free or animal-free. Additional matrix materials can include poly-L-lysine, laminin, gelatin, collagen, keratin, fibronectin, elastin, heparin sulfate, dextran, dextran sulfate, chondroitin sulfate or a mixture of laminin, collagen I, heparin sulfate proteoglycan and entactin 1, and derivatives or fragments thereof. The ECM component can be attached or connected or applied to the culture vessel by conventional methods. For example, any coupling agent or cross-linking agent mentioned above can be used for connecting the ECM component to the culture vessel.

[0398] The additional ECM components may include macromolecules, such as polysaccharides, proteins, proteoglycans, glycoproteins, glycosaminoglycans (GAGs), which are usually covalently linked to proteins in the form of proteoglycans, fibrous proteins, including elastin, fibronectin and laminin, collagens (such as collagen I, collagen III, collagen IV, collagen VI), etc.

[0399] The matrix coating may include glycosaminoglycans (GAGs). Glycosaminoglycans include unbranched polysaccharide chains consisting of repeating disaccharide units. One of the two sugars in the repeating disaccharides is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which is sulfated in most cases. The second sugar is typically uric acid (glucuronic acid or iduronic acid).

[0400] The matrix coating may include hyaluronic acid (hyaluronan, also known as hyaluronic acid or hyaluronate) or a derivative thereof. The hyaluronic acid may be derived from several sources, such as from bovine vitreous. A salt or base of hyaluronic acid may be used, such as sodium hyaluronate. It may be from streptococci.

[0401] The matrix coating may include laminin and / or fibronectin and / or collagen and / or keratin and / or gelatin.

[0402] The matrix coating may include examples of GAGs such as chondroitin sulfate, dermatan sulfate, heparan sulfate and keratan sulfate, for example, proteoglycans linked to proteins. The matrix coating may include aggrecans, decolins, and the like.

[0403] The matrix coating may include heparan or a derivative thereof such as a base or a salt. The matrix coating may include heparan sulfate proteoglycans. The heparan sulfate proteoglycans may be derived from several sources, such as from bovine kidney.

[0404] The matrix coating may include dextran such as dextran sulfate or sodium dextran sulfate. The matrix coating may include fibronectin, laminin, nidogen or type IV collagen. The matrix coating may include chondroitin sulfate.

[0405] The matrix may include gelatin, polyornithine, or a binding motif of the fibronectin RGD binding domain.

[0406] ECM matrix materials include: laminin, fibronectin, vitronectin, collagen, elastin, aggrecan, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, and hyaluronic acid.

[0407] The matrix coating may include a mixture of any two or more of the above components mixed in any proportion. The matrix coating may include a purified or substantially purified component such as ECM. The matrix coating may include a partially purified component of ECM. It may include an ECM extract such as Matrigel™.

[0408] and vitronectin as a transduction enhancer

[0409] Various aspects of the present invention relate to viral-mediated gene transduction. Transduction is the process of introducing foreign DNA into cells via a virus or viral vector (Bouard et al., (2009) 157(2): 153-165). Transduction does not require physical contact between the cell providing the DNA and the cell receiving the DNA (occurs during conjugation) and is DNase-resistant (transformation is susceptible to DNase). Transduction is a common tool used by molecular biologists to stably introduce foreign genes into the genome of host cells (including bacteria and mammalian cells).

[0410] Viral vector transduction can be used to insert or modify genes in mammalian cells. When constructing a plasmid, the gene to be transferred is flanked by viral sequences, which are used by viral proteins to recognize the viral genome and package it into viral particles. This plasmid is inserted into production cells (usually by transfection) along with other plasmids (DNA constructs) carrying the viral genes required to form infectious viral particles. In these production cells, the viral proteins expressed by these packaging constructs bind to sequences on the DNA / RNA to be transferred (depending on the type of viral vector) and insert it into viral particles. The viruses collected from these cells are applied to the cells to be changed. This will result in the expression of the transferred gene and (in the case of lentiviral / retroviral vectors) the insertion of the DNA to be transferred into the cell genome.

[0411] In order to make cell transformation as efficient as possible, it is necessary to ensure the efficiency of transduction. To achieve this goal, it is necessary to use additives to enhance transduction. The inventors have shown Vitronectin, derived from Polypeptides derived from vitronectin can serve as transduction enhancers.

[0412] It has been demonstrated that transduction can be enhanced by a number of methods. and / or vitronectin (or derived from and / or vitronectin polypeptides) can be present in the culture medium, added to the culture medium, or as a coating on the culture vessel. Vitronectin, derived from The details of how the polypeptides derived from vitronectin are present in the culture medium, added to the culture medium, or as a coating on the culture vessel are described. Determining transduction efficiency by any method is known in the art. One method is shown in the examples (such as Example 3), where flow cytometry is used to determine the percentage of transduced cells. The percentage of transduced cells indicates the transduction efficiency. The skilled person is aware of a variety of tests for determining transduction efficiency.

[0413] For example, if cells are transduced with CV-hCD19 LV vector and subsequently 25% of the cells express CD19CAR on the cell surface, the transduction efficiency is 25%.

[0414] For example, if the transduction efficiency of a cell population is 25% without the use of a transduction enhancer, and when the polypeptide is used as a transduction enhancer, the transduction efficiency of the cell population is 27.5%, then the polypeptide improves the transduction efficiency. In fact, the polypeptide improves the transduction efficiency by 1.1 times (i.e., by 10%), and the transduction efficiency of the cell population when the polypeptide is used as a transduction enhancer is 1.1 times the transduction efficiency of the cell population when the transduction enhancer is not used.

[0415] In some embodiments, the polypeptide-enhanced transduction efficiency is compared to the transduction efficiency without the transduction enhancer.

[0416] In some embodiments, the polypeptide increases the transduction efficiency by at least 1.1 times compared to the transduction efficiency without the transduction enhancer. In some embodiments, the polypeptide increases the transduction efficiency by at least 1.2 times compared to the transduction efficiency without the transduction enhancer, preferably, wherein the polypeptide increases the transduction efficiency by at least 1.3, 1.4, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times compared to the transduction efficiency without the transduction enhancer.

[0417] In some embodiments, the transduction efficiency is at least 1.1 times the transduction efficiency without a transduction enhancer. In some embodiments, the transduction efficiency is 1.2, 1.3, 1.4, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times the transduction efficiency without a transduction enhancer.

[0418] Cytokine secretion inhibitors

[0419] It is well known that cytokines such as IL-6 are core mediators of cytokine release syndrome (CRS) toxicity associated with CAR-T cell therapy (Tanaka et al., Immunotherapy (2016) 8(8):959-70). A challenge in the development of cell therapies such as CAR-T cell therapies is the need to reduce the impact of CRS without compromising efficacy.

[0420] The examples illustrate that the polypeptides disclosed herein can inhibit cells from secreting cytokines.

[0421] The examples also demonstrate that the polypeptides disclosed herein are capable of inhibiting cytokine secretion from cells without negatively affecting the cell killing ability / toxicity of the cells.

[0422] In some embodiments, the polypeptides described in the present disclosure can inhibit cells from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit immune cells from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit effector immune cells from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit cells suitable for adoptive cell transfer from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit cells for adoptive cell transfer from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit cells for cell therapy from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit T cells or NK cells from secreting cytokines. In some embodiments, the polypeptides described in the present disclosure can inhibit CART cells or CAR NK cells from secreting cytokines.

[0423] In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells while maintaining the cell killing ability of the cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by immune cells while maintaining the cell killing ability of the immune cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by effector immune cells while maintaining the cell killing ability of the effector immune cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells suitable for adoptive cell transfer while maintaining the cell killing ability of the cells suitable for adoptive cell transfer. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells used for adoptive cell transfer while maintaining the cell killing ability of the cells used for adoptive cell transfer. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells used for cell therapy while maintaining the cell killing ability of the cells used for cell therapy. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by T cells or NK cells while maintaining the cell killing ability of the T cells or NK cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by CAR T cells or CAR NK cells while maintaining the cell killing ability of the CAR T cells or CAR NK cells.

[0424] In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by immune cells without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by effector immune cells without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells suitable for adoptive cell transfer without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells used for adoptive cell transfer without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by cells used for cell therapy without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by T cells or NK cells without inhibiting the killing of target cells. In some embodiments, the polypeptides described in the present disclosure can inhibit the secretion of cytokines by CAR T cells or CAR NK cells without inhibiting the killing of target cells.

[0425] In one aspect, a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a polypeptide derived from SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1 is contacted with a cell.

[0426] In some embodiments, a polypeptide comprising an amino acid sequence having at least 70% sequence identity to either SEQ ID NO:3 or SEQ ID NO:2, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a polypeptide derived from SEQ ID NO:3 or SEQ ID NO:2 is contacted with a cell.

[0427] In one aspect, a method for inhibiting and / or reducing the secretion of cytokines by cells is provided, the method comprising contacting the cells with a polypeptide comprising an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity with a polypeptide fragment of SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:1.

[0428] In some embodiments, a method for inhibiting and / or reducing the secretion of cytokines by cells is provided, the method comprising contacting the cells with a polypeptide comprising an amino acid sequence having at least 70% sequence identity with either SEQ ID NO:3 or SEQ ID NO:2, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity with a polypeptide fragment of SEQ ID NO:3 or SEQ ID NO:2.

[0429] It is also contemplated to use a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any of SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 1, in a method of inhibiting and / or reducing cytokine secretion.

[0430] It is also contemplated to use a polypeptide comprising an amino acid sequence having at least 70% sequence identity to either SEQ ID NO: 3 or SEQ ID NO: 2, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO: 3 or SEQ ID NO: 2, in a method of inhibiting and / or reducing cytokine secretion.

[0431] In some embodiments, the cell is a cell for adoptive cell transfer. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an effector immune cell. In some embodiments, the cell is a T cell or a NK cell. In some embodiments, the cell is a CAR T cell or a CAR NK cell. In some embodiments, the method of inhibiting and / or reducing cytokine secretion cannot reduce the cell killing ability of the cell. In some embodiments, there is no reduction / inhibition of killing of target cells. In some embodiments, cytotoxicity is not reduced / inhibited. In some embodiments, cytotoxicity is maintained.

[0432] In some embodiments, a method for inhibiting and / or reducing cytokine secretion while maintaining cytotoxicity is provided, wherein the method comprises contacting a cell with a polypeptide comprising an amino acid sequence having at least 70% sequence identity to either SEQ ID NO:3 or SEQ ID NO:2, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a polypeptide fragment of SEQ ID NO:3 or SEQ ID NO:2.

[0433] In some embodiments, the cytokine is one or more of IL-6, TNF-α, IFN-γ, and IL-8. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is TNF-α. In some embodiments, the cytokine is IFN-γ. In some embodiments, the cytokine is IL-8.

[0434] In some embodiments, the method is performed in glassware, in vitro, or in vivo. In some embodiments, the method is a method in glassware. In some embodiments, the method is a method in vitro. In some embodiments, the method is a method in vivo.

