Method for preparing immune cells

Abstract

A method for preparing immune cells in a completely closed system is provided. Specifically, the method includes the steps of pretreatment, cell sorting, activation, transduction, and expansion. This method significantly improves the efficiency of immune cell preparation and reduces preparation costs.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to China Patent Application No. 2020 / 105366876, filed on 12 June 2020, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to the field of biotechnology, and more specifically, to a method for the automated preparation of immune cells, for example, in a completely closed system. [Background technology]

[0003] In cellular immunotherapy, also known as adoptive cell therapy, a patient's immune cells are collected for genetic modification or selective proliferation to enhance antigen-specific immune responses. In recent years, tumor immunotherapy has proven successful in clinical practice. However, the preparation of immune cells is relatively complex. Any deficiency in any component of the process, such as process flow, equipment and facilities, and reagents, can significantly impact the quality of the final cell preparation, which in turn affects clinical efficacy. Therefore, a fully automated, completely closed preparation process can maximize product safety and batch-to-batch consistency, reduce impacts on personnel and the environment, and improve the production efficiency of immunotherapy products.

[0004] The conventional immunotherapy preparation cycle is approximately 10-14 days. This relatively long period can not only cause excessive differentiation and aging of cells during the culture process, but can also significantly impact the progression of the patient's disease. Therefore, a faster preparation process is more in line with clinical needs. It can improve clinical efficacy, reduce costs, and increase production capacity. To meet the characteristics and needs of each cell type, the culture system may contain serum, be serum-low, or serum-free.

[0005] Therefore, there is an urgent need to develop more robust and efficient methods for generating immune cells for clinical use. [Overview of the project]

[0006] This disclosure provides a method for culturing genetically modified immune cells. The method may include (a) providing a sample containing immune cells; (b) optionally washing the sample to obtain pre-treated immune cells; (c) sorting the pre-treated immune cells to obtain concentrated immune cells; (d) activating the concentrated immune cells with microbeads coated with an activator to obtain activated immune cells; (e) genetically modifying the activated immune cells to obtain genetically modified immune cells; and (f) optionally growing the genetically modified immune cells.

[0007] Microbeads can have diameters ranging from approximately 1 μm to 10 μm, approximately 2 μm to 8 μm, or approximately 4 μm to 5 μm.

[0008] In step (d), activation may be performed with a microbead-to-cell ratio in the range of approximately 0.1 to 10, approximately 0.2 to 8, approximately 0.5 to 8, approximately 0.1 to 8, approximately 0.5 to 5, approximately 0.5 to 4, approximately 0.5 to 3, approximately 0.5 to 2, approximately 0.5 to 1, approximately 1 to 8, approximately 1 to 6, approximately 1 to 5, approximately 1 to 3, approximately 0.5 to 5, approximately 1 to 2, approximately 0.1, approximately 0.2, approximately 0.5, approximately 0.8, approximately 1, approximately 1.2, approximately 1.5, approximately 1.8, approximately 2, approximately 2.5, approximately 3, approximately 3.5, approximately 4, approximately 4.5, or approximately 5.

[0009] The activator may be an antibody or a fragment thereof, a cytokine, a recombinant costimulatory molecule, a small drug inhibitor, or a combination thereof. In certain embodiments, the activator is an anti-CD3 and / or anti-CD28 antibody or a fragment thereof.

[0010] In step (d), activation occurs in approximately 2 hours to 1 week, approximately 2 hours to 6 days, approximately 2 hours to 5 days, approximately 2 hours to 4 days, approximately 2 hours to 3 days, approximately 2 hours to 2 days, approximately 2 hours to 1 day, approximately 2 hours to 20 hours, approximately 2 hours to 16 hours, approximately 4 hours to 5 days, approximately 4 hours to 96 hours, approximately 4 hours to 48 hours, approximately 4 hours to 36 hours, approximately 4 hours to 24 hours, approximately 4 hours to 20 hours, approximately 4 hours to 16 hours, approximately 16 hours to 48 hours, and approximately 1 It can be carried out over 6 to 40 hours, 16 to 36 hours, 16 to 24 hours, 2 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, 50 hours, 55 hours, 60 hours, 65 hours, 72 hours, 84 hours, 96 hours, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, or over a week.

[0011] Steps (d), (e), and (f) of the method, or all steps of the method, may be carried out over approximately 2 to 5 days, 3 to 4 days, 2 to 10 days, 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, 2 to 4 days, 3 to 10 days, 3 to 9 days, 3 to 8 days, 3 to 7 days, 3 to 6 days, 3 to 5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0012] Steps (c) to (f) of the method, steps (d) to (f) of the method, or all steps of the method may be carried out in a sealed sterile system.

[0013] Immune cells can be T cells, T cell subsets, or natural killer (NK) cells.

[0014] In step (d), activation occurs approximately 0.5 × 10⁻⁶ times. 6 cells / ml ~ approx. 10×10 6 cells / ml, approximately 2×10 6 cells / ml ~ approx. 3×10 6 cells / ml, approximately 0.5×106 cells / ml to approximately 8×10 6 cells / ml, approximately 0.5×10 6 cells / ml to approximately 5×10 6 cells / ml, approximately 1×10 6 cells / ml to approximately 8×10 6 cells / ml, approximately 1×10 6 cells / ml to approximately 6×10 6 cells / ml, approximately 1×10 6 cells / ml to approximately 5×10 6 cells / ml, approximately 1×10 6 cells / ml to approximately 4×10 6 cells / ml, approximately 1×10 6 cells / ml to approximately 3×10 6 in the range of cells / ml, approximately 0.5×10 6 cells / ml, approximately 1×10 6 cells / ml, approximately 1.5×10 6 cells / ml, approximately2×10 6 cells / ml, approximately 2.5×10 6 cells / ml, approximately 3×10 6 cells / ml, approximately 3.5×10 6 cells / ml, approximately 4×10 6 cells / ml, approximately 4.5×10 6 cells / ml, approximately 5×10 6 cells / ml, approximately 5.5×10 6 cells / ml, approximately 6×10 6 cells / ml, approximately 6.5×10 6 cells / ml, approximately 7×10 6 cells / ml, approximately 7.5×10 6 cells / ml, approximately 8×10 6 cells / ml, approximately 8.5× 6 cells / ml, approximately 9×10 6 cells / ml, approximately 9.5×10 6 cells / ml, or approximately 10×10 6 It can be carried out at an immune cell density of cells / ml.

[0015] In step (e), gene modification can be transduction or transfection.

[0016] It should be noted that there seems to be a small error in the original text where "1×10" in line 23 should probably be "1×10", and "8×10 " in line 59 should probably be "8×10 " with a closing parenthesis or some other appropriate ending. The translation has been done based on the provided text as accurately as possible.In step (e), gene modification may include introducing a polynucleotide encoding a chimeric antigen receptor (CAR) or T cell receptor (TCR) into activated immune cells.

[0017] In step (e), gene modification may include transducing activated immune cells with a lentiviral vector, a gamma-retroviral vector, an alpha-retroviral vector, or an adenoviral vector. In certain embodiments, gene modification may include transducing activated immune cells with a lentiviral vector.

[0018] In step (b), washing may involve using a human serum albumin (HSA) solution having a final HAS concentration of approximately 0.1% to 30%, 0.1% to 10%, 0.1% to 25%, 0.1% to 20%, 0.1% to 15%, 0.1% to 8%, 0.1% to 6%, or 0.1% to 5%.

[0019] In step (b), washing may include centrifugation of the sample using centrifugal force in the range of approximately 100 × g to approximately 1,000 × g, approximately 200 × g to approximately 400 × g, approximately 100 × g to approximately 800 × g, approximately 100 × g to approximately 600 × g, approximately 100 × g to approximately 500 × g, approximately 200 × g to approximately 800 × g, approximately 200 × g to approximately 600 × g, or approximately 200 × g to approximately 500 × g.

[0020] In step (b), washing could include centrifugation of the sample for approximately 100 to 600 seconds, 300 to 400 seconds, 50 to 1,000 seconds, 100 to 800 seconds, 100 to 500 seconds, 200 to 800 seconds, 200 to 600 seconds, 200 to 500 seconds, 300 to 800 seconds, or 300 to 500 seconds.

[0021] In step (b), washing may include diluting the sample by approximately 0 to 5 times, 1 to 5 times, 1 to 4 times, 1 to 3 times, or 2 to 3 times.

[0022] In step (b), washing may include performing / repeating the washing cycle 1 to 5 times, 1 to 4 times, 1 to 3 times, 1 to 2 times, 2 to 5 times, 2 to 4 times, or 2 to 3 times.

