Composition for cell culture

Abstract

Disclosed are a composition for cell culture, a method for preparing the composition, a method for culturing cells by using the composition, and cells obtained via the method. Culturing cells by using the cell culture composition has the effects of achieving large-scale cell expansion, and maintaining high cell viability, early memory phenotype and low exhaustion phenotype.

Description

A composition for cell culture

[0001] This application claims priority to Chinese patent application No. 202410389898X, filed April 2, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present disclosure relates to a composition for cell culture, a method for preparing the composition, and a method for efficiently culturing cells using the composition. Background Art

[0003] Various cell therapy approaches, such as CAR-T and TCR-T, 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 TexMACS and T-VIVO, wherein the volume ratio of TexMACS to T-VIVO is 1:0.1-10; the composition exhibits a higher immune cell expansion fold than TexMACS or T-VIVO. 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%, 400%, 450%, 500%, or more compared to TexMACS or T-VIVO. In some embodiments, the combined volume of TexMACS and T-VIVO accounts for more than 50% of the volume of the composition. In some embodiments, the combined volume of TexMACS and T-VIVO accounts for more than 65% of the volume of the composition. In some embodiments, the combined volume of TexMACS and T-VIVO accounts for more than 80% of the volume of the composition. In some embodiments, the composition is composed of TexMACS and T-VIVO. In some embodiments, the volume ratio of TexMACS to T-VIVO is 1:0.1-5. In some embodiments, the volume ratio of TexMACS to T-VIVO is 1:0.25-4. In some embodiments, the volume ratio of TexMACS to T-VIVO is 1:0.5-4. In some embodiments, the volume ratio of TexMACS to T-VIVO is about 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, or 1:4.

[0006] In one aspect, the present disclosure provides a composition comprising a culture medium matrix comprising TexMACS and T-VIVO, wherein the combined volume of the TexMACS and T-VIVO accounts for more than 50% of the volume of the culture medium matrix, and the volume ratio of the TexMACS to T-VIVO is 1:0.1-10. In some embodiments, the combined volume of the TexMACS and T-VIVO accounts for more than 65% of the volume of the culture medium matrix. In some embodiments, the combined volume of the TexMACS and T-VIVO accounts for more than 80% of the volume of the culture medium matrix. In some embodiments, the culture medium matrix is ​​composed of TexMACS and T-VIVO. In some embodiments, the volume ratio of the TexMACS to T-VIVO is 1:0.1-5. In some embodiments, the volume ratio of the TexMACS to T-VIVO is 1:0.25-4. In some embodiments, the volume ratio of the TexMACS to T-VIVO is 1:0.5-4. In some embodiments, the volume ratio of TexMACS to T-VIVO is about 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, or 1:4.

[0007] In some embodiments, the composition of any of the preceding items, wherein the composition comprises a supplement added to the culture medium, 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.

[0008] 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.

[0009] 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. In some embodiments, the concentration of the IL-2 supplemented in the composition is 60-200 IU / mL. In some embodiments, the concentration of the IL-2 supplemented in the composition is about 100 IU / mL. 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 supplemented IL-2 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.

[0010] 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.

[0011] 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.

[0012] In some embodiments, the composition of any of the preceding items comprises a culture medium matrix, wherein the culture medium matrix comprises TexMACS and T-VIVO, the combined volume of the TexMACS and T-VIVO accounts for more than 80% of the volume of the culture medium matrix, and the volume ratio of the TexMACS to T-VIVO is 1:0.25-1. In some embodiments, the culture medium matrix is ​​composed of TexMACS and T-VIVO, and the volume ratio of the TexMACS to T-VIVO is 1:0.25-1. In some embodiments, the culture medium matrix is ​​composed of TexMACS and T-VIVO, and the volume ratio of the TexMACS to T-VIVO is about 1:0.5.

[0013] 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.

[0014] In one aspect, the present disclosure provides a method of culturing cells, comprising incubating the cells in the composition as described in any one of the preceding items.

[0015] 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. DETAILED DESCRIPTION

[0016] 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.

[0017] I. Terminology

[0018] "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..."

[0019] 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.

[0020] 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% (w / w) serum, containing only trace amounts of serum, or containing no measurable serum.

[0021] 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.

[0022] 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).

[0023] The term "amino acid" refers to all naturally occurring alpha 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.