[0435] In some embodiments, the cell is contacted with the polypeptide in a glass vessel, in vitro, or in vivo. In some embodiments, the cell is contacted with the polypeptide in a glass vessel. In some embodiments, the cell is contacted with the polypeptide in vitro. In some embodiments, the cell is contacted with the polypeptide in vivo.

[0436] The level of cytokine secretion from the cells can be compared to a suitable control group. For example, the level of cytokine secretion from cells contacted with a polypeptide according to the disclosure can be compared to cells that have not been contacted with a polypeptide according to the disclosure.

[0437] Alternatively, the levels of cytokine secretion from cells contacted with a polypeptide according to the present disclosure can be compared to cells contacted with a known transduction enhancer. Alternatively, the levels of cytokine secretion from cells contacted with a polypeptide according to the present disclosure can be compared to cells contacted with a known transduction enhancer. The exposed cells were compared.

[0438] Methods for detecting the level of cytokines secreted by cells are known in the art, for example, using the method used in Example 9 of the present disclosure. In this experiment, the cells can be incubated in a suitable culture medium for a period of time (such as 24 hours) before the supernatant is analyzed. Technicians already know a variety of suitable methods for analyzing the supernatant. In the experiment in Example 9, CAR-expressing T cells under each condition were co-cultured with NALM-6-Luc tumor cells expressing luciferase 4 days or 14 days after transduction. After 24 hours of co-culture, the cell culture supernatant was collected. The cell culture supernatant samples were analyzed using a fully automated ELISA (ELLA immunoassay system, ProteinSimple, USA) to determine the cytokine levels of the transduction enhancer-treated group or the control group. Technicians can easily adjust this experiment to evaluate the levels of cytokines secreted by many different types of cells. Technicians know many methods for evaluating the level of cytokines secreted by cells.

[0439] Production of cells for adoptive cell transfer

[0440] The method disclosed herein may involve the stimulation and / or amplification of immune cells. The method may involve the stimulation and / or amplification of peripheral blood cells, such as peripheral blood mononuclear cells. The method may involve the amplification of an immune cell group (such as a T cell group) in an immune cell group (such as a PBMC). For example, by stimulating the T cells in the PBMC group, the T cell group in the PBMC group can be amplified. Therefore, in the embodiments of the method in the present disclosure, stimulating and / or amplifying T cells may involve stimulating the PBMC group. In some embodiments, by stimulating the T cells in the tumor infiltrating lymphocyte group, the T cell group in the tumor infiltrating lymphocyte group can be amplified. Therefore, in the embodiments of the method in the present disclosure, stimulating and / or amplifying T cells may involve stimulating the tumor infiltrating lymphocyte group. In some embodiments, a T cell group can be amplified from a T cell group (such as a heterogeneous specific T cell group), and the T cell group can be obtained from a blood sample, a PBMC group, or a tumor infiltrating lymphocyte group. The method may involve stimulating and / or amplifying T cells. The cells can be obtained from the patient to be treated (i.e., autologous cells) or from other individuals (i.e., allogeneic cells). In a specific embodiment, the method involves stimulating and / or amplifying isolated immune cells. That is, a specific method can be performed in a cell population (e.g., red blood cells) that is substantially free of non-immune cells. In some cases, the immune cells are isolated PBMCs or isolated T cells.

[0441] The cells can be obtained from a blood sample, such as a blood sample obtained from a patient or individual. The cells can be obtained from a tissue sample or a biopsy. The cells can be obtained from a tumor (such as tumor infiltrating lymphocytes). The specific method disclosed herein relates to the step of obtaining PBMC and / or T cells from a sample from a patient. The specific embodiment of the method does not involve the step of obtaining a blood or cell sample from a patient or individual, but instead processes the sample or cells previously obtained. The method may involve processing the sample, such as enriching the sample to obtain immune cells, such as PBMC and / or T cells. This method may involve removing or substantially reducing the content of red blood cells, platelets, serum and / or plasma in the sample. This may result in the immune cell population being substantially free of other cells such as red blood cells. The method disclosed herein can be performed in isolated immune cells, or in a sample containing immune cells in addition to other cells.

[0442] In some embodiments, the generated / expanded immune cell population is prepared for use in a subject from which the generated / expanded immune cell population is obtained / derived. In some embodiments, the generated / expanded immune cell population can be used in any subject, e.g., a subject different from the subject from which the initial immune cell population was derived, from which the generated / expanded immune cell population is obtained / derived.

[0443] In some embodiments, cells are modified to express specific non-natural receptors so that T cells can effectively or more effectively target desired target cells, such as cells expressing specific antigens. Cells can be modified to express chimeric antigen receptors (CARs), such as T cell receptors, etc. Cells can be modified to express expression vectors (which can be viruses (including retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, etc.) or non-viruses) at specific time points in the method, such as introducing vectors after 2 to 5 days of culture. In some embodiments, cells are contacted with expression vectors 3 days after each stimulation, but in this case, the modification occurs in more differentiated T cells, which have shorter long-term potentiation effects (which is desirable in certain circumstances).

[0444] In certain embodiments, cells are engineered to express CARs targeting tumor antigens, such as EphA2, HER2, GD2, Glypican-3, 5T4, 8H9, αvβ6 integrin, B cell maturation antigen (BCMA) B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor alpha, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11 Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA, VEGF receptor, and / or other antigens typically present in the extracellular matrix of tumor cells, such as fibronectin, tenascin, or carcinoembryonic variants in tumor necrotic areas, as well as other tumor-associated antigens or actionable mutations discovered through tumor genomic analysis and or differential expression studies.

[0445] Enhancers of cell killing

[0446] When preparing cells for adoptive cell transfer, it is necessary to maintain the cell killing ability of the therapeutic cells. The inventors have discovered that the polypeptides of the present disclosure are able to enhance the cell killing ability of cells for adoptive cell transfer.

[0447] In some embodiments, the polypeptides described in the present disclosure can enhance the cell killing ability of cells. In some embodiments, the polypeptides described in the present disclosure can enhance the cell killing ability of immune cells. The polypeptides described in the present disclosure can enhance the cell killing ability of effector immune cells. The polypeptides described in the present disclosure can enhance the cell killing ability of cells used for adoptive cell transfer. The polypeptides described in the present disclosure can enhance the cell killing ability of cells used for cell therapy. In some embodiments, the polypeptides described in the present disclosure can enhance the cell killing ability of T cells or NK cells. In some embodiments, the polypeptides described in the present disclosure can enhance the cell killing ability of CAR T cells or CAR NK cells.

[0448] In one aspect, a method for enhancing or improving cell killing of a target cell is provided. In some embodiments, the cell is contacted with a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1.

[0449] In some embodiments, the method comprises contacting the cell with a polypeptide having an amino acid sequence having at least 70% sequence identity to either SEQ ID NO:3 or SEQ ID NO:2, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3 or SEQ ID NO:2.

[0450] In some embodiments, the cell is a cell for adoptive cell transfer. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an effector immune cell. In some embodiments, the cell is a T cell or a NK cell. In some embodiments, the cell is a CAR T cell or a CAR NK cell. In some embodiments, during cell culture or transduction, the cell for adoptive cell transfer is contacted with a polypeptide of the present disclosure. In some embodiments, during cell culture, the cell for adoptive cell transfer is contacted with a polypeptide of the present disclosure. In some embodiments, during transduction, the cell for adoptive cell transfer is contacted with a polypeptide of the present disclosure.

[0451] In some embodiments, the target cell is a tumor cell. In some embodiments, the cell for adoptive cell transfer is a CAR T cell or a CAR NK cell, and the target cell includes an antigen targeted by a CAR expressed by a cell for adoptive cell transfer. In some embodiments, the cell for adoptive cell transfer is a CAR T cell, and the target cell includes an antigen targeted by a CAR expressed by a cell for adoptive cell transfer. For example, in some embodiments, the cell for adoptive cell transplantation is a CD19 CAR T cell, and the target cell is CD19 positive (i.e., the target cell expresses CD19 targeted by CD19 CAR T cells).

[0452] The increase or enhancement of cell killing can be compared to an appropriate control group. For example, the level of cell killing of cells for adoptive cell transfer contacted with a polypeptide according to the disclosure can be compared to cells that have not been contacted with a polypeptide according to the disclosure. Alternatively, the level of cell killing of cells for adoptive cell transfer contacted with a polypeptide according to the disclosure can be compared to cells contacted with a known transduction enhancing agent. Alternatively, the level of cell killing of cells for adoptive cell transfer contacted with a polypeptide according to the disclosure can be compared to cells contacted with a known transduction enhancing agent. The exposed cells were compared.

[0453] Methods for detecting the level of cell killing are known in the art, for example, using the method used in Example 9 of the present disclosure. In this experiment, the target cells are modified to express a reporter factor, and the reporter factor is analyzed to determine the level or total amount of cell killing. In Example 9 of the present disclosure, luciferase is used as the reporter factor. In this experiment, tumor cells expressing luciferase are incubated with CAR T cells under different treatment conditions. According to Bright-Glo TM Tumor cell killing was measured by luciferase activity detected by the luciferase assay system (Promega, USA). The specific cell killing rate was calculated based on the ratio of total cell lysis to spontaneous cell lysis under each condition. Fig.12 D. The skilled person will appreciate that other reporter genes may also be used, and the same method may be used to assess different cells suitable for adoptive cell transfer. The skilled person will also appreciate that other techniques may be used to assess the level of cell killing. Microscopic in vivo imaging, commercial detection techniques, and other methods in the art may also be used to assess the level of target cell killing.

[0454] Chimeric Antigen Receptors (CARs)

[0455] The present invention relates to cells comprising / expressing a chimeric antigen receptor (CAR).

[0456] CAR is a recombinant receptor molecule that provides antigen binding and T cell activation functions. The structure and engineering of a team of CARs are reviewed, for example, Dotti et al. described in "Immunol Rev" (2014) 257 (1), the contents of which are incorporated herein by reference. CAR includes an antigen binding domain connected to a signaling domain via a transmembrane domain. An optional hinge domain can provide a separation between the antigen binding domain and the transmembrane domain and can act as a flexible joint. When expressed by a cell, the antigen binding domain is located outside the cell, while the signaling domain is located inside the cell.

[0457] The antigen binding domain mediates binding to the CAR-specific target antigen. The antigen binding domain of CAR can be based on the antigen binding region of an antibody that is specific to the CAR-targeted antigen. For example, the antigen binding domain of CAR may include the amino acid sequence of the complementary determining region (CDR) of an antibody that specifically binds to the target antigen. The antigen binding domain of CAR may include or consist of the light chain and heavy chain variable region amino acid sequences of an antibody that specifically binds to the target antigen. The antigen binding domain may be used as a single-chain variable fragment (scFv), which includes the light chain and heavy chain variable region amino acid sequences of the antibody. Based on protein: protein interactions, such as ligand: receptor binding, the antigen binding domain of CAR can target antigens; for example, using an IL-13-based antigen binding domain, an IL-13Rα2-targeted CAR has been developed (see, for example, Kahlon et al., described in Cancer Res, (2004) 64 (24): 9160-9166).

