End-to-end cell therapy automation

Abstract

To provide an automated method for the translation of important immunotherapies, such as T cell immunotherapies, to the broad patient population.SOLUTION: The present disclosure provides an automated method of producing genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, utilizing a fully-enclosed cell engineering system.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001]

[0001] This disclosure provides an automated method for generating genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, using a completely sealed cell engineering system. [Background technology]

[0002]

[0002] As expectations for the accelerated clinical adoption of advanced cell therapies grow, more attention is being paid to the underlying manufacturing strategies that will enable these therapies to benefit patients worldwide. While cell therapies hold great clinical promise, high manufacturing costs compared to reimbursement pose a significant obstacle to commercialization. Thus, the need for cost-effectiveness, process efficiency, and product consistency is driving automation efforts in many cell therapy fields, particularly in T-cell immunotherapy (see, for example, 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 untreatable cancers (see, e.g., Lu, 2017; Berdeja, 2017; Kebriae, 2016). As these novel therapeutics move from clinical trials to commercial scale, challenges related to cell manufacturing arise (see, e.g., Morrissey, 2017).

[0004]

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

[0005]

[0005] To translate these important immunotherapies to a broad patient population, it is crucial to integrate cell activation, transduction, and expansion into a commercial manufacturing platform. Applying these life-saving treatments to a global patient population requires a shift in manufacturing technologies to support personalized medicine. The benefits of automation have been previously described (see, e.g., Trainor, 2014; Mahdavi, 2015). These benefits include labor time savings associated with the use of automation, as well as improved product consistency, reduced room classification, reduced cleanroom footprint, reduced training complexity, and improved scale-up and traceability logistics. Furthermore, software can be used to streamline the documentation process with automatically generated electronic batch records, providing a history of all processing equipment, reagents, patient identification, operator identification, and sensor data during processing. [Overview of the Initiative] [Means for solving the problem]

[0006]

[0006] In some embodiments, methods are provided herein for the automated production of genetically modified immune cell cultures, the methods comprising: activating an immune cell culture using an activating reagent to produce an activated immune cell culture; transducing the activated immune cell culture using a vector to produce a transduced immune cell culture; expanding the transduced immune cell culture; concentrating the expanded immune cell culture from (c); and recovering the concentrated immune cell culture from (d) to produce 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) to (e) are performed by a fully sealed cell engineering system, and (a) to (e) are optimized through the process of producing a genetically modified immune cell culture.

[0007]

[0007] In further embodiments, methods are provided herein for promoting a preferred phenotype of a genetically modified immune cell culture, the methods comprising: activating the 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 (c); and recovering the concentrated immune cell culture (d) to produce a genetically modified immune cell culture, wherein (a) to (e) are performed by a fully sealed automated cell engineering system.

[0008]

[0008] In additional embodiments, methods for the automated production of genetically modified immune cell cultures are provided herein, which include: 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 from (c); and recovering the concentrated immune cell culture from (d) to produce a genetically modified immune cell culture, wherein (a) to (e) are performed by a fully sealed automated cell engineering system, and each of (a) to (e) is performed with an optimized cell density (cells / mL) and an optimized cell density (cells / cm²). 2 This procedure is performed using an immunocellular culture containing )

[0009]

[0009] In additional embodiments, the Specified herein provides a method for the automated production of genetically modified immune cell cultures, 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 without shaking during expansion; concentrating the expanded immune cell culture from (c); and recovering the concentrated immune cell culture from (d) to produce a genetically modified immune cell culture, wherein (a) to (e) are performed by a fully sealed automated cell engineering system.

[0010]

[0010] In further embodiments, a method for the automated production of genetically modified immune cell cultures is provided herein, which is carried out by a cell engineering system and includes: activating an immune cell culture with an activating reagent to produce an activated immune cell culture in a first chamber of the cell engineering system; transducing the activated immune cell culture, which 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 produce 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 from (c); and recovering the concentrated immune cell culture from (d) to produce a genetically modified cell culture.

[0011]

[0011] In additional embodiments, the Specified Provisions Provide a cassette for use in an automated cell engineering system, comprising: a cryogenic chamber for storing cell culture media; a high-temperature chamber for activating, transducing and expanding immunocellular cultures, the high-temperature chamber being separated from the cryogenic chamber by a thermal barrier, the high-temperature chamber comprising a cell culture chamber; and one or more fluid pathways connected to the cell culture chamber, the fluid pathways providing recirculation, waste removal, uniform gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber.

[0012]

[0012] In further embodiments, the Specified Provisions provide a cassette for use in an automated cell engineering system, comprising 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, a satellite volume for increasing the working volume of the chamber by providing an additional volume of culture medium 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 fluid pathways so that the culture medium is exchanged with the culture chamber without disturbing the immune cell culture. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows a generalized manufacturing process for chimeric antigen receptor (CAR) T cells. [Figure 2] This figure shows a laboratory space including an exemplary cell engineering system described in the embodiments of this specification. [Figure 3] This figure shows a CAR T cell generation process that can be carried out using the cell engineering system described in the embodiments of this specification. [Figure 4] This figure shows a comparison between the COCOON system and control methods for maintaining populations of CD8+ and CD4+ cells. [Figure 5]This figure shows a comparison of the Cocoon System and methods for controlling the quantity of CAR T cells in CD8+ and CD4+ cell populations. [Figure 6A] Figures 6A-6C show an overview of the cocoon system used in Example 1. Figure 6A shows the cocoon system in a closed configuration. [Figure 6B] Figures 6A-6C show an overview of the cocoon system used in Example 1. Figure 6B shows a cassette that can be inserted into the cocoon. [Figure 6C] Figures 6A-6C show an overview of the cocoon system used in Example 1. Figure 6C shows the cocoon system in an open configuration. [Figure 6DE] This figure shows the position and orientation of the cell culture chambers used in the Cocoon System. [Figure 6F] A more detailed diagram of the cell culture chamber used in the Cocoon system is shown. [Figure 6G] This figure shows the legend for the process flow of the Cocoon system. [Figure 6H] This figure shows gas transfer data using the Cocoon system. [Figure 7A] Figures 7A-7C show the results of the experiment described in Example 1, comparing GFP transduction and manual operation in the Cocoon system. Figure 7A shows a comparison of average yields. [Figure 7B] Figures 7A-7C show the results of the experiment described in Example 1, comparing GFP transduction and manual manipulation in the Cocoon system. Figure 7B shows a comparison of average recovery survival rates. [Figure 7C] Figures 7A-7C show the results of the experiment described in Example 1, comparing GFP transduction and manual operation in the Cocoon system. Figure 7C shows a comparison of average transduction efficiencies. [Figure 8A] Figures 8A-8B show the results of the experiment described in Example 1, comparing HER-2 CAR T transduction in the Cocoon System and Permalife bags. Figure 8A shows a comparison of live cell yields. [Figure 8B] Figures 8A-8B show the results of the experiment described in Example 1, comparing HER-2 CAR T transduction in the Cocoon System and the Permalife bag. Figure 8B shows a comparison of survival rate and transduction efficiency. [Figure 9A] Figures 9A-9D show the results of the experiment described in Example 1 and compare the Cocoon System and the PermaLife Bag. Figure 9A shows a comparison of relative CAR T purity. [Figure 9B] Figures 9A-9D show the results of the experiment described in Example 1, comparing the Cocoon System and the PermaLife Bag. Figure 9B shows a comparison of the percentage of CD8+ cells. [Figure 9C] Figures 9A-9D show the results of the experiment described in Example 1, comparing the Cocoon System and the PermaLife Bag. Figures 9C and 9D show the production of TNFα and INFγ, respectively. [Figure 9D] Figures 9A-9D show the results of the experiment described in Example 1, comparing the Cocoon System and the PermaLife Bag. Figures 9C and 9D show the production of TNFα and INFγ, respectively. [Figure 10A] This figure shows the results of the experiment described in Example 1, comparing the killing of target tumor cells by CAR T cells cultured in the Cocoon System (Figure 10A) and Permalife Bags (Figure 10B). [Figure 10B] This figure shows the results of the experiment described in Example 1, comparing the killing of target tumor cells by CAR T cells cultured in the Cocoon System (Figure 10A) and Permalife Bags (Figure 10B). [Figure 11A] Figures 11A–11E show another configuration of the Cocoon System as described in the embodiments of this specification. Figure 11A shows a disposable T cell cassette that can be filled into the Cocoon System. [Figure 11B] Figures 11A-11E show another configuration of the cocoon system described in the embodiments of this specification. Figure 11B shows the cocoon system in an open configuration. [Figure 11C] Figures 11A-11E show another configuration of the cocoon system described in the embodiments of this specification. Figure 11C shows a cassette filled in the cocoon. [Figure 11D] Figures 11A-11E show another configuration of the cocoon system described in the embodiments of this specification. Figure 11D shows the cocoon in a closed configuration. [Figure 11E] Figures 11A-11E show another configuration of the Cocoon system described in the embodiments of this specification. Figure 11E shows a detailed diagram of a cassette for use with the Cocoon. [Figure 11F] This diagram shows the use of a syringe and bag for sampling from a cassette. [Figure 12A] Figure 12A shows an overview of the CAR T cell generation process. [Figure 12B] Figure 12B shows a cocoon cassette cell proliferation chamber containing an ongoing CAR T cell culture. [Figure 12C] Figure 12C shows a manually operated CAR T cell generation process using cell culture bags in an incubator. [Figure 13A] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation using DYNABEADS or OKT3. Figure 13A shows a comparison of live cell yields. [Figure 13B] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation by DYNABEADS or OKT3. Figure 13B shows a comparison of population doubling levels (PDL). [Figure 13C]Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation using DYNABEADS or OKT3. Figure 13C shows a comparison of the yield of viable CD3+ T cells. [Figure 13D] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation using DYNABEADS or OKT3. Figure 13D shows a comparison of CD3+ cell PDL. [Figure 13E] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation by DYNABEADS or OKT3. Figure 13E shows a comparison of the proportion of CD3+ subsets (CD4+ and CD8+). [Figure 13F] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation by DYNABEADS or OKT3. Figure 13F shows a comparison of cell depletion measured with anti-PD-1. [Figure 13G] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation using DYNABEADS or OKT3. Figures 13G and 13H show cytometry plots of CD8+CD3+ T cells activated with DYNABEADS or OKT3, respectively. [Figure 13H] Figures 13A-13H show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system with T cell activation using DYNABEADS or OKT3. Figures 13G and 13H show cytometry plots of CD8+CD3+ T cells activated with DYNABEADS or OKT3, respectively. [Figure 14A]Figures 14A-14F show the results of the experiment described in Example 2 and compare the Permalife bag and the Cocoon system. Figure 14A compares the transduction efficiency of CD3+ cells. [Figure 14B] Figures 14A-14F show the results of the experiment described in Example 2, comparing the PermaLife bag and the Cocoon system. Figure 14B compares the total number of surviving CAR T cells. [Figure 14C] Figures 14A-14F show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system. Figure 14C compares the transduction efficiency of T cell subsets (CD4+ and CD8+). [Figure 14D] Figures 14A-14F show the results of the experiment described in Example 2, comparing the PermaLife bag and the Cocoon system. Figure 14D compares total CAR T cells for each subset. [Figure 14E] Figures 14A-14F show the results of the experiment described in Example 2 and compare the PermaLife bag and the Cocoon system. Figures 14E and 14F show cytometry plots of CD3+OKT3 activated cells in the Cocoon and PermaLife bag, respectively. [Figure 14F] Figures 14A-14F show the results of the experiment described in Example 2 and compare the PermaLife bag and the Cocoon system. Figures 14E and 14F show cytometry plots of CD3+OKT3 activated cells in the Cocoon and PermaLife bag, respectively. [Figure 15A] Figures 15A-15F show the results of the experiment described in Example 2 and compare the Permalife bag and the Cocoon system. Figure 15A compares the percentage of cells that produce TNFα. [Figure 15B] Figures 15A-15F show the results of the experiment described in Example 2, comparing the PermaLife bag and the Cocoon system. Figure 15B compares the percentage of cells that produce IFNγ. [Figure 15C]Figures 15A-15F show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system. Figures 15C and 15D show cytometry plots of cells that were activated by DynaBeads secreting TNFα and IFNγ, respectively, and that produced cocoons. [Figure 15D] Figures 15A-15F show the results of the experiment described in Example 2, comparing the Permalife bag and the Cocoon system. Figures 15C and 15D show cytometry plots of cells that were activated by DynaBeads secreting TNFα and IFNγ, respectively, and that produced cocoons. [Figure 15E] Figures 15A-15F show the results of the experiment described in Example 2 and compare the PermaLife bag and the Cocoon system. Figures 15E and 15F show the tumor-killing efficiency of CAR T cells generated from the PermaLife bag or the Cocoon system, respectively. [Figure 15F] Figures 15A-15F show the results of the experiment described in Example 2 and compare the PermaLife bag and the Cocoon system. Figures 15E and 15F show the tumor-killing efficiency of CAR T cells generated from the PermaLife bag or the Cocoon system, respectively. [Figure 16] This figure shows a comparison of Cocoon and Permalife, and an overview of activation by DynaBeads or OKT3. [Figure 17] This figure shows the integration of an electroporation unit with a cell engineering system according to an embodiment of this specification. [Figure 18] This diagram shows the flow of immune cell cultures from the cell engineering system to the electroporation unit. [Figure 19] This figure shows the results of human stem cell experiments described herein. [Figure 20] This figure shows the results of human stem cell experiments described herein. [Figure 21] Figure 21 shows the differentiated cell phenotype in Example 4. [Figure 22]Figure 22 shows that a single colony in Example 4 can form polyphyletic differentiation. [Modes for carrying out the invention]