[0023] Step (b) may have an output volume in the range of approximately 5 ml to 400 ml, approximately 20 ml to 100 ml, approximately 10 ml to 300 ml, approximately 10 ml to 200 ml, approximately 10 ml to 100 ml, approximately 20 ml to 400 ml, approximately 20 ml to 300 ml, approximately 20 ml to 200 ml, approximately 50 ml to 400 ml, approximately 50 ml to 300 ml, approximately 50 ml to 200 ml, or approximately 50 ml to 100 ml.

[0024] In step (c), the sorting may include using an anti-CD4 and / or anti-CD8 antibody or a fragment thereof.

[0025] The sample may be, for example, peripheral blood, immune cells, monocytes, or peripheral blood mononuclear cells (PBMCs) from a subject (e.g., a patient) or from multiple subjects.

[0026] This disclosure provides genetically modified immune cells prepared by the present method.

[0027] This disclosure also provides cell preparations including genetically modified immune cells.

[0028] This disclosure also includes pharmaceutical compositions containing genetically modified immune cells. The pharmaceutical compositions may further include a pharmaceutically acceptable carrier.

[0029] The objective is, for example, to provide a method for the completely closed automated preparation of immune cells in a completely closed system.

[0030] In the first embodiment, a method for culturing immune cells is provided, the method comprising the following steps: (a) To provide a sample containing immune cells, (b) Optionally, pre-treat a sample containing immune cells by washing to obtain pre-treated immune cells. (c) Perform cell sorting on pre-treated immune cells to obtain sorted immune cells. (d) Obtaining activated immune cells by activating selected immune cells, wherein the activation process is carried out by using an activator selected from TransAct®, Dynabeads®, and OKT3 antibody. (e) Transfecting activated immune cells to obtain transfected immune cells, (f) Culture transfected immune cells to prepare the desired immune cells.

[0031] In another embodiment, steps (c), (d), (e), and (f) are performed using a Prodigy® apparatus.

[0032] In another embodiment, in step (d), the activator is Dynabeads®, preferably in an amount of Dynabeads:cell = 0.1 to 10, more preferably Dynabeads:cell = 0.5 to 5.

[0033] In another embodiment, in step (d), the activator is TransAct®, preferably in an amount of 0.1 to 30 ml, more preferably 1 to 10 ml.

[0034] In another embodiment, in step (d), the activator is an OKT3 antibody, preferably in an amount of 10 ng / ml to 50 mg / ml (final concentration).

[0035] In another embodiment, in step (d), the density of immune cells subjected to the activation treatment is (0.1~20) × 10 6Cells / ml, preferably (0.5~10) × 10 6 The value is cells / ml.

[0036] In another embodiment, in step (d), the activation treatment time is 4 to 96 hours, preferably 16 to 48 hours.

[0037] In another embodiment, in step (d), the number of activated cells is 1 × 10 5 ~20×10 9 That is the case.

[0038] In another embodiment, the immune cells include T cells and natural killer (NK) cells.

[0039] In another embodiment, the sample containing immune cells is selected from peripheral blood, immune cells, monocyte collections, and PBMCs.

[0040] In another embodiment, the sample includes fresh and frozen samples.

[0041] In another embodiment, the amount of sample is 5 to 500 ml, preferably 10 to 100 ml.

[0042] In another embodiment, in step (b), the pre-wash treatment is carried out using an apparatus selected from Sepax 2, Sepax C-pro, Sefia, Lovo, CS 5+, CS Elite, and a centrifuge.

[0043] In another embodiment, in step (b), the centrifugal force of the pre-wash treatment is 100 to 1,000 g, preferably 200 to 400 g.

[0044] In another embodiment, the centrifugal time for the pre-wash treatment in step (b) includes, but is not limited to, 100 to 600 seconds, preferably 300 to 400 seconds.

[0045] In another embodiment, in step (b), the dilution ratio of the sample for pre-wash treatment is 0 to 5 times, preferably 1 to 3 times.

[0046] In another embodiment, in step (b), the number of washing cycles of the pre-wash treatment is 1 to 5, preferably 1 to 3.

[0047] In another embodiment, in step (b), the output volume of the sample after washing includes, but is not limited to, 5 to 400 ml, preferably 20 to 100 ml.

[0048] In another embodiment, in step (c), the amount of immune cells for cell sorting is 1 × 10⁻⁶ 5 ~50×10 9 Cells, preferably (1-10) × 10 9 It is a cell.

[0049] In another embodiment, step (c) includes positive screening and / or negative screening.

[0050] In another embodiment, the positive screening marker is CD4 + CD8 + CD62L + CD3 + CD56 + or a combination thereof.

[0051] In another embodiment, the marker for the negative screening marker is CD14 + CD19 + CD269 + or a combination thereof.

[0052] In another embodiment, sorting is performed by using an anti-CD4 antibody and / or an anti-CD8 antibody, or fragments thereof. For example, the anti-CD4 antibody reagent is a CliniMACS CD4 reagent diluted 3 to 5 times, and the anti-CD8 antibody reagent is a CliniMACS CD8 reagent diluted 3 to 5 times. In another preferred embodiment, dilution is performed by using PBS-EDTA containing 0.1% to 10% HSA, preferably by using PBS-EDTA containing 0.2% to 1% HSA, and more preferably by using PBS-EDTA containing 0.4% to 0.6% HSA.

[0053] In another embodiment, the volumes of the anti-CD4 antibody reagent and the anti-CD8 antibody reagent used in step (c) are 1.5 to 10 ml, preferably 4 to 6 ml.

[0054] In another embodiment, in step (c), the solvent of the sorting reagent containing the anti-CD4 antibody reagent and the anti-CD8 antibody reagent is PBS-EDTA containing 0.1% to 10% HSA.

[0055] In another embodiment, the proportions of the anti-CD4 antibody reagent, the anti-CD8 antibody reagent, and the immune cells are 100 × 10⁻⁶. 6 ~1,000 x 10 6 The concentration is cells / mL.

[0056] In another embodiment, the method for diluting the anti-CD4 antibody reagent and the anti-CD8 antibody reagent is as follows: 7.5 ml (entire bottle) of CliniMACS CD4 reagent or CliniMACS CD8 reagent is divided into 1.5 ml / bottles, and then 3.5 ml of PBS-EDTA containing 0.1% to 10% HSA is added to prepare a total of 5 ml of diluted reagent for sorting.

[0057] In another embodiment, in step (e), the transfection process is selected from non-viral transfection (including electroporation systems such as the Neon transfection system and the Maxcyte transfection system) and viral transfection (including lentivirus systems, adenovirus systems, and adeno-associated virus vectors).

[0058] In another embodiment, in step (e), the virus is used for transduction, and the infection multiplicity or MOI is 0 to 1,000, preferably 1 to 10.

[0059] In another embodiment, the viral titer is 1 × 10⁻⁶ 5 ~10×10 10 It is TU / ml.

[0060] In another embodiment, in step (f), the culture medium used for culturing includes serum medium, low-serum medium, or serum-free medium.

[0061] In another preferred embodiment, in step (f), the seeded cell density of the culture is 0.01 × 10⁻⁶. 6 / ml~20×10 6 It is / ml.

[0062] In another embodiment, in step (f), the cell culture time is 4 to 96 hours, preferably 16 to 48 hours.

[0063] In another embodiment, after step (f), the method further includes the following steps: (g) washing and concentrating the cultured immune cells to obtain concentrated immune cells, and / or (h) distributing the (concentrated) immune cells.

[0064] In another embodiment, washing and concentration are performed using an apparatus selected from Sepax 2, Sepax C-pro, Sefia, Lovo, CS 5+, CS Elite, and a centrifuge.

[0065] In another embodiment, the volume of the sample to be washed and concentrated is 5 to 500 ml.

[0066] In another embodiment, in step (g), the centrifugal force for washing the sample is 100 × g to 1,000 × g.

[0067] In another embodiment, the centrifugation time for washing the sample in step (g) is 100 to 600 seconds.

[0068] In another embodiment, in step (g), the dilution ratio of the sample is 0 to 5 times.

[0069] In another embodiment, in step (g), the number of washing cycles is 1 to 5.

[0070] In another embodiment, in step (g), the output volume after concentration is 5 to 400 ml.

[0071] In another embodiment, in step (h), dispensing is performed using an apparatus selected from Sepax 2, Sepax C-pro, Sefia, and Prodigy®.

[0072] In another embodiment, the volume of the sample dispensed in step (h) is between 5 ml and 500 ml.

[0073] In another embodiment, in step (h), the output volume of the dispensed portion is 5 ml to 400 ml.

[0074] In another embodiment, the proportion of T (naive) cells in the sorted / enriched immune cells obtained in step (c) is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, or greater than 50%.

[0075] In another embodiment, the sorting recovery rate in step (c) is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0076] In another embodiment, the positive rate of activated immune cells obtained in step (d) is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90%.