[0458] The transmembrane domain is located in the antigen binding domain and signaling domain of CAR. The transmembrane domain uses the antigen binding domain in the extracellular space and the signaling domain in the cell to anchor CAR to the cell membrane of the cell expressing CAR. The transmembrane domain of CAR can be derived from the transmembrane region sequence of the cell membrane-bound protein (such as CD28, CD8, etc.).

[0459] The signal domain includes the amino acid sequence required for activating immune cell function.The CAR domain may include the amino acid sequence of the CD3-ζ extracellular domain, which provides for phosphorylation and activation of the immunoreceptor tyrosine activation motif (ITAM) of cells expressing CAR.The signal domain includes other protein sequences containing ITAM that are also used for CAR, such as the domain containing ITAM region of FcγRI (Haynes et al. in "J Immunol 1" (2001) 66 (1): 182-187 description). CAR including the signal domain derived from the CD3-ζ intracellular domain is generally referred to as the first generation CAR.

[0460] The signal domain of CAR generally also includes the signal domain of costimulatory proteins (such as CD24, 4-1BB, etc.), so as to provide the costimulatory signal necessary for enhancing immune cell activation and effector function.CAR with a signal domain including an additional costimulatory sequence is generally referred to as second-generation CAR.In some cases, CAR is modified to provide costimulation of different intracellular signaling pathways.For example, CD28 costimulation preferentially activates phosphatidylinositol 3-kinase (P13K) pathways, while 4-1BB costimulation is triggered by TNF receptor-associated factor (TRAF) adapter protein signal.Therefore, the signal domain of CAR sometimes includes a costimulatory sequence derived from the signal domain of more than one costimulatory molecule.CAR including a signal domain with a variety of costimulatory sequences is generally referred to as third-generation CAR.

[0461] The optional hinge region can provide separation between the antigen binding domain and the transmembrane domain and can serve as a flexible linker. The hinge region can be a flexible domain that allows the binding molecule to be oriented in different directions, for example, the CH1-CH2 hinge region can be derived from IgG.

[0462] By modifying to express a specific CAR for a specific target antigen, immune cells (usually T cells, but other immune cells such as NK cells) can be guided to kill cells expressing the target antigen. CAR-expressing T cells (CAR-T cells) specifically bind to the target antigen to trigger intracellular signals and thus activate T cells. Stimulate the activated CAR-T cells to separate and produce factors that kill cells expressing the target antigen.

[0463] "Antigen binding domain" refers to a molecule that can bind to a target antigen. The target antigen can be a peptide / polypeptide, glycoprotein, lipoprotein, polysaccharide, glycolipid, lipid, or a fragment thereof. According to the present disclosure, the antigen binding domain can be derived from an antibody fragment (such as Fv, scFv, Fab, single-chain Fab (scFab), single domain antibody (such as VhH), etc.) of an antibody / target antigen or other target antigen binding agent (such as a target antigen binding peptide or nucleic acid aptamer, ligand or other molecule).

[0464] The CAR of the present disclosure includes a transmembrane domain. A transmembrane domain refers to any three-dimensional structure formed by a thermodynamically stable amino acid sequence in a biological membrane (such as a cell membrane). For the purposes of the present disclosure, a transmembrane domain can be an amino acid sequence that spans the cell membrane of a cell expressing CAR.

[0465] The transmembrane domain may include or be composed of an amino acid sequence forming a hydrophobic alpha helix or a beta fold. The amino acid sequence of the transmembrane domain of the CAR of the present invention may be, or may be derived from the amino acid sequence of the transmembrane domain of the protein including the transmembrane domain. The transmembrane domain is recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or is identified / predicted by amino acid sequence analysis tools such as TMHMM (Krogh et al., JMolBiol (2001) 305:567-580 description).

[0466] The chimeric antigen receptor of the present invention includes a signaling domain. The signaling domain provides a sequence that initiates an intracellular signal in a cell expressing CAR.

[0467] The signaling domain includes a sequence containing ITAM. The sequence containing ITAM includes one or more immunoreceptor tyrosine activation motifs (ITAM). ITAM includes an amino acid sequence of YXXL / I (SEQ ID NO: 4), wherein "X" represents any amino acid. In proteins containing ITAM, the sequence is usually separated by 6 to 8 amino acids as shown in SEQ ID NO: 4; YXXL / I (X) 6-8YXXL / I (SEQ ID NO: 5). When a phosphate group is added to the tyrosine residue of ITAM by a tyrosine kinase, a signaling cascade in the cell is initiated.

[0468] In some embodiments, the signaling domain comprises one or more copies of the amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the signaling domain comprises at least 1, 2, 3, 4, 5, or 6 copies of the amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the signaling domain comprises at least 1, 2, or 3 copies of the amino acid sequence as set forth in SEQ ID NO: 5.

[0469] The CAR may also include a hinge region. The hinge region is located between the antigen binding domain and the transmembrane domain. The hinge region is an amino acid sequence that flexibly connects the antigen binding domain and the transmembrane domain of the CAR.

[0470] The presence, absence, and length of the hinge region have been shown to affect the function of CAR (reviewed by Dotti et al. in Immunol Rev (2014) 257(1):supra).

[0471] In some embodiments, the CAR includes a dimerization domain. As used herein, a "dimerization domain" refers to an amino acid sequence that can connect a protein to another protein to form a dimer, oligomer, or polymer. In some embodiments, other proteins can be membrane-bound molecules, such as receptors or ligands. In some embodiments, the dimerization domain can make CAR self-connected to form a homodimer, or can make it form a heterodimer with another different protein.

[0472] In some embodiments, the CAR includes one or more joints between different domains (such as antigen binding domains, hinge regions, transmembrane domains, signaling domains). In some embodiments, the CAR includes one or more joints between domain subsequences (such as between antigen binding domains VH and VL).

[0473] CAR-expressing immune cells

[0474] In some embodiments, immune cells expressing CARs are contemplated.

[0475] The cell may be a eukaryotic cell, such as a mammalian cell. The mammal may be a human, or a non-human mammal (such as a rabbit, guinea pig, rat, mouse or other rodent (including any animal from the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows, or any animal from the genus Bos), horse (including any animal from the family Equidae), donkey, and non-human primate).

[0476] In some embodiments, the cells may be derived from, or may be obtained from, a human subject. In some embodiments, the cells may be derived from, or may be obtained from, blood or an umbilical cord.

[0477] The cell may be a hematopoietic cell, such as a neutrophil, an eosinophil, a basophil, a lymphocyte, or a monocyte. The lymphocyte may be a T cell, a B cell, or a NK cell or a precursor thereof. The cell may express a CD3 polypeptide (such as CD3γ, CD3ε, CD3ζ, or CD3δ), a TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8.

[0478] In some embodiments, the cell is a T cell. In some embodiments, the T cell is a CD3+T cell. In some embodiments, the T cell is a CD3+, CD8+T cell. In some embodiments, the T cell is a cytotoxic T cell (such as a cytotoxic T lymphocyte (CTL)). In some embodiments, the T cell is a γδT cell, a cytotoxic T cell, or a helper T cell.

[0479] In some embodiments, the cell is a target protein reactive CAR-T cell. In this embodiment, a "target protein reactive" CAR-T cell is a cell with T cell-specific functional properties for a target protein (such as expressed on the cell surface) specific to the CAR antigen binding domain. In some embodiments, the property is a functional property associated with effector T cells (such as cytotoxic T cells).

[0480] The present invention also provides a method for producing a cell comprising a nucleic acid or a vector according to the present invention, comprising introducing a nucleic acid or a vector according to the present invention into a cell. The present invention also provides a method for producing a cell expressing a CAR according to the present invention, comprising introducing a nucleic acid or a vector according to the present invention into a cell. In some embodiments, the method further comprises culturing the cell under suitable conditions so that the cell expresses the nucleic acid or the vector. In some embodiments, the method is performed in vitro.

[0481] Virus-mediated gene transfer In some embodiments, the introduction of the isolated nucleic acid or vector according to the present invention into the cell comprises viral transduction, such as lentiviral transduction. Therefore, in some embodiments, the isolated nucleic acid or vector is contained in a viral vector, or the vector is a viral vector. In some embodiments, the method comprises introducing the nucleic acid or vector according to the present invention into the cell using electroporation, such as Koh et al. in Molecular Therapy-Nucleic Acids (2013) 2: e114, the contents of which are incorporated herein by reference.

[0482] Viral vector transduction can be used to insert or modify genes in mammalian cells. When constructing a plasmid, the gene to be transferred is flanked by viral sequences, which are used by viral proteins to recognize the viral genome and package it into viral particles. This plasmid is inserted into production cells (usually by transfection) along with other plasmids (DNA constructs) carrying the viral genes required to form infectious viral particles. In these production cells, the viral proteins expressed by these packaging constructs bind to sequences on the DNA / RNA to be transferred (depending on the type of viral vector) and insert it into viral particles. The viruses collected from these cells are applied to the cells to be changed. This will result in the expression of the transferred gene and (in the case of lentiviral / retroviral vectors) the insertion of the DNA to be transferred into the cell genome.

[0483] The invention also provides a cell obtained or obtainable according to the method for producing a cell according to the invention.

[0484] Methods of using transduced cells

[0485] The transduced cells (e.g., immune cells) (e.g., T cells expressing CAR, such as CD19.CAR T cells) described herein can be used in therapeutic and / or preventive methods.

[0486] The present disclosure also provides a composition comprising one or more (such as a group) CAR-expressing immune cells according to the present disclosure.

[0487] The cells described herein can be formulated into pharmaceutical compositions or drugs for clinical use, and can include pharmaceutically acceptable carriers, diluents, excipients or adjuvants. The composition can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal administration, which may include injection or infusion.

[0488] Suitable preparations may include cells in sterile or isotonic culture media. Drugs and pharmaceutical compositions may be formulated into fluid (including gel) forms. Liquid preparations may be formulated into dosing agents by injection or infusion (e.g., via catheters) for selected areas in the human or animal body.

[0489] In some embodiments, the composition is formulated for injection or infusion, such as into a blood vessel or a tumor.

[0490] The present disclosure also provides a method for preparing an effective pharmaceutical composition, which preparation method may include one or more steps selected from the following group: producing the cells described herein; isolating the cells described herein; and / or mixing the cells described herein with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0491] For example, another aspect of the present disclosure relates to a method for formulating or producing a medicament or pharmaceutical composition for the treatment of a disease / symptom (such as cancer), the method comprising formulating the pharmaceutical composition or medicament by mixing the cells described herein with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0492] "Cancer" may include any one or more of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, female reproductive system cancer, male reproductive system cancer, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon and rectal cancer, colon cancer, endometrial cancer, endometrial stromal sarcoma, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, Leukemia, leukemia, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penis cancer, pharyngeal cancer, pituitary tumor, plasma cell tumor, primary central nervous system lymphoma, prostate cancer, rectal cancer, respiratory system cancer, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, gastric cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0493] The cancer may be of a specific type. Examples of cancer types include: astrocytoma, carcinoma (e.g., adenocarcinoma, hepatocellular carcinoma, medullary carcinoma, papillary carcinoma, squamous cell carcinoma), glioma, lymphoma, medulloblastoma, melanoma, myeloma, meningioma, neuroblastoma, sarcoma (e.g., angiosarcoma, chondrosarcoma, osteosarcoma).

