Methods for producing immune cell cultures

Abstract

The present disclosure provides a method for producing immune cell cultures utilizing a completely closed system.

Description

[Technical field]

[0001] The present disclosure provides methods for producing immune cell cultures in a completely closed system. In particular, the present disclosure relates to processes for the upstream production and downstream processing of immune cell cultures. [Background technology]

[0002] Immune cell therapy is a class of disease treatment that uses genetically engineered immune cells to efficiently target and destroy cancer cells. For example, adoptive cell therapy uses CAR T cells that express chimeric antigen receptors designed to bind to certain proteins on cancer cells. The use of CAR T cells in the treatment of cancer has shown remarkable tumor specificity and robust antitumor immune responses, resulting in complete responses. To date, the FDA has approved four autologous CAR T cell therapy products: tisagenlecleucel for acute lymphoblastic leukemia (Novartis, 2017), axicabtagene siloreucel for large B-cell lymphoma (Gilead, 2017), brexcabtagene outrucel (Gilead) for mantle cell lymphoma in 2020 and relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL) in 2021, and risocabtagene maraleucel for relapsed or refractory large B-cell lymphoma (Bristol Myers Squibb, 2021) (Young, C. M., C. Quinn, and M. R. Trusheim, Durable cell and gene therapy potential patient and financial impact: US projections of product approvals, patients treated, and product revenues. Drug Discov Today, 2021).

[0003] Although autologous CAR T cell therapy shows remarkable efficacy, it suffers from several limitations. Autologous CAR T cell therapy requires T cell harvesting from the patient, followed by genetic modification to express CAR and expansion, which can take at least two weeks. During this process, T cell expansion depends on the quality characteristics of the input, and the inability to optimize the quality of the patient's T cells can lead to reduced yields, so invasive tumor progression can be fatal. Expansion of tumor-infiltrating leukocytes offers an attractive strategy, as lymphocytes are primed against multiple tumor-associated antigens. However, their ex vivo expansion is ineffective, as they are phenotypically exhausted and have limited replicative capacity. In addition to the above, the prohibitive cost of surgery poses a significant challenge to autologous cell therapy.

[0004] In contrast, allogeneic cell therapy may be available as an "off-the-shelf" product addressing the challenges of autologous cell therapy. With HLA-A, -B, and -DR matching potentially negating graft-versus-host disease (GVHD), cell therapy products generated from selected individuals may be applicable to a broader population. Thus, cell banks can be generated from optimal T cell subpopulations of healthy individuals, decreasing production costs while increasing applicability and efficacy. Thus, allogeneic cell therapy has the potential to break through the limitations of autologous cell therapy. Due to the promise of allogeneic cell therapy, clinical trials are underway to evaluate the efficacy of allogeneic, "off-the-shelf", CAR T cells targeting various tumor-associated antigens. Given the advances in cell therapy, especially on the allogeneic front, the need for industrial-scale production of T cell products is inevitable.

[0005] Although the number of CAR T cells per dose varies, estimates suggest a need for 3 trillion CAR T cells per year to treat hematological malignancies and 150 trillion CAR T cells for solid tumor malignancies. Scaling up static 2D flask-based cultures to meet the required batch sizes would result in additional labor, laboratory footprint, variability, risk of contamination, and poor process control. Scale-up transition kinetics have been well characterized for stirred tank bioreactors (STRs), thus providing an excellent option to meet the demand. Furthermore, because STRs are closed, automated, GMP-compatible systems with in-process controls, T cell manufacturing in STRs significantly reduces labor, batch-to-batch variability, and contamination risk. T cells are non-adherent and are essentially cultured in suspension as single cells. This makes suspension-based bioreactors suitable for their expansion without the need to artificially adapt them to suspension culture conditions and culture them in aggregates or attached to carriers. Like other cell types, T cell proliferation is sensitive to the accumulation of cellular metabolites, such as ammonia and lactate, in the culture medium and therefore requires medium replenishment. Despite the fact that fresh nutrients are supplied and metabolites are removed using fed-batch culture, inhibitors can accumulate and inhibit growth in the intervals between fed-batch medium exchanges, ensuring continuous medium exchange via perfusion. The properties that make T cells suitable for growth in 3D-based suspension cultures also present challenges for automated continuous medium exchange. Perfusion of T cell cultures is challenging, as T cells range in diameter from 5 to 10 mm, the main contributing factors being frequent filter fouling and cell leakage. Thus, there is an urgent need for scale-up technologies with continuous medium perfusion that enable T cell proliferation. Summary of the Invention

[0006] In some embodiments, a method for producing an immune cell culture in a completely closed system, comprising obtaining immune cells, introducing the immune cells into a stirred tank bioreactor containing immune cell complete medium, activating the immune cells with an activation reagent in the stirred tank bioreactor to produce activated immune cells, expanding the activated immune cells in the stirred tank bioreactor to produce an expanded immune cell culture, replacing a defined amount of fresh medium with spent medium via an alternating tangential flow filtration (ATF) connected to the bioreactor, depleting the expanded immune cell culture in the stirred tank bioreactor to produce an depleted immune cell culture, harvesting the depleted immune cell culture in the completely closed system to produce a harvested immune cell culture, and concentrating the harvested immune cell culture in the completely closed system, wherein the method results in a loss of less than 1% of the immune cell culture. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows a flow diagram for the production of immune cell cultures according to embodiments herein. [Figure 2A] 1 shows the proliferation of T cells in agitated compared to 2D static culture as described in the embodiments herein. [Figure 2B] 1 shows the proliferation of T cells in agitated compared to 2D static culture as described in the embodiments herein. [Figure 2C] 1 shows the proliferation of T cells in agitated compared to 2D static culture as described in the embodiments herein. [Figure 3A] 1 shows the expansion of activated T cells in a stirred tank bioreactor and continuous cell culture medium perfusion as described in embodiments herein. [Figure 3B] 1 shows the expansion of activated T cells in a stirred tank bioreactor and continuous cell culture medium perfusion as described in embodiments herein. [Figure 3C]1 shows the expansion of activated T cells in a stirred tank bioreactor and continuous cell culture medium perfusion as described in embodiments herein. [Figure 3D] 1 shows the expansion of activated T cells in a stirred tank bioreactor and continuous cell culture medium perfusion as described in embodiments herein. [Figure 3E] 1 shows the expansion of activated T cells in a stirred tank bioreactor and continuous cell culture medium perfusion as described in embodiments herein. [Figure 4A] 1 shows the phenotypic characteristics of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described in embodiments herein. [Figure 4B] 1 shows the phenotypic characteristics of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described in embodiments herein. [Figure 4C] 1 shows the phenotypic characteristics of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described in embodiments herein. [Figure 5A] 1 shows the functional status of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described herein. [Figure 5B] 1 shows the functional status of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described herein. [Figure 5C] 1 shows the functional status of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described herein. [Figure 5D] 1 shows the functional status of T cells activated in a stirred tank bioreactor with ATF-mediated continuous perfusion as described herein. [Figure 6A] 1 shows the efficiency of T cell depletion in a closed system according to embodiments herein. [Figure 6B] 1 shows the efficiency of T cell depletion in a closed system according to embodiments herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Allogeneic T cells are key immunotherapeutic cells to combat cancer and other clinical indications. High T cell doses per patient and increasing patient numbers will result in clinical demand for large numbers of allogeneic T cells. This requires a manufacturing platform that can be scaled up while retaining cell quality. Allogeneic CAR T cells can be used as an "off-the-shelf" cell therapy, and are expected to increase the population applicability of cell therapy products on a broader scale. Current estimates suggest the need for batch sizes of 2000L to meet the demand for CAR T cells used in hematological and solid tumor malignancies. Considering the large footprint, risk of contamination, variability, and poor process control in static 2D culture, stirred tank bioreactors provide an excellent platform for cell expansion of T cells, offering well-characterized scale-up kinetics, in-process control, and low risk of contamination. Converting the 2D growth process in stirred tank bioreactors to 3D growth has been successfully demonstrated in the case of adherent cells. However, the absence of intrinsic perfusion capacity of STRs, and the small size of T cells (5-10 mm diameter), proved to be formidable obstacles to achieving high yields of T cells in STRs.

