Cell culture composition

Abstract

The present disclosure relates to compositions for cell culture, methods of making the compositions, methods of culturing cells using the compositions, and cells obtained thereby. When the cell culture composition disclosed by the invention is used for culturing cells, the effects of realizing large-scale amplification of the cells and maintaining high motility rate and early memory phenotype of the cells as well as low-depletion phenotype can be realized.

Description

A cell culture composition

[0001] This application claims priority to Chinese Patent Application No. 2022115433522 filed on December 2, 2022, and Chinese Patent Application No. 2023115733235 filed on November 22, 2023. This application incorporates the entirety of the aforementioned Chinese patent applications. Technical Field

[0002] The present disclosure relates to compositions for cell culture, methods of preparing the compositions, methods of culturing cells using the compositions, and cells obtained therefrom. Background Art

[0003] A variety of cell therapy approaches have been used to treat diseases. However, obtaining genetically engineered or unengineered cells requires efficient cell culture processes, including culture media that can achieve large-scale cell expansion while maintaining high cell viability, an early memory phenotype, and a low exhaustion phenotype. Compositions and methods that address this need are provided herein.

[0004] Summary of the Invention

[0005] Provided herein is a composition capable of efficiently expanding cells. In one aspect, the composition comprises a first serum-free culture medium and a second serum-free culture medium, wherein the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-10; the composition has a higher immune cell expansion fold than either the first serum-free culture medium or the second serum-free culture medium. In some embodiments, the cells are cultured for at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days. In some embodiments, the cells are cultured at 37.0±1.0°C and 5.0±1.0% CO2. In some embodiments, after 9, 11, or 13 days of culturing T cells with the composition, the expansion fold is increased by at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% or more compared to a single serum-free culture medium. In some embodiments, the combined volume of the first serum-free culture medium and the second serum-free culture medium accounts for more than 50% of the volume of the composition. In some embodiments, the combined volume of the first serum-free culture medium and the second serum-free culture medium accounts for more than 65% of the volume of the composition. In some embodiments, the combined volume of the first serum-free culture medium and the second serum-free culture medium accounts for more than 80% of the volume of the composition. In some embodiments, the composition is composed of the first serum-free culture medium and the second serum-free culture medium. In some embodiments, the first serum-free culture medium is TexMACS; the second serum-free culture medium comprises one or more culture media selected from the group consisting of X-VIVO 10, X-VIVO 15, and X-VIVO 20. In some embodiments, the first serum-free culture medium is TexMACS; the second serum-free culture medium is X-VIVO 15. In some embodiments, the composition further comprises a third serum-free culture medium, which is AIM-V.

[0006] In one aspect, the present disclosure provides a composition comprising a culture medium matrix, the culture medium matrix comprising a first serum-free culture medium and a second serum-free culture medium, the combined volume of the first serum-free culture medium and the second serum-free culture medium accounting for more than 50% of the volume of the culture medium matrix, the volume ratio of the first serum-free culture medium to the second serum-free culture medium being 1:0.1-10, the first serum-free culture medium being TexMACS, and the second serum-free culture medium comprising one or more culture media selected from the group consisting of X-VIVO 10, X-VIVO 15, and X-VIVO 20. In some embodiments, the combined volume of the first serum-free culture medium and the second serum-free culture medium accounting for more than 65% of the volume of the culture medium matrix. In some embodiments, the culture medium matrix is ​​composed of the first serum-free culture medium and the second serum-free culture medium. In some embodiments, the first serum-free culture medium is TexMACS; the second serum-free culture medium is X-VIVO 15.

[0007] In some embodiments, the composition comprises a culture medium matrix, wherein the culture medium matrix comprises a first serum-free culture medium and a second serum-free culture medium, the sum of the volumes of the first serum-free culture medium and the second serum-free culture medium accounts for more than 80% of the volume of the culture medium matrix, the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-10, the first serum-free culture medium is TexMACS, and the second serum-free culture medium is one or more culture media selected from the group consisting of X-VIVO 10, X-VIVO 15, and X-VIVO 20.

[0008] In some embodiments, the composition comprises a culture medium matrix consisting of a first serum-free culture medium and a second serum-free culture medium, wherein the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-10, the first serum-free culture medium is TexMACS, and the second serum-free culture medium is composed of one or more culture media selected from the group consisting of X-VIVO 10, X-VIVO 15, and X-VIVO 20. In some embodiments, the composition comprises a culture medium matrix consisting of a first serum-free culture medium and a second serum-free culture medium, wherein the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-10, the first serum-free culture medium is TexMACS, and the second serum-free culture medium is X-VIVO 15.

[0009] In some embodiments, the composition of any of the preceding items, the volume ratio of the first serum-free medium to the second serum-free medium is 1:0.1-2. In some embodiments, the volume ratio of the first serum-free medium to the second serum-free medium is 1:0.25-1. In some embodiments, the volume ratio of the first serum-free medium to the second serum-free medium is 1:0.1, 1:0.25, 1:0.5, or 1:1. In some embodiments, the volume ratio of the first serum-free medium to the second serum-free medium is about 1:0.5.

[0010] In some embodiments, the composition of any of the preceding items comprises a culture medium matrix, wherein the culture medium matrix comprises TexMACS and X-VIVO 15, wherein the volume of TexMACS and X-VIVO 15 accounts for more than 80% of the volume of the culture medium matrix, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.1-2. In some embodiments, the composition is composed of TexMACS and X-VIVO 15, wherein the volume ratio of TexMACS to X-VIVO 15 is 1:0.25-1. In some embodiments, the composition is composed of TexMACS and X-VIVO 15, wherein the volume ratio of TexMACS to X-VIVO 15 is 1:0.5.

[0011] In some embodiments, the composition of any of the preceding items comprises a third serum-free medium AIM-V, and the ratio of the sum of the volumes of the first and second serum-free medium to the volume of the third serum-free medium is no less than 2: 1. In some embodiments, the ratio of the sum of the volumes of the first and second serum-free medium to the volume of the third serum-free medium is no less than 4: 1. In some embodiments, the ratio of the sum of the volumes of the first and second serum-free medium to the volume of the third serum-free medium is no less than 10: 1.

[0012] In some embodiments, the composition of any of the preceding items, wherein the culture medium comprises a third serum-free culture medium, AIM-V, and the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is no less than 2: 1. In some embodiments, the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is no less than 4: 1. In some embodiments, the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is no less than 10: 1.

[0013] In some embodiments, the composition as described in any of the preceding items, wherein the composition comprises a supplement added to the culture medium matrix, the supplement comprising one or more components selected from the group consisting of inorganic salts, sugars, vitamins, albumin, lipids, amino acids, cytokines and antioxidants. In some embodiments, the supplement comprises one or more components selected from the group consisting of amino acids, cytokines and antioxidants. In some embodiments, the supplement is or comprises amino acids and cytokines. In some embodiments, the supplement is or comprises amino acids, cytokines and antioxidants.

[0014] In some embodiments, the composition as described in any of the preceding items, the cytokine comprises one or more components selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferon and tumor necrosis factor. In some embodiments, the cytokine comprises one or more components selected from the group consisting of IL-2, IL-7 and IL-15. In some embodiments, the cytokine is or comprises IL-2. In some embodiments, the cytokine is or comprises IL-2 and IL-7. In some embodiments, the cytokine is or comprises IL-7 and IL-15. In some embodiments, the cytokine is or comprises IL-2, IL-7 and IL-15.

[0015] In some embodiments, the composition of any of the preceding items, the IL-2 is supplemented at a concentration of 10-1000 IU / mL. In some embodiments, the IL-2 is supplemented at a concentration of 60-200 IU / mL. In some embodiments, the IL-2 is supplemented at a concentration of about 100 IU / mL.

[0016] In some embodiments, the composition as described in any of the preceding items, the concentration of the IL-2 supplemented in the composition is 10-1000 IU / mL, the concentration of the L-7 is 100-5000 IU / mL, and the concentration of the IL-15 is 0-500 IU / mL. In some embodiments, the concentration of the IL-2 supplemented in the composition is 60-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, and the concentration of the IL-15 is 25-100 IU / mL. In some embodiments, the concentration of the IL-2 supplemented in the composition is about 100 IU / mL, the concentration of the IL-7 is about 700 IU / mL, and the concentration of the IL-15 is about 46 IU / mL.

[0017] In some embodiments, the composition of any of the preceding items, wherein the amino acid is L-alanyl-L-glutamine. In some embodiments, the concentration of the L-alanyl-L-glutamine added to the composition is 1 mM to 5 mM. In some embodiments, the concentration of the L-alanyl-L-glutamine added to the composition is about 2 mM.

[0018] In some embodiments, the composition of any of the preceding items, wherein the antioxidant is N-acetyl-L-cysteine. In some embodiments, the concentration of the supplemented N-acetyl-L-cysteine ​​in the composition is 0.5 mg / mL to 2 mg / mL. In some embodiments, the concentration of the supplemented N-acetyl-L-cysteine ​​in the composition is 0.5 mg / mL to 1 mg / mL. In some embodiments, the concentration of the supplemented N-acetyl-L-cysteine ​​in the composition is about 0.8 mg / mL.

[0019] In some embodiments, the composition of any of the preceding items comprises a culture medium matrix comprising a first serum-free culture medium, a second serum-free culture medium, and optionally a third serum-free culture medium AIM-V, wherein the combined volume of the first and second serum-free culture mediums accounts for greater than 80% of the total volume of the culture medium matrix, and the volume ratio of the first and second serum-free culture mediums is 1:0.25-1. In some embodiments, the culture medium matrix is ​​composed of the first and second serum-free culture mediums, and the volume ratio of the first and second serum-free culture mediums is 1:0.25-1. In some embodiments, the culture medium matrix is ​​composed of the first, second, and third serum-free culture mediums, and the volume ratio of the first and second serum-free culture mediums is 1:0.25-1, and the ratio of the combined volume of the first and second serum-free culture mediums to the volume of the third serum-free culture medium is no less than 10:1. In some embodiments, the culture medium matrix is ​​composed of the first and second serum-free culture mediums, and the volume ratio of the first and second serum-free culture mediums is approximately 1:0.5. In some embodiments, the culture medium matrix is ​​composed of a first serum-free culture medium, a second serum-free culture medium, and a third serum-free culture medium, wherein the volume ratio of the first serum-free culture medium, the second serum-free culture medium, and the third serum-free culture medium is about 5:4:1.