[0014]

[0037] This disclosure provides an automated method for generating chimeric antigen receptor T (CAR T) cells. CAR T cell generation typically requires manual intervention for patient-specific products. Automating CAR T cell culture is particularly challenging due to the multiple highly sensitive unit operations, such as cell activation, transduction, and expansion. Therefore, this disclosure provides an automated method for CAR T cell generation utilizing a fully sealed cell engineering system.

[0015] Automated cell processing

[0038] In autologous cell therapies such as T-cell therapy, the need for cost-effectiveness, process efficiency, and purified product consistency is particularly critical because batches producing microlots (one patient per lot) lack the economies of scale available to homogeneous (multiple patients per lot) processes (see, e.g., Jones, 2012; Trainor, 2014). The larger, more localized workforce and facilities required for microlots impose considerable demands on logistics and GMP compliance for manual production, particularly regarding staff availability and training. Furthermore, the potential for variability in operator skill can pose an undesirable risk to ensuring consistent meeting of release standards and the provision of safe and reliable products.

[0016]

[0039] As described herein, the introduction and comprehensive validation of automated manufacturing are these logistics and provide solutions to operational challenges. A key approach to introducing automation into the production process is to identify key modular steps in which the operator applies physical or chemical modifications to the production material, referred to as “unit operations.” In the case of cell manufacturing, these include steps such as cell separation, genetic manipulation, proliferation, washing, concentration, and cell harvesting. Manufacturers often identify bottlenecks in the focus process as direct opportunities to introduce automation. This is reflected in the technical operating range of most commercially available bioreactors, which tend to focus on individual process steps. Process challenges in cell manufacturing (from maintaining sterility to sample tracking) are addressed herein by end-to-end automation that produces consistent cell output while mitigating inevitable process variability. The methods described herein also provide simplification, and the associated electronic records assist in compliance with GMP standards (see, e.g., Trainor, 2014).

[0017] Automation of unit operations and key process sensitivity

[0040] Recent rapid advances in the clinical development of modified autologous T cells for cancer immunotherapy have led to planning of related transitions and scale-up / scale-out implications.

[0018]

[0041] While specific protocols may vary with respect to T cell production, a generalized chimeric antigen receptor T cell (CAR T) process is shown in Figure 1. Figure 1 describes the unit operation of CAR T cell production, from initial processing of patient blood samples to formulation of output cells for autologous T cell therapy.

[0019]

[0042] As described herein, to achieve automation of cell production, the methods described herein provide an understanding of the state of cells at each transition point and the effects of specific unit operations. Microlot production for patient-specific therapies requires respecting important process sensitivities that affect the feasibility of automation. The automation described herein encompasses a variety of process steps.

[0020]

[0043] Table 1 below highlights the challenges of several process steps identified for T cell automation and notes the impact of susceptibility on automation strategies. Note that open cell transfers between instruments are a critical susceptibility in all unit operations due to the risk of contamination. [Table 1] JPEG2023153817000003.jpg134149

[0021]

[0044] Adjusting the automation of manual processes, particularly those related to sensitivity as listed in Table 1, can support the success of transforming, maintaining, or improving the performance of cell therapies.

[0022] Integration of automated unit operations

[0045] Considering GMP logistics, economics, and the impact of automation on patient safety, unit operations can be evaluated in relation to typical labor hours per unit operation (including labor hours for both operators and quality assurance monitors). Table 2 shows the nominal manual processing timelines for typical steps of CAR T automation. This table highlights the resource commitment 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] JPEG2023153817000005.jpg181149

[0023]

[0046] Based on the methods described herein, the automation of unit operations can reduce the nominal manual process to approximately 40 hours, about a quarter of the original time.

[0024] Individual vs. fully integrated automation

[0047] While there is compelling evidence for the value of automation (see, e.g., Trainor 2014; Levine 2017), further analysis is needed regarding the value and practicality of integrating these automation steps in end-to-end sequences with automated transfer. There are differing perspectives on the merits of individual process automation versus the merits of end-to-end integration.

[0025]

[0048] The main advantage of individual automation is flexibility. This relates to the following areas: 1) Maintenance of specific process operations 2) Accelerating conversion activities based on operational verification of individual units 3) Ability to modify processing steps to accommodate variations between donors

[0026]

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

[0027]

[0050] The use of equipment is a separate discussion for individual automation. Some unit operations may take significantly longer than others. In an end-to-end processing system, multiple unit operations must all be performed on a single system, thus occupying equipment throughout the culture process.

[0028]

[0051] While individual automation has its advantages, an end-to-end approach offers something different, though the benefits are not as compelling. Firstly, a fully integrated system significantly reduces the risk of contamination. Individual approaches require more handling, making product variability more likely due to operator intervention. Secondly, as mentioned earlier, this inevitably leads to increased labor costs.

[0029]

[0052] The flexibility provided by an individualized approach is crucial. Where the process is critical to defining the product, the end-to-end system should have the flexibility to integrate unique sensitivities. This could include specific supply strategies, oxygen levels, surface treatments, etc. Such an approach requires flexibility in both software and disposable components. The system should offer options to withdraw cells and culture samples at various points in the process to ensure that specific unit operations meet product specification checkpoints. If changes are necessary, the software should be able to implement these changes to provide ideal conditions. While user-friendly and flexible software is highly beneficial for conversion purposes, 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 modify the protocol should be restricted. However, to address the issue of inherent donor variability, there should be an option to select from a range of validated protocols based on cell growth rates. For example, if cells are growing rapidly, the system should be able to respond and adjust feeding or harvesting timing 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 footprint required 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 expensive GMP cleanroom space. Figure 2 shows a 96-patient-specific end-to-end unit running in a standard laboratory space.

[0031]

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

[0032] Commercial platform for integrating unit operations

[0055] The success of numerous autologous cell therapies, particularly in immunotherapy for hematological cancers, underscores the importance of translating new clinical protocols into robust production platforms to meet anticipated clinical demand (see, e.g., Levine, 2017; Locke, 2017). In the case of autologous therapies, the processing of each patient-specific cell therapy appropriately utilizes comprehensive manufacturing activities and operational controls. The methods herein link unit operations of a turnkey automated system to achieve process optimization, security, and economic efficiency.

[0033]

[0056] There are two challenges in designing a self-processing system. First, unlike homogeneous processes, where individual processing steps may occur in parts of physically separated and optimized equipment, a scaled-out self-processing platform must properly perform all necessary steps in a single, closed, self-contained automated environment. Second, unlike homogeneous processes, where theoretically all executions begin with high-quality vials from a cell bank, with known quality and predictable process behavior, the starting materials for a self-processing system are highly diverse and generally come from individuals with compromised health.

[0034]

[0057] Therefore, this specification provides a method for sensing culture conditions and responding accordingly as a sophisticated bioreactor by controlling factors such as physical agitation, pH, supply, and gas treatment. Furthermore, technology transfer associated with autotherapy presents significantly different challenges compared to allogeneic therapy. Auto-manufactured products may have greater limitations in terms of stability between the manufacturing process and patient treatment. Sites can be located globally rather than as a single center. Using a lockdown (e.g., fully sealed) all-in-one system significantly improves the technology transfer process between sites.

[0035]

[0058] While supply chain variability cannot be eliminated, automation helps to eliminate variability in the final self-manufactured product through standardization and reproducibility. This approach is employed by leading cell system providers to obtain a baseline for cell performance via biosensors that monitor the state of active cell cultures. In end-to-end integration, the output from a particular stage of the process must fall within acceptable parameters for the progress of the process.

