CN117965445B - Composition for cell culture - Google Patents

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. Culturing cells using the cell culture compositions of the present disclosure includes the effect of being able to achieve a large number of cell expansion, and maintaining a high cell viability and early memory phenotype, as well as a low depletion phenotype.

Description

Composition for cell culture

Technical Field

The present disclosure relates to compositions for cell culture, methods of making the compositions, and methods of efficiently culturing cells using the compositions.

Background

A variety of cell therapy approaches, such as CAR-T, TCR-T have been used to treat diseases. Whereas the acquisition of cells that have not been or have been genetically engineered requires efficient cell culture processes, including media that enable large cell expansion and maintain high cell viability and early memory phenotypes, as well as low depletion phenotypes. Compositions and methods thereof that meet this need are provided herein.

Disclosure of Invention

Provided herein is a composition capable of efficiently expanding cells. In one aspect, the composition comprises TexMACS and T-VIVO in a volume ratio of 1:0.1-10; the composition has an immune cell expansion factor higher than that of 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, 13 days. In some embodiments, the cells are cultured under conditions of 37.0±1.0 ℃, 5.0±1.0% CO ₂. In some embodiments, the expansion of T cells 9, 11, or 13 days after culturing with the composition is increased by at least 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more over TexMACS or T-VIVO. In some embodiments, the sum of the volumes of the TexMACS and the T-VIVO is greater than 50% of the volume of the composition. In some embodiments, the sum of the volumes of the TexMACS and T-VIVO is greater than 65% of the volume of the composition. In some embodiments, the sum of the volumes of the TexMACS and T-VIVO is greater than 80% of the volume of the composition. In some embodiments, the composition consists of TexMACS and T-VIVO. In some embodiments the volumetric ratio of TexMACS to T-VIVO is 1:0.1-5. In some embodiments, the volumetric ratio of TexMACS to T-VIVO is 1:0.25-4. In some embodiments, the volumetric ratio of TexMACS to T-VIVO is 1:0.5-4. In some embodiments, the volumetric ratio of TexMACS to T-VIVO is about 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, or 1:4.

In one aspect, the present disclosure provides a composition comprising a culture medium matrix comprising TexMACS and T-VIVO, the sum of the volumes of the TexMACS and T-VIVO accounting for greater than 50% of the volume of the culture medium matrix, the volume ratio of the TexMACS and T-VIVO being from 1:0.1 to 10. In some embodiments, the sum of the volumes of the TexMACS and the T-VIVO is greater than 65% of the volume of the medium matrix. In some embodiments, the sum of the volumes of the TexMACS and the T-VIVO is greater than 80% of the volume of the medium matrix. In some embodiments, the medium matrix is composed of TexMACS and T-VIVO. In some embodiments the volumetric ratio of TexMACS to T-VIVO is 1:0.1-5. In some embodiments, the volumetric ratio of TexMACS to T-VIVO is 1:0.25-4. In some embodiments, the volumetric ratio of TexMACS to T-VIVO is 1:0.5-4. In some embodiments, the volumetric ratio of TexMACS to T-VIVO is about 1:0.1, 1:0.25, 1:0.5, 1:1, 1:2, or 1:4.

In some embodiments, the composition of any one of the preceding claims, wherein the composition comprises a supplement comprising one or more ingredients selected from the group consisting of inorganic salts, sugars, vitamins, albumin, lipids, amino acids, cytokines, and antioxidants added to the medium matrix. In some embodiments, the supplement comprises one or more ingredients 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.

In some embodiments, the composition of any one of the preceding claims, 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 cytokines are or include IL-2, IL-7 and IL-15.

In some embodiments, the composition of any one of the preceding claims, the concentration of the IL-2 added in the composition is 10-1000 IU/mL. In some embodiments, the concentration of the IL-2 added in the composition is 60-200 IU/mL. In some embodiments, the concentration of the additional IL-2 added to the composition is about 100 IU/mL. In some embodiments, the composition of any one of the preceding claims, the concentration of the additional added IL-2 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 additional IL-2 added to 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 additional IL-2 added to 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.

In some embodiments, the composition of any one of the preceding claims, wherein the amino acid is L-alanyl-L-glutamine. In some embodiments, the concentration of the L-alanyl-L-glutamine added in the composition is 1 mM-5 mM. In some embodiments, the concentration of the L-alanyl-L-glutamine added in the composition is about 2 mM.

In some embodiments, the composition of any one of the preceding claims, wherein the antioxidant is N-acetyl-L-cysteine. In some embodiments, the concentration of the N-acetyl-L-cysteine added in the composition is 0.5 mg/mL to 2 mg/mL. In some embodiments, the concentration of the N-acetyl-L-cysteine added in the composition is from 0.5 mg/mL to 1 mg/mL. In some embodiments, the concentration of the N-acetyl-L-cysteine added in the composition is about 0.8 mg/mL.

In some embodiments, the composition of any one of the preceding claims, comprising a culture medium matrix comprising TexMACS and T-VIVO, the sum of the volumes of the TexMACS and T-VIVO accounting for 80% or more of the volume of the culture medium matrix, the volume ratio of the TexMACS and T-VIVO being 1:0.25-1. In some embodiments, the medium matrix is composed of TexMACS and T-VIVO in a volume ratio of 1:0.25-1. In some embodiments, the medium matrix is composed of TexMACS and T-VIVO in a volume ratio of about 1:0.5.

In one aspect, the present disclosure provides a method of preparing a culture medium comprising mixing a culture medium matrix as described in any one of the preceding claims with a supplement.

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

In one aspect, the present disclosure provides a cell produced by the method of culturing a cell as described in any one of the preceding claims. In some embodiments, the cell is a mammalian cell or a human cell. In some embodiments, the cell is an immune cell, such as an innate or adaptive immune cell, e.g., a bone marrow or lymphoid cell (including lymphocytes, e.g., T cells and/or NK cells). In some embodiments, the cell is a primary cell. 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 whole T cell populations, CD4 ⁺ cells, CD8 ⁺ cells, and subpopulations thereof.