[0020] In some embodiments, the composition of any of the preceding items comprises a supplement added to the culture medium, wherein the supplement comprises IL-2, IL-7, IL-15, L-alanyl-L-glutamine, and optionally N-acetyl-L-cysteine. In some embodiments, the concentration of the IL-2 supplemented in the composition is 50-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, the concentration of the IL-15 is 25-100 IU / mL, the concentration of the L-alanyl-L-glutamine is 1 mM-5 mM, and the concentration of the N-acetyl-L-cysteine ​​is 0.5 mg / mL-2 mg / mL. In some embodiments, the composition is supplemented with IL-2 at a concentration of about 100 IU / mL, IL-7 at a concentration of about 700 IU / mL, IL-15 at a concentration of about 46 IU / mL, L-alanyl-L-glutamine at a concentration of about 2 mM, and N-acetyl-L-cysteine ​​at a concentration of about 0.8 mg / mL.

[0021] In some embodiments, the composition as described in any of the preceding items comprises a culture medium matrix and a supplement added to the culture medium matrix, wherein the culture medium matrix is ​​composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.25-1; the supplement comprises IL-2, IL-7, IL-15 and L-alanyl-L-glutamine; the concentration of the IL-2 supplemented in the composition is 50-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, the concentration of the IL-15 is 25-100 IU / mL, and the concentration of the L-alanyl-L-glutamine is 1 mM-5 mM.

[0022] In some embodiments, the composition as described in any of the preceding items comprises a culture medium matrix and a supplement added to the culture medium matrix, wherein the culture medium matrix is ​​composed of TexMACS and X-VIVO 15, the volume ratio of TexMACS to X-VIVO15 is 1:0.25-1, and the supplement comprises IL-2, IL-7, IL-15, L-alanyl-L-glutamine and N-acetyl-L-cysteine, and the concentration of the IL-2 supplemented in the composition is 50-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, the concentration of the IL-15 is 25-100 IU / mL, the concentration of the L-alanyl-L-glutamine is 1 mM-5 mM, and the concentration of the N-acetyl-L-cysteine ​​is 0.5 mg / mL-2 mg / mL.

[0023] In some embodiments, the composition as described in any of the preceding items is composed of a culture medium matrix, and IL-2, IL-7, IL-15 and L-alanyl-L-glutamine added to the culture medium matrix, wherein the culture medium matrix is ​​composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.5; the concentration of the IL-2 supplemented in the composition is about 100 IU / mL, the concentration of the IL-7 is about 700 IU / mL, the concentration of the IL-15 is about 46 IU / mL, and the concentration of the L-alanyl-L-glutamine is about 2 mM.

[0024] In some embodiments, the composition as described in any of the preceding items is composed of a culture medium matrix, and IL-2, IL-7, IL-15, L-alanyl-L-glutamine and N-acetyl-L-cysteine ​​added to the culture medium matrix, wherein the culture medium matrix is ​​composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO15 is 1:0.5; the concentration of the IL-2 supplemented in the composition is about 100 IU / mL, the concentration of the IL-7 is about 700 IU / mL, the concentration of the IL-15 is about 46 IU / mL, the concentration of the L-alanyl-L-glutamine is about 2 mM, and the concentration of the N-acetyl-L-cysteine ​​is about 0.8 mg / mL.

[0025] In one aspect, the present disclosure provides a multi-unit composition comprising two or three selected from the group consisting of a first culture medium, a second culture medium, and a third culture medium. In some embodiments, the multi-unit composition comprises a first culture medium and a second culture medium. In some embodiments, the multi-unit composition comprises a first culture medium and a third culture medium. In some embodiments, the multi-unit composition comprises a second culture medium and a third culture medium. In some embodiments, the multi-unit composition comprises a first culture medium, a second culture medium, and a third culture medium. In some embodiments, the first culture medium comprises or consists of a culture medium matrix, the second culture medium comprises or consists of a culture medium matrix, a cytokine, an amino acid, and an antioxidant, and the third culture medium comprises or consists of a culture medium matrix, a cytokine, and an amino acid.

[0026] In some embodiments, the multi-unit composition of any of the preceding items, the culture medium matrix of the first culture medium, the second culture medium, and the third culture medium all comprises a first serum-free culture medium and a second serum-free culture medium. In some embodiments, the first serum-free culture medium is TexMACS, and the second serum-free culture medium is composed of one or more culture media in the group consisting of X-VIVO 10, X-VIVO 15, and X-VIVO 20. In some embodiments, the volume of the first serum-free culture medium and the second serum-free culture medium accounts for more than 80% of the volume of the culture medium matrix, and the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-2. In some embodiments, the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.25-1. In some embodiments, the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1, 1:0.25, 1:0.5, or 1:1.

[0027] In some embodiments, the first culture medium is composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.25-1. In some embodiments, the volume ratio of TexMACS to X-VIVO 15 is 1:0.5.

[0028] In some embodiments, the multi-unit composition of any of the preceding items, the second culture medium comprises a culture medium matrix and a supplement added to the culture medium matrix. In some embodiments, the culture medium matrix of the second culture medium is composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.25-1. In some embodiments, the volume ratio of TexMACS to X-VIVO 15 is 1:0.5. In some embodiments, the supplement comprises IL-2, IL-7, IL-15, L-alanyl-L-glutamine and N-acetyl-L-cysteine, wherein the concentration of the IL-2 supplemented in the composition is 50-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, the concentration of the IL-15 is 25-100 IU / mL, the concentration of the L-alanyl-L-glutamine is 1 mM-5 mM, and the concentration of the N-acetyl-L-cysteine ​​is 0.5 mg / mL-2 mg / mL. In some embodiments, the second culture medium is composed of a culture medium matrix, and IL-2, IL-7, IL-15, L-alanyl-L-glutamine and N-acetyl-L-cysteine ​​added to the culture medium matrix, wherein the culture medium matrix is ​​composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.5; the concentration of the IL-2 supplemented in the composition is about 100 IU / mL, the concentration of the IL-7 is about 700 IU / mL, the concentration of the IL-15 is about 46 IU / mL, the concentration of the L-alanyl-L-glutamine is about 2 mM, and the concentration of the N-acetyl-L-cysteine ​​is about 0.8 mg / mL.

[0029] In some embodiments, the multi-unit composition of any of the preceding items, the third culture medium comprises a culture medium matrix and a supplement added to the culture medium matrix. In some embodiments, the culture medium matrix of the third culture medium is composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.25-1. In some embodiments, the volume ratio of TexMACS to X-VIVO 15 is 1:0.5. In some embodiments, the supplement comprises IL-2, IL-7, IL-15 and L-alanyl-L-glutamine, and the concentration of the IL-2 supplemented in the composition is 50-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, the concentration of the IL-15 is 25-100 IU / mL, and the concentration of the L-alanyl-L-glutamine is 1 mM-5 mM. In some embodiments, the third culture medium is composed of a culture medium matrix, and IL-2, IL-7, IL-15 and L-alanyl-L-glutamine added to the culture medium matrix, wherein the culture medium matrix is ​​composed of TexMACS and X-VIVO 15, and the volume ratio of TexMACS to X-VIVO 15 is 1:0.5; the concentration of the IL-2 supplemented in the composition is about 100 IU / mL, the concentration of the IL-7 is about 700 IU / mL, the concentration of the IL-15 is about 46 IU / mL, and the concentration of the L-alanyl-L-glutamine is about 2 mM.

[0030] In one aspect, the present disclosure provides a method for preparing a culture medium, comprising mixing the culture medium matrix according to any one of the preceding items with a supplement.

[0031] In one aspect, the present disclosure provides a method of culturing cells, comprising incubating the cells in the composition or multi-unit composition as described in any of the preceding items.

[0032] In one aspect, the present disclosure provides a method for culturing cells, comprising a step of expanding T cells, the step comprising expanding the T cells in a third culture medium as described in any of the preceding items. In some embodiments, the method for culturing cells comprises a step of activating the T cells before expanding the T cells, the step comprising activating the T cells in a second culture medium as described in any of the preceding items. In some embodiments, the method for culturing cells comprises a step of washing the T cells before activating the T cells, the step comprising washing the T cells in a first culture medium as described in any of the preceding items.

[0033] In one aspect, the present disclosure provides a method for culturing cells, comprising: 1) isolating and washing the cells, comprising washing the T cells in a first culture medium as described in any of the preceding items or in DPBS; 2) activating the T cells, comprising activating the T cells in a second culture medium as described in any of the preceding items; and 3) expanding the T cells, comprising expanding the T cells in a third culture medium as described in any of the preceding items. In some embodiments, step 2) comprises adding magnetic beads conjugated to anti-CD3 antibodies and anti-CD28 antibodies to the second culture medium and co-incubating the cells. In some embodiments, the method comprises a step of genetically engineering the T cells between steps 2) and 3).

[0034] In one aspect, the present disclosure provides a cell produced by a method of culturing cells as described in any of the preceding items. In some embodiments, the cell is a mammalian cell or a human cell. In some embodiments, the cell is an immune cell, such as a cell of innate immunity or adaptive immunity, such as a bone marrow or lymphoid cell (including lymphocytes, such as T cells and / or NK cells). In some embodiments, the cell is a primary cell. In some embodiments, the cell is allogeneic and / or autologous. In some embodiments, the cell includes one or more subsets of T cells or other cell types, such as the entire T cell population, CD4 T cells, or a combination thereof. + cells, CD8 + cells and their subpopulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A. Effect of cytokine concentration on cumulative expansion fold; Figure 1B. Effect of cytokine concentration on cell viability; Figure 1C. Effect of cytokine concentration on T SCM Figure 1D. Effect of cytokine concentration on PD-1 expression ratio.

[0036] Figure 2. Effect of serum-free medium ratio on T cell expansion DETAILED DESCRIPTION

[0037] After reading this specification, it will become apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, the embodiments herein are presented by way of example only and not by way of limitation. As such, this detailed description should not be construed as limiting the scope or breadth of the present invention as set forth below.

[0038] I. Terminology

[0039] "A" or "an" may refer to one or more or one or more. "About", "substantially" and "approximately" refer to a range in which the corresponding data value is increased or decreased by up to 5%. "Including", "including" and "containing" should be understood as including any other feasible elements in addition to the elements included, as long as the addition of the element does not make the technical solution containing the element unimplementable. "Composed of..." means that it only has the elements described by "...". "Comprising" covers the situation of "composed of..."

[0040] The term "culture medium" refers to a nutrient source for growing or maintaining cells. This nutrient source contains essential components required for cell growth and / or survival and can be used alone for cell culture.

[0041] The term "serum-free medium" refers to a medium that contains no or substantially no serum. As used herein, "substantially serum-free" means containing less than about 1% serum by weight, containing only trace amounts of serum, or containing undetectable amounts of serum.

