Automation of End-to-End Cell Therapy

Abstract

To provide an automated method of producing genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, utilizing a fully-enclosed cell engineering system.SOLUTION: An automated cell engineering system includes the following: a first chamber for storing cell culture media at a first temperature; a second chamber for activating and expanding immune cell cultures at a second temperature; a thermal barrier for insulating the first chamber from the second chamber; and one or more fluidic pathways for establishing fluidic connection of the second chamber to the first chamber. The one or more fluidic pathways provide recirculation, waste removal, and consistent gas exchange and nutrient distribution to the second chamber, ensuring that the cells within the second chamber remain uncontaminated.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001]

[0001] The present disclosure provides an automated method for generating genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, using a fully enclosed cell engineering system.

Background Art

[0002]

[0002] As expectations grow for the accelerated clinical adoption of advanced cell therapies, there has been increased attention on the manufacturing strategies that can underlie these therapies to benefit patients worldwide. Although cell therapies hold great clinical promise, high manufacturing costs relative to reimbursement are a formidable barrier to commercialization. Therefore, the need for cost-effectiveness, process efficiency, and product consistency is driving automation efforts in many cell therapy areas, particularly T cell immunotherapy (see, e.g., Wang, 2016).

[0003]

[0003] Recent successful clinical results from immunotherapy trials using chimeric antigen receptor (CAR) T cells offer new hope to patients suffering from previously incurable cancers (see, e.g., Lu, 2017; Berdeja, 2017; Kebriaei, 2016). As these novel therapeutics transition from the clinical trial stage to commercial scale, challenges related to cell manufacturing have arisen (see, e.g., Morrissey, 2017).

[0004]

[0004] The generation of these cells may require significant manual involvement for patient-specific products. Automation of CAR T cell culture is particularly difficult due to multiple highly sensitive unit operations such as cell activation, transduction, and expansion. Activation is particularly important because the efficiency of this process can affect transduction and expansion.

[0005]

[0005] To translate these important immunotherapies to a broad patient population, integration into commercial manufacturing platforms for cell activation, transduction, and expansion is crucial. To apply these life-saving treatments to the global patient population, a shift in manufacturing technology is needed to support personalized medicine. The advantages of automation have been described previously (see, e.g., Trainor, 2014; Mahdavi, 2015). These advantages include labor time savings associated with the use of automation, as well as improvements in product consistency, reduction of room classification, reduction of cleanroom footprint, reduction of training complexity, and improvement of scale-up and tracking logistics. Furthermore, software can be used to rationalize the documentation process using automatically generated electronic batch records and provide a history of all processing equipment, reagents, patient identification, operator identification, sensor data during processing, etc.

Summary of the Invention

Means for Solving the Problems

[0006]

[0006] In some embodiments, provided herein is a method for the automated generation of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture; transducing the activated immune cell culture with a vector to generate a transduced immune cell culture; expanding the transduced immune cell culture; concentrating the expanded immune cell culture of (c); and recovering the concentrated immune cell culture of (d) to generate a genetically modified immune cell culture, further comprising washing either or both of the expanded immune cell culture and the concentrated immune cell culture, wherein (a)-(e) are performed by a fully enclosed cell engineering system and (a)-(e) are optimized through the process of generating the genetically modified immune cell culture.

[0007]

[0007] In a further embodiment, provided herein is a method for promoting a preferred phenotype of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activating reagent to produce an activated immune cell culture, wherein the activating reagent and activation conditions promote the phenotype of the genetically modified immune cell culture; transducing the activated immune cell culture with a vector to produce a transduced immune cell culture; expanding the transduced immune cell culture; concentrating the expanded immune cell culture of (c); and recovering the concentrated immune cell culture of (d) to produce a genetically modified immune cell culture, wherein (a)-(e) are performed by a fully enclosed automated cell engineering system.

[0008]

[0008] In an additional embodiment, provided herein is a method for the automated production of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activating reagent to produce an activated immune cell culture; transducing the activated immune cell culture with a vector to produce a transduced immune cell culture; expanding the transduced immune cell culture; concentrating the expanded immune cell culture of (c); and recovering the concentrated immune cell culture of (d) to produce a genetically modified immune cell culture, wherein (a)-(e) are performed by a fully enclosed automated cell engineering system, and each of (a)-(e) is performed using an immune cell culture having an optimized cell density (cells / mL) and an optimized cell packing density (cells / cm 2 ).

[0009]

[0009] In a further embodiment, provided herein is a method for the automated generation of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture; transducing the activated immune cell culture with a vector to generate a transduced immune cell culture; expanding the transduced immune cell culture, wherein the transduced immune cell culture is not shaken during expansion; concentrating the expanded immune cell culture of (c); and recovering the concentrated immune cell culture of (d) to generate a genetically modified immune cell culture, wherein (a)-(e) are performed by a fully enclosed automated cell engineering system.

[0010]

[0010] In yet a further embodiment, provided herein is a method for the automated generation of a genetically modified immune cell culture, the method being performed by a cell engineering system and comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture in a first chamber of the cell engineering system; transducing the activated immune cell culture, wherein transduction comprises transferring the activated immune cell culture from the first chamber to an electroporation unit; electroporating the activated immune cell culture with a vector to generate a transduced immune cell culture; transferring the transduced immune cell culture to a second chamber of the cell engineering system; expanding the transduced immune cell culture; concentrating the expanded immune cell culture of (c); and recovering the concentrated immune cell culture of (d) to generate a genetically modified cell culture.

[0011]

[0011] In an additional embodiment, provided herein is a cassette for use in an automated cell engineering system, including a cryogenic chamber for storing cell culture media; a high-temperature chamber for activating, transducing, and expanding an immune cell culture, the high-temperature chamber being separated from the cryogenic chamber by a thermal barrier, the high-temperature chamber including a cell culture chamber; and one or more fluidic paths connected to the cell culture chamber, the fluidic paths providing recirculation, waste removal, uniform gas exchange, and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber.

[0012]

[0012] In yet a further embodiment, provided herein is a cassette for use in an automated cell engineering system, including a cell culture chamber for activating, transducing, and / or expanding an immune cell culture having a chamber volume configured to contain the immune cell culture, and a satellite volume for increasing the working volume of the chamber by providing additional volume for media and other working fluids without containing the immune cell culture, the satellite volume being fluidly connected to the cell culture chamber via one or more fluidic paths such that media is exchanged between the culture chamber and the satellite volume without disturbing the immune cell culture.

Brief Description of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014]

[0037] The present disclosure provides an automated method for generating chimeric antigen receptor T (CAR T) cells. The generation of CAR T cells typically requires manual involvement for patient-specific products. The automation of CAR T cell culture is particularly difficult due to multiple highly sensitive unit operations such as cell activation, transduction, and expansion. Accordingly, an automated method for generating CAR T cells using a fully enclosed cell engineering system is disclosed herein.

[0015] Automated cell processing

[0038] In the case of autologous cell therapy such as T cell therapy, the batch production of micro-lots (one patient per lot) lacks the economies of scale to utilize an allogeneic (multiple patients per lot) process, so the need for cost-effectiveness, process efficiency, and consistency of the product is particularly critical (see, for example, Jones, 2012; Trainor, 2014). The larger scale and localized labor force and facilities required for micro-lots impose significant requirements on logistics and manual production GMP compliance, particularly with respect to staff availability and training. Furthermore, the potential for variability in techniques among operators can pose an undesirable risk of consistently meeting release criteria and ensuring a safe and reliable product.

[0016]

[0039] As described herein, the introduction of automated manufacturing and comprehensive verification are these logistics and provide solutions to operational challenges. An important approach to introducing automation into the production process is for the operator to identify the main modular steps in which physical or chemical changes called "unit operations" are applied to the production materials. In the case of cell manufacturing, this includes steps such as cell separation, genetic manipulation, proliferation, washing, concentration, and cell recovery. Manufacturers often identify bottlenecks in the focal process as a direct opportunity to introduce automation. This is reflected in the technical operating ranges of most commercially available bioreactors and tends to focus on individual process steps. Process challenges in cell manufacturing (from maintaining sterility to tracking samples) are addressed herein by end-to-end automation that produces consistent cell output while improving inevitable process variations. The methods described herein also provide simplification, and the associated electronic records assist in compliance with GMP standards (see, for example, Trainor, 2014).

[0017] Automation of Unit Operations and Key Process Sensitivities

[0040] The recent rapid progress in the clinical development of modified autologous T cells for cancer immunotherapy has led to the planning of the associated translational and scale-up / scale-out implications.

[0018]

[0041] Specific protocols can vary with respect to T cell manufacturing, but a generalized chimeric antigen receptor T cell (CAR T) process is shown in FIG. 1. FIG. 1 describes the unit operations of CAR T cell manufacturing from the initial processing of a patient's blood sample to the formulation of the output cells for autologous T cell therapy.

[0019]

[0042] As described herein, to achieve the automation of cell manufacturing, the methods described herein provide an understanding of the cell state at each transition point and the impact of specific unit operations. Micro-lot generation for patient-specific therapies needs to respect the important process sensitivities that affect the feasibility of automation. The automation described herein encompasses various process steps.

[0020]

[0043] Table 1 below highlights the challenges of some of the process steps identified for T cell automation and notes the impact on sensitivity to the automation strategy. It should be noted that in all unit operations, there is a risk of contamination, so the open transfer of cells between each piece of equipment is an important sensitivity. [Table 1] JPEG0007714010000002.jpg134149

[0021]

[0044] Adjusting the automation of manual processes centered on the sensitivities listed in Table 1 can support the success of transforming, maintaining, or improving the performance of cell therapy.

[0022] Integration of automated unit operations

[0045] Considering the impact of GMP logistics, economy, and automation on patient safety, unit operations can be evaluated in terms of the typical labor hours per unit operation (including the labor hours of both the operator and the quality assurance monitor). Table 2 shows the nominal manual processing timeline for representative steps of CAR T automation. This table highlights the resource commitments required for each unit operation of the generalized CAR T cell process. For each step, the estimated remaining labor hours of the automated process are identified along with the reasons for reduction. [Table 2] JPEG0007714010000004.jpg181149

[0023]

[0046] Based on the method described in this specification, the automation of unit operations can reduce the nominal manual process by approximately 40 hours, to about one quarter of the original time.

[0024] Individual to fully integrated automation

[0047] There is compelling evidence of the value of automation (see, for example, Trainor 2014; Levine 2017), but subsequent analysis is needed regarding the value and practicality of integrating these automation steps in the sequence between endpoints. There are different perspectives on the advantages of individual process automation versus end-to-end integration.

[0025]

[0048] The main advantage of individual automation is flexibility. This is relevant in the following areas: 1) Maintenance of unique process operations 2) Acceleration of conversion activities based on the operation verification of individual units 3) The ability to change processing steps to accommodate variations between donors

[0026]

[0049] The first aspect related to the improvement of flexibility is to provide the operator with more control over the process. This is important in situations where there are very sensitive steps in the process that can affect the final product. Switching to an all-in-one system may impose constraints that can affect the outcome of the product. An individual approach provides flexibility in choosing how each step is performed, which can be particularly important in highly sensitive unit operations. Also, an individual approach allows for a gradual transition from manual processing to automation, which helps to demonstrate equivalence when each unit operation can be tested individually. Furthermore, automating specific unit operations provides flexibility in decisions based on cell performance. For example, if the cells are growing rapidly, it may be necessary to expand from one cell culture bag to two. Finally, the approach to automation using individual systems also allows the group to select which equipment to use for each unit operation.

[0027]

[0050] The utilization of equipment is another consideration for individual automation. There may be unit operations that take significantly more time than other unit operations. In an end-to-end processing system, all of the multiple unit operations need to be performed in a single system and thus the equipment is occupied during the culture process.

[0028]

[0051] While there are advantages to individual automation, the end-to-end approach offers different, though perhaps less attractive, benefits. First, a fully integrated system significantly reduces the risk of contamination. With the increased handling required in an individual approach, there is a greater likelihood of product variation due to operator intervention. Second, as mentioned above, this necessarily results in an increase in labor costs.

[0029]

[0052] The flexibility provided by an individual approach is important. In situations where the process is important in defining the product, the end-to-end system should be made flexible enough to integrate unique sensitivities. This can include specific supply strategies, oxygen levels, surface treatments, etc. Such an approach requires flexibility in both software and disposable components. The system should provide the option to withdraw cells and culture samples at various points in the process to ensure that specific unit operations meet the product specification checkpoints. If changes are needed, the software should be able to implement these changes to provide the ideal conditions. User-friendly and flexible software is highly beneficial for conversion purposes, but it is important that the software can be easily locked down to comply with clinical standards (FDA 21 CFR Part 11). Once locked down, any ability of the operator to change the protocol should be restricted. However, to address the issue of inherent donor variability, there needs to be an option to select from a range of validated protocols based on the cell growth rate. For example, if the cells are growing rapidly, the system should respond and adjust the feeding or harvesting times accordingly.

[0030]

[0053] The choice between end-to-end integration and individual automation also depends on the long-term vision of the clinical process. A single all-in-one system can significantly improve space efficiency and minimize the required footprint in an expensive GMP cleanroom. For example, as shown in Figure 2, a fully integrated automation system is designed to maximize the required footprint and reduce the expensive GMP cleanroom space. Figure 2 shows 96 patient-specific end-to-end units operating in a standard laboratory space.

[0031]

[0054] A single system also makes data tracking easier, but individual systems may not provide compliance software to link all electronic data files together. Using software platforms such as VINETI (Vineti Ltd) and TRAKCEL (TrakCel Ltd) enables the electronic monitoring and orchestration of supply chain logistics. However, a single all-in-one culture system can further evolve by incorporating the history of both processing events and biomonitoring of culture conditions associated with each unit operation into batch records. Thus, the advantages of end-to-end integration provide a significant competitive advantage.

[0032] Commercial platform for unit operation integration

[0055] The success of clinical trials in many autologous cell therapies, particularly immunotherapies for blood cancers, emphasizes the importance of being able to convert new clinical protocols into robust production platforms to meet the predicted clinical need (see, for example, Levine, 2017; Locke, 2017). In the case of autologous therapies, the processing of each patient-specific cell therapy appropriately utilizes comprehensive manufacturing activities and operations management. The methods herein link the unit operations of a turnkey automated system to achieve process optimization, security, and economy.

[0033]

[0056] The challenges in designing an autologous process are twofold. First, unlike allogeneic processes where individual processing steps can occur in physically separate and optimized pieces of equipment, a scaled-out autologous platform appropriately performs all the steps required in a single closed, self-contained automated environment. Second, unlike allogeneic processes where, in theory, all runs start from high-quality vials in a cell bank with known quality and predictable process behavior, the starting materials for autologous processes are highly diverse and generally come from individuals with compromised health.

[0034]

[0057] Accordingly, provided herein is a method that can sense culture conditions by controlling factors such as physical agitation, pH, feeding, and gas treatment, and respond as a sophisticated bioreactor accordingly. Furthermore, there are significantly different challenges in technology transfer related to autologous therapy compared to allogeneic therapy. Autologous products may have greater limitations due to the stability between the manufacturing process and patient treatment. Sites can be located globally rather than at a single center. Using a lockdown (e.g., fully enclosed) all-in-one system significantly improves the technology transfer process between sites.

[0035]

[0058] Although variability in the source cannot be eliminated, automation helps remove variability in the final autologous product through standardization and reproducibility. This approach is adopted by major cell system providers to obtain a reference point for cell performance via biosensors that monitor the state of active cell culture. In end-to-end integration, the output from a particular stage of the process needs to fall within acceptable parameters for the progress of the process.

[0036]

[0059] As described herein, in an embodiment, the method provided utilizes the Octane Biotech (Kingston, ON) cocoon platform that integrates multiple unit operations into a single turnkey platform. Multiple cell protocols are provided for very specific cell processing purposes. To provide an efficient and effective automation conversion, the method described herein utilizes the concept of application / sponsor-specific disposable cassettes that combine multiple unit operations - all focused on the core requirements of the final cell therapy product.

[0037]

[0060] The method described herein has been used to expand CAR T cells (including activation, viral transduction and expansion, concentration and washing) in a fully integrated, closed automation system (Figure 3).

[0038]

[0061] In the experiments conducted, the fold expansion of CAR T cells in cultures over 10 - 14 days reached approximately 40 - 60. Both CD4+ and CD8+ T cell subsets are required for the success of CAR T therapy. Therefore, the ability to maintain the culture of both T cell subsets was evaluated by flow cytometry, along with the relevant controls. Figure 4 shows that all runs, as well as all controls, were able to maintain both T cell subsets. The percentage of CAR T cells present was also evaluated in each population of the T cell subsets (Figure 5). In all samples, detection of NGFR (indicating the CAR construct) was higher in the CD4+ fraction compared to the CD8+ fraction, but in all samples, the NGFR+ fraction of the CD8+ portion exceeded 50% of the fraction found in the paired CD4+ population. In summary, the automated CAR T process using the methods described herein results in a healthy population of T cell subsets.

[0039] Advantages of Automation

[0062] Automation of unit operations in the generation of cell therapies provides opportunities for universal benefits across allogeneic and autologous cell therapy applications. In the context of patient - specific autologous cell products, the recent clinical success of these therapies has further highlighted this, but the advantages of automation are particularly attractive with very complex micro - lots in terms of small - lot GMP compliance, economics, patient traceability, and early identification of process deviations. The emergence of the associated complex manufacturing protocols has drawn attention to the fact that the value of end - to - end integration of automated unit operations in micro - lot cell generation has not been a major research focus. However, the expected demand for these therapies following imminent approvals indicates that the implementation of fully closed end - to - end systems can provide much - needed solutions to manufacturing bottlenecks such as hands - on time and footprint.

[0040]

[0063] Developers of advanced therapies are recommended to consider automation early in the deployment of clinical translation and scale up clinical trial protocols. Early automation can influence protocol development, avoid the need for a comparability study when switching from a manual to an automated process at a later stage, and deepen the understanding of the long-term commercialization route.

[0041] Method for generating genetically modified immune cells including CAR T cells

[0064] In embodiments, provided herein is a method for the automated generation of a genetically modified immune cell culture. As used herein, a "genetically modified immune cell culture" (or genetically modified immune cell) refers to a cell of the immune system that has been modified or primed (e.g., through co-culture with antigen-presenting cells) and results in a cell having a desired phenotype useful for the treatment, prevention, or amelioration of one or more diseases in animals including humans. As used herein, an "immune cell culture" refers to a collection of cells prepared by the methods described herein and can include a cell population for use in an investigation or clinical trial and for administration to a mammal including a human patient for medical therapy. Genetically modified immune cell cultures that can be generated using the methods described herein can include mast cells, dendritic cells, natural killer cells, B cells, T cells, and the like.

[0042]

[0065] The various methods described herein can also be extended to other genetically modified cell cultures, including, for example, the generation of genetically modified human stem cell cultures including hematopoietic stem cells.

[0043]

[0066] In an exemplary embodiment, the method comprises activating an immune cell culture with an activation reagent to generate an activated immune cell culture, transducing the activated immune cell culture with a vector to generate a transduced immune cell culture, expanding the transduced immune cells, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to generate a genetically modified immune cell culture. Optionally, the method further comprises either or both of the expanded immune cell culture and the concentrated immune cell culture. In embodiments, the various steps of the method are performed by a fully closed cell engineering system and optimized through the process of generating a genetically modified immune cell culture.

[0044]

[0067] Methods for optimizing the process for generating genetically modified immune cells include optimizing cell culture conditions prior to initiating an automated method, and using feedback from various sensors, such as those that assist in real-time modification of growth conditions (e.g., gas concentration, media conditions, temperature, pH, waste and nutrient concentrations, etc.).

[0045]

[0068] In embodiments, the optimization process is a self-regulating process, which does not require input from an external (human) user and can determine the modifications necessary for cell culture or other characteristics that optimize the automated process through various computer programs and conditions. In embodiments, the self-regulating process includes monitoring with 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. As described herein, the use of these various sensors in a fully closed cell engineering system occurs at various times and locations within the system and works in concert to provide optimization. For example, the self-regulating process can adjust (e.g., increase or decrease) one or more of temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density of the transduced T cell culture based on the monitoring.

[0046]

[0069] The optimization process can also be based on the unique characteristics of the starting cell population, including, for example, the total cell number, the source of the cells, the cell density, the age of the cells, etc. Before initiating the automated method, the characteristics of the starting cell population can be input into a computer-controlled system. In this case, the system makes various initial changes to optimize the method, such as, for example, the concentration, flow rate of oxygen and carbon dioxide, the incubation time, the pH, etc. Alternatively, by monitoring the cell process, it becomes possible to automatically characterize the progression of the cell culture sequence from the starting population, and the conditions for the optimized final cell culture characteristics can be adjusted on a case-by-case basis.

[0047]

[0070] In an exemplary embodiment, the methods described herein generate at least about 50 million viable genetically modified immune cells. In suitable embodiments, the methods described generate at least about 100 million viable genetically modified immune cells, or at least about 200 million cells, at least about 300 million cells, at least about 400 million cells, at least about 500 million cells, at least about 600 million cells, at least about 700 million cells, at least about 800 million cells, at least about 1 billion cells, at least about 1.1 billion cells, at least about 1.2 billion cells, at least about 1.3 billion cells, at least about 1.4 billion cells, at least about 1.5 billion cells, at least about 1.6 billion cells, at least about 1.7 billion cells, at least about 1.8 billion cells, at least about 1.9 billion cells, at least about 2 billion cells, at least about 2.1 billion, at least about 2.2 billion cells, at least about 2.3 billion cells, at least about 2.4 billion cells, at least about 2.5 billion cells, at least about 2.6 billion cells, at least about 2.7 billion cells, at least about 2.8 billion cells, at least about 2.9 billion cells, or at least about 3 billion genetically modified immune cells.

[0048]

[0071] As described herein, the genetically modified immune cell cultures generated by the present method are suitably T cell cultures, including chimeric antigen receptor T (CAR T) cell cultures. In such embodiments, the vectors utilized to generate such CAR T cells are vectors encoding a chimeric antigen receptor. Suitably, the immune cell cultures include peripheral blood mononuclear cells and / or purified T cells. In embodiments, the immune cell cultures include at least one accessory cell, suitably monocytes or monocyte-derived cells. As described herein, in embodiments, the accessory cells include antigens for T cell receptors, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0049]

[0072] Suitably, the activation reagent includes an antibody or dendritic cells. In embodiments, the antibody can be immobilized on a surface including polystyrene plastic, silicone, or other surfaces, such as the surface of beads.