[0077] In another embodiment, steps (d), (e), and (f), or all steps of the method, are completed in 2 to 12 days, 2 to 10 days, 2 to 8 days, 2 to 6 days, 2 to 5 days, 3 to 12 days, 3 to 10 days, 3 to 8 days, 3 to 6 days, 3 to 4 days, 2 to 5 days, 3 to 5 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0078] In another embodiment, the ratio (V1 / V2) of the volume of immune cells V1 obtained in step (f) to the volume of immune cells V2 before activation in step (d) is 6 to 30, preferably 8 to 12.

[0079] In another embodiment, the proportion of T naive cells among the immune cells obtained in step (f) is greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, or greater than 60%.

[0080] In another embodiment, the ratio of the amount of T naive cells, T1, in the immune cells obtained in step (f) to the amount of T naive cells, T2, in the immune cells obtained in step (d) (T1 / T2) is (0.8~1.2):(0.8~1.2). The ratio of the amount of central memory T (Tcm) cells, T3, in the immune cells obtained in step (f) to the amount of Tcm cells, T4, in the immune cells obtained in step (d) (T3 / T4) is (0.8~1.2):(0.8~1.2).

[0081] In a second embodiment, cultured immune cells are provided, and the immune cells are prepared by the present method.

[0082] In a third embodiment, a cell preparation is provided, which comprises (a) the immune cells and (b) a pharmaceutically acceptable carrier.

[0083] In another embodiment, the cell preparation is a liquid preparation (such as an injectable drug).

[0084] It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., embodiments) can be combined to form new or preferred technical solutions. Due to space limitations, they will not be repeated here one by one. [Brief explanation of the drawing]

[0085] [Figure 1] This shows the quantity and positivity rate of cells sorted by Prodigy®. [Figure 2] This shows a subset of cells after activation with Dynabeads®. [Figure 3] This shows a subset of cells after activation using TransAct®. [Figure 4] This shows the cell volume and positivity rate during the culture process after activation by different activators (or activating factors). [Modes for carrying out the invention]

[0086] This method is robust and yields an equivalent or greater quantity of clinically suitable immune cells (e.g., genetically modified T cells) compared to, for example, a standard CliniMACS Prodigy® automated process. This method can provide better transduction efficiency and robust production of a clinically relevant number of genetically modified T cells.

[0087] The clinical production of genetically modified T cells is currently a complex process that typically begins with obtaining peripheral blood mononuclear cells (PBMCs) from a patient. Current protocols feature a leukocyte depletion step. PBMCs are often enriched for T cells and activated before genetic modification with a viral or non-viral vector. The modified T cells are then proliferated to reach the number required for treatment, and the cells are subsequently formulated and / or cryopreserved before reinfusion into the patient. Cell products are subjected to numerous quality control assays and must meet shipping standards and Good Manufacturing Practice (GMP) guidelines.

[0088] This disclosure provides a method for culturing genetically modified immune cells. The method may include (a) providing a sample containing immune cells; (b) optionally washing the sample to obtain pre-treated immune cells; (c) sorting the pre-treated immune cells to obtain concentrated immune cells; (d) activating the concentrated immune cells with microbeads coated with an activator to obtain activated immune cells; (e) genetically modifying the activated immune cells to obtain genetically modified immune cells; and (f) optionally growing the genetically modified immune cells.

[0089] In certain embodiments, the method is carried out through an automated process in a device suitable for cell processing in a closed GMP-compliant environment (closed sterile cell culture system).

[0090] Cell processing in a closed, GMP-compliant system can be performed, for example, using CliniMACS Prodigy® and associated tube sets (Miltenyi Biotec GmbH, Germany). CliniMACS Prodigy® provides a flexible platform for cell processing applications, enabling magnetic separation and cell processing protocols for different cell types.

[0091] CliniMACS Prodigy® is a device capable of providing fully closed, integrated production of various cell products. The culture mode differs from the typical manual preparation of immune cells. Automated machine control reduces the time spent on manual operations. The fully closed system reduces the risk of contamination, and the standardized preparation process reduces manual errors, improving the production efficiency and quality of immunotherapy products. See U.S. Patents US10,131,876, US10,119,970, US10,620,212, US10,705,090, US10,705,091, US8,727,132, US8,747,290, US9,625,463, and US9,714,945 for cell preparation systems such as CliniMACS Prodigy®.

[0092] This method may include automated cell preparation, selection (isolation) of immune cells, cell activation, cell proliferation, cell transduction, and cell formulation (washing), for example, for subsequent clinical use.

[0093] In certain embodiments, all steps are performed using a single device, such as CliniMACS Prodigy®, with a set of disposable, closed, sterile tubes and programmed software. This method can result in high transduction efficiency of the produced T cells and high transgene expression in the genetically modified immune cells.

[0094] Furthermore, a large number of highly viable immune cells can be robustly generated in less than 5 days.

[0095] Immune cells can be enriched through magnetic separation using antigen-binding molecules (e.g., antibodies or fragments thereof) that are specific to cell surface markers on the surface of immune cells, such as markers CD2, CD3, CD4, CD8, CD25, CD28, CD27, CD45RA, CD45RO, CD62L, CD95, CD127, CD137, alpha / beta TCR, gamma / delta TCR, CCR7, PD-1, or Lag3.

[0096] The activator may be an agonist antibody, cytokine, recombinant costimulatory molecule, small drug inhibitor, or a combination thereof. In certain embodiments, the activator is an anti-CD3 and / or anti-CD28 antibody or fragment thereof bound to a microbead, microparticle, microsphere, or microstructure. In certain embodiments, the activator is a microbead coated with an anti-CD3 and / or anti-CD28 antibody or fragment thereof.

[0097] In certain embodiments, the microbeads are polymer microbeads. In certain embodiments, the microbeads are magnetic microbeads. In certain embodiments, the microbeads are magnetic polymer microbeads. In certain embodiments, the microbeads are superparamagnetic polymer microbeads. The polymer may include polystyrene, polyester, polyether, polyacrylate, polyacrylamide, polyamine, polyethyleneimine, polyquaternium polymer, polyphosphazene, polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymer, or polyurethane.

[0098] Microbeads are approximately 1μm to 50μm, 1μm to 40μm, 1μm to 30μm, 1μm to 20μm, 1μm to 15μm, 1μm to 12μm, 1μm to 10μm, 1μm to 8μm, 1μm to 6μm, 1μm to 5μm, 2μm to 10μm, 2μm to 8μm, 2μm to 6μm, and 3μm to approx. It may have a diameter (or central diameter) of 10 μm, approximately 3 μm to approximately 8 μm, approximately 3 μm to approximately 6 μm, approximately 3 μm to approximately 5 μm, approximately 4 μm to approximately 5 μm, approximately 1 μm, approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, approximately 9 μm, approximately 10 μm, approximately 11 μm, approximately 12 μm, approximately 13 μm, approximately 14 μm, approximately 15 μm, or approximately 4.5 μm.

[0099] Genetic modification of immune cells can be performed by transduction, transfection, or electroporation.

[0100] Transduction can be performed with lentiviruses, gamma-retroviruses, alpha-retroviruses, or adenoviruses. Cellular electroporation or transfection can be performed by introducing nucleic acids (DNA, mRNA, miRNA, antagonists, ODNs), proteins, site-specific nucleases (zinc finger nucleases, TALEN, CRISP / R), self-replicating RNA viruses (e.g., equine encephalopathy virus), or integration-deficient lentiviral vectors into cells.

[0101] In one embodiment, genetic modification of immune cells may be performed by transducing the cells with a lentiviral vector.

[0102] The proliferation of genetically modified immune cells can be achieved by adding a cell culture medium suitable for cell growth, such as TexMACS GMP medium (Miltenyi Biotec GmbH), to the culture chamber.

[0103] In certain embodiments, activation, gene modification, and / or proliferation of immune cells may be performed under shaking or rotational conditions. In one embodiment, shaking is performed during the proliferation of immune cells. Shaking (rotation) in the culture chamber may be performed sporadically or periodically by rotating the culture chamber (centrifugation chamber) every 1 to 120 seconds, every 15 to 60 seconds, or every 30 seconds. The centrifugal force may be (>) greater than 0 to a maximum of 70 × g (1 to 1000 rpm in a chamber with a radius of 6 cm) in one or two directions, 0.2 to 17 × g (50 to 500 rpm in a chamber with a radius of 6 cm) in one or two directions, or 6 × g (300 rpm in a chamber with a radius of 6 cm) in two directions. Importantly, the shaking conditions may be adapted during culture to best support immune cell proliferation (e.g., increased with increasing cell density).

[0104] Cell activation is 0.2 × 10 6 cells / ml~4×106 cells / ml, 0.5×10 6 cells / ml~2×10 6 cells / ml, or approximately 1 × 10⁶ 6 This can be done by using a cell density of cells / ml. Alternatively, activation can be performed using 4 × 10⁶ cells. 6 cells / ml~2×10 7 cells / ml, or 4 × 10 6 cells / ml~1×10 7 This can be done by using a high cell density of cells / ml.