[0009] Presented in this disclosure is a closed, scalable platform for T cell manufacturing to meet clinical demand. The upstream manufacturing steps of T cell activation and expansion are performed in-vessel in a stirred tank bioreactor. Selection of T cells required for CAR-T based therapy is performed in the bioreactor itself, thus maintaining optimal culture conditions throughout the selection step. The automated nature of the platform and performing the T cell activation, expansion, and selection steps in-vessel contributes greatly to improved process control, cell quality, and reduced manual labor and contamination risks. In addition, the feasibility of integrating a closed, automated, downstream process of cell enrichment is demonstrated. The presented T cell manufacturing platform has scale-up capabilities while retaining the critical factors of cell quality and process control. The present disclosure provides a GMP compatible, closed, scalable platform for T cell expansion in a perfusable STR. In addition, the present disclosure provides an in-unit and potentially scalable cell depletion magnetic technology, avoiding unit operations and lowering contamination and labor risks.

[0010] Published patents, patent applications, websites, company names, and scientific literature mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Similarly, any conflict between the art definition of a word or phrase specifically taught in this specification and the definition of the word or phrase shall be resolved in favor of the latter.

[0011] The use of the words "a" or "an," when used in conjunction with the term "comprising" in the claims and / or specification, may mean "one," but may also be consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0012] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value. Typically, the term is meant to include a variation of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, depending on the context.

[0013] Although use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0014] As used in the specification and claim(s), the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of including, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, device, system, and / or composition of the invention.

[0015] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Reference is made herein to various methodologies and materials known to those skilled in the art.

[0016] In embodiments, provided herein is a method for producing immune cell cultures in a completely closed system. Suitably, the method is for automated upstream production and downstream processing of immune cell cultures.

[0017] In embodiments, provided herein are methods for introducing immune cells into a stirred tank bioreactor containing immune cell complete medium.

[0018] As referred to herein, the word "introducing" may mean adding immune cells to a stirred tank bioreactor, or it may refer to the presence of immune cells in the stirred tank bioreactor prior to starting the method.

[0019] "Immune cells" produced and processed by the upstream production and downstream processing of the methods, respectively, refer to cells of the immune system that have been modified or primed (e.g., via co-culture with antigen-presenting cells) to result in cells with a desired phenotype useful for the treatment, prevention, or amelioration of one or more diseases in animals, including humans. As used herein, "immune cell culture" refers to a collection of cells prepared by the methods described herein and can include cell populations for use in research or clinical trials, and for administration to mammals, including human patients, for medical therapy. Genetically modified immune cell cultures that can be produced using the methods described herein can include mast cells, dendritic cells, natural killer cells (NK cells), B cells, T cells, and the like.

[0020] In an exemplary embodiment, the method includes activating immune cells with an activation reagent to produce activated immune cells, expanding the immune cells, exchanging a defined amount of fresh medium with spent medium via an alternating tangential flow filtration (ATF) connected to the bioreactor, depleting the expanded immune cell culture in a stirred tank bioreactor to produce a depleted immune cell culture, harvesting the depleted immune cell culture in a fully closed system to produce a harvested immune cell culture, and concentrating the harvested immune cell culture in a fully closed system, wherein the method results in a loss of less than 1% of the immune cell culture.

[0021] In embodiments, the resulting immune cell culture loss is less than 0.99%, less than 0.95%, 0.9%, less than 0.85%, less than 0.80%, less than 0.75%, less than 0.70%, less than 0.65%, less than 0.60%, less than 0.55%, less than 0.50%, less than 0.45%, less than 0.40%, less than 0.35%, less than 0.30%, less than 0.25%, less than 0.20%, less than 0.15%, and less than 0.10%.

[0022] In embodiments, the immune cells are isolated from a population of peripheral blood mononuclear cells (PBMCs) immediately prior to obtaining the immune cells, or are isolated from a population of PBMCs and stored for an extended period of time and then thawed prior to obtaining the immune cells.

[0023] As referred to herein, "isolating" immune cells means separating immune cells from the matrix (cells, tissues, fluids, etc.) in which the product is produced. Suitably, isolating immune cells may include subjecting immune cells to a series of mechanisms including, but not limited to, washing, magnetic application, columns, filtration, membranes, centrifuges, and other isolation processes known in the art. The word "isolate" is synonymous with the word "purify" in this application.

[0024] In embodiments, the immune cells are derived from a population of pluripotent stem cells. In further embodiments, the population of pluripotent stem cells is a population of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or a combination thereof.

[0025] As referred to herein, "deriving" immune cells from a population of pluripotent stem cells means generating immune cells in vitro from hematopoietic progenitor cells present in the hematopoietic zone within the bone marrow.

[0026] In the embodiment, 0.25×10 6 T cells / mL~2×10 6 In another embodiment, 0.1×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~0.5×10 6 In another embodiment, 0.2×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~0.5×10 6 In another embodiment, 0.3×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~0.6×10 6 In another embodiment, 0.4×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~0.7×10 6 In another embodiment, 0.5×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~0.8×10 6 In another embodiment, 0.6×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~0.9×10 6 In another embodiment, 0.7×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~1.0×10 6 In another embodiment, 1.0×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~1.5×10 6 In another embodiment, 1.5×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~1.7×10 6In another embodiment, 1.7×10 T cells / mL are introduced into the bioreactor. 6 T cells / mL~2.0×10 6 T cells / mL are introduced into the bioreactor.

[0027] In an embodiment, the method comprises: 6 cells / mL ~ approx. 90×10 6 In an embodiment, the method produces an immune cell culture comprising about 90×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 100×10 6 In an embodiment, the method produces an immune cell culture comprising about 100×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 200×10 6 In an embodiment, the method produces an immune cell culture comprising about 200×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 300×10 6 In an embodiment, the method produces an immune cell culture comprising about 300×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 400×10 6 In an embodiment, the method produces an immune cell culture comprising about 500×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 600×10 6 In an embodiment, the method produces an immune cell culture comprising about 600×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 700×10 6 In an embodiment, the method produces an immune cell culture comprising about 800×10 cells / mL of viable immune cells. 6 cells / mL ~ approx. 900×10 6 In an embodiment, the method produces an immune cell culture comprising about 1.0×10 cells / mL of viable immune cells. 7 cells / mL ~ approx. 2.0×10 7 Produce immune cell cultures containing 1000 cells / mL of viable immune cells.

[0028] In embodiments, immune cells are activated with an activation reagent in a stirred tank bioreactor to produce activated immune cells. In further embodiments, activating the immune cells comprises stirring the medium with the activation reagent for a period of about 72 hours at 37° C. In embodiments, the immune cell culture is stirred at a tip speed of 0.15 to 0.5 revolutions per minute (RPM). In embodiments, the immune cell culture is stirred at a tip speed of 0.6 to 0.8 revolutions per minute (RPM). In embodiments, the immune cell culture is stirred at a tip speed of 0.8 to 1.0 revolutions per minute (RPM).

[0029] In an embodiment, the immune cell culture medium has a pH of about pH 5.0 to about pH 7.5 during the activation period. In an embodiment, the immune cell culture medium has a pH of about pH 5.0 to about pH 5.5 during the activation period. In an embodiment, the immune cell culture medium has a pH of about pH 5.5 to about pH 6.0 during the activation period. In an embodiment, the immune cell culture medium has a pH of about pH 6.0 to about pH 6.5 during the activation period. In an embodiment, the immune cell culture medium has a pH of about pH 6.5 to about pH 7.0 during the activation period. In an embodiment, the immune cell culture medium has a pH of about pH 7.0 to about pH 7.5 during the activation period.

[0030] Preferably, the activation reagent comprises a soluble antibody complex. In embodiments, the activation reagent comprises an antibody that is a soluble antibody, including at least one of an anti-CD3 antibody and an anti-CD28 antibody. Exemplary antibodies include OKT3.