[0050]

[0073] In other embodiments, the activation reagent includes an antibody, which is a soluble antibody including at least one of an anti-CD3 antibody and an anti-CD28 antibody. Exemplary antibodies include OKT3.

[0051]

[0074] Various methods for transducing cells can be utilized in automated methods, including, for example, viral infection, electroporation, membrane disruption, or combinations thereof.

[0052]

[0075] In an exemplary embodiment, the vector utilized in the present method is a lentiviral vector or a retrovirus. Suitably, transduction includes mixing the vector in cell culture medium and uniformly delivering the vector in the medium to the activated immune cell culture. As described herein, uniform delivery of the vector in a uniform manner to the cells provides optimization of various cell characteristics of high output of the desired genetically modified immune cells.

[0053]

[0076] As described herein, methods of expanding cells appropriately include at least one or more of the supply, washing, monitoring, and selection of the transduced immune cell culture.

[0054]

[0077] The various methods described herein are carried out in such a way that the oxygen level of the transduced immune cell culture is optimized for the immune cell culture. This optimization enables the generation of a large number of viable cells with the desired phenotypic characteristics, including the promotion of the desired cell phenotype, as described herein. In embodiments, the oxygen level or concentration is optimized by a cell engineering system that recirculates the cell culture medium through an oxygenation component during one or more of steps (a) through (e). As described herein, oxygenation occurs appropriately through one or more fluid pathways that include silicone-based tube components.

[0055]

[0078] In further embodiments, the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during the various method processes. This recirculation aids in the generation of a large number of viable cells with the desired phenotype(s). Appropriately, the carbon dioxide level provided by the cell engineering system decreases during the expansion step to optimize cell growth and the like. In other embodiments, for example, when a complete medium exchange is utilized, the CO2 level can be increased.

[0056]

[0079] Other mechanisms for optimizing the growth conditions of cells include modifying and controlling the flow rate of the medium provided to the cells. When the cells begin to grow, the circulation rate of the provided medium increases, thereby improving gas exchange and allowing oxygen and carbon dioxide to be introduced into or removed from the cell culture according to the state of the cells and the requirements at that time.

[0057]

[0080] In an embodiment, the cell engineering system is configured to perform one or more multiple rounds of supply, washing, and monitoring, and in an embodiment, is configured to select a transfected immune cell culture. These various activities can be performed in any order and can be performed alone or in combination with another activity. In an embodiment, cell concentration includes centrifugation, removal of supernatant after sedimentation, or filtration. Appropriately, the optimization process further includes adjusting the parameters of centrifugation or filtration, appropriately in an autoregulatory process. Selection of transfected cells can be performed, for example, by magnetic separation, filtration, attachment to plastic or other substrates, and the like.

[0058]

[0081] In the embodiments described herein, the cell engineering system includes a plurality of chambers, and each step of the method is performed in a different chamber of the plurality of chambers of the cell engineering system.

[0059]

[0082] Appropriately, the method further includes removing an activation reagent from the activated immune cell culture after step (a) and can include removing the vector following the transfection step. The activation reagent is appropriately removed from the immune cell culture by washing, discharging, or physically removing the cell or the activation reagent. The vector can be removed by washing or by binding the vector to a surface (e.g., a surface coated with retronectin or fibronectin) and then transferring the cells to another chamber.

[0060]

[0083] In an exemplary embodiment, the cell engineering system includes a cell culture, an activation reagent, a vector, and a cell culture medium before starting the method. In other embodiments, the activation reagent and / or the vector can be added separately after the start of the production method or at any appropriate time during the process.

[0061]

[0084] In a further embodiment, provided herein is a method for promoting a preferred phenotype of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activating reagent to produce an activated immune cell culture, wherein the activating reagent and activation conditions promote the phenotype of the genetically modified immune cell culture, transducing the activated immune cell culture with a vector to produce a transduced immune cell culture, expanding the transduced immune cell culture, concentrating the expanded immune cell culture, and (d) recovering the concentrated immune cell culture to produce a genetically modified immune cell culture. As described herein, the method is suitably performed by a fully enclosed automated cell engineering system.

[0062]

[0085] As described herein, the selection of an appropriate activating reagent and appropriate activation conditions provides for the promotion of the desired phenotype of the genetically modified immune cell culture. That is, the phenotype of the immune cell culture can be specifically selected and promoted such that a suitable majority of the cells produced by the method have the desired preferred phenotype. In other embodiments, the desired ratio of one cell phenotype to another phenotype can be controlled and promoted to provide a desired preferred phenotype balance.

[0063]

[0086] As described herein, it has been found that the use of an activating reagent that is an antibody, particularly a soluble antibody, can promote the desired phenotype of a genetically modified immune cell. Suitably, the antibody utilized is at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, including the soluble antibody OKT3.

[0064]

[0087] In an embodiment, the activation conditions provide an immunocyte culture that enables stable contact between the activation reagent and the immunocyte culture and is substantially unimpeded. As described herein, it has been found that cells can be activated under conditions that are substantially unimpeded and through the use of a flat and substantially inflexible cell culture chamber. This provides an environment in which the cells are uniformly contacted with the activation reagent and interact with the necessary nutrients, dissolved gases, etc. to achieve the desired enhanced phenotype.

[0065]

[0088] The methods described herein can affect the properties of the final immunocyte 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 CD8 population than CD4. Other levels of CD8 and CD4 can also be provided using the methods described herein. In an exemplary embodiment, as described herein, the methods can be utilized to prepare CAR T cells. Suitably, the methods can be utilized to promote a phenotype of CAR T cells having a CD8+ cell to CD4+ cell ratio of from about 0.1:1 to about 10:1, including from about 0.5:1 to about 5:1, from about 0.8 to about 3:1, or about 1:1, about 2:1.

[0066]

[0089] In a further embodiment, a method for the automated production of a genetically modified immune cell culture is provided, the method comprising activating an immune cell culture with an activating reagent to produce an activated immune cell culture, transforming the activated immune cell culture with a vector to produce a transduced immune cell culture, expanding the transduced immune cell culture, concentrating the expanded immune cell culture of (c), and recovering the concentrated immune cell culture of (d) to produce a genetically modified immune cell culture. As described herein, the method is suitably performed by a fully enclosed automated cell engineering system. In an embodiment, each step of the method is performed with an immune cell culture having an optimized cell density (cells / mL) and an optimized cell confluency (cells / cm 2 ).

[0067]

[0090] As described herein, by utilizing an optimized cell density (cells per 1 mL of cell culture medium) and / or cell confluency (cells per area (cm 2 ) of the cell culture chamber in which the cells act and proliferate), it has been determined that increased production of viable cells and better control of cell phenotypes can be provided, among other things.

[0068]

[0091] In an embodiment, the optimized cell density is from about 0.05×10 6 cells / mL to about 60×10 6 cells / mL, from about 0.05×10 6 cells / mL to about 40×10 6 cells / mL, or from about 0.05×10 6 cells / mL to about 20×10 6 cells / mL. The optimized cell density can vary throughout the course of the production method, and thus, at each stage of the method (i.e., activation, transduction, expansion, concentration), the cell density is controlled or manipulated to provide the best cell density for a particular step of the method. The cell density can be optimized, for example, by selection of an optimal starting cell density, increasing or decreasing the oxygen and / or carbon dioxide concentration, pH, temperature, nutrient regulation, waste removal, and the like. Exemplary cell densities include about 0.05×10 6cells / mL, approximately 0.08×10 6 cells / mL, approximately 1×10 6 cells / mL, approximately 5×10 6 cells / mL, approximately 10×10 6 cells / mL, approximately 20×10 6 cells / mL, approximately 30×10 6 cells / mL, approximately 40×10 6 cells / mL, approximately 50×10 6 cells / mL, or approximately 60×10 6 cells / mL, etc. are included.

[0069]

[0092] In an embodiment, the optimized cell density is about 0.1×10 6 cells / cm 2 ~ about 60×10 6 cells / cm 2 、or about 0.1×10 6 cells / cm 2 ~ about 40×10 6 cells / cm 2 、or about 0.1×10 6 cells / cm 2 ~ about 20×10 6 cells / cm 2 is. The optimized cell density can vary throughout the process of the production method, and at each stage of the method (i.e., activation, transduction, expansion, concentration), the cell density is controlled or manipulated to provide the best cell density for a particular step of the method. The cell density can be optimized by selection of an optimal starting cell density, selection of the material of the cell culture chamber, increase or decrease of oxygen and / or carbon dioxide concentration, pH, temperature, adjustment of nutrients, removal of waste, etc. Exemplary cell densities include about 0.1×10 6 cells / cm 2 、about 0.5×10 6 cells / cm 2 、about 1×10 6 cells / cm 2 、about 0.5×10 6 cells / cm 2 、about 10×10 6 cells / cm 2 、about 20×10 6 cells / cm 2 、about 30×106 cells / cm 2 、 about 40×10 6 cells / cm 2 、 about 50×10 6 cells / cm 2 、 or about 60×10 6 cells / cm 2 etc. are included.

[0070]

[0093] In an embodiment, the method provides that the recirculation of nutrients, waste products, released cytokines, and / or dissolved gases is about 0.05×10 6 cells / mL to about 20×10 6 cells / mL in density, and about 0.1×10 6 cells / cm 2 ~ about 20×10 6 cells / cm 2 and is uniformly provided to cells having a density of.

[0071]

[0094] In a further embodiment, a method for the automated generation of a genetically modified immune cell culture is provided, the method comprising activating an immune cell culture with an activating reagent to produce an activated immune cell culture, transducing the activated immune cell culture with a vector to produce a transduced immune cell culture, expanding the transduced immune cell culture, wherein the transduced cell culture is not shaken during expansion, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to produce a genetically modified immune cell culture. As described herein, suitably, the method is performed by a fully enclosed automated cell engineering system.

[0072]

[0095] As described herein, surprisingly, it has been found that cells can be expanded under conditions where the cells are not agitated (i.e., not rotated or agitated to cause the cells to flow on top of each other). This method provides high viable cell yields and optimal cell characteristics including the desired phenotype. Significantly, the non-agitated cell culture chamber need not agitate or disturb the cells and can provide uniform access of the cells to the necessary reagents, nutrients, gas exchange, etc. while removing cell waste to achieve the desired results. Indeed, as described herein, such a method for the automated generation of genetically modified immune cells results in a greater number of viable cells, a greater number / ratio of the desired cell type, and more robust cell characteristics as compared to methods that utilize cell agitation, such as those described in Miltenyi et al., "Sample Processing System and Methods", U.S. Patent No. 8,727,132.

[0073]

[0096] Suitably, the expansion step of the method includes at least one or more of the supply, washing, monitoring, and selection of the transduced immune cell culture without agitating the immune cell culture.

[0074]

[0097] Also provided herein is a method for the automated generation of a genetically modified immune cell culture, the method being performed by a cell engineering system and including activating an immune cell culture with an activating reagent to generate an activated immune cell culture in a first chamber of the cell engineering system and transducing the activated immune cell culture. In an exemplary method, transduction includes transferring the activated immune cell culture from the first chamber to an electroporation unit, electroporating the activated immune cell culture with a vector to generate a transduced immune cell culture, and transferring the transduced immune cell culture to a second chamber of the cell engineering system. The method further includes expanding the transduced immune cell culture, concentrating the expanded immune cell culture, and (d) recovering the concentrated immune cell culture to generate a genetically modified cell culture.

[0075]

[0098] For example, as shown in FIG. 17, an activated immune cell culture is transferred from the cassette 602 of the cell engineering system 600 to the electroporation unit 1706 via, for example, the connection tube 1704. The electroporation unit 1706 suitably includes an electroporation cartridge 1708 that holds the cell culture during the electroporation process. After the electroporation process, the transfected immune cell culture is returned to the cell engineering system 600 via the connection tube 1704. FIG. 17 also shows the use of two optional reservoirs 1710 and 1712 used to hold the cell culture before and after electroporation, which assist in the transfer between the cell engineering system and the electroporation unit as a result of different pump speeds, required pressures and flow rates. However, such reservoirs can be removed and the cell culture can be transferred directly from the cell engineering system 1702 to the electroporation unit 1706.

[0076]

[0099] FIG. 18 shows a flow diagram of the cell culture from 1) the cell engineering system to the first reservoir, 2) the electroporation unit, 3) the second reservoir, and finally 4) the cell engineering system.

[0077]

[0100] In an exemplary embodiment, as shown in FIGS. 17 and 18, the electroporation unit 1706 is disposed outside the cell engineering system 1702. In such an embodiment, transfection transfers the activated immune cell culture from the first chamber to the electroporation unit via a first sterile closed connection (e.g., connection tube 1704), electroporates the activated immune cell culture with a vector to produce a transfected immune cell culture, and transfers the transfected immune cell culture to a second chamber of the cell engineering system via a second sterile closed connection (e.g., connection tube 1704).

[0078]

[0101] Also, it should be understood that multiple separate cell engineering systems 600 (see, e.g., FIG. 2) can be connected to a single electroporation unit and run in a suitable order such that a cell culture is transferred from the cell engineering system to the electroporation unit and then back to a suitable cell engineering system.

[0079]

[0102] In other embodiments, the electroporation unit 1706 can be disposed within the cell engineering system 600 such that the overall system is a closed self - contained system. Methods of including the electroporation unit 1706 inside the cell engineering system 600 are known to those skilled in the art and utilize various miniaturization strategies and the like.

[0080]

[0103] The various methods described herein enable the generation of genetically modified immune cell cultures, and the transduction efficiency of the methods is at least 20% higher than the transduction efficiency of methods that utilize a flexible gas - permeable bag for cell culture. As described herein and as shown in the examples, the methods that utilize the cell engineering systems described herein are superior to conventional methods that rely on the use of a flexible gas - permeable bag for performing cell culture. In further embodiments, the transduction efficiency of the method is at least 10% higher, more suitably at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, or in embodiments, at least 40% higher than the transduction efficiency of methods that utilize a flexible gas - permeable bag for cell culture.

[0081]

[0104] Suitably, the methods described herein generate at least 20% more genetically modified immune cells than methods that utilize a manual cell culture with a flexible gas - permeable bag. More suitably, the method generates at least 25% more, at least 30% more, at least 35% more, or at least 40% more genetically modified immune cells than methods that utilize a manual cell culture with a flexible gas - permeable bag.

[0082]

[0105] In an exemplary embodiment, the cell engineering system described herein includes a plurality of chambers, each step of the various methods described herein is performed in a different chamber of the plurality of chambers of the cell engineering system, each of the activation reagent, the vector, and the cell culture medium is included in a different chamber of the plurality of chambers before initiating the method, at least one of the plurality of chambers is maintained at a temperature for growing cells (e.g., about 37° C.), and at least one of the plurality of chambers is maintained at a refrigeration temperature (e.g., about 4-8° C.).

[0083]

[0106] In some embodiments, the present disclosure provides a method of generating chimeric antigen receptor T cells, the method comprising: (a) appropriately activating a peripheral blood mononuclear cell culture in a culture medium comprising at least one of an anti-CD3 antibody and an anti-CD28 antibody to generate an activated T cell culture; (b) transducing the activated T cell culture with a lentiviral vector, which is a vector encoding a chimeric antigen receptor, to generate a transduced T cell culture; (c) expanding the transduced T cell culture to a pre-defined culture size; (d) concentrating the expanded T cell culture of (c) to a volume of from about 20 mL to about 500 mL, appropriately from about 50 mL to about 200 mL; and (e) recovering the concentrated T cell culture of (d) to generate a chimeric antigen receptor T (CAR T) cell culture, wherein the activated T cell culture is not substantially disrupted between steps (a) and (b), and the method is performed by a fully enclosed cell engineering system that appropriately has instructions for performing steps (a) to (e). Appropriately, steps (a) to (e) are performed within one or more chambers of the cell engineering system. As described herein, in embodiments, the method generates at least 20% more CAR T cells than a method that utilizes a flexible gas-permeable bag for cell culture. In an exemplary embodiment, the method generates at least 2 billion viable CAR T cells.

[0084]

[0107] Chimeric antigen receptor T cells, or "CAR T cells," are T cells modified with a chimeric antigen receptor (CAR) to more specifically target cancer cells. Generally, a CAR contains three parts: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain is the region of the receptor that is exposed to the extracellular fluid and contains three parts: a signal peptide, an antigen recognition region, and a spacer. The signal peptide directs the nascent protein to the endoplasmic reticulum. In a CAR, the signal peptide is a single-chain variable fragment (scFv). The scFv contains the light chain (VL) and heavy chain (VH) of an immunoglobulin linked by a short linker peptide. In some embodiments, the linker contains glycine and serine. In some embodiments, the linker contains glutamic acid and lysine.

[0085]

[0108] The transmembrane domain of the CAR is a hydrophobic α-helix that spans the membrane. In some embodiments, the transmembrane domain of the CAR is the CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain results in a highly expressed CAR. In some embodiments, the transmembrane domain of the CAR is the CD3-ζ transmembrane domain. In some embodiments, the CD3-ζ transmembrane domain results in a CAR that is incorporated into the native T cell receptor.

[0086]

[0109] The endodomain of the CAR is generally considered the "functional" end of the receptor. After antigen recognition by the antigen recognition region of the ectodomain and CAR clustering, a signal is transmitted to the cell. In some embodiments, the endodomain is the CD3-ζ endodomain and contains three immunoreceptor tyrosine-based activation motifs (ITAMs). In this case, the ITAMs transmit an activation signal to the T cell after antigen binding, triggering a T cell immune response.

[0087]

[0110] During the generation of CAR T cells, T cells are removed from a human subject, genetically modified, and reintroduced into the patient to attack cancer cells. CAR T cells can be derived from the patient's own blood (autologous) or another healthy donor (allogeneic). Generally, CAR T cells are generated to be specific for an antigen that is expressed in tumors but not in healthy cells.

[0088]

[0111] Activation of T cells. In some embodiments, the immune cell cultures generated by the methods described herein are CAR T cell cultures. CAR T cells can be activated to form an activated T cell culture. In vivo, antigen-presenting cells (APCs) such as dendritic cells act as a stimulus for T cell activation through the interaction of the T cell receptor (TCR) with the APC major histocompatibility complex (MHC). The TCR associates with CD3, a T cell coreceptor that aids in the activation of both cytotoxic T cells (e.g., CD8+ naive T cells) and helper T cells (e.g., CD4+ naive T cells). Generally, T cell activation requires stimulation of the TCR / CD3 complex and costimulatory receptors according to a two-signal model. T cell activation is further described, for example, in Kochenderfer 2015; Callos 2011.

[0089]

[0112] In the absence of a co-stimulatory signal, cells are susceptible to anergy and become unresponsive. Therefore, co-stimulation of T cells may be important for T cell proliferation, differentiation, and survival. Non-limiting examples of T cell co-stimulatory molecules include CD28, which is a receptor for CD80 and CD86 on the membrane of APCs; and CD278 or ICOS (inducible T cell co-stimulator), which is a CD28 superfamily molecule expressed on activated T cells that interacts with ICOS-L. Thus, in some embodiments, the co-stimulatory molecule is CD28. In other embodiments, the co-stimulatory molecule is ICOS. In vivo, the co-stimulatory signal is presented by the B7 molecule of APCs and binds to the CD28 receptor of T cells. B7 is a peripheral membrane-spanning protein found on activated APCs that can interact with CD28 or CD152 surface proteins on T cells to generate a co-stimulatory signal. Thus, in some embodiments, the co-stimulatory molecule is B7. Co-stimulatory receptors are further described, for example, in Lafferty, 1975; Harding, 1992; Clavreul, 2000; Charron, 2015; Fathman, 2007; Greenwald, 2005. Co-stimulation is further described, for example, in Carpenter, 2000; Andris, 2004. B7 molecules are further described, for example, in Fleischer, 1996; Schwartz, 2003.

[0090]

[0113] Various methods of activation are utilized in vitro to simulate T cell activation. In embodiments, the T cell culture is activated with an activating reagent. In further embodiments, the activating reagent is an antigen-presenting cell (APC). In still further embodiments, the activating reagent is a dendritic cell. Dendritic cells are APCs that process antigen and present it to T cells on the cell surface. In some embodiments, the activating reagent is co-cultured with the T cell culture. Co-culture may require separate purification and culturing of a second cell type, which can increase labor requirements and sources of variability. Thus, in some embodiments, alternative activation methods are used.

[0091]

[0114] In an embodiment, the cells maintain stable contact with the activation reagent during the activation step. One way to maintain stable contact between the cells and the activation reagent is by preventing unnecessary or excessive movement of the cells. Thus, in an embodiment, cell culture is not substantially disrupted during the activation step. "Not substantially disrupted" means that while the cell culture medium is being exchanged, the cells generally remain in the same region of the cell culture chamber, for example, at the bottom of the chamber. If the cells are moved between different containers, for example, from one culture flask to another, the cells can be disturbed, or if the container is flexible, the cells can be disturbed. For example, a flexible container such as a culture bag can move the cells when handling the bag. As described herein, the method appropriately utilizes a substantially flat and low cell culture chamber to allow uniform access of the cells to various nutrients and gases, and also facilitates the removal of waste products and the movement of the medium. The substantially flat cell culture chamber also allows the cells to contact each other during various stages of the method that can enhance cell growth and the generation of the desired cell phenotype(s).

[0092]

[0115] In some embodiments, the activation reagent is an antibody. In some embodiments, the cell culture is activated with an antibody bound to a surface such as a polymer surface that includes beads. In further embodiments, the one or more antibodies are anti-CD3 and / or anti-CD28 antibodies. For example, the beads can be magnetic beads such as Dynabeads that are coated with anti-CD3 and anti-CD28. Anti-CD3 and anti-CD28 beads can appropriately provide the stimulatory signals that support the activation of T cells. See, for example, Riddell, 1990; Trickett, 2003.

[0093]

[0116] In other embodiments, the cell culture is activated with a soluble antibody. In further embodiments, the soluble antibody is a soluble anti-CD3 antibody. OKT3 is a murine monoclonal antibody of the immunoglobulin IgG2a isotype that targets CD3. Thus, in some embodiments, the soluble anti-CD3 antibody is OKT3. OKT3 is further described, for example, in Dudley, 2003; Manger, 1985; Ceuppens, 1985; Van Wauwe, 1980; Norman, 1995.