[0036]

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

[0037]

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

[0038]

[0061] In the experiments conducted, the polyploidization of CAR T cells in cultures for 10–14 days reached approximately 40–60. Both CD4+ and CD8+ T cell subsets are necessary for successful CAR T therapy. Therefore, the ability of the run and associated controls to maintain cultures of both T cell subsets was evaluated by flow cytometry. Figure 4 shows that all runs, as well as all controls, were able to maintain both T cell subsets. The proportion of CAR T cells present was also evaluated in each population of 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 in the CD8+ portion exceeded 50% of the fraction found in the paired CD4+ population. In summary, the automated CAR T process using the method described herein results in a healthy population of T cell subsets.

[0039] Advantages of automation

[0062] The automation of unit operations in the production of cell therapies offers opportunities for universal benefits across allogeneic and autologous cell therapy applications. While the recent clinical successes of these therapies further highlight the unique scenarios of patient-specific autologous cell products, the benefits of automation are particularly attractive with highly complex microlots, offering 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 microlot cell production has not been a key point of research. However, the anticipated demand for these therapies following imminent approval indicates that the implementation of fully closed end-to-end systems could provide crucial solutions to manufacturing bottlenecks such as hands-on time and footprint.

[0040]

[0063] Developers of advanced therapies are advised to consider automation early in the deployment of clinical translation and to scale up clinical trial protocols. Early automation can impact protocol development, avoid the need for comparability studies when switching from manual to automated processes at later stages, 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, this specification provides an automated method for producing genetically modified immune cell cultures. As used herein, “genetically modified immune cell culture” (or genetically modified immune cells) refers to cells of the immune system that have been modified or primed (e.g., through co-culture with antigen-presenting cells) to result in cells having a desired phenotype useful for treating, preventing, or improving one or more diseases in animals, including humans. As used herein, “immune cell culture” refers to a collection of cells prepared by the method described herein and may include a population of cells for use in investigation or clinical trials, and for administration to mammals, including human patients, for medical therapy. Genetically modified immune cell cultures that can be produced using the method described herein may 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 containing hematopoietic stem cells.

[0043]

[0066] In exemplary embodiments, the method includes 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 cells, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to produce a genetically modified immune cell culture. Preferably, the method further includes either or both of the expanded immune cell culture and the concentrated immune cell culture. In embodiments, various steps of the method are performed by a fully sealed cell engineering system and optimized through the process of producing a genetically modified immune cell culture.

[0044]

[0067] Methods for optimizing the process for generating genetically modified immune cells include optimizing cell culture conditions before initiating automated methods, as well as using feedback from various sensors to support real-time modifications to growth conditions (e.g., gas concentration, medium conditions, temperature, pH, waste and nutrient concentrations).

[0045]

[0068] In embodiments, the optimization process is a self-regulating process that does not require input from an external (human) user and can determine the modifications required 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 the following: 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 sealed cell engineering system occurs at various times and places within the system and works in coordination to provide optimization. For example, the self-regulating process can adjust (e.g., increase or decrease) one or more of the following: temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density of the transduced T cell culture, based on monitoring.

[0046]

[0069] The optimization process can also be based on the unique characteristics of the starting cell population, including, for example, total cell number, cell source, cell density, and cell age. Before starting the automated method, the characteristics of the starting cell population can be input into a computer control system. In this case, the system makes various initial changes to optimize the method, such as oxygen and carbon dioxide concentrations, flow rates, incubation time, and pH. Alternatively, monitoring the cell process allows for automated characterization of the progression of the cell culture sequence from the starting population, enabling case-by-case adjustment of the conditions for optimized final cell culture characteristics.

[0047]

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

[0048]

[0071] As described herein, the genetically modified immune cell culture produced by this method is, appropriately, a T cell culture comprising a chimeric antigen receptor T (CAR T) cell culture. In such embodiments, the vector used to produce such CAR T cells is a vector encoding a chimeric antigen receptor. Preferably, the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells. In embodiments, the immune cell culture comprises at least one accessory cell, preferably a monocyte or monocyte-derived cell. As described herein, in embodiments, the accessory cell comprises an antigen for T cell receptors, including CD28, CD40, CD2, CD40L and / or ICOS.

[0049]

[0072] Suitablely, the activating reagent includes an antibody or dendritic cells. In embodiments, the antibody is immobilized on a surface that may include polystyrene plastic, silicone, or other surfaces, such as the surface of beads.

[0050]

[0073] In other embodiments, the activating reagent includes an antibody which is a soluble antibody comprising at least one of an anti-CD3 antibody and an anti-CD28 antibody. An example antibody is OKT3.

[0051]

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

[0052]

[0075] In exemplary embodiments, the vector used in this method is a lentiviral vector or a retrovirus. Preferably, transduction involves mixing the vector in a cell culture medium and uniformly delivering the vector in the medium to an activated immune cell culture. As described herein, uniform delivery of the vector in a uniform manner to cells provides optimization of various high-output cellular properties of the desired genetically modified immune cells.

[0053]

[0076] As described herein, a method for expanding cells appropriately includes at least one or more of the supply, washing, monitoring, and selection of transduced immune cell cultures.

[0054]

[0077] The various methods described herein are carried out in such a manner that the oxygen level of the transduced immunocell culture is optimized for the immunocell culture. This optimization enables the generation of a large number of viable cells with desired phenotypic characteristics, including the enhancement 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) to (e). As described herein, oxygenation is appropriately achieved through one or more fluid pathways including a silicone-based tubular component.

[0055]

[0078] In further embodiments, the cell engineering system recirculates nutrients, waste products, released cytokines, and / or lysed gases during various method processes. This recirculation assists in the generation of a large number of viable cells having a desired phenotype(s). Appropriately, the carbon dioxide levels provided by the cell engineering system are reduced during expansion steps to optimize cell proliferation, etc. In other embodiments, for example, the CO2 level can be increased when a complete culture medium change is utilized.

[0056]

[0079] Other mechanisms for optimizing cell growth conditions include modifying and controlling the flow rate of the culture medium supplied to the cells. As cells begin to grow, the circulation rate of the supplied medium increases, thereby improving gas exchange, and oxygen and carbon dioxide may be added to or removed from the cell culture depending on the state of the cells and their requirements at that time.

[0057]

[0080] In embodiments, the cell engineering system is configured to perform one or more rounds of feeding, washing, and monitoring, and in embodiments, it is configured to perform selection of transduced immunocell cultures. These various activities can be performed in any order and can be performed individually or in combination with other activities. In embodiments, cell enrichment includes centrifugation, removal of supernatant after sedimentation, or filtration. Preferably, the optimization process further includes adjusting the parameters of centrifugation or filtration, preferably in a self-regulating process. Selection of transduced cells may be performed, for example, by magnetic separation, filtration, or attachment to a plastic or other substrate.

[0058]

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

[0059]

[0082] Suitablely, the method may further include removing the activating reagent from the activated immune cell culture after step (a), and may include removing the vector following the transduction step. The activating reagent can be adequately removed from the immune cell culture by washing, draining, or physically removing the cells or the activating 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 activating reagent, a vector, and a cell culture medium before initiating the method. In other embodiments, the activating reagent and / or vector may be added separately after the initiation of the production method or at any appropriate time during the process.

[0061]

[0084] In further embodiments, this specification provides a method for promoting a preferred phenotype of a genetically modified immune cell culture, the method comprising activating the 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 recovering the concentrated immune cell product of (d) to produce a genetically modified immune cell culture. As described herein, the method is appropriately carried out by a fully sealed automated cell engineering system.

[0062]

[0085] As described herein, the selection of appropriate activating reagents and appropriate activation conditions provides the enhancement of a desired phenotype in the genetically modified immunocell culture. That is, the phenotype of the immunocell culture can be specifically selected and enhanced, and as a result, a suitable majority of the cells produced by this method will have the desired preferred phenotype. In other embodiments, a desired ratio of one cell phenotype to another can be controlled and enhanced to provide a desired preferred phenotypic balance.

[0063]

[0086] As described herein, it has been found that the use of activating reagents, particularly soluble antibodies, can promote a desired phenotype in genetically modified immune cells. Preferably, the antibodies used include at least one of the following: an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody, including the soluble antibody OKT3.

[0064]

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

[0065]

[0088] The methods described herein may influence the characteristics of the final immune cell culture product by selecting an appropriate activation method to provide a preferred phenotype. For example, activation utilizing the bead-based process described herein promotes a more balanced CD4:CD8 ratio, while the use of soluble anti-CD3 promotes a higher CD8 population than CD4. Other levels of CD8 and CD4 can also be provided using the methods described herein. In exemplary embodiments, CAR T cells can be prepared using the methods described herein. Appropriately, the methods can be used to promote phenotypes of CAR T cells having CD8+ cell to CD4+ cell ratios of about 0.1:1 to about 10:1, including CD8+ cell to CD4+ cell ratios of about 0.5:1 to about 5:1, 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 generation of a genetically modified immune cell culture is provided, the method comprising activating an immune cell culture with an activation reagent to generate an activated immune cell culture, transforming 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. 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 [[ID=二十]]cells / mL to about 40×10 6 cells / mL, or from about 0.05×10<00,00008>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, increase or decrease of oxygen and / or carbon dioxide concentration, pH, temperature, adjustment of nutrients, removal of waste, etc. Exemplary cell densities include about 0.05×10 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 This includes cells / mL, etc.

[0069]

[0092] In this embodiment, the optimized cell density is approximately 0.1 × 10⁻⁶. 6 cells / cm 2 ~Approx. 60×10 6 cells / cm 2 , or approximately 0.1 × 10 6 cells / cm 2 ~About 40×10 6 cells / cm 2 , or approximately 0.1 × 10 6 cells / cm 2 ~About 20×10 6 cells / cm 2 That is the case. The optimized cell density can vary throughout the entire process of the generation method, and at each stage of the method (i.e., activation, transduction, expansion, and enrichment), the cell density can be controlled or manipulated to provide the best cell density for a specific step of the method. Cell density can be optimized by selecting an optimal starting cell density, selecting materials for the cell culture chamber, increasing or decreasing oxygen and / or carbon dioxide concentrations, adjusting pH, temperature, nutrients, and removing waste products. An example cell density is approximately 0.1 × 10⁻⁶. 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 approximately 60 x 10 6 cells / cm 2 This includes things like:

[0070]

[0093] In this embodiment, the method recirculates nutrients, waste products, released cytokines, and / or dissolved gases for approximately 0.05 × 10⁻⁶ times. 6 cells / mL ~ approx. 20×10 6 Cell density / mL, and approximately 0.1 × 10⁻⁶ 6 cells / cm 2 ~Approx. 20×10 6 cells / cm 2 This includes being uniformly supplied to cells having a certain density.

[0071]

[0094] In further embodiments, an automated method for generating genetically modified immune cell cultures is provided, which includes activating an immune cell culture with an activating 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 without shaking during expansion, concentrating the expanded immune cell culture, and recovering the concentrated immune cell culture to generate a genetically modified immune cell culture. As appropriately described herein, the method is carried out by a fully sealed automated cell engineering system.