Detailed Description

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

I. terminology

"A" or "an" may mean one or more or one or more. "about", "substantially" and "approximately" refer to ranges where their corresponding data values are increased or decreased by up to 5%. It should be understood that "comprising," "including," and "containing" are intended to include any viable elements other than the elements so long as the addition of such elements does not render the solution containing such elements impractical. "consisting of … …" means having only the elements described by "… …". "comprising" encompasses the situation "consisting of … …".

The term "culture medium matrix" refers to a nutrient source used to grow cells or to maintain cells. The nutrient source contains essential components required for cell growth and/or survival, and can be used for cell culture alone.

The term "serum-free medium" refers to a medium that is free or substantially free of 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.

The term "supplement" refers to a substance that is added in addition to the medium matrix for maintaining, activating and/or expanding cells. The supplement herein may comprise one or more substances, which may or may not have been included in the medium matrix. The concentration of the supplement herein is a concentration other than the concentration of the corresponding substance in the medium matrix, for example, the total concentration of L-alanyl-L-glutamine in the composition is 5mM, the concentration of the original L-alanyl-L-glutamine in the medium matrix is 3mM, then the concentration of the supplemental added L-alanyl-L-glutamine is 2 mM; the total concentration of L-alanyl-L-glutamine in the composition is 2mM, and the concentration of the supplemented L-alanyl-L-glutamine is 2mM when the medium matrix does not contain L-alanyl-L-glutamine. The concentration units of the supplements herein are calculated as the original volume of the medium matrix.

The term "cytokine" refers to biomolecules that affect cells of the immune system, including natural cytokines and fragments and functional variants that have at least 10%, 30%, 50% or 80% of the activity (e.g., naturally occurring cytokine immunomodulatory activity) as compared to the natural cytokine.

The term "amino acid" refers to all naturally occurring alpha amino acids and synthetic oligopeptides, analogs and derivatives. Analogues refer to the substitution of atoms in amino acids with different atoms that generally have similar properties. A derivative refers to a molecule having an amino acid attached to another structure, for example, a product obtained by propionylating an amino acid. Amino acids herein include L-alanyl-L-glutamine.

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

The term "culturing" refers to the maintenance of cells in vitro under conditions conducive to growth and/or differentiation and/or sustained viability. "culturing" may be used interchangeably with "cell culturing".

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.

The term "multi-unit composition" refers to a composition comprising two or more independently present compositions, such as media.

The terms "immune cell" and "immune system cell" refer to a cell that is involved in an immune response intended to protect an organism from foreign substances, viruses and cells. Immune cells may be derived from a number of organs and tissues, such as thymus, spleen, lymph nodes, lymphoid tissue clusters (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 progenitor cells that develop into these cells.

II culture medium

The present disclosure provides a composition comprising at least two serum-free media. Surprisingly, the compositions of the present disclosure have significantly improved cell expansion, more sustainable cell viability, lower low PD-1 expression, higher CD25 ⁺CD69⁺ T cells, and/or higher Tscm ratios when used in the culture of immune cells (e.g., T cells) than in serum-free medium alone. For example, after culturing T cells with the composition for 7, 9, 11, or 13 days, the expansion ratio is increased by 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% or more as compared to a single serum-free medium.

II-a Medium matrix

In some embodiments, the composition as described above comprises a culture medium matrix. In some embodiments, the medium matrix comprises a first serum-free medium and a second serum-free medium that are different from each other. In some embodiments, the serum-free medium is well known to those skilled in the art and commercially available, such as RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, texMACS ^TM、X-VIVO^TM10、X-VIVO^TM, and X-VIVO ^TM20、T-VIVO^TM or OpTmizer ^TM. In some embodiments, the first serum-free medium is TexMACS ^TM and the second serum-free medium is one or more media selected from the group consisting of RPMI 1640、AIM-V、DMEM、MEM、a-MEM、F-12、X-VIVO^TM10、X-VIVO^TM15、X-VIVO^TM20、T-VIVO^TM and OpTmizer ^TM. In some embodiments, the first serum-free medium is TexMACS ^TM and the second serum-free medium is one or more media selected from the group consisting of X-VIVO ^TM10、X-VIVO^TM15、X-VIVO^TM 20 and T-VIVO ^TM. In a specific embodiment, the first serum-free medium is TexMACS ^TM and the second serum-free medium is T-VIVO ^TM. In some embodiments, the first serum-free medium and the second serum-free medium in the composition are to be or have been formulated as media. In some embodiments, the composition is composed of a culture medium matrix.

In some embodiments, the volume of the first serum-free medium and the second serum-free medium as described above comprises 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%, greater than 99% of the volume of the medium matrix. The volumes of the first serum-free medium and the second serum-free medium account for 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% of the volume of the medium matrix. In a specific embodiment, the medium matrix consists of a first serum-free medium and a second serum-free medium. Those skilled in the art will appreciate that there may be minor differences in the sum of the volumes of the different media and other solutions after mixing and the volumes before mixing. When there is a difference, the volume of the medium matrix herein is the sum of the volumes of the different media and other solutions before mixing.

In some embodiments, the volume ratio (v: v) of the first serum-free medium to the second serum-free medium as described previously 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 at or about 1:1 or 1:4. In some embodiments, the volume of the first serum-free medium and the second serum-free medium comprises 100% of the volume of the medium matrix, and the volume ratio of the first serum-free medium and 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 comprises 100% of the volume of the medium matrix, and the volume ratio of the first serum-free medium and the second serum-free medium is at or about 1:0.25, 1:0.5, 1:1, 1:2, or 1:4. Herein, the volume ratio of the first serum-free medium and the second serum-free medium means the volume ratio before mixing the two.