[0094]

[0117] In some embodiments, the co-stimulatory signal for T cell activation is provided by accessory cells. Accessory cells can include, for example, Fc receptors that enable cross-linking of the TCR / CD3 complex of T cells and the CD3 antibody. In some embodiments, the cell culture is a mixed population of peripheral blood mononuclear cells (PBMCs). PBMCs can include accessory cells that can support the activation of T cells. For example, the CD28 co-stimulatory signal can be provided by B7 molecules present on monocytes of PBMCs. Thus, in some embodiments, accessory cells include monocytes or monocyte-derived cells (e.g., dendritic cells). In additional embodiments, accessory cells include B7, CD28, and / or ICOS. Accessory cells are further described, for example, in Wolf, 1994; Chai, 1997; Verwilghen, 1991; Schwartz, 1990; Ju, 2003; Baroja, 1989; Austyn, 1987; Tax, 1983.

[0095]

[0118] As described herein, the activation reagent can determine the phenotype of the generated CAR T cells and enable the promotion of the desired phenotype. In some embodiments, the activation reagent determines the ratio of T cell subsets, i.e., CD4+ helper T cells and CD8+ cytotoxic T cells. Cytotoxic CD8+ T cells are typically involved in killing cancer cells (i.e., anti-tumor response), cells infected (e.g., with a virus), or cells damaged in other ways. CD4+ T cells typically produce cytokines, assist in regulating the immune response, and in some cases, can support cell lysis. CD4+ cells activate APCs, which then stimulate naive CD8+ T cells for the anti-tumor response. Thus, in embodiments, the methods of the present disclosure further include generating CAR T cells of a predetermined phenotype (i.e., promoting cells of the desired phenotype). The predetermined phenotype can be, for example, a predetermined ratio of CD8+ cells to CD4+ cells. In some embodiments, the ratio of CD8+ cells to CD4+ cells in the population of CAR T cells is about 1:1, about 0.25:1, or about 0.5:1. In other embodiments, the ratio of CD8+ cells to CD4+ cells in the population of CAR T cells is about 2:1, about 3:1, about 4:1, or about 5:1.

[0096]

[0119] In embodiments, the activation reagent is removed from the activated T cell culture after the activation step. The activation reagent, e.g., anti-CD3 antibody and / or anti-CD28 antibody, can be present in the cell culture medium. Thus, in some embodiments, the cell culture medium containing the activation reagent, e.g., anti-CD3 antibody and / or anti-CD28 antibody, is removed from the activated T cell culture after the activation step. In some embodiments, the removal of the activation reagent includes the removal of soluble antibodies. For example, soluble antibodies can be removed by exchanging the cell culture medium. Soluble antibodies can also be removed by affinity methods specific to the soluble antibody. In other embodiments, the removal of the activation reagent includes the removal of beads containing the antibody. The removal of the beads can include, for example, filtration of the beads or removal by a magnet.

[0097]

[0120] Transduction of activated T cells. In some embodiments, the genetically modified immune cell culture is an activated T cell culture transfected with a vector encoding a chimeric antigen receptor to produce a transduced T cell culture. In some embodiments, transduction includes viral infection, transposons, mRNA transfection, electroporation, or combinations thereof. In some embodiments, transduction includes electroporation. Thus, in embodiments, the cell engineering system includes an electroporation system or electroporation unit as described herein. In additional embodiments, transduction includes viral infection. The vector can be a viral vector such as, for example, a lentiviral vector, a gamma-retroviral vector, an adeno-associated viral vector, or an adenoviral vector. In embodiments, transduction includes introducing a viral vector into the activated T cells of the cell culture. In additional embodiments, the vector is delivered as viral particles.

[0098]

[0121] In some embodiments, the transduction step comprises transducing activated T cells with a lentiviral vector, and the lentiviral vector is introduced at a multiplicity of infection (MOI) of about 0.5 to about 50, about 0.5 to about 30, or about 0.5 to about 20. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 8. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 6. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 4. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 2. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.6 to about 1.5. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.7 to about 1.3. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.8 to about 1.1. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.

[0099]

[0122] In some embodiments, after the activation step, the cell culture medium is removed from the T cell culture, and then the medium is mixed with a vector (e.g., a lentiviral vector) and uniformly distributed to the cells. In some embodiments, the removed cell culture medium is used to dilute and to uniformly deliver the vector to the activated T cell culture. The uniform distribution of the vector (e.g., lentiviral vector) in the T cell culture and the resulting uniform exposure improve the transduction efficiency. In some embodiments, the volume of the cell culture decreases after activation and before addition of the vector. The decrease in volume can allow for a higher degree of cell-vector contact. In some embodiments, the activated T cell culture is not substantially disrupted during transduction. In some embodiments, the cell culture is not substantially disrupted during the activation and transduction steps, i.e., the cells generally remain in the same region of the chamber (e.g., the bottom of the cell culture chamber) while the activation reagent or vector is being provided to the cells. This promotes a uniform distribution and uniform exposure of the activation reagent and / or vector to the cells and thus can improve the activation and / or transduction efficiency.

[0100]

[0123] Thus, in some embodiments, the transduction efficiency of the methods using a cell engineering system is higher than the transduction efficiency of the methods using a flexible gas permeable bag for cell culture. In some embodiments, the transduction efficiency of the automated method for generating CAR T cells described herein is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, or at least 100% greater than the transduction efficiency of the method utilizing a flexible gas permeable bag.

[0101]

[0124] Expansion of Transduced T Cells. In some embodiments, a transduced T cell culture (or other immune cell culture) is expanded to a predetermined culture size (i.e., number of cells). The predetermined culture size may include a sufficient number of cells suitable for clinical use, i.e., infusion into a patient, research and development work, etc. In some embodiments, the clinical dose or therapeutic dose of CAR T cells for administration to a patient is about 10 5 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells, or about 10 10 cells. In some embodiments, the method generates at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 clinical doses of CAR T cells. In some embodiments, the transduced T cell culture is expanded to a total volume of from about 0.1 L to about 5 L, from about 0.1 L to about 2 L, or from about 0.2 L to about 2 L. In some embodiments, the transduced T cell culture is expanded to a total volume of about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L or about 1.0 L. Also, depending on the stage of the cell generation process, the volume can be changed through the process as needed. In some embodiments, the predetermined culture size is input by the user of the cell engineering system. The user can input the predetermined culture size as the desired number of cells to be generated (e.g., 10 10 CAR T cells) or as the desired number of clinical doses or therapeutic doses to be generated (e.g., 10 clinical doses or therapeutic doses of CAR T cells). In embodiments, the number of CAR T cells generated by the methods described herein is at least about 100 million (i.e., 1×10 6) cells, or at least about 300 million, at least about 500 million, at least about 600 million, at least about 700 million, at least about 800 million, at least about 900 million, at least about 1 billion (i.e., 1×10 9 ) cells, at least about 1.1 billion, at least about 1.2 billion, at least about 1.3 billion, at least about 1.4 billion, at least about 1.5 billion, at least about 1.6 billion, at least about 1.7 billion, at least about 1.8 billion, at least about 1.9 billion, at least about 2 billion (i.e., 2×10 9 ) cells, for example, at least about 2.1 billion, at least about 2.2 billion, at least about 2.3 billion, at least about 2.4 billion, at least about 2.5 billion, at least about 2.6 billion, at least about 2.7 billion, at least about 2.8 billion, at least about 2.9 billion, or at least about 3 billion CAR T cells.

[0102]

[0125] In some embodiments, expansion of the transduced T cell culture comprises at least one round of feeding, washing, monitoring, and selection of the transduced T cell culture. Feeding of the cell culture may comprise supplementing the cell culture with media and / or additional nutrients. Washing of the cell culture comprises removing spent media (i.e., media that is nutrient-depleted and / or contains cellular waste products) and replenishing the cell culture with fresh media. Monitoring of the cell culture comprises monitoring the temperature, pH, glucose, oxygen level, carbon dioxide level, and / or optical density of the cell culture. Selection of the cell culture may comprise, for example, selection of cells having desired characteristics such as viability, type, and / or morphology, and removal of cells not having the desired characteristics. In some embodiments, the cell engineering system is configured to perform multiple rounds of feeding, washing, monitoring, and / or selection of the transduced T cell culture to achieve a predetermined culture size. In some embodiments, the cell engineering system performs at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or at least 100 rounds of feeding, washing, monitoring, and / or selection of the transduced T cell culture to achieve a predetermined culture size.

[0103]

[0126] In embodiments, one or more of feeding, washing, and monitoring can be removed, or the order of events can be changed, depending on, for example, the desired cell phenotype or number of cells.

[0104]

[0127] In an embodiment, monitoring includes monitoring by a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor. Thus, in some embodiments, the cell engineering system includes one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor. In additional embodiments, the cell engineering system is configured to adjust the temperature, pH, glucose, oxygen level, carbon dioxide level, and / or optical density of the cell culture based on a predetermined culture size. For example, if the cell engineering system detects that the current oxygen level of the cell culture is too low to achieve the growth required for the desired cell culture size, the cell engineering system can, for example, introduce an oxygenated cell culture medium, replace the cell culture medium with an oxygenated cell culture medium, or flow the cell culture medium through an oxygen supply component (i.e., a silicone tube) to automatically increase the oxygen level of the cell culture. In another example, if the cell engineering system detects that the current temperature of the cell culture is too high and the cells are growing too rapidly (e.g., the potential for cell overcrowding can lead to undesirable characteristics), the cell engineering system automatically lowers the temperature of the cell culture to maintain a stable growth rate (or an exponential growth rate if desired) of the cells. In still further embodiments, the cell engineering system automatically adjusts the cell supply schedule (i.e., providing fresh medium and / or nutrients to the cell culture) based on the cell growth rate and / or the number of cells, or other monitoring factors such as pH, oxygen, glucose. The cell engineering system is configured to store the medium (and other reagents such as wash solutions) in a low temperature chamber (e.g., 4 °C or -20 °C) and can be configured to warm the medium in a room temperature chamber or a high temperature chamber (e.g., 25 °C or 37 °C respectively) before introducing the warmed medium into the cell culture.

[0105]

[0128] In an embodiment, washing includes washing the cells by filtration or sedimentation. In some embodiments, the washing step does not require moving the cell culture vessel or flask, i.e., the cells can be washed within the same cell culture vessel or flask. In further embodiments, the cells remain substantially unperturbed during the washing step. In an embodiment, selection includes mixing the cell culture with one or more selection reagents. The selection reagent can be beads specific to the desired cell type, such as magnetic beads, and then the cells bound to the beads are separated from the unbound cells, for example, by passing through a magnetic chamber. For example, the selection beads include an antibody specific to the desired cell type, such as an anti-CD8 antibody or an anti-CD4 antibody. Selection can also be performed by filtration to remove or select a specific cell type based on size. Selection of cells by plastic adhesion (i.e., the cells start in one chamber, the unwanted cells adhere to the surface, and then the desired cells still in suspension are transferred to another chamber) can also be utilized.

[0106]

[0129] Suitably, during the expansion phase, the cells are not agitated and rotated. Maintaining the cells in a relatively stationary position during expansion has been determined to assist overall cell production and to help provide the desired cell phenotype.

[0107]

[0130] Concentration of the expanded culture. In some embodiments, the expanded T cell culture (or other immune cell culture) is concentrated to a predetermined concentration. The predetermined concentration is a volume that can be appropriately injected into a patient. For example, the expanded T cell culture can be concentrated to about 1 ml, about 2 ml, about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, or about 100 ml. In some embodiments, the concentration is performed by centrifugation. In some embodiments, the concentration is performed by filtration. In some embodiments, the filtration is ultrafiltration and / or diafiltration. In some embodiments, the predetermined concentration is input by the user of the cell engineering system. In other embodiments, the predetermined concentration is determined by the cell engineering system based on different parameters input by the user, such as the number or volume of the clinical dose or treatment dose to be generated; or the number of cells to be generated. In some embodiments, the cell engineering system automatically adjusts the volume or number of the clinical dose or treatment dose to be generated based on the input parameters. In some embodiments, the cell engineering system automatically adjusts the parameters of centrifugation (e.g., speed, duration of centrifugation) or filtration (e.g., filter size, volume, duration) based on the predetermined concentration.

[0108]

[0131] Sedimentation based on port location and chamber design can also be utilized. That is, without removing the cells, the volume of the liquid in the chamber can be reduced to about 0.5 mL.

[0109]

[0132] CAR T Cell Culture and Recovery. In some embodiments, a concentrated T cell culture (or other immune cell culture) is recovered to appropriately generate a chimeric antigen receptor (CAR) T cell culture. In some embodiments, recovery includes agitation, fluid flow, and washing of the CAR T cells. In some embodiments, recovery includes separation of the cells from unwanted products such as, for example, cellular waste, beads (e.g., beads containing antibodies and / or beads used for cell separation), or excess viral vectors. In some embodiments, recovery includes uniform distribution of the CAR T cells into one or more flasks, vials, or containers. In some embodiments, recovery includes resuspending the CAR T cells in a formulation reagent, e.g., a solution that stabilizes the CAR T cells for long-term storage. In some embodiments, recovery includes cryopreserving the CAR T cells.

[0110]

[0133] Further Downstream Processes. In some embodiments, the CAR T cells undergo further downstream processing prior to therapeutic use in a patient. For example, cryopreserved CAR T cells can be filtered by sterile filtration to remove residues of potential viral particles. After sterile filtration, the CAR T cells can undergo at least one or more concentration steps before being packaged into one or more vials, flasks, vessels, or containers. The packaged CAR T cells may be subjected to quality assessment and / or quality control testing. In some embodiments, the CAR T cells undergo minimal downstream processing prior to administration to a patient. For example, in some embodiments, the recovered CAR T cells are not cryopreserved but are transferred to the patient within a short time after recovery. Avoiding the cryopreservation step may improve the cell viability.

[0111]

[0134] Cell engineering system. In some embodiments, the methods described herein are performed by a fully enclosed cell engineering system 600 (see FIGS. 6A, 6B), and appropriately have instructions for performing activation, transduction, expansion, enrichment, and modification steps. A cell engineering system for the automated generation of genetically modified immune cells, including CAR T cells, is described herein and is also referred to herein as an automated cell engineering system, a cocoon, or a cocoon system. For example, a user can provide a cell engineering system pre-filled with cell cultures and reagents (e.g., activation reagents, vectors, cell culture media, nutrients, selection reagents, etc.) and cell generation parameters (e.g., starting cell number, type of media, type of activation reagent, type of vector, number of cells or dose to be generated, etc.). The cell engineering system can execute a method for generating a genetically modified immune cell culture, including CAR T cells, without further input from the user. At the end of the automated generation process, the cell engineering system can alert the user to collect the generated cells (e.g., by playing a warning message or sending a mobile app alert). In some embodiments, the fully enclosed cell engineering system includes a sterile cell culture chamber. In some embodiments, the fully enclosed cell engineering system minimizes contamination of the cell culture by reducing exposure of the cell culture to a non-sterile environment. In additional embodiments, the fully enclosed cell engineering system minimizes contamination of the cell culture by reducing handling of the cells by the user.

[0112]

[0135] As described herein, the cell engineering system appropriately includes a cassette 602. Thus, in embodiments, provided herein is a cassette for use in an automated cell engineering system. As used herein, "cassette" refers to an element of the cell engineering system that includes one or more chambers for performing various elements of the methods described herein and appropriately also includes one or more of cell culture media, activation reagents, vectors, etc., and is a largely self-contained, removable, and replaceable element.

[0113]

[0136] Figure 6B shows an embodiment of cassette 602 according to an embodiment of the present specification. In the embodiment, cassette 602 includes a cryogenic chamber 604 suitable for storing cell culture medium, and a high-temperature chamber 606 for appropriately performing activation, transduction, and / or expansion of immune cell cultures. Appropriately, the high-temperature chamber 606 is separated from the cryogenic chamber 606 by a thermal barrier 1102 (see FIG. 11B). As used herein, a "cryogenic chamber" refers to a chamber that is appropriately maintained at a temperature below room temperature, more appropriately at about 4°C to about 8°C, in order to maintain cell culture medium, etc. at a refrigerated temperature. The cryogenic chamber can include a bag or other holder for the medium that contains about 1L, about 2L, about 3L, about 4L, or about 5L of fluid. An additional medium bag or other fluid source can be connected outside the cassette and connected to the cassette via an access port.

[0114]

[0137] As used herein, a "high-temperature chamber" refers to a chamber that is appropriately maintained above room temperature and at a temperature that enables cell proliferation and growth, i.e., about 35 - 40°C, more appropriately about 37°C.

[0115]

[0138] In the embodiment, as shown in FIGS. 6D and 6E, the high-temperature chamber 606 appropriately includes a cell culture chamber 610 (also referred to as a proliferation chamber or a cell proliferation chamber).

[0116]

[0139] The cassette further includes one or more fluid pathways connected to the cell culture chamber, and the fluid pathways provide recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber. Cassette 602 also includes one or more pumps 605, including a peristaltic pump for driving fluid through the cassette, and one or more valves 607 for controlling the flow through the various fluid pathways, as described herein.

[0117]

[0140] In an exemplary embodiment, as shown in FIG. 6D, the cell culture chamber 610 is a flat, non-flexible chamber that does not easily bend or curve (i.e., made of a substantially non-flexible material such as plastic). By using a non-flexible chamber, the cells can be maintained in a substantially unobstructed state. As shown in FIG. 6E, the cell culture chamber 610 is oriented such that the immune cell culture can spread across the bottom 612 of the cell culture chamber. As shown in FIG. 6E, the cell culture chamber 610 is properly maintained in a position parallel to the floor or table, maintaining the cell culture in an unobstructed state and allowing the cell culture to spread across a large area of the bottom 612 of the cell culture chamber. In an embodiment, the overall thickness of the cell culture chamber 610 (i.e., the height 642 of the chamber) is thin, on the order of about 0.5 cm to about 5 cm. Appropriately, the cell culture chamber has a volume of about 0.50 ml to about 300 ml, more appropriately about 50 ml to about 200 ml, or the cell culture chamber has a volume of about 180 ml. Using a low chamber height 642 (less than 5 cm, appropriately less than 4 cm, less than 3 cm, or less than 2 cm) allows for effective medium and gas exchange close to the cells. The ports are configured to allow mixing via fluid recirculation without disturbing the cells. A static container with a higher height may generate a concentration gradient, and the region near the cells may be limited in oxygen and fresh nutrients. Controlled hydrodynamics allows for medium exchange without disturbing the cells. The medium can be removed from an additional chamber (where there are no cells) without the risk of losing cells.

[0118]

[0141] As described herein, in an exemplary embodiment, the cassette is pre-filled with one or more of a cell culture, a culture medium, an activation reagent, and / or a vector, including any combination of the following. In further embodiments, these various elements can be added later via a suitable injection port or the like.

[0119]

[0142] As described herein, in embodiments, the cassette may further suitably include one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, a lactate sensor / monitor, and / or an optical density sensor. The cassette can also include one or more sampling ports and / or injection ports. An example of such sampling and injection ports (1104) is shown in FIG. 11. This can include access ports for connecting the cartridge to external devices such as an electroporation unit or an additional media source. FIG. 11A also shows the cell input 1105, a reagent warming bag 1106 that can be used to warm the cell culture medium, etc., and the arrangement of a culture zone 1107 that holds various components for use in the culture medium, including, for example, cell culture medium, vectors, nutrients, and waste products.

[0120]

[0143] FIG. 11B shows the cocoon cell engineering system with the cassette 602 removed. In FIG. 11B, components of the cell engineering system can be seen, including a gas control seal 1120, a warming zone 1121, an actuator 1122, a pivot 1123 for rocking or tilting the cell engineering system as desired, and a cryogenic zone 1124 for holding the cryogenic chamber 606. Also shown is an exemplary user interface 1130 that can include a barcode reader, and a function for receiving input using a touchpad or other similar device. FIG. 11E shows additional details of the cassette 602, including the arrangement of a secondary chamber 1150 that can be used if additional cell culture volume is required, and a recovery chamber 1152 that can be used to recover the final cell culture produced herein.

[0121]

[0144] In an exemplary embodiment, as shown in FIG. 6F, the cell culture chamber 610 further includes at least one of the following: a distal port 620 configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port; an intermediate port 622 configured to function as a recirculation inlet port; and a proximal port 624 configured to function as a discharge port for cell removal.

[0122]

[0145] In yet a further embodiment, provided herein is a cassette 602 for use in an automated cell engineering system 600, including a cell culture chamber 610 having a chamber volume configured to house an immune cell culture and perform activation, transduction, and / or expansion of the immune cell culture, and a satellite volume 630 (i.e., the satellite volume contains no cells) for increasing the working volume of the cell culture chamber by providing an additional volume to the medium and other working fluids without housing the immune cell culture. Suitably, the satellite volume is fluidly connected to the cell culture chamber, such that the medium is exchanged with the culture chamber without disturbing the immune cell culture. In an exemplary embodiment, the satellite volume is a bag, and in other embodiments, the satellite volume is a non-yield chamber. In an embodiment, the satellite volume is between about 0.50 ml and about 300 ml, more suitably between about 50 ml and about 200 ml. FIGS. 6D - 6E show the location of the satellite volume 630 of the cassette 602.

[0123]

[0146] Figure 6G shows a schematic diagram depicting the connection between the cell culture chamber 610 and the satellite volume 630. Also shown in Figure 6G are various sensors (e.g., pH sensor 650, dissolved oxygen sensor 651), as well as a sampling / sample port 652, and various valves (control valve 653, bypass check valve 654), and the arrangement of one or more fluid paths 640, appropriately including silicone-based tube components that connect the components. As described herein, the use of silicone-based tube components enables oxygen supply through the tube components, facilitating gas transfer and optimal oxygen supply for cell culture. Also shown in Figure 6G is the use of one or more hydrophobic filters 655 or hydrophilic filters 656 in the flow path of the cassette, along with the pump tube 657 and the bag / valve module 658.

[0124]

[0147] Figure 6H shows gas exchange data using the cocoon system, compared to a conventional bag.

[0125]

[0148] In an embodiment, the satellite volume 630 is further configured to be able to remove the culture medium without losing the cells of the immune cell culture. That is, the medium exchange between the satellite volume and the cell culture chamber is performed in such a way that the cells are not disturbed and are not removed from the cell culture chamber.

[0126]

[0149] In an additional embodiment, as shown in Figure 6G, the cassette 602 appropriately further includes, as needed, a cross-flow reservoir 632 for holding additional culture medium. Appropriately, the cross-flow reservoir has a volume between about 0.50 ml and about 300 ml, more appropriately between about 100 ml and about 150 ml.