[0072]

[0095] As described herein, it has been found that cells can be expanded under conditions where the cells are not shaken (i.e., not rotated or shaken to allow cells to flow over each other). This method provides high viable cell yield and optimal cellular characteristics, including the desired phenotype. A large, non-shaking cell culture chamber was found to be able to achieve the desired results by providing uniform access to necessary reagents, nutrients, gas exchange, etc., while removing cellular waste, without the need to shake or disturb the cells. In fact, as described herein, such a method for automated generation of genetically modified immune cells has been found to produce a larger number of viable cells, a greater number / ratio of the desired cell type, and more robust cellular characteristics compared to methods utilizing cell shaking, such as Miltenyi et al., "Sample Processing System and Methods," U.S. Patent No. 8,727,132.

[0073]

[0096] Appropriately, the extended step of the method includes at least one or more of the transduced immune cell cultures: supplying, washing, monitoring, and selecting, without shaking the immune cell cultures.

[0074]

[0097] Furthermore, this specification provides an automated method for generating genetically modified immune cell cultures, which is performed by a cell engineering system and includes 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 recovering the concentrated immune cell culture from (d) to generate a genetically modified cell culture.

[0075]

[0098] For example, as shown in Figure 17, activated immunocellular cultures are transferred from cassette 602 of the cell engineering system 600 to the electroporation unit 1706, for example, via connecting tube 1704. The electroporation unit 1706 appropriately contains an electroporation cartridge 1708 that holds the cell cultures during the electroporation process. After the electroporation process, the transduced immunocellular cultures are returned to the cell engineering system 600 via connecting tube 1704. Figure 17 also shows the use of two optional reservoirs 1710 and 1712 used to hold the cell cultures before and after electroporation, assisting the transfer between the cell engineering system and the electroporation unit as a result of different pump speeds, required pressure and flow rates. However, such reservoirs can be removed, and the cell cultures can be transferred directly from the cell engineering system 1702 to the electroporation unit 1706.

[0076]

[0099] Figure 18 shows the flow of cell cultures 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 exemplary embodiments, as shown in Figures 17 and 18, the electroporation unit 1706 is located outside the cell engineering system 1702. In such embodiments, transduction involves transferring an activated immune cell culture from a first chamber to the electroporation unit via a first sterile closed connector (e.g., a connecting tube 1704), electroporating the activated immune cell culture with a vector to produce a transduced immune cell culture, and transferring the transduced immune cell culture to a second chamber of the cell engineering system via a second sterile closed connector (e.g., a connecting tube 1704).

[0078]

[0101] Furthermore, it should be understood that multiple separate cell engineering systems 600 (see, for example, Figure 2) can be connected to a single electroporation unit and executed in the appropriate sequence so that cell cultures are transferred from the cell engineering system to the electroporation unit and then back to the appropriate cell engineering system.

[0079]

[0102] In other embodiments, the electroporation unit 1706 can be placed within the cell engineering system 600 so that the entire system is a closed, self-contained system. Methods for incorporating the electroporation unit 1706 inside the cell engineering system 600 are known to those skilled in the art and utilize various miniaturization strategies, etc.

[0080]

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

[0081]

[0104] More appropriately, the methods described herein generate at least 20% more genetically modified immune cells than methods utilizing manual cell cultures with flexible gas-permeable bags. More appropriately, the methods generate at least 25% more, at least 30% more, at least 35% more, and at least 40% more genetically modified immune cells than methods utilizing manual cell cultures with flexible gas-permeable bags.

[0082]

[0105] In exemplary embodiments, the cell engineering system described herein comprises 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, and each of the activating reagents, vectors, and cell culture media are placed in a different chamber of the plurality of chambers before the method is initiated, at least one of the plurality of chambers is maintained at a temperature for cell growth (e.g., about 37°C), and at least one of the plurality of chambers is maintained at a refrigerated temperature (e.g., about 4-8°C).

[0083]

[0106] In some embodiments, the Disclosure provides a method for generating chimeric antigen receptor T cells, the method comprising: (a) appropriately activating a peripheral blood mononuclear cell culture with a culture medium containing at least one 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 predefined culture size; (d) concentrating the expanded T cell culture from (c) to a volume of about 20 mL to about 500 mL, preferably about 50 mL to about 200 mL; and (e) recovering the concentrated T cell culture from (d) to generate a chimeric antigen receptor T (CAR T) cell culture, wherein the activated T cell culture is substantially undisturbed between steps (a) to (b), and the method is carried out by a fully sealed cell engineering system which has appropriate instructions for carrying out steps (a) to (e). Preferably, steps (a) through (e) are performed within one or more chambers of a cell engineering system. In embodiments as described herein, the method generates at least 20% more CAR T cells than a method utilizing 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 receptor region exposed to the extracellular fluid and contains three parts: a signaling peptide, an antigen recognition region, and a spacer. The signaling peptide induces the nascent protein into the endoplasmic reticulum. In CARs, the signaling peptide is a single-chain variable fragment (scFv). The scFv contains a light chain (VL) and a heavy chain (VH) of 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 spanning 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 a CAR is generally considered the "functional" end of the receptor. Following antigen recognition by the CAR cluster in the antigen-recognition region of the ectodomain, a signal is transmitted to the cell. In some embodiments, the endodomain is a CD3-ζ endodomain containing three immune receptor tyrosine-based activation motifs (ITAMs). In this case, the ITAMs transmit an activation signal to T cells after antigen binding, triggering a T cell immune response.

[0087]

[0110] During CAR T cell generation, T cells are extracted from a human subject, genetically modified, and reintroduced into the patient to attack cancer cells. CAR T cells can originate from the patient's own blood (autologous) or from another healthy donor (allogeneic). Generally, CAR T cells are generated to be specific to antigens expressed in tumors that are not expressed in healthy cells.

[0088]

[0111] T cell activation. In some embodiments, the immune cell cultures produced by the methods described herein are CAR T cell cultures. CAR T cells can be activated to form activated T cell cultures. In vivo, antigen-presenting cells (APCs), such as dendritic cells, act as stimuli for T cell activation through the interaction of the T cell receptor (TCR) with the APC major histone compatibility complex (MHC). The TCR associates with CD3, a T cell coreceptor that facilitates 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 follows a two-signal model and requires stimulation of the TCR / CD3 complex, as well as the costimulatory receptor. Further information on T cell activation can be found, for example, in Kochenderfer 2015; Kalos 2011.

[0089]

[0112] Without a co-stimulatory signal, cells become more susceptible to anergy and become unresponsive. Therefore, T cell co-stimulation may be important for T cell proliferation, differentiation, and survival. Non-limiting examples of T cell co-stimulatory molecules include CD28, the CD80 and CD86 receptors on the APC membrane; and CD278 or ICOS (Inducible T Cell Co-stimulator), CD28 superfamily molecules expressed on activated T cells that interact with ICOS-L. Therefore, 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 on the APC, which binds to the CD28 receptor on T cells. B7 is a peripheral transmembrane protein found on activated APCs that can interact with the CD28 or CD152 surface proteins on T cells to generate a co-stimulatory signal. Therefore, in some embodiments, the co-stimulatory molecule is B7. Co-stimulatory receptors have been further described, for example, by Lafferty, 1975; Harding, 1992; Clavreul, 2000; Charron, 2015; Fathman, 2007; and Greenwald, 2005. Co-stimulation has been further described, for example, by Carpenter, 2000; and Andris, 2004. The B7 molecule has been further described, for example, by Fleischer, 1996; and Schwartz, 2003.

[0090]

[0113] Various activation methods are used in vitro to simulate T cell activation. In some embodiments, T cell cultures are activated with an activation reagent. In further embodiments, the activation reagent is antigen-presenting cells (APCs). In even further embodiments, the activation reagent is dendritic cells. Dendritic cells are APCs that process antigens and present them to T cells on their cell surface. In some embodiments, the activation reagent is co-cultured with the T cell culture. Co-culture may require separate purification and culture of the second cell type, potentially increasing labor requirements and sources of variation. Therefore, in some embodiments, alternative activation methods are used.

[0091]

[0114] In embodiments, cells maintain stable contact with the activating reagent during the activation step. One way to maintain stable contact between cells and the activating reagent is to prevent unnecessary or excessive movement of cells. Thus, in embodiments, cell culture is substantially undisturbed during the activation step. "Substantially undisturbed" means that while the cell culture medium is being changed, the cells generally remain in the same area of ​​the cell culture chamber, e.g., at the bottom of the chamber. If cells are moved between different containers, e.g., from one culture flask to another, the cells may be disturbed, or if the container is flexible, the cells may be disturbed. For example, flexible containers such as culture bags may move cells when the bag is handled. As described herein, the method appropriately utilizes a substantially flat, low cell culture chamber to allow uniform access of cells to various nutrients and gases, and also facilitates the removal of waste products and the movement of the medium. A substantially flat cell culture chamber also allows cells to come into contact with each other during the various stages of the method, which can enhance cell proliferation and the development of a desired cell phenotype(s).

[0092]

[0115] In some embodiments, the activating agent is an antibody. In some embodiments, the cell culture is activated with an antibody bound to a surface, such as a polymer surface, including beads. In further embodiments, one or more antibodies are anti-CD3 and / or anti-CD28 antibodies. For example, the beads could be magnetic beads coated with anti-CD3 and anti-CD28, such as DynaBeads. Anti-CD3 and anti-CD28 beads can appropriately provide stimulating signals that support T cell activation. 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 mouse monoclonal antibody of the immunoglobulin IgG2a isotype that targets CD3. Therefore, in some embodiments, the soluble anti-CD3 antibody is OKT3. OKT3 is further described, for example, by Dudley, 2003; Manger, 1985; Ceuppens, 1985; Van Wauwe, 1980; and Norman, 1995.

[0094]

[0117] In some embodiments, the co-stimulatory signal for T cell activation is provided by accessory cells. Accessory cells may include, for example, Fc receptors that enable crosslinking between the TCR / CD3 complex of T cells and CD3 antibodies. In some embodiments, the cell culture is a mixed population of peripheral blood mononuclear cells (PBMCs). PBMCs may include accessory cells that can support T cell activation. For example, the CD28 co-stimulatory signal may be provided by the B7 molecule present in 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; and Tax, 1983.