[0042] The term "supplement" refers to a substance added to the culture medium to maintain, activate, and / or expand cells. A supplement herein may include one or more substances, the types of which may or may not be contained in the culture medium. The concentration of a supplement herein is in addition to the concentration of the corresponding substance in the culture medium. For example, if the total concentration of L-alanyl-L-glutamine in the composition is 5 mM and the concentration of L-alanyl-L-glutamine already in the culture medium is 3 mM, the concentration of the supplemented L-alanyl-L-glutamine is 2 mM. If the total concentration of L-alanyl-L-glutamine in the composition is 2 mM and the culture medium does not contain L-alanyl-L-glutamine, the concentration of the supplemented L-alanyl-L-glutamine is 2 mM. The concentration of a supplement herein is calculated based on the original volume of the culture medium.

[0043] The term "cytokine" refers to biological molecules that affect cells of the immune system, including native cytokines and fragments and functional variants that have at least 10%, 30%, 50% or 80% of the activity of the native cytokine (e.g., the immunomodulatory activity of a naturally occurring cytokine).

[0044] The term "amino acid" refers to all naturally occurring α-amino acids, as well as synthetic oligopeptides, analogs, and derivatives. Analogs are molecules in which an atom in an amino acid is replaced with a different atom, generally with similar properties. Derivatives are molecules in which other structures are attached to an amino acid, for example, products obtained by propionylating an amino acid. Amino acids herein include L-alanyl-L-glutamine.

[0045] The term "optionally" refers to the presence or absence of N-acetyl-L-cysteine. For example, optional N-acetyl-L-cysteine ​​refers to the presence or absence of N-acetyl-L-cysteine ​​in the supplement.

[0046] The term "culturing" refers to maintaining cells in vitro under conditions that are conducive to growth and / or differentiation and / or sustained viability. "Cultivating" can be used interchangeably with "cell culture."

[0047] The term "expansion" refers to the growth of cells in culture to increase the number of cells from an initial cell number to a larger cell number after culture.

[0048] The term "multi-unit composition" refers to a composition comprising two or more independently existing compositions, such as culture media.

[0049] The terms "immune cell" and "immune system cell" refer to cells that participate in the immune response designed to protect an organism from foreign substances, viruses, and cells. Immune cells can be derived from many organs and tissues, such as the thymus, spleen, lymph nodes, lymphoid tissue clusters (such as in the gastrointestinal tract and bone marrow). Such cells include T cells, B cells, natural killer cells, macrophages, neutrophils, tumor-infiltrating lymphocytes, dendritic cells, mast cells, eosinophils and basophils, and the progenitor cells that develop into these cells.

[0050] II. Culture Medium

[0051] The present disclosure provides a composition comprising at least two serum-free culture media. Surprisingly, when the composition of the present disclosure is used to culture immune cells (such as T cells), it has significantly improved cell expansion multiples, more sustainable cell viability, lower PD-1 expression, higher CD25 expression, and the like compared to a single serum-free culture medium. + CD69 + T cells and / or a higher proportion of Tscm. For example, after culturing T cells with the composition for 7, 9, 11, or 13 days, the expansion fold of the T cells is increased by 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% or more compared to a single serum-free medium.

[0052] II-a. Culture medium matrix

[0053] In some embodiments, the composition as described above comprises a culture medium matrix. In some embodiments, the culture medium matrix comprises a first serum-free culture medium and a second serum-free culture medium that are different from each other. In some embodiments, the serum-free culture medium is well known to those skilled in the art and is commercially available, such as RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, TexMACSTM 、X-VIVO TM 10. X-VIVO TM 15 and X-VIVO TM 20 or OpTmizer TM In some embodiments, the first serum-free medium is TexMACS TM The second serum-free culture medium is selected from RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-VIVO TM 10. X-VIVO TM 15. X-VIVO TM 20 and OpTmizer TM In some embodiments, the first serum-free medium is TexMACS TM The second serum-free medium is selected from X-VIVO TM 10. X-VIVO TM 15 and X-VIVO TM In a specific embodiment, the first serum-free medium is TexMACS TM , the second serum-free medium is AIM-V. In a specific embodiment, the first serum-free medium is TexMACS TM , the second serum-free medium is F-12. In a specific embodiment, the first serum-free medium is TexMACS TM , the second serum-free medium is X-RPMI 1640. In a specific embodiment, the first serum-free medium is TexMACS TM , the second serum-free medium is DMEM. In a specific embodiment, the first serum-free medium is TexMACS TM The second serum-free medium is X-VIVO TM 15. In some embodiments, the first serum-free culture medium and the second serum-free culture medium in the composition are to be prepared or have been prepared as culture media.

[0054] In some embodiments, the volume of the first serum-free medium and the second serum-free medium as described above accounts for more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the volume of the culture medium matrix. The volume of the first serum-free medium and the second serum-free medium accounts for 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the volume of the culture medium matrix. In specific embodiments, the volume of the first serum-free medium and the second serum-free medium accounts for 100% of the volume of the culture medium matrix, that is, the culture medium matrix consists of the first serum-free medium and the second serum-free medium. Those skilled in the art will understand that the volume of different culture media and other solutions after mixing may differ slightly from the sum of their volumes before mixing. When differences exist, the volume of the culture medium matrix herein is the sum of the volumes of the different culture media and other solutions before mixing.

[0055] In some embodiments, the volume ratio (v:v) of the first serum-free culture medium and the second serum-free culture medium as described above is 1:0.1-10, 1:0.2-5, 1:0.2-4, 1:0.2-3, 1:0.2-2, 1:0.2-1, 1:0.25-2, 1:0.25-1, 1:0.3-3, 1:0.3-2, 1:0.3-1, 1:0.4-2, 1:0.4-1, 1:0.5-2, 1:0.5- 1. In some embodiments, the volume ratio of the first serum-free culture medium to the second serum-free culture medium is or is about 1:1 or 1:0.5. In some embodiments, the volume of the first serum-free medium and the second serum-free medium accounts for 100% of the volume of the culture medium matrix, and the volume ratio of the first serum-free medium to the second serum-free medium is 1:0.1-10, 1:0.2-5, 1:0.2-4, 1:0.2-3, 1:0.2-2, 1:0.2-1, 1:0.25-2, 1:0.25-1, 1:0.3-3, 1:0.3-2, 1:0.3-1, 1:0.4-2, 1:0.4-1, 1:0.5-2, 1:0.5-1. In some embodiments, the volume of the first serum-free medium and the second serum-free medium accounts for 100% of the volume of the culture medium matrix, and the volume ratio of the first serum-free medium to the second serum-free medium is or is about 1:1, 1:0.5, or 1:0.25. Herein, the volume ratio of the first serum-free medium and the second serum-free medium refers to the volume ratio before mixing the two.

[0056] In some embodiments, the culture medium comprises a first serum-free culture medium, a second serum-free culture medium, and a third serum-free culture medium that are different from each other, wherein the first serum-free culture medium is TexMACS TM The second serum-free medium is composed of X-VIVO TM 10. X-VIVO TM 15 and X-VIVO TM 20, wherein the third serum-free medium is AIM-V. In some embodiments, the volume of the first, second, and third serum-free mediums described above accounts for greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% of the volume of the culture medium. In some embodiments, the volume of the first, second, and third serum-free mediums accounts for 100% of the volume of the culture medium. In some embodiments, the ratio of the sum of the volumes of the first and second serum-free medium to the volume of the third serum-free medium is no less than 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In specific embodiments, the volume ratio of the first, second, and third serum-free mediums is approximately 5:4:1.

[0057] II-b. Supplements

[0058] In some embodiments, the composition as described above comprises a supplement added to the culture medium matrix, the supplement comprising one or more components selected from the group consisting of inorganic salts, sugars, vitamins, albumin, lipids, amino acids, stimulants (e.g., cytokines) and antioxidants. In some embodiments, the supplement comprises one or more components selected from the group consisting of amino acids, cytokines and antioxidants. In some embodiments, the supplement is or comprises amino acids, cytokines and antioxidants.

[0059] In some embodiments, the supplement as described above comprises a cytokine. In some embodiments, the cytokine is a recombinant cytokine. In a specific embodiment, the cytokine is a recombinant human cytokine. In some embodiments, the cytokine is capable of binding to a receptor expressed by an immune cell (e.g., a T cell). In some embodiments, the cytokine includes interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony stimulating factor (G-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the cytokine includes interferon, tumor necrosis factor. In some embodiments, the cytokine is selected from one or more components in the group consisting of IL-2, IL-7, and IL-15. In some embodiments, the cytokine is or comprises IL-2. In a specific embodiment, the cytokine is or comprises IL-2, IL-7, and IL-15. These cytokines are used as stimulants in the culture medium to activate the cells. Once the cells have been activated, the culture medium without the addition of cytokines can be used.

[0060] In some embodiments, the cytokine as described above comprises IL-2. In some embodiments, the concentration of the IL-2 (e.g., human recombinant IL-2) is 10-1000 IU / mL, 50-500 IU / mL, 50-200 IU / mL, or 50-150 IU / mL. In specific embodiments, the concentration of the IL-2 is or is about 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL, 110 IU / mL, 120 IU / mL, 130 IU / mL, 140 IU / mL, or 150 IU / mL. In some embodiments, the concentration of the IL-2 is or is about 100 IU / mL.

[0061] In some embodiments, the cytokine as described above comprises IL-7. In some embodiments, the concentration of the IL-7 (e.g., human recombinant IL-7) is 100-5000 IU / mL, 200-2000 IU / mL, 500-1000 IU / mL, or 500-800 IU / mL. In specific embodiments, the concentration of the IL-7 is or is about 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, 1000 IU / mL. In some embodiments, the concentration of the IL-7 is or is about 700 IU / mL.

[0062] In some embodiments, the cytokine as described above comprises IL-15. In some embodiments, the concentration of the IL-15 (e.g., human recombinant IL-15) is 0-500 IU / mL, 10-200 IU / mL, 20-100 IU / mL, 30-100 IU / mL, 40-80 IU / mL, or 40-60 IU / mL. In specific embodiments, the concentration of the IL-15 is or is about 25 IU / mL, 30 IU / mL, 40 IU / mL, 46 IU / mL, 50 IU / mL, 60 IU / mL, 70 IU / mL, 80 IU / mL, 90 IU / mL, 100 IU / mL. In some embodiments, the concentration of the IL-15 is or is about 46 IU / mL.

[0063] In some embodiments, the supplement as described above comprises amino acids. In some embodiments, the amino acids are selected from aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, norvaline, tryptophan, phenylalanine, isoleucine, lysine, hydroxyproline, sarcosine, proline or a combination thereof. In some embodiments, the supplement comprises glutamine. In some embodiments, the composition comprises synthetic amino acids. In some embodiments, the synthetic amino acids can be converted into free form L-glutamine in a cell culture comprising cells. In some embodiments, the synthetic amino acids are dipeptides. In some embodiments, the synthetic amino acids are L-alanyl-L-glutamine (GlutaMax).