[0127]

[0150] The cell engineering system described herein appropriately has three related volumes: a cell culture chamber volume, a working volume, and a total volume. Appropriately, the working volume used in the cassette ranges from 180 mL to 460 mL based on the process step and can be increased up to a maximum of about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, or about 1 L. In embodiments, the cassette can readily achieve 4×10 9 cells to 10×10 9 cells. The cell concentration during the process varies from 0.3×10 6 cells / ml to about 10×10 6 cells / ml. As described herein, the cells are placed in the cell culture chamber, but the medium is continuously recycled through additional chambers (e.g., a cross-flow reservoir and a satellite volume) to increase the working volume.

[0128]

[0151] Unlike a flexible bag that changes shape when filled with a liquid (e.g., a cell culture) and when picked up or moved, a “substantially non-yielding chamber” (e.g., the exemplary cell culture chamber 610) remains filled with liquid under typical handling conditions and does not change shape (e.g., bend, curve, deform, etc.) when picked up or moved. Thus, in some embodiments, the substantially non-yielding chamber allows the cells to remain substantially in the same region of the chamber even when the chamber is picked up or moved. The substantially non-yielding chamber also does not have the curvature associated with a bag. Thus, in some embodiments, the cells are more uniformly distributed within the substantially non-yielding chamber compared to a bag. In some embodiments, the activation reagent and / or vector are more uniformly distributed within the substantially non-yielding chamber compared to a bag.

[0129]

[0152] In some embodiments, the cell engineering system includes a plurality of chambers. In further embodiments, each step of activation, transduction, expansion, concentration, and recovery of the methods for cells described herein is performed in a different chamber of the plurality of chambers of the cell engineering system. In some embodiments, the cells are not substantially disturbed during movement from one chamber to another. In other embodiments, the steps of the method are performed within the same chamber of the cell engineering system, and the cell engineering system automatically adjusts the chamber environment required for each step of the method. Thus, the cells can be prevented from being disturbed during various steps.

[0130]

[0153] In some embodiments, the cell engineering system has improved gas exchange compared to a flexible gas-permeable bag for cell culture. In some embodiments, the cell engineering system includes a gas exchange line. The gas exchange line can be made of a gas-permeable material such as silicone, for example. In some embodiments, the gas permeability coefficient of the gas exchange line is higher than the permeability coefficient of the material used in a flexible gas-permeable bag. In some embodiments, the cell engineering system recirculates oxygen throughout the substantially non-yield chamber during the cell generation method. Thus, in some embodiments, the oxygen level of the cell culture in the cell engineering system is higher than the oxygen level of the cell culture in a flexible gas-permeable bag. Increasing the oxygen level may support increased cell growth and proliferation, so it may be important to increase the oxygen level during the expansion step of cell culture.

[0131]

[0154] In some embodiments, the cell engineering system continuously recirculates the medium throughout the chamber without disturbing the cells. For example, the cell engineering system can replenish nutrients, remove waste products, and continuously circulate the released cytokines and dissolved gases within the chamber while the cells remain in the same region of the chamber. Continuous circulation improves the uniform distribution of positive factors and the uniform removal of negative factors, reducing local effects caused by non-uniform distribution without disturbing the cells.

[0132]

[0155] In some embodiments, the cell engineering system provides carbon dioxide throughout the chamber during a cell generation method (including CAR T generation). CO2 helps maintain the target pH of the cell culture, which is important for cell growth and proliferation. In some embodiments, the cell engineering system monitors the CO2 level of the cell culture and adjusts the amount of CO2 provided based on the measured CO2 level. For example, as the cell culture increases, there is an increase corresponding to the amount of CO2 produced by the cells, and the cell engineering system reduces the amount of CO2 provided. The desired CO2 level for the cell culture can be, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% CO2 and can be defined by the user. Since the cell engineering system continuously adjusts the amount of CO2 provided based on the measured CO2 level of the cell culture, the cell engineering system can maintain the desired CO2 level throughout the production process. Dissolved CO2 generally acidifies the solution (to react with water to form carbonic acid), so the amount of CO2 in the cell culture can also affect the pH of the culture. Therefore, maintaining a stable CO2 level in the cell culture may result in a more stable pH. Thus, in embodiments, the pH level of the cell culture remains substantially constant during the production process. In further embodiments, the pH level of the transduced cell culture remains substantially constant during the expansion step.

[0133]

[0156] Yields from genetically modified immune cell production, including CAR T cell generation, may be affected by activation and transduction efficiency, as well as cell growth conditions. Activation efficiency can be improved by more stable contact between the cells and the activation reagent. Cell movement throughout the culture vessel can lead to a non-uniform distribution of the cells and, thus, when the activation reagent is added to the cell culture chamber, can produce local effects. In contrast to a flexible culture bag, cells grown in a non-yield chamber remain unaffected during the activation process, which can contribute to a higher activation efficiency.

[0134]

[0157] Improved activation efficiency can also result in a greater vector transduction efficiency. When the cells are activated and actively dividing, a vector (e.g., a lentiviral vector) can be more effectively incorporated into the cells. A uniform distribution of the cells within the cell culture chamber 610 can promote uniform exposure of the cells to the vector, while the cells are non-uniformly distributed and, thus, may receive different vector exposures within a flexible cell culture bag. Thus, in some embodiments, the transduction efficiency of the methods for the automated generation of genetically modified immune cells, including CAR T cells, described herein is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, or at least 100% greater than the transduction efficiency of the methods that utilize a flexible gas permeable bag.

[0135]

[0158] The growth conditions of cell cultures can also improve cell yields. For example, higher oxygen levels in a cell engineering system, facilitated by high gas permeability tubes and continuous oxygen recirculation within the cell culture chamber, may increase cell growth. The ability of a cell engineering system to continuously monitor the state of cell culture and make adjustments accordingly can also be advantageous. For example, a cell engineering system can monitor the CO2, O2, N2, and / or pH levels of a cell culture and adjust the levels of CO2, O2, or N2. Nutrients are also provided in a timely and consistent manner and are evenly distributed to the cell culture. Thus, the automated methods for generating genetically modified immune cells, including CAR T cells, described herein advantageously result in higher cell yields compared to manual methods or methods that utilize flexible culture bags. Thus, in some embodiments, the methods for the automated generation of genetically modified immune cells, including CAR T cells, that utilize the cell engineering systems described herein produce at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40% more, at least 45% more, at least 50% more, at least 55% more, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, or at least 100% more cells than methods that utilize flexible gas permeable bags for cell culture. In embodiments, the number of cells generated by the methods described herein is at least about 2 billion, at least about 2.2 billion, at least about 2.3 billion, at least about 2.4 billion, at least about 2.5 billion, at least about 2.6 billion, at least about 2.7 billion, at least about 2.8 billion, at least about 2.9 billion, or at least about 3 billion cells, including at least about 2 billion (i.e., 2×10 9 ) cells.

[0136] Additional exemplary embodiments

[0159] Embodiment 1 is a method for the automated generation of a genetically modified immune cell culture. The method activates an immune cell culture using an activation reagent to generate an activated immune cell culture, transduces the activated immune cell culture using a vector to generate a transduced immune cell culture, expands the transduced immune cell culture, concentrates the expanded immune cell culture, and collects the concentrated immune cell culture to generate a genetically modified immune cell culture, and further includes washing either or both of the expanded immune cell culture and the concentrated immune cell culture. The steps are performed by a completely enclosed cell engineering system, and the steps are optimized through the process of generating a genetically modified immune cell culture.

[0137]

[0160] Embodiment 2 includes the method of Embodiment 1, wherein the process is a self-regulating process, monitored using 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; and based on the monitoring, adjusting one or more of the temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density of the transduced T cell culture.

[0138]

[0161] Embodiment 3 includes the methods of Embodiments 1-2, which generate at least about 100 million viable genetically modified immune cells.

[0139]

[0162] Embodiment 4 includes the methods of Embodiments 1-3, which generate at least about 2 billion viable genetically modified immune cells.

[0140]

[0163] Embodiment 5 includes the methods of Embodiments 1-4, wherein the immune cell culture is a T cell culture.

[0141]

[0164] Embodiment 6 includes the method of Embodiment 5, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0142]

[0165] Embodiment 7 includes the method of Embodiment 6, wherein the vector encodes a chimeric antigen receptor.

[0143]

[0166] Embodiment 8 includes the methods of Embodiments 1 to 7, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells.

[0144]

[0167] Embodiment 9 includes the methods of Embodiments 1 to 8, wherein the immune cell culture comprises at least one accessory cell.

[0145]

[0168] Embodiment 10 includes the method of Embodiment 9, wherein the accessory cell comprises monocytes or monocyte-derived cells.

[0146]

[0169] Embodiment 11 includes the method of Embodiment 9, wherein the accessory cell comprises an antigen for a T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0147]

[0170] Embodiment 12 includes the methods of Embodiments 1 to 11, wherein the activation reagent comprises an antibody or dendritic cells.

[0148]

[0171] Embodiment 13 includes the method of Embodiment 12, wherein the antibody is immobilized on the surface.

[0149]

[0172] Embodiment 14 includes the method of Embodiment 13, wherein the surface is the surface of beads.

[0150]

[0173] Embodiment 15 includes the method of Embodiment 12, wherein the antibody is a soluble antibody.

[0151]

[0174] Embodiment 16 includes the methods of Embodiments 12 to 15, wherein the antibody comprises at least one of an anti-CD3 antibody and an anti-CD28 antibody.

[0152]

[0175] Embodiment 17 includes the methods of Embodiments 1 to 16, wherein the transduction comprises viral infection, electroporation, membrane disruption, or a combination thereof.

[0153]

[0176] Embodiment 18 includes the methods of Embodiments 1-17, wherein the vector is a lentiviral vector or a retrovirus.

[0154]

[0177] Embodiment 19 includes the methods of Embodiments 1-18, wherein transduction includes mixing the vector in a cell culture medium and uniformly delivering the vector in the medium to an activated immune cell culture.

[0155]

[0178] Embodiment 20 includes the methods of Embodiments 1-19, wherein expansion includes at least one or more of supplying, washing, and monitoring the transduced immune cell culture.

[0156]

[0179] Embodiment 21 includes the methods of Embodiments 2-20, wherein the oxygen level of the transduced immune cell culture is optimized for the immune cell culture.

[0157]

[0180] Embodiment 22 includes the methods of Embodiments 1-21, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a)-(e).

[0158]

[0181] Embodiment 23 includes the methods of Embodiments 1-22, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during steps (a)-(e).

[0159]

[0182] Embodiment 24 includes the methods of Embodiments 2-23, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c).

[0160]

[0183] Embodiment 25 includes the methods of Embodiments 1-24, wherein the cell engineering system is configured to perform one or more rounds of one or more of supplying, washing, monitoring, and selecting the transduced immune cell culture.

[0161]

[0184] Embodiment 26 includes the method of Embodiments 1 to 25, wherein the concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration.

[0162]

[0185] Embodiment 27 includes the method of Embodiment 26, wherein the process further includes adjusting the parameters of centrifugation or filtration.

[0163]

[0186] Embodiment 28 includes the method of Embodiments 1 to 27, wherein the cell engineering system includes a plurality of chambers, and each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system.

[0164]

[0187] Embodiment 29 includes the method of Embodiments 1 to 28, further including removing the activation reagent from the activated immune cell culture after step (a).

[0165]

[0188] Embodiment 30 includes the method of Embodiments 1 to 29, wherein the cell engineering system includes the cell culture of (a), the activation reagent, the vector, and the cell culture medium before starting the method.

[0166]

[0189] Embodiment 31 is a method for promoting a preferred phenotype of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture, wherein the activation reagent and the activation conditions promote the phenotype of the genetically modified immune cell culture, transducing the activated immune cell culture with a vector to generate a transduced immune cell culture, expanding the transduced immune cell culture; concentrating the expanded immune cell culture; and recovering the concentrated immune cell culture to generate a genetically modified immune cell culture, and the steps are performed by a fully enclosed automated cell engineering system.

[0167]

[0190] Embodiment 32 includes the method of Embodiment 31, wherein the activation reagent includes an antibody or a dendritic cell.

[0168]

[0191] Embodiment 33 includes the method of Embodiment 32 in which the antibody is immobilized on the surface.

[0169]

[0192] Embodiment 34 includes the method of Embodiment 33 in which the surface is the surface of beads.

[0170]

[0193] Embodiment 35 includes the method of Embodiment 32 in which the antibody is a soluble antibody.

[0171]

[0194] Embodiment 36 includes the methods of Embodiments 32 - 35 in which the antibody includes at least one of an anti - CD3 antibody, an anti - CD28 antibody, and an anti - CD2 antibody.

[0172]

[0195] Embodiment 37 includes the method of Embodiment 36 in which the soluble antibody is OKT3.

[0173]

[0196] Embodiment 38 includes the methods of Embodiments 31 - 37 in which the activation conditions provide a substantially unperturbed immune cell culture that allows for stable contact between the activation reagent and the immune cell culture.

[0174]

[0197] Embodiment 39 includes the methods of Embodiments 31 - 38 that generate at least about 100 million viable genetically modified immune cells.

[0175]

[0198] Embodiment 40 includes the method of Embodiment 39 that generates at least about 2 billion viable genetically modified immune cells.

[0176]

[0199] Embodiment 41 includes the methods of Embodiments 31 - 40 in which the immune cell culture is a T - cell culture.

[0177]

[0200] Embodiment 42 includes the method of Embodiment 41 in which the T - cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0178]

[0201] Embodiment 43 includes the method of Embodiment 42 in which the vector encodes a chimeric antigen receptor.

[0179]

[0202] Embodiment 44 includes the methods of Embodiments 31-43, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells.

[0180]

[0203] Embodiment 45 includes the methods of Embodiments 31-44, wherein the cell culture comprises at least one accessory cell.

[0181]

[0204] Embodiment 46 includes the method of Embodiment 45, wherein the accessory cell comprises monocytes or monocyte-derived cells.

[0182]

[0205] Embodiment 47 includes the method of Embodiment 45, wherein the accessory cell comprises an antigen for the T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0183]

[0206] Embodiment 48 includes the methods of Embodiments 41-47, wherein the phenotype of the T cell culture has a CD8+ cell:CD4+ ratio of about 0.1:1 to about 10:1.

[0184]

[0207] Embodiment 49 includes the methods of Embodiments 31-48, wherein transduction includes viral infection, electroporation, membrane disruption, or combinations thereof.

[0185]

[0208] Embodiment 50 includes the methods of Embodiments 31-49, wherein the vector is a lentiviral vector or a retrovirus.

[0186]

[0209] Embodiment 51 includes the methods of Embodiments 31-50, wherein transduction includes mixing the vector in the cell culture medium and uniformly delivering the vector in the medium to the activated immune cell culture.

[0187]

[0210] Embodiment 52 includes the methods of Embodiments 31-51, wherein expansion includes at least one or more of supplying, washing, and monitoring the transduced immune cell culture.

[0188]

[0211] Embodiment 53 includes the methods of Embodiments 31-52, wherein the oxygen level of the transfected immune cell culture is optimized for the promoted phenotype.

[0189]

[0212] Embodiment 54 includes the methods of Embodiments 31-53, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a)-(e).

[0190]

[0213] Embodiment 55 includes the methods of Embodiments 31-54, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during steps (a)-(e).

[0191]

[0214] Embodiment 56 includes the methods of Embodiments 31-55, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c).

[0192]

[0215] Embodiment 57 includes the methods of Embodiments 31-56, wherein the cell engineering system is configured to perform multiple rounds of supply, washing, monitoring, and selection of the transfected immune cell culture.

[0193]

[0216] Embodiment 58 includes the methods of Embodiments 31-57, wherein concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration.

[0194]

[0217] Embodiment 59 includes the methods of Embodiments 31-58, wherein the cell engineering system includes multiple chambers, and each of steps (a)-(e) is performed in a different chamber of the multiple chambers of the cell engineering system.

[0195]

[0218] Embodiment 60 includes the methods of Embodiments 31-59, further comprising removing the activation reagent from the activated immune cell culture after step (a).

[0196]

[0219] Embodiment 61 includes the methods of Embodiments 31 to 60, further comprising removing the vector after transfection in (b).

[0197]

[0220] Embodiment 62 includes the methods of Embodiments 31 to 61, wherein the cell engineering system includes the cell culture of (a), an activation reagent, a vector, and a cell culture medium before starting the method.

[0198]

[0221] Embodiment 63 is a method for the automated generation of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture, transfecting the activated immune cell culture with a vector to generate a transfected immune cell culture, expanding the transfected immune cell culture, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to generate a genetically modified immune cell culture, the steps being performed by a fully enclosed automated cell engineering system, and each of the steps being performed using an immune cell culture having an optimized cell density (cells / mL) and an optimized cell packing density (cells / cm 2 ).

[0199]

[0222] Embodiment 64 includes the method of Embodiment 63, wherein the optimized cell density in (a) is about 0.05×10 6 cells / mL to about 60×10 6 cells / mL.

[0200]

[0223] Embodiment 65 includes the method of Embodiment 63 or 64, wherein the optimized cell packing density in (a) is about 0.1×10 6 cells / cm 2 to about 60×10 6 cells / cm 2 .

[0201]

[0224] Embodiment 66 includes the methods of Embodiments 63 to 65, wherein the activation reagent includes an antibody or dendritic cells.

[0202]

[0225] Embodiment 67 includes the method of Embodiment 66 in which an antibody is immobilized on the surface.

[0203]

[0226] Embodiment 68 includes the method of Embodiment 67 in which the surface is the surface of beads.

[0204]

[0227] Embodiment 69 includes the method of Embodiment 66 in which the antibody is a soluble antibody.

[0205]

[0228] Embodiment 70 includes the method of Embodiments 66 - 69 in which the antibody includes at least one of an anti - CD3 antibody and an anti - CD28 antibody.

[0206]

[0229] Embodiment 71 includes the method of Embodiments 63 - 70 in which at least about 100 million viable genetically modified immune cells are generated.

[0207]

[0230] Embodiment 72 includes the method of Embodiments 63 - 71 in which at least about 2 billion viable genetically modified immune cells are generated.

[0208]

[0231] Embodiment 73 includes the method of Embodiments 63 - 72 in which the immune cell culture is a T - cell culture.

[0209]

[0232] Embodiment 74 includes the method of Embodiment 73 in which the T - cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0210]

[0233] Embodiment 75 includes the method of Embodiment 74 in which the vector encodes a chimeric antigen receptor.

[0211]

[0234] Embodiment 76 includes the method of Embodiments 64 - 75 in which the immune cell culture includes peripheral blood mononuclear cells and / or purified T cells.

[0212]

[0235] Embodiment 77 includes the method of Embodiments 64 - 76 in which the cell culture includes at least one accessory cell.

[0213]

[0236] Embodiment 78 includes the method of Embodiment 77, wherein the accessory cells include monocytes.

[0214]

[0237] Embodiment 79 includes the method of Embodiment 77, wherein the accessory cells include an antigen for the T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0215]

[0238] Embodiment 80 includes the methods of Embodiments 63 - 79, wherein the transduction includes viral infection, electroporation, membrane disruption, or a combination thereof.

[0216]

[0239] Embodiment 81 includes the methods of Embodiments 63 - 80, wherein the vector is a lentiviral vector or a retrovirus.

[0217]

[0240] Embodiment 82 includes the methods of Embodiments 63 - 81, wherein the transduction includes mixing the vector in the cell culture medium and uniformly delivering the vector in the medium to the activated immune cell culture.

[0218]

[0241] Embodiment 83 includes the methods of Embodiments 63 - 82, wherein the expansion includes at least one or more of supplying, washing, monitoring, and selecting the transduced immune cell culture.

[0219]

[0242] Embodiment 84 includes the methods of Embodiments 63 - 83, wherein the oxygen level of the transduced immune cell culture is optimized for cell density and cell confluency.

[0220]

[0243] Embodiment 85 includes the methods of Embodiments 63 - 84, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a) - (e).

[0221]

[0244] Embodiment 86 includes the method of Embodiment 85, wherein the oxygen recirculation is provided by a silicone tube during steps (a) - (c).

[0222]

[0245] Embodiment 87 includes the methods of Embodiments 63 - 86, wherein the cell engineering system recycles nutrients, waste products, released cytokines, and / or dissolved gases during steps (a) - (e).

[0223]

[0246] Embodiment 88 includes the methods of Embodiments 63 - 87, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c).

[0224]

[0247] Embodiment 89 includes the methods of Embodiments 63 - 88, wherein the recycling of nutrients, waste products, released cytokines, and / or dissolved gases is uniformly provided using cells having a density of about 0.05×10 6 cells / mL to about 60×10 6 cells / mL and a confluency of about 0.1×10 6 cells / cm 2 to about 60×10 6 cells / cm 2

[0225]

[0248] Embodiment 90 includes the methods of Embodiments 63 - 89, wherein the cell engineering system is configured to perform multiple rounds of feeding, washing, monitoring, and selection of the transfected immune cell culture.

[0226]

[0249] Embodiment 91 includes the methods of Embodiments 63 - 90, wherein concentration includes centrifugation, removal of supernatant after sedimentation, or filtration.

[0227]

[0250] Embodiment 92 includes the methods of Embodiments 63 - 91, wherein the cell engineering system includes multiple chambers, and each of steps (a) - (e) is performed in a different chamber of the multiple chambers of the cell engineering system.

[0228]

[0251] Embodiment 93 includes the methods of Embodiments 63 - 92, further comprising removing the activation reagent from the activated immune cell culture after step (a).

[0229] ​

[0252] Embodiment 94 includes the methods of Embodiments 63-93, further comprising removing the vector after transduction in (b).

[0230]

[0253] Embodiment 95 includes the methods of Embodiments 63-94, wherein the cell engineering system includes, before starting the method, the cell culture of (a), an activation reagent, a vector, and a cell culture medium.

[0231]

[0254] Embodiment 96 is a method for the automated generation of a genetically modified immune cell culture, the method comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture, transducing the activated immune cell culture with a vector to generate a transduced immune cell culture, expanding the transduced immune cell culture, wherein the transduced cell culture is not shaken during expansion, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to generate a genetically modified immune cell culture, the steps being performed by a fully enclosed automated cell engineering system.

[0232]

[0255] Embodiment 97 includes the method of Embodiment 96, wherein the activation reagent includes an antibody or dendritic cells.