[0031] In other embodiments, the activating reagent comprises an antibody or a dendritic cell. In embodiments, the antibody is immobilized on a surface, which may include polystyrene plastic, silicone, or other surfaces, including, for example, the surface of a bead.

[0032] In an embodiment, the activated immune cells are expanded in a stirred tank bioreactor to produce an expanded immune cell culture. As described herein, the method of expanding cells preferably includes at least one or more of adding fresh medium to the bioreactor, feeding, washing, monitoring, and adjusting the conditions of the immune cell culture. In a further embodiment, the expanding further includes sampling the expanding immune cell culture, determining the cell growth and expansion fold of the expanding T cell culture, and replacing a defined amount of fresh medium with spent medium based on the cell growth and expansion fold. Exemplary conditions include temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density.

[0033] Various methods described herein are carried out in such a way that oxygen levels of the expanding immune cell cultures are optimized for the immune cell cultures. This optimization allows for the production of large numbers of viable cells having desired phenotypic characteristics, including promotion of a desired cell phenotype, as described herein. In embodiments, oxygen levels or concentrations are optimized during one or more of steps (d)-(f) by an alternating tangential flow filtration system that recirculates T cell complete medium through an oxygenation component.

[0034] In further embodiments, the alternating tangential flow filtration system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during various method processes, which aids in the production of large numbers of viable cells having the desired phenotype(s).

[0035] Other mechanisms for optimizing cell growth conditions include modifying and controlling the flow rate of the medium provided to the cells: as the cells begin to grow, the circulation rate of the medium provided increases, improving gas exchange and allowing oxygen and carbon dioxide to enter and leave the cell culture as conditions require.

[0036] In embodiments, the system is configured to perform several rounds of one or more of feeding, washing, and monitoring, and in embodiments, selection of the expanded immune cell culture. These various operations may be performed in any order, alone or in combination with other actions. In embodiments, the concentration of cells includes centrifugation, removal of supernatant after sedimentation, or filtration. Preferably, the optimization process further includes adjusting the parameters of centrifugation or filtration in a self-regulating process. Depletion of the expanded cell culture may be performed, for example, by magnetic separation, filtration, adhesion to beads, plastic, or other substrates, etc.

[0037] In embodiments, fresh medium is added to grow immune cell cultures at a viable cell density of 1.5×10 6 The spent medium is replaced at a rate of 1 vessel volume per day (VVD) when the viable cell density of the growing immune cell culture is greater than 1.4×10 cells / mL. In embodiments, fresh medium is added when the viable cell density of the growing immune cell culture is greater than 1.4×10 cells / mL. 6 The spent medium is replaced at a rate of 1 vessel volume per day (VVD) when the viable cell density of the growing immune cell culture is greater than 1.3×10 cells / mL. In embodiments, fresh medium is added when the viable cell density of the growing immune cell culture is greater than 1.3×10 6 The spent medium is replaced at a rate of 1 vessel volume per day (VVD) when the viable cell density of the growing immune cell culture is greater than 1.2×10 cells / mL. In embodiments, fresh medium is added when the viable cell density of the growing immune cell culture is greater than 1.2×10 6 The spent medium is replaced at a rate of 1 vessel volume per day (VVD) when the viable cell density of the growing immune cell culture is greater than 1.0×10 cells / mL. In embodiments, fresh medium is added when the viable cell density of the growing immune cell culture is greater than 1.0×10 6 If greater than cells / mL, spent medium is replaced at a rate of 1 vessel volume / day (VVD).

[0038] In an embodiment, depleting the immune cell culture comprises adding surface-activated magnetic beads to the immune cell culture after expansion, agitating the expanded immune cell culture and beads for about 30 minutes, and isolating the population of target cells from the expanded immune cell culture with a magnet. In another embodiment, depleting the immune cell culture comprises physical separation methods known in the art, including using counterflow centrifugal elution, fractionation in a density gradient, or differential agglutination with lectins followed by resetting with sheep red blood cells. In another embodiment, depleting the immune cell culture comprises immunological methods known in the art that utilize antibodies, either alone or in conjunction with homologous, heterologous, or rabbit complement factors directed against T cells. In another embodiment, depleting the immune cell culture comprises using a combination of physical separation and immunological methods described herein.

[0039] In an exemplary embodiment, the stirred tank bioreactor contains immune cell culture medium prior to starting the process, in other embodiments, fresh immune cell culture medium can be added separately after the start of the production process or at any suitable time during the process.

[0040] In other embodiments, provided herein is a method for promoting a preferred phenotype of an immune cell culture, the method comprising activating an immune cell culture in a stirred tank bioreactor to produce an activated immune cell culture, and expanding the activated immune cells in the stirred tank bioreactor to produce an expanded immune cell culture, the activation and expansion conditions promoting a phenotype and functional state of the immune cell culture. Exemplary phenotypes include stemness, ability to produce cytokines, central memory, effector memory, and naive / stem memory. Exemplary functional states include cytokine production. As described herein, the method is suitably performed by a fully closed automated cell engineering system.

[0041] In embodiments, the activation conditions provide a substantially undisturbed immune cell culture that allows for stable contact between the activation reagent and the immune cell culture. As described herein, it has been found that allowing the cells to be activated under substantially undisturbed conditions, through the use of a stirred tank bioreactor, provides an environment in which the cells can be homogenously contacted with the activation reagent and can interact with the necessary nutrients, dissolved gases, etc. to achieve the desired promoted phenotype.

[0042] The methods described herein can affect the properties of the final immune cell culture product by selecting an appropriate activation method to provide a preferred phenotype. For example, activation utilizing the bead-based process described herein promotes a more balanced CD4:CD8 ratio, while the use of soluble anti-CD3 promotes a higher population of CD8 than CD4. Other levels of CD8 and CD4 may also be provided using the methods described herein. In an exemplary embodiment, the methods may be utilized to prepare CAR T cells as described herein. Suitably, the methods may be utilized to promote a phenotype of CAR T cells having a ratio of CD8+ cells to CD4+ cells of about 0.1:1 to about 10:1, including ratios of CD8+ cells to CD4+ cells of about 0.5:1 to about 5:1, about 0.8: to about 3:1, or about 1:1, about 2:1, etc.

[0043] As described herein, it has been surprisingly found that by allowing cells to grow under conditions where the cells are not shaken (i.e., not rotated or shaken so that the cells flow over each other), the method provides high viable cell yields and optimal cell characteristics, including the desired phenotype. It has been determined that large, non-shaking cell culture chambers can provide cells with homogenous access to necessary reagents, nutrients, gas exchange, etc., without the need to shake or disturb the cells to achieve the desired outcome, while removing cellular waste products. Indeed, as described herein, such methods for automated production of genetically modified immune cells have been found to produce more viable cells, greater numbers / ratios of desired cell types, and more robust cell characteristics, as compared to methods utilizing cell shaking, such as those described in, for example, Miltenyi et al., “Sample Processing System and Methods,” U.S. Patent No. 8,727,132.

[0044] In embodiments, the various steps of the method are performed in a completely closed system and optimized throughout the process of producing immune cell cultures.

[0045] Suitably, the methods described herein include one or more sensors and / or mechanisms for detecting and / or regulating one or more of the following: temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density of the immune cell culture.

[0046] As used herein, a "bioreactor" may include a fermenter or fermentation unit, or any other reaction vessel. For example, in some aspects, an exemplary bioreactor unit may perform one or more or all of the following: feeding nutrients and / or carbon sources, injection of a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. The methods described herein may be utilized in conjunction with any suitable bioreactor, including, but not limited to, stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or entrained bed bioreactors. Any suitable reactor diameter may be used. In some embodiments, the bioreactor allows for agitation or mixing of liquid media by continuous and repetitive movement therethrough.

[0047] In embodiments, the bioreactor can have a volume capacity of about 1 L to about 2000 L. Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, and the like. Examples of volumetric capacities include: 1.5 torr, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters. Additionally, suitable reactors may be multi-use, single use, disposable, or non-disposable and may be formed of any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastic, and / or glass.