[0024] 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.

[0025] 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."

[0026] 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.

[0027] The term "multi-unit composition" refers to a composition comprising two or more independently existing compositions, such as culture media.

[0028] 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.

[0029] II. Culture Medium

[0030] 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.

[0031] II-a. Culture medium matrix

[0032] 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. T-VIVO TM 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. T-VIVO TM 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. X-VIVO TM 20 and T-VIVO TM In a specific embodiment, the first serum-free medium is TexMACS TM The second serum-free medium is T-VIVO TM 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. In some embodiments, the composition is composed of a culture medium matrix.

[0033] 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 culture medium matrix consists of the first serum-free medium and the second serum-free medium. It will be appreciated by those skilled in the art that the volume of different culture media and other solutions after mixing may differ slightly from the sum of their volumes before mixing. When a difference exists, the volume of the culture medium matrix herein refers to the sum of the volumes of the different culture media and other solutions before mixing.

[0034] In some embodiments, the volume ratio (v:v) of the first serum-free medium and the second serum-free medium as described above is 1:0.1-10, 1:0.2-5, 1:0.2-4, 1:0.25-4, 1:0.3-4, 1:0.4-4, 1:0.5-4. In some embodiments, the volume ratio of the first serum-free medium to the second serum-free medium is or is about 1:1 or 1:4. 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 1:0.25, 1:0.5, 1:1, 1:2 or 1:4. 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 1:0.25, 1:0.5, 1:1, 1:2 or 1:4. Herein, the volume ratio of the first serum-free medium to the second serum-free medium refers to the volume ratio before the two are mixed.

[0035] II-b. Supplements

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] 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.

[0044] 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.

[0045] II-c. Multi-unit compositions

[0046] 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.

[0047] 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.

[0048] In some embodiments, the second culture medium comprises a culture medium matrix as described in any of the preceding items and a supplement. In some embodiments, the second culture medium comprises a culture medium matrix as described in any of the preceding items and a cytokine. In some embodiments, the second culture medium comprises a culture medium matrix 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 matrix 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 cell activation or cell transduction steps.

[0049] 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 following cell activation, such as cell transduction or cell expansion.

[0050] II-d. Formulation of the composition

[0051] 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.

[0052] III. Cell Culture

[0053] 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.

[0054] 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 +In some embodiments, the cell comprises a genetically engineered cell or an enriched genetically engineered cell colony. In some embodiments, the cell comprises a cell to be genetically engineered or being genetically engineered. In some embodiments, the cell comprises a cell colony to be genetically engineered or being genetically engineered. In some embodiments, the cell comprises a genetically engineered T cell or an enriched genetically engineered T cell colony. In some embodiments, the cell comprises a chimeric antigen receptor (CAR) expressing T cell or an enriched CAR expressing T cell. In some embodiments, the cell has previously been cryopreserved. In some embodiments, the cell has been cultured in serum-free medium and cryopreserved after culture. In some embodiments, the cell specifically targets tumor cells.

[0055] 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 present 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 1, 2, 3, 4, 5, 6, 7, 8, or 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.

[0056] 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 preparation of cells, and freezing. 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.

[0057] 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 within a closed system.

[0058] III-a. Cell separation

[0059] In some embodiments, the cell culture method includes separating cells or its compositions from biological samples.In some embodiments, the biological sample is blood or a sample derived from blood, or or derived from apheresis or leukocyte apheresis product, or other tissue samples.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 the cell derived from them.In some embodiments, biological sample includes the sample from autologous and allogeneic sources.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] III-b. Cell Activation

[0067] 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.

[0068] 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 comprises IL-2, IL-7, and / or IL-15. In some embodiments, the stimulatory conditions comprise 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.

[0069] 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.

[0070] 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.

[0071] III-c. Genetic Engineering

[0072] 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.

[0073] 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.

[0074] 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 (IU / cell) of viral vector particle copies or its infectious units (IU) to the total number of cells to be transduced. In some embodiments, the viral particles are present as or at least 0.1, 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.

[0075] 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.

[0076] 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.

[0077] III-d. Cell Expansion

[0078] 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.

[0079] 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, 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.

[0080] 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 rate 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 rate is between 4 rpm and 12 rpm, for example, between 4 rpm and 6 rpm, inclusive. In some embodiments, the constant rocking speed is between 5 rpm and 15 rpm, for example, 6 rpm or 10 rpm. In some embodiments, the bioreactor or culture vessel is in a stationary state. 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.