[0105] Genetically modified immune cells can be genetically modified to express chimeric antigen receptors (CARs), T cell receptors (TCRs), or any auxiliary molecules on their cell surface.

[0106] For final formulation, the proliferated and genetically modified immune cells can be washed by centrifugation and replacement of the culture medium with a buffer suitable for subsequent application (e.g., injection of the resulting cell composition into the patient).

[0107] If necessary, genetically modified immune cells can be separated from unmodified immune cells, for example, using magnetic separation.

[0108] In another embodiment, the method provides a substantially pure composition of genetically modified immune cells. In a particular embodiment, the method generates about 80% to about 100%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% of the desired immune cells (e.g., genetically modified immune cells) in the cell composition.

[0109] In a further embodiment, the disclosure provides a pharmaceutical composition comprising genetically modified immune cells.

[0110] In one embodiment, CliniMACS Prodigy® and associated tube set (Miltenyi Biotec GmbH, Germany) are used as a closed cell sample processing system in which the automated process described herein is carried out. However, the use of this method is not intended to be limited to the CliniMACS Prodigy® system.

[0111] The CliniMACS Prodigy® system is designed to automate and standardize the entire cell product manufacturing process. It combines CliniMACS® separation technology (Miltenyi Biotec GmbH, Germany) with a wide range of sensor-controlled cell processing capabilities. The device features include disposable CentriCult® chambers enabling standardized cell processing and culture, cell enrichment and depletion functions, cell culture and cell proliferation functions, final product formulation in predefined media and volumes, the possibility of programming the device using a Flexible Programming Suite (FPS) and GAMP5 compatible programming language for customizing cell processing, and tube sets tailored to various applications, either alone or in combination with CliniMACS® reagents (Miltenyi Biotec GmbH).

[0112] The immune cell sorting / isolation steps may include one, several (two or more), or a combination of positive enrichment steps, i.e., isolation (direct magnetic labeling) of T cells, T cell subsets, and / or T cell precursors. T cells can be selected for CD4+ and / or CD8+ T cells by using antigen-binding molecules bound to particles such as magnetic beads specific to CD4 and / or CD8. Subpopulations of T cells, such as naive and central memory T cells, can be isolated, for example, by using the marker CD62L.

[0113] The immune cell sorting / isolation step may also involve negative enrichment of T cells (direct labeling of non-T cells) or depletion of a subset of cells to be removed from the preparation. For example, B cells may be removed via the CD19 marker. Inhibitory cells such as regulatory T cells (high CD25) and monocytes (CD14) can also be removed using the markers CD25 and CD14, respectively.

[0114] Viral transduction of immune cells can be enhanced by the use of transduction enhancer reagents, particularly, but not limited to, polycationic reagents (polybrene, protamine sulfate, poly-L-lysine, peptides with a net positive charge), adhesion molecules such as poloxamers, fibronectin, or modified fibronectin (RetroNectin), or protein-targeting domains such as antibodies, antibody complexes, or magnetic particles. Transduction enhancers are provided in solution and can be coated onto culture chambers or onto carrier materials present in suspensions / solutions within culture chambers.

[0115] In one embodiment, the centrifugation chamber and the culture chamber may be identical. The centrifugation chamber and the culture chamber can be used under various conditions for, for example, separation or transduction, and high rotational speeds (i.e., high gravity) can be applied, but the culture step, for example, can be carried out at slow rotation or idle. In one embodiment, the chamber changes the direction of rotation in an oscillating manner, resulting in shaking of the chamber and maintenance of cells in suspension. Thus, T cell activation, gene modification, and / or culture steps can be carried out under steady or shaking conditions of the centrifugation or culture chamber.

[0116] In one embodiment, for example, a patient sample containing the target immune cells is introduced into a chamber of a closed, sterile culture system such as CliniMACS Prodigy®. The sample is preferentially centrifuged using optical density phase detection to remove excess red blood cells, and the cell sample is washed with, for example, CliniMACS buffer (Miltenyi Biotec GmbH) to avoid cell aggregation, and magnetically labeled with magnetic cell separation reagents such as CliniMACS CD4 and CD8 reagents (Miltenyi Biotec GmbH). After labeling, the cells are washed, magnetically concentrated via an integrated magnetic cell selection column, and returned to the cell culture chamber.

[0117] In a cell culture chamber, immune cells may be activated under steady-state or shaking culture conditions with one or a combination of activators capable of inducing the proliferation of immune cells (e.g., T cells), such as agonist antibodies (e.g., anti-CD3 and anti-CD28), cytokines (e.g., IL-1b, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-17, IL-21, IL-22, IL-23, IL-35, TGF-b, IFN-alpha, IFN-gamma, TNF-alpha), recombinant proteins, costimulatory molecules, lectins, ionophores, synthetic molecules, antigen-presenting cells (APCs), artificial APCs, feeders, and combinations thereof. These activators are supplied in solution and can be coated onto the culture chamber or onto a carrier material present in the culture chamber or in a suspension / solution on large particles.

[0118] Immune cells can be cultured under steady-state or shaking culture conditions. In certain embodiments, after a period of culture, a viral vector is added to the culture chamber and the cells are transduced. After a further cell culture period, the cells can be transduced again or thoroughly washed and harvested (formulated). Prior to in vivo transfer of the genetically modified immune cell product, the cells can be washed, concentrated, and resuspended in a buffer compliant with the clinical requirements for in vivo injection. Two or more (e.g., all) of the above steps can be performed automatically.

[0119] In one embodiment, immune cells are labeled by binding to antibody-conjugated magnetic beads that attach to cell surface markers present on the surface of the immune cells, and then enriching the labeled cells by magnetic separation (positive enrichment).

[0120] In another embodiment, immune cells are enriched by binding antibody-conjugated magnetic beads to cell surface markers that are not present on the surface of T cells or a defined cell subset, and then depleting the labeled cells by magnetic separation (positive enrichment).

[0121] In further embodiments, in addition to the first enrichment of immune cells, genetically modified immune cells are enriched in a second enrichment step by magnetic labeling of the genetically modified immune cells and magnetic separation before or after culture, thereby obtaining a higher frequency of genetically modified immune cells in the final cell composition obtained by this method. For example, if the genetically modified cells are T cells expressing CAR or TCR, the second separation step may be performed by using antigen-binding molecules bound to magnetic particles specific to the recombinantly expressed CAR or TCR on the cell surface of the genetically modified T cells.

[0122] In one embodiment, a sample, for example, whole blood from a patient containing immune cells, is provided. The sample may be connected to a sealed sterile cell culture system, for example, the sample may be connected to a CliniMACS Prodigy® device via a set of tubes. The cell sample may be prepared by centrifugation in the device's centrifugation chamber, resulting in the separation of red blood cells and platelets from other cells, including immune cells. Magnetic separation of immune cells may be performed by using antibodies conjugated to magnetic particles specific to immune cell markers such as CD2, CD3, CD4, CD8, CD25, CD28, CD27, CD45RA, CD45RO, CD62L, CD95, CD127, CD137, alpha / beta TCR, gamma / delta TCR, CCR7, PD-1, or Lag3. Passing the labeled cells through a magnetic unit equipped with the device's separation column results in the enrichment of immune cells. After transferring the separated immune cells to the device's culture chamber (which may be identical to the centrifugation chamber), the cells are activated by using activators described herein. 6 cells / ml~2×10 6 The cells are then set to a given density of cells / ml. The activated immune cells are then genetically modified in the device's culture chamber, for example, they are transduced with a lentiviral vector containing a polynucleotide sequence encoding a CAR. The genetically modified immune cells are then grown in culture under shaking conditions. Shaking can be performed by sporadic or periodic centrifugation of the culture chamber (in this case, the culture chamber is identical to the centrifugation chamber) under conditions that allow the cells to be in suspension (and as disclosed herein). Finally, the cultured cells are washed by centrifugation, thereby allowing the culture medium to be replaced with a buffer suitable for subsequent applications such as injection of the resulting cell composition into a patient.

[0123] In one embodiment of the present invention, higher purity of transduced immune cells, such as T cells expressing transgenes such as CARs or TCRs on their cell surface, is obtained at the end of the manufacturing process. An additional cell selection step may be used to specifically enrich the genetically modified immune cells. For example, magnetic particles coated with antibodies against surface molecules encoded by the transgenes may be used in the selection step. The enrichment step may also be carried out in an automated manner by using the magnetic separation unit of the device and is performed before final formulation.

[0124] In certain embodiments, a selective agent is used that can be removed from the surface of selected cells after this second concentration and before application to a patient or downstream use.