[0233]

[0256] Embodiment 98 includes the method of Embodiment 97, wherein the antibody is immobilized on the surface.

[0234]

[0257] Embodiment 99 includes the method of Embodiment 98, wherein the surface is the surface of beads.

[0235]

[0258] Embodiment 100 includes the method of Embodiment 97, wherein the antibody is a soluble antibody.

[0236]

[0259] Embodiment 101 includes the methods of Embodiments 96-100, wherein the antibody includes at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody.

[0237]

[0260] Embodiment 102 includes the method of Embodiments 96-101 that generates at least about 100 million viable genetically modified immune cells.

[0238]

[0261] Embodiment 103 includes the method of Embodiment 102 that generates at least about 2 billion viable genetically modified immune cells.

[0239]

[0262] Embodiment 104 includes the method of Embodiments 96-103 where the immune cell culture is a T cell culture.

[0240]

[0263] Embodiment 105 includes the method of Embodiment 104 where the T cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0241]

[0264] Embodiment 106 includes the method of Embodiment 105 where the vector encodes a chimeric antigen receptor.

[0242]

[0265] Embodiment 107 includes the method of Embodiments 96-106 where the immune cell culture includes peripheral blood mononuclear cells and / or purified T cells.

[0243]

[0266] Embodiment 108 includes the method of Embodiments 96-107 where the cell culture includes at least one accessory cell.

[0244]

[0267] Embodiment 109 includes the method of Embodiment 108 where the accessory cell includes monocytes or monocyte-derived cells.

[0245]

[0268] Embodiment 110 includes the method of Embodiment 109 where the accessory cell includes an antigen for the T cell receptor that includes CD28, CD40, CD2, CD40L, and / or ICOS.

[0246]

[0269] Embodiment 111 includes the method of Embodiments 96-110 where transduction includes viral infection, electroporation, membrane disruption, or a combination thereof.

[0247]

[0270] Embodiment 112 includes the methods of Embodiments 96-111, wherein the vector is a lentiviral vector or a retrovirus.

[0248]

[0271] Embodiment 113 includes the methods of Embodiments 96-112, wherein transduction includes mixing the vector in a cell culture medium and uniformly delivering the vector in the medium to an activated immune cell culture.

[0249]

[0272] Embodiment 114 includes the methods of Embodiments 96-113, wherein expansion includes at least one or more of supplying, washing, monitoring, and selecting the transduced immune cell culture without agitating the immune cell culture.

[0250]

[0273] Embodiment 115 includes the methods of Embodiments 96-114, wherein the oxygen level of the transduced immune cell culture is optimized for the immune cell culture.

[0251]

[0274] Embodiment 116 includes the methods of Embodiments 96-115, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a)-(e).

[0252]

[0275] Embodiment 117 includes the methods of Embodiments 96-116, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases.

[0253]

[0276] Embodiment 118 includes the methods of Embodiments 96-117, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c).

[0254]

[0277] Embodiment 119 includes the methods of Embodiments 96-118, wherein the cell engineering system is configured to perform multiple rounds of supplying, washing, monitoring, and selecting the transduced immune cell culture.

[0255]

[0278] Embodiment 120 includes the methods of Embodiments 96-119, where the concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration.

[0256]

[0279] Embodiment 121 includes the methods of Embodiments 96-120, where the cell engineering system includes a plurality of chambers, and each of steps (a)-(e) is performed in a different chamber of the plurality of chambers of the cell engineering system.

[0257]

[0280] Embodiment 122 includes the methods of Embodiments 96-121, further including removing the activation reagent from the activated immune cell culture after step (a).

[0258]

[0281] Embodiment 123 includes the methods of Embodiments 96-122, further including removing the vector after transduction in (b).

[0259]

[0282] Embodiment 124 includes the methods of Embodiments 96-123, where the cell engineering system includes the cell culture of (a), the activation reagent, the vector, and the cell culture medium before starting the method.

[0260]

[0283] Embodiment 125 is a method for the automated generation of a genetically modified immune cell culture, where the method performed by the cell engineering system activates the immune cell culture with an activation reagent to generate an activated immune cell culture in a first chamber of the cell engineering system; transduces the activated immune cell culture, where transduction includes transferring the activated immune cell culture from the first chamber to an electroporation unit, electroporating the activated immune cell culture with a vector to generate a transduced immune cell culture, and transferring the transduced immune cell culture to a second chamber of the cell engineering system, expanding the transduced immune cell culture, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to generate a genetically modified cell culture.

[0261]

[0284] Embodiment 126 includes the method of Embodiment 125, wherein transduction transfers an activated immune cell culture from a first chamber to an electroporation unit via a first sterile closed connection, subjects the activated immune cell culture to electroporation with a vector to generate a transduced immune cell culture, and transfers the transduced immune cell culture to a second chamber of a cell engineering system via a second sterile closed connection.

[0262]

[0285] Embodiment 127 includes the method of Embodiment 126, wherein the electroporation unit is located outside the cell engineering system.

[0263]

[0286] Embodiment 128 includes the methods of Embodiments 125-127, which generate at least about 100 million viable genetically modified immune cells.

[0264]

[0287] Embodiment 129 includes the method of Embodiment 128, which generates at least about 2 billion viable genetically modified immune cells.

[0265]

[0288] Embodiment 130 includes the methods of Embodiments 125-129, wherein the immune cell culture is a T cell culture.

[0266]

[0289] Embodiment 131 includes the method of Embodiment 130, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0267]

[0290] Embodiment 132 includes the method of Embodiment 131, wherein the vector encodes a chimeric antigen receptor.

[0268]

[0291] Embodiment 133 includes the methods of Embodiments 125-132, wherein the immune cell culture includes peripheral blood mononuclear cells and / or purified T cells.

[0269]

[0292] Embodiment 134 includes the methods of Embodiments 125-132, wherein the cell culture includes at least one accessory cell.

[0270]

[0293] Embodiment 135 includes the method of Embodiment 134, wherein the accessory cells include monocytes or monocyte-derived cells.

[0271]

[0294] Embodiment 136 includes the method of Embodiment 134, wherein the accessory cells include an antigen for a T cell receptor, including CD28, CD40, CD40L, and / or ICOS.

[0272]

[0295] Embodiment 137 includes the methods of Embodiments 125-136, wherein the activation reagent includes an antibody or dendritic cells.

[0273]

[0296] Embodiment 138 includes the method of Embodiment 137, wherein the antibody is immobilized on the surface.

[0274]

[0297] Embodiment 139 includes the method of Embodiment 138, wherein the surface is the surface of beads.

[0275]

[0298] Embodiment 140 includes the method of Embodiment 137, wherein the antibody is a soluble antibody.

[0276]

[0299] Embodiment 141 includes the methods of Embodiments 138-140, wherein the antibody includes at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody.

[0277]

[0300] Embodiment 142 includes the methods of Embodiments 125-141, wherein the vector is a lentiviral vector or a retrovirus.

[0278]

[0301] Embodiment 143 includes the methods of Embodiments 125-142, wherein the expansion includes at least one or more of the supply, washing, monitoring, and selection of the transduced immune cell culture.

[0279]

[0302] Embodiment 144 includes the methods of Embodiments 125-143, wherein the oxygen level of the transduced immune cell culture is optimized for the immune cell culture.

[0280]

[0303] Embodiment 145 includes the method of Embodiments 125-144, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a)-(e).

[0281]

[0303] Embodiment 146 includes the method of Embodiments 125-145, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during steps (a)-(e).

[0282]

[0305] Embodiment 147 includes the method of Embodiments 125-146, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c).

[0283]

[0306] Embodiment 148 includes the method of Embodiments 125-147, wherein the cell engineering system is configured to perform multiple rounds of feeding, washing, monitoring, and selection of the transduced immune cell culture.

[0284]

[0307] Embodiment 149 includes the method of Embodiments 125-148, wherein concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration.

[0285]

[0308] Embodiment 150 includes the method of Embodiments 125-149, wherein the cell engineering system includes a plurality of chambers, and each of steps (a)-(e) is performed in a different chamber of the plurality of chambers of the cell engineering system.

[0286]

[0309] Embodiment 151 includes the method of Embodiments 125-150, further comprising removing the activation reagent from the activated immune cell culture after step (a).

[0287]

[0310] Embodiment 152 includes the method of Embodiments 125-151, further comprising removing the vector after transduction in (b).

[0288]

[0311] Embodiment 153 includes the methods of Embodiments 125-152, wherein the cell engineering system includes, prior to initiating the method, the cell culture of (a), an activation reagent, a vector, and a cell culture medium.

[0289]

[0312] Embodiment 154 includes the methods of Embodiments 1-153, wherein the transduction efficiency in step (c) of the method is flexible for cell culture and at least 20% higher than the transduction efficiency of the method using a gas-permeable bag.

[0290]

[0313] Embodiment 155 includes the methods of Embodiments 1-154, which produce at least 20% more genetically modified immune cells than the method using manual cell culture with a flexible, gas-permeable bag.

[0291]

[0314] Embodiment 156 includes the methods of Embodiments 1-155, wherein the cell engineering system includes a plurality of chambers, each of steps (a)-(e) is performed in a different chamber of the plurality of chambers of the cell engineering system, each of (a), the activation reagent, the vector, and the cell culture medium is included in a different chamber of the plurality of chambers prior to initiating the method, at least one of the plurality of chambers is maintained at a temperature for growing cells, and at least one of the plurality of chambers is maintained at a refrigeration temperature.

[0292]

[0315] Embodiment 157 is a cassette for use in an automated cell engineering system, including a low-temperature chamber for storing a cell culture medium and a high-temperature chamber for activating, transducing, and expanding an immune cell culture, wherein the high-temperature chamber is separated from the low-temperature chamber by a thermal barrier, the high-temperature chamber includes a cell culture chamber; and one or more flow paths connected to the cell culture chamber, the flow paths providing recirculation, waste removal, uniform gas exchange, and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber.

[0293]

[0316] Embodiment 158 includes the cassette of Embodiment 157, where the cell culture chamber is flat with a low chamber height and has no flexibility.

[0294]

[0317] Embodiment 159 includes the cassette of Embodiment 157 or Embodiment 158, where the cell culture chamber is oriented such that the immune cell culture can spread across the entire bottom of the cell culture chamber.

[0295]

[0318] Embodiment 160 includes the cassettes of Embodiments 157 - 159, which are pre - filled with a cell culture, a culture medium, an activation reagent, and a vector.

[0296]

[0319] Embodiment 161 includes the cassettes of Embodiments 157 - 160, which further include one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor.

[0297]

[0320] Embodiment 162 includes the cassettes of Embodiments 157 - 161, which further include one or more sampling ports and / or injection ports.

[0298]

[0321] Embodiment 163 includes the cassettes of Embodiments 157 - 162, which further include at least one of a distal port configured as a recirculation port, an intermediate port configured to function as a recirculation inlet port, and a proximal port configured to function as a discharge port for cell removal, where the cell culture chamber is configured to allow removal of air bubbles from the cell culture chamber.

[0299]

[0322] Embodiment 164 includes the cassettes of Embodiments 157 - 163, which further include an access port for connecting the cartridge to an external device.

[0300]

[0323] Embodiment 165 includes the cassette of Embodiment 164, where the external device includes an electroporation unit or an additional medium source.

[0301]

[0324] Embodiment 166 is a cassette for use in an automated cell engineering system, having a chamber volume configured to contain an immune cell culture for activating, transducing, and / or expanding an immune cell culture, and including a satellite volume for increasing the working volume of the chamber by providing an additional volume to the medium and other working fluids without containing the immune cell culture, wherein the satellite volume is fluidly connected to the cell culture chamber via one or more fluid pathways such that the medium is exchanged with the culture chamber without disturbing the immune cell culture.

[0302]

[0325] Embodiment 167 includes the cassette of Embodiment 166, wherein the satellite volume is a bag.

[0303]

[0326] Embodiment 168 includes the cassette of Embodiment 166, wherein the satellite volume is a non-deformable chamber.

[0304]

[0327] Embodiment 169 includes the cassettes of Embodiments 166 - 168, wherein the satellite volume is further configured to enable medium removal without loss of cells of the immune cell culture.

[0305]

[0328] Embodiment 170 includes the cassettes of Embodiments 166 - 169 and further includes a crossflow reservoir.

[0306]

[0329] Embodiment 171 includes the cassettes of Embodiments 166 - 170, wherein the cell culture chamber has a volume between about 0.50 ml and about 300 ml.

[0307]

[0330] Embodiment 172 includes the cassette of Embodiment 171, wherein the cell culture chamber has a volume between about 50 ml and about 200 ml.

[0308]

[0331] Embodiment 173 includes the cassette of Embodiment 172, wherein the cell culture chamber has a volume of about 180 ml.

[0309]

[0332] Embodiment 174 includes the cassettes of Embodiments 166 - 173 where the satellite volume is between about 0.50 ml and about 300 ml.

[0310]

[0333] Embodiment 175 includes the cassette of Embodiment 174 where the satellite volume is between about 150 ml and about 200 ml.

[0311]

[0334] Embodiment 176 includes the cassettes of Embodiments 166 - 175 where the cross - flow reservoir has a volume between about 0.50 ml and about 300 ml.

[0312]

[0335] Embodiment 177 includes the cassette of Embodiment 176 where the cross - flow reservoir has a volume between about 100 ml and about 150 ml.

[0313]

[0336] Embodiment 178 includes the cassettes of Embodiments 166 - 177 where the working volume is between about 180 mL and about 1 L.

[0314]

[0337] Embodiment 179 includes the cassette of Embodiment 178 where the working volume is between about 180 mL and about 460 mL.

[0315]

[0338] Embodiment 180 includes the cassettes of Embodiments 157 - 179 where one or more fluid pathways include silicone - based tube components that allow oxygen supply through tube components.

Example

[0316] Example 1 - Automated Generation of CAR T Cells Using a Cocoon System

[0339] In this example, GFP and HER-2 lentiviruses were used to transduce T cells using the following process parameters: initiation of inoculation of 60 million peripheral blood mononuclear cells (PBMCs), CD3 / CD28 activation, and supplementation of IL-2 and IL-7 to the T cell growth medium for culture expansion. A single-use sensor of a disposable cassette was used to monitor temperature, pH, and optical density (OD) in real time. Multiple cassette chambers connected via fluid channels enabled automatic supply and addition of process components. Some chambers are temperature-controlled at 4°C for medium and reagent storage, while other chambers contain elements for cell warming, mixing, washing, and concentration, enabling a fully enclosed process. Samples during processing were made for cell count and viability. At the end of the recovery process, FACS analysis was performed using the following panels: CD4, CD8, NGFR, IFN-γ, TNF-α, etc. The overview of the cocoon system used in this example is shown in FIG. 6. FIG. 6A shows a closed configuration of the cocoon system together with an external user control display that can be used to adjust parameters or monitor cell culture. A sterile, single-use cell culture "cassette" can be filled into the cocoon (FIG. 6C). As shown in the detailed view of the cassette (FIG. 6B), each cassette contains an upper chamber maintained at 37°C for cell growth, and a lower chamber maintained at 4°C for storing medium, viral vectors, and other temperature-responsive reagents. The cassette is configured such that fluid can be exchanged via internal fluid pathways and pumped into or out of the cassette. Sensors attached to the cassette can monitor the pH and optical density of the cell culture, etc.

[0317]

[0340] The results are shown in Figures 7 to 10. Figures 7A, 7B, and 7C show, respectively, the average recovery amount, average recovery survival rate, and average transduction efficiency of GFP transduction using the automated cocoon system, compared with the manual operation and magnification of cells using a G-REX (WilsonWolf) cell culture plate as a control. The G-REX plate has a gas-permeable bottom, and typically, when a user uses G-REX, the medium is changed every 4 to 5 days.

[0318]

[0341] Figures 8A and 8B show, respectively, viable cells, and the survival rate and transduction efficiency of HER-2 CAR-T transduction. After 10 days of culture, the HER-2 CAR-T cells reached approximately 2.2 billion, with a survival rate of 97% and a transduction rate of 65% (n = 4) in the cocoon system.

[0319]

[0342] Also, the performance of the automated cocoon system was compared with the manual operation and proliferation of cells using a PermaLife cell culture bag (OriGen) as a control. The PermaLife bag is a sealable, gas-permeable cell culture bag made of inert fluorinated ethylene propylene (FEP), and is equipped with a valve that facilitates the supply and recovery of cells by the user. Figure 9A shows the relative T cell purity level using the cocoon system compared with the PermaLife bag, evaluated by the proportion of CD3+ cells. Figure 9B shows that the proportion of CD8+ cells cultured in the cocoon system is higher compared with the PermaLife Bag control. Figures 9C and 9D show that the transfected cells produce TNF-α and INF-γ, respectively.

[0320]

[0343] Figures 10A and 10B show the effective and specific killing of target tumor cells by CAR T cells cultured in the cocoon system and the PermaLife bag, respectively.

[0321]

[0344] In conclusion, the cocoon system is a completely sealed cell engineering system and a feasible solution to transform the labor-intensive CAR T process into a fully automated and highly controlled system, thus enabling scalability, high yield, reduction of manufacturing costs, and improvement of process control to produce high-quality CAR-T cells.

[0322] Example 2 - Comparison of Activation Methods in the Cocoon System

[0345] This example compares the cell culture performance using different activation methods in the clinical-scale generation of CAR T cells in a cocoon automated manufacturing system and a PermaLife bag.

[0323]

[0346] T cells can be activated using magnetic anti-CD3 / anti-CD28 Dynabeads activation factor beads. These beads provide the two stimulatory signals necessary to support effective T cell activation. Another way to activate naive T cells is to utilize soluble anti-CD3 antibody (OKT3). OKT3 is a monoclonal IgG2a antibody that was originally used as an immunosuppressant. The co-stimulatory signal can be provided by accessory cells. When starting T cell culture from a mixed population of peripheral blood mononuclear cells (PBMCs), OKT3 can provide the accessory cells necessary to support T cell activation.

[0324]

[0347] Since OKT3 and Dynabeads utilize distinct activation mechanisms, the choice of one method over the other can affect the properties of the final product; specifically, the ratio of T cell subsets, CD4+ helper T cells, and CD8+ cytotoxic T cells. Cytotoxic CD8 T cells are involved in anti-tumor responses. CD4 cells produce cytokines and assist in the regulation of the immune response. Although killing is delayed compared to CD8 cells, CD4 cells have also been demonstrated to support cytolysis. CD4 cells send signals to APCs and thus activate APCs, subsequently priming naive CD8 T cells. Due to limited clinical data, the ideal target ratio of CD8 cells to CD4 cells is not well understood. Studies have shown that a combination of CD8 and CD4 cells is preferred over delivery of CD8 cells alone (see, e.g., Church, 2014; Feldmann, 2012; Reusch, 2015).

[0325]

[0348] Both methods of in vitro activation have advantages and disadvantages. Antibody-conjugated beads provide consistency and ensure stable co-activation of the TCR / CD3 complex and the CD28 co-stimulation pathway. The main drawback of the bead approach is the high cost associated with this product. Beads may also need to be effectively removed from the culture prior to transplantation. OKT3 provides a low-cost option for activating T cells. The main drawbacks associated with the soluble anti-CD3 approach are the dependence on accessory cells and the sensitivity to the culture environment. Patient samples may have a very diverse set of accessory cells and negative interactions that can potentially inactivate T cells functionally after previous stimulation. To understand the impact of each activation method on cell proliferation, phenotype, and function, T cells activated by Dynabeads and OKT3 were cultured on a clinical-scale automated platform.

[0326]

[0349] The cocoon provides environmental control of gas and temperature. This includes not only the linked refrigerated zone but also the 37 °C zone. Since there is no fluid contact between the cocoon and the cassette, the cleaning required during operation is minimized. All reagents can be filled into the cassette on the day of seeding and stored in the refrigerated zone of the cocoon until needed. Before delivery to the cells, the fluid is warmed to 37 °C. Due to the stability of the lentivirus, this is thawed on the day of transduction and delivered to the cassette via a sterile connector. Gas exchange (oxygen supply and CO2 buffering) is achieved via recirculation of the culture medium through gas permeable tubing. The embedded biosensor provided real-time data on dissolved oxygen and pH. Since T cells require stable contact with other cells or activation reagents, medium exchange, washing and recirculation for gas exchange can be performed via perfusion without disturbing the cells. Rocking can be used to facilitate efficient recovery.

[0327] Method

[0350] Cell culture. Peripheral blood mononuclear cells (PBMC) (Lonza) were thawed with DNase (Sigma) and <2×10 6They were allowed to recover overnight at a density of cells / mL. The cells were counted using a NUCLEOCOUNTER 200 equipped with a Blood Assay protocol and containing Solution 17 (Chemometec). The cells were transduced using a third-generation lentiviral vector encoded with the low-affinity nerve growth factor receptor (NGFR) as a transduction marker. This lentivirus was produced at the cGMP virus manufacturing facility of Lonza (Houston, Texas) based on the protocol and primers from the Bramson Lab at McMaster University (Hamilton, Canada). A multiplicity of infection (MOI) of 1 was used for all conditions. The virus titer was determined by detecting NGFR using flow cytometry with HEK293TM cells. The activation medium consisted of X-VIVO 15 (Lonza) supplemented with 22 IU / mL of IL-2 (Cedarlane) and 1% penicillin-streptomycin (Sigma). In the condition activated with soluble anti-CD3, OKT3 (Biolegend) was added to the activation medium at a final concentration of 50 ng / mL. In the condition activated with Dynabeads, beads and cells at a ratio of 1:1 were added to the activation medium. The expansion medium consisted of X-VIVO 15 (Lonza) supplemented with 29 IU / mL of IL-2 (Cedarlane), 5% human serum from male AB plasma (Sigma), 1% GLUTAMAX (Thermo Fisher), and 1% penicillin-streptomycin (Sigma).