[0081] 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.

[0082] IV. Recombinant Protein

[0083] In one aspect, the cells cultured as the compositions disclosed herein express recombinant proteins. In some embodiments, the cells include one or more nucleic acids encoding recombinant proteins introduced by genetic engineering, and thus express the recombinant protein. In some embodiments, the expression of the recombinant protein is achieved by first activating the cells, then transducing the activated cells, and amplifying in the culture medium to a quantity sufficient for clinical application. In some embodiments, the cells are not activated and are directly transduced, or activation and transduction are performed simultaneously. In some embodiments, the recombinant protein is a chimeric receptor, a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a combination thereof.

[0084] IV-a. Chimeric Antigen Receptor (CAR)

[0085] In some embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some embodiments, the antigen is selectively expressed or overexpressed on the cells (e.g., tumors or pathogenic cells) of the disease or illness compared to normal or non-targeted cells. In some embodiments, the disease and illness 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] IV-bT cell receptor (TCR)

[0091] In some embodiments, the recombinant protein is a TCR. In some embodiments, the TCR is naturally occurring or modified. In some embodiments, the TCR contains 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 to 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 containing two separate chains connected, for example, by one or more disulfide bonds. In some embodiments, the TCR also contains a constant domain, a transmembrane domain, and / or a short cytoplasmic tail. In some embodiments, The TCR chain contains one or more constant domains, for example, the extracellular domain of the TCR chain contains two immunoglobulin-like domains adjacent to the cell membrane, such as a variable domain and a constant domain. In some embodiments, the extracellular domain of the TCR formed by the two chains contains two membrane proximal constant domains and two membrane distal variable domains. The constant domain of the TCR can contain a short connecting sequence in which the cysteine ​​residues form a disulfide bond, thereby connecting the two chains of the TCR. In some embodiments, the TCR can have additional cysteine ​​residues in each of the α chain and the β chain so that the TCR contains two disulfide bonds in the constant domain.

[0092] In a particular embodiment, the recombinant protein disclosed herein includes the aforementioned CAR or TCR. Those skilled in the art will appreciate that the culture medium disclosed herein has the effect of increasing cell proliferation capacity and is applicable to cells that have not been transduced or have been transduced with any of the recombinant proteins.

[0093] V. Examples

[0094] Example 1: Effects of Mixed Compositions of Various Culture Media and TexMACS Culture Media on T Cell Culture

[0095] 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 then frozen. T cells from two different donors (donor 1 and donor 2) were revived, grouped after staining and counting, and washed twice by centrifugation with DPBS (Corning, 21-031-CV, the same below). The T cell pellet was resuspended in DPBS and the cell viability was detected. T cells were cultured in a G-Rex 24 culture plate, and medium-2 was added to make the cell density 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.

[0096] After T cell activation, each group was supplemented with medium-2 to the same volume. The cells were transferred to a 37.0±1.0°C, 5.0±1.0% CO2 incubator and incubated with medium-3 as the supplemental medium. Starting from day 5, cell passages were performed, with a cell density of approximately 0.625×10 6 cells / mL.

[0097] The above-mentioned medium-1 was prepared according to Table 1. Medium-3 was prepared by adding 100 IU / mL IL-2, 700 IU / mL IL-7, 46 IU / mL IL-15, and 2 mM GlutaMAX to medium-1; medium-2 was prepared by adding 0.8 mg / mL NAC to medium-3. The concentrations of cytokines, GlutaMAX, or NAC were calculated based on the volume of medium-1 before addition. The cumulative expansion fold, viability, and early memory phenotype T cell counts at day 13 of culture were analyzed. SCM The parameters of ratio and PD-1 expression ratio are shown in Table 1.

[0098] Table 1. Culture medium formulation and amplification efficiency

[0099] Note: NA means the cells died during the culture process and could not be cultured to day 13.

[0100] The amplification factor is a key indicator of culture medium efficiency. The combined culture medium obtained by compounding TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20 is significantly better than the combined culture medium or single culture medium obtained by compounding TexMACS with OpTmizer, AIM-V, RPMI 1640, DMEM or MEM in this indicator. Surprisingly, among the culture media tested in this example, 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 combined 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.