[0048] Stirred tank bioreactors utilize agitation as a mechanism to ensure uniform distribution of gases and nutrients. To determine whether T cells can proliferate with agitation, T cells activated and cultured at different agitation rates in spinner flasks were compared to cells grown in 2D static flasks. Cell counts performed at various time points throughout the run showed that the viable cell density (VCD) and total number of T cells in 2D static cultures were consistently lower than stirred conditions (Figures 2A and 2B). Agitation at a constant 75 RPM resulted in a higher VCD of T cells compared to the VCD when agitation was initiated at 50 RPM and increased to 100 RPM on day 5 (Figure 2A). Furthermore, agitation at 75 RPM resulted in a 15-fold increase in T cell proliferation compared to the 10-fold increase in cell proliferation observed when agitation was initiated at 50 RPM and increased to 100 RPM (Figure 2B). Maintaining the fed-batch medium exchange regime and the range of tip speeds tested in spinner flasks, T cell proliferation in a 1 L stirred tank bioreactor (STR) was evaluated at two different agitation speeds. As shown in Figure 2C, agitation at 88 RPM resulted in a higher viable cell density of T cells compared to agitation at 65 RPM.

[0049] Increased cell density is accompanied by nutrient depletion and accumulation of inhibitory metabolites. Continuous medium perfusion provides optimal culture conditions and allows cell proliferation. To evaluate whether continuous medium perfusion can be performed using alternating tangential flow (ATF) without cell loss, 3.0 × 10 cells were cultured in 2 L of culture medium in a 3 L STR. 6The effect of 24-h medium perfusion with ATF on T cells inoculated at 1000 cells / mL was tested. As shown in Table 1, no significant decrease in the VCD of T cells was observed after 24 h of perfusion. This was accompanied by the absence of T cells in the waste bag, indicating that ATF-mediated medium exchange did not result in cell loss. Furthermore, evaluation of the weight of the waste bag after 24 h of perfusion indicated that continuous medium perfusion of one vessel volume per day (1 VVD) was achieved without filter fouling. To evaluate whether T cell expansion could be achieved in STR with continuous medium perfusion, CD3+ T cells were isolated from peripheral blood mononuclear cells (PBMNC), inoculated into a stirred tank bioreactor, and activated as described in Materials and Methods. Phenotypic evaluation of the cells after isolation indicated that 98% of the cells were CD3+ (Figure 3A) and the viability was greater than 98% (data not shown). After inoculation and activation in the STR, T cell proliferation was monitored over a 14-day period, with a T cell VCD of 2.0 × 10 6 The results showed that the viable cell density reached 33.5 × 10 cells / mL on day 14 (Figure 3B). With the increase in cell density, a concomitant increase in lactate levels was observed (Figure 3C). Cell maintenance and medium exchange with ATF at 1VVD suppressed lactate accumulation, with viable cell densities reaching 33.5 × 10 6 In comparison, T cells isolated from the same donor and cultured in static mode in G-Rex® reached 3.4 × 10 cells / mL on day 14 (Figure 3C). 6 This resulted in a VCD of 100 cells / mL (Figure 3B). In addition to avoiding lactate accumulation, ATF-mediated perfusion at 1VVD allowed for nutrient replenishment, as shown by the stable glucose levels during cell growth in Figure 3D. Furthermore, agitation in a stirred tank bioreactor combined with ATF-mediated cell transfer did not result in cell death, as demonstrated via a stable >96% cell viability (Figure 3E).

[0050] Achieving a high cell expansion fold is key to process scale-up. However, this characteristic does not promise to achieve performance when cell quality is suboptimal. Evaluation of the CD4:CD8 T cell ratio of T cells expanded in stirred tank bioreactors showed that the CD4:CD8 T cell ratio at inoculation was maintained during expansion with a gradual shift toward CD8+ T cells over time (Figure 4A). To determine the impact of expansion in stirred tank bioreactors on T cell phenotype, T cell phenotype during and after expansion was evaluated compared to T cell phenotype before inoculation. As shown in Figure 4B, T cell expansion resulted in approximately 80% central memory T cells, less than 10% effector memory subsets, and approximately 15% naive / stem memory subsets. Furthermore, expansion did not result in the accumulation of terminally differentiated T cells (Figure 4B), senescent T cells, or exhausted T cells (Figure 4C).

[0051] To assess the functional status of T cells following expansion in stirred tank bioreactors, the ability of T cells to produce cytokines following stimulation was evaluated. Isolation of CD4+ and CD8+ T cells from T cell samples obtained on days 0 and 14 yielded >98% pure populations (Figure 5A). Cells were stimulated, stained, and assessed for cytokine production as described in Materials and Methods. As shown in Figure 5B, the number of cells producing multiple cytokines, indicative of multifunctionality, is maintained throughout expansion. Compared to day 0, day 14 samples showed an increase in the number of cells producing more than 5 cytokines. Although at a low frequency, day 14 T cells produced the most cytokines, with CD4+ T cells producing 11 cytokines and CD8+ T cells producing 9 cytokines (Figure 5C). Furthermore, cytokine signature-based classification of cell types suggests an increase in effector multifunctional strength index while retaining the stimulatory signature (Figure 5D).

[0052] As mentioned above, depletion of TCR positive cells is necessary to enable allogeneic CAR T cell based therapy. The concentration of unwanted T cells varies based on the cell editing technology and delivery platform, and can result in low or high concentrations after expansion. As a proof of concept, depletion of CD4+ T cells at low (17%) and high (52%) concentrations was evaluated. Depletion of CD4+ T cells for 30 minutes using the proprietary magnetic technology resulted in over 99% depletion at lower cell concentrations and approximately 97% depletion at higher concentrations (Table 2 and Figure 6A). To evaluate the time required to deplete CD4+ T cells and beads at higher densities, a time course of bead depletion was performed. As shown in Figure 5B, application of a magnetic field for 90 minutes depletes the beads as indicated by visual bead counts after sample collection. Assessment of the remaining bead percentage showed that after 120 minutes of bead depletion with a magnet, less than 0.001% beads were present, with no loss of cell viability and CD8+ T cells (Table 3 and Figure 6B).

[0053] Two mutually exclusive systems, ekko™ (Millipore-Sigma) and kSep400 (Sartorius), were evaluated for closed post-harvest cell concentration. Closed cell harvest from the STR into harvest bags was performed as described in Materials and Methods. Cells from the bags were transferred to each of the concentrators in a closed manner. As shown in Table 4, cell concentration using ekko™ resulted in a 7-fold concentration, 0% loss of cell viability, with a final cell viability of 98.5% and cell recovery of 79%. Similarly, cell concentration using kSep 400 resulted in a 7.65-fold concentration, 6% loss of cell viability, with a final cell viability of 89.4% and cell recovery of 69%. EXAMPLES

[0054] Isolation of T cells from PBMCs Human PBMCs (Lonza Cat. No. 4W-270C) were thawed quickly in a 37°C water bath until a small amount of ice remained in the vial. Thawed cells were added dropwise to EasySep™ buffer (Stem Cell Technologies Cat. No. 20144). Cells were centrifuged at 300 RCF for 5 min at room temperature (RT). Supernatant was discarded and cells were reconstituted in 50 mL EasySep™ buffer. Cell concentration and viability were assessed using a NucleoCounter NC-200 (Chemometec, Denmark). Cells were centrifuged again at 300 RCF for 5 min at RT. Supernatant was discarded and cells were reconstituted at 50×106 cells / mL in X-VIVO™15 serum-free hematopoietic cell medium (Lonza Cat. No. 04-418Q). Samples were aliquoted for immunophenotypic staining. T cells were isolated using a T cell isolation kit (Stem Cell Technologies catalog number 17951) according to the manufacturer's protocol. After T cell isolation, cell concentration and viability were assessed using a NucleoCounter NC-200 (Chemometec, Denmark) and samples were aliquoted for immunophenotypic staining.