[0095]

[0118] As described herein, activating reagents can determine the phenotype of the CAR T cells produced and enable the promotion of a desired phenotype. In some embodiments, the activating reagent determines a T cell subset, namely the ratio of CD4+ helper T cells to CD8+ cytotoxic T cells. Cytotoxic CD8+ T cells are typically involved in killing cancer cells (i.e., antitumor responses), infected cells (e.g., with viruses), or otherwise damaged cells. CD4+ T cells typically produce cytokines, assist in the modulation of immune responses, and may, in some cases, support cell lysis. CD4+ cells activate APCs, which then stimulate naive CD8+ T cells for antitumor responses. Thus, in embodiments, the methods of the present disclosure further include generating CAR T cells of a predetermined phenotype (i.e., promoting cells of a desired phenotype). The predetermined phenotype may be, for example, a predetermined ratio of CD8+ cells to CD4+ cells. In some embodiments, the CD8+ cell to CD4+ cell ratio in the CAR T cell population is approximately 1:1, approximately 0.25:1, or approximately 0.5:1. In other embodiments, the CD8+ cell to CD4+ cell ratio in the CAR T cell population is approximately 2:1, approximately 3:1, approximately 4:1, or approximately 5:1.

[0096]

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

[0097]

[0120] Transduction of activated T cells. In some embodiments, a genetically modified immune cell culture is transduced with a vector encoding a chimeric antigen receptor to produce an activated T cell culture. In some embodiments, transduction includes viral infection, transposonization, mRNA transfection, electroporation, or a combination thereof. In some embodiments, transduction includes electroporation. Therefore, 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 may be a viral vector such as, for example, a lentiviral vector, a gamma retrovirus vector, an adeno-associated virus vector, or an adenovirus vector. In embodiments, transduction includes introducing a viral vector into activated T cells in a cell culture. In additional embodiments, the vector is delivered as a viral particle.

[0098]

[0121] In some embodiments, the transduction step includes transducing a lentiviral vector into activated T cells, wherein the lentiviral vector is introduced at an infection multiplicity (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 the medium is then mixed with the vector (e.g., a lentiviral vector) and uniformly distributed to the cells. In some embodiments, the removed cell culture medium is used for dilution and uniform delivery of the vector to the activated T cell culture. Uniform distribution of the vector (e.g., a lentiviral vector) in the T cell culture and the resulting uniform exposure improves transduction efficiency. In some embodiments, the volume of the cell culture is reduced after activation and before the addition of the vector. The reduction in volume can allow for greater cell-vector contact. In some embodiments, the activated T cell culture is substantially undisturbed during transduction. In some embodiments, the cell culture is substantially undisturbed during the activation and transduction steps, i.e., the cells generally remain in the same area of ​​the chamber (e.g., the bottom of the cell culture chamber) while the activating reagent or vector is being supplied to the cells. This promotes uniform distribution and uniform exposure of the activating reagent and / or vector to the cells and can therefore improve activation and / or transduction efficiency.

[0100]

[0123] Therefore, in some embodiments, the transduction efficiency of the method using the cell engineering system is higher than the transduction efficiency of the method using a flexible gas-permeable bag for cell culture. In some embodiments, the transduction efficiency of the automated CAR T cell generation method described herein is 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 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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, the 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 contain a sufficient number of cells for clinical use, i.e., transfusion to a patient, research and development work, etc. In some embodiments, the clinical or therapeutic dose of CAR T cells for administration to a patient is about 10⁵ cells, about 10⁶ cells, about 10⁷ cells, about 10⁸ cells, about 10⁹ cells, or about 10¹⁰ 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 approximately 0.1 L to approximately 5 L, approximately 0.1 L to approximately 2 L, or approximately 0.2 L to approximately 2 L. In some embodiments, the transduced T cell culture is expanded to a total volume of approximately 0.1 L, approximately 0.2 L, approximately 0.3 L, approximately 0.4 L, approximately 0.5 L, approximately 0.6 L, approximately 0.7 L, approximately 0.8 L, approximately 0.9 L, or approximately 1.0 L. The volume can also be varied throughout the process as needed, depending on the stage of the cell generation process. In some embodiments, a predetermined culture size is input by the user of the cell engineering system. The user can input a predetermined culture size in advance as a desired number of cells to be generated (e.g., 10¹⁰ CAR T cells) or as a desired number of clinical or therapeutic doses to be generated (e.g., 10 clinical or therapeutic doses of CAR T cells). In embodiments, the number of CAR T cells generated by the method described herein is at least approximately 100 million (i.e., 1 × 10¹⁰ 6 ) cells, or at least approximately 300 million, at least approximately 500 million, at least approximately 600 million, at least approximately 700 million, at least approximately 800 million, at least approximately 900 million, at least approximately 1 billion (i.e., 1 × 10⁶ 9) pieces, at least approximately 1.1 billion, at least approximately 1.2 billion, at least approximately 1.3 billion, at least approximately 1.4 billion, at least approximately 1.5 billion, at least approximately 1.6 billion, at least approximately 17, at least approximately 1.8 billion, at least approximately 1.9 billion, at least approximately 2 billion (i.e., 2 × 10⁶) 9 These are cells, for example, at least approximately 2.1 billion, at least approximately 2.2 billion, at least approximately 2.3 billion, at least approximately 2.4 billion, at least approximately 2.5 billion, at least approximately 2.6 billion, at least approximately 2.7 billion, at least approximately 2.8 billion, at least approximately 2.9 billion, or at least approximately 3 billion CAR T cells.

[0102]

[0125] In some embodiments, expanding a transduced T cell culture includes at least one round of feeding, washing, monitoring, and selection of the transduced T cell culture. Feeding the cell culture may include supplementing the cell culture with medium and / or additional nutrients. Washing the cell culture includes removing used medium (i.e., medium that is nutrient-depleted and / or contains cellular waste) and replenishing the cell culture with fresh medium. Monitoring the cell culture includes monitoring the temperature, pH, glucose, oxygen level, carbon dioxide level, and / or optical density of the cell culture. Selection of the cell culture may include selecting cells that have desired characteristics, such as viability, type, and / or morphology, and removing cells that do not have 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 transduced T cell cultures to achieve a predetermined culture size.

[0103]

[0126] In the embodiment, one or more of the supply, washing, and monitoring steps can be removed or the order of the events can be changed, depending on the desired cell phenotype or the number of cells.

[0104]

[0127] In some embodiments, monitoring includes monitoring by temperature sensors, pH sensors, glucose sensors, oxygen sensors, carbon dioxide sensors, and / or optical density sensors. Thus, in some embodiments, the cell engineering system includes one or more of the temperature sensors, pH sensors, glucose sensors, oxygen sensors, carbon dioxide sensors, and / or optical density sensors. 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 to reach the desired cell culture size, the cell engineering system automatically increases the oxygen level of the cell culture, for example, by introducing oxygenated cell culture medium, by replacing the cell culture medium with oxygenated cell culture medium, or by flowing the cell culture medium through an oxygenated component (i.e., a silicone tube). In another embodiment, if the cell engineering system detects that the current temperature of the cell culture is too high and that the cells are growing too rapidly (e.g., cell overcrowding may lead to undesirable characteristics), the cell engineering system automatically lowers the temperature of the cell culture to maintain a stable growth rate (or exponential growth rate if necessary) of the cells. In yet another embodiment, 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 cell number, or other monitoring factors such as pH, oxygen, and glucose. The cell engineering system may be configured to store the medium (and other reagents such as washing solutions) in a low-temperature chamber (e.g., 4°C or -20°C) and 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 embodiments, washing includes washing 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 in the same cell culture vessel or flask. In further embodiments, the cells remain substantially undisturbed during the washing step. In embodiments, selection includes mixing the cell culture with one or more selection reagents. The selection reagent may be beads specific to the desired cell type, such as magnetic beads, and cells bound to the beads are then separated from unbound cells, for example, by passing through a magnetic chamber. For example, the selection beads may contain antibodies specific to the desired cell type, such as anti-CD8 antibodies or anti-CD4 antibodies. Selection can also be performed by filtration to remove or select specific cell types based on size. Cell selection by plastic adhesion (i.e., cells start in one chamber, unwanted cells adhere to the surface, and then desired cells still in suspension are moved to another chamber) can also be utilized.

[0106]

[0129] Ideally, cells should not be shaken or rotated during the expansion phase. Maintaining cells in a relatively stationary position during expansion was determined to aid overall cell generation and help provide the desired cellular 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 may 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, concentration is performed by centrifugation. In some embodiments, concentration is performed by filtration. In some embodiments, 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, a predetermined concentration is determined by the cell engineering system based on different parameters entered by the user, such as the number or volume of clinical or therapeutic doses 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 clinical or therapeutic doses 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 a predetermined concentration.

[0108]

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

[0109]

[0132] CAR T cell culture recovery. In some embodiments, concentrated T cell cultures (or other immune cell cultures) are recovered to appropriately produce chimeric antigen receptor (CAR) T cell cultures. In some embodiments, recovery includes stirring, fluid flow, and washing of CAR T cells. In some embodiments, recovery includes separation of cells from undesirable products, such as cellular waste, selective reagents such as 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 CAR T cells into one or more flasks, vials, or containers. In some embodiments, recovery includes resuspending CAR T cells in formulation reagents, such as solutions that stabilize CAR T cells for long-term storage. In some embodiments, recovery includes cryopreservation of CAR T cells.

[0110]

[0133] Further downstream processes. In some embodiments, CART cells undergo further downstream processing before therapeutic use in a patient. For example, cryopreserved CAR T cells may be filtered by sterile filtration to remove residual potential viral particles. After sterile filtration, CAR T cells may undergo at least one enrichment step before being packaged in one or more vials, flasks, vessels, or containers. Packaged CAR T cells may be subjected to quality assessment and / or quality control testing. In some embodiments, CART cells undergo minimal downstream processing before administration to a patient. For example, in some embodiments, 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 cell viability.

[0111]

[0134] Cell engineering system. In some embodiments, the methods described herein are carried out by a fully sealed cell engineering system 600 (see Figures 6A, 6B) which appropriately has 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 collectively referred to as the automated cell engineering system, cocoon, or 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 parameters for cell generation (e.g., starting cell number, type of medium, type of activation reagent, type of vector, number of cells, or amount to be produced, etc.). The cell engineering system can perform a method for generating genetically modified immune cell cultures, 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 warning). In some embodiments, the fully sealed cell engineering system includes a sterile cell culture chamber. In some embodiments, a fully sealed cell engineering system minimizes contamination of cell cultures by reducing their exposure to a non-sterile environment. In additional embodiments, a fully sealed cell engineering system minimizes contamination of cell cultures by reducing user handling of cells.

[0112]

[0135] As described herein, the cell engineering system appropriately includes cassette 602. Accordingly, in embodiments, what is provided herein is a cassette for use in an automated cell engineering system. As used herein, “cassette” means one or more chambers for carrying out various elements of the method described herein, and preferably also including one or more of the cell culture medium, activating reagents, vectors, etc., and most of the cell engineering system is self-contained, removable, and replaceable.