[0101] Example 2: TexMACS+T-VIVO medium composition

[0102] T cells from different donors (Donor 3, Donor 4, Donor 5, Donor 6) were cultured using the same method as in Example 1. Medium-1 was prepared according to Table 2. The supplements added to Medium-2 and Medium-3 were also the same as in Example 1. The cumulative expansion times, viability, and early memory phenotype T cells at day 13 of culture were significantly correlated with the T cell viability. SCM The parameters of the ratio and PD-1 expression ratio are shown in Table 2 .

[0103] Table 2. Culture medium formula

[0104] The results showed that compared with the single T-VIVO medium, the expansion multiple and viability of T cells in the TexMACS:X-VIVO combination medium were significantly higher, further verifying the synergistic effect of the combination medium. But surprisingly, when TexMACS was combined with T-VIVO, the expansion multiple and viability of T cells could be further improved. SCM The ratio was slightly higher than that in the TexMACS + X-VIVO 15 combination medium. The proportion of T cells expressing the exhaustion marker PD-1 was lower in the TexMACS + T-VIVO combination medium than in the TexMACS + X-VIVO 15 combination medium. These data indicate that T cells in the TexMACS + T-VIVO combination medium have higher expansion capacity and better differentiated and exhausted phenotypes.

[0105] Example 3: Optimization of TexMACS+T-VIVO medium composition based on cell expansion

[0106] T cells from donor 7 were cultured using the same method as in Example 1. Medium-1 was prepared according to Table 3. The supplements added to Medium-2 and Medium-3 were also the same as in Example 1. The cumulative expansion fold of T cells on day 13 of culture is shown in Table 3.

[0107] Table 3. Culture medium formula

[0108] The results showed that TexMACS + T-VIVO medium demonstrated superior expansion efficiency compared to both T-VIVO medium alone and the combined medium TexMACS + X-VIVO 15. This synergistic effect was achieved over a wide range of dilution ratios, from 4:1 to 1:4, and was maintained after dilution.

[0109] Exemplary embodiments

[0110] 1. A composition comprising TexMACS and T-VIVO, wherein the volume ratio of TexMACS to T-VIVO is 1:0.1-10; the composition has a higher immune cell amplification factor than TexMACS or T-VIVO.

[0111] 2. The composition according to embodiment 1, wherein the volume ratio of TexMACS to T-VIVO is 1:0.25-4.

[0112] 3. The composition according to embodiment 1 or 2, wherein the volume ratio of TexMACS to T-VIVO is 1:0.5-4.

[0113] 4. The composition of any one of embodiments 1 to 3, wherein the volume ratio of TexMACS to T-VIVO is 1:0.5, 1:1, 1:2 or 1:4.

[0114] 5. A composition comprising a culture medium matrix, wherein the culture medium matrix comprises TexMACS and T-VIVO, the sum of the volumes of the TexMACS and T-VIVO accounts for more than 50% of the volume of the culture medium matrix, and the volume ratio of the TexMACS to T-VIVO is 1:0.1-10.

[0115] 6. The composition according to embodiment 5, wherein the combined volume of TexMACS and T-VIVO accounts for more than 65% of the volume of the culture medium matrix.

[0116] 7. The composition according to embodiment 5 or 6, wherein the combined volume of TexMACS and T-VIVO accounts for more than 80% of the volume of the culture medium matrix.

[0117] 8. The composition of any one of embodiments 5 to 7, wherein the culture medium matrix consists of TexMACS and T-VIVO.

[0118] 9. The composition according to any one of embodiments 5 to 8, wherein the volume ratio of TexMACS to T-VIVO is 1:0.25-4.

[0119] 10. The composition according to any one of embodiments 5 to 9, wherein the volume ratio of TexMACS to T-VIVO is 1:0.5-4.

[0120] 11. The composition of any one of embodiments 5 to 10, wherein the volume ratio of TexMACS to T-VIVO is 1:0.5, 1:1, 1:2 or 1:4.

[0121] 12. The composition of any one of embodiments 5 to 11, 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.

[0122] 13. The composition of embodiment 12, wherein the supplement comprises one or more ingredients selected from the group consisting of amino acids, cytokines, and antioxidants.

[0123] 14. The composition of embodiment 12 or 13, wherein the supplement comprises a cytokine.