[0055] T cell activation and expansion in spinner flasks T cell complete medium was prepared by adding human AB serum (Sigma Cat. No. H4522) to a final concentration of 5% and recombinant human IL-2 (Peprotech Cat. No. 200-02) to a final concentration of 50 ng / mL to X-VIVOTM15 (Lonza Cat. No. 04-418Q). After T cell isolation (as described in 4.1), cells were seeded at 1×106 cells / mL in 40 mL of T cell complete medium in spinner flasks. Cells were stimulated with ImmunoCultTM Human CD3 / CD28 T cell activator (Stem Cell Technologies Cat. No. 10991) for 3 days according to the manufacturer's instructions. Medium was added on day 5 to adjust the cell density to 1×106 cells / mL and medium was doubled on day 8. Agitation in the spinner flasks in one condition was maintained at 75 RPM for the entire 11 days, while agitation in the other condition was increased to 50 RPM starting from days 1 to 4, then 100 RPM from days 5 to 11. Cell numbers and viability were determined by sampling on days 4, 7, 8, and 11 of culture.

[0056] T Cell Activation and Proliferation in G-Rex® T cell complete medium was prepared by adding human AB serum (Sigma Cat. No. H4522) to a final concentration of 5% and recombinant human IL-2 (Peprotech Cat. No. 200-02) to a final concentration of 50 ng / mL to X-VIVOTM15 (Lonza Cat. No. 04-418Q). After T cell isolation (described in 4.1), 1 L of G-Rex® (Wilson Wolf Cat. No. G-Rex® 100M-CS) was inoculated with T cells at 0.5×106 cells / mL in 300 mL of T cell complete medium. T cell activation was performed in G-Rex® using ImmunoCult™ human CD3 / CD28 T cell activator (Stem Cell Technologies Cat. No. 10991) according to the manufacturer's instructions for 3 days. T cell cultures in G-Rex® were maintained at 37° C. in a humidified atmosphere with 5% CO2. After activating the T cells for 3 days, 700 mL of complete T cell medium was added and cultured until the 14th day.

[0057] T cell activation and proliferation in stirred tank bioreactors A BioBlu single-use bioreactor vessel was configured according to the manufacturer's instructions (Eppendorf, 1386000300). Briefly, a 1 L vessel was equipped with the necessary probes (Mettler Toledo) for online monitoring of key parameters including percentage of dissolved oxygen (DO), pH, and temperature. The bioreactor was controlled using a G3Lab universal control device (Thermo Fisher Scientific). T cell complete medium was prepared by adding human AB serum (Sigma catalog no. H4522) to a final concentration of 5% and recombinant human IL-2 (Peprotech catalog no. 200-02) to a final concentration of 50 ng / mL to X-VIVOTM15 (Lonza catalog no. 04-418Q). Prior to inoculation, 400 mL of T cell complete medium was introduced into the bioreactor and equilibrated with air. After T cell isolation, the bioreactor was seeded with 200×106 T cells at a seeding density of 0.5×106 cells / mL. T cell activation was performed in the bioreactor for 3 days using ImmunoCult™ Human CD3 / CD28 T Cell Activator (Stem Cell Technologies Catalog No. 10991) according to the manufacturer's instructions. T cell cultures in the bioreactor were maintained at 37° C., 88 RPM agitation (unless otherwise stated), and pH<7.2. After 3 days of T cell activation, 600 mL of T cell complete medium was added. To determine cell growth and fold expansion, 15 mL samples were taken in duplicate at various time points along the run, and cell counts and viability were measured using a Nucle-oCounter NC-200 (Chemometec, Denmark). When the viable cell density of T cells reached 2.0×106 cells / mL, perfusion with fresh T cell complete medium was started at a rate of 1 vessel volume per day (VVD). On the days indicated, samples were used for immunophenotyping and single cell secretome analysis. To monitor changes in key metabolites, 5 mL samples were taken from the bioreactor at various time points along the run.Offline monitoring to determine changes in parameters such as pH and major nutrients was performed using a BioProfile FLEX Analyzer (Nova Biomedical). Cell concentration, viability, presence of cells in the waste bag, and volumes inside and outside the bioreactor were assessed, as shown in Table 1. [Table 1]

[0058] Continuous medium perfusion Cell retention and continuous media perfusion was performed using an Xcell™ ATF 2 single-use device (Repligen catalog number suATF2-S02PES). The ATF was sterilely connected to the bioreactor on day 0 and the ATF column was wetted using X-VIVO™ 15 serum-free hematopoietic cell medium (Lonza catalog number 04-418Q). On day 8, when the cells reached 2.0×106 cells / mL, perfusion was allowed at an ATF rate of 0.5 liters per minute (LPM). Media in and out were set at 0.7 mL / min to maintain 1 VVD.

[0059] CD4+ T cell depletion After cell expansion, on day 14, perfusion was stopped and the required number of Dynabeads™ CD4 (Thermo Fisher Scientific Catalogue No. 11145D) was washed in isolation buffer (DPBS supplemented with 0.1% human AB serum and 2 mM EDTA) according to the manufacturer's instructions. The beads were added to the bioreactor and incubated for 30 min with stirring. Incubation on the magnet was performed for the required period. At the indicated times, 10 mL samples were withdrawn from the bioreactor for photography under 20x magnification in a Rebel light microscope (Echo, USA), calculation of the remaining bead concentration (see 4.9), staining for immunophenotypic analysis (see 4.8), evaluation of viable cell concentration and cell viability (NucleoCounter NC-200). The efficiency of depletion of CD4+ T cells at low and high concentrations for 30 min was evaluated, as seen in Table 2. [Table 2]

[0060] Table 3 shows the efficiency of bead depletion. The percentage of remaining beads, loss of cell viability, and time course of CD8+ T cells were assessed. [Table 3]

[0061] Harvesting T cells from stirred tank bioreactors The T cells in the 1 L bioreactor were harvested on day 14 of the total cell culture. The connection between the bioreactor and the ATF was closed and the cell solution in the bioreactor was pumped into a sterile 1 L bag with continuous stirring. The same 1 L bag was connected to the harvest line of the ATF to harvest the cell solution present in the ATF. 15 mL of the cell solution in the harvest bag was sampled for immunophenotyping (see 4.8), viable cell concentration and cell viability assessment (NucleoCounter NC-200).

[0062] Downstream Processing Bags containing T cell suspensions harvested from the bioreactor were sampled in triplicate and then viability and cell density were determined using a NucleoCounter NC-200. The mean viable cell density (VCD) was used to calculate the concentrated volume that would be harvested by the kSep (Equation 1, see Appendix A). The kSep (Sartorius) was fitted with a 400.50 rotor and serves as a 1 / 3.5 scaled down model of the kSep400. The associated 400.50 single use kit (chamber set and valve set) was then installed. A solution of PlasmaLyte-A (Baxter) and (0.25%) human AB serum (Sigma catalogue no. H4522) was used to prime the system. A static centrifugation speed of 1000 g was used. The fluidized bed was established for 60 min at a flow rate of 24 mL / min and harvested into the harvest bag at 120 mL / min. Throughout the enrichment process, 5 mL samples were drawn from the flow exiting the kSep chamber and tested using a NucleoCounter NC-200 (Chemometec, Denmark) to monitor the amount of cells leaking out of the fluidized bed. After processing 1 L of cell suspension, the enriched cells were harvested. The volume of the concentrate was verified and samples were taken to determine viability and cell density. The remaining concentrate was stored frozen.

[0063] For enrichment with ekko™, an ekko™ single-use cartridge was attached and the chamber was primed with 100 mL of a solution of wash buffer (PlasmaLyte-A (Baxter) and (0.25%) human AB serum (Sigma catalogue no. H4522). The feed was recirculated from the acoustic fluidic bed at 120 W and a flow rate of 70 mL / min. After processing 1 L of cell suspension, the enriched cells were harvested in two cycles. The volume of the concentrate was verified and samples were taken to determine viability and cell density. The remaining concentrate was stored frozen. Table 4 shows the concentration of cells using the closed system. The concentration factor, cell viability, and % recovery were evaluated. [Table 4]

[0064] cryopreservation Human T cells were suspended in cryopreservation medium (CS10, Biolife Solutions Inc, 210102). Cryovials were cryopreserved in a Cryomed™ controlled rated freezer (Thermo Fisher Scientific, model 7456) and subsequently stored in liquid nitrogen until use.