[0113]

[0136] Figure 6B shows an embodiment of cassette 602 according to the embodiments herein. In the embodiments, cassette 602 includes a low-temperature chamber 604 suitable for storing cell culture media, and a high-temperature chamber 606 for appropriately performing activation, transduction, and / or expansion of immunocellular cultures. Preferably, the high-temperature chamber 606 is separated from the low-temperature chamber 606 by a thermal barrier 1102 (see Figure 11B). As used herein, “low-temperature chamber” refers to a chamber appropriately maintained at below room temperature, more preferably about 4°C to about 8°C, for storing cell culture media and the like at refrigerated temperatures. The low-temperature chamber may include a bag or other holder for culture media containing about 1 L, about 2 L, about 3 L, about 4 L, or about 5 L of fluid. Additional culture media bags or other fluid sources may be connected to the outside of the cassette and connected to the cassette via an access port.

[0114]

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

[0115]

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

[0116]

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

[0117]

[0140] In exemplary embodiments, as shown in Figure 6D, the cell culture chamber 610 is a flat, non-flexible chamber that does not easily flex or bend (i.e., made of a substantially non-flexible material such as plastic). By using a non-flexible chamber, the cells can be kept substantially undisturbed. As shown in Figure 6E, the cell culture chamber 610 is oriented to allow the immunotherapy cell culture to spread across the bottom 612 of the cell culture chamber. As shown in Figure 6E, the cell culture chamber 610 is properly maintained in a position parallel to the floor or table, keeping the cell culture undisturbed and allowing the cell culture to spread across a wide area of ​​the bottom 612 of the cell culture chamber. In embodiments, the overall thickness of the cell culture chamber 610 (i.e., the height of the chamber 642) is thin, on the order of about 0.5 cm to about 5 cm. Preferably, the cell culture chamber has a volume of about 0.50 ml to about 300 ml, more preferably about 50 ml to about 200 ml, or the cell culture chamber has a volume of about 180 ml. Using a low chamber height of 642 (less than 5 cm, preferably less than 4 cm, less than 3 cm, or less than 2 cm) allows for effective medium and gas exchange in close proximity to the cells. The ports are configured to allow mixing via fluid recirculation without disturbing the cells. Higher height static vessels may create concentration gradients, and the area near the cells is restricted in terms of oxygen and fresh nutrients. Controlled hydrodynamics allow for medium exchange without disturbing the cells. The medium can be removed from the additional chamber without the risk of cell loss (cells are not present).

[0118]

[0141] As described herein, in exemplary embodiments, the cassette is pre-filled with one or more of the following, including any combination: cell cultures, culture media, activating reagents, and / or vectors. In further embodiments, these various elements may be added later via a suitable injection port or the like.

[0119]

[0142] As described herein, in embodiments, the cassette may further appropriately include one or more of the following: 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 may also include one or more sampling ports and / or injection ports. An example of such sampling ports and injection ports (1104) is shown in Figure 11. This may include an access port for connecting the cartridge to an external device such as an electroporation unit or an additional culture medium source. Figure 11A also shows the arrangement of a cell input 1105, a reagent warming bag 1106 which can be used to warm cell media, etc., and a culture zone 1107 which holds various components for use in the culture medium, such as cell media, vectors, nutrients, and waste products.

[0120]

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

[0121]

[0144] In an exemplary embodiment, as shown in Figure 6F, the cell culture chamber 610 further includes at least one of the following: a distal port 620 configured to allow for the removal of bubbles from the cell culture chamber and / or to function 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 an outlet port for cell removal.

[0122]

[0145] In further embodiments, this specification provides a cassette 602 for use in an automated cell engineering system 600, comprising a cell culture chamber 610 for performing activation, transduction and / or expansion of an immune cell culture having a chamber volume configured to contain the immune cell culture, and a satellite volume 630 for increasing the working volume of the cell culture chamber by providing an additional volume of culture medium and other working fluids without containing the immune cell culture (i.e., the satellite volume does not contain any cells). Preferably, the satellite volume is fluidly connected to the cell culture chamber so that the culture medium is exchanged with the culture chamber without disturbing the immune cell culture. In exemplary embodiments, the satellite volume is a bag, and in other embodiments, the satellite volume is a non-yielding chamber. In embodiments, the satellite volume is between approximately 0.50 ml and approximately 300 ml, more preferably between approximately 50 ml and approximately 200 ml. Figures 6D–6E show the location of the satellite volume 630 in cassette 602.

[0123]

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

[0124]

[0147] Figure 6H shows gas exchange data using the Cocoon system compared to conventional bags.

[0125]

[0148] In this embodiment, the satellite volume 630 is further configured to allow the removal of the culture medium without loss of cells in the immune cell culture. That is, the exchange of the culture medium between the satellite volume and the cell culture chamber is carried out in a manner that does not disturb the cells or remove them from the cell culture chamber.

[0126]

[0149] In additional embodiments, as shown in Figure 6G, the cassette 602 may optionally further include a cross-flow reservoir 632 for holding additional culture medium. Preferably, the cross-flow reservoir has a volume between about 0.50 ml and about 300 ml, more preferably between about 100 ml and about 150 ml.

[0127]

[0150] The cell engineering systems described herein appropriately have three related volumes: cell culture chamber volume, working volume, and total volume. Preferably, the working volume used in the cassette ranges from 180 mL to 460 mL based on the process step and can be increased to a maximum of approximately 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1 L. In embodiments, the cassette is 4 × 10 9 cells ~10×10 9 Cells can be easily achieved. The cell concentration during the process is 0.3 × 10⁻⁶. 6 cells / ml ~ approx. 10×10 6 The volume changes to cells / ml. As described herein, the cells are placed in a cell culture chamber, but the culture medium is continuously recirculated through additional chambers (e.g., cross-flow reservoirs and satellite volumes) to increase the working volume.

[0128]

[0151] Unlike flexible bags, as described herein, which change shape when filled with liquid (e.g., cell cultures) and picked up or moved, a "substantially low yield chamber" (e.g., exemplary cell culture chamber 610) does not change shape (e.g., bend, curve, deform, etc.) when filled with liquid, picked up or moved under typical handling conditions. Therefore, in some embodiments, a substantially low yield chamber allows cells to remain substantially in the same area of ​​the chamber even when the chamber is picked up or moved. A substantially low yield chamber also does not have the curvature associated with bags. Therefore, in some embodiments, cells are distributed more uniformly in a substantially low yield chamber compared to a bag. In some embodiments, activating reagents and / or vectors are distributed more uniformly in a substantially low yield chamber compared to a bag.

[0129]

[0152] In some embodiments, the cell engineering system includes multiple chambers. In further embodiments, the activation, transduction, expansion, enrichment, and harvesting steps of the cell method described herein are performed in different chambers of the multiple chambers of the cell engineering system. In some embodiments, the cells are substantially undisturbed during transfer 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 kept undisturbed during the 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 may be made from a gas-permeable material such as silicone. In some embodiments, the gas permeability coefficient of the gas exchange line is higher than that of the material used in a flexible gas-permeable bag. In some embodiments, the cell engineering system recirculates oxygen throughout a substantially non-yielding chamber during the cell generation method. Therefore, in some embodiments, the oxygen level of the cell culture in the cell engineering system is higher than that of the cell culture in a flexible gas-permeable bag. Since an increased oxygen level may support increased cell growth and proliferation, it may be important to increase the oxygen level in the cell culture expansion step.

[0131]

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

[0132]

[0155] In some embodiments, the cell engineering system supplies 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 supplied 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 supplied. The desired CO2 level of the cell culture may be, for example, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% CO2, and may be defined by the user. Because the cell engineering system constantly adjusts the amount of CO2 supplied based on the measured CO2 level of the cell culture, the cell engineering system can maintain the desired CO2 level throughout the generation process. Since dissolved CO2 generally acidifies solutions (by reacting with water to form carbonic acid), the amount of CO2 in a 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 some embodiments, the pH level of the cell culture remains substantially constant throughout the production process. In further embodiments, the pH level of the transduced cell culture remains substantially constant throughout the expansion step.

[0133]

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

[0134]

[0157] Improved activation efficiency can also lead to greater vector transduction efficiency. When cells are activated and actively dividing, vectors (e.g., lentiviral vectors) can be incorporated into the cells more effectively. Uniform distribution of cells within the cell culture chamber 610 can promote uniform exposure of cells to the vector, whereas cells may be unevenly distributed and therefore receive different vector exposures within a flexible cell culture bag. Accordingly, in some embodiments, the transduction efficiency of the methods for the automated generation of genetically modified immune cells, including CAR T cells, as described herein, is 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 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% greater than the transduction efficiency of the methods utilizing flexible gas-permeable bags.

[0135]

[0158] Growth conditions in cell cultures can also improve cell yield. For example, higher oxygen levels in a cell engineering system, facilitated by high gas permeability tubing and continuous oxygen recirculation within the cell culture chamber, can increase cell proliferation. The ability of a cell engineering system to constantly monitor the state of the cell culture and adjust accordingly can also be advantageous. For example, a cell engineering system can monitor and adjust the CO2, O2, N2, and / or pH levels of the cell culture. Nutrients can also be supplied in a timely and consistent manner and uniformly distributed to the cell culture. Thus, the automated method for generating genetically modified immune cells, including CAR T cells, described herein, advantageously results in higher cell yields compared to manual methods or methods utilizing flexible culture bags. Therefore, in some embodiments, a method for the automated generation of genetically modified immune cells, including CAR T cells, utilizing the cell engineering system described herein, produces 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 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% more cells than a method utilizing a flexible, gas-permeable bag for cell culture. In embodiments, the number of cells produced by the method described herein includes 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 cells, with at least about 2 billion (i.e., 2 × 10⁶) cells. 9 ) These are cells.

[0136] Additional exemplary embodiments

[0159] Embodiment 1 is a method for the automated production of genetically modified immune cell cultures, the method comprising: activating an immune cell culture using an activating reagent to produce an activated immune cell culture; transducing the activated immune cell culture using a vector to produce 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 produce a genetically modified immune cell culture, further comprising washing either or both of the expanded immune cell culture and the concentrated immune cell culture, the steps being carried out by a fully sealed cell engineering system, and the steps being optimized through a process for producing 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 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 based on the monitoring.

[0138]

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

[0139]

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

[0140]

[0163] Embodiment 5 includes the methods of Embodiments 1 to 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 comprises 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 comprises the methods of Embodiments 1 to 8, wherein the immune cell culture includes at least one accessory cell.

[0145]

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

[0146]

[0169] Embodiment 11 comprises the method of Embodiment 9, wherein the accessory cells contain antigens for T cell receptors, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0147]

[0170] Embodiment 12 includes the methods of Embodiments 1 to 11, wherein the activating 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 a bead.