[0494] According to some aspects of the present invention, the cells, drugs or pharmaceutical compositions can be used for adoptive cell transfer.

[0495] Adoptive cell transfer generally refers to the process of obtaining cells (such as immune cells) from a subject, usually by extracting a blood sample. The cells are usually subsequently transformed and / or amplified and applied to the same subject (in the case of adoptive transfer of autologous / self-derived cells) or different subjects (in the case of adoptive transfer of allogeneic cells). The purpose of treatment is usually to provide a cell population with certain desired characteristics to the subject, or to increase the frequency of cells with these characteristics in the subject. The purpose of adoptive transfer is to introduce cells or cell populations into the subject, and / or to increase the frequency of cells or cell populations in the subject. The adoptive transfer of immune cells has been described in, for example, Kalos and June in "Immunity" (2013) 39 (1): 49-60 and Davis et al. in "Cancer J." (2015) 21 (6): 486–491, the contents of which are incorporated herein by reference. A skilled person can determine appropriate reagents and procedures for adoptive cell transfer based on the present disclosure, for example, referring to the description by Dai et al. in J Nat Cancer Inst (2016) 108(7): djv439, the contents of which are incorporated herein by reference.

[0496] The present disclosure provides a method comprising administering to a subject an immune cell comprising / expressing a CAR according to the present disclosure, or an immune cell comprising / expressing a nucleic acid encoding a CAR according to the present disclosure.

[0497] In some embodiments, the method includes generating immune cells, or generating / amplifying immune cell populations. In some embodiments, the method includes modifying immune cells to include / express CAR according to the present disclosure. In some embodiments, the method includes modifying immune cells to include / express nucleic acids encoding CAR according to the present disclosure. In some embodiments, the method includes administering to the subject an immune cell modified to express / include CAR according to the present disclosure (or modified to express / include nucleic acids encoding such CAR).

[0498] In some embodiments, the method comprises:

[0499] (a) engineering an immune cell to express or include a CAR according to the present disclosure, or to express or include a nucleic acid encoding a CAR according to the present disclosure, and

[0500] (b) administering to a subject an immune cell engineered to express or include a CAR according to the present disclosure, or an immune cell engineered to express or include a nucleic acid encoding a CAR of the present disclosure.

[0501] In some embodiments, the method comprises:

[0502] (a) isolating or obtaining immune cells;

[0503] (b) modifying immune cells to express or include a CAR according to the present disclosure, or to express or include a nucleic acid encoding a CAR according to the present disclosure, and

[0504] (c) administering to a subject an immune cell engineered to express or include a CAR according to the present disclosure, or an immune cell engineered to express or include a nucleic acid encoding a CAR of the present disclosure.

[0505] In some embodiments, the method comprises:

[0506] (a) isolating immune cells (such as PBMC) from a subject;

[0507] (b) generating / expanding immune cell populations;

[0508] (c) engineering an immune cell to express or include a CAR according to the present disclosure, or to express or include a nucleic acid encoding a CAR according to the present disclosure, and

[0509] (d) administering to a subject an immune cell engineered to express or include a CAR according to the present disclosure, or an immune cell engineered to express or include a nucleic acid encoding a CAR of the present disclosure.

[0510] In some embodiments, the subject to which the immune cells are isolated is the same as the subject to which the cells are administered (i.e., adoptive transfer can be performed with autologous / self-derived cells). In some embodiments, the subject to which the immune cells are isolated is different from the subject to which the cells are administered (i.e., adoptive transfer can be performed with allogeneic cells).

[0511] In some embodiments, the method may include one or more of the following:

[0512] obtaining a blood sample from the subject;

[0513] Isolating immune cells (such as PBMC) from the blood sample obtained from the subject;

[0514] Generate / expand immune cell populations;

[0515] Growing immune cells in glassware or in vitro cell culture;

[0516] Modifying immune cells to express or include a CAR according to the present disclosure, or to express or include a nucleic acid encoding a CAR according to the present disclosure (such as by transducing a viral vector encoding such a CAR, or a viral vector including such a nucleic acid);

[0517] Cultivating immune cells expressing / including a CAR according to the present disclosure, or immune cells expressing / including a nucleic acid encoding a CAR according to the present disclosure in glassware or in vitro cell culture;

[0518] Collecting / isolating immune cells expressing / comprising the CAR according to the present disclosure, or immune cells expressing / comprising the nucleic acid encoding the CAR according to the present disclosure;

[0519] The immune cells expressing / including the CAR according to the present disclosure, or the immune cells expressing / including the nucleic acid encoding the CAR according to the present disclosure are prepared into a pharmaceutical composition, such as mixing the cells with a pharmaceutically acceptable adjuvant, diluent or carrier; and administering the immune cells expressing / including the CAR according to the present disclosure, or the immune cells expressing / including the nucleic acid encoding the CAR according to the present disclosure to a subject.

[0520] In some embodiments, the method may further include treating the cell or subject to reduce / increase the expression of CAR and / or to reduce / increase the proliferation or survival of immune cells comprising / expressing the CAR.

[0521] The treatment and / or prevention methods can effectively reduce the development / progression of the disease / disorder, alleviate the symptoms of the disease / disorder, or alleviate the pathological characteristics of the disease / disorder. The method can effectively prevent the progression of the disease / disorder, such as preventing the disease / disorder from getting worse or slowing the rate of development of the disease / disorder. In some embodiments, the method can improve the disease / disorder, such as reducing the severity of the symptoms of the disease / disorder, or reducing the severity / activity of some other symptoms related to the disease / disorder. In some embodiments, the method can prevent the disease / disorder from developing to subsequent stages (such as chronic stages or metastases).

[0522] It will be appreciated that, in accordance with the present disclosure, the therapeutic and prophylactic uses of CAR-expressing immune cells can be extended to the treatment / prevention of any disease / disorder that may benefit from reducing the number / activity of cells expressing / overexpressing the CAR target antigen and / or reducing the number / activity of cells infected with a virus.

[0523] In some embodiments, the disease / disorder to be treated / prevented according to the present disclosure, wherein the target antigen of the CAR is associated with the pathology. That is, in some embodiments, the disease / disorder is a disease / disorder caused or aggravated by the expression / overexpression of the target antigen, a disease / disorder for which the expression / overexpression of the target antigen is a risk factor, and / or a disease / disorder in which the expression / overexpression of the target antigen is positively correlated with the onset, development, progression and / or severity of the disease / disorder.

[0524] In certain embodiments, the cells produced by the disclosed methods are provided to an individual in need of treatment for a condition. As used herein, "treating" or "treated" includes a beneficial or desirable effect on the symptoms or pathological features of a disease, or a condition, and may include a minimal decrease in one or more measurable markers of the disease or condition (such as cancer) being treated. Treatment may involve alleviating or ameliorating the symptoms of a disease or condition, or it may be slowing the progression of a disease or condition. "Treatment" does not necessarily mean complete eradication or cure of a disease or condition or its associated symptoms.

[0525] The individual being treated is known to have cancer, is suspected of having cancer, or is at risk of developing cancer (e.g., personal or family history; active sex life, including promiscuity; and / or genetic predisposition, including one or more specific markers). The patient may have a benign or malignant tumor. The individual may have early or advanced cancer, and the technician knows that the method of producing cells can be customized for these different stages of cancer, such as by using peptides from antigens associated with early and advanced cancers to produce APCs. In specific embodiments, the cancer can be primary, metastatic, recurrent, refractory, etc.

[0526] In some embodiments, administration of one or more cells produced by the disclosed methods is provided to individuals in need. The length of time between different administrations can be any suitable duration, including 1-7 days, 1-4 weeks, 1-12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some cases, multiple injections can be made within one year. In the case of providing more than one cell administration to the patient, the cell-targeted antigen may or may not be the cell-targeted antigen at the time of the earlier administration. For example, for refractory cancers, additional administration may be required. Additional stimulation can be used in combination with one or more other types of cancer treatment methods.

[0527] The method of medical treatment may include treating cancer by improving, treating or preventing human malignant tumors, wherein the steps of the method help or promote the immune system to eradicate cancer cells. This method may include administering cells according to the present invention to call for active (or passive) immune responses to eliminate cancer cells. The treatment method may optionally include the co-administration of biological adjuvants (e.g., interleukins, cytokines, BCG, monophosphoryl lipid A, etc.) with traditional therapies for treating cancer (e.g., chemotherapy, radiotherapy or surgery). The treatment method may include administering a composition according to the present invention in the form of a vaccine to prevent or destroy tumor cell growth by activating the immune system. The method of medical treatment may also include in glassware, in vitro, and adoptive immunotherapy, including the use of autologous and / or allogeneic cells or immortalized cell lines.

[0528] According to the present disclosure, the term "pharmaceutical composition" relates to a composition administered to an individual. In a specific embodiment, the pharmaceutical composition includes a composition containing therapeutic immune cells for parenteral, transdermal, intracavitary, intraarterial, intrathecal or intravenous administration, or direct injection into a tumor (such as cancer). It is particularly contemplated that the pharmaceutical composition is administered to an individual by infusion or injection. Suitable compositions can be administered in different ways, such as intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intradermal administration.

[0529] The pharmaceutical composition of the present disclosure may also include a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are known in the art, including phosphate buffered solutions, water, emulsions (such as oil / water emulsions), various wetting agents, sterile solutions, etc. Compositions including these carriers may be configured by well-known traditional methods. These pharmaceutical compositions may be administered to a subject in a suitable dose.

[0530] The dosage regimen is determined by the attending physician and clinical factors. Medical technicians are well aware that the dosage for a patient depends on many factors, including the patient's size, body surface area, age, specific compound being administered, sex, time and period of administration, general health, and other medications being taken concurrently. The specific dosage administered may range from 2×10 per square meter of body surface area to 10×10 per square meter of body surface area. 7 cells to 1×10 10 . Monitor progress through periodic evaluations.

[0531] The compositions of the present disclosure can be administered topically or systemically. In a preferred embodiment, the pharmaceutical composition is administered by subcutaneous injection, and in a more preferred embodiment, by intravenous injection. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous solution carriers include water, alcohol / aqueous solvents, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose, sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous carriers include fluid and nutritional supplements, electrolyte supplements (such as supplements based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases, etc. In addition, the pharmaceutical compositions of the present disclosure may also include protein carriers, such as serum albumin or immunoglobulin, preferably human serum albumin or immunoglobulin. It is contemplated that the pharmaceutical compositions of the present disclosure may contain other biologically active agents in addition to the cells described herein, depending on the intended use of the pharmaceutical composition.