[0124] 15. The composition of any one of embodiments 12 to 14, wherein the supplement comprises amino acids and cytokines.

[0125] 16. The composition of any one of embodiments 12 to 15, wherein the supplement comprises amino acids, cytokines, and antioxidants.

[0126] 17. The composition of any one of embodiments 12 to 16, wherein the cytokines comprise 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.

[0127] 18. The composition of any one of embodiments 12 to 17, wherein the cytokine comprises one or more components selected from the group consisting of IL-2, IL-7, and IL-15.

[0128] 19. The composition of any one of embodiments 12 to 18, wherein the cytokine comprises IL-2.

[0129] 20. The composition of any one of embodiments 12 to 19, wherein the cytokines comprise IL-2, IL-7, and IL-15.

[0130] 21. The composition according to any one of embodiments 12 to 20, wherein the concentration of the IL-2 supplemented in the composition is 10-1000 IU / mL.

[0131] 22. The composition of any one of embodiments 12 to 21, wherein the concentration of the IL-2 supplemented in the composition is 60-200 IU / mL.

[0132] 23. The composition of any one of embodiments 12 to 22, wherein the IL-2 supplemented in the composition is at a concentration of about 100 IU / mL.

[0133] 24. The composition according to any one of embodiments 12 to 23, wherein the concentration of the IL-7 supplemented in the composition is 100-5000 IU / mL.

[0134] 25. The composition of any one of embodiments 12 to 24, wherein the concentration of the IL-7 supplemented in the composition is 500-800 IU / mL.

[0135] 26. The composition of any one of embodiments 12 to 25, wherein the IL-7 supplemented in the composition has a concentration of about 700 IU / mL.

[0136] 27. The composition of any one of embodiments 12 to 26, wherein the concentration of the IL-15 supplemented in the composition is 0-500 IU / mL.

[0137] 28. The composition of any one of embodiments 12 to 27, wherein the concentration of the IL-15 supplemented in the composition is 40-60 IU / mL.

[0138] 29. The composition of any one of embodiments 12 to 28, wherein the concentration of the supplemented IL-15 in the composition is about 46 IU / mL.

[0139] 30. The composition of any one of embodiments 12 to 29, wherein the amino acid is L-alanyl-L-glutamine.

[0140] 31. The composition of any one of embodiments 12 to 30, wherein the concentration of the supplemented L-alanyl-L-glutamine in the composition is 1 mM to 5 mM.

[0141] 32. The composition of any one of embodiments 12 to 31, wherein the concentration of the supplemented L-alanyl-L-glutamine in the composition is about 2 mM.

[0142] 33. The composition of any one of embodiments 12 to 32, wherein the antioxidant is N-acetyl-L-cysteine.

[0143] 34. The composition of any one of embodiments 12 to 33, wherein the concentration of the supplemented N-acetyl-L-cysteine ​​in the composition is 0.5 mg / mL to 2 mg / mL.

[0144] 35. The composition of any one of embodiments 12 to 34, wherein the concentration of the supplemented N-acetyl-L-cysteine ​​in the composition is about 0.8 mg / mL.

[0145] 36. The composition of any one of embodiments 5 to 35, wherein the culture medium matrix comprises TexMACS and T-VIVO, the sum of the volumes of TexMACS and T-VIVO accounts for more than 50% of the volume of the culture medium matrix, and the volume ratio of TexMACS to T-VIVO is 1:0.25-4.

[0146] 36. The composition of any one of embodiments 5 to 35, wherein the culture medium matrix comprises TexMACS and T-VIVO, the combined volume of TexMACS and T-VIVO accounts for more than 80% of the volume of the culture medium matrix, and the volume ratio of TexMACS to T-VIVO is 1:0.25-4.

[0147] 37. The composition of any one of embodiments 5 to 36, wherein the culture medium matrix is ​​composed of TexMACS and T-VIVO, and the volume ratio of TexMACS to T-VIVO is 1:0.5-4.

[0148] 38. The composition of any one of embodiments 5 to 37, 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.

[0149] 39. The composition of any one of embodiments 5 to 38, wherein the IL-2 supplemented in the composition has a concentration of 50-200 IU / mL, the IL-7 has a concentration of 500-1000 IU / mL, the IL-15 has a concentration of 25-100 IU / mL, the L-alanyl-L-glutamine has a concentration of 1 mM-5 mM, and the N-acetyl-L-cysteine ​​has a concentration of 0.5 mg / mL-2 mg / mL.