[0328]

[0351] Automated CAR T cell generation. On day 0, 60×10 6 PBMCs were placed in the input bag of the cassette. Also, in the condition activated using anti-CD3 / anti-CD28 beads, 60×10 6Anti-CD3 / anti-CD28 Dynabeads (ThermoFisher) were added to the seeding bag at a 1:1 bead:cell ratio with the cells. The seeding bag was connected to the cassette and brought into the Cocoon (Octane Biotech Inc.). Following the operator's sign-in, the cassette was filled with the Cocoon. On day 1, the virus (Lonza Houston) was thawed and then transferred to the cell culture chamber via the cassette access port at an MOI of 1. Prior to delivering the virus to the cells, the medium was diluted using activation medium. The activation medium was removed from the culture chamber and returned with the virus without disturbing the cells. On day 4, expansion medium was added and the total working volume increased. Partial medium exchanges were performed with expansion medium on days 6 and 8. After the expansion step, the Cocoon reduced the final volume to less than 100 mL before removing the cells. Throughout the culture, data was continuously collected by the Cocoon. This included steps of all pumps and actuators, such as each time the door was opened and closed. Comprehensive sensor data such as heat value, gas concentration, fluid pH, and dissolved oxygen was collected. The operator was able to remotely monitor the status of the culture using a phone or an external computer.

[0329]

[0352] Manual CAR T cell generation. Manual generation of CAR T cells was performed in parallel with the Cocoon in a PermaLife cell culture bag. On day 0, 60×10 6 PBMCs were seeded in activation medium at 0.27×10 6 cells / mL on day 0. These cultures utilized the same donor cells and the same medium for activation and expansion as the automated culture. The cultures were initiated in PermaLife bags (PL240, Origen) and transferred to larger PermaLife bags (PL325, Origen) as the cells expanded on day 6. As the volume increased, the cells were expanded into PL240 and PL325 bags on day 8. On day 1, lentivirus was added to the bags at an MOI of 1. It was supplied to the cells at a volume equivalent to the Cocoon culture. However, unlike the Cocoon conditions, there was no medium sent to waste. The volume used was such that the culture was brought to 2×10 6It was maintained below cells / mL. On the 10th day, the culture volume was obtained by mass, and a sample of all cells was removed from the bag for counting and analysis. Before use in functional assays, the cells were centrifuged to reduce the residue and volume.

[0330]

[0353] Non-transduced and non-activated conditions. Non-transduced and non-activated negative controls used for fluorescence-activated cell sorting (FACS) analysis were cultured on a small scale according to the previously described protocol. Briefly, 1×10 5 cells were seeded in 96-well plates containing X-VIVO 15 (Lonza) supplemented with 5% human AB serum (Sigma) and 22 ng / mL of IL-2 (Cedarlane). Activated but non-transduced controls were set up using a similar protocol. After seeding the cells, an equal volume of medium was added. For conditions activated with soluble anti-CD3, it was supplemented with 100 ng / mL OKT3 (Biolegend) at a final concentration of 50 ng / mL. For conditions activated with anti-CD3 / anti-CD28 beads, Dynabeads were added at a ratio of 1:1. The activated cultures were expanded from 96-well plates to 24-well plates on the 4th day and transferred to T25 and T75 flasks based on their growth and fed every 2 days from the 4th day.

[0331]

[0354] Flow cytometry. To examine the phenotype of the starting population, cells were stained with the following primary antibodies: Pacific Blue CD3 (clone UCHT1, BD Biosciences), PE CD14 (clone 61D3, ThermoFisher), APCeFluor780 CD4 (clone OKT4, ThermoFisher), PerCP-Cy5.5 CD8a (clone RPA-T8, ThermoFisher), BV605 CD279 (PD-1, clone EH12.2H7 BioLegend), and LIVE / DEAD Fixable Violet dead cell stain (ThermoFisher). To evaluate the efficiency of HER2 transduction, cells were stained as above except instead of staining for monocytes (CD14), and cells were stained with BV421 CD271 (C40-1457 NGFR, BD Biosciences) and LIVE / DEAD Fixable Green dead cell stain (ThermoFisher). Next, the cells were fixed and washed. More than 20,000 events per condition were acquired with a SA3800 Sony Spectral Analyzer. FACS analysis was performed using FlowJo 10.4.2. Gates were set with fluorescence minus one (FMO) controls using non-transduced conditions and non-activated conditions.

[0332]

[0355] Tumor cell lines. LOX-IMVI cells (National Cancer Institute), which are HER2-negative tumor cells, are derived from metastatic melanoma and were expanded in RPMI (Sigma) containing 10% FBS (Sigma) as described above. SKOV-3 (ATCC) cells, which are HER2-positive tumor cells, are derived from ovarian serous cystadenocarcinoma and were expanded in McCoy’s 5a (modified) medium (ThermoFisher) containing 10% FBS as described above. Cells were passaged before confluence using 0.25% trypsin for 5 - 10 minutes. Low passage numbers were cryopreserved and the tumor lines were passaged 2 - 3 times before use in the ALAMARBLUE or ICS assays.

[0333]

[0356] Cytokine secretion assay. As described above (e.g., Atkuri, 2005; Avgoustiniatos, 2008), 50,000 LOX IMVI or SKOV-3 tumor cells were seeded in triplicate in round-bottom 96-well plates under each culture condition. The next day, T cells were seeded at a ratio of 8:1 per well of the tumor strain together with the protein transport inhibitor brefeldin A (Golgi Plug, BD Biosciences) at 37°C for 4 hours. The cells were stored overnight at 4°C. Next, the cells were pooled for staining and analysis. As described above, the cells were stained with the surface phenotypes CD3, CD4, CD8a, NGFR, and LIVE / DEAD Fixable Green Dead Cell Stain. Intracellular cytokine staining (ICS) was completed after fixation and permeabilization with the BD Cytofix / Cytoperm Fixation / Permeabilization Solution Kit (554714, BD Biosciences). The activated cytokines tested included APC IFNγ (clone B27, BD Biosciences) and PE TNFα (clone MAb11, BD Biosciences). On the Sony SA3800, more than 230,000 events (maximum 500,000) were collected for ICS analysis. The difference in cytokine production between the SKOV-3 and LOX-IMVI tumor strains was reported as the percentage of the population secreting TNFα or IFNγ. Non-transduced and non-activated conditions were used to set the gates together with FMO controls.

[0334]

[0357] Cytotoxicity assay. Cytotoxicity was tested as described above (e.g., Atkuri, 2005; Avgoustiniatos, 2008). Adherent tumor cell lines were seeded at 2×10 4Plated overnight in cells / wells (SKOV-3 or LOX-IMVI). CAR T cells from cocoon and control conditions were added to the wells of tumor cells at various effector (E) T cell to tumor (T) E:T ratios (0.25:1 to 8:1) and co-incubated overnight at 37°C. The wells were washed three times with warmed PBS or RPMI medium to remove any non-adherent cells. A 10% solution of ALAMARBLUE cell viability reagent (Life Technologies) was added at 100 μL and the wells were incubated at 37°C for 3 hours. ALAMARBLUE, a metabolic indicator of live cells that fluoresces upon reduction of mitochondria, was measured by fluorescence (excitation 530 nm, emission 595 nm) using a Tecan Infinite M200 Pro plate reader (Tecan, Maennendorf, Switzerland). Tumor cell viability was calculated as the loss of fluorescence in experimental wells compared to untreated target cells. Each condition was tested in triplicate.

[0335] Results

[0358] Using the Cocoon, an automation platform, we demonstrated the feasibility of achieving clinical-scale generation of CAR T cells using two different activation methods. The platform consists of a single-use disposable Cocoon cassette (Figures 11A, 11E) and a Cocoon control system (Figure 11B). Figure 11F shows how syringes 1170 or bags 1172 are used for cassette 602 sampling. The cassette is designed with multiple reagent bags to pre-fill and store all the reagents required for the process in the refrigerated zone of the cassette and enable cell processing in the culture zone. The cassette supports multiple unit operations linked as a closed system, such as cell activation, transduction, expansion, real-time dissolved oxygen and pH monitoring, washing, and cell concentration. At the bottom of the cassette, there are multiple bags for holding various reagents and waste required for culture. The Cocoon provides the control system for the cassette. This includes control of fluid and cell movement, as well as oscillation, agitation, and remote monitoring of control sensors. The actuator enables automated valve control without contacting the fluid. Without actuator interaction, the valve remains closed, allowing the cassette to be moved between rooms or to a microscope while preventing uncontrolled fluid movement. After filling the required reagents into the fluid reservoir of the cassette, it is snapped into the culture zone of the cassette where various unit operations are performed. For removal or injection of a sterile sample of the virus, an ICU Spiros connector is utilized. Before sample removal or virus addition, the operator proceeds at a specific time as defined by a pre-programmed protocol. After the operator signs in and confirms the notification, the Cocoon automatically opens, enabling sample removal or virus addition. The operator recognizes that the action is complete before the door automatically closes and environmental control resumes. When the cassette is filled in the Cocoon (Figure 11C) and the outer shell is closed (Figure 11D), the bottom of the cassette is separated from the top by a thermal barrier. The bottom is maintained at refrigerated temperature, and the top is maintained at 37°C. The closed Cocoon enables control of gas and heat. The cells are maintained at 37°C, and the reagents are maintained in the cold zone, extending their stability.The opaque shell prevents photoinduced toxicity associated with the degradation of medium components. A preheating chamber is positioned in the 37 °C zone to warm the medium before transferring it to the cells. All culture steps can be automated, from PBMC seeding to final concentration and cell recovery. As shown in Figure 11A, the cassette has a series of access ports that can be used to fill the virus after activation. Real-time dissolved oxygen and pH sensors are incorporated into the cassette and provide feedback to the Cocoon software. Real-time data and history graphs can be monitored to confirm that these factors are maintained within the target ranges.

[0336]

[0359] An overview of the Cocoon process steps is shown in Figure 12A. Gas-permeable PermaLife bags were used for parallel control cultures and expansion of CAR T cells (e.g., Lu, 2016). Figures 12B (Cocoon) and 12C (PermaLife bag) demonstrate two formats of cell distribution containing cells within the Cocoon cultured in the upper chamber of the cassette. The same volume of medium was used in both systems. The PermaLife cell culture bag utilized a fed-batch culture process where the area expanded as the total volume increased, as is commonly done. The Cocoon cassette utilized a fixed area, employed an initial fed-batch feeding strategy, and used partial medium exchanges on days 6 and 8 of culture.

[0337]

[0360] To evaluate the impact of the activation method and the performance of the automated platform, the following criteria were used: viability, cell number, phenotype, exhaustion, transduction efficiency, functional intracellular cytokine secretion, and cytotoxicity. The results are summarized in Figure 16 and discussed herein.

[0338]

[0361] Unless otherwise indicated for donor 2, the same donor cells were used for all conditions. All conditions were at 60×10 6PBMCs were seeded and given the same media volume and composition. The starting cell population contained 66.6% CD3+ T cells and 12.0% CD14+ cells. Of the CD3+ cells, 71.2% were CD4+ and 28.1% were CD8+ cells. A second donor was used to determine the effect of donor-to-donor variation. This second population of PBMCs originally contained 75.0% CD3+ T cells and 4.5% CD14+ cells. Of the CD3+ cells, 65.0% were CD4+ and 32.9% were CD8+ cells.

[0339]

[0362] The viable cell yields on day 10 from the cocoon cultures activated with OKT3 and Dynabeads were 2.55×10 9 ±0.1×10 9 and 2.15×10 9 ±0.1×10 9 respectively. The viable cell yields from the PermaLife bag cultures activated with OKT3 and Dynabeads were 2.08×10 9 ±0.1×10 9 and 1.53×10 9 ±0.1×10 9 respectively (Figure 13A). The viability at all conditions exceeded 95% (Figure 13A). The population doubling levels (PDLs) were 5.2 - 5.4 (36 - 43 fold) in the cocoon and 4.7 - 5.1 (25 - 35 fold) in the PermaLife bag (Figure 13B).

[0340]

[0363] All conditions showed a high level of purity of T cells, with over 88% of the viable cells expressing CD3. Except for the bead-activated PBMCs grown in the PermaLife bag, the total viable T cells generated in 10 days exceeded 2 billion (Figure 13C). Regardless of the activation method, the total T cell yield was higher in the cocoon conditions compared to the bag. The yield of cocoon cassette T cells on day 10 was 2.0 - 2.4×10 9 respectively. The PermaLife bag was 1.5 - 2.0×10 9T cells were generated (Figure 13C). Using the same donor cells, the PDL of CD3+ cells was 5.7 and 5.9 (51- and 60-fold) in cocoons activated with Dynabeads or OKT3, respectively (Figure 13D). In permalife bags activated with Dynabeads and OKT3, respectively, the PDL of CD3+ cells was 5.2 and 5.6 (38- and 49-fold).

[0341]

[0364] Figure 13E shows the percentages of CD3+ T cells expressing the glycoproteins CD4 and CD8, which represent helper T cells or cytotoxic T cells, respectively. The most important result related to the T cell subpopulation was the increase in the number of CD8 cells under conditions activated with OKT3 compared to Dynabeads-activated cells. OKT3 activation resulted in 83-86% CD8+ and 6-11% CD4+ cells, while the Dynabeads activation condition resulted in subpopulations of 48-56% CD8+ and 41-48% CD4+ cells. In all cultures by the same donor, the exhaustion-related marker PD-1 was less than 10%, indicating low-level cell exhaustion (Figure 13F). The second donor expressed PD-1 in 21% of the cells when cultured in a cocoon containing Dynabeads. Figures 13G and 13H show representative contour plots highlighting the significant difference in CD8+ cells under the Dynabeads activation condition compared to the OKT3 activation condition.

[0342]

[0365] High transduction efficiency was determined by the expression of the surrogate surface marker CD271 (NGFR) of T cell HER2 specificity, which had 62-78% of CD3+ cells in the cocoon and 42-60% of CD3+ cells in the permalife bag expressing NGFR (Figure 14A). The transduction efficiency was greater in the cocoon compared to the bag culture. Due to high transduction and expansion, the total number of viable CAR T cells was 1.26-1.66×10 9 in the cocoon and 0.62-1.20×10 9was in the range (Figure 14B). The percentages and total numbers of CAR T cells in the CD4 and CD8 subpopulations are shown in Figures 14C and 14D, respectively. The percentage of transduced CD4 cells was 75.4 - 80.9% of CD4 cells and 64 - 73.2% of CD8 cells in the cocoon expressing NGFR, which was greater than that of CD8 cells. In the PermaLife bag, 54.7 - 79.9% of CD4 cells and 36.1 - 58.9% of CD8 cells expressed NGFR. Since the expansion of CD8 cells was significantly greater than that of CD4 cells, the total number of CD8+ transduced cells was significantly greater than that of CD4+ transduced cells under all conditions except for the Dynabeads-activated bag culture (Figure 14D). In the cocoon, there were 0.25 - 0.64×10 9 transduced CD4 cells and 0.66 - 1.43×10 9 transduced CD8 cells. Under the PermaLife bag conditions, there were 0.09 - 0.41×10 9 transduced CD4 cells and 0.25 - 1.06×10 9 transduced CD8 cells. Representative contour plots of transduction efficiency under cocoon conditions and PermaLife bag conditions are shown in Figures 14E and 14F, respectively.

[0343]

[0366] Functional tests of the cells were performed using an intracellular cytokine release assay and an ALAMARBLUE killing assay (see Nociari, 1998) (Figure 15). In all cases, the cells demonstrated production of TNFα and IFNγ characteristic of type 1 helper T CD4+ cells and cytotoxic CD8+ cells (Figure 15A and 15B) (see, for example, Romagnani, 1991). A higher percentage of CD4+ cells secreted TNFα. The generation of TNFα-secreting cells was greater under cocoon conditions compared to bag culture of the same donor cells. The Dynabeads activation condition generated TNFα- and IFNγ-secreting transduced cells at a higher rate than the OKT3 activation condition. The ALAMARBLUE killing assay demonstrated effective killing of the ovarian cancer cell line SKOV-3 HER2+ tumor cells by the CAR T cells (Figure 15C and 15D). The trend of the killing effect showed a strong response from both Permalife and cocoon-generated cells following serial dilution of the effector T cells. LOX IMVI, a HER2 tumor cell, was also exposed to T cells to demonstrate HER2 specificity. No killing trend was confirmed in HER2-negative cultures in response to the CAR T cells.

[0344] Examination

[0367] Activation methods. The evaluation of CAR T cell production included activation using soluble anti-CD3 (OKT3), as well as bead-bound anti-CD3 / anti-CD28 Dynabeads. Cultures activated with OKT3 demonstrated a 19–36% improved proliferation compared to cultures activated with Dynabeads (Figure 13A). The method of activation also produced a significant difference in the final phenotype (Figure 13E). Under the Dynabeads activation condition, the CD3+CD8+ cells were on average 52.7% compared to 84.5% under the OKT3 activation condition. This represents a CD8+ to CD4+ ratio of approximately 1.2:1 compared to 9.8:1 for activation with OKT3 for the condition activated with Dynabeads. The increase in CD8+ cell number was found regardless of whether the cells were cultured under bag or cocoon conditions.

[0345]

[0368] The improved yields due to OKT3 activation were an unexpected result. Dynabeads activate T cells by binding to the TCR / CD3 complex and the CD28 costimulatory receptor. Unlike Dynabeads with costimulatory anti-CD28 antibodies, activation with soluble anti-CD3 is monocyte dependent and presents the B7 receptors, CD80 and CD86, which are ligands for CD28 (see, e.g., Fleischer, 1996). However, the B7 receptor also binds to CTLA-4 and stimulates this inhibitory pathway and can thus inhibit T cell proliferation. The improved total cell yields based on the activation method were found regardless of whether the cells were cultured in bag or cocoon conditions. Bead-bound anti-CD3 / anti-CD28 antibodies promote the expansion of helper T cells (CD4+ cells), and OKT3 can promote the expansion of cytotoxic T cells (CD8+ cells) (see, e.g., Fleischer, 1996; Laux, 2000; Li, 2010; Zhu, 2007).

[0346]

[0369] Higher cell yields, and specifically, CD8+ cell predominance, may result from the stimulation of additional receptors when activated using OKT3 and monocytes. It has previously been reported that 95% of CD4+ T cells express CD28, while only 50% of CD8 cells express CD28 (see Ledbetter, 1990). As a result, Dynabeads can activate only up to 50% of CD8+ cells. Cultures activated with OKT3 may benefit from other costimulatory ligands present on monocytes rather than on the beads.

[0347]

[0370] For example, monocytes express CD58 (LFA-3) and the CD40 receptor, which are ligands for CD2 and CD40L. Stimulation of these receptors is known to promote T cell proliferation. These accessory cells may also interact with CD137 and express CD137L, which can stimulate the expansion of CD8+ cells. Interaction with these other receptors may represent more physiological antigen presentation compared to Dynabead activation.

[0348]

[0371] Since OKT3 activation depends on other cells, donor variability may be more important than Dynabeads activation. The starting cell population for this study consisted of 12.0% CD14+ cells and 66.6% CD3+ cells on day 0. A dose study could be performed to examine the effect of monocyte sensitivity on the final yield and phenotype.

[0349]

[0372] Automation. The cocoon produced a greater yield of viable CAR T cells when activated with either OKT3 or Dynabeads compared to manual conditions. When activated with Dynabeads, COCOO culture produced 40% more growth than bag culture. In OKT3 culture, the cocoon produced 23% more cells than bag culture. The cocoon condition also demonstrated a higher transduction efficiency and, as a result, a higher total yield of CAR T cells (Figure 14). Under Dynabeads activation conditions, the total CAR T cell yield of the cocoon was more than twice that of the bag. Under OKT3 activation conditions, the yield of CAR T cells was approximately 40% higher in the cocoon than in the bag.

[0350]

[0373] The improved yield of the cocoon compared to the permalife bag could be an increase in activation. This could be due to the distribution throughout the culture area. The cocoon utilizes a solid non-yielding chamber, while the bag is flexible. After cell sedimentation, it was observed that the curvature of the bag caused a non-uniform distribution of cells. This could have caused a non-uniform distribution of the activator and / or cells. Another possible cause could be related to the amount of agitation during activation. Since the cells were transduced the day after activation, it is possible that activation was still ongoing or the activator was not taken up by the cells. During the transduction step, the bag culture is moved from the incubator to the biosafety cabinet and the cells are delivered using sterile techniques. The movement of the bag promotes virus distribution within the bag; however, the cells are also disturbed during the movement of the bag from incubator to incubator. This movement could have had a negative impact on the cells since stable contact may be important for cell activation. The cells of the cocoon culture are not disturbed between activation or transduction steps. In the cocoon, the medium used for activation is removed from the culture prior to transduction. Since a small amount of medium remains in the chamber, the cells remain at the bottom of the chamber and are not disturbed during volume transfer. The medium removed from the chamber is used to dilute and mix the virus and is then transferred back to the cell population. The cells remain unaffected during this process.

[0351]

[0374] Efficient activation can correlate with more efficient transduction. That is, when cells are activated and actively dividing, the lentivirus may integrate more effectively. To evaluate this, samples can be taken prior to transduction to determine the activation efficiency. Improved transduction efficiency may also be related to the uniform distribution of the virus to the cells. In cocoon culture, the virus is mixed with the medium and uniformly distributed to the cells. Using a flat, non-flexible container improves the uniform distribution and, as a result, serves for uniform exposure of the virus among the cell population.

[0352]

[0375] Another reason for the improved performance may be related to gas exchange. An increase in oxygen levels may support an increase in growth. High oxygen levels were maintained in the automated platform by using recirculation of the culture supernatant through silicone gas exchange lines. Gas exchange was achieved in the bag state by diffusion through fluorinated ethylene propylene (FEP), the material of the bag. The permeability coefficient of silicone is significantly greater than that of FEP (see, e.g., Avgoustiniatos, 2008). The cocoon protocol was created to ensure sufficient oxygen concentration. This was confirmed by data from biosensors generated throughout the culture period.

[0353]

[0376] Gas exchange through silicone tubing also supports the pH level. That is, at the start of the culture, the medium maintains the target pH by gas exchange with an environment rich in CO2. As the number of cells in the culture increases, the cells produce lactic acid and CO2, removing the need for a CO2 environment. The CO2 in the cocoon environment decreased during the culture period and helped maintain the pH. The PermaLife bag followed the conventional protocol of being stored in a 5% CO2 environment throughout the culture process.

[0354]

[0377] A further advantage of continuous recirculation without disturbing the cells is a more uniform distribution of positive and negative factors. This includes nutrients, waste products, released cytokines, and dissolved gases. Continuous recirculation helps reduce local effects and improve the efficiency of the medium by evenly dispersing the factors.

[0355]

[0378] Automated conversion. In this example, a cocoon, a closed automated production system, was used to generate CAR T cells activated by either bead-bound antibody or soluble OKT3. The results demonstrate that clinically relevant yields can be generated from the cocoon with high transduction efficiency using low concentrations of virus. Furthermore, the phenotype of the cells can be driven by the activation method.