[0125] In another embodiment, the automated manufacturing process can be initiated at a higher cell density by activating the immune cells under suspension conditions. If a sufficient number of target immune cells can be obtained from the starting material, the automated manufacturing process can be initiated directly at a high cell density of 4e6-1e7 immune cells under shaking conditions, which allow the cells to be in suspension for activation at the start of the culture. The immune cells can be further modified using lentiviral vectors and grown under suspension. In this embodiment, shaking conditions may be maintained during the activation, gene modification, and growth steps of the process disclosed herein in order to maintain the high-density cell culture in suspension.

[0126] In one embodiment, immune cells may be genetically modified using a lentiviral vector. In one embodiment, a lentiviral vector having a VSVG pseudotype allows for efficient transduction under an automated manufacturing method. Other types of lentiviral vectors may also be used, such as pseudotyped envelopes derived from measles virus (ML-LV), gibbon leukemia virus (GALV), feline endogenous retrovirus (RD114), and baboon endogenous retrovirus (BaEV). Other viral vectors, such as gamma or alpha retrovirus vectors, may be used. Transduction enhancer reagents may be added.

[0127] The terms "closed cell sample processing system" and "closed sterile (cell culture) system" can be used interchangeably.

[0128] As used herein, the term “closed sterile (cell culture) system” refers to any closed system that reduces the risk of cell culture contamination while performing culture processes, such as the introduction of new materials by transduction, and cell culture steps such as cell proliferation, differentiation, activation, and / or isolation. Such systems enable operation under GMP or GMP-like conditions ("sterile") that result in clinically applicable cell compositions. For example, CliniMACS Prodigy® (Miltenyi Biotec GmbH, Germany) can be used as a closed cell sample processing system. This system is disclosed in WO2009 / 072003. However, this method is not intended to be limited to CliniMACS Prodigy®.

[0129] This method may be carried out in a closed, sterile system (closed cell sample processing system) including a centrifuge chamber containing a cylinder, pump, valve, magnetic cell separation column, and a base plate and cover plate connected by a tube set. Blood samples or other sources containing T cells, T cell subpopulations, and / or T cell precursors may be transferred to and from the tube set by sterile docking or sterile welding.

[0130] A closed cell sample processing system may include multiple tube sets in which cells are transferred between tube sets by sterile docking or sterile welding.

[0131] As used herein, the terms “automated method” or “automated process” refer to any process that is automated through the use of devices and / or computers and computer software. Automated methods (processes) require less human intervention and less human time. In some cases, a method is automated if at least one step of the method is performed without any human assistance or intervention. Preferably, automated processes are carried out in closed cell sample processing systems such as the CliniMACS Prodigy® disclosed herein.

[0132] A closed cell sample processing system may include: a) a sample processing unit comprising input and output ports coupled to a rotating vessel (or centrifugation chamber) having at least one sample chamber, wherein the sample processing unit is configured to provide a first processing step to a sample or to apply centrifugal force to a sample deposited in the chamber and rotate the vessel to separate at least a first component from a second component of the deposited sample; and b) a sample separation unit coupled to the output port of the sample processing unit, wherein the sample separation unit comprises a separation column holder, a pump, and a plurality of valves configured to at least partially control the flow of fluid through a separation column arranged in a fluid circuit and holder, wherein the separation column is configured to separate labeled and unlabeled components of a sample flowing through the column.

[0133] Rotating vessels can also be used as temperature-controlled cell incubation and culture chambers (CentriCult Units = CCUs). These chambers can be filled with a defined gas mixture, provided by an attached gas mixing unit (e.g., using pressurized air / N2 / CO2 or N2 / CO2 / O2).

[0134] All agents may be connected to a closed system before the process begins. This includes all buffers, solutions, culture media and supplements, and microbeads used for washing, transporting, suspending, culturing, harvesting, or immunomagnetic cell sorting of cells within the closed system. Alternatively, such agents may be welded or connected by sterile means at any point during the process.

[0135] The sample may be provided in a transport bag or other suitable container that can be connected to a system sealed by sterile means.

[0136] The sample may be a human cell sample derived from blood. For example, a cell sample may consist of blood cells from a donor or patient. Such a blood product may be in the form of whole blood, pia mater, leukocyte apheresis, PBMC, or any clinical collection of blood products. It may be from a fresh or frozen source.

[0137] The centrifugation step may include one, more, or all of the following: gradient separation, erythropenia, platelet removal, and cell washing.

[0138] In certain embodiments, washing means replacing the culture medium or buffer in which the cells are held. The replacement of the supernatant may be partial or complete. Several washing steps may be combined to obtain a more complete replacement of the original culture medium in which the cells are held. The washing steps may include pelletizing the cells by centrifugal force and removing the supernatant. The cells may be pelletized, for example, at 300 × g by rotating the chamber, and the supernatant is removed during the rotation of the chamber. The culture medium is added during rotation or at a steady state.

[0139] The term "shaking conditions" can refer to any means by which cells can be kept in a suspension state. Shaking may be performed by rotating (or sporadic centrifugation) the culture chamber of a closed sterile cell culture system, and the rotation may be continuous or periodic. Shaking may also be performed by using, for example, a foaming device, propulsion device, or fluid flow (e.g., a channel) integrated into the closed sterile cell culture system used to prevent cell sedimentation.

[0140] The term "marker" can refer to a cellular antigen specifically expressed by a particular cell type. Preferably, a marker is a cell surface marker that allows for the enrichment, isolation, and / or detection of living cells. A marker may be a positive select marker such as CD4, CD8, and / or CD62L, or a negative select marker (e.g., depletion of cells expressing CD14, CD16, CD19, CD25, CD56).

[0141] As used herein, the term “antigen-binding molecule” refers to any desired target molecule of a cell, i.e., any molecule that preferably binds to or is specific to an antigen. The term “antigen-binding molecule” includes, for example, antibodies or antibody fragments. As used herein, the term “antibody” refers to polyclonal or monoclonal antibodies. Antibodies may be of any species, e.g., mouse, rat, sheep, human, etc. When non-human antigen-binding fragments are used for therapeutic purposes, they may be humanized by any method known in the art. Antibodies may also be modified antibodies (e.g., oligomeric, reduced, oxidized, and labeled antibodies).

[0142] The term “antibody” includes both intact molecules and antibody fragments, such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies. Additionally, the term “antigen-binding molecule” includes any molecule other than antibodies or antibody fragments that preferentially bind to a desired target molecule in a cell. Suitable molecules include, without limitation, oligonucleotides known as aptamers that bind to a desired target molecule, carbohydrate, lectin, or any other antigen-binding protein (e.g., receptor-ligand interactions). The binding (coupling) between the antibody (or antibody fragment) and the microbeads may be covalent or non-covalent. Covalent bonding may, for example, be binding to a carboxyl group on a polystyrene bead, or to an NH2 or SH2 group on a modified bead. Non-covalent bonding may, for example, be via biotin-avidin or fluorophore-binding particles bound to an anti-fluorophore antibody.

[0143] A powerful sorting technique is magnetic cell sorting. Methods for magnetically separating cells are commercially available, for example, from Invitrogen, Stem cell Technologies (Cellpro, Seattle, or Advanced Magnetics, Boston). For example, monoclonal antibodies can be directly bound to magnetic polystyrene particles such as Dynabeads® or similar magnetic particles and used, for example, for cell separation. Cells are isolated by placing tubes on a magnetic rack. These microbeads can be directly bound to monoclonal antibodies or used in combination with anti-immunoglobulin, avidin, or anti-hapten specific microbeads. Cells can be separated by incubating them with magnetic microbeads coated with antibodies against one or more specific surface antigens. This causes cells expressing this antigen to adhere to the magnetic microbeads. In this method, cells can be separated as positive or negative with respect to a specific antigen. The procedure can be carried out using direct or indirect magnetic labeling. For direct labeling, the specific antibody is directly bound to the magnetic microbead. Indirect labeling is a convenient alternative when direct magnetic labeling is not possible or undesirable. A primary antibody against any cell surface marker, a specific monoclonal or polyclonal antibody, or a combination of primary antibodies can be used in this labeling strategy. The primary antibody may be unconjugated, biotinylated, or fluorophore-conjugated. Magnetic labeling is then achieved with anti-immunoglobulin microbeads, anti-biotin microbeads, or anti-fluorophore microbeads. The process described above can also be carried out in a closed cell sample processing system such as CliniMACS® (Miltenyi Biotec GmbH, Germany) or CliniMACS Prodigy® (Miltenyi Biotec GmbH, Germany).

[0144] The term “genetically modified cell” means a cell that contains and / or expresses an exogenous gene or nucleic acid sequence that modifies the genotype or phenotype of the cell or its offspring. In particular, the term refers to the fact that cells can be manipulated by recombinant methods known in the art to stably or transiently express peptides or proteins, such as CARs, that are not expressed in nature in these cells. Genetic modification of cells may include, but is not limited to, transfection, electroporation, nucleofection, retroviral vectors, lentiviral vectors, non-integrated retro or lentiviral vectors, transposons, designer nucleases including zinc finger nucleases, TALENs, or transduction using CRISPR / Cas.