II-b. supplement

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

In some embodiments, the supplement as described previously comprises a cytokine. In some embodiments, the cytokine is a recombinant cytokine. In a particular 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 cytokines include 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 comprises interferon, tumor necrosis factor. In some embodiments, the cytokine is selected from one or more components of the group consisting of IL-2, IL-7, and IL-15. In some embodiments, the cytokine is or comprises IL-2. In particular embodiments, the cytokine is or comprises IL-2, IL-7 and IL-15. These cytokines are used as stimulators in the medium to activate the cells. When the cells have been activated, a medium without cytokine addition can be used.

In some embodiments, the cytokine as described above comprises IL-2. In some embodiments, the IL-2 (e.g., human recombinant IL-2) concentration is 10-1000 IU/mL, 50-500 IU/mL, 50-200 IU/mL or 50-150 IU/mL. In particular embodiments, the concentration of IL-2 is at or 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 IL-2 concentration is at or about 100 IU/mL.

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

In some embodiments, the cytokine as described above comprises IL-15. In some embodiments, the IL-15 (e.g., human recombinant IL-15) concentration 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 particular embodiments, the concentration of IL-15 is at or 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 IL-15 concentration is at or about 46 IU/mL.

In some embodiments, the supplement as described previously comprises amino acids. In some embodiments, the amino acid is 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 a synthetic amino acid. In some embodiments, the synthetic amino acid is capable of being converted to L-glutamine in free form in a cell culture comprising cells. In some embodiments, the synthetic amino acid is a dipeptide. In some embodiments, the synthetic amino acid is L-alanyl-L-glutamine (GlutaMax).

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

In some embodiments, the supplement as described previously comprises an antioxidant. In some embodiments, the antioxidant comprises N-acetyl-L-cysteine (NAC), 2-mercaptoethanol, or D, L-tocopheryl acetate, or derivatives or mixtures thereof. In some embodiments, the antioxidant is N-acetyl-L-cysteine.

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、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、5 mg/mL., in some embodiments, the concentration of N-acetyl-L-cysteine is at or about 0.8 mg/mL.

II-c. Multiunit compositions

In some embodiments, the composition as described above is a multi-unit composition. Each unit in the multi-unit composition (e.g., each independently present composition or medium) may be used alone for cell culture. In some embodiments, the multi-unit composition comprises a plurality of independent media. In some embodiments, the multi-unit composition comprises a first medium, a second medium, a third medium, or any combination thereof. In some embodiments, the multi-unit composition includes a first medium, a second medium, and a third medium.

In some embodiments, the first medium comprises the medium matrix of any one of the preceding claims, and optionally a supplement. In some embodiments, the first medium is the medium matrix of any one of the preceding claims. In some embodiments, the first medium comprises the medium matrix of any one of the preceding claims, and optionally a supplement other than a stimulating agent (e.g., a cytokine). In some embodiments, the first medium comprises the medium matrix of any one of the preceding claims, and optionally an amino acid and/or an antioxidant. The first medium may also be referred to as basal medium. In some embodiments, the first medium may be used for cell culture before or after cell activation, such as isolation, washing, or expansion of cells. In some embodiments, the first medium may be used for a step prior to cell activation.

In some embodiments, the second medium comprises the medium matrix and supplement of any one of the preceding claims. In some embodiments, the second medium comprises the medium matrix and cytokines of any one of the preceding claims. In some embodiments, the second medium comprises the medium matrix of any one of the preceding claims, a cytokine, and optionally an amino acid and/or an antioxidant. In some embodiments, the second medium comprises the medium matrix of any one of the preceding claims, a cytokine, an amino acid, and an antioxidant. This second medium, which may also be referred to as an activation medium, may be used for the step of cell activation or cell transduction.

In some embodiments, the third medium comprises the medium matrix of any one of the preceding claims, and optionally a supplement. In some embodiments, the third medium comprises the medium matrix of any one of the preceding claims, a cytokine, and optionally a cytokine and/or an amino acid. In some embodiments, the third medium comprises the medium matrix, cytokines, and amino acids of any one of the preceding claims. This third medium may also be referred to as an amplification medium. In some embodiments, the third medium may be used in a step following cell activation, such as a step of cell transduction or cell expansion.

II-d. formulation of compositions

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

III cell culture

In one aspect, the present disclosure provides a method of culturing cells employing the composition of any one of the preceding or a combination thereof. In some embodiments, the cell is an immune cell, such as a T cell. In some embodiments, the cell is a genetically engineered cell, such as a genetically engineered T cell. In some embodiments, the cells are cultured for 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, the cells are cultured for 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 inclusive. In some embodiments, the cells are cultured under conditions of 37.0±1.0 ℃. In some embodiments, the cells are cultured under conditions of 5.0±1.0% CO ₂. In some embodiments, the culturing may be performed for greater than or greater than 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 medium when culturing the cells. In some embodiments, perfusion (e.g., semi-continuous perfusion) methods may be used in conjunction with culturing cells.

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 cell or an enriched CD4 ⁺ T cell. In some embodiments, the cell is a CD8 ⁺ T cell or an enriched CD8 ⁺ T cell. In some embodiments, the cells are CD4 ⁺ T cells and CD8 ⁺ T cells. In some embodiments, the cells are enriched CD4 ⁺ T cells and enriched CD8 ⁺ T cells. In some embodiments, the cells comprise genetically engineered cells or an enriched population of genetically engineered cells. In some embodiments, the cells comprise cells to be genetically engineered or being genetically engineered. In some embodiments, the cells comprise an enriched population of cells to be genetically engineered or being genetically engineered. In some embodiments, the cells comprise genetically engineered T cells or an enriched population of genetically engineered T cells. 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 cells have been previously cryopreserved. In some embodiments, the cells have been cultured in serum-free medium and cryopreserved after culturing. In some embodiments, the cells specifically target tumor cells.