[0356]

[0379] The results were mainly generated from a single donor to compare the effects of activation methods. The variability between conditions was very low. When the tests were repeated using different donors, the results were similar among donors when the same activation method was used. This study demonstrates an efficient method to effectively automate the generation of CAR T cells in a clinically relevant, scalable, and user-friendly manner.

[0357] Example 3 - Transduction by Electroporation Using a Cell Engineering System Background

[0380] The Octane Cocoon™ system is an automated, closed end-to-end bioreactor system for manufacturing cell therapy products. The Octane Automated Cell and Tissue Engineering System (ACTES) consists of three main components: basic equipment, software, and customizable disposable cassettes. The Cocoon™ system enables automated isolation, expansion, concentration, and buffer exchange in both upstream and downstream cell culture processes.

[0358]

[0381] The electroporation unit enables the transfection of cells that are conventionally known to have low transfection efficiency via electroporation and other non-viral methods, including primary cells, stem cells, neurons, and quiescent or non-proliferating cells. The system includes an electroporation unit, an electroporation solution, an electroporation cartridge, and an optimized electroporation protocol. The electroporation unit consists of a core unit and one to three additional functional add-on units to accommodate various needs. For example, using the electroporation unit, transfection can be performed with various cell numbers from 20 μL to 100 μL and volumes from 1 mL to 20 mL at 1×10 7 ~1×10 9 .

[0359]

[0382] This specification describes an automated, fully enclosed, sterile, and robust transfection and cell expansion procedure using an electroporation unit and an Octane Cocoon (trademark) system. In a proof-of-concept (PoC) evaluation, each electroporation software and Octane Cocoon (trademark) ACTES software operate independently of each other. In other embodiments, the software is fully integrated across the systems. Method

[0383] The evaluation of peripheral blood mononuclear cell (PBMC) transfection and expansion using an electroporation unit and a Cocoon (trademark) system was divided into three main focus areas.

[0360]

[0384] Cell concentration in the Cocoon (trademark) cassette, cell transfer between the Octane Cocoon (trademark) and the electroporation unit, expansion of transfected cells that have moved between the Cocoon (trademark) and the electroporation unit, and cell concentration within the Cocoon (trademark) cassette.

[0361]

[0385] The Cocoon (trademark) ACTES cassette recirculates approximately 450 mL of culture medium within its culture chamber. The cell growth chamber typically holds a fixed volume of up to 180 mL of medium within a 260 cm 2 area. An additional medium volume exceeding the 180 mL capacity of the 260 cm 2 growth chamber is provided from various satellite reservoirs and chambers of the Cocoon (trademark) cassette. This additional medium from these satellite reservoirs recirculates within the disposable Cocoon (trademark) culture portion to provide fresh nutrients and remove waste from the cells within the 260 cm 2 growth chamber.

[0362]

[0386] An exemplary volume that can be transfected by the electroporation unit is 20 mL. The 20 mL volume needs to be appropriately constituted with at least 90% of a suitable electroporation solution. Thus, in the PoC study, the original culture volume was reduced to 10 mL, then diluted with an additional 90 mL of supplemented P3 primary cell electroporation solution and concentrated to a final volume of 10 mL - 18 mL.

[0363]

[0387] The described proof-of-concept study utilized the following:

[0388] A 20-gauge, 0.024-inch inner diameter / 0.036-inch inner diameter flow restrictor from Nordson EFD, added at the end of the permeation line.

[0364]

[0389] 1×10 8 of PBMC were stimulated with 1×10 8 of CD3+:CD28+ Dynabeads (Invitrogen) and expanded in complete T cell medium composed of X-VIVO 15 medium (Lonza) supplemented with 5% human serum A / B (Sigma) and 10 ng / mL of IL-2 (Peprotech) using multiple GREX 100 (Wilson Wolf) culture vessels for up to 10 days. The test concentration of the cells was transferred to a 250 mL conical vial and allowed to acclimate in a 37 °C incubator with 5% CO2 and humidified air for 2 - 4 hours. The supernatant of the acclimated cell suspension was reduced to 10 mL and the excess supernatant was discarded. 90 mL of supplemented P3 primary cell electroporation solution (Lonza) was added to the concentrated cell suspension to bring the final volume to 100 mL. Next, the 100 mL cell suspension was concentrated to a volume of 10 mL. The control sample of the cells was incubated at 37 °C.

[0365]

[0390] A double count was performed on the undiluted cell culture, the diluted culture, and the final concentrated cell suspension using a Nucleocounter NC-200 (Chemometec). Volumes were measured using a serum pipette and a KrosFlo scale. Residual test samples were obtained from the initial pre-culture dilution, the supernatant, and the final concentrated cell suspension. A human serum ELISA kit (Bethyl Laboratories) was used to determine the percentage of serum remaining after dilution and concentration. FACS analysis was performed on control cells and the concentrated cell suspension for CD4+ and CD8+ expression.

[0366]

[0391] A successful demonstration of the volume reduction of the Cocoon (trademark) transfection protocol was defined as follows: ≧85% recovery of cells, ≦10% decrease in cell viability, and ≦10% residual human serum of the initial concentration.

[0367]

[0392] Cell transfer between OctaCocoon (trademark) and the electroporation unit

[0393] Several disposable consumables are required for cell transfer between the Cocoon (trademark) and the electroporation unit: a Cocoon (trademark) cassette, an electroporation cartridge, two modified electroporation reservoirs, and two connection tube sets (see Figure 17).

[0368]

[0394] The modified electroporation reservoir includes an inlet and outlet weldable tube with a Luer lock connection end, a cell input port within a reservoir housing connected to an external inlet reservoir tube for aseptic cell transfer to the reservoir, a Luer lock substrate addition port for the input tube of the LV reservoir, and a vent filter on the cap for releasing air during volume transfer. A Cocoon (trademark) cassette is designed with ports that can automate the transfer of fluid and cell suspension out of the Cocoon (trademark) in a controlled manner without compromising the sterility of the culture or the health of the cells.

[0369]

[0395] The successful demonstration of the aseptic transfer of cells between the Cocoon (trademark) and the electroporation unit demonstrated the following: the supernatant of the transferred and transfected cells passed the sterility test, mycoplasma was not detected in the culture samples before and after transfection, after transfection, in the Cocoon (trademark) cassette after delivery to the Cocoon (trademark) cassette growth chamber, a recovery of more than 90% of the cells / volume up to transfection, a change of 5% or less in the viability of non-transfected cells during the transfer movement of Cocoon (trademark) and electroporation unit cells, and a change of 20% or less in CD3+, CD4+, and CD8+ cells when compared to transfected cells regardless of the presence or absence of automated transfer between the Cocoon (trademark) and the electroporation unit.

[0370]

[0396] The Cocoon (trademark) ACTES cassette has two sampling ports with BD Q-Syte mesurlock ends, and inlet and outlet ports with cannulas that enable the automated transfer of cell suspensions via a connection tube set aseptically connected to these locations from the Cocoon (trademark) cassette. During the PoC study, the connections (connections) between the Cocoon (trademark) cassette, the electroporation reservoir, the electroporation cartridge, and the connection tube set were aseptically connected, and a sterilization loop between the Cocoon (trademark) and the electroporation system was created as follows.

[0371]

[0397] A connection tube set with a Spiros® male luer lock end connector from ICU Medical was connected to two BD Q-Syte female luer lock sampling ports of the COCoon™. To create a sterilization path from the COCoon™ cassette to the electroporation reservoir, another Spiros® male luer lock connection (ICU Medical) of the connection tube set was connected to the female luer lock inlet tube of the electroporation reservoir. To connect the modified electroporation reservoir to the electroporation cartridge, the female luer lock end of the modified electroporation reservoir drain line was attached to the Spiros® male luer lock connection (ICU Medical) at the inlet of the electroporation cartridge. To recover the transfected cells, the Spiros® male luer lock output connection of the electroporation cartridge was connected to the female luer lock connector inlet of the second electroporation reservoir. The female luer lock end of the second electroporation reservoir drain line was connected to the Spiros® male luer lock connector of the connection tube set at the second automated sampling port of the COCoon™ cassette.

[0372]

[0398] In an embodiment, the COCoon™ pump transfers the transfected cells to the COCoon™ growth chamber and recovers the newly transfected cells prior to delivery to the growth chamber of the COCoon™ cassette using a second electroporation reservoir or other collection container that allows for aseptic transfer of the cells. Instead of a sterile luer lock connection between the PVC tubing lines at the inlet and outlet of the modified electroporation reservoir, aseptic welding techniques can be used and a connection tube set with PVC tubing is possible.

[0373]

[0399] The cell engineering system (Cocoon) described herein also enables a sterile, closed connection between the Cocoon cassette and the electroporation unit via a tube that is derived from the internal Cocoon environment through the hollow shaft of the Cocoon instrument. This hollow shaft, called the "trumpet arm," provides access from the external environment to the internal environment of the Cocoon culture chamber without loss of control of the main process parameters. Cell movement between the Cocoon and the electroporation unit used a peristaltic pump and the software of two independent control systems, but the software of the combined system can also be used to control the independent pump system.

[0374]

[0400] Prior to transfection, the cells / fluid were manually transferred to a sterile electroporation reservoir to mimic the pre-amplification (day 0) transfection procedure, or using the Cocoon pump, software, and connection tubing set (described above), transferred from the Cocoon cassette growth chamber to mimic the post-amplification transfection procedure in the sterile electroporation reservoir. Next, the Cocoon pump and software automated the movement of the cells / fluid from the Cocoon cassette to the inlet of the electroporation reservoir. The electroporation system performed a pre-programmed pump movement of up to 20 mL from the electroporation reservoir, through the electroporation cartridge, to a second electroporation reservoir. Thereafter, the Cocoon pump transferred the recovered, transfected cells / buffer from the second electroporation reservoir to the growth chamber of the Cocoon cassette.

[0375]

[0401] A second electroporation reservoir was incorporated to harvest the transfected cells and hold them until ready to transfer them to the COCoon™ growth chamber by the COCoon™ pump. To move the transfected cells from the electroporation unit to the COCoon™ growth chamber using only the electroporation unit pump, a "connecting tube set clearing" program can be utilized. Additionally, the length of the connecting tube set needs to be consistent.

[0376]

[0402] Using the aforementioned COCoon™ cassette, connecting tube set, and modified electroporation reservoir connection (Figure 17), 11 mL of phosphate buffered saline (Lonza) was transferred from the COCoon™ cassette to the modified electroporation reservoir using the COCoon™ pump. A simulated transfection of the PBS solution was performed using the electroporation program and the 11 mL volume was transferred to the second modified electroporation reservoir. Next, using the COCoon™ pump and software, the 11 mL volume was transferred from the second modified electroporation reservoir to the output bag of the COCoon™ cassette. The amount transferred from the COCoon™ reservoir to the satellite bag was estimated to be 11 mL per run. The actual volume was measured using a serum pipette after transferring to the first modified electroporation reservoir, the second modified electroporation reservoir, and the COCoon™ output bag. The pass criteria was established at a fluid recovery rate of ≥90% from the first modified electroporation reservoir to the COCoon™ output bag. Cell suspension test

[0403] 1×10 8 and 5×10 8Total viable PBMCs are expanded in 450 mL of complete T cell medium consisting of X-VIVO 15 medium (Lonza) supplemented with 5% human serum A / B (Sigma) and 10 ng / mL of IL-2 (Peprotech) in a sterile Cocoon™ ACTES cassette. On day 3, 440 mL of the culture supernatant is removed and retained for sterility and mycoplasma testing. The cells are diluted in 90 mL of supplemented P3 electroporation solution (Lonza). Next, the cells are concentrated in the Cocoon™ cassette to approximately 10 mL of cell suspension and transferred to a Cocoon™ satellite bag. The option of washing the growth chamber with an additional 10 mL of supplemented P3 electroporation solution and adding it to the cell suspension in the Cocoon™ satellite bag is evaluated. Samples are taken from the concentrated cells in the satellite bag using a Nucleocounter NC-200 (Chemometec) to measure the duplicate cell counts while maintaining sterility. Next, as described above, the cells are transferred to the modified electroporation reservoir via the Cocoon™ pump and connecting tubing set. Using an electroporation unit pump and the EO-210 program, the T cells are transfected with the pmax GFP vector (Lonza) and the transfected cells are transferred to a second modified electroporation reservoir. Next, the Cocoon™ pump transfers the cells from the modified electroporation reservoir to the growth chamber of the Cocoon™ ACTES cassette. Samples of the cells are taken from the ACTES cassette growth chamber for duplicate cell counts, mycoplasma, and sterility testing. This procedure is repeated with a control culture where the cells are not transfected but instead passed through the electroporation unit using a mock electroporation program CA-100. This procedure is evaluated using three different donors; both newly isolated and cryopreserved PBMC lots.

[0377]

[0404] Changes in cell viability are measured in untransfected cell cultures. Cell recovery, sterility, and mycoplasma load are evaluated in all cultures. Flow cytometry is used to evaluate GFP, CD3+, CD4+, CD8+, and additional marker expression.

[0378]

[0405] Sterile transfer of the cell suspension between the Cocoon™ and the electroporation unit is sterile before and after the transfer, provides a mycoplasma-free supernatant, and results in a cell recovery of ≥90% before transfection of the Cocoon™ cassette into the Cocoon™ cassette growth chamber, a change in viability of non-transfected cells of ≤5%, and a change in the CD3+, CD4+, and CD8+ cell ratios of ≤20% after transfection.

[0379] Expansion of transfected cells transferred between the Cocoon™ and the electroporation unit

[0406] 1×10 8 and 5×10 8 Total viable PBMCs are expanded and concentrated in a Cocoon™ cassette, transfected via a sterile connection to the electroporation LV unit, and transferred aseptically to the Cocoon™ as described in the method section for "Intercellular transfer between the Octane Cocoon™ and the electroporation LV unit, cell suspension test". Transfected cells are cultured in the Cocoon™ cassette growth chamber for up to 15 days using the most relevant, optimized automated Cocoon™ protocol. Controls are expanded for 3 days in a T-225 flask (Corning) or GREX 100 (Wilson Wolf) culture vessel. On day 3, the control cultures are aseptically and manually concentrated, transfected via the electroporation LV unit EO-210 program, and returned to the original vessel for continued expansion for up to 15 days. This procedure is evaluated using three different donors from newly isolated or cryopreserved PBMC lots.

[0380]

[0407] The expansion of the transfected cells transferred between the Cocoon (trademark) and the electroporation unit, when determined via FACS, showed a variation of ±10% in transfection efficiency, a final cell concentration of ≧80% of the control culture, a variation of ±5% in the final cell viability when compared to the control culture, a variation of ±10% in GFP+, CD3+, CD4+, and CD8+ expression, when compared to the control culture. The supernatant of the transferred transfected cells passed the sterility test, and mycoplasma was not detected in the culture samples before and after transfection.

[0381] Results

[0408] Cell concentration of the Cocoon (trademark) cassette

[0409] Cells from two donors were concentrated and sedimented to a volume of 10 mL with a total viable cell count of 4.4×10 8 and 4.2×10 8 These two cell suspensions were then diluted and concentrated with 90 mL of supplemented electroporation solution (NFS). The cell recovery rates after concentration were 92% and 87%. The cell viabilities before transfection were 92% and 74%, with a decrease of less than 5%. In both runs, 6% and 8% of the initial culture supernatant was detected in the final concentrated cell suspension.

Table 3

[0382]

[0410] There was no difference in the CD4+:CD8+ profile after concentration compared to the non-concentrated control culture.

[0383]

[0411] The results demonstrated the recovery of fluid from the Cocoon (trademark) satellite bag to the Cocoon (trademark) output bag. The expansion of the transfected cells was transferred between the Cocoon (trademark) and the electroporation unit. Electroporation was successfully performed in the electroporation unit, resulting in transduced cells.

[0384]

[0412] This specification provides an automated, fully closed transfection using a closed loop between an electroporation unit and a COCoon (trademark) system. A method of concentrating cells in the COCoon (trademark) system can be used.

[0385] Example 4 - Expansion of Hematopoietic Stem Cells

[0413] CD34+ was focused on the expansion of umbilical cord blood. This particular application was the expansion of CD34+ from umbilical cord blood samples containing low CD34+ numbers for use of a single well - matched umbilical cord in adult treatment. Thus, compared to some other protocols, the starting cell numbers and concentrations were very low. It is expected that as the starting numbers and concentrations increase, the cell expansion will decrease.

[0386]

[0414] Selected and expanded CD34+ cells

[0415] Total nucleated cells (TNC) tracked over time

[0416] The starting cell concentration was lower than many other protocols (0.1M cells / ml)

[0417] Cell expansion was found to vary based on the recovery protocol (Figure 19).

[0387]

[0418] Changes in cell phenotype are tracked during the culture period

[0419] 25.3% of the TNC is CD34+ after 12 days of expansion (Figure 20).

[0388]

[0420] The differentiated cell phenotypes are shown in Figure 21. Figure 22 shows that a single colony can form multi - lineage differentiation.

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[0390]

[0421] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and uses described herein can be made without departing from the scope of the embodiments.

[0391]

[0422] Although specific embodiments have been illustrated and described herein, it should be understood that the claims are not limited to the specific forms or arrangements of parts that have been illustrated and described. Although exemplary embodiments are disclosed herein and specific terms are used, they are generic and are used only in a descriptive sense and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. Accordingly, it should be understood that the embodiments may be practiced otherwise than as specifically described.

[0392]