[0532] Function Definition

[0533] The polypeptides described herein can be characterized by reference to specific functional properties. In some embodiments, the polypeptide is vitronectin, and / or a polypeptide having at least 70% sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a polypeptide comprising an amino acid sequence having at least 70% sequence identity to any of the fragments of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the polypeptides described herein may have one or more of the following properties:

[0534] Improve cell adhesion

[0535] Improves cell attachment to surfaces

[0536] Improve cell attachment to culture vessels

[0537] Improves cell attachment to the inner surface of culture vessels.

[0538] Cell attachment is enhanced as compared to cell attachment of control cells cultured in the same manner but without the polypeptide, as assessed by microscopy, flow chamber pressure measurement, non-invasive label-free real-time impedance measurement, or measurement using anti-cell attachment antibodies.

[0539] Improves cell attachment by 1.5-fold compared to control cells cultured in the same manner but without the peptide

[0540] Compared to the cell adhesion of control cells cultured in the same manner but without the polypeptide, the cell adhesion is increased by 2-fold, 3-fold, 4-fold, or 5-fold

[0541] Improves cell attachment in the absence of fetal bovine serum (FBS)

[0542] Improved cell attachment in FBS-free medium

[0543] Improve cell attachment in the absence of serum

[0544] Improved cell attachment in serum-free medium

[0545] Improve cell attachment without animal products

[0546] Improved cell attachment in animal product-free media

[0547] Improved cell attachment in xeno-free media

[0548] Reduce cell aggregation

[0549] By microscopic observation, cell aggregation is reduced compared to aggregation of control cells cultured in the same manner but without the polypeptide.

[0550] Cell aggregation was reduced 1.5-fold compared to aggregation of control cells cultured in the same manner but without the polypeptide.

[0551] Cell aggregation was reduced 2-fold compared to aggregation of control cells cultured in the same manner but without the polypeptide.

[0552] Increase virus titer

[0553] Increase the virus titer of culture supernatant

[0554] Increase the viral titer of the culture supernatant after transfection

[0555] Increase the viral titer of culture supernatant after transfection with a plasmid containing the gene of interest

[0556] Increase the viral titer of the culture supernatant 6 hours after transfection incubation

[0557] Increase the viral titer of the culture supernatant 12 hours after transfection incubation

[0558] Increase the viral titer of the culture supernatant 24 hours after transfection incubation

[0559] Increase the viral titer of the culture supernatant 48 hours after transfection incubation

[0560] Increase the viral titer of the culture supernatant 72 hours after transfection incubation

[0561] The increase in viral titer when the polypeptide is used is compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide

[0562] The viral titer was increased more than 1.5-fold when the polypeptide was used compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0563] The viral titer is increased more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold when the polypeptide is used compared to the viral titer produced by control cells cultured in the same manner but without the polypeptide.

[0564] Improve / enhance virus-mediated gene transfer

[0565] Improve / enhance virus-mediated gene transduction in mammalian cells

[0566] Improve transduction efficiency compared to transduction without the polypeptide

[0567] Improves transduction efficiency compared to transduction without transduction enhancers

[0568] Compared to transduction without the use of a transduction enhancer, the transduction efficiency is increased by at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times

[0569] Compared to transduction without a transduction enhancer, the transduction efficiency is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times. Compared to transduction without a transduction enhancer, the transduction efficiency is increased by at least 1.1 times.

[0570] Compared to transduction without the polypeptide, the transduction efficiency is increased by at least 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times

[0571] Compared to transduction without the polypeptide, the transduction efficiency is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, or 4.0 times; Compared to transduction without the polypeptide, the transduction efficiency is increased by 1.1 times

[0572] Reduce cell secretion of cytokines

[0573] Reduced cytokine secretion by effector immune cells

[0574] Reduced cytokine secretion from cells transduced with chimeric antigen receptor (CAR)

[0575] Reduced cytokine secretion by cells used for adoptive cell transfer

[0576] Reduce CAR-T cell secretion of cytokines

[0577] Reduces cytokine secretion while maintaining cytotoxicity

[0578] Reduced cytokine secretion without reducing target cell killing

[0579] Improved cell-killing capacity of effector immune cells

[0580] Improved cell killing ability of CAR-transduced cells

[0581] Improving the cell killing ability of cells used for adoptive cell transfer

[0582] Improving the cell-killing ability of CAR T cells

[0583] Reduction of cytokine secretion by effector immune cells compared to cells not contacted with the polypeptide according to the invention

[0584] Reducing the secretion of cytokines by cells transduced with a chimeric antigen receptor (CAR) compared to cells not contacted with a polypeptide according to the invention

[0585] Reducing the secretion of cytokines by cells used for adoptive cell transfer compared to cells not contacted with the polypeptide according to the invention

[0586] Reducing the secretion of cytokines by CAR-T cells compared to cells not contacted with the polypeptide according to the invention

[0587] Reduce cell secretion of IL-6, TNF-α, IFN-γ, and / or IL-8

[0588] Reduced secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by effector immune cells

[0589] Reduce the secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by cells transduced with chimeric antigen receptor (CAR)

[0590] Reduce the secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by cells used for adoptive cell transfer

[0591] Reduce the secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by CAR-T cells

[0592] Reduces secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by cells that maintain cytotoxicity

[0593] Reduced target cell killing without reduced cell secretion of IL-6, TNF-α, IFN-γ, and / or IL-8

[0594] Reduction of secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by effector immune cells compared to cells not contacted with the polypeptide according to the invention

[0595] Reducing the secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by cells transduced with a chimeric antigen receptor (CAR) compared to cells not contacted with a polypeptide according to the invention

[0596] Reducing the secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by cells used for adoptive cell transfer compared to cells not contacted with the polypeptide according to the invention

[0597] Reducing the secretion of IL-6, TNF-α, IFN-γ, and / or IL-8 by CAR-T cells compared to cells not contacted with the polypeptide according to the present invention

[0598] Improved cell killing capacity of effector immune cells compared to cells not contacted with the polypeptide according to the invention

[0599] Improved cell killing ability of cells transduced with CAR compared to cells not contacted with the polypeptide according to the present invention

[0600] Improved cell killing ability of cells used for adoptive cell transfer compared to cells not contacted with a polypeptide according to the invention

[0601] Improve the cell killing ability of CAR T cells compared to cells not contacted with the polypeptide according to the present invention

[0602] Improved target cell killing ability of cells transduced with CAR compared to cells not contacted with the polypeptide according to the present invention

[0603] Improved target cell killing ability of cells used for adoptive cell transfer compared to cells not contacted with a polypeptide according to the invention

[0604] Improved target cell killing ability of CAR T cells compared to cells not contacted with the polypeptide according to the present invention The polypeptides described herein may have one or more of the above-mentioned functional properties, which may be combined.

[0605] Sequence identity

[0606] As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to the nucleotides / amino acid residues in a reference sequence, after the sequences have been aligned, introducing gaps as necessary, to achieve the maximum percentage of sequence identity between the sequences. To determine the percentage of sequence identity between two or more nucleic acid or amino acid sequences, a variety of methods for performing double and multiple sequence alignments are known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega ( J. Bioinformatics (2005) 21, 951-960), T-coffee (Notredame et al. J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer 2005, BMC Bioinformatics, 6 (298)) and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30 (4) 772-780) software. When using such software, it is preferred to use default parameters, such as gap penalties and extension penalties.

[0607] sequence

[0608]

[0609]

[0610]

[0611]

[0612] The features disclosed in the above description or in the following claims or in the drawings, expressed in their specific form, or in the form of a method for performing a disclosed function, or a method or process for obtaining a disclosed result, may be used to implement the invention in various forms, alone or in any combination of these features, as appropriate.

[0613] Although the present invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations are apparent to those skilled in the art for the disclosure of the present invention. Therefore, the exemplary embodiments of the present invention described above are considered to be illustrative rather than restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the present invention.

[0614] To avoid any doubt, any theoretical explanations provided herein are intended to deepen the reader's understanding. The inventors do not wish to be bound by these theoretical explanations.

[0615] Any section headings used herein are for structural purposes only and are not to be construed as limiting the subject matter described.

[0616] In this specification (including the following claims), unless the context indicates otherwise, "comprises" and "comprising" and variations such as "containing", "including" and "comprising", will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0617] It must be noted that the singular forms "a", "an", and "the" used in the specification and the appended claims include plural referents unless the context clearly dictates otherwise. Ranges herein may be expressed as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the preposition "about", it can be understood that the particular value constitutes another embodiment. The term "about" in relation to a value is optional, for example, meaning + / -10%.

[0618] Number statement

[0619] 1. A cell culture method, comprising attaching and culturing cells in a culture container, wherein the cells are cultured in a culture medium without fetal bovine serum (FBS), the culture container comprising a polypeptide having at least 70% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein one or more of the cells express a viral vector.

[0620] 2. The method of statement 1, wherein the viral vector is a lentiviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, or an adenoviral vector.

[0621] 3. The method according to claim 1 or claim 2, wherein the cell is a mammalian cell or an insect cell.

[0622] 4. A method as described in any of the above statements, wherein the polypeptide has at least 75% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, preferably, wherein the polypeptide has at least 80%, 85%, 90%, 95% or 99% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0623] 5. The method according to any of the preceding statements, wherein the polypeptide is present in the culture medium.

[0624] 6. A method as claimed in any preceding statement, wherein the polypeptide is provided in the form of a liquid solution.

[0625] 7. The method according to any of the preceding statements, wherein the polypeptide is coated on the surface of the culture container.

[0626] 8. The method according to any of the preceding statements, wherein the culture container is a culture plate, a culture dish, a culture flask, or a bioreactor.

[0627] 9. The method according to any of the preceding statements, wherein the polypeptide is provided as a supplement to FBS-free culture medium.

[0628] 10. The method according to any of the preceding statements, wherein the culture medium does not contain animal serum.

[0629] 11. The method of any of the preceding statements, wherein the cells are transfected with a packaging helper plasmid.

[0630] 12. The method of any of the preceding statements, wherein the cells are transfected with a plasmid encoding a gene of interest.

[0631] 13. The method of statement 12, wherein the target gene is a chimeric antigen receptor (CAR) gene.

[0632] 14. The method of any one of statements 3 to 13, wherein the mammalian cell is a human cell.

[0633] 15. The method of statement 14, wherein the human cells are human embryonic kidney cells, HeLa cells or HT-1080 cells.

[0634] 16. The method of statement 15, wherein the human embryonic kidney cells are HEK293 cells, HEK293T cells, or HEK293FT cells.

[0635] 17. A method for producing a viral vector, wherein the method comprises culturing cells according to any of the above statements, and the method further comprises the step of collecting viruses, viral vectors, and / or viral particles from the culture.