[0145] Genetically modified immune cells, obtainable by the methods disclosed herein, may be used in subsequent steps such as research, diagnostic, pharmacological, or clinical applications, as known to those skilled in the art.

[0146] Genetically modified immune cells can also be used as pharmaceutical compositions in therapies, such as cell therapy, or in the prevention of disease. Pharmaceutical compositions can be transplanted into animals or humans, for example, human patients. Pharmaceutical compositions can be used for the treatment and / or prevention of diseases in mammals, particularly humans, and may include the administration of a pharmaceutically effective amount of the pharmaceutical composition to a mammal. The pharmaceutical compositions of this disclosure may be administered in a manner appropriate to the disease being treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, as well as the type and severity of the patient's disease, although an appropriate dose may be determined by clinical trials.

[0147] The composition of genetically modified immune cells obtained by this method may be administered either alone or as a pharmaceutical composition in combination with a diluent and / or other components such as cytokines or cell populations. In short, the pharmaceutical composition of the present invention may comprise genetically modified immune cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline or sulfate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0148] This method can efficiently produce high-quality immune cell preparations in a short period of 3-4 days. This method can be carried out in a closed system for immune cell preparation. In certain embodiments, this method uses CliniMACS Prodigy® as the primary culture system and Dynabeads® to activate the cells.

[0149] The TCR / CD3 complex is present on the surface of T cells. The first signal of cell activation is transmitted via CD3, thereby activating various signaling pathways in T cells. Subsequently, the second signal of T cell activation is generated by the interaction between CD28-binding molecules and CD28-costimulatory molecules on the surface of T cells, thereby inducing T cell proliferation and differentiation. The activation reagents Dynabeads®, TransAct®, and OKT3 all activate T cells through this activation principle.

[0150] Compared to the standard CliniMACS Prodigy® method, this method allows for a significant increase in sample volume. A sufficient amount of cell product with a high percentage of T-naive and T-cm cells can be obtained in as little as 3-4 days.

[0151] Samples that can be processed include, but are not limited to, fresh peripheral blood, fresh apheresis collections, cryopreserved apheresis collectibles, PBMC collectibles, T cells, NK cells, and the like.

[0152] The culture media that can be used in the process system include, but are not limited to, serum-containing media, low-serum media, and serum-free media.

[0153] The sample preparation process may use instruments including, but not limited to, Sepax 2, Sepax C-pro, Sepia, Lovo, CS 5+, CS Elite, and centrifuges.

[0154] The sample volume that can be processed may range from 5 to 500 ml. The centrifugal force for washing the sample may range from 100 to 1,000 g. The centrifugation time for washing the sample may range from 100 to 600 seconds. The sample dilution ratio may range from 0 (no dilution) to 5 times. The number of washing cycles may range from 1 to 5. The output volume after washing may range from 5 to 400 ml.

[0155] A fully automated, closed culture process can have short cycles of 3-4 days.

[0156] The initial loading of a single ball into the system is 1 × 10⁻⁶ 5 ~50×10 9 It can be within the range of cells. Cell sorting is, for example, CD4 + CD8 + CD62L + CD3 + CD56 + , and positive screening based on combinations thereof, as well as, for example, CD14 + CD19 + CD269 + This includes, but is not limited to, negative screening based on, and combinations thereof. The dose ratio is 50 to 5,000 × 10⁻⁶. 6 The number of labeled cells / ml may be in the range of [value missing].

[0157] The cell activation density is 0.1 × 10⁻⁶. 6 / ml~20×10 6 It could be in the range of / ml.

[0158] The activating reagents include, but are not limited to, TransAct®, microbeads described herein (e.g., Dynabeads®), anti-CD3 antibodies (e.g., OKT3 antibodies), and other activating reagents. The dosage range for TransAct® includes, but is not limited to, 0.1 ml to 30 ml. The microbead-to-cell ratio may be in the range of 0.1 to 10. The dosage range for OKT3 antibodies includes, but is not limited to, 10 ng / ml to 50 mg / ml.

[0159] The activation time can range from approximately 4 hours to approximately 96 hours. The amount of activated cells is 1 × 10⁻⁶. 5 ~20×10 9 It could be within the range.

[0160] Genetic modification of cells can be performed using non-viral transfection systems (electroporation, e.g., Neon transfection system, Maxcyte, etc.) or viral transduction systems (lentiviral systems, adenovirus systems, and adeno-associated virus vectors, etc.).

[0161] Regarding transduction, the MOI can range from 0 to 1,000. The titer of the viral vector is approximately 1 × 10⁻⁶. 5 TU / ml ~ approximately 10 x 10 10 It can be in the range of TU / ml. The cell density is 0.01 × 10⁻⁶. 6 / ml~20×10 6 It could be in the range of / ml.

[0162] The sample output volume during cell washing and concentration may range from 50 to 200 ml.

[0163] Cell washing and concentration devices include, but are not limited to, Sepax 2, Sepax C-pro, Sefia, Lovo, CS 5+, CS Elite, Prodigy, etc. The volume of the sample that can be processed includes, but is not limited to, 5 - 500 ml. The centrifugal force for washing the sample includes, but is not limited to, 100 - 1,000 g. The centrifugation time for washing the sample includes, but is not limited to, 100 - 600 seconds. The dilution rate of the sample includes, but is not limited to, 0 - 5 times. The number of washing cycles includes, but is not limited to, 1 - 5. The output volume after concentration includes, but is not limited to, 5 - 400 ml.

[0164] Cell dispensing devices include, but are not limited to, Sepax 2, Sepax C-pro, Sefia, Prodigy dispensing devices, etc. The volume of the sample that can be processed includes, but is not limited to, 5 ml - 500 ml. The output volume of dispensing includes, but is not limited to, 5 ml - 400 ml.

[0165] A comparison of the technical effects between this method and the standard CliniMACS Prodigy® method is shown in Table A below.

Table 1

[0166] Compared with the standard CliniMACS Prodigy® method, this method may include additional pretreatment steps. This method may include an improved selection process that significantly increases the selection recovery rate (from 35.7% to 80.2%) and the proportion of T-naïve after selection (from 31.2% to 68.4%). This also reduces the cost of selecting reagents. This method may improve the cell activation step. The number of cells to be processed is doubled (from 200×10 6 to 400×10 6When this is done, the percentage of activated cells (from 70.4% to 95.1%) and the positivity rate (from 31.2% to 68.4%) can still be significantly increased. Importantly, the duration of this method is shortened from 10-14 days (for the standard CliniMACS Prodigy® method) to 3-4 days. The number of positive cells and the T-naive / T-cm ratio at collection are significantly greater than those obtained by the standard CliniMACS Prodigy® method.

[0167] Table B compares each step of the present invention with the standard CliniMACS Prodigy® method. [Table 2]

[0168] In certain embodiments, the microbeads (e.g., Dynabeads®) are monodisperse / homogeneous, superparamagnetic, and polymer microspheres comprising γFe2O3 and Fe3O4 magnetic materials. The microbeads are coated with a layer of polymer material that acts as a carrier for adsorbing or binding antibodies specific to CD3 and / or CD28 cell surface molecules.

[0169] The main advantages of this method are as follows: This method can significantly increase the sample processing volume. A sufficient amount of cell products with a high percentage of T-naive and T-cm can be obtained in just 3-4 days. This method uses microbeads (e.g., Dynabeads®) to activate the cells. Compared with the activation reagent TransAct®, the number of cells produced and the positive rate are higher. This method can be used with a fully automated, closed system. The efficiency of immune cell preparation is high, and quality control is also good.

[0170] This disclosure provides a highly efficient integrated method for preparing immunocells. Compared to the standard CliniMACS Prodigy® method, this method includes additional pretreatment of apheresis collectibles, optimized cell sorting, optimized cell activation, and additional cell enrichment and dispensing steps.

[0171] Example 1 describes the sample pretreatment. Example 2 describes further treatment of the pretreated sample using an embodiment of this method. Example 3 describes further treatment of the pretreated sample using a method recommended by CliniMACS Prodigy®. In Example 4, an embodiment of this method was used, except that cell sorting was performed according to a method recommended by CliniMACS Prodigy®. In Example 5, an embodiment of this method was used, except that cell activation was performed according to a method recommended by CliniMACS Prodigy®.

[0172] This disclosure is further detailed below in relation to specific embodiments. These embodiments are used solely to illustrate the invention, but should not be understood as limiting the scope of the invention. Experimental methods in the following embodiments that do not specify particular conditions generally follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0173] Example 1: Sample Pretreatment Sample preparation was carried out as follows: 1) 10-100 ml of frozen apheresis collection was thawed. The total number of cells was determined. For use in Examples 3 and 6, 2 / 5 of the cell suspension was divided into two parts. 2) The cells were washed using the Sepax Pro "Culture Wash" program. The parameters were set as follows: "Initial volume" = 40 ml, "Final volume" = 60 ml, "Dilution ratio" = 2, "Number of wash cycles" = 2, "Intermediate volume" = 20 ml, "g-force" = 300 g, "Dilution rate" = 60 ml / min, "Settling time" = 360 seconds. The washing solution was HSA solution, and the final concentration of HSA was 0.1% to 30%. 3) After washing, a new sample bag was removed and the total number of cells was measured. 4) The cells were divided into three groups, which were to be used in Example 2, Example 4, and Example 5, respectively.