[0065] Flow cytometry Flow cytometry was used for quantitative detection of T cell differentiation, senescence, and exhaustion states. Briefly, 300,000 cells were live stained for the following cell surface markers: CD62L (Biolegend Catalog No. 304806), CD45RA (Biolegend Catalog No. 304108), CD45RO (Biolegend Catalog No. 304218), CD3 (BD ​​Biosciences Catalog No. 564713), CD4 (BD Biosciences Catalog No. 560158), CD8 (Biolegend Catalog No. 301028), KLRG1 (Biolegend Catalog No. 367716), CTLA4 (BD Bio-sciences Catalog No. 563931), PD-1 (BD Biosciences Catalog No. 564324), CD57 (Biolegend Catalog No. 393304), and ZOMBIE421 (Biolegend Catalog No. 423114). Samples were processed using a FACS Celesta™ (Becton Dickinson) and data acquired using BD FACS Diva software and subsequently analyzed using FlowJo v10 software (FlowJo).

[0066] Percentage of beads remaining From each sample, 1 ml was dispensed into each of three tubes. The tubes were centrifuged at 700 RCF for 5 minutes at room temperature. The cell pellet was resuspended in 1 mL of DPBS. To the cell solution, 4 mL of lysis buffer (1:1 ratio of DPBS and sodium hypochlorite) was added, mixed, and left at room temperature for 5 minutes. The tubes were centrifuged at 700 RCF for 5 minutes at room temperature, and 4.95 mL of the supernatant was removed. The remaining 50 μL was mixed well and 10 μL was dispensed to each side of the hemocytometer. The number of beads in the four corner squares was counted. The count was repeated for the second side of the hemocytometer. The remaining solution in the tube was mixed well and counting was performed until the solution in the tube was completely counted. The number of beads remaining was calculated using Equation 2, see Appendix A. The percentage of beads remaining relative to the viable cell density of the sample was calculated using Equation 3, see Appendix A.

[0067] T cell polyfunctionality analysis using Isoplexis During the bioreactor run, 15 ml samples were taken for multiple analyses. At the indicated time points, 1 mL of the solution was centrifuged at 500 RCF for 5 min at room temperature. The supernatant was transferred to a new sterile 1.5 mL tube and the pellet was cryopreserved as described in section 4.7. Cells were thawed as described in 4.1 and recovered overnight at 5% CO2 and 37°C in T cell complete medium supplemented with 10 ng / mL human recombinant IL-2. CD4+ and CD8+ cell enrichment was performed as described in 4.13. Two distinct T cell populations were stimulated in 50 ng / mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich Catalog No. P8139) and 1 ug / mL ionomycin (Sigma-Aldrich Catalog No. I0634) for 5 hours. Cells were loaded onto single-cell secretome barcode chips (Isoplexis catalog number PANEL-1001-8) for single-cell secretomics assessment. Single cell functional profiles were determined for each T cell type. Profiles were classified into effector (granzyme B, IFN-g, MIP-1a, perforin, TNF-a, TNF-b), stimulatory (GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21), regulatory (IL-4, IL-10, IL-13, IL-22, TGF-b1, sCD137, sCD40L), chemoattractant (CCL-11, IP-10, MIP-1b, RANTES), and inflammatory (IL-1b, IL-6, IL-17A, IL-17F, MCP-1, MCP-4) groups.

[0068] Isolation of CD4+ and CD8+ T cells CD4+ and CD8+ T cells were isolated from CD3+ T cells using CD4+ MicroBeads, human (Miltenyi Biotec Catalog No. 130-045-101) and CD8+ MicroBeads, human (Miltenyi Biotec Catalog No. 130-045-201). Briefly, cells were labeled with specific microparticles and passed through a MACS LS column (Miltenyi Biotec Catalog No. 130-042-303) placed on a mi-diMACS separator (Miltenyi Biotec Catalog No. 130-042-401) and a MACS Multistand (Miltenyi Biotec Catalog No. 130-042-302). Enriched cells captured on the column were loaded into a fresh collection tube, washed, and resuspended in T cell complete medium. Samples were collected before and after separation for flow cytometry analysis as described in 4.10.

[0069] We found that T cell proliferation under stirred conditions in spinner flasks was higher compared to static 2D culture (Figures 2A and 2B). showed that in an automated STR, proliferation of primary T cells increased with agitation speed. In agreement, an increase in T cell proliferation yield at higher agitation speeds was observed in the STR (Figure 2C). Cell growth accompanied by nutrient uptake leads to lactate accumulation in the cell culture, inhibiting efficient cell growth and the quality of the final cell therapy product. Continuous medium perfusion allows a fresh supply of nutrients and removal of harmful metabolic products while retaining the cells. However, due to the 5-10 mm diameter of T cells, cell leakage and filter fouling are major issues associated with T cell culture medium perfusion. Tangential flow filtration systems offer an attractive solution as a cell retention system, where fluid movement allows medium perfusion without fouling. Furthermore, ATFs offer the added benefit of self-cleaning induced by alternating flow backflushing. Because the hollow fiber pore size in Repligen's ATF is 0.2 mm, the ATF was used as a cell retention device for continuous medium perfusion in the STR. Initial testing of the ATF for cell retention and medium exchange resulted in successful perfusion of 1 VVD without filter fouling and cell loss (Table 1). 6 Following inoculation of STR at 1000 cells / mL of T cells, T cells were activated for 3 days in the presence of recombinant human IL-2 in CD3+28. After 3 days of activation, cells were expanded in X-VIVO™ 15 medium containing recombinant human IL-2. Lactate levels were monitored and a 3-fold increase in lactate levels was observed from day 7 to day 8 (Figure 3C). This was consistent with a viable cell density of 2.1 x 10 6 Cells / mL (Figure 3B), and a decrease in glucose concentration from 6.3 mM on day 7 to 5.4 mM on day 8 (Figure 3D) to ensure medium perfusion. Enabling medium perfusion on day 8 resulted in steady-state levels of glucose and lactate (Figures 3C and 3D). Furthermore, continuous medium perfusion allowed exponential growth of T cells, resulting in a final VCD of approximately 35 x 10 6In contrast, T cells from the same donor were activated and expanded in 1 L of G-Rex®. T cell expansion in G-Rex® was 3.4×10 cells / mL at day 14. 6 A 14-day culture in the STR with ATF-mediated perfusion resulted in a 167-fold expansion of T cells (Figure 3B). In addition, it was observed that agitation in the STR combined with ATF-mediated cell transfer did not reduce cell viability or cell proliferation (Figure 3E).

[0070] Turtle et al. reported in a clinical trial that CAR T cell therapy with a 1:1 CD4:CD8 T cell ratio led to the achievement of bone marrow remission in 93% of patients

[18] . Figure 4A shows the maintenance of the CD4:CD8 T cell ratio in the STR over 14 days of expansion, suggesting that the 1:1 seeding ratio is maintained until harvest. Increasing evidence suggests that the presence of many naive, stem cell memory, and central memory T cells in the final CAR T cell therapy product led to relapse-free remission, compared to the lower efficacy observed in CAR T cell products containing a higher percentage of effector memory subsets. It has been shown that T cell expansion in the tested conditions leads to a final T cell subtype composition containing approximately 80% central memory subsets, approximately 15% naive and stem cell memory subsets, and less than 10% effector memory and terminally differentiated subsets (Figure 4B). This phenotype can be explained by the higher replicative capacity of naive and stem cell memory T cells, which replicate and differentiate upon activation. Higher differentiation from naive and T memory stem cells (Tscm) combined with higher replicative capacity of central memory T cells may result in a higher percentage of central memory T cells. Although the final T cell subtype concentration depends on the initial T cell composition, expansion of T cells in quanta led to similar phenotypic outcomes. The presence of senescent and exhausted T cells in the final drug product leads to lower efficacy explained by reduced replicative capacity and dysfunctional state of T cells, respectively. It was shown that expansion of T cells in STR leads to more than 5% senescent (CD57+KLRG1+) or exhausted T cells (CTLA4+ / PD-1+) (Figure 4C). The polyfunctional strength index™ (PSI) of T cells is predictive of clinical response in acute myeloid leukemia and could be used as a biomarker in immunotherapy. The PSI of immune cells is calculated by multiplying the number of cytokines produced by each cell by the amount of each cytokine. Assessment of PSI of CD4+ and CD8+ T cells after 5 h of stimulation suggested an increase in effector CD4+ and CD8+ T cells from day 0 to day 14. In addition to an increase in effector signatures, an increase in stimulatory signatures was observed, especially in CD8+ T cells (Figure 5D).Polyfunctional T cells are capable of producing 2+ cytokines upon stimulation with antigen, which is considered an important functional characteristic. Evaluation of the proportion of cells producing multiple cytokines suggested an increase in the percentage of CD4+ and CD8+ T cells producing multiple cytokines, specifically 5+ cytokines (Figure 5B). Furthermore, about 1% of CD4+ T cells produced 11 cytokines and about 1% of CD8+ T cells produced 9 cytokines upon activation (Figure 5C). As assessed by the phenotypic characteristics of T cells, the increase in the central memory phenotype could be translated into an increase in the number of cells producing multiple cytokines.