[0064] In some embodiments, the concentration of the amino acid (eg, L-alanyl-L-glutamine) is about 0.5 mM-5 mM. In some embodiments, the concentration of L-alanyl-L-glutamine is at or about 0.5 mM-1 mM, 0.5 mM-1.5 mM, 0.5 mM-2 mM, 0.5 mM-2.5 mM, 0.5 mM-3 mM, 0.5 mM-3.5 mM, 0.5 mM-4 mM, 0.5 mM-4.5 mM, 0.5 mM-5 mM, 1 mM-1.5 mM, 1 mM-2 mM, 1 mM-2.5 mM, 1 mM-3 mM, 1 mM-3.5 mM, 1 mM-4 mM, 1 mM-4.5 mM, 1 mM-5 mM, 1.5 mM-2 mM, 1.5 mM-2.5 mM, 1.5 mM-3 .5mM, 1.5mM-4mM, 1.5mM-4.5mM, 1.5mM-5mM, 2mM-2.5mM, 2mM-3mM, 2mM-3 .5mM, 2mM-4mM, 2mM-4.5mM, 2mM-5mM, 2.5mM-3mM, 2.5mM-3.5mM, 2.5mM-4 mM, 2.5mM-4.5mM, 2.5mM-5mM, 3mM-3.5mM, 3mM-4mM, 3mM-4.5mM, 3mM-5mM , 3.5mM-4mM, 3.5mM-4.5mM, 3.5mM-5mM, 4mM-4.5mM, 4mM-5mM or 4.5mM-5mM. In some embodiments, the concentration of L-alanyl-L-glutamine is at or about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM. In a specific embodiment, the concentration of L-alanyl-L-glutamine is 2 mM.

[0065] In some embodiments, the supplements described above include an antioxidant. In some embodiments, the antioxidant includes N-acetyl-L-cysteine ​​(NAC), 2-mercaptoethanol, or D,L-tocopheryl acetate, or a derivative or mixture thereof. In some embodiments, the antioxidant is N-acetyl-L-cysteine.

[0066] In some embodiments, the concentration of N-acetyl-L-cysteine ​​is at or about 0.1-10 mg / mL, 0.2-10 mg / mL, 0.5-10 mg / mL, 0.1-5 mg / mL, 0.5-1 mg / mL, 0.2-5 mg / mL, 0.5-5 mg / mL, 0.5-2 mg / mL, or 0.6-1 mg / mL. In some embodiments, the concentration of N-acetyl-L-cysteine ​​is at or about 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL. In some embodiments, the concentration of N-acetyl-L-cysteine ​​is at or about 0.8 mg / mL.

[0067] II-c. Multi-unit compositions

[0068] In some embodiments, the composition as described above is a multi-unit composition. Each unit in the multi-unit composition (e.g., each independently existing composition or culture medium) can be used alone for cell culture. In some embodiments, the multi-unit composition includes multiple independent culture media. In some embodiments, the multi-unit composition includes a first culture medium, a second culture medium, a third culture medium, or any combination thereof. In some embodiments, the multi-unit composition includes a first culture medium, a second culture medium, and a third culture medium.

[0069] In some embodiments, the first culture medium comprises a culture medium matrix as described in any of the preceding items, and optional supplements. In some embodiments, the first culture medium is a culture medium matrix as described in any of the preceding items. In some embodiments, the first culture medium comprises a culture medium matrix as described in any of the preceding items, and optionally supplements other than stimulants (e.g., cytokines). In some embodiments, the first culture medium comprises a culture medium matrix as described in any of the preceding items, and optionally amino acids and / or antioxidants. This first culture medium may also be referred to as a basal medium. In some embodiments, this first culture medium can be used for cell culture before or after cell activation, such as separation, washing, or amplification of cells. In some embodiments, this first culture medium can be used for steps before cell activation.

[0070] In some embodiments, the second culture medium comprises a culture medium as described in any of the preceding items and a supplement. In some embodiments, the second culture medium comprises a culture medium as described in any of the preceding items and a cytokine. In some embodiments, the second culture medium comprises a culture medium as described in any of the preceding items, a cytokine, and optionally an amino acid and / or an antioxidant. In some embodiments, the second culture medium comprises a culture medium as described in any of the preceding items, a cytokine, an amino acid, and an antioxidant. This second culture medium may also be referred to as an activation medium and may be used in a cell activation step.

[0071] In some embodiments, the third culture medium comprises the culture medium matrix described in any of the preceding items, and optionally, supplements. In some embodiments, the third culture medium comprises the culture medium matrix described in any of the preceding items, cytokines, and optionally, cytokines and / or amino acids. In some embodiments, the third culture medium comprises the culture medium matrix described in any of the preceding items, cytokines, and amino acids. This third culture medium may also be referred to as an expansion medium. In some embodiments, this third culture medium may be used for steps subsequent to cell activation, such as cell expansion steps.

[0072] II-d. Formulation of the composition

[0073] In one aspect, the present disclosure provides a method for preparing a composition, comprising combining the aforementioned components simultaneously or sequentially. In some embodiments, the method comprises combining a first serum-free medium and a second serum-free medium in a culture medium matrix, and then adding a supplement, such as a cytokine, L-alanyl-L-glutamine, N-acetyl-L-cysteine, or a combination thereof.

[0074] III. Cell Culture

[0075] In one aspect, present disclosure provides a kind of method of culturing cell, it adopts any one of the aforementioned compositions or its combination.In some embodiments, described cell is immune cell, such as T cell.In some embodiments, described cell is genetically engineered cell, such as genetically engineered T cell.In some embodiments, culture cell at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 days.In some embodiments, culture cell about 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days, 8-9 days, 9-10 days, 10-11 days, 11-12 days, 12-13 days, 13-14 days, 14-15 days, 15-16 days, 16-17 days, 17-18 days, 18-19 days or 19-20 days, each comprises end value. In some embodiments, the cells are cultured at 37.0 ± 1.0°C. In some embodiments, the cells are cultured at 5.0 ± 1.0% CO2. In some embodiments, the culture can be performed for greater than or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days without changing the culture medium. In some embodiments, perfusion (e.g., semi-continuous perfusion) can be used in conjunction with culturing the cells.

[0076] In some embodiments, the cell is an immune cell or an enriched immune cell. In some embodiments, the cell is a T cell or an enriched T cell. In some embodiments, the cell is a CD4 + T cells or enriched CD4 + In some embodiments, the cells are CD8 + T cells or enriched CD8 + In some embodiments, the cells are CD4 + T cells and CD8 + In some embodiments, the cells are enriched for CD4 + T cells and enriched CD8 +T cells. In some embodiments, the cells include genetically engineered cells or enriched genetically engineered cell colonies. In some embodiments, the cells include cells to be genetically engineered or being genetically engineered. In some embodiments, the cells include cells to be genetically engineered or being genetically engineered enriched cell colonies. In some embodiments, the cells include genetically engineered T cells or enriched genetically engineered T cell colonies. In some embodiments, the cells include chimeric antigen receptor (CAR) expressing T cells or enriched CAR expressing T cells. In some embodiments, the cells include T cells (e.g., Examples 1 and 3) expressing caTCR and CSR in conjunction with AFP and GPC-3. In some embodiments, the cells include T cells (e.g., Example 2) expressing caTCR and CSR in conjunction with GPC-3. In some embodiments, the cells have previously been cryopreserved. In some embodiments, the cells have been cultured in serum-free medium and cryopreserved after culture. In some embodiments, the cells specifically target tumor cells.

[0077] In some embodiments, cells are expanded at least or about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, or more after being cultured using the cell culture methods of the disclosure for or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days. In some embodiments, cells are expanded at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more after being cultured in a serum-free medium formulation for about 9 days. In some embodiments, cell culture using the composition of the present disclosure has a higher immune cell expansion fold than the first serum-free medium and the second serum-free medium alone.

[0078] In some embodiments, the cell culture method involves one or more steps or processes. In some embodiments, the one or more steps include separation, selection, activation, transduction, cultivation, expansion, washing, suspension, dilution, concentration and / or formulation of cells. In some embodiments, the method includes isolating cells, preparing, processing, and culturing cells under one or more stimulating conditions. In some embodiments, the method includes processing steps performed in the following order: first, the cells are separated from the biological sample (such as selected); the selected cells are incubated with viral vector particles for transduction, such as after the step of stimulating the isolated cells in the presence of a stimulating agent; the transduced cells are cultured, such as amplifying the cells.

[0079] In some embodiments, the one or more processing steps may include one or more of the following: (a) pre-washing a biological sample containing cells (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cell (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a leukocyte sample, an apheresis product, or a leukapheresis product); (b) isolating (e.g., selecting) desired cells (e.g., CD4 + and / or CD8 + T cells), for example, by incubating the cells with an immunoaffinity reagent; (c) introducing a vector encoding a recombinant receptor into the isolated or selected cells, such as by incubating the isolated (e.g., selected) cells with viral vector particles encoding the recombinant receptor; and (d) culturing or expanding the cells. In some embodiments, the method may further include (f) activating the cells by exposing them to stimulating conditions, which may be performed before, during, and / or after incubating the cells with the viral vector particles, for example, between step (b) and step (c). In some embodiments, step (f) is performed before incubating the cells with the viral vector particles. In some embodiments, washing and / or suspension steps may also be performed before or after any of the above steps, for example, between step (b) and step (f). In some embodiments, one, more, or all steps of the cell culture method are performed under sterile conditions. In some embodiments of this method, cell separation, transduction, washing, optional activation or stimulation, and formulation are all performed in a closed system.

[0080] III-a. Cell separation

[0081] In some embodiments, the cell culture method includes separating cells or its compositions from a biological sample. In some embodiments, the biological sample is blood or a sample derived from blood, or is or is derived from apheresis or leukocyte apheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs and / or cells derived from them. In some embodiments, the biological sample includes a sample from autologous and allogeneic sources.

[0082] In some embodiments, the cells are mammalian cells or human cells. In some embodiments, the cells are immune cells, such as cells of innate immunity or adaptive immunity, such as bone marrow or lymphoid cells (including lymphocytes, such as T cells and / or NK cells). In some embodiments, the cells are primary cells. In some embodiments, the cells are allogeneic and / or autologous. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4 T cells, or other cell types. + cells, CD8 + Cells and their subsets. T cells and / or CD4 + and / or CD8 + T cell subtypes and subsets include naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes (such as stem cell memory T (T SCM ), central memory T(T CM ), effect memory T(T EM ) or terminally differentiated effector memory T cells), tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated constant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), α / β T cells and δ / γ T cells. In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or granulocyte, such as a bone marrow cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil and / or a basophil. In some embodiments, the cell is a tumor infiltrating lymphocyte.