[0636] 18. A method for producing cells for adoptive cell therapy, comprising the steps of:

[0637] (i) producing a viral vector according to the method of statement 17, and

[0638] (ii) transducing immune cells with the viral vector to generate cells for adoptive cell therapy.

[0639] 19. The method of statement 18, wherein steps (i) and (ii) are performed without changing the culture medium.

[0640] 20. The method of statement 18 or statement 19, wherein FBS-free medium is used in steps (i) and (ii).

[0641] 21. The method of any one of statements 18 to 20, wherein step (ii) is performed in the presence of a polypeptide having at least 70% sequence identity to any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0642] 22. A method for transducing immune cells with a viral vector encoding a target gene, the method comprising contacting immune cells with a viral vector encoding a target gene and a polypeptide having at least 70% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 under conditions suitable for transducing cells with the viral vector, thereby producing transduced cells for adoptive cell therapy.

[0643] 23. The method of statement 22, wherein the polypeptide is vitronectin.

[0644] 24. The method of statement 22 or statement 23, wherein the immune cells are mammalian immune cells.

[0645] 25. The method of any one of statements 22 to 24, wherein the mammalian immune cells are human immune cells.

[0646] 26. The method of any one of statements 22 to 25, wherein the cells used for adoptive cell therapy are cells expressing CAR.

[0647] 27. The method of claim 26, wherein the CAR-expressing immune cell is a CAR T cell or a CAR-natural killer (CAR-NK) cell.

[0648] 28. The method of any one of statements 22 to 27, further comprising the step of collecting one or more transduced cells.

[0649] 29. The method of any one of statements 22 to 28, further comprising the step of expanding the transduced cells.

[0650] 30. The method of any one of statements 22 to 29, further comprising formulating the transduced mammalian cells with a pharmaceutically acceptable excipient into a medicament. Example

[0651] Example 1 Promote cell attachment for viral vector production

[0652] Human platelet lysate (5%; Stem Cell Technologies), fetal bovine serum (pure; Life Technologies) and (8 μg / mL; Takara Bio) at 37°C overnight. To investigate whether these reagents could support cell attachment in the absence of serum, human embryonic kidney HEK293T cells (also abbreviated as 293T) adapted with 10% and 5% (v / v) fetal bovine serum (FBS) were seeded into pre-coated wells at a density of 280K per 6-well dish. 72 hours after seeding, cells were transfected with PE1 reagent and pre-optimized CellVec packaging helper plasmids (plasmids 1-3). Briefly, plasmid #1 carries HIV1 Rev protein, plasmid #2 carries VSV-G envelope protein, and plasmid #3 carries HIV1 gag / pol protein. The target gene plasmid #4 mimics the chimeric antigen receptor, and for the purpose of viral vector titration, the target was replaced by 6×HisTag. After optimizing the antibody binding detection by flow cytometry, titration was performed using PE-conjugated anti-6×HisTag antibody (Abcam, 1:200). As expected, there were no significant differences in viral production from 293T cells cultured in DMEM supplemented with 10% FBS under various conditions. However, when DMEM supplemented with FBS reduced to 5% was used, viral production from 293T cells was significantly higher than that from the other groups. Significantly improved the attachment of 293T cells ( Figure 1 ).exist Figure 1 In the experiment, the plates of the FBS group were coated with pure FBS and incubated at 37°C overnight, and the plates of the hPL group were coated with 5% human platelet lysate / PBS and incubated at 37°C overnight. The plate was smeared with 8 μg / mL The cells were incubated at 4°C overnight, and only PBS was used for the control plate. Can replace factors in serum that are required to promote cell attachment and / or LV production.

[0653] The lentiviral vector supernatant was diluted in different volumes (0, 0.1 μL, 0.2 μL, 0.4 μL, 1 μL, 2 μL, 4 μL, 8 μL, etc.) and added to 293T cells. After 72 hours, the number of positive cells expressing the surface target was detected by flow cytometry. The titer was calculated by dividing the total number of positive cells (i.e., the percentage of positive cells multiplied by the total number of cells) by the volume of the viral vector supernatant used, and then multiplied by 1000 (TU / mL).

[0654] This example further tested the following method in several commercially available serum-free culture media: To promote the ability of 293T cells to attach, the culture medium is as follows: ImmunoCult TM-XF T cell expansion medium (Stem Cell Technologies), 4Cell Nutri-T medium (abbreviated as NutriT; Sartorius), and TexMacs medium (Miltenyi Biotech GmbH). The cells were incubated at 37°C and 5% CO 2 The cells were cultured under the conditions of

[0655] In the absence of serum, 293T aggregated and formed aggregates ( Figure 2 A). Briefly, HEK293T cells were cultured under serum-free conditions and cultured using Nikon TM EclispeTS100 / Leica TM DMIL or equivalent was used to evaluate the culture characteristics over time. Compared with the case without coating (e.g., PBS alone), cells spread evenly in a monolayer. In addition, in different types of serum-free culture media, cells coated with The proliferation of 293T cells in 6-well plates coated with (8 μg / mL PBS) was significantly increased within 3 days. LV production was significantly increased in all serum-free media tested in tissue culture dishes ( Figure 2 C) NutriT can be used as a universal serum-free medium to support LV production and T cell expansion, as independent evidence shows that NutriT not only supports robust expansion of T cells, but also that the expansion is biased towards the CD8+ cell population, thereby improving the potency of CAR-T in vitro and in vivo.

[0656] In PBS Dilute to a certain concentration range (0, 1, 4, 8 and 16 μg / mL) Figure 2 D) Each concentration of coating was repeated. The total volume per well was 0.8 mL, and the culture dish was sealed with a sealing film and incubated at 4°C overnight. This study also illustrates the use of After that, place the coating at room temperature or in an incubator for 2-3 hours to improve cell adhesion. The solution was removed and 293T was inoculated in serum-free NutriT medium, followed by transfection with CellVec packaging helper plasmid and plasmid #4 described above. Crude viral supernatant was collected 48 h after transfection and used from each replicate (0, 1, 4, 8 and 16 μg / mL under different conditions) 2 μL and 4 μL of crude virus supernatant collected from 2 μL of 293T cells were infected and then titrated by flow cytometry. The results showed that The effect of improving virus production in serum-free medium was dose-dependent.

[0657] This example subsequently investigated whether adherent LV production could be obtained from 6-well plates (volume of approximately 9.6 mL, containing approximately 1.5 mL of culture medium; Figure 3 A) Expand into a T75 flask (volume approximately 75 mL, containing approximately 10 mL of medium; Figure 3 B) Expand to a T175 flask (volume approximately 175 mL, containing approximately 25 mL of culture medium; Figure 3 C) in an incubator at 37°C and 5% CO 2 The cells were cultured under the conditions of

[0658] The results showed that compared with Compared with cells coated with LV production from 293T cells seeded in DMEM supplemented with 10% FBS was increased 4-7 fold. The effect is small / not obvious ( Figure 3 A-3C). Thus, crude LVs can be produced on an adherent platform in serum-free medium in different culture vessels.

[0659] The choice of serum-free medium for Mustang Q ion exchange chromatography is pH-dependent. Kutner and Reiser reported that the amount of virus binding to Mustang Q ion exchange chromatography increased at pH 8.0 compared to pH 7.0 or pH 6.0 (Molecular Therapy, Vol. 15, Supplement 1, S313, May 1, 2007). This example also independently found that the pH buffer range of TexMacs could not be adjusted to pH 8.0. Since LV could not bind effectively to the Mustang Q ion exchange chromatography, the recovery rate of viral vector was very low. In view of this, the recovery rate of viral vector was very low when pre-applied with 8 μg / mL 293T cells were seeded in serum-free NutriT medium in a two-layer cell factory. 72h after seeding, the cells were transfected with the CellVec packaging helper plasmid with an in-house designed humanized CD19-CAR (CV-hCD19). The crude LV supernatant was filtered with a 0.45μm filter, purified by Mustang Q ion exchange chromatography, ultrafiltration and diafiltration steps (UF / DF), and subsequently transformed in NutriT medium. Similar experiments were performed, in which 293T cells were seeded in DMEM medium supplemented with 10% FBS and underwent a similar purification process, but the final transformation was performed in TexMacs medium. The results showed that 293T seeded in serum-free NutriT medium maintained the morphology and growth characteristics of the parental HEK293T ( Figure 4Aii) Compared with the traditional attachment, serum-dependent culture method, the same amount of lentivirus can be produced. The total virus yield before centrifugation was 2.22×10 8 and 2.33×10 8 TU( Figure 4 B).

[0660] Example 2 - Vitronectin promotes cell attachment to produce viral vectors

[0661] Although it has been clarified that Separated from its usual function as a transduction enhancer, the feasibility of producing viral vectors in serum-free medium, but with Pre-coating the cell factory is still an additional step and requires more effort. A study by Matsushita and colleagues explored the effects of shear stress on the adhesion and productivity of parental 293 cells in culture medium containing FBS (Matsushita T et al., 2000, Cytotechnology 32(3):181-190). Among the various additives tested, vitronectin can robustly increase the yield of 293 cells exposed to shear stress when cultured in FBS-containing culture medium. Therefore, this example explores whether vitronectin can be added as a liquid solution to FBS-free culture medium to promote the attachment of 293T cells. This study used recombinant vitronectin ACF produced by Sartorius. It is a single-chain monomer protein of 459 amino acids that can be used as a chemically precise matrix component for the attachment of human embryonic stem cells and induced pluripotent stem cells in a feeder-free culture system. The results were compared with Figure 2 Similar experiments were performed. 10 μg / mL vitronectin solution was added to 293T cells in serum-free NutriT medium. The results showed that vitronectin and Has similar effects ( Figure 5 A). In the absence of vitronectin, 293T cells aggregated and formed cell clusters. Furthermore, in different types of serum-free media, 293T cell proliferation was significantly increased in 6-well plates coated with vitronectin (5 μg / mL PBS) compared to controls with similar media without any coating in the corresponding wells (i.e., the same volume of PBS). LV production was also significantly improved in all serum-free media tested ( Figure 5 C). Soluble vitronectin was added in a certain concentration range (0.5 μg / ml; 1 μg / mL; 5 μg / mL; 10 μg / mL and 20 μg / mL) during the inoculation of 293T into NutriT. Figure 2The results showed that vitronectin increased LV production in a dose-dependent manner ( Figure 5 D) In ​​summary, this example demonstrates that soluble vitronectin can also be used to improve the adhesion of 293T cells under serum-free conditions.