[0071] The generation of allogeneic CAR T cells requires the deletion of the TCR alpha-encoding TRAC locus. Various strategies are used to delete the TRAC locus, which vary widely in efficiency. CRISPR / Cas9 shows an efficiency of 70-80%, TALEN-60-80%, zinc finger nuclease-20-40%, and megaTAL-75% efficiency. The presence of TCR-positive T cells in allogeneic cell therapy products results in GVHD and needs to be depleted before enrichment and formulation. Depletion of TCR-positive T cells requires taking T cells from the STR and processing them through an exclusive unit, which increases the possibility of contamination and incubation of cells under non-optimized conditions. A proprietary magnetic technology was developed that facilitates the depletion of cells in the STR while maintaining optimized conditions. In addition, depletion of cells in the STR avoids the need for additional unit operations and reduces the possibility of contamination. As a proof of principle, CD4+ T cells were depleted after T cell expansion. As shown in Figure 6A and Table 2, magnetic cell depletion for 30 minutes effectively depletes lower (>99%) and higher (97%) percentages of CD4+ T cells. FDA guidelines require a percentage of residual beads of less than 0.003% in the final cell therapy product, ensuring minimal effects of beads on the system. Using magnetic depletion for 120 minutes, a reduction in the percentage of residual beads to 0.001% was shown without significant cell loss and loss of viability (Figure 6B and Table 3). Furthermore, the proprietary magnetic technology could potentially be scaled to obtain similar results at different scales.

[0072] After depletion of unwanted T cells, concentration and formulation are performed to reduce the volume to a therapeutically friendly volume. Two automated, mutually exclusive closed systems for cell processing and concentration were evaluated. The ekko™ performs concentration using acoustic technology, while the kSep concentrates cells by fluidized bed formation using centrifugal force. As shown in Table 4, cells were concentrated at least 7-fold and over 70% of the cells were recovered without significant loss of cell viability.

[0073] To date, this is the first study describing the successful application of ATF for cell retention and medium perfusion in T cell manufacturing. Collectively, the data demonstrate the capability of a closed GMP-compatible end-to-end platform for expanding T cells to clinically relevant doses, purifying the cell culture, and concentrating and formulating the cell product (Figure 1).

[0074] Additional Exemplary Embodiments Embodiment 1 is a method of producing an immune cell culture in a completely closed system, comprising obtaining immune cells, introducing the immune cells into a stirred tank bioreactor containing immune cell complete medium, activating the immune cells with an activation reagent in the stirred tank bioreactor to produce activated immune cells, expanding the activated immune cells in the stirred tank bioreactor to produce an expanded immune cell culture, exchanging a defined amount of fresh medium with spent medium via an alternating tangential flow filtration (ATF) connected to the bioreactor, depleting the expanded immune cell culture in the stirred tank bioreactor to produce an depleted immune cell culture, harvesting the depleted immune cell culture in the completely closed system to produce a harvested immune cell culture, and concentrating the harvested immune cell culture of (g) in the completely closed system, wherein the method results in a loss of less than 1% of the immune cell culture.

[0075] Embodiment 2 includes the method of embodiment 1, wherein the immune cell complete medium comprises a buffer, amino acids, trace elements, vitamins, inorganic salts, glucose, and serum.

[0076] Embodiment 3 includes the method of embodiment 1, wherein the stirred tank bioreactor has a volumetric capacity of about 1 L to about 2000 L.

[0077] Embodiment 4 includes the method of embodiment 1, wherein the immune cells are isolated from a population of peripheral blood mononuclear cells (PBMCs) immediately prior to obtaining the immune cells, or are isolated from a population of PBMCs, stored for an extended period of time, and then thawed prior to obtaining the immune cells.

[0078] Embodiment 5 includes the method of embodiment 1, wherein the immune cells are derived from a population of pluripotent stem cells.

[0079] Embodiment 6 includes the method of embodiment 5, wherein the population of pluripotent stem cells is a population of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or a combination thereof.

[0080] Embodiment 7 includes the method of embodiment 4, wherein isolating the immune cells includes washing the PBMCs with a solution comprising the antibody complexes and the magnetic particles to produce a solution comprising the PBMCs and isolated T cells, and separating the isolated T cells from the solution with a magnet.

[0081] In the eighth embodiment, 0.25×10 6 T cells / mL~2×10 6 The method of embodiment 1, wherein T cells / mL are introduced into the bioreactor.

[0082] Embodiment 9 is a method comprising: 6 cells / mL ~ approx. 90×10 6 The method of embodiment 1, wherein the immune cell culture comprises 100 cells / mL of viable immune cells.

[0083] Embodiment 10 includes the method of embodiment 1, wherein the method produces an immune cell culture comprising at least about 100 million viable immune cells.

[0084] Embodiment 11 includes the method of embodiment 1, wherein the activating reagent comprises a soluble antibody complex.

[0085] Embodiment 12 includes the method of embodiment 1, wherein activating the immune cells includes agitating the medium with the activation reagent for a period of about 72 hours at 37°C.

[0086] Embodiment 13 includes the method of embodiment 12, wherein the immune cell culture is agitated at a tip speed of 0.15 to 0.5 revolutions per minute (RPM).

[0087] Embodiment 14 includes the method of embodiment 1, wherein the medium has a pH of about pH 5.0 to about pH 7.5 during the activation period.

[0088] Embodiment 15 includes the method of embodiment 1, wherein the growing includes adding fresh medium to the bioreactor after the activation period; monitoring one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor of the immune cell culture; and adjusting one or more of a temperature, a pH level, a glucose level, an oxygen level, a carbon dioxide level, and an optical density of the immune cell culture based on the monitoring.

[0089] Embodiment 16 includes the method of embodiment 15, wherein the expanding further includes sampling the expanding immune cell culture, determining the cell growth and fold expansion of the expanding T cell culture, and replacing a defined amount of fresh medium with the spent medium based on the cell growth and fold expansion.

[0090] Embodiment 17 includes the method of embodiment 16, wherein fresh medium is exchanged for spent medium at a rate of 1 vessel volume per day (VVD) when the viable cell density of the growing immune cell culture is greater than 1.5×10 cells / mL.

[0091] Embodiment 18 includes any of the methods of embodiment 1, wherein depleting the immune cell culture includes adding surface-activated magnetic beads to the immune cell culture after expansion, agitating the expanded immune cell culture and beads for about 30 minutes, and isolating the population of target cells from the expanded immune cell culture with a magnet.

[0092] Embodiment 19 includes the method of embodiment 18, wherein the beads are added to the bioreactor at a 1:1 ratio of beads to cells.

[0093] Embodiment 20 includes the method of embodiment 1, wherein the harvesting includes pumping the immune cell culture from the stirred tank bioreactor into a sterile vessel connected to the stirred tank bioreactor, and pumping the immune cell culture from the ATF into a sterile vessel connected to a harvest line of the ATF.