[0150] 40. The composition of any one of embodiments 5 to 39, wherein the IL-2 supplemented in the composition has a concentration of about 100 IU / mL, the IL-7 has a concentration of about 700 IU / mL, the IL-15 has a concentration of about 46 IU / mL, the L-alanyl-L-glutamine has a concentration of about 2 mM, and the N-acetyl-L-cysteine ​​has a concentration of about 0.8 mg / mL.

[0151] 41. A method of culturing cells, the method comprising incubating the cells in the composition of any one of embodiments 5-40.

[0152] 42. The method of embodiment 41, wherein the cell is a bone marrow or lymphoid cell.

[0153] 43. The method of embodiment 41 or 42, wherein the cell is a T cell.

[0154] 44. The method of any one of embodiments 41 to 43, comprising the steps of cell activation and cell expansion.

[0155] 45. The method according to any one of embodiments 41 to 44, wherein the composition used before cell activation comprises a culture medium matrix, the composition used during cell activation comprises 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, and the composition used during cell expansion comprises a culture medium matrix and IL-2, IL-7, IL-15, L-alanyl-L-glutamine and optionally N-acetyl-L-cysteine ​​added to the culture medium matrix.

Claims

1. A composition comprising a culture medium matrix, wherein the culture medium matrix comprises TexMACS and T-VIVO, wherein the volume ratio of TexMACS to T-VIVO is 1:0.1-10; the composition has a higher immune cell expansion factor than TexMACS or T-VIVO.

2. A composition comprising a culture medium matrix, wherein the culture medium matrix comprises TexMACS and T-VIVO, wherein the combined volume of the TexMACS and T-VIVO accounts for more than 50% of the volume of the culture medium matrix, and the volume ratio of the TexMACS to T-VIVO is 1:0.1-10; Preferably, the combined volume of the TexMACS and T-VIVO accounts for more than 80% of the volume of the culture medium matrix; More preferably, the culture medium matrix consists of TexMACS and T-VIVO.

3. The composition according to claim 1 or 2, wherein the volume ratio of TexMACS to T-VIVO is 1:0.25-4; Preferably, the volume ratio of TexMACS to T-VIVO is 1:0.5-4; More preferably, the volume ratio of TexMACS to T-VIVO is 1:0.5, 1:1, 1:2 or 1:

4.

4. The composition according to any one of claims 1 to 3, wherein the composition comprises a supplement added to the culture medium, the supplement comprising one or more components selected from the group consisting of amino acids, cytokines and antioxidants; 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.

5. The composition according to claim 4, 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.

6. The composition of claim 4, wherein the amino acid is L-alanyl-L-glutamine; Preferably, the concentration of the L-alanyl-L-glutamine supplemented in the composition is 1 mM-5 mM; More preferably, the concentration of the supplemented L-alanyl-L-glutamine in the composition is about 2 mM.

7. The composition of claim 4, wherein the antioxidant is N-acetyl-L-cysteine; Preferably, the concentration of the N-acetyl-L-cysteine ​​supplemented in the composition is 0.5 mg / mL-2 mg / mL; More preferably, the concentration of the supplemented N-acetyl-L-cysteine ​​in the composition is about 0.8 mg / mL.

8. The composition according to any one of claims 1 to 7, wherein the culture medium matrix comprises TexMACS and T-VIVO, the sum of the volumes of the TexMACS and T-VIVO accounts for more than 50% of the volume of the culture medium matrix, and the volume ratio of the TexMACS to T-VIVO is 1:0.25-4; Preferably, the culture medium matrix comprises TexMACS and T-VIVO, the sum of the volumes of TexMACS and T-VIVO accounts for more than 80% of the volume of the culture medium matrix, and the volume ratio of TexMACS to T-VIVO is 1:0.25-4; More preferably, the culture medium matrix is ​​composed of TexMACS and T-VIVO, and the volume ratio of TexMACS to T-VIVO is 1:0.5-4.

9. The composition of claim 8, 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.

10. A method of culturing cells, comprising incubating cells in the composition according to any one of claims 1 to 9; Preferably, the cells are myeloid or lymphoid cells; More preferably, the cell is a T cell.

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