[0083] In some embodiments, the cell separation step is non-affinity-based. In some examples, cells are washed and / or centrifuged in the presence of one or more reagents to remove unwanted components, enrichment of required components, cracking or removal of cells sensitive to specific reagents. In some examples, cells are separated based on one or more characteristics (such as density, adhesion properties, size, sensitivity and / or resistance to specific components). In some embodiments, the method includes a density-based cell separation method, such as by dissolving red blood cells and preparing white blood cells from peripheral blood by gradient centrifugation.

[0084] In some embodiments, the cell separation step includes incubating and separating the cells with a selection reagent. In some embodiments, the selection reagent is a reagent for binding cells based on immunoaffinity. In some embodiments, the selection reagent is an antibody or its conjugate specifically bound to a cell marker. In some embodiments, the selection reagent is a particle (such as a magnetic bead) conjugated with a specific selection agent (such as an antibody). In some embodiments, the magnetic bead comprises a magnetic response material bound to an antibody. In some embodiments, the magnetic response material is coated with an antibody (antibody binding to cells), a second antibody, a lectin, an enzyme, or a streptavidin, and is attached to the cell by an antibody. The cell separation step separates cells by expression or expression levels of one or more markers (typically cell surface markers) in cells, for example, cells are incubated with an antibody or its conjugate specifically binding to the marker, and then separated by washing to obtain cells (positive selection) or cells (negative selection) that have been bound to the antibody. In some embodiments, the cell separation step enriches a specific cell population via positive selection, or exhausts a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed or expressed at relatively high levels on the positively or negatively selected cells, respectively. The separation need not achieve 100% enrichment or removal of a particular cell population or cells expressing a particular marker. In some embodiments, the steps include multiple rounds, such as subjecting the positively or negatively selected cells to another round of positive or negative selection. Specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers (e.g., CD28 + 、CD62L + 、CCR7 + 、CD27 + 、CD127 + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO +T cells) can be isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibodies or conjugates thereof that specifically bind to such markers. For example, CD4 can be positively selected using magnetic beads conjugated to anti-CD4 and anti-CD8 antibodies. + and CD8 + T cells.

[0085] In some embodiments, the cell separation step is carried out in a buffer. In some embodiments, the buffer and the selection reagent are mixed before adding the cells. In some embodiments, the buffer and the selection reagent are selected to be added to the cell sample separately. In some embodiments, the total duration of hatching together with the selection reagent is from or from about 5 minutes to or to about 6 hours, such as 30 minutes to 3 hours, for example, at least or at least about 30 minutes, 60 minutes, 120 minutes or 180 minutes.

[0086] In some embodiments, the cell separation step further comprises pre-washing a sample containing cells (such as a single collection sample). In some embodiments, the separation system based on immunoaffinity is or contains a magnetic separation column. The separation step comprises placing the sample in a magnetic field, and the cells bound to the magnetic beads will be attracted to the magnetic separation column and separated from the cells that are not bound to the magnetic beads. For positive selection, cells attracted by the magnet are retained; for negative selection, cells that are not attracted are retained. In some embodiments, affinity-based selection is carried out via magnetic activated cell sorting (MACS, such as CliniMACS system, Miltenyi Biotec). In some embodiments, the steps are carried out in the inner cavity of a centrifugal chamber, for example, under centrifugal rotation. In some embodiments, the steps are carried out in an automated manner, for example, using an automated program to complete the steps of pre-washing, binding and separation in a single closed system.

[0087] In some embodiments, the cell separation step includes a step of freezing (e.g., cryopreserving) the cells after separation. In some embodiments, the freezing step removes granulocytes from the cell population and, to a certain extent, monocytes. In some embodiments, the cells are frozen to -80°C at a rate of 1°C / minute for storage.

[0088] III-b. Cell Activation

[0089] In some embodiments, the steps include stimulating the isolated cells (e.g., the cell colony of selection). In some embodiments, the activation of the cells is performed before genetic engineering or simultaneously with genetic engineering (e.g., transduction). In some embodiments, the activation of the cells is performed before genetic engineering.

[0090] In some embodiments, the composition or cell is incubated under stimulation and / or activation conditions. In some embodiments, the conditions for stimulation and / or activation may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, medicine (e.g., nutrients, amino acids, antibiotics, ions and / or stimulants, such as cytokines, chemokines, antigens, fusion proteins, recombinant soluble receptors and any other medicament capable of activating cells). In some embodiments, stimulation conditions include one or more medicines capable of stimulating or activating the intracellular signaling domain of the TCR complex. In some embodiments, the medicine can start the TCR / CD3 intracellular signaling cascade in T cells, for example, medicines (e.g., anti-CD3 antibodies) suitable for transmitting primary signals to, for example, start the activation of ITAM-induced signals, and / or agents promoting costimulatory signals (e.g., costimulatory signals specific to T cell costimulatory receptors), such as anti-CD28 or anti-4-1BB antibodies (e.g., bound to a solid support) and / or one or more cytokines. The stimulators include anti-CD3 antibodies and anti-CD28 antibodies, or magnetic beads conjugated with anti-CD3 / anti-CD28 antibodies (e.g., Dynabeads TM CD3 / CD28 T cell expander). In some embodiments, the stimulatory agent includes IL-2, IL-7 and / or IL-15. In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25°C, at least about 30°C, or about 37°C.

[0091] In some embodiments, the cell activation step is carried out in the lumen of a centrifugal chamber. In some embodiments, cells are mixed with stimulating conditions or stimulants in a centrifugal chamber. In some embodiments, cells are added to an activation medium containing cytokines and incubated. In some embodiments, the volume of the activation medium is 10mL to 200mL, or about 10mL, 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, 90mL, 100mL, 150mL or 200mL. In some embodiments, the activation medium comprises a culture medium matrix and supplements. In some embodiments, the supplements comprise cytokines, and optionally amino acids and antioxidants. In some embodiments, the cytokines are IL-2, IL-7 and IL-15. In some embodiments, the activation medium is pre-formulated. In some embodiments, the culture medium matrix and supplements are added to the cells separately.

[0092] In some embodiments, the total duration of the activation process is between or about between 1 hour and 96 hours, between 1 hour and 72 hours, between 1 hour and 48 hours, between 4 hours and 36 hours, between 8 hours and 30 hours, or between 12 hours and 24 hours, such as at least or about at least 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, or 72 hours.

[0093] III-c. Genetic Engineering

[0094] In one aspect, the cell culture method includes a genetic engineering step. In some embodiments, this step introduces a nucleic acid molecule encoding a recombinant protein into the cell. In some embodiments, the nucleic acid molecule encoding the recombinant protein is introduced into the cell before, simultaneously with, or after cell activation. In some embodiments, the nucleic acid molecule encoding the recombinant protein is introduced into the cell after cell activation. In some embodiments, the introduction is carried out by contacting the cell with a nucleic acid molecule encoding the recombinant protein. For the cell culture method of the present disclosure, this step is optional.

[0095] In some embodiments, the genetic engineering step includes introducing the recombinant protein into the cell via a vector. Such vectors include viral and non-viral systems. In some embodiments, the viral system includes recombinant infectious virus particles, such as vectors of adenovirus, adeno-associated virus (AAV) and human immunodeficiency virus (HIV), and recombinant lentiviral vectors or retroviral vectors (such as γ-retroviral vectors). In some embodiments, the non-viral system includes a transposon system, such as the gene transfer system of PiggyBac or Sleeping Beauty. In some embodiments, this step is performed by electroporation. In some embodiments, this step is performed by transduction, transposon, electroporation or a combination thereof. Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microparticle bombardment and strontium phosphate DNA co-precipitation.

[0096] In some embodiments, the concentration of cells to be transduced is at or about 1.0×10 5 cells / mL to 1.0×10 8 cells / mL, such as at least or at least about or about 1.0×10 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL or 1×10 8Cells / mL. In some embodiments, the viral particles are provided as a ratio of viral vector particle copies or their infectious units (IU) to the total number of cells to be transduced (IU / cell). In some embodiments, the viral particles are present as or at least 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or 60 IU of viral vector particles per cell. In some embodiments, transduction can be achieved at a multiplicity of infection (MOI) of less than 100, for example, typically less than 60, 50, 40, 30, 20, 10, 5 or less.

[0097] In some embodiments, the step comprises contacting the cells with the viral particles. In some embodiments, the duration of the contact is 30 minutes to 72 hours, 30 minutes to 48 hours, 30 minutes to 24 hours, or 1 hour to 24 hours, or at least about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, or 36 hours. In some embodiments, the contacting is performed in a solution or culture medium. In some embodiments, the contacting is performed in a second culture medium. In some embodiments, the cells and viral particles are contacted in a volume of 0.5 mL to 500 mL, such as, for example, or about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL, or 200 mL to 500 mL. In some embodiments, the contacting is accomplished by centrifugation. In some embodiments, the centrifugation speed is lower than the speed used to pellet the cells, for example, from or about 600 rpm to or to about 1700 rpm, for example, at or about or at least 600 rpm, 1000 rpm or 1500 rpm or 1700 rpm.

[0098] In some embodiments, after the genetic engineering step, the cells are transferred to other containers for culturing the genetically engineered cells, such as for expanding the cells. In some embodiments, the container for expanding the cells is a bioreactor bag, such as a perfusion bag.

[0099] III-d. Cell Expansion

[0100] In one aspect, the cell culture method includes a step of expanding the cells in an expansion medium. In some embodiments, the cells are expanded after the genetic engineering step. In some embodiments, the expansion medium is any of the compositions described in II. Culture medium. The composition has a surprising cell expansion effect.

[0101] In some embodiments, the time of the expansion step is greater than or is 24 hours, 48 ​​hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days. In some embodiments, the temperature of the expansion step is at least about 25 ° C, at least about 30 ° C, or about 37 ° C. In some embodiments, the temperature is 25 to 38 ° C, 36 to 38 ° C. In some embodiments, the cells are expanded under conditions that maintain a target amount of carbon dioxide (CO2) in the cell culture. In some embodiments, the amount of CO2 accounts for 10% to 0% (v / v), 8% to 2% (v / v), or is or is about 5% (v / v) of the culture environment gas.