[0174] result Sample pretreatment with Sepax Pro resulted in good recovery rates for both monocytes and lymphocytes. Specific results are shown in Table 1. [Table 3]

[0175] Example 2: Short-cycle culture and harvesting experiment The pre-treated samples from Example 1 were subjected to a fully automated, closed culture system for a short period of 3-4 days. The specific procedure was as follows: 1) Cell preparation. Pre-treated samples from Example 1 were connected to the Prodigy® line. 2) Cell sorting. Apheresis samples were sorted positively using anti-CD4 and anti-CD8 antibodies. The sorting reagent was diluted with PBS-EDTA containing 0.1% to 10% HSA. The dilution method was as follows: 7.5 ml of antibody was dispensed into 1.5 ml / bottle, and then 3.5 ml of PBS-EDTA containing 0.1% to 10% HSA was added to prepare a 5 ml diluted sorting system. During sorting, positive sorting was performed using anti-CD4 and anti-CD8 antibodies. 3) Cell activation. Selected cells were activated with coated microbeads (e.g., Dynabeads®), and the cell activation state was analyzed by flow cytometry 24 hours after activation. The specific activation method was as follows: 10 × 10 6 ~1000×10 6 Individual cells (for example, 400 x 10 6 Cells were activated with coated microbeads (e.g., Dynabeads®), and the bead-to-cell ratio was in the range of 0.5–5 (e.g., approximately 1:1). The activation density was 0.5–10 × 10⁻⁶. 6 It was / ml. 4) Viral transduction. One to two days after cell activation, lentivirus was added at an MOI of 1 to 10 (e.g., 3). 5) Virus removal. After 1-2 days of incubation, the virus was removed using Prodigy®. 6) Cell culture and harvesting. The cells were cultured for a further 1-2 days and then harvested using Prodigy®. The harvested volume was 50-200 ml. 7) Cell concentration. Cells were concentrated using Sepax Pro with the following parameters: "Initial volume" = 100 ml, "Final volume" = 21 ml, "Dilution ratio" = 0, "Number of wash cycles" = 1, "Intermediate volume" = 20 ml, "g-force" = 300 g, "Dilution rate" = 60 ml / min, "Settling time" = 360 seconds. After the concentration step, the sample bag was removed and the concentration and volume of the cell suspension were measured. 8) Cell dispensing. Cells were dispensed using Sepax Pro, with dispensing volumes ranging from 10 to 80 ml. After the dispensing step, the sample bag was removed and the volume and cell density were measured.

[0176] result The above method achieved cell sorting, activation, transduction, and proliferation within 3-4 days, yielding the desired number of cells. The cell sorting recovery rate is shown in Table 2. [Table 4]

[0177] When Dynabeads® was used for activation, 95.1% of the cells were activated after 24 hours. Further culture allowed the cells to continue proliferating. Specific results are shown in Table 3. [Table 5]

[0178] T cells were cultured for a short period of 3-4 days in a completely closed, automated system. Cell subsets were assayed in apheresis collectibles after sorting on days 0, 1, 3, and 4. This method was found to generate a high proportion of T naive cells and Tcm cells. Specific results are shown in Table 4. [Table 6]

[0179] Cell samples were concentrated using Sepax Pro, which ensured a sufficient output volume and good recovery of monocytes and lymphocytes. Specific results are shown in Tables 5 and 6 below. The results indicate that the recovery rate of monocytes and lymphocytes was higher than 98%. [Table 7] [Table 8]

[0180] Cell samples were dispensed using Sepax Pro, which ensured consistency in output volume and cell density during dispensing. Specific results are shown in Table 7 below. The results demonstrate that the sample volume and concentration after dispensing substantially matched the theoretical values. [Table 9]

[0181] Example 3: Standard CliniMACS Prodigy® Method The standard CliniMACS Prodigy (registered trademark) method was performed without the pretreatment described in Example 1 as follows: 1) Cell preparation: The sample was connected to the Prodigy (registered trademark) line. 2) Cell sorting: The apheresis collection was positively sorted with anti-CD4 antibody and anti-CD8 antibody. The sorting reagent was used without dilution. 3) Cell activation: The sorted cells were activated with TransAct (trademark). The cell activation state was analyzed by flow cytometry 24 hours after activation. Specifically, 10×10 6 ~1,000×10 6 cells (e.g., 200×10 6 cells) were activated with TransAct (trademark). 4) Viral transduction: After culturing for 1 - 2 days, the required volume of lentivirus was added to achieve an MOI of 1 - 10 (e.g., 3). 5) Virus removal: After 1 - 2 days of incubation, the virus was removed using Prodigy (registered trademark). 6) Cell culture and collection: The cells were cultured for 14 days and then collected using Prodigy (registered trademark). The collection volume was 50 - 200 ml.

[0182] The experimental results are shown in Table 8.

Table 10

[0183] When TransAct (trademark) was used for activation, 70.4% of the cells were activated after 24 hours of culture. When further cultured, the cells continued to proliferate. The specific results are shown in Table 9.

Table 11

[0184] Cell culture was performed using the standard CliniMACS Prodigy® method. Cell subsets were assayed in apheresis collections after sorting on days 0, 1, 3, and 4. Specific results are shown in Table 10. [Table 12]

[0185] Compared to the embodiment of the present method described in Example 2, the method of this example did not include a cell pretreatment step and used the standard CliniMACS Prodigy® method for cell sorting and activation. The results showed that the method of this example yielded a lower cell sorting recovery rate after sorting, a lower cell activation percentage, a significantly lower final positivity rate, a significantly lower cell proliferation rate, and a significantly lower proportion of T naive and Tcm cells compared to the embodiment of the present method described in Example 2. In addition, the cost of cell sorting and activation in Example 2 was significantly lower than that of the standard CliniMACS Prodigy® method. Regarding culture time, Example 2 significantly reduced the culture time compared to the standard CliniMACS Prodigy® method.

[0186] Example 4 The culture experiment was performed using the sample pretreated in Example 1. The specific method is as follows: 1) Cell preparation: Pre-treated samples from Example 1 were connected to the Prodigy® line. 2) Cell sorting: Apheresis samples were sorted using anti-CD4 and anti-CD8 antibodies. The sorting reagents were used without dilution. 3) Cell Activation: Selected cells were activated with coated microbeads (e.g., Dynabeads®), and the cell activation state was analyzed by flow cytometry 24 hours after activation. The specific activation method was as follows: 10 × 10 6 ~1000×10 6 Individual cells (for example, 400 x 10 6Cells were activated with coated microbeads (e.g., Dynabeads®), and the bead-to-cell ratio was in the range of 0.5–5 (e.g., approximately 1:1). The activation density was 0.5–10 × 10⁻⁶. 6 It was / ml. 4) Viral transduction. One to two days after cell activation, lentivirus was added at an MOI of 1 to 10 (e.g., 3). 5) Virus removal: After 1-2 days of incubation, the virus was removed using Prodigy®. 6) Cell culture and collection: Cells were cultured for 4 days and then collected using Prodigy®. The collected volume ranged from 50 to 200 ml.

[0187] The experimental results are shown in Table 11. [Table 13]

[0188] When Dynabeads® was used for activation, 89.2% of cells were activated after 24 hours of culture. Further culture resulted in continued cell proliferation. Specific results are shown in Table 12. [Table 14]

[0189] Cell culture was performed using the standard CliniMACS Prodigy® method. Cell subsets were assayed in apheresis collections after sorting on days 0, 1, 3, and 4. Specific results are shown in Table 13. [Table 15]

[0190] Compared to the embodiment of the method described in Example 2, the method in this example used the standard CliniMACS Prodigy® cell sorting protocol. The results show that the cell sorting and recovery rate in this example was not significantly different from that of Example 2. Subsequent cell activation, cell proliferation, final positivity rate, T-naiveté at collection, and Tcm were also not significantly different from those in Example 2. In the sorting step of Example 2, the dispensing and sorting of magnetic beads significantly reduced costs and did not affect the subsequent state of the cells.