[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 anti-CD3 antibody and an anti-CD28 antibody.

[0152]

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

[0153]

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

[0154]

[0177] Embodiment 19 includes the methods of Embodiments 1 to 18, 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.

[0155]

[0178] Embodiment 20 includes the methods of Embodiments 1 to 19, further comprising at least one or more of the supply, washing, and monitoring of transduced immune cell cultures.

[0156]

[0179] Embodiment 21 includes the methods of Embodiments 2 to 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 to 21, wherein the cell engineering system recirculates the cell culture medium through an oxygenation component during one or more of steps (a) to (e).

[0158]

[0181] Embodiment 23 includes the methods 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).

[0159]

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

[0160]

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

[0161]

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

[0162]

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

[0163]

[0186] Embodiment 28 includes the methods of Embodiments 1 to 27, 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.

[0164]

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

[0165]

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

[0166]

[0189] Embodiment 31 is a method for promoting a preferred phenotype of a genetically modified immune cell culture, the method comprising the steps of: activating the immune cell culture using 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 using a vector to produce 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 produce a genetically modified immune cell culture, the steps of which are carried out by a fully sealed automated cell engineering system.

[0167]

[0190] Embodiment 32 includes the method of Embodiment 31, wherein the activating reagent comprises an antibody or dendritic cells.

[0168]

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

[0169]

[0192] Embodiment 34 includes the method of Embodiment 33, wherein the surface is the surface of a bead.

[0170]

[0193] Embodiment 35 includes the method of Embodiment 32, wherein the antibody is a soluble antibody.

[0171]

[0194] Embodiment 36 includes the methods of Embodiments 32 to 35, wherein the antibody comprises 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, wherein the soluble antibody is OKT3.

[0173]

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

[0174]

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

[0175]

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

[0176]

[0199] Embodiment 41 includes the methods of Embodiments 31 to 40, wherein the immune cell culture is a T cell culture.

[0177]

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

[0178]

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

[0179]

[0202] Embodiment 44 includes the methods of Embodiments 31 to 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 to 44, wherein the cell culture comprises at least one accessory cell.

[0181]

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

[0182]

[0205] Embodiment 47 comprises the method of Embodiment 45, wherein the accessory cells contain antigens for T cell receptors, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0183]

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

[0184]

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

[0185]

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

[0186]

[0209] Embodiment 51 includes the methods 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.

[0187]

[0210] Embodiment 52 includes the methods of Embodiments 31 to 51, further comprising supplying, washing, and monitoring transduced immune cell cultures.

[0188]

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

[0189]

[0212] Embodiment 54 includes the methods of Embodiments 31-53, wherein the cell engineering system recirculates the cell culture medium through an oxygenation 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 levels provided by the cell engineering system are reduced 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 supplying, washing, monitoring, and selecting transduced immune cell cultures.

[0193]

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

[0194]

[0217] Embodiment 59 includes the methods 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.

[0195]

[0218] Embodiment 60 includes the methods of Embodiments 31 to 59, further comprising removing the activating 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 transduction in (b).

[0197]

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

[0198]

[0221] Embodiment 63 is a method for the automated production of genetically modified immune cell cultures, the method comprising: activating an immune cell culture using an activating reagent to produce an activated immune cell culture; transducing the activated immune cell culture using a vector to produce 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 produce a genetically modified immune cell culture, the steps being performed by a fully sealed automated cell engineering system, and each step being optimized for an optimized cell density (cells / mL) and optimized cell density (cells / cm²). 2 This procedure is performed using an immunocellular culture containing )

[0199]

[0222] Embodiment 64 has an optimized cell density of approximately 0.05 × 10⁻⁶ in (a). 6 cells / mL~about 60×10 6 The method of Embodiment 63 includes a cell / mL ratio.

[0200]

[0223] Embodiment 65 has an optimized cell density of approximately 0.1 × 10⁻⁶ in (a). 6 cells / cm 2 ~Approx. 60×10 6 cells / cm 2 This includes the method of embodiment 63 or 64.

[0201]

[0224] Embodiment 66 includes the methods of Embodiments 63 to 65, wherein the activating reagent comprises 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 methods 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 methods of Embodiments 63 - 70 that generate at least about 100 million viable genetically modified immune cells.

[0207]

[0230] Embodiment 72 includes the methods of Embodiments 63 - 71 that generate at least about 2 billion viable genetically modified immune cells.

[0208]

[0231] Embodiment 73 includes the methods 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 methods 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 methods 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 comprises the method of Embodiment 77, wherein the accessory cells contain antigens for T cell receptors, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0215]

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

[0216]

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

[0217]

[0240] Embodiment 82 includes the methods of Embodiments 63 to 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.

[0218]

[0241] Embodiment 83 includes the methods of Embodiments 63-82, further comprising supplying, washing, monitoring, and selecting transduced immune cell cultures.

[0219]

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

[0220]

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

[0221]

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

[0222]

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

[0223]

[0246] Embodiment 88 includes the methods of Embodiments 63-87, in which 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, in which 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

[0225]

[0248] Embodiment 90 includes the methods of Embodiments 63-89, in which 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, in which concentration includes centrifugation, removal of the supernatant after sedimentation, or filtration.

[0227]

[0250] Embodiment 92 includes the methods of Embodiments 63-91, in which 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, which further includes removing the activation reagent from the activated immune cell culture after step (a).

[0229] ​

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

[0230]

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

[0231]

[0254] Embodiment 96 is a method for the automated production of genetically modified immune cell cultures, the method comprising: activating an immune cell culture using an activating reagent to produce an activated immune cell culture; transducing the activated immune cell culture using a vector to produce a transduced immune cell culture; expanding the transduced immune cell culture; the transduced cell culture not being shaken during expansion; concentrating the expanded immune cell culture; and recovering the concentrated immune cell culture to produce a genetically modified immune cell culture, the steps of which are carried out by a fully sealed automated cell engineering system.

[0232]

[0255] Embodiment 97 includes the method of Embodiment 96, wherein the activating reagent comprises 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 a bead.

[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 comprises at least one of an anti-CD3 antibody, an anti-CD28 antibody, and an anti-CD2 antibody.

[0237]

[0260] Embodiment 102 includes the methods of Embodiments 96-101 for generating at least about 100 million viable, genetically modified immune cells.

[0238]

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

[0239]

[0262] Embodiment 104 includes the methods of Embodiments 96-103, wherein the immune cell culture is a T cell culture.

[0240]

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

[0241]

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

[0242]

[0265] Embodiment 107 comprises the methods of Embodiments 96-106, wherein the immune cell culture comprises peripheral blood mononuclear cells and / or purified T cells.

[0243]

[0266] Embodiment 108 includes the methods of Embodiments 96-107, wherein the cell culture comprises at least one accessory cell.

[0244]

[0267] Embodiment 109 includes the method of Embodiment 108, wherein the accessory cells include monocytes or monocyte-derived cells.

[0245]

[0268] Embodiment 110 includes the method of Embodiment 109, wherein the accessory cells contain antigens for T cell receptors, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0246]

[0269] Embodiment 111 includes the methods of Embodiments 96 to 110, wherein the transduction includes viral infection, electroporation, membrane disruption, or a combination thereof.

[0247]

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

[0248]

[0271] Embodiment 113 includes the methods of Embodiments 96-112, 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.

[0249]

[0272] Embodiment 114 includes the methods of Embodiments 96-113, wherein the expansion includes supplying, washing, monitoring, and selecting transduced immune cell cultures without shaking the immune cell cultures.

[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 oxygenation 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 levels provided by the cell engineering system are reduced 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 transduced immunocell cultures.

[0255]

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

[0256]

[0279] Embodiment 121 includes the methods of Embodiments 96-120, 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.

[0257]

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

[0258]

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

[0259]

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

[0260]

[0283] Embodiment 125 is a method for the automated production of genetically modified immune cell cultures, the method, performed by a cell engineering system, includes activating an immune cell culture using an activating reagent to produce an activated immune cell culture in a first chamber of the cell engineering system; transducing the activated immune cell culture, the transduction of which 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 produce 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, and recovering the concentrated immune cell culture to produce a genetically modified cell culture.

[0261]

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

[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 for generating at least about 100 million viable, genetically modified immune cells.

[0264]

[0287] Embodiment 129 includes the method of Embodiment 128 for generating 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 comprises peripheral blood mononuclear cells and / or purified T cells.

[0269]

[0292] Embodiment 134 includes the methods of Embodiments 125-132, wherein the cell culture comprises 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 comprises the method of Embodiment 134, wherein the accessory cells contain antigens for T cell receptors, including CD28, CD40, CD40L, and / or ICOS.

[0272]

[0295] Embodiment 137 includes the methods of Embodiments 125-136, wherein the activating reagent comprises 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 a bead.

[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 comprises 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 to 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 transduced immune cell cultures.

[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 methods of Embodiments 125-144, wherein the cell engineering system recirculates the cell culture medium through an oxygenation 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 levels provided by the cell engineering system are reduced during step (c).

[0283]

[0306] Embodiment 148 includes the methods of Embodiments 125-147, wherein the cell engineering system is configured to perform multiple rounds of supplying, washing, monitoring, and selecting transduced immunocell cultures.

[0284]

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

[0285]

[0308] Embodiment 150 includes the methods of Embodiments 125-149, 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.

[0286]

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

[0287]

[0310] Embodiment 152 includes the methods of Embodiments 125 to 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 comprises (a) the cell culture, activating reagent, vector, and cell culture medium before initiating the method.

[0289]

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

[0290]

[0313] Embodiment 155 includes the methods of Embodiments 1 to 154, which generate at least 20% more genetically modified immune cells than methods utilizing manual cell culture with flexible, gas-permeable bags.

[0291]

[0314] Embodiment 156 comprises the methods of Embodiments 1 to 155, wherein the cell engineering system comprises a plurality of chambers, each of steps (a) to (e) is performed in a different chamber of the plurality of chambers of the cell engineering system, and in each of (a), the activating reagent, vector, and cell culture medium are placed in a different chamber of the plurality of chambers before the method is started, at least one of the plurality of chambers is maintained at a temperature for cell growth, and at least one of the plurality of chambers is maintained at a refrigerated temperature.

[0292]

[0315] Embodiment 157 is a cassette for use in an automated cell engineering system, comprising a low-temperature chamber for storing cell culture media, a high-temperature chamber for activating, transducing, and expanding immune cell cultures, 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 fluid pathways connected to the cell culture chamber, the fluid pathways providing recirculation, waste removal, uniform gas exchange, and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber.

[0293]

[0316] Embodiment 158 ​​includes the cassette of Embodiment 157, wherein the cell culture chamber is a flat, non-flexible chamber with a low chamber height.