[0102] In some embodiments, cells are expanded using a container used in conjunction with a bioreactor. In some embodiments, the bioreactor is subjected to motion or sway. Moving the bioreactor can increase oxygen transfer, including but not limited to rotating along a horizontal axis, rotating along a vertical axis, swaying motion along the tilted horizontal axis of the bioreactor, or any combination thereof. In some embodiments, the sway angle is or is about 20 °, 19 °, 18 °, 17 °, 16 °, 15 °, 14 °, 13 °, 12 °, 11 °, 10 °, 9 °, 8 °, 7 °, 6 °, 5 °, 4 °, 3 °, 2 °, or 1 °. In certain embodiments, the sway angle is between 6-16 °. In other embodiments, the sway angle is between 7-16 °. In other embodiments, the sway angle is between 8-12 °. In some embodiments, the sway angle is 5 °-10 °, for example, 6 °. In some embodiments, the rocking speed is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 112, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 rpm. In some embodiments, the rocking speed is between 4 rpm and 12 rpm, such as between 4 rpm and 6 rpm, inclusive. In some embodiments, the constant rocking speed is between 5 rpm and 15 rpm, such as 6 rpm or 10 rpm. In some embodiments, the bioreactor is maintained at a temperature of or near 37° C. and a CO level of or near 5%, with a constant air flow rate of or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In certain embodiments, expansion is performed under perfusion.

[0103] In some embodiments, the expanding step is performed in a closed system. In some embodiments, the expanding step is performed in the same closed system as one or more other steps of the cell culture method.

[0104] IV. Recombinant Protein

[0105] In one aspect, cells cultured with the compositions disclosed herein express recombinant proteins. In some embodiments, the cells comprise one or more nucleic acids encoding recombinant proteins introduced by genetic engineering, and thereby express the recombinant proteins. In some embodiments, expression of the recombinant protein is achieved by first activating the cells, then transducing the activated cells, and amplifying them in culture to a quantity sufficient for clinical application.

[0106] In some embodiments, the recombinant protein is a chimeric receptor, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell construct (caTCR), a chimeric signaling receptor (CSR), or a combination thereof.

[0107] IV-a. Chimeric Antigen Receptor (CAR)

[0108] In some embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some embodiments, the antigen is selectively expressed or overexpressed on cells (e.g., tumors or pathogenic cells) of a disease or condition compared to normal or non-targeted cells. In some embodiments, the disease and condition include proliferative, neoplastic and malignant diseases and disorders, including cancer and tumors, including blood cancers, immune system cancers, such as lymphomas, leukemias and / or myeloma, such as B leukemia, T leukemia and myeloid leukemia, lymphoma and multiple myeloma. In some embodiments, the antigen is a tumor antigen or a cancer marker. In some embodiments, the antigen (including the ligand) is or includes B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9), cancer-testis antigen, cancer / testis antigen 1B (NY-ESO-1), carcinoembryonic antigen (CEA), cyclin, cyclin A2, CC motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, MAGE-A4, DLL3, CEA, Claudin18.2 (CLDN18.2), chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), epidermal growth factor receptor type III mutant (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin B2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5), fetal acetylcholine receptor (fetal AchR), folate binding protein (FBP), folate receptor α, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), glypican-3 (GPC3), G protein-coupled receptor class C 5 member D (GPRC5D), GUCY2C, Her2 / neu (receptor tyrosine kinase erb-B2), H er3 (erb-B3), Her4 (erb-B4), erbB dimer, human high molecular weight melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-A1), human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha (IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domain receptor (KDR), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, leucine-rich repeat-containing protein 8 family member A (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1,MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer cell group 2 member D (NKG2D) ligand, melanin A (MART-1), neural cell adhesion molecule (NCAM), carcinoembryonic antigen, preferentially expressed antigen in melanoma (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblast glycoprotein (TPBG, also known as 5T4), tumor-associated glycoprotein 72 (TAG72), tyrosinase-related protein 1 (TRP1, also known as TYRP1 or gp75), tyrosinase-related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with universal tags, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. In some embodiments, the receptor-targeted antigen includes an antigen associated with a B-cell malignancy, such as any of a variety of known B-cell markers. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igκ, Igλ, CD79a, CD79b or CD30.

[0109] In some embodiments, the CAR contains an extracellular antigen recognition domain that specifically binds to an antigen. Therefore, the extracellular antigen recognition domain that specifically binds to an antigen includes one or more antigen binding molecules, such as one or more antigen binding fragments, domains or portions, or one or more antibody variable domains, and / or antibodies. In some embodiments, the antigen binding molecule is a full-length antibody, such as a single-domain antibody, a monospecific antibody or a multispecific antibody; or an antibody fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab')2, scFv.

[0110] In some embodiments, the CAR includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof, such as a hinge region and / or CH1 / CL and / or Fc region. In some embodiments, the spacer is located between the extracellular antigen recognition domain and the transmembrane domain.

[0111] In some embodiments, the CAR includes a transmembrane domain. In some embodiments, the transmembrane domain is derived from a natural source or from a synthetic source. When the source is natural, the transmembrane domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane domain includes one or more of the following, α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic amino acid residues, such as leucine and valine.

[0112] In some embodiments, the CAR contains an intracellular signaling domain, and the intracellular signaling domain includes a cytoplasmic signaling domain, for example, an intracellular domain capable of inducing a primary activation signal in a T cell, for example, a ζ chain of a CD3ζ chain; and / or the intracellular signaling domain includes an activation motif (ITAM) based on immunoreceptor tyrosine. In some embodiments, the CAR contains a costimulatory domain. In some embodiments, the costimulatory domain is a signaling region and / or a transmembrane portion of CD28, 4-1BB, OX40, DAP10 or ICOS. In some aspects, the same CAR includes both a primary activation signaling region and a costimulatory component. In some embodiments, the same CAR includes both a primary activation signaling domain and a costimulatory domain.

[0113] IV-bT cell receptor (TCR)

[0114] In some embodiments, the recombinant protein is a TCR. In some embodiments, the TCR is naturally occurring or modified. In some embodiments, the TCR comprises variable α and β chains (TCRα and TCRβ (or variable γ and δ chains (TCRγ and TCRδ) or antigen binding portions thereof, and is capable of specifically binding a peptide bound to an MHC molecule. In some embodiments, the TCR can be a heterodimer of two chains α and β, or the TCR can be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two separate chains connected, for example, by one or more disulfide bonds. In some embodiments, the TCR further comprises a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. In some embodiments, the TCR chain comprises one or more constant domains, for example, the extracellular domain of the TCR chain comprises two immunoglobulin-like domains, such as a variable domain and a constant domain, adjacent to the cell membrane. In some embodiments, the extracellular domain of the TCR formed by the two chains comprises two membrane proximal constant domains and two membrane distal variable domains. The constant domain of the TCR can contain a short linker sequence in which cysteine ​​residues form a disulfide bond, thereby connecting the TCR In some embodiments, the TCR can have additional cysteine ​​residues in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domain.

[0115] IV-c. Chimeric Antibody-T Cell Constructs (caTCRs)

[0116] In some embodiments, the recombinant protein is a chimeric antibody-T cell construct (caTCR). In some embodiments, the caTCR includes a naturally occurring TCR domain or at least one non-naturally occurring TCR domain. In some embodiments, the caTCR includes an antigen binding module that provides antigen specificity and a T cell receptor module (TCRM) that allows CD3 recruitment and signaling. In some embodiments, the antigen binding module is connected to the amino terminus of TCRM. In some embodiments, the antigen binding module is an antigen binding molecule, including a full-length antibody, such as a single-domain antibody, a monospecific antibody, or a multispecific antibody; or an antibody fragment, such as Fv, Fab, Fab', Fab'-SH, F(ab')2, scFv. In some embodiments, TCRM includes a transmembrane module (TCR-TM) derived from a transmembrane domain of one or more TCRs, such as αβ and / or γδTCR, and optionally further includes a connecting peptide of TCR or its fragment and / or one or more TCR intracellular domains or its fragment or both.

[0117] In some embodiments, TCR comprises two polypeptide chains, each polypeptide chain including a connecting peptide, a transmembrane domain and an optionally present TCR intracellular domain from the amino terminus to the carboxyl terminus. In some embodiments, TCR comprises one or more non-naturally occurring TCR domains. In some embodiments, the caTCR may include a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains form an antigen binding moiety and TCR together. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and caTCR is a polymer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked such as by a peptide bond or by another chemical bond (such as a disulfide bond). In some embodiments, the first polypeptide chain and the second polypeptide chain are connected by at least one disulfide bond.

[0118] In some embodiments, caTCR further includes one or more T cell costimulatory signaling sequences. The one or more costimulatory signaling sequences may be individually all or part of the intracellular domain of a costimulatory molecule, including, for example, CD27, CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand specifically binding CD83, and the like. In some embodiments, one or more costimulatory signaling sequences are between the first TCR-TM and the first TCR intracellular domain and / or between the second TCR-TM and the second TCR intracellular domain. In some embodiments, one or more costimulatory signaling sequences are at the carboxyl end of the first TCR intracellular domain and / or the second TCR intracellular domain. In some embodiments, the caTCR lacks a T cell costimulatory signaling sequence.

[0119] In some embodiments, the caTCR further comprises a stabilizing module comprising a first stabilizing domain and a second stabilizing domain, wherein the first and second stabilizing domains have binding affinity to each other that stabilizes the caTCR. In some embodiments, the stabilizing module is located between the antigen binding module and the TCR. In some embodiments, the caTCR further comprises a spacer module between any two caTCR modules or domains. In some embodiments, the spacer module comprises one or more peptide linkers connecting two caTCR modules or domains.

[0120] IV-d. Chimeric Signaling Receptors (CSRs)

[0121] In some embodiments, the recombinant protein is a chimeric costimulatory receptor (CSR). In some embodiments, the CSR includes a ligand binding module, a transmembrane module and a costimulatory immune cell signaling module that provide ligand binding specificity. In some embodiments, the CSR lacks a primary activation signaling sequence. In some embodiments, CSR includes a single polypeptide chain containing a ligand binding module, a transmembrane module and a costimulatory signaling module. In some embodiments, CSR includes a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains form a ligand binding module, a transmembrane module and a costimulatory signaling module together. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and CSR is a polymer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by a peptide bond or by another chemical bond (such as a disulfide bond). In some embodiments, the first polypeptide chain and the second polypeptide chain are connected by at least one disulfide bond.

[0122] In some embodiments, the target ligand is a cell surface antigen. In some embodiments, the target ligand is a peptide / MHC complex. In some embodiments, the target ligand is the same as the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is different from the target antigen of the caTCR expressed in the same immune cell. In some embodiments, the target ligand is a molecule presented on the surface of a cell presenting the target antigen. In some embodiments, the target ligand is a disease-associated ligand. In some embodiments, the target ligand is a cancer-associated ligand. In some embodiments, the cancer-associated ligand is CD19, CD20, CD22, CD47, IL4, GPC-3, ROR1, ROR2, BCMA, GPRC5D, or FCRL5. In some embodiments, the cancer-associated ligand is a peptide / MHC complex comprising peptides derived from proteins comprising WT-1, AFP, HPV16-E7, NY-ESO-1, PRAME, EBV-LMP2A, and PSA. In some embodiments, the target ligand is a virus-associated ligand. In some embodiments, the target ligand is an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule includes PD-L1, PD-L2, CD80, CD86, ICOSL, B7-H3, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GAL9. In some embodiments, the target ligand is an apoptosis molecule. In some embodiments, the apoptosis molecule includes FasL, FasR, TNFR1, and TNFR2.