[0094] Embodiment 21 includes the method of embodiment 20, wherein the connection between the stirred tank bioreactor and the ATF is closed.

[0095] Embodiment 22 includes the method of embodiment 20, wherein pumping of the cell culture from the stirred tank bioreactor to the sterile vessel is performed while the immune cell culture is continuously stirred in the stirred tank bioreactor.

[0096] Embodiment 23 includes the method of embodiment 1, wherein the harvesting occurs after about 14 days of whole cell culture.

[0097] Embodiment 24 includes the method of embodiment 1, wherein concentrating comprises centrifugation of the harvested cell culture, removal of supernatant after precipitation, filtration, acoustic cell treatment, or a combination thereof.

[0098] Embodiment 25 includes the method of embodiment 24, wherein the harvested cell culture is centrifuged at about 250G to 3,000G for about 60 minutes.

[0099] Embodiment 26 includes the method of embodiment 25, wherein centrifugation of the harvested cell culture produces a fluidized bed comprising a biomass of the desired cells at a flow rate of about 20 mL / min to about 30 mL / min.

[0100] Embodiment 27 includes the method of embodiment 26, wherein the fluidized bed is pumped into a sterile and sealed container for collection and / or storage.

[0101] Embodiment 28 includes the method of embodiment 24, wherein the acoustic cell treatment includes circulating the harvested cell culture through an acoustic fluidized bed at 120 watts and a flow rate of about 50 mL / min to about 80 mL / min.

[0102] Embodiment 29 includes the method of embodiment 1, wherein activation and expansion of immune cells in a stirred tank bioreactor results in a T cell culture that produces more than five cytokines and has more than 75% central memory T cells, less than 10% effector memory T cells, and more than 10% naive / stem memory T cells.

[0103] Embodiment 30 includes any of the methods of embodiment 1, wherein the enriched T cells are cryopreserved.

[0104] Embodiment 31 is a population of T cells produced by the method of embodiment 1.

[0105] Embodiment 32 is a T cell-based therapy comprising T cells produced by the method of embodiment 1.

[0106] Although specific embodiments have been illustrated and described herein, it should be understood that the claims should not be limited to the specific forms or arrangements of parts described and illustrated. Although exemplary embodiments are disclosed herein and specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It should be understood, therefore, that the embodiments may be practiced otherwise than as specifically described.

[0107] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for producing immune cell cultures in a completely closed system, comprising: a) obtaining immune cells; b) introducing the immune cells into a stirred tank bioreactor containing complete immune cell medium; c) activating the immune cells with an activation reagent in the stirred tank bioreactor to produce activated immune cells; d) expanding the activated immune cells in the stirred tank bioreactor to produce an expanded immune cell culture; e) exchanging a defined amount of fresh medium for spent medium via an alternating tangential flow filtration (ATF) connected to said bioreactor; f) depleting the expanded immune cell culture of (d) in the stirred tank bioreactor to produce a depleted immune cell culture; g) harvesting the depleted immune cell culture of (f) in the completely closed system to produce a harvested immune cell culture; h) concentrating the collected immune cell culture of (g) in the completely closed system; The method, wherein the method results in a loss of less than 1% of the immune cell culture.

2. 10. The method of claim 1, wherein the immune cell complete medium comprises a buffer, amino acids, trace elements, vitamins, inorganic salts, glucose, and serum.

3. 10. The method of claim 1, wherein the stirred tank bioreactor has a volumetric capacity of 1 L to 2000 L.

4. 2. The method of claim 1, wherein the immune cells are isolated from a population of peripheral blood mononuclear cells (PBMCs) immediately prior to obtaining the immune cells in a), or are isolated from a population of PBMCs, stored for an extended period of time, and then thawed prior to obtaining the immune cells in a).

5. The method of claim 1 , wherein the immune cells are derived from a population of pluripotent stem cells.

6. 6. The method of claim 5, wherein the population of pluripotent stem cells is a population of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or a combination thereof.

7. 5. The method of claim 4, wherein isolating the immune cells comprises: a) washing the PBMCs with a solution comprising antibody complexes and magnetic particles to produce a solution comprising the PBMCs and isolated T cells; and b) separating the isolated T cells from the solution with a magnet.

8. 0.25 x 10 ６ T cells / mL ~ 2 x 10 ６ 10. The method of claim 1, wherein T cells / mL are introduced into the bioreactor.

9. The method is ６ cells / mL~90×10 ６ 10. The method of claim 1, wherein the method produces an immune cell culture comprising 100 cells / mL of viable immune cells.

10. 10. The method of claim 1, wherein the method produces an immune cell culture comprising at least 100 million viable immune cells.

11. The method of claim 1 , wherein the activating reagent comprises a soluble antibody complex.

12. 10. The method of claim 1, wherein activating the immune cells comprises agitating the medium with the activation reagent for a period of 72 hours at 37°C.

13. 13. The method of claim 12, wherein the immune cell culture is agitated at a tip speed of 0.15 to 0.5 revolutions per minute (RPM).

14. 2. The method of claim 1, wherein the medium has a pH between pH 5.0 and pH 7.5 during the activation period.

15. The growing i) adding fresh medium to the bioreactor after the activation period in (c); ii) monitoring one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor of said immune cell culture; iii) adjusting one or more of the temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density of the immune cell culture based on the monitoring.

16. said expanding comprising: iv) sampling said expanding immune cell culture; v) determining cell growth and fold expansion of said expanding T cell culture; and vi) replacing a defined amount of fresh medium with spent medium based on said cell growth and fold expansion.

16. The method of claim 15, further comprising:

17. The fresh medium is added to the growing immune cell culture until the viable cell density of the culture reaches 1.5×10 ６ 17. The method of claim 16, wherein the spent medium is replaced at a rate of 1 vessel volume per day (VVD) if the volume is greater than 100 cells / mL.

18. The depleting i) adding surface-activated magnetic beads to the immune cell culture after the expansion in d); ii) agitating the expanded immune cell culture and beads for 30 minutes; iii) isolating a population of target cells from said expanded immune cell culture using a magnet.

19. 20. The method of claim 18, wherein the beads are added to the bioreactor at a 1:1 ratio of beads to cells.

20. 2. The method of claim 1, wherein said harvesting comprises: i) pumping the immune cell culture from the stirred tank bioreactor into a sterile vessel connected to the stirred tank bioreactor; and ii) pumping the immune cell culture from the ATF into the sterile vessel connected to a harvest line of the ATF.

21. 21. The method of claim 20, wherein the connection between the stirred tank bioreactor and the ATF is closed.

22. 21. The method of claim 20, wherein the pumping of the cell culture from the stirred tank bioreactor to the sterile vessel is performed while the immune cell culture is continuously stirred in the stirred tank bioreactor.

23. 10. The method of claim 1, wherein said harvesting occurs after 14 days of total cell culture.

24. 10. The method of claim 1, wherein said concentrating comprises centrifugation of said harvested cell culture, removal of supernatant after sedimentation, filtration, acoustic cell treatment, or a combination thereof.

25. 25. The method of claim 24, wherein the harvested cell culture is centrifuged at 250G to 3,000G for 60 minutes.

26. 26. The method of claim 25, wherein centrifugation of the harvested cell culture produces a fluidized bed containing the desired cellular biomass at a flow rate of 20 mL / min to 30 mL / min.

27. 27. The method of claim 26, wherein the fluidized bed is pumped into a sterile and sealed container for collection and / or storage.

28. 25. The method of claim 24, wherein the acoustic cell treatment comprises circulating the harvested cell culture through an acoustic fluidized bed at 120 watts and a flow rate of 50 mL / min to 80 mL / min.

29. 10. The method of claim 1, wherein the activation and expansion of the immune cells in the stirred tank bioreactor results in a T cell culture that produces more than five cytokines and has more than 75% central memory T cells, less than 10% effector memory T cells, and more than 10% naive / stem memory T cells.

30. 10. The method of claim 1, wherein the enriched T cells are cryopreserved.

31. A population of T cells produced by the method of claim 1.

32. 10. A T cell-based therapy comprising T cells produced by the method of claim 1.

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