[0662] Example 3 - Vitronectin enhances virus-mediated gene transduction

[0663] The present invention then examined the effects of NutriT medium and ImmunoCult (which has been used as a standard T cell culture medium) on T cell proliferation and T cell subsets, both of which were supplemented with 100 units / mL of IL-2 and then transduced with the test bed construct of the present invention, CV-hCD19 LV. The results showed that NutriT robustly supported T cell expansion ( Figure 6 In addition, under normal cell culture conditions ( Figure 6 ), and when transduced with CV-hCD19 LV vector, NutriT can preferentially expand CD8 cell populations. Various cell populations were stained with corresponding antibodies in FACS analysis. CAR surface expression was detected with human CD19 recombinant protein labeled Atto 488 (R&D Systems). Transduction efficiency under various conditions was evaluated by flow cytometry. In brief, cell populations were stained with corresponding antibodies in FACS analysis. CAR surface expression was detected with human CD19 recombinant protein labeled Atto488 (R&D Systems). Based on the BD FACS Suite TM or FlowJo TM (BD Biosciences) to calculate the corresponding differences in CAR expression of T cells cultured under different conditions (as described above).

[0664] because As a transduction enhancer, we therefore investigated whether soluble vitronectin had a similar effect. In the same experiment, vitronectin was applied to untreated 24-well dishes and PBMCs were transduced with LVs under the same experimental conditions as described above. The results showed that vitronectin can also be used as a transduction enhancer and improve the transduction efficiency of PBMCs ( Figure 6 ).

[0665] Example 4 - Efficacy of viral vectors

[0666] Next, the present invention tested the efficacy of lentiviral vector encoding humanized CD19-CAR (CV-hCD19) CAR LV, which was produced from 293T cultured in DMEM supplemented with 10% (v / v) FBS or NutriT using a traditional attachment process. Human peripheral blood mononuclear cells (PBMCs) were activated with CD3 / CD28 (72 h) and then labeled with Under different conditions of coating the surface of 24-well plates with vitronectin, they were transduced with CV-hCD19 at a 5-fold multiplicity of infection (MOI) or not transduced (NTC). Afterwards, CAR-T lymphocytes expressing CD19 were co-cultured with Raji cells (Burkitt lymphoma) and Nalm6 cells (acute lymphoblastic leukemia) at various effector to target (E:T) ratios labeled ( Figure 7 A), thereby evaluating the cytotoxic potential.

[0667] Briefly, Nalm6–Luc or Raji-Luc cells (Nalm-6 or Raji cells expressing luciferase) were incubated with CAR-T cells at a specific time point. TM The luciferase detection system (Promega, Madison, USA) records the luciferase activity of Raji-Luc or Nalm6-Luc as an indicator of tumor cell activity. After co-culture with Raji-Luc or Nalm6-Luc tumor cells for a period of time, the efficacy of the cytotoxic function of CAR-T cells (preparation and culture conditions as described above) is evaluated based on tumor cell activity and luciferase activity.

[0668] The cytolytic activity of CAR T cells was expressed as percentage of specific cell lysis. The results showed that CV-hCD19 LV prepared using the novel serum-free attachment (NSAP) was as efficient in killing Raji or Nalm6 cells as CV-hCD19 LV prepared using the standard method in DMEM containing 10% FBS. Raji cell model ( Figure 7 C) and Nalm cell model ( Figure 7 D) also secreted IFN-γ levels comparable to those of LV produced by conventional methods in 10% FBS. In conclusion, the data of the present invention show that NSAP-produced LVs are as potent as LVs produced by standard conventional methods.

[0669] Example 5 - Long-term coating

[0670] The inventors unexpectedly discovered that the longer the coating is incubated, the better the effect.

[0671] To evaluate the cell attachment properties of 293T cells resuspended in serum-free NutriT medium, (8 μg / mL) over approximately 3 weeks in 24-well plates compared to uncoated The cells (1×10 5 After 3 hours, the inventors were surprised to find that 293T cells were A monolayer ( Fig. 9 A). Without coating on plates (DMEM supplemented with 10% FBS or NutriT- Fig. 9 A) No such effect was observed.

[0672] The results showed reproducibility. It was also found that the monolayer formed within 3 hours can support virus production. The standard method for virus production is to plate 293T cells in DMEM supplemented with 10% FBS and incubate for 72 hours before transfection. This 72 hour period allows attachment to occur. To ensure comparability of the data, 293T cells were plated using the following methods: (1) the standard 72 hour method in DMEM supplemented with 10% FBS, and (2) plated 3 hours before transfection to allow for long-term attachment. The results showed that cells seeded in the presence of long-term After 3 hours in the coated wells, virus production efficiency was the same as that of cells seeded 72 hours before transfection. This finding has the potential to significantly shorten the overall LV production time.

[0673] Example 6 - Summary and Discussion

[0674] The inventors have developed a novel serum-free attachment process (NSAP) for the production of LVs (eg Figure 8 As shown). According to the prior art, there are no public reports on the production of attached LVs under serum-free conditions. Not using bovine serum in the production process reduces the risk of cattle-related disease transmission and is therefore more suitable for the clinical production of cell therapy products. Compared with traditional bovine serum, the protein content is lower and the impact on the efficiency of the downstream LV purification process is smaller because the processing time is shorter, which not only makes the product safer but also higher in quality. In addition, this method can also solve other challenges faced by the use of bovine serum, such as global shortages, high costs, and batch-to-batch differences.

[0675] Example 7 - Derived from vitronectin and Generation of peptides

[0676] 7.1 Design of recombinant peptides

[0677] The inventor speculates that A region within the heparin binding domain may serve as a viral binding moiety, but the sequence responsible is unknown. Two recombinant proteins, RV#1 (SEQ ID NO: 16) and RV#2 (SEQ ID NO: 17), were designed, containing different lengths of the predicted viral binding sequence (e.g. Fig.10 B). RV#1 contains The sequence is a homologue of fibronectin type III domains 12 and 13 (SEQ ID NO:20); RV#2 contains a sequence corresponding to fibronectin type III domains 12-14 (SEQ ID NO:19).

[0678] right Comparison with the vitronectin sequence revealed similar functional motifs around the "RGD" sequence. Therefore, the vitronectin RGD sequence region consisting of the signal peptide (SEQ ID NO: 22) and the first 51 amino acids at the N-terminus of vitronectin (SEQ ID NO: 23) was incorporated into the designed recombinant protein. This sequence was incorporated into the N-terminus of RV#1 and RV#2.

[0679] These sequences were fused to human IgG via an EPKSA linker sequence (SEQ ID NO: 27). Attempts to fuse IgG to recombinant proteins were made to further improve stability and half-life, and potentially reduce immunogenicity.

[0680] 7.2 Design of peptides derived from vitronectin

[0681] A series of polypeptides (SEQ ID NO: 2 and SEQ ID NO: 3) were designed based on the predicted functional domains of vitronectin. Figure 1 As shown in P1, P2, P6 and P7 in C.

[0682] P1 (SEQ ID NO: 6) and P7 (SEQ ID NO: 12) are fragments of vitronectin, and P2 (SEQ ID NO: 7) and P6 (SEQ ID NO: 9) are both derived from fragments of vitronectin.

[0683] The P1 (SEQ ID NO: 6) peptide corresponds to the sequence of vitronectin containing the RGD motif, including the boundary between the somatomedin-like domain and the central hemopexin-like domain.

[0684] The P2 (SEQ ID NO: 7) peptide is based on amino acid residues 40-52 of vitronectin and also contains the RGD domain. The N-terminus of this sequence is adjacent to the CCRR and the C-terminus is adjacent to the WLC.

[0685] The P6 (SEQ ID NO:9) peptide is based on the vitronectin signal peptide sequence and has been further engineered to improve specific properties. A "KKA" residue was added to the N-terminus of the sequence to improve solubility during preparation of the peptide. An insertion of an "H" residue was added to the sequence because computational analysis found that the addition of an "H" residue increased the predicted biological activity from 0.81 to 0.82.

[0686] The P7 (SEQ ID NO: 12) peptide corresponds to a vitronectin sequence belonging to the C-terminal heparin binding domain.

[0687] P4 (SEQ ID NO: 25) polypeptide was used as a control. P4, also known as LAH4 peptide or Vectofusin-1, is known to enhance viral infection and has been used in Hcd34 cell-based gene therapy (Fenard, D. et al., Mol Ther Nucleic Acids. (2013) 2(5): e90).

[0688] Additional peptides, P3 (SEQ ID NO:24) and P5 (SEQ ID NO:26) were also studied but were not found to be functional.

[0689] 7.3 Derived from Peptide design

[0690] Sequence alignment with the N-terminal region of vitronectin confirmed Conserved groups within the cell-binding domain.

[0691] It was then determined sequences that correspond to polypeptides derived from vitronectin, in particular peptides P1 and P7. The polypeptides (PR1 (SEQ ID NO: 13), PR7.1 (SEQ ID NO: 14) and PR7.2 (SEQ ID NO: 15), such as Fig.10 C) is predicted to be a transduction enhancer in either adherent or soluble transduction methods.

[0692] Example 8 - Derived from vitronectin and Transduction-enhancing properties of peptides

[0693] Commercial human PBMCs (Stem Cell Technologies, Canada) were thawed and activated for 72 h using a CD3 / CD28 cocktail (Miltenyi Biotec, Germany).

[0694] In existence Activated PBMCs were transduced with hCD19-CAR at a 5-fold multiplicity of infection (MOI) or not transduced (NTC) in 24-well plates with (standard transduction enhancer control), vitronectin, RV#1, RV#2, peptides P1, P2, P4, P6, or P7, or without added reagents.

[0695] 72 hours after transduction, the cultures were analyzed to obtain the percentage of CD19 CAR+ cells. Fig.11 As shown in A.

[0696] Cells transduced in the presence of recombinant protein RV#1 or RV#2 showed higher transduction efficiency compared to cells transduced without added reagents. The transduction efficiency was also significantly increased in cells transduced in the presence of RV#1 and RV#2 compared to cells transduced in the presence of RV#2.

[0697] Cells transduced in the presence of polypeptides P1, P2, P6 and P7 showed higher transduction efficiency compared to cells transduced without added agents. Interestingly, cells transduced in the presence of polypeptide P4 (a polypeptide known to enhance viral infection) did not show higher transduction efficiency compared to cells transduced without added agents.

[0698] It has been shown that in the presence of various transduction enhancers, the CAR-T expression capacity of transduced cells is increased in a dose-dependent manner, which has been demonstrated when vitronectin and peptide P1 were used. Fig.11 B and 11C.

[0699] Example 9 - Effects of transduction enhancers on cytokine secretion after transduction

[0700] IL-6 is a core mediator of cytokine release syndrome (CRS) toxicity known to be associated with CAR-T cell therapy (Tanaka et al., Immunotherapy (2016) 8(8):959-70). A challenge in the development of CAR-T cell therapies is the need to reduce the impact of CRS without reducing the efficacy of CAR-T cell therapy. To evaluate how the use of vitronectin affects CRS, vitronectin-enhanced IL-6 secretion from hCD19-CAR transduced cultures was evaluated.

[0701] Commercial human PBMCs (Stemcell Technologies, Canada) were thawed and activated with a CD3 / CD28 cocktail (Miltenyi Biotec, Germany) and IL-7 and IL-15 (Miltenyi Biotec, Germany) at 37°C and 5% CO. 2The cells were cultured in an incubator for 24 hours using Nutri-T cell culture medium (Sartorius, Singapore).