[0123] In some embodiments, the transmembrane module includes one or more transmembrane domains derived from, for example, CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0124] In some embodiments, the costimulatory immune cell signaling module is part of the intracellular domain of a costimulatory molecule, including CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

[0125] In a particular embodiment, the recombinant protein disclosed herein includes both the aforementioned CAR, TCR, or caTCR and CSR. Those skilled in the art will appreciate that the culture medium disclosed herein has the effect of increasing cell proliferation and is suitable for cells that have not been transduced or have been transduced with any of the recombinant proteins.

[0126] V. Examples

[0127] Example 1: Effect of the composition on T cell culture

[0128] After centrifugation and washing, the human leukocyte apheresis product was incubated with anti-CD4 magnetic beads and CD8 magnetic beads (Miltenyi biotec, item number 276-01 and item number 275-01, the same below). The incubated product was sorted using a magnetic sorting system (Miltenyi biotec, model CliniMACS Plus instrument, the same below), and the sorted T cells were then frozen. T cells from two different donors (donor 1 and donor 2) were revived, and the T cells were grouped after staining and counting, and the T cells were centrifuged and washed twice with culture medium-1. The T cell pellet was resuspended in culture medium-1, and the cell viability and CD25 + CD69 + T cell ratio and T SCM Take T cells and add them into medium-2 to make the cell density no less than 2.5×10 6 cells / mL, and activated T cells using magnetic beads coupled with anti-CD3 and anti-CD28 antibodies. The cells were then incubated in a 37.0±1.0°C, 5.0±1.0% CO2 incubator for 24±6 hours.

[0129] Detect the density, viability, and CD25 of activated T cells + CD69 + T cell ratio and TSCM Ratio. T cells were transduced and each group was supplemented with medium-3 to the same volume. The cells were transferred to a 37.0±1.0℃, 5.0±1.0% CO2 incubator for incubation. T cells were detected and supplemented with medium-3 at the corresponding time points. Starting from Day 5, cells were passaged, and the cell density of each passage was approximately 0.625×10 6 cells / mL.

[0130] The above-mentioned Medium-1, Medium-2, and Medium-3 were prepared according to Table 1. The medium matrix of Medium-2 and Medium-3 was the Medium-1 of the corresponding group, and the concentration of cytokines, GlutaMAX, or NAC was calculated based on the volume of Medium-1 before the addition of cytokines, GlutaMAX, and NAC.

[0131] Table 1. Culture medium formula

[0132] Note: T+X (1:1) refers to the mixture of TexMACS and X-VIVO 15, with a volume ratio of 1:1, the same below.

[0133] The cumulative expansion times, viability, activation status, and differentiation phenotype parameters during T cell culture are shown in the table below.

[0134] Table 2. Cumulative amplification fold

[0135] Note: NA stands for cell death

[0136] Although the T+X medium of this example does not contain stimuli and nutrients such as IL-2, GlutaMAX, and NAC, the cumulative expansion times of the two donor cells cultured in T+X medium were significantly higher than those of the cells in other groups, resulting in a surprising synergistic effect.

[0137] Table 3. Cell viability (%)

[0138] Note: NA stands for cell death

[0139] Although the T+X medium in this example does not contain stimulants and nutrients such as IL-2, GlutaMAX, and NAC, both donor cells maintained high viability when cultured in T+X medium. Due to the source of the donor cells, a medium containing only X-VIVO 15 was not suitable for culturing Donor 1 cells; and a medium containing only TexMACS struggled to maintain high viability for Donor 2 cells in the later stages of culture.

[0140] Table 4. CD25 + CD69 +T cell ratio (%)

[0141] Note: NA stands for cell death

[0142] By analyzing the expression levels of activation marker molecules of T cells from two donors, it was found that CD25 + CD69 + The proportion of double-positive T cells was higher than that in the culture medium alone, indicating that T cells had a better activation state in the T+X culture medium, which was beneficial to the expansion of T cells.

[0143] Table 5. Early memory phenotype T cells SCM Proportion(%)

[0144] Note: NA stands for cell death

[0145] Further analysis of the differentiation phenotype revealed that the early memory phenotype of T cells cultured in T+X medium was T SCM The higher ratio maintained a less differentiated state, comparable to that of medium containing only TexMACS.

[0146] Example 2: Effect of the ratio of serum-free culture medium on T cell culture

[0147] After centrifugation and washing, the human leukocyte apheresis product was incubated with anti-CD4 magnetic beads and CD8 magnetic beads. The incubated product was sorted using a magnetic sorting system, and the sorted T cells were subsequently frozen. T cells from two different donors (donor 3 and donor 4) were revived, grouped after staining and counting, and washed twice by centrifugation with culture medium-1. The T cell pellet was resuspended in culture medium-1, and the cell density, viability, and PD-1 expression ratio were detected (recorded as Day 0). T cells were added to culture medium-2 to a cell density of not less than 2.5×10 6 cells / mL, and activated T cells using magnetic beads coupled with anti-CD3 and anti-CD28 antibodies. The cells were then incubated in a 37.0±1.0°C, 5.0±1.0% CO2 incubator for 24±6 hours.

[0148] The density, viability, and PD-1 expression ratio of activated T cells were measured. T cells were transduced and each group was supplemented with medium-3 to the same volume. The cells were transferred to a 37.0±1.0°C, 5.0±1.0% CO2 incubator for incubation. T cells were tested and supplemented with medium-3 at the corresponding time points. Starting from day 5, cells were passaged, with a cell density of approximately 0.625×10 6 cells / mL.

[0149] The above-mentioned medium-1, medium-2, and medium-3 were prepared according to Table 6. The concentrations of cytokines, GlutaMAX, or NAC are based on the volume of the medium before the addition of cytokines, GlutaMAX, or NAC.

[0150] Table 6. Culture medium formula

[0151] The cumulative expansion times, viability, and PD-1 expression ratio parameters during T cell culture are shown in the table below.

[0152] Table 7. Cumulative amplification fold

[0153] By analyzing the cumulative amplification multiples, all culture media can efficiently culture cells. When the volume ratio of T+X is 4:1-1:1, the amplification effect is significantly improved; and when the volume ratio of T+X is 2:1, the amplification effect is the best.

[0154] Table 8. Cell viability (%)

[0155] By analyzing cell viability, all culture media can effectively culture cells and maintain high cell viability during the culture period, and the best effect is achieved when the volume ratio of T+X is 2:1.

[0156] Table 9. PD-1 expression ratio (%)

[0157] A low PD-1 expression ratio indicates that cells cultured under these conditions have a lower level of exhaustion. All culture media in this example can achieve low PD-1 expression in cells, maintaining a very low level of exhaustion.

[0158] Example 3: Effect of cytokine concentration on T cell culture

[0159] After centrifugation and washing, the human leukocyte apheresis product was incubated with anti-CD4 magnetic beads and CD8 magnetic beads. The incubated product was sorted using a magnetic sorting system, and the sorted T cells were subsequently frozen. T cells from two different donors (donor 5 and donor 6) were revived, grouped after staining and counting, and washed twice by centrifugation with culture medium-1. The T cell pellet was resuspended in culture medium-1, and the cell density, viability, and T SCM The expression ratio of T cells and PD-1 was recorded as Day 0. T cells were added to medium-2 to make the cell density no less than 2.5×10 6cells / mL, and activated T cells using magnetic beads coupled with anti-CD3 and anti-CD28 antibodies. The cells were then incubated in a 37.0±1.0°C, 5.0±1.0% CO2 incubator for 24±6 hours.

[0160] Detect the density, viability, and T cell activation SCM The ratio of PD-1 expression was quantified. T cells were transduced and each group was supplemented with medium-3 to the same volume. The cells were transferred to a 37.0±1.0℃, 5.0±1.0% CO2 incubator for incubation. T cells were tested and supplemented with medium-3 at the corresponding time points. Starting from day 5, cells were passaged, and the cell density for each passage was approximately 0.625×10 6 cells / mL.

[0161] The above-mentioned Medium-1, Medium-2, and Medium-3 were prepared according to Table 10. The medium base of Medium-2 and Medium-3 was the Medium-1 of the corresponding group, and the concentration of cytokines, GlutaMAX, or NAC was calculated based on the volume of Medium-1 before the addition of cytokines, GlutaMAX, and NAC.

[0162] Table 10. Culture medium formula

[0163] The cumulative expansion times, viability, and T SCM The parameters of the PD-1 expression ratio are shown in the table below.

[0164] Table 11. Summary of T cell culture test indicators

[0165] The analysis of the data is shown in Figure 1A to Figure 1D. Increasing the concentration of IL-2 is beneficial to achieving efficient cell expansion, maintaining high cell viability, and can improve T to a certain extent. SCM When the concentration of IL-2 reaches 60 IU / m or more, a good cell culture effect can be achieved. The concentrations of IL-7 and IL-15 have no significant effect on the cumulative expansion times and cell viability. When the concentration of IL-7 increases, it can improve T cell proliferation to a certain extent. SCM ratio and reduced the PD-1 expression ratio; while IL-15 had no significant effect on these two indicators.

[0166] Example 4: Effects of Mixed Compositions of Various Culture Media and TexMACS Culture Media on T Cell Culture

[0167] After centrifugation and washing, the human leukocyte apheresis product was incubated with anti-CD4 magnetic beads and CD8 magnetic beads. The incubated product was sorted using a magnetic sorting system, and the sorted T cells were subsequently frozen. T cells from two different donors (donor 7 and donor 8) were revived, grouped after staining and counting, and washed twice by centrifugation with DPBS (Corning, 21-031-CV). The T cell pellet was resuspended in DPBS and the cell viability was detected. T cells were cultured in G-Rex 24 culture plates and medium-2 was added to keep the cell density at not less than 2.5×10 6 cells / mL, and activated T cells using magnetic beads coupled with anti-CD3 and anti-CD28 antibodies. The cells were then incubated in a 37.0±1.0°C, 5.0±1.0% CO2 incubator for 24±6 hours.

[0168] After T cell activation, each group was supplemented with medium-3 to the same volume. The cells were transferred to a 37.0 ± 1.0°C, 5.0 ± 1.0% CO2 incubator for incubation. T cells were detected and supplemented with medium-3 at the corresponding time points. Starting from Day 5, cells were passaged, with the cell density of each passage being approximately 0.625 × 10 6 cells / mL.