In some embodiments, cells are expanded at least or at least 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 culturing for at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more using the cell culture methods of the present disclosure. In some embodiments, the cells expand at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more after culturing in a serum-free medium formulation for about 1, 2, 3, 4, 5,6, 7, 8, or 9 days. In some embodiments, cell culture with the compositions of the present disclosure has a higher expansion of immune cells than the first serum-free medium and the second serum-free medium alone.

In some embodiments, the cell culture method involves one or more steps or processes. In some embodiments, the one or more steps include isolation, selection, activation, transduction, incubation, expansion, washing, suspension, dilution, concentration, and/or formulation of the cells, cryopreservation. In some embodiments, the methods comprise isolating cells, preparing, processing, culturing the cells under one or more stimulating conditions. In some embodiments, the method comprises the processing steps performed in the following order: first separating (e.g., selecting) cells from a biological sample; following the step of incubating the selected cells with viral vector particles for transduction, e.g., stimulating the isolated cells in the presence of a stimulating agent; culturing the transduced cells, e.g., expanding cells.

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 leukocyte apheresis product); (b) Isolating (e.g., selecting) desired cells (e.g., CD4 ⁺ and/or CD8 ⁺ T cells) from the sample, e.g., by incubating the cells with an immunoaffinity reagent; (c) Introducing a vector encoding a recombinant receptor into the isolated or selected cell, such as by incubating the isolated (e.g., selected) cell with a viral vector particle encoding a recombinant receptor; and (d) culturing or expanding the cells. In some embodiments, the method may further comprise (f) activating the cells by exposing the cells to a stimulating condition, 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 prior to incubating the cells with the viral vector particles. In some embodiments, a washing and/or suspending step 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 of the steps of the cell culture method are performed under sterile conditions. In some embodiments of this method, cell isolation, transduction, washing, optional activation or stimulation, and formulation are all performed within a closed system.

III-a cell separation

In some embodiments, the cell culture method comprises isolating cells or a composition thereof from a biological sample. In some embodiments, the biological sample is blood or a blood-derived sample, or is derived from apheresis or a leukocyte apheresis product, or other tissue sample. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemias, lymphomas, lymph nodes, intestine-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and/or cells derived therefrom. In some embodiments, the biological sample includes samples from autologous and allogeneic sources.

In some embodiments, the cell is a mammalian cell or a human cell. In some embodiments, the cell is an immune cell, such as an innate or adaptive immune cell, e.g., a bone marrow or lymphoid cell (including lymphocytes, e.g., T cells and/or NK cells). In some embodiments, the cell is a primary cell. 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 whole T cell populations, CD4 ⁺ cells, CD8 ⁺ cells, and subpopulations thereof. Subtypes and subsets of T cells and/or CD4 ⁺ and/or CD8 ⁺ T cells include naive T (T _N) cells, effector T cells (T _EFF), memory T cells and subtypes thereof (e.g., stem cell memory T (T _SCM), central memory T (T _CM), effector 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, mucosa-associated constant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), alpha/beta T cells, and delta/gamma T cells. In some embodiments, the cell is a Natural Killer (NK) cell. In some embodiments, the cells are monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. In some embodiments, the cell is a tumor-infiltrating lymphocyte.

In some embodiments, the cell separation step is non-affinity based. In some examples, the cells are washed and/or centrifuged in the presence of one or more reagents to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to a particular reagent. In some examples, the cells are isolated based on one or more characteristics (e.g., density, adhesion characteristics, size, sensitivity to a particular component, and/or resistance). In some embodiments, the method includes a density-based cell separation method, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes and by gradient centrifugation.

In some embodiments, the cell separation step comprises incubating and separating the cells with a selection agent. In some embodiments, the selection agent is an immune affinity based cell binding agent. In some embodiments, the selection agent is an antibody or conjugate thereof that specifically binds to a cell marker. In some embodiments, the selection reagent is a particle (e.g., a magnetic bead) conjugated with a specific selection agent (e.g., an antibody). In some embodiments, the magnetic beads comprise magnetically responsive material bound to an antibody. In some embodiments, the magnetically responsive material is coated with a primary antibody (an antibody that binds to a cell), a secondary antibody, lectin, an enzyme, or streptavidin, and is attached to the cell by the primary antibody. The cell separation step separates the cells by the expression or level of expression of one or more markers (typically cell surface markers) in the cells, e.g. incubating the cells with an antibody or a conjugate thereof that specifically binds to the marker, followed by washing to separate cells that have bound the antibody (positive selection) or cells that have not bound the antibody (negative selection). In some embodiments, the cell separation step enriches the specific cell population via positive selection or depletes the 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 on the positively or negatively selected cells or at relatively high levels, respectively. The isolation need not achieve 100% enrichment or removal of a particular cell population or cells expressing a particular marker. In some embodiments, the step comprises multiple rounds, e.g., subjecting the positively or negatively selected cells to another positive or negative selection. Specific subpopulations of T cells, such as cells that are positive or highly expressed for one or more surface markers (e.g., CD28⁺、CD62L⁺、CCR7⁺、CD27⁺、CD127⁺、CD4⁺、CD8⁺、CD45RA⁺ and/or CD45RO ⁺ T cells), may 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 ⁺ and CD8 ⁺ T cells may be positively selected using magnetic beads conjugated with anti-CD 4 antibodies and anti-CD 8 antibodies.

In some embodiments, the cell separation step is performed in a buffer. In some embodiments, the buffer and selection reagent are mixed prior to addition of the cells. In some embodiments, the selection buffer and the selection reagent are added separately to the cell sample. In some embodiments, the total duration of incubation with the selection agent 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.