[0191] Example 5 The culture experiment was performed using samples from Example 1. The specific procedure was as follows: 1) Cell preparation: Pre-treated samples from Example 1 were connected to the Prodigy® line. 2) Cell sorting: Apheresis samples were positively sorted using anti-CD4 and anti-CD8 antibodies. The sorting reagent was diluted with PBS-EDTA containing 0.1% to 10% HSA. The dilution method was as follows: 7.5 ml of antibody was dispensed into 1.5 ml / bottle, and then 3.5 ml of PBS-EDTA containing 0.1% to 10% HSA was added to prepare a 5 ml diluted sorting system. During sorting, positive sorting was performed using anti-CD4 and anti-CD8 antibodies. 3) Cell activation: Selected cells were activated with TransAct (trademark). The cell activation state was analyzed by flow cytometry 24 hours after activation. Specifically, 10 × 10 6 ~1,000 x 10 6 Individual cells (for example, 200 × 10 6 Individual cells were activated with TransAct (trademark). 4) Viral transduction. One to two days after cell activation, lentivirus was added at an MOI of 1 to 10 (e.g., 3). 5) Virus removal. After 1-2 days of incubation, the virus was removed using Prodigy®. 6) Cell culture and collection. Cells were cultured for 4 days and then collected using Prodigy®. The collected volume ranged from 50 to 200 ml.

[0192] The experimental results are shown in Table 14. [Table 16]

[0193] When TransAct® was used for activation, 72.6% of cells were activated after 24 hours of culture. When cultured further, the cells continued to proliferate. Specific results are shown in Table 15. [Table 17]

[0194] Cell culture was performed using the standard CliniMACS Prodigy® method. Cell subsets were assayed in apheresis collections after sorting on days 0, 1, 3, and 4. Specific results are shown in Table 16. [Table 18]

[0195] The difference between the methods used in this example and Example 2 lies in the cell activation method. In this example, TransAct® was used for cell activation, while in Example 2, Dynabeads® was used. In this example, the cell activation ratio, cell proliferation, final positivity rate, and T-naive and T-cm at collection were significantly lower than in Example 2.

[0196] Example 6 The pre-treated samples from Example 1 were subjected to a fully automated, closed culture system for a short period of 3-4 days. The specific procedure was as follows: 1) Cell preparation. Pre-treated samples from Example 1 were connected to the Prodigy® line. 2) Cell sorting. Apheresis samples were sorted positively using anti-CD4 and anti-CD8 antibodies. The sorting reagent was diluted with PBS-EDTA containing 0.1% to 10% HSA. The dilution method was as follows: 7.5 ml of antibody was dispensed into 1.5 ml / bottle, and then 3.5 ml of PBS-EDTA containing 0.1% to 10% HSA was added to prepare a 5 ml diluted sorting system. During sorting, positive sorting was performed using anti-CD4 and anti-CD8 antibodies. 3) Cell Activation: Selected cells were activated with coated microbeads (e.g., Dynabeads®), and the cell activation state was analyzed by flow cytometry 24 hours after activation. The specific activation method was as follows: 10 × 10 6 ~1000×10 6 Individual cells (for example, 317 × 10⁻¹⁰ 6 Cells were activated with coated microbeads (e.g., Dynabeads®), and the bead-to-cell ratio was in the range of 0.5–5 (e.g., approximately 1:1). The activation density was 0.5–10 × 10⁻⁶. 6 It was / ml. 4) Viral infection. Lentivirus was added 1-2 days after cell activation at an MOI of 1-10 (e.g., 3). 5) Virus removal. After 1-2 days of incubation, the virus was removed using Prodigy®. 6) Cell culture and harvesting. The cells were cultured for a further 1-2 days and then harvested using Prodigy®. The harvested volume was 50-200 ml.

[0197] The experimental results are shown in Table 17. [Table 19]

[0198] When Dynabeads® was used for activation, 75.8% of the cells were activated after 24 hours of culture. Further culture resulted in continued cell proliferation. Specific results are shown in Table 18.

Table 20

[0199] The culture was carried out in an automatic mode that was completely closed for a short period of 3 to 4 days. Cell subsets were assayed in the apheresis collections after sorting on days 0, 1, 3, and 4. The specific results are shown in Table 19.

Table 21

[0200] The difference between the method used in this Example and Example 2 was that the cells were not pretreated in this Example. The results showed that the cell sorting recovery rate was significantly lower than that in Example 2. There was no significant difference in the cell activation ratio, final positive rate, T naive and Tcm at the time of collection. Due to the low recovery rate of cell sorting, the total number of cells at the time of collection was low during the 3 - 4 day culture period.

[0201] Example 7 This example further compares the effects of Dynabeads® and TransAct™ on cell activation and subsequent proliferation. The specific method was as follows: 1) Cell preparation. T cells were manually sorted from 1,000×10 6 apheresis collections. 400×10 6 cells were collected and equally divided into two parts for follow - up experiments. 2) Cell activation. 200×10 6 cells were activated with Dynabeads®. The specific activation method was as follows. 10×10 6 ~1,000×10 6 cells (for example, 200×10 6 cells) were activated with Dynabeads®. The bead - to - cell ratio was in the range of 0.5 - 5 (for example, about 1:1). The activation density was 0.5 - 10×10 6 / ml (for example, 2.86×10 6 / ml). Half of the cells from the other party (200 x 10 6 cells) were activated with TransAct (trademark). The specific activation method was as follows. 0.2 x 10 6 ~500 x 10 6 cells (for example, 200 x 10 6 cells) were activated with the bottle of TransAct (trademark). The activation density was 0.5~10 x 10 6 / ml (for example, 2.86 x 10 6 / ml). 3) Virus transduction. One to two days after cell culture (for example, transduction was performed one day after activation), lentivirus was added at an MOI of 1~10 (for example, 2). 4) Virus removal: After 1~2 days of incubation (for example, about 1 day), the sample was centrifuged to remove the lentivirus vector. 5) Cell culture and collection. The cells were cultured for an additional 1~2 days. Information on cell subsets during culture is shown in Tables 20, 21, Figures 2, and 3. Information on the number of cells during culture is shown in Tables 22 and Figure 4. The positive rate during culture is shown in Tables 23 and Figure 4.

Table 22

Table 23

Table 24

Table 25

[0202] The scope of this disclosure is not limited to what is specifically shown and described above. Those skilled in the art will recognize that there are suitable alternatives to the examples of materials, configurations, structures and dimensions shown. Numerous references, including patents and various publications, are cited and discussed in this description of the invention. Such citations and discussions are provided solely to clarify the description of this disclosure and do not constitute an endorsement that any reference is prior art to the invention described herein. All references cited and discussed herein are incorporated herein by reference in their entirety. Changes, modifications and other practices of what is described herein will be conceivable to those skilled in the art without departing from the spirit and scope of the invention. While embodiments of this disclosure have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the spirit and scope of the invention. Matters described herein and in the accompanying drawings are provided for illustrative purposes only and not as limitations.

Claims

1. A method for culturing genetically modified immune cells, (a) A step of washing a sample containing immune cells to obtain pre-treated immune cells, wherein the sample is peripheral blood, immune cells, monocyte collection, or peripheral blood mononuclear cells (PBMCs), (b) A step of selecting the pretreated immune cells to obtain concentrated immune cells, wherein the selection is a positive selection using anti-CD4 and / or anti-CD8 antibodies or fragments thereof, (c) A step of activating the concentrated immune cells with microbeads coated with an activator to obtain activated immune cells, wherein the activation is performed for 16 to 48 hours. (d) The step of obtaining genetically modified immune cells by genetically modifying the activated immune cells, (e) a step of increasing the gene-modified immune cells, The aforementioned immune cells are T cells, All steps of the above method are carried out over 2 to 5 days. method.

2. The method according to claim 1, wherein the microbeads have a diameter in the range of 1 μm to 10 μm, 2 μm to 8 μm, or 4 μm to 5 μm.

3. The method according to claim 1, wherein in step (c), the activation is performed in a microbead-to-cell ratio in the range of 0.1 to 10, 0.5 to 5, or 1 to 2.

4. The method according to claim 1, wherein the activator is selected from the group consisting of an antibody or a fragment thereof, a cytokine, a recombinant costimulatory molecule, a small drug inhibitor, and a combination thereof.

5. The method according to claim 1, wherein the activator is an anti-CD3 and / or anti-CD28 antibody or a fragment thereof.

6. The method according to claim 1, wherein steps (c), (d), and (e) are performed over 2 to 5 days, or 3 to 4 days.

7. The method according to claim 1, wherein all steps of the method are performed over three to four days.

8. In step (c), the activation is 0.5 × 10 ６ cells / ml~10×10 ６ Cells / ml range, or 2 × 10 ６ cells / ml~3×10 ６ The method according to claim 1, wherein the immune cell density is in the range of cells / ml.

9. The method according to claim 1, wherein in step (d), the gene modification is transfection or transfection.

10. The method according to claim 1, wherein step (d) involves introducing a polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) into the activated immune cells.

11. The method according to claim 1, wherein step (d) includes transducing the activated immune cells with a lentiviral vector, a gamma-retroviral vector, an alpha-retroviral vector, or an adenovirus vector.

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