[0423] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0393] Preferred Embodiments Preferred embodiments of the present invention are as follows in 1 to 26. 1. (a) Activating an immune cell culture with an activating reagent to produce an activated immune cell culture; (b) Transducing the activated immune cell culture with a vector to produce a transduced immune cell culture; (c) Expanding the transduced immune cell culture; (d) Concentrating the expanded immune cell culture of (c); and (e) Recovering the concentrated immune cell culture of (d) to produce a genetically modified immune cell culture A method for the automated production of a genetically modified immune cell culture, which further comprises washing either or both of the expanded immune cell culture and the concentrated immune cell culture, (a) to (e) are performed by a fully enclosed cell engineering system, and (a) to (e) are optimized through a process of producing a genetically modified immune cell culture, a method for automated production. 2. The process is a self-regulating process, (a) Monitoring using 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; and (b) Adjusting one or more of the temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density of the transfected T cell culture based on the monitoring The method according to embodiment 1, comprising the above. 3. The method according to embodiment 1 or 2, generating at least about 100 million viable genetically modified immune cells. 4. The method according to any one of embodiments 1 to 3, generating at least about 2 billion viable genetically modified immune cells. 5. The method according to any one of embodiments 1 to 4, wherein the immune cell culture is a T cell culture. 6. The method according to embodiment 5, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture. 7. The method according to embodiment 6, wherein the vector encodes a chimeric antigen receptor. 8. The method according to any one of embodiments 1 to 7, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells. 9. The method according to any one of embodiments 1 to 8, wherein the immune cell culture comprises at least one accessory cell. 10. The method according to embodiment 9, wherein the accessory cell comprises monocytes or monocyte-derived cells. 11. The method according to embodiment 9, wherein the accessory cell comprises an antigen for the T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS. 12. The method according to any one of embodiments 1 to 11, wherein the activation reagent comprises an antibody or dendritic cells. 13. The method according to embodiment 12, wherein the antibody is immobilized on the surface. 14. The method according to embodiment 13, wherein the surface is the surface of beads. 15. The method according to embodiment 12, wherein the antibody is a soluble antibody. 16. The method according to any one of embodiments 12 to 15, wherein the antibody comprises at least one of an anti-CD3 antibody and an anti-CD28 antibody. 17. The method according to any one of embodiments 1 to 16, wherein the transduction comprises viral infection, electroporation, membrane disruption, or a combination thereof. 18. The method according to any one of embodiments 1 to 17, wherein the vector is a lentiviral vector or a retrovirus. 19. The method according to any one of embodiments 1 to 18, wherein the transduction comprises mixing the vector in a cell culture medium and uniformly delivering the vector in the medium to an activated immune cell culture. 20. The method according to any one of embodiments 1 to 19, wherein the expansion comprises at least one or more of supplying, washing, and monitoring the transduced immune cell culture. 21. The method according to any one of embodiments 2 to 20, wherein the oxygen level of the transduced immune cell culture is optimized for the immune cell culture. 22. The method according to any one of embodiments 1 to 21, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a) to (e). 23. The method according to any one of embodiments 1 to 22, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during steps (a) to (e). 24. The method according to any one of embodiments 2 to 23, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c). 25. The method according to any one of embodiments 1 to 24, wherein the cell engineering system is configured to perform one or more rounds of one or more of supplying, washing, monitoring, and selecting the transduced immune cell culture. 26. The method according to any one of embodiments 1 to 25, wherein the concentration comprises centrifugation, removal of the supernatant after sedimentation, or filtration. 27. The method according to embodiment 26, wherein the process further comprises adjusting the parameters of centrifugation or filtration. 28. The method according to any one of embodiments 1 to 27, wherein the cell engineering system includes a plurality of chambers, and each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system. 29. The method according to any one of embodiments 1 to 28, further comprising removing the activation reagent from the activated immune cell culture after step (a). 30. The method according to any one of embodiments 1 to 29, wherein the cell engineering system includes the cell culture of (a), the activation reagent, the vector, and the cell culture medium before starting the method. 31. (a) Activating an immune cell culture with an activation reagent to generate an activated immune cell culture, wherein the activation reagent and the activation conditions promote the phenotype of the genetically modified immune cell culture; (b) Transducing the activated immune cell culture with a vector to generate a transduced immune cell culture; (c) Expanding the transduced immune cell culture; (d) Concentrating the expanded immune cell culture of (c); and (e) Recovering the concentrated immune cell culture of (d) to generate a genetically modified immune cell culture A method for promoting a preferred phenotype of a genetically modified immune cell culture, comprising: (a) to (e) are performed by a fully enclosed automated cell engineering system. 32. The method according to embodiment 31, wherein the activation reagent includes an antibody or dendritic cells. 33. The method according to embodiment 32, wherein the antibody is immobilized on the surface. 34. The method according to embodiment 33, wherein the surface is the surface of beads. 35. The method according to embodiment 32, wherein the antibody is a soluble antibody. 36. The method according to any one of embodiments 32 to 35, wherein the antibody includes at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody. 37. The method according to embodiment 36, wherein the soluble antibody is OKT3. 38. The method according to any one of embodiments 31 to 37, providing an immunocyte culture in which the activation conditions enable substantially unimpeded stable contact between the activation reagent and the immunocyte culture. 39. The method according to any one of embodiments 31 to 38, generating at least about 100 million viable genetically modified immunocytes. 40. The method according to embodiment 39, generating at least about 2 billion viable genetically modified immunocytes. 41. The method according to any one of embodiments 31 to 40, wherein the immunocyte culture is a T cell culture. 42. The method according to embodiment 41, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture. 43. The method according to embodiment 42, wherein the vector encodes a chimeric antigen receptor. 44. The method according to any one of embodiments 31 to 43, wherein the immunocyte culture comprises peripheral blood mononuclear cells and / or purified T cells. 45. The method according to any one of embodiments 31 to 44, wherein the cell culture comprises at least one accessory cell. 46. The method according to embodiment 45, wherein the accessory cell comprises monocytes or monocyte-derived cells. 47. The method according to embodiment 45, wherein the accessory cell comprises an antigen for the T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS. 48. The method according to any one of embodiments 41 to 47, wherein the phenotype of the T cell culture has a CD8+ cell:CD4+ ratio of about 0.1:1 to about 10:1. 49. The method according to any one of embodiments 31 to 48, wherein transduction comprises viral infection, electroporation, membrane disruption, or a combination thereof. 50. The method according to any one of embodiments 31 to 49, wherein the vector is a lentiviral vector or a retrovirus. 51. The method according to any one of embodiments 31 to 50, wherein transduction comprises mixing the vector in a cell culture medium and uniformly delivering the vector in the medium to an activated immune cell culture. 52. The method according to any one of embodiments 31 to 51, wherein expansion comprises at least one or more of supplying, washing, and monitoring the transduced immune cell culture. 53. The method according to any one of embodiments 31 to 52, wherein the oxygen level of the transduced immune cell culture is optimized for the promoted phenotype. 54. The method according to any one of embodiments 31 to 53, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a) to (e). 55. The method according to any one of embodiments 31 to 54, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during steps (a) to (e). 56. The method according to any one of embodiments 31 to 55, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c). 57. The method according to any one of embodiments 31 to 56, wherein the cell engineering system is configured to perform multiple rounds of supplying, washing, monitoring, and selecting the transduced immune cell culture. 58. The method according to any one of embodiments 31 to 57, wherein concentration comprises centrifugation, removal of the supernatant after sedimentation, or filtration. 59. The method according to any one of embodiments 31 to 58, wherein the cell engineering system comprises a plurality of chambers, and each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system. 60. The method according to any one of embodiments 31 to 59, further comprising removing the activation reagent from the activated immune cell culture after step (a). 61. The method according to any one of embodiments 31 to 60, further comprising removing the vector after transduction in (b). 62. A method according to any one of embodiments 31 to 61, wherein the cell engineering system comprises, prior to initiating the method, a cell culture, an activation reagent, a vector, and a cell culture medium of (a). 63. (a) Activating an immune cell culture with an activation reagent to produce an activated immune cell culture; (b) Transducing the activated immune cell culture with a vector to produce a transduced immune cell culture; (c) Expanding the transduced immune cell culture; (d) Concentrating the expanded immune cell culture of (c); and (e) Recovering the concentrated immune cell culture of (d) to produce a genetically modified immune cell culture A method for the automated production of a genetically modified immune cell culture, comprising: (a)-(e) are performed by a fully enclosed automated cell engineering system, and each of (a)-(e) is performed using an immune cell culture having an optimized cell density (cells / mL) and an optimized cell confluency (cells / cm 2 ). 64. The method according to embodiment 63, wherein the optimized cell density of (a) is about 0.05×10 6 cells / mL to about 60×10 6 cells / mL. 65. The method according to embodiment 63 or 64, wherein the optimized cell confluency of (a) is about 0.1×10 6 cells / cm 2 to about 60×10 6 cells / cm 2 . 66. The method according to any one of embodiments 63 to 65, wherein the activation reagent comprises an antibody or dendritic cells. 67. The method according to embodiment 66, wherein the antibody is immobilized on a surface. 68. The method according to embodiment 67, wherein the surface is the surface of beads. 69. The method according to embodiment 66, wherein the antibody is a soluble antibody. 70. The method according to any one of embodiments 66 - 69, wherein the antibody comprises at least one of an anti - CD3 antibody and an anti - CD28 antibody. 71. The method according to any one of embodiments 63 - 70, which generates at least about 100 million viable genetically modified immune cells. 72. The method according to any one of embodiments 63 - 71, which generates at least about 2 billion viable genetically modified immune cells. 73. The method according to any one of embodiments 63 - 72, wherein the immune cell culture is a T - cell culture. 74. The method according to embodiment 73, wherein the T - cell culture is a chimeric antigen receptor T (CAR T) cell culture. 75. The method according to embodiment 74, wherein the vector encodes a chimeric antigen receptor. 76. The method according to any one of embodiments 64 - 75, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells. 77. The method according to any one of embodiments 64 - 76, wherein the cell culture comprises at least one accessory cell. 78. The method according to embodiment 77, wherein the accessory cell comprises monocytes. 79. The method according to embodiment 77, wherein the accessory cell comprises an antigen for a T - cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS. 80. The method according to any one of embodiments 63 - 79, wherein transduction comprises viral infection, electroporation, membrane disruption, or a combination thereof. 81. The method according to any one of embodiments 63 - 80, wherein the vector is a lentiviral vector or a retrovirus. 82. The method according to any one of embodiments 63 - 81, wherein transduction comprises mixing the vector in a cell culture medium and uniformly delivering the vector in the medium to an activated immune cell culture. 83. The method according to any one of embodiments 63 - 82, wherein expansion comprises at least one or more of supplying, washing, monitoring, and selecting a transduced immune cell culture. 84. The method according to any one of embodiments 63 - 83, wherein the oxygen level of the transfected immune cell culture is optimized with respect to cell density and cell confluency. 85. The method according to any one of embodiments 63 - 84, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a) - (e). 86. The method according to embodiment 85, wherein the oxygen recirculation is provided by a silicone tube during steps (a) - (c). 87. The method according to any one of embodiments 63 - 86, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases during steps (a) - (e). 88. The method according to any one of embodiments 63 - 87, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c). 89. The recirculation of nutrients, waste products, released cytokines, and / or dissolved gases is uniformly provided using cells having a density of about 0.05×10 6 cells / mL to about 60×10 6 cells / mL and a confluency of about 0.1×10 6 cells / cm 2 to about 60×10 6 cells / cm 2 The method according to any one of embodiments 63 - 88. 90. The method according to any one of embodiments 63 - 89, wherein the cell engineering system is configured to perform multiple rounds of feeding, washing, monitoring, and selection of the transfected immune cell culture. 91. The method according to any one of embodiments 63 - 90, wherein the concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration. 92. The method according to any one of embodiments 63 - 91, wherein the cell engineering system includes a plurality of chambers, and each of steps (a) - (e) is performed in a different chamber of the plurality of chambers of the cell engineering system. 93. The method according to any one of embodiments 63 to 92, further comprising removing the activation reagent from the activated immune cell culture after step (a). 94. The method according to any one of embodiments 63 to 93, further comprising removing the vector after transduction in (b). 95. The method according to any one of embodiments 63 to 94, wherein the cell engineering system comprises, prior to initiating the method, the cell culture of (a), the activation reagent, the vector, and the cell culture medium. 96. (a) Activating an immune cell culture with an activation reagent to produce an activated immune cell culture; (b) Transducing the activated immune cell culture with a vector to produce a transduced immune cell culture; (c) Expanding the transduced immune cell culture, wherein the transduced cell culture is not shaken during expansion. (d) Concentrating the expanded immune cell culture of (c); and (e) Recovering the concentrated immune cell culture of (d) to produce a genetically modified immune cell culture A method for the automated production of a genetically modified immune cell culture, comprising (a) to (e), which are performed by a fully enclosed automated cell engineering system. 97. The method according to embodiment 96, wherein the activation reagent comprises an antibody or dendritic cells. 98. The method according to embodiment 97, wherein the antibody is immobilized on a surface. 99. The method according to embodiment 98, wherein the surface is the surface of beads. 100. The method according to embodiment 97, wherein the antibody is a soluble antibody. 101. The method according to any one of embodiments 96 to 100, wherein the antibody comprises at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody. 102. The method according to any one of embodiments 96 to 101, which produces at least about 100 million viable genetically modified immune cells. 103. The method according to embodiment 102, which generates at least about two billion viable genetically modified immune cells. 104. The method according to any one of embodiments 96 to 103, wherein the immune cell culture is a T cell culture. 105. The method according to embodiment 104, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture. 106. The method according to embodiment 105, wherein the vector encodes a chimeric antigen receptor. 107. The method according to any one of embodiments 96 to 106, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells. 108. The method according to any one of embodiments 96 to 107, wherein the cell culture comprises at least one accessory cell. 109. The method according to embodiment 108, wherein the accessory cell comprises monocytes or monocyte-derived cells. 110. The method according to embodiment 109, wherein the accessory cell comprises an antigen for the T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS. 111. The method according to any one of embodiments 96 to 110, wherein transduction comprises viral infection, electroporation, membrane disruption, or a combination thereof. 112. The method according to any one of embodiments 96 to 111, wherein the vector is a lentiviral vector or a retrovirus. 113. The method according to any one of embodiments 96 to 112, wherein transduction comprises mixing the vector in the cell culture medium and uniformly delivering the vector in the medium to the activated immune cell culture. 114. The method according to any one of embodiments 96 to 113, wherein expansion comprises at least one or a plurality of supply, washing, monitoring, and selection of the transduced immune cell culture without shaking the immune cell culture. 115. The method according to any one of embodiments 96 to 114, wherein the oxygen level of the transduced immune cell culture is optimized for the immune cell culture. 116. The method according to any one of embodiments 96 to 115, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a) to (e). 117. The method according to any one of embodiments 96 to 116, wherein the cell engineering system recirculates nutrients, waste products, released cytokines, and / or dissolved gases. 118. The method according to any one of embodiments 96 to 117, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c). 119. The method according to any one of embodiments 96 to 118, wherein the cell engineering system is configured to perform multiple rounds of feeding, washing, monitoring, and selection of the transduced immune cell culture. 120. The method according to any one of embodiments 96 to 119, wherein the concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration. 121. The method according to any one of embodiments 96 to 120, wherein the cell engineering system includes a plurality of chambers, and each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system. 122. The method according to any one of embodiments 96 to 121, further comprising removing the activation reagent from the activated immune cell culture after step (a). 123. The method according to any one of embodiments 96 to 122, further comprising removing the vector after transduction in (b). 124. The method according to any one of embodiments 96 to 123, wherein the cell engineering system includes the cell culture of (a), the activation reagent, the vector, and the cell culture medium before starting the method. 125. A method for the automated generation of a genetically modified immune cell culture, wherein the method performed by the cell engineering system is: (a) activating an immune cell culture with an activation reagent to generate an activated immune cell culture in a first chamber of the cell engineering system; (b) transducing the activated immune cell culture, wherein the transduction is i. Transfer the activated immune cell culture from the first chamber to the electroporation unit; ii. Electroporate the activated immune cell culture with a vector to generate a transduced immune cell culture; iii. Transfer the transduced immune cell culture to the second chamber of the cell engineering system comprising; (c) Expand the transduced immune cell culture; (d) Concentrate the expanded immune cell culture of (c); and (e) Recover the concentrated immune cell culture of (d) to generate a genetically modified cell culture A method for automated generation, comprising. 126. The transduction is i. Transfer the activated immune cell culture from the first chamber to the electroporation unit through a first sterile closed connection; ii. Electroporate the activated immune cell culture with a vector to generate a transduced immune cell culture; iii. Transfer the transduced immune cell culture to the second chamber of the cell engineering system through a second sterile closed connection The method according to embodiment 125, comprising. 127. The method according to embodiment 126, wherein the electroporation unit is located outside the cell engineering system. 128. The method according to any one of embodiments 125 to 127, generating at least about 100 million viable genetically modified immune cells. 129. The method according to embodiment 128, generating at least about 2 billion viable genetically modified immune cells. 130. The method according to any one of embodiments 125 to 129, wherein the immune cell culture is a T cell culture. 131. The method according to embodiment 130, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture. 132. The method according to embodiment 131, wherein the vector encodes a chimeric antigen receptor. 133. The method according to any one of embodiments 125 to 132, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells. 134. The method according to any one of embodiments 125 to 132, wherein the cell culture comprises at least one accessory cell. 135. The method according to embodiment 134, wherein the accessory cell comprises monocytes or monocyte-derived cells. 136. The method according to embodiment 134, wherein the accessory cell comprises an antigen for the T cell receptor, including CD28, CD40, CD40L, and / or ICOS. 137. The method according to any one of embodiments 125 to 136, wherein the activation reagent comprises an antibody or dendritic cells. 138. The method according to embodiment 137, wherein the antibody is immobilized on the surface. 139. The method according to embodiment 138, wherein the surface is the surface of beads. 140. The method according to embodiment 137, wherein the antibody is a soluble antibody. 141. The method according to any one of embodiments 138 to 140, wherein the antibody comprises at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody. 142. The method according to any one of embodiments 125 to 141, wherein the vector is a lentiviral vector or a retrovirus. 143. The method according to any one of embodiments 125 to 142, wherein the expansion comprises at least one or more of the supply, washing, monitoring, and selection of the transduced immune cell culture. 144. The method according to any one of embodiments 125 to 143, wherein the oxygen level of the transduced immune cell culture is optimized for the immune cell culture. 145. The method according to any one of embodiments 125 to 144, wherein the cell engineering system recirculates the cell culture medium through an oxygen supply component during one or more of steps (a) to (e). 146. The method according to any one of embodiments 125 to 145, wherein the cell engineering system recycles nutrients, waste products, released cytokines, and / or dissolved gases during steps (a) to (e). 147. The method according to any one of embodiments 125 to 146, wherein the carbon dioxide level provided by the cell engineering system decreases during step (c). 148. The method according to any one of embodiments 125 to 147, wherein the cell engineering system is configured to perform multiple rounds of supply, washing, monitoring, and selection of the transduced immune cell culture. 149. The method according to any one of embodiments 125 to 148, wherein the concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration. 150. The method according to any one of embodiments 125 to 149, wherein the cell engineering system includes a plurality of chambers, and each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system. 151. The method according to any one of embodiments 125 to 150, further comprising removing the activation reagent from the activated immune cell culture after step (a). 152. The method according to any one of embodiments 125 to 151, further comprising removing the vector after transduction in (b). 153. The method according to any one of embodiments 125 to 152, wherein the cell engineering system includes the cell culture of (a), the activation reagent, the vector, and the cell culture medium before starting the method. 154. The method according to any one of embodiments 1 to 153, wherein the transduction efficiency in step (c) of the method is flexible for cell culture and at least 20% higher than the transduction efficiency of the method using a gas-permeable bag. 155. The method according to any one of embodiments 1 to 154, which generates genetically modified immune cells and is at least 20% more than the method using manual cell culture using a flexible and gas-permeable bag. 156. The cell engineering system includes a plurality of chambers, and each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system. In each of (a), the activation reagent, vector, and cell culture medium are included in different chambers of the plurality of chambers before starting the method. At least one of the plurality of chambers is maintained at a temperature for growing cells, and at least one of the plurality of chambers is maintained at a refrigeration temperature. The method according to any one of Embodiments 1 to 155. 157. (a) A low-temperature chamber for storing a cell culture medium; (b) A high-temperature chamber for activating, transducing, and expanding an immune cell culture, The high-temperature chamber is separated from the low-temperature chamber by a heat barrier, The high-temperature chamber includes a cell culture chamber; and (c) One or a plurality of flow paths connected to the cell culture chamber, the flow paths providing recirculation, waste removal, uniform gas exchange, and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber A cassette for use in an automated cell engineering system, comprising. 158. The cassette according to Embodiment 157, wherein the cell culture chamber is flat with a low chamber height and is inflexible. 159. The cassette according to Embodiment 157 or Embodiment 158, wherein the cell culture chamber is oriented such that the immune cell culture can spread over the entire bottom of the cell culture chamber. 160. The cassette according to any one of Embodiments 157 to 159, pre-filled with a cell culture, culture medium, activation reagent, and vector. 161. The cassette according to any one of Embodiments 157 to 160, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor. 162. The cassette according to any one of Embodiments 157 to 161, further comprising one or more sampling ports and / or injection ports. 163. The cell culture chamber is configured to enable removal of bubbles from the cell culture chamber and / or a distal port as a recirculation port; an intermediate port configured to function as a recirculation inlet port; and a proximal port configured to function as a discharge port for cell removal The cassette according to any one of Embodiments 157 to 162, further comprising at least one of the above. 164. The cassette according to any one of Embodiments 157 to 163, further comprising an access port for connecting the cartridge to an external device. 165. The cassette according to Embodiment 164, wherein the external device includes an electroporation unit or an additional medium source. 166. (a) A cell culture chamber for activating, transducing, and / or expanding an immune cell culture having a chamber volume configured to contain the immune cell culture, (b) A satellite volume for increasing the working volume of the chamber by providing an additional volume to the medium and other working fluids without containing the immune cell culture. A cassette for use in an automated cell engineering system, the satellite volume being fluidly connected to the cell culture chamber via one or more fluid paths such that the medium is exchanged with the culture chamber without disturbing the immune cell culture. 167. The cassette according to Embodiment 166, wherein the satellite volume is a bag. 168. The cassette according to Embodiment 166, wherein the satellite volume is a non-deformable chamber. 169. The cassette according to any one of Embodiments 166 to 168, wherein the satellite volume is further configured to enable medium removal without losing the cells of the immune cell culture. 170. The cassette according to any one of Embodiments 166 to 169, further comprising a crossflow reservoir. 171. The cassette according to any one of embodiments 166 to 170, wherein the cell culture chamber has a volume of about 0.50 ml to about 300 ml. 172. The cassette according to embodiment 171, wherein the cell culture chamber has a volume between about 50 ml and about 200 ml. 173. The cassette according to embodiment 172, wherein the cell culture chamber has a volume of about 180 ml. 174. The cassette according to any one of embodiments 166 to 173, wherein the satellite volume is between about 0.50 ml and about 300 ml. 175. The cassette according to embodiment 174, wherein the satellite volume is between about 150 ml and about 200 ml. 176. The cassette according to any one of embodiments 166 to 175, wherein the cross-flow reservoir has a volume between about 0.50 ml and about 300 ml. 177. The cassette according to embodiment 176, wherein the cross-flow reservoir has a volume between about 100 ml and about 150 ml. 178. The cassette according to any one of embodiments 166 to 177, wherein the working volume is about 180 mL to about 1 L. 179. The cassette according to embodiment 178, wherein the working volume is about 180 mL to about 460 mL. 180. The cassette according to any one of embodiments 157 to 179, wherein one or more fluid paths include a silicone-based tube component that enables oxygen supply through the tube component.

Claims

1. A first chamber for storing a cell culture medium at a first temperature; A second chamber for activating, transducing, and expanding an immune cell culture at a second temperature; A thermal barrier for insulating the first chamber from the second chamber; and One or more fluid paths fluidly connecting the second chamber to the first chamber An automated cell engineering system comprising The one or more fluid paths provide recirculation, waste removal, and uniform gas exchange and nutrient distribution to the second chamber without contaminating the cells within the second chamber, The one or more fluid paths include a silicone-based tube component that enables oxygen supply through a tube component, The system wherein the second chamber is a flat and non-flexible chamber.

2. The system according to claim 1, wherein the thermal barrier thermally isolates the first chamber from the second chamber, and the first temperature is lower than the second temperature.

3. The system according to claim 1, wherein the second chamber is a cell culture chamber.

4. The system according to claim 1, comprising 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.

5. The system according to claim 4, further comprising a pump configured to drive fluid to and / or from the second chamber in response to monitoring one or more of the temperature sensor, the pH sensor, the glucose sensor, the oxygen sensor, the carbon dioxide sensor, and the optical density sensor.

6. The system according to claim 5, wherein the system automatically operates the pump and is configured to introduce the medium from the first chamber through the one or more fluid paths to the second chamber in response to monitoring one or more of the temperature sensor, the pH sensor, the glucose sensor, the oxygen sensor, the carbon dioxide sensor, and the optical density sensor. **Claim 7**: The system according to claim 1, further comprising a pump configured to drive fluid through the one or more fluid paths, wherein the one or more fluid paths are fluidly connected to the first chamber via one or more ports configured to allow mixing of the fluid without disturbing the cells in the second chamber during activation and transduction of the immune cell culture. **Claim 8** The system according to claim 1, further comprising a cassette having a plurality of chambers including the first chamber and the second chamber. **Claim 9** A first chamber for activating, transducing, and expanding an immune cell culture; and A second chamber for storing a cell culture medium; A thermal barrier for insulating the first chamber from the second chamber A cassette for use in an automated cell engineering system, comprising: The first chamber One or more fluid paths connecting the first chamber to the second chamber, the one or more fluid paths including one or more fluid paths configured to provide recirculation, waste removal, and uniform gas exchange and nutrient distribution to the first chamber without contaminating the cells in the first chamber. The one or more fluid paths include a silicone-based tube component that allows oxygen supply through a tube component. The first chamber is a flat and non-flexible chamber, the cassette. **Claim 10** The cassette according to claim 9, further comprising a plurality of chambers containing one or more reagents, media, and / or vectors. **Claim 11** The cassette according to claim 10, wherein the one or more fluid paths fluidly connect each of the plurality of chambers to the first chamber. **Claim 12** The cassette according to claim 9, further comprising a valve configured to control the fluid moving through the one or more fluid paths without contacting the fluid. **Claim 13**: The cassette according to claim 9, further comprising a pump configured to drive fluid through the one or more fluid paths, wherein the one or more fluid paths are fluidly connected to the second chamber via one or more ports configured to allow mixing of the fluid without disturbing the cells in the first chamber during activation and transduction of the immune cell culture. **Claim 14**: The cassette further includes 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, and is configured to automatically operate a pump to introduce the medium from the second chamber to the first chamber through the one or more fluid paths in response to monitoring one or more of the temperature sensor, the pH sensor, the glucose sensor, the oxygen sensor, the carbon dioxide sensor, and the optical density sensor. The cassette according to claim 13.

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