In some embodiments, the cell separation step further comprises pre-washing the cell-containing sample (e.g., a apheresis sample). In some embodiments, the immunoaffinity-based separation system is or contains a magnetic separation column. The separation step comprises subjecting the sample to a magnetic field and the magnetic bead-bound cells will be attracted to the magnetic separation column and separated from the unbound cells. For positive selection, cells attracted by the magnet were retained; for negative selection, cells that were not attracted were retained. In some embodiments, affinity-based selection is performed via magnetically activated cell sorting (MACS, e.g., clinic MACS system, miltenyiBiotec). In some embodiments, the step is performed in the lumen of a centrifugal chamber, e.g., under centrifugal rotation. In some embodiments, the steps are performed in an automated manner, e.g., using an automated procedure to complete the steps of pre-washing, combining, and separating in a single closed system.

In some embodiments, the cell separation step comprises the step of freezing (e.g., cryopreserving) the cells after separation of the cells. In some embodiments, the freezing step removes granulocytes and to some extent monocytes from the cell population. In some embodiments, the cells are frozen at a rate of 1 ℃ per minute to-80 ℃ for storage.

III-b activation of cells

In some embodiments, the step comprises the step of stimulating the isolated cells (e.g., the selected cell population). In some embodiments, activation of the cell occurs prior to or concurrent with genetic engineering (e.g., transduction). In some embodiments, activation of the cells occurs prior to genetic engineering.

In some embodiments, the composition or cell is incubated under stimulating and/or activating conditions. In some embodiments, the conditions for stimulation and/or activation may include one or more of the following: specific media, temperature, oxygen content, carbon dioxide content, time, drugs (e.g., nutrients, amino acids, antibiotics, ions, and/or stimulators such as cytokines, chemokines, antigens, fusion proteins, recombinant soluble receptors, and any other agent capable of activating cells). In some embodiments, the stimulation conditions include one or more agents capable of stimulating or activating the intracellular signaling domain of the TCR complex. In some embodiments, the agent is capable of initiating a TCR/CD3 intracellular signaling cascade in T cells, e.g., an agent (e.g., an anti-CD 3 antibody) suitable for delivering a primary signal, e.g., to initiate activation of an ITAM-induced signal, and/or an agent that promotes a co-stimulatory signal (e.g., a co-stimulatory signal specific for a T cell co-stimulatory receptor), e.g., an anti-CD 28 or anti-4-1 BB antibody (e.g., bound to a solid support) and/or one or more cytokines. The stimulatory agents include anti-CD 3 antibodies and anti-CD 28 antibodies, or magnetic beads conjugated with anti-CD 3/anti-CD 28 antibodies (e.g., dynabead ^TM CD3/CD 28T cell expansion agent). In some embodiments, the stimulatory agent includes IL-2, IL-7 and/or IL-15. In some embodiments, the stimulation conditions include a temperature suitable for growth of human T lymphocytes, for example at least about 25 ℃, at least about 30 ℃, or about 37 ℃.

In some embodiments, the cell activation step is performed in the lumen of a centrifugal chamber. In some embodiments, the cells are mixed with a stimulating condition or agent in the centrifugal chamber. In some embodiments, the cells are incubated with an activation medium comprising cytokines. In some embodiments, the volume of 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 medium matrix and a supplement. In some embodiments, the supplement comprises a cytokine, and optionally an amino acid and an antioxidant. 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 medium matrix and the supplement are added to the cells separately.

In some embodiments, the total duration of the activation process is between or between about 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 at least about 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, or 72 hours.

III-c. genetic engineering

In one aspect, the cell culture method comprises a step of genetic engineering. 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 prior to, simultaneously with, or after activation of the cell. In some embodiments, the nucleic acid molecule encoding the recombinant protein is introduced into the cell after activation of the cell. In some embodiments, the introducing is performed by contacting the cell with a nucleic acid molecule encoding a recombinant protein. This step is optional for the cell culture methods of the present disclosure.

In some embodiments, the genetic engineering step comprises 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 viral particles, such as vectors of adenovirus, adeno-associated virus (AAV) and Human Immunodeficiency Virus (HIV), as well as recombinant lentiviral vectors or retroviral vectors (e.g., gamma retroviral vectors). In some embodiments, the non-viral system comprises a transposon system, such as a PiggyBac or a Sleeping Beauty gene transfer system. 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 of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection, tungsten particle-promoted microprojectile bombardment, and strontium phosphate DNA co-precipitation.

In some embodiments, the concentration of cells to be transduced is from or from about 1.0X10 ⁵ cells/mL to 1.0X10 ⁸ cells/mL, such as at least or at least about or about 1.0X10 ⁵ cells/mL, 5X 10 ⁵ cells/mL, 1X 10 ⁶ cells/mL, 5X 10 ⁶ cells/mL, 1X 10 ⁷ cells/mL, 5X 10 ⁷ cells/mL, or 1X 10 ⁸ cells/mL. In some embodiments, the viral particles are provided as a ratio of copies of the viral vector particles or units of Infection (IU) thereof to the total number of cells to be transduced (IU/cells). 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 60IU of viral vector particles per cell. In some embodiments, transduction may be achieved at a multiplicity of infection (MOI) of less than 100, e.g., typically less than 60, 50, 40, 30, 20, 10, 5, or less.

In some embodiments, the step comprises contacting the cell with a viral particle. In some embodiments, the duration of the contacting is from 30 minutes to 72 hours, from 30 minutes to 48 hours, from 30 minutes to 24 hours, or from 1 hour to 24 hours, or at least about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours. In some embodiments, the contacting is performed in solution or medium. In some embodiments, the contacting is performed in a second medium. In some embodiments, the cells and virions are contacted in a volume of 0.5mL to 500mL, e.g., or about 0.5mL to 200mL, 0.5mL to 100mL, 0.5mL to 50mL, 0.5mL to 10mL, 0.5mL to 5mL, 5mL to 500mL, 5mL to 200mL, 5mL to 100mL, 5mL to 50mL, 5mL to 10mL, 10mL to 500mL, 10mL to 200mL, 10mL to 100mL, 10mL to 50mL, 50mL to 500mL, 50mL to 200mL, 50mL to 100mL, 100mL to 500mL, 100mL to 200mL, or 200mL to 500mL. In some embodiments, the contacting is achieved by centrifugation. In some embodiments, the speed of centrifugation is lower than the speed used to precipitate the cells, e.g., from or about 600rpm to or to about 1700rpm, e.g., at or about or at least 600rpm, 1000rpm, or 1500rpm or 1700rpm.