[0169] Medium-1 was prepared according to Table 12. Medium-2 consisted of Medium-1 supplemented with 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, 2 mM GlutaMAX, and 0.8 mg / mL NAC. Medium-3 consisted of Medium-1 supplemented with 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, and 2 mM GlutaMAX. The concentrations of cytokines, GlutaMAX, or NAC were calculated based on the volume of Medium-1 before the addition of cytokines, GlutaMAX, and NAC. The cumulative expansion fold, viability, proportion of early memory phenotype cells (TSCMs), and PD-1 expression on day 13 of T cell culture are shown in Table 12.

[0170] Table 12. Medium formulation and expansion efficiency

[0171] Note: NA means the cells died during culture and could not be cultured until day 13.

[0172] The expansion multiple is a key indicator of culture medium efficiency. The combination culture medium obtained by compounding TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20 is significantly better than the combination culture medium or single culture medium obtained by compounding TexMACS with OpTmizer, AIM-V, RPMI 1640, DMEM or MEM in this indicator. Surprisingly, except for the combination of TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20, TexMACS has no synergistic effect with other culture media. At the same time, in the combination culture medium obtained by compounding TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20, the cell viability is also maintained at a high level, and the early memory phenotype cells T SCM The expression ratio of the exhaustion marker molecule PD-1 was similar to that of other groups.

[0173] At the same time, this study evaluated the effects of other components on the composition of the culture medium. The T cell collection and culture process of donor 8 was the same as above. Culture medium-1 was prepared according to Table 13, where T+X (2:1) indicates that the mixed volume ratio of TexMACS and X-VIVO 15 is 2:1. Culture medium-2 is culture medium-1 with the addition of 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, 2 mM GlutaMAX, and 0.8 mg / mL NAC; culture medium-3 is culture medium-1 with the addition of 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, and 2 mM GlutaMAX. The concentrations of the above cytokines, GlutaMAX, or NAC are based on the volume of culture medium-1 before the addition of cytokines, GlutaMAX, and NAC. The cumulative expansion times, viability, proportion of early memory phenotype cells TSCM, and PD-1 expression ratio of T cells on day 13 are shown in Table 13.

[0174] Table 13. Culture medium formula and amplification efficiency

[0175] Note: NA means the cells died during culture and could not be cultured until day 13.

[0176] The T+X (2:1) combination medium can be diluted moderately, and the diluted medium is still significantly better than the single medium. The combination medium of TexMACS, X-VIVO 15 and AIM-V medium can achieve better expansion effect. When the T+X (2:1) combination medium is compounded with AIM-V medium at a volume ratio of 4:1, the expansion multiple and cell viability of T cells are higher than those of T+X (2:1) medium, and the early memory phenotype cells TSCM The ratio of the expression of the exhaustion marker molecule PD-1 was also comparable to that of the T+X (2:1) culture medium.

[0177] Example 5: Effect of the ratio of three-phase culture medium on T cell culture

[0178] After centrifugation and washing, the human leukocyte apheresis product was incubated with anti-CD4 magnetic beads and CD8 magnetic beads. The incubated product was sorted using a magnetic sorting system, and the sorted T cells were subsequently frozen. T cells from two different donors (donor 9 and donor 10) were revived, grouped after staining and counting, and washed twice by centrifugation with DPBS. The T cell pellet was resuspended in DPBS and the cell viability was detected. T cells were cultured in G-Rex 24 culture plates and medium-2 was added to keep the cell density at not less than 2.5×10 6 cells / mL, and activated T cells using magnetic beads coupled with anti-CD3 and anti-CD28 antibodies. The cells were then incubated in a 37.0±1.0°C, 5.0±1.0% CO2 incubator for 24±6 hours.

[0179] After activation, each group of T cells was supplemented with Medium-3 to the same volume. The cells were transferred to a 37.0±1.0°C, 5.0±1.0% CO2 incubator for incubation. T cells were tested and supplemented with Medium-3 at the corresponding time points. Starting from Day 5, cells were passaged, with the cell density of each passage being approximately 0.625×10 6 cells / mL.

[0180] Medium-1 was prepared according to Table 14. Medium-2 consisted of Medium-1 supplemented with 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, 2 mM GlutaMAX, and 0.8 mg / mL NAC. Medium-3 consisted of Medium-1 supplemented with 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, and 2 mM GlutaMAX. The concentrations of the above cytokines, GlutaMAX, or NAC were based on the volume of Medium-1 before the addition of cytokines, GlutaMAX, and NAC. The cumulative expansion fold, viability, proportion of early memory phenotype cells (TSCM), and PD-1 expression ratio of T cells on day 13 of culture are shown in Table 14.

[0181] Table 14. Culture medium formula and amplification efficiency

[0182] Analysis of cumulative expansion fold data is shown in Figure 2. Data fitting analysis revealed that the optimal ratio of TexMACS, X-VIVO15, and AIM-V culture media is 5:4:1. Comparison of experimental data from the same donors (Groups 9-1 and 9-3, and Groups 10-1 and 10-3) revealed that at this optimal ratio, T cell expansion folds were significantly higher than those achieved with the T+X (2:1) combination culture medium. Cell viability, the proportion of early memory cells, and the exhaustion marker PD-1 were comparable to those achieved with the T+X (2:1) combination culture medium.

Claims

1. A composition comprising a first serum-free culture medium and a second serum-free culture medium, wherein the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-10; the composition has a higher immune cell expansion multiple than the first serum-free culture medium or the second serum-free culture medium.

2. A composition comprising a culture medium matrix, wherein the culture medium matrix comprises a first serum-free culture medium and a second serum-free culture medium, wherein the sum of the volumes of the first serum-free culture medium and the second serum-free culture medium accounts for more than 65% of the volume of the culture medium matrix, and the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.1-10, wherein the first serum-free culture medium is TexMACS, and the second serum-free culture medium is composed of one or more culture media selected from the group consisting of X-VIVO 10, X-VIVO 15, and X-VIVO 20; Preferably, the sum of the volumes of the first serum-free culture medium and the second serum-free culture medium accounts for more than 80% of the volume of the culture medium matrix; More preferably, the culture medium matrix consists of a first serum-free culture medium and a second serum-free culture medium.

3. The composition of claim 1 or 2, wherein the volume ratio of the first serum-free medium to the second serum-free medium is 1:0.1-2; preferably, the volume ratio of the first serum-free medium to the second serum-free medium is 1:0.25-1; more preferably, the volume ratio of the first serum-free medium to the second serum-free medium is about 1:0.

5.

4. The composition of claim 2, wherein the culture medium comprises a third serum-free culture medium AIM-V, and the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is not less than 2:1; preferably, the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is not less than 4:1; more preferably, the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is not less than 10:

1.

5. The composition of claim 2, wherein the composition comprises a supplement added to the culture medium matrix, the supplement comprising one or more components selected from the group consisting of inorganic salts, sugars, vitamins, albumin, lipids, amino acids, cytokines and antioxidants; preferably, the supplement comprises one or more components selected from the group consisting of amino acids, cytokines and antioxidants; more preferably, the supplement comprises amino acids, cytokines and antioxidants.

6. The composition of claim 5, wherein the cytokine comprises one or more components selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferon and tumor necrosis factor; preferably, the cytokine comprises one or more components selected from the group consisting of IL-2, IL-7 and IL-15; more preferably, the cytokine comprises IL-2.

7. The composition of claim 6, wherein the concentration of the IL-2 supplemented in the composition is 10-1000 IU / mL; preferably, the concentration of the IL-2 supplemented in the composition is 60-200 IU / mL; more preferably, the concentration of the IL-2 supplemented in the composition is about 100 IU / mL.

8. The composition of claim 5, wherein the amino acid is L-alanyl-L-glutamine; preferably, the L-alanyl-L-glutamine is supplemented in the composition at a concentration of 1 mM to 5 mM; more preferably, the L-alanyl-L-glutamine is supplemented in the composition at a concentration of about 2 mM.

9. The composition of claim 5, wherein the antioxidant is N-acetyl-L-cysteine; preferably, the N-acetyl-L-cysteine ​​is supplemented in the composition at a concentration of 0.5 mg / mL to 2 mg / mL. More preferably, the N-acetyl-L-cysteine ​​is supplemented in the composition at a concentration of about 0.8 mg / mL.

10. The composition of claim 2, comprising a culture medium matrix, wherein the culture medium matrix comprises a first serum-free culture medium, a second serum-free culture medium, and optionally a third serum-free culture medium AIM-V, wherein the sum of the volumes of the first serum-free culture medium and the second serum-free culture medium accounts for more than 80% of the volume of the culture medium matrix, and the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.25-1; Preferably, the culture medium matrix is ​​composed of a first serum-free culture medium and a second serum-free culture medium, and the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.25-1; or the culture medium matrix is ​​composed of a first serum-free culture medium, a second serum-free culture medium, and a third serum-free culture medium, and the volume ratio of the first serum-free culture medium to the second serum-free culture medium is 1:0.25-1, and the ratio of the sum of the volumes of the first and second serum-free culture media to the volume of the third serum-free culture medium is not less than 10:1; More preferably, the culture medium matrix consists of a first serum-free culture medium and a second serum-free culture medium, and the volume ratio of the first serum-free culture medium to the second serum-free culture medium is about 1:0.5; or the culture medium matrix consists of a first serum-free culture medium, a second serum-free culture medium and a third serum-free culture medium, and the volume ratio of the first serum-free culture medium, the second serum-free culture medium and the third serum-free culture medium is about 5:4:

1.

11. The composition of claim 10, comprising a supplement added to the culture medium, the supplement comprising IL-2, IL-7, IL-15, L-alanyl-L-glutamine, and optionally N-acetyl-L-cysteine; Preferably, the concentration of the IL-2 supplemented in the composition is 50-200 IU / mL, the concentration of the IL-7 is 500-1000 IU / mL, the concentration of the IL-15 is 25-100 IU / mL, the concentration of the L-alanyl-L-glutamine is 1 mM-5 mM, and the concentration of the N-acetyl-L-cysteine ​​is 0.5 mg / mL-2 mg / mL; More preferably, the concentration of the IL-2 supplemented in the composition is about 100 IU / mL, the concentration of the IL-7 is about 700 IU / mL, the concentration of the IL-15 is about 46 IU / mL, the concentration of the L-alanyl-L-glutamine is about 2 mM, and the concentration of the N-acetyl-L-cysteine ​​is about 0.8 mg / mL.

12. A method for preparing a culture medium, comprising mixing the culture medium matrix according to any one of claims 5 to 9 with a supplement.

13. A method for culturing cells, comprising incubating cells in the composition according to any one of claims 1 to 11; preferably, the cells are bone marrow or lymphoid cells; more preferably, the cells are T cells.

14. A cell produced by the method of culturing cells according to claim 13.

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