[0702] In existence Activated PBMCs were transduced with hCD19-CAR or hCD19Del-CAR (a non-functional control group without the signaling endodomain) by a spinfection procedure in the absence of CAR (TakaraBio, Japan), vitronectin (Sartorius, Singapore), or no added reagent (control group).

[0703] To measure IL-6 secretion levels, CAR-expressing T cells from each condition were co-cultured with luciferase-expressing NALM-6-Luc tumor cells 4 or 14 days after transduction. After 24 hours of co-culture, the cell culture supernatant was collected. Cell culture supernatant samples were analyzed using an automated ELISA (ELLA Immunoassay System, ProteinSimple, USA) to determine IL-6 levels in the transduction enhancer-treated or control group. Statistical analysis was performed using a two-tailed paired Student's t-test. P<0.05 was significant; P<0.01 ( ＊＊ ); P<0.001( ＊＊＊ ). The result is Fig.12 A and 12B.

[0704] The results show that Compared with CAR-T cells transduced in the presence of vitronectin, CAR-T cells transduced in the presence of vitronectin secreted significantly less IL-6 on day 4 ( Fig.12 A).

[0705] exist There was no significant difference between CAR-T cells transduced in the presence and absence of transduction enhancers.

[0706] In PBMCs alone and in cells transduced with a non-functional hCD19Del-CAR, IL-6 secretion was low. At day 14, the results remained consistent ( Fig.12 B), with Compared with CAR-T cells treated with vitronectin, the IL-6 secretion of CAR-T cells treated with vitronectin was significantly reduced (P<0.01).

[0707] To confirm that this effect is due to the presence of vitronectin, this example analyzed the secretion of IL-6 in T cells transduced with hCD19-CAR in the presence of increasing amounts of vitronectin. As described above, cells were co-cultured with NALM-6-Luc tumor cells and the supernatant was analyzed by ELISA. The results are shown in Fig.12As shown in C.

[0708] The results showed that decreased IL-6 secretion was associated with increased vitronectin concentrations.

[0709] To evaluate whether the reduction of IL-6 secretion would affect the potential therapeutic effect of CAR-T cells, this example evaluated the cell killing ability of hCD19-CAR-T cells transduced in the presence of vitronectin.

[0710] 72 hours after transduction, CAR-expressing T cells from each condition were co-cultured with luciferase-expressing NALM-6-Luc tumor cells. TM Tumor cell killing was measured by luciferase activity detected by the luciferase assay system (Promega, USA). The specific cell killing rate was calculated based on the ratio of total cell lysis to spontaneous cell lysis under each condition. Fig.12 As shown in D.

[0711] Importantly, the reduction in IL-6 secretion did not impair the cell killing function of hCD19-CAR transduced T cells. In fact, the results showed that the percentage of specific cell killing mediated by hCD19-CAR transduced T cells enhanced by vitronectin was significantly increased (P<0.01) compared with hCD19-CAR transduced T cells enhanced by Retronectin (recombinant human fibronectin fragment) or hCD19-CAR transduced T cells without added reagent ( Fig.12 D).

[0712] Similar results were observed with another NKG2D CAR construct used as an internal control, indicating that this result was independent of the CAR construct used.

[0713] The presence of vitronectin was assessed after transduction. Briefly, the presence of vitronectin was determined using the human vitronectin EIA kit (TakaraBio, USA). Fig.12 Residual vitronectin associated with vitronectin-treated samples (treated with 10 μg / mL) for the experiments shown. Samples without vitronectin were used as negative controls (as shown), while empty medium and vitronectin-containing samples were used as experimental controls (not shown). Trace amounts of vitronectin (approximately 20 ng / mL) were detected in CAR T cells collected on day 4, but no vitronectin was detected when collected on day 14 ( Fig.12 A).

[0714] These results show that vitronectin can not only enhance CAR expression, but also minimize the dose of viral vector used, inhibit the release of cytokines by CAR-T cells and peripheral blood monocytes, and maintain CAR-T functionality. In order to confirm that other cytokines are also regulated by vitronectin in a similar manner, the inventors evaluated the levels of three other cytokines: tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and interleukin 8 (IL-8). The experimental method is the same as the above-mentioned IL-6 analysis method. The results showed that compared with untreated CAR-T cells and Compared with CAR-T cells treated with vitronectin, the secretion of TNF-α, IFN-γ and IL-8 by CAR-T cells treated with vitronectin was significantly reduced ( Fig.12 F to 12H).

[0715] The inventors also evaluated the granzyme levels in the different treatment groups ( Fig.12 I). Granzymes contribute to the function of CAR T cells. Granzyme levels measured by multiplex ELISA were similar in hCD19-CAR transduced T cells enhanced with vitronectin and hCD19-CAR transduced T cells enhanced with Retronectin (recombinant human fibronectin fragment), indicating that although cytokines were reduced, granzymes were still active in the vitronectin-treated group.

[0716] References

[0717] Some publications are cited above to more fully describe and disclose the present invention and the state of the art to which the present invention relates. The complete citations of the references are provided below. The entire contents of these references are incorporated herein.

[0718] Gandara C, Affleck V, and Stoll EA. Manufacturing third-generation lentivirus for preclinical use and process development considerations for transition to good manufacturing practice. 2018; 2018 Feb; 29(1): 1-15. doi: 10.1089 / hgtb.2017.098. Epub 2018 Jan 24. PMID: 29212357

[0719] Matshushita T, Kawakubo Y, Sawano M, and Funatsu K. 2000. "Vitronectin enhances adhesion and t-PA production in weakly adherent 293 cells exposed to shear stress." Cytotechnology, 32(3):181-190.

[0720] Merten OW, Schweizer M, Chahal P, Kamen AA. “Manufacturing of viral vectors for gene therapy: Part I: Upstream processing.” Pharm. Bioprocess (2014) 2:183-203.

[0721] Zhou A, Huntington JA, Pannu NS, Carrell RW, and Read RJ. 2003. How vitronectin binds PAI-1 to regulate fibrinolysis and cell migration. Nature Structural Biology, 10(7):541–4. doi:10.1038 / nsb943

[0722] For standard molecular biology techniques, reference is made to Sambrook, J., Russel, DW, Molecular Cloning, A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Claims

1. A cell culture method, comprising attaching and culturing cells in a culture container, characterized in that: The cells are cultured in a culture medium without fetal bovine serum (FBS), and the culture container contains a polypeptide, which includes an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:

3.

2. The method according to claim 1, characterized in that The polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3, SEQ ID NO:2, or a fragment of SEQ ID NO:1 has at least 70% sequence identity to one or more of SEQ ID NOs:6-23.

3. The method according to claim 1 or claim 2, characterized in that: One or more of the cells express the viral vector.

4. The method according to claim 3, characterized in that The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated virus (AAV) vector, or an adenoviral vector.

5. A method according to any one of the preceding claims, characterized in that The cells are mammalian or insect cells.

6. A method according to any one of the preceding claims, characterized in that The polypeptide has at least 75% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23, preferably, the polypeptide has at least 75% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:

23. NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23 have at least 80%, 85%, 90%, 95%, or 99% sequence identity.

7. A method according to any one of the preceding claims, characterized in that The polypeptide is present in the culture medium.

8. A method according to any one of the preceding claims, characterized in that The polypeptide is provided in the form of a liquid solution.

9. A method according to any one of the preceding claims, characterized in that The polypeptide is coated on the surface of the culture container.

10. A method according to any one of the preceding claims, characterized in that The culture container is a culture plate, a culture dish, a culture bottle, or a bioreactor.

11. A method according to any one of the preceding claims, characterized in that The polypeptide is provided as a supplement to the FBS-free medium.

12. A method according to any one of the preceding claims, characterised in that The culture medium does not contain animal serum.

13. A method according to any one of the preceding claims, characterised in that The cells are transfected with a packaging helper plasmid.

14. A method according to any one of the preceding claims, characterised in that The cells are transfected with a plasmid encoding a gene of interest.

15. The method according to claim 14, characterized in that The target gene is a chimeric antigen receptor (CAR) gene.

16. The method according to any one of claims 5 to 15, characterized in that The mammalian cells are human cells.

17. The method according to claim 16, characterized in that The human cells are human embryonic kidney cells, HeLa cells or HT-1080 cells.

18. The method according to claim 17, characterized in that The human embryonic kidney cells are HEK293 cells, HEK293T cells, or HEK293FT cells.

19. A method for producing a viral vector, the method comprising culturing cells according to the method of any preceding claim, the method further comprising the step of collecting viruses, viral vectors, and / or viral particles from the culture.

20. A method for producing cells for adoptive cell therapy, comprising the steps of: (i) producing a viral vector according to the method of claim 19, and (ii) transducing immune cells with the viral vector to generate cells for adoptive cell therapy.

21. The method of claim 20, wherein: Steps (i) and (ii) were performed without changing the culture medium.

22. The method of claim 20 or claim 21, wherein: Use FBS-free medium in steps (i) and (ii).

23. The method according to any one of claims 20 to 22, characterized in that Step (ii) is performed in the presence of a polypeptide having at least 70% sequence identity to any one of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1, or comprising an amino acid sequence having at least 70% sequence identity to a fragment of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:

1.

24. A method for producing cells for adoptive cell therapy, the method comprising contacting cells with a polypeptide and a viral vector encoding a target gene, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1, or comprises an amino acid sequence having at least 70% sequence identity with a fragment of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:

1.

25. A method for transducing immune cells with a viral vector encoding a target gene, the method comprising contacting immune cells with a viral vector encoding a target gene and a polypeptide having at least 70% sequence identity with any one of SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:1 under conditions suitable for transducing cells with the viral vector, thereby producing transduced cells for adoptive cell therapy.

26. The method according to any one of claims 23 to 25, characterized in that The polypeptide has 75% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:

23. Preferably, the polypeptide has 75% sequence identity to any of SEQ ID NO:3, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:

23. NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and / or SEQ ID NO:23 have at least 80%, 85%, 90%, 95%, or 99% sequence identity.

27. The method of claim 26, wherein: The polypeptide is vitronectin or a vitronectin fragment.

28. The method of claim 26 or claim 27, wherein: The immune cells are mammalian immune cells.

29. The method of claim 28, wherein: The mammalian immune cells are human immune cells.

30. The method according to any one of claims 24 to 29, characterized in that The cells used for adoptive cell therapy are cells expressing CAR.

31. The method of claim 30, wherein: The CAR-expressing immune cells are CAR T cells or CAR-natural killer (CAR-NK) cells.

32. The method of any one of claims 25 to 31, further comprising the step of collecting one or more transduced cells.

33. The method of any one of claims 25 to 32, further comprising the step of expanding the transduced cells.

34. The method of any one of claims 25 to 33, further comprising formulating the transduced mammalian cells with a pharmaceutically acceptable excipient into a medicament.

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