In some embodiments, after the genetic engineering step, the cells are transferred to other containers for culturing the genetically engineered cells, e.g., for expanding the cells, and in some embodiments, the containers for expanding the cells are bioreactor bags, such as perfusion bags.

III-d expansion of cells

In one aspect, the cell culture method comprises the step of expanding cells, which is performed in an expansion medium. In some embodiments, the cells are expanded after the step of genetically engineering. In some embodiments, the amplification medium is any of the compositions as described in medium ii. The composition has a surprising cell expansion effect.

In some embodiments, the time of the amplification step is greater than or 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 amplification step is at least about 25 ℃, at least about 30 ℃, or about 37 ℃. In some embodiments, the temperature is 25 to 38 ℃, 36 to 38 ℃. In some embodiments, the cells are expanded under conditions that maintain a target amount of carbon dioxide (CO ₂) in the cell culture. In some embodiments, the amount of CO ₂ is 10% to 0% (v/v), 8% to 2% (v/v), or at or about 5% (v/v) of the culture ambient gas.

In some embodiments, the cells are expanded using a vessel used in conjunction with a bioreactor. In some embodiments, the bioreactor is subjected to motion or rocking. Moving the bioreactor may increase oxygen transfer, including but not limited to rotation along a horizontal axis, rotation along a vertical axis, rocking movement along a tilted horizontal axis of the bioreactor, or any combination thereof. In some embodiments, the rocking 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 rocking angle is between 6-16 °. In other embodiments, the rocking angle is between 7-16 °. In other embodiments, the rocking angle is between 8-12 °. In some embodiments, the rocking 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、1 12、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、40rpm. in some embodiments, the rocking rate is between 4rpm and 12rpm, for example between 4rpm and 6rpm, inclusive. In some embodiments, the constant rocking speed is 5 rpm-15rpm, such as 6rpm or 10rpm. In some embodiments, the bioreactor or culture vessel is in a stationary state. In some embodiments, the bioreactor is maintained at a temperature at or near 37 ℃ and at or near 5% CO ₂ level, with a stable air flow as follows: is or at least 0.01L/min, 0.05L/min, 0.1L/min, 0.2L/min, 0.3L/min, 0.4L/min, 0.5L/min, 1.0L/min, 1.5L/min or 2.0L/min or more than 2.0L/min. In certain embodiments, the amplification is performed under perfusion.

In some embodiments, the amplification step is performed in a closed system. In some embodiments, the amplifying step is performed in the same closed system as the other step or steps of the cell culture method.

IV recombinant proteins

In one aspect, cells cultured as in the compositions of the present disclosure express recombinant proteins. In some embodiments, the cell comprises one or more nucleic acids encoding a recombinant protein introduced by genetic engineering, and thereby expresses the recombinant protein. In some embodiments, expression of the recombinant protein is achieved by: cells are first activated and then transduced and expanded in culture to an amount sufficient for clinical use. In some embodiments, the cell is not activated, is transduced directly, or is activated and transduced simultaneously. In some embodiments, the recombinant protein is a chimeric receptor, chimeric Antigen Receptor (CAR), T Cell Receptor (TCR), or a combination thereof.

IV-a Chimeric Antigen Receptor (CAR)

In some embodiments, the recombinant protein is a Chimeric Antigen Receptor (CAR). In some embodiments, the antigen is selectively expressed or over-expressed on cells of a disease or disorder (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells. In some embodiments, the diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including hematological cancers, cancers of the immune system, such as lymphomas, leukemias and/or myelomas, such as B-leukemia, T-leukemia and myelogenous leukemia, lymphomas and multiple myelomas. In some embodiments, the antigen is a tumor antigen or a cancer marker. In some embodiments, the antigen (including ligand) is or includes B Cell Maturation Antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA 9), cancer-testis antigen, cancer/testis antigen 1B (NY-ESO-1), carcinoembryonic antigen (CEA), cyclin A2, C-C 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 (CSPG 4), 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 (EPHa 2), estrogen receptor, fc receptor-like protein 5 (FCRL 5), fetal acetylcholine receptor (fetal AchR), folate Binding Protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD 2), ganglioside GD3, glycoprotein 100 (gp 100), phosphatidylinositol proteoglycan-3 (GPC 3), g protein coupled receptor class C group 5 member D (GPRC 5D), GUCY2C, her2/neu (receptor tyrosine kinase erb-B2), her3 (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-22 Ralpha), IL-13 receptor alpha 2 (IL-13 Ralpha 2), kinase insertion domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM), CE7 epitope of L1-CAM, protein 8 family member A containing leucine-rich repeat (LRRC 8A), lewis Y, melanomA-Associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine Cytomegalovirus (CMV), mucin 1 (MUC 1), MUC16, natural killer cell group 2 member D (NKG 2D) ligand, melanin A (MART-1), neural Cell Adhesion Molecule (NCAM), Cancer embryo antigen, melanoma preferential expression antigen (PRAME), progesterone receptor, prostate specific antigen, prostate Stem Cell Antigen (PSCA), prostate Specific Membrane Antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR 1), survivin, trophoblast glycoprotein (TPBG, also known as 5T 4), tumor-associated glycoprotein 72 (TAG 72), tyrosinase-associated protein 1 (TRP 1, also known as TYRP1 or gp 75), tyrosinase-associated protein 2 (TRP 2, also known as dopachrome tautomerase, dopachrome delta isomerase or DCT), and, Vascular Endothelial Growth Factor Receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR 2), wilms tumor 1 (WT-1), pathogen-specific or pathogen-expressed antigens, or antigens associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. In some embodiments, the receptor-targeted antigen comprises 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 comprises CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30.

In some embodiments, the CAR contains an extracellular antigen recognition domain that specifically binds to an antigen. Thus, the extracellular antigen-recognition domain that specifically binds 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, e.g., a single domain antibody, a monospecific antibody, or a multispecific antibody; or antibody fragments, e.g., fv, fab, fab ', fab ' -SH, F (ab ') ₂, scFv.

In some embodiments, the CAR comprises a spacer, which may be or comprise 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.

In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from a natural source or from a synthetic source. Where 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 α, β or ζ chains of T cell receptors, CD28, CD3 epsilon, 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 comprises predominantly hydrophobic amino acid residues, such as leucine and valine.

In some embodiments, the CAR contains an intracellular signaling domain comprising a cytoplasmic signaling domain, e.g., an intracellular domain capable of inducing a primary activation signal in a T cell, e.g., the zeta chain of the CD3 zeta chain; and/or the intracellular signaling domain comprises an immune receptor tyrosine based activation motif (ITAM). In some embodiments, the CAR contains a co-stimulatory domain. In some embodiments, the co-stimulatory domain is the signaling region and/or transmembrane portion of CD28, 4-1BB, OX40, DAP10 or ICOS. In some aspects, the same CAR includes both the primary activation signaling region and the co-stimulatory component. In some embodiments, the same CAR comprises both a primary activation signaling domain and a co-stimulatory domain.

IV-b T Cell Receptor (TCR)

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 alpha and beta chains (TCR alpha and beta (or variable gamma and delta chains (TCR gamma and TCR delta)) or antigen binding portions thereof, and is capable of specifically binding peptides bound to MHC molecules.

In particular embodiments, the recombinant proteins of the disclosure include the aforementioned CAR or TCR. The skilled artisan will appreciate that the culture media of the present disclosure have the effect of increasing the expansion capacity of cells, and are suitable for cells that have not transduced or transduced any of the recombinant proteins.

V. examples

Example 1: effect of multiple Medium and TexMACS Medium Mixed compositions on T cell culture

After centrifugal cleaning, anti-CD 4 magnetic beads and CD8 magnetic beads are added to the single-harvest product of the human leucocytes for incubation. The incubated products are separated by a magnetic separation system, and then the separated T cells are frozen. T cells from two different donors (donor 1, donor 2) were resuscitated, stained, counted, T cells were grouped and washed twice by centrifugation with DPBS (Corning, 21-031-CV, supra). The T cell pellet was resuspended with DPBS and the viability of the cells was detected. T cells are taken and cultured in a G-Rex 24 culture plate, and medium-2 is added to ensure that the cell density is not lower than 2.5X10 ⁶ cells/mL, and magnetic beads coupled with an anti-CD 3 antibody and an anti-CD 28 antibody are adopted to activate the T cells. The cells were then incubated in a 37.0.+ -. 1.0 ℃ 5.0.+ -. 1.0% CO ₂ incubator for 24.+ -. 6 hours.

After T cell activation, each group was supplemented with medium-2 to the same volume. Cells were transferred to 37.0.+ -. 1.0 ℃ and incubated in a 5.0.+ -. 1.0% CO ₂ incubator using medium-3 as the supplemental medium. Starting on Day 5 (Day 5), cell passages were performed with a cell density of approximately 0.625×10 ⁶ cells/mL per passage.

The above medium-1 was prepared according to Table 1. Medium-3 was supplemented with 100 IU/mL IL-2, 700 IU/mL IL-7, 46 IU/mL IL-15, and 2mM GlutaMAX to Medium-1; medium-2 was supplemented with 0.8 mg/mL NAC to medium-3. The concentration of cytokine, glutaMAX or NAC is calculated as the volume of medium-1 before addition. The cumulative expansion fold, the activation rate, the early memory phenotype cell T _SCM proportion and the PD-1 expression proportion parameters at day 13 of T cell culture are shown in Table 1.

TABLE 1 Medium formulation and amplification efficiency

Note that: NA represents the death of the cells during the culture and was not cultured until day 13.

Amplification factors are key indicators of the efficiency of the culture medium. The combined culture medium obtained by compounding TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20 is obviously superior to the combined culture medium or a single culture medium obtained by compounding TexMACS with Optmizer, AIM-V, RPMI 1640, DMEM or MEM. Surprisingly, in the media tested in this example, texMACS had no synergistic effect with other media except for the combination of TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20. Meanwhile, in a combined culture medium obtained by compounding TexMACS with X-VIVO 10, X-VIVO 15 or X-VIVO 20, the cell activity rate is also kept at a high level, and the proportion of the early memory phenotype cells T _SCM and the expression proportion of the depletion marker molecule PD-1 are similar to those of other groups.

Example 2: texMACS+T-VIVO medium composition

T cells from different donors (donor 3, donor 4, donor 5, donor 6) were cultured in the same manner as in example 1. Wherein, medium-1 was formulated according to Table 2. The supplements added to medium-2 and medium-3 were also the same as in example 1. The cumulative expansion fold, the activation rate, the early memory phenotype cell T _SCM ratio and the PD-1 expression ratio parameters at day 13 of T cell culture are shown in Table 2.

TABLE 2 Medium formulation Table

The results show that the expansion times and the activation rate of T cells in the TexMACS: X-VIVO combined medium are obviously higher than those of a single T-VIVO medium, and the synergistic effect of the combined medium is further verified. However, surprisingly, when TexMACS is combined with T-VIVO, the expansion and activation rate of T cells can be further improved. The proportion of T cells in the TexMACS+T-VIVO combined medium was slightly higher than the proportion of T _SCM in the TexMACS+X-VIVO 15 combined medium. The expression rate of the PD-1 marker molecules of the T cells in the TexMACS+T-VIVO combined culture medium is lower than that of the T cells in the TexMACS+X-VIVO 15 combined culture medium. The data indicate that T cells have higher expansion capacity and better differentiation phenotype and depletion phenotype in the TexMACS+T-VIVO combined medium.

Example 3: optimizing TexMACS+T-VIVO medium composition based on cell expansion

T cells from donor 7 were cultured in the same manner as in example 1. Wherein, medium-1 was formulated 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 at day 13 of T cell culture is shown in table 3.

TABLE 3 Medium formulation Table

The results show that the TexMACS+T-VIVO medium shows better amplification rate than the single T-VIVO medium and the TexMACS+X-VIVO 15 combined medium. The synergistic effect of this formulation is achieved over a wide range of conditions from 4:1 to 1:4 and remains after dilution.

Exemplary embodiments

1. A composition comprising TexMACS and T-VIVO in a volume ratio of 1:0.1-10; the composition has an immune cell expansion factor higher than that of TexMACS or T-VIVO.

2. The composition of embodiment 1, wherein the volumetric ratio of TexMACS to T-VIVO is 1:0.25-4.

3. The composition of embodiment 1 or 2, wherein the volumetric ratio of TexMACS to T-VIVO is 1:0.5-4.

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

5. A composition comprising a culture medium matrix comprising TexMACS and T-VIVO, wherein the sum of the volumes of TexMACS and T-VIVO is greater than 50% of the volume of the culture medium matrix, and wherein the volume ratio of TexMACS to T-VIVO is 1:0.1-10.

6. The composition of embodiment 5, wherein the sum of the volumes of the TexMACS and the T-VIVO is 65% or more of the volume of the medium matrix.

7. The composition of embodiment 5 or 6, wherein the sum of the volumes of the TexMACS and the T-VIVO is greater than 80% of the volume of the medium matrix.

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

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

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

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

12. The composition of any one of embodiments 5 to 11, wherein the composition comprises a supplement comprising one or more ingredients selected from the group consisting of inorganic salts, sugars, vitamins, albumin, lipids, amino acids, cytokines, and antioxidants added to a medium matrix.

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.

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

15. The composition of any one of embodiments 12 to 14, wherein the supplement comprises an amino acid and a cytokine.

16. The composition of any one of embodiments 12 to 15, wherein the supplement comprises an amino acid, a cytokine, and an antioxidant.

17. The composition of any one of embodiments 12-16, 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.

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

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

20. The composition of any one of embodiments 12 to 19, wherein the cytokine comprises IL-2, IL-7, and IL-15.

21. The composition of any one of embodiments 12-20, wherein the concentration of the IL-2 added in the composition is 10-1000 IU/mL.

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

23. The composition of any one of embodiments 12-22, wherein the concentration of the IL-2 added in the composition is about 100 IU/mL.

24. The composition of any one of embodiments 12-23, wherein the concentration of the IL-7 added in the composition is 100-5000 IU/mL.

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

26. The composition of any one of embodiments 12-25, wherein the concentration of the IL-7 added in the composition is about 700 IU/mL.

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

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

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

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

31. The composition of any one of embodiments 12-30, wherein the concentration of the L-alanyl-L-glutamine added in the composition is 1 mM-5 mM.

32. The composition of any one of embodiments 12-31, wherein the concentration of the L-alanyl-L-glutamine added in the composition is about 2mM.

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

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

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

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 accounting for more than 50% of the volume of the culture medium matrix, the volume ratio of TexMACS and T-VIVO being 1:0.25-4.

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 accounting for 80% or more of the volume of the culture medium matrix, the volume ratio of TexMACS and T-VIVO being 1:0.25-4.

37. The composition of any one of embodiments 5 to 36, wherein the medium matrix consists of TexMACS and T-VIVO in a volume ratio of 1:0.5-4.

38. The composition of any one of embodiments 5 to 37, comprising a supplement comprising IL-2, IL-7, IL-15, L-alanyl-L-glutamine and optionally N-acetyl-L-cysteine added to a medium matrix.

39. The composition of any one of embodiments 5-38, wherein the concentration of the additional IL-2 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.

40. The composition of any one of embodiments 5-39, wherein the concentration of the additional IL-2 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.

41. A method of culturing cells, the method comprising incubating cells in a composition according to any one of embodiments 5-40.

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

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

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

45. The method of any one of embodiments 41-44, wherein the composition used prior to cell activation comprises a culture medium matrix, and wherein the composition used upon 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 wherein the composition used upon 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 (9)

1. A composition comprising a culture medium matrix comprising TexMACS and T-VIVO, wherein the sum of the volumes of TexMACS and T-VIVO is 80% or more of the volume of the culture medium matrix, and wherein the volume ratio of TexMACS to T-VIVO is 1:0.25-4.

2. The composition of claim 1, wherein the medium matrix consists of TexMACS and T-VIVO.

3. The composition of claim 1, wherein the volumetric ratio of TexMACS to T-VIVO is 1:0.5-4.

4. A composition according to any one of claims 1 to 3, wherein the composition comprises a supplement to the medium matrix, the supplement comprising one or more ingredients selected from the group consisting of amino acids, cytokines and antioxidants.

5. The composition of claim 4, wherein the cytokine comprises IL-2.

6. The composition of claim 4, wherein the cytokine comprises IL-2, IL-7, and IL-15.

7. The composition of claim 4, wherein the amino acid is L-alanyl-L-glutamine.

8. The composition of claim 4, wherein the antioxidant is N-acetyl-L-cysteine.

9. A method of culturing T cells comprising incubating the cells in the composition of any one of claims 1-8.