[0294]

[0317] Embodiment 159 includes the cassette of Embodiment 157 or Embodiment 158, wherein the cell culture chamber is oriented to allow the immune cell culture to 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 cell cultures, culture medium, activation reagents, and vectors.

[0296]

[0319] Embodiment 161 includes a cassette of Embodiments 157-160, 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.

[0297]

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

[0298]

[0321] Embodiment 163 includes a cassette of Embodiments 157-162, further comprising a cell culture chamber comprising at least one of a distal port configured to allow for the removal of bubbles from the cell culture chamber and / or as a recirculation port, an intermediate port configured to function as a recirculation inlet port, and a proximal port configured to function as an outlet port for cell removal.

[0299]

[0322] Embodiment 164 includes a cassette of embodiments 157-163, further including an access port for connecting the cartridge to an external device.

[0300]

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

[0301]

[0324] Embodiment 166 is a cassette for use in an automated cell engineering system, comprising a cell culture chamber for activating, transducing, and / or expanding an immune cell culture, having a chamber volume configured to contain an immune cell culture, and a satellite volume for increasing the working volume of the chamber by providing an additional volume of culture 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 so that the culture 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 allow removal of the culture medium without loss of cells in the immune cell culture.

[0305]

[0328] Embodiment 170 includes the cassettes of embodiments 166-169, further including a cross-flow reservoir.

[0306]

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

[0307]

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

[0308]

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

[0309]

[0332] Embodiment 174 includes the cassettes of Embodiments 166 to 173, wherein the satellite volume is between approximately 0.50 ml and approximately 300 ml.

[0310]

[0333] Embodiment 175 includes the cassette of Embodiment 174, wherein the satellite volume is between approximately 150 ml and approximately 200 ml.

[0311]

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

[0312]

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

[0313]

[0336] Embodiment 178 includes the cassettes of Embodiments 166 to 177, with a working volume of approximately 180 mL to approximately 1 L.

[0314]

[0337] Embodiment 179 includes the cassette of Embodiment 178, with a working volume of approximately 180 mL to approximately 460 mL.

[0315]

[0338] Embodiment 180 includes the cassettes of Embodiments 157-179, wherein one or more fluid pathways include silicone-based tubular components that allow oxygenation through the tubular components. [Examples]

[0316] Example 1 - Automated generation of CAR T cells using the Cocoon System

[0339] In this example, T cells were transduced using GFP and HER-2 lentivirus with the following process parameters: initiation of inoculation of 60 million peripheral blood mononuclear cells (PBMCs), CD3 / CD28 activation, and supplementation of T cell proliferation medium with IL-2 and IL-7 for culture expansion. Temperature, pH, and optical density (OD) were monitored in real time using single-use sensors in disposable cassettes. Multiple cassette chambers connected via fluid channels enabled automated supply and addition of process components. Some chambers were temperature-controlled to 4°C for medium and reagent storage, while others contained elements for cell warming, mixing, washing, and concentration, allowing for a completely sealed process. Samples were prepared during processing 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. An overview of the cocoon system used in this example is shown in Figure 6. Figure 6A shows the Cocoon system in a closed configuration, along with an external user control display that can be used to adjust parameters or monitor cell culture. Sterile, single-use cell culture "cassettes" can be filled into the Cocoon (Figure 6C). As shown in a detailed diagram of the cassette (Figure 6B), each cassette includes an upper chamber maintained at 37°C for cell growth, and a lower chamber maintained at 4°C for storing culture media, viral vectors, and other temperature-responsive reagents. The cassette is configured so that fluids can be exchanged through 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, among other things.

[0317]

[0340] The results are shown in Figures 7-10. Figures 7A, 7B, and 7C show the average GFP transduction yield, average transduction viability, and average transduction efficiency using the automated cocoon system, compared to manual manipulation and expansion of cells using G-REX (WilsonWolf) cell culture plates as a control, respectively. G-REX plates have a gas-permeable bottom, and typically, users change the medium every 4-5 days when using G-REX.

[0318]

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

[0319]

[0342] Furthermore, the performance of the automated Cocoon system was compared to manual cell handling and proliferation using PermaLife cell culture bags (OriGen) as a control. PermaLife bags are sealable, gas-permeable cell culture bags made of inert fluorinated ethylene propylene (FEP) and equipped with valves to facilitate cell supply and retrieval by the user. Figure 9A shows the relative T cell purity levels using the Cocoon system compared to PermaLife bags, evaluated by the percentage of CD3+ cells. Figure 9B shows a higher percentage of CD8+ cells cultured in the Cocoon system compared to the PermaLife bag control. Figures 9C and 9D show that transfected cells produce TNF-α and INF-γ, respectively.

[0320]

[0343] Figures 10A and 10B demonstrate the effective and specific killing of target tumor cells by CAR T cells cultured in the Cocoon System and Permalife bags, respectively.

[0321]

[0344] In conclusion, the Cocoon System is a fully enclosed cell engineering system and a viable solution that transforms the labor-intensive CAR-T process into a fully automated and highly controlled system, thus enabling the production of high-quality CAR-T cells with improved scalability, high yield, reduced manufacturing costs, and enhanced process control.

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

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

[0323]

[0346] T cells can be activated using magnetic anti-CD3 / anti-CD28 DynaBeads Activator beads. These beads provide two stimulating signals necessary to support effective T cell activation. Another method for activating naive T cells is to utilize soluble anti-CD3 antibody (OKT3). OKT3 is a monoclonal IgG2a antibody originally used as an immunosuppressant. Co-stimulatory signals can be provided by accessory cells. When initiating T cell culture from a mixed population of peripheral blood mononuclear cells (PBMCs), OKT3 can provide the necessary accessory cells to support T cell activation.

[0324]

[0347] Because OKT3 and DynaBeads utilize distinct activation mechanisms, the choice of one method over the other can affect the characteristics 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 the antitumor response. CD4 cells produce cytokines and assist in regulating the immune response. Although killing is delayed compared to CD8 cells, CD4 cells have also been shown to support cytolysis. CD4 cells signal to APCs, thus activating APCs and subsequently primed naive CD8 T cells. Due to limited clinical data, the ideal target ratio of CD8 to CD4 cells is not well understood. Studies have shown that combinations of CD8 and CD4 cells are preferable to delivery of CD8 cells alone (see, e.g., Church, 2014; Feldmann, 2012; Reusch, 2015).

[0325]

[0348] Both in vitro activation methods have advantages and disadvantages. Antibody-conjugated beads provide consistency and ensure stable simultaneous activation of the TCR / CD3 complex and CD28 costimulatory pathway. The main disadvantage of the bead approach is the high cost associated with this product. Beads may also need to be effectively removed from the culture before transplantation. OKT3 offers a low-cost option for activating T cells. The main disadvantages associated with the soluble anti-CD3 approach are its dependence on accessory cells and sensitivity to the culture environment. Patient samples may have a wide variety of accessory cells and negative interactions that can functionally inactivate T cells after prior stimulation. To understand the effects 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 a 37°C zone as well as a linked refrigerated zone. Because there is no fluid contact between the cocoon and the cassette, the cleaning required between runs is minimized. All reagents can be filled into the cassette on the seeding day and stored in the cocoon's refrigerated zone 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 transduction day and delivered to the cassette via a sterile connector. Gas exchange (oxygenation and CO2 buffering) is achieved via recirculation of the culture medium through gas-permeable tubing. An embedded biosensor provided real-time data on dissolved oxygen and pH. Because 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. Locking can be used to facilitate efficient recovery.

[0327] method

[0350] Cell culture. Peripheral blood mononuclear cells (PBMCs) (Lonza) were thawed with DNase (Sigma) and cultured at 37°C for <2 × 10⁻⁶ days. 6Cells were restored overnight at a density of cells / mL. Cell counting was performed using a NUCLEOCOUNTER 200 equipped with a Blood Assay protocol including Solution 17 (Chemometec). Cells were transduced using a third-generation lentiviral vector encoded with low affinity neuron growth factor receptor (NGFR) as a marker of transduction. This lentivirus was manufactured at the cGMP virus manufacturing facility in Lonza (Houston, Texas) based on protocols and primers from the Bramson Lab at McMaster University (Hamilton, Canada). A multiple of infection (MOI) of 1 was used under all conditions. Viral titers were determined using HEK293™ cells and by detecting NGFR using flow cytometry. The activation medium consisted of X-VIVO 15 (Lonza) supplemented with 22 IU / mL of IL-2 (Cedarlane) and 1% penicillin-streptomycin (Sigma). Under conditions activated with soluble anti-CD3, OKT3 (Biolegend) was added to the activated medium at a final concentration of 50 ng / mL. Under conditions activated with DynaBeads, beads and cells were added to the activated medium in a 1:1 ratio. The expansion medium consisted of X-VIVO 15 (Lonza) supplemented with 29 IU / mL of IL-2 (Cedarlane), 5% human serum derived 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 x 10 6 The PBMC was placed in the cassette's input bag. Also, under conditions activated using anti-CD3 / anti-CD28 beads, 60 × 10 6Anti-CD3 / anti-CD28 DynaBeads (ThermoFisher) were added to the cells in a 1:1 bead:cell ratio in an ingestion bag. The ingestion bag was connected to a cassette and brought into the cocoon (Octane Biotech Inc.). Following operator sign-in, the cassette was filled into 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 a MOI of 1. The medium was diluted with activated medium before delivering the virus to the cells. The activated medium was removed from the culture chamber and returned with the virus without disturbing the cells. On day 4, expansion medium was added, increasing the total working volume. Partial medium changes 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 all pump and actuator steps, such as each time the door was opened and closed. Comprehensive sensor data, including thermal values, gas concentrations, fluid pH, and dissolved oxygen, was collected. Operators could remotely monitor the culture status using a telephone or external computer.

[0329]

[0352] Manual CAR T cell generation. Manual generation of CAR T cells was performed in Permalife cell culture bags in parallel with the cocoon method. On day 0, 60 × 10⁶ cells were generated. 6 Place the PBMC in the activated medium in a 0.27 × 10⁶ medium. 6 Cells were seeded on day 0 at a rate of cells / mL. These cultures utilized the same donor cells and the same medium for activation and expansion as the automated cultures. Cultures were started in permalife bags (PL240, Origen) and transferred to larger permalife bags (PL325, Origen) on day 6 as the cells expanded. As the volume increased, cells were expanded into PL240 and PL325 bags on day 8. Lentivirus was added to the bags at an MOI of 1 on day 1. Cells were supplied in volumes equivalent to those used in cocoon cultures. However, unlike cocoon conditions, no medium was sent to waste. The volume used was 2 × 10⁶ of culture. 6The cell volume was maintained below cells / mL. On day 10, 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 residue and volume.

[0330]

[0353] Non-transduced and inactivated conditions. Negative controls for non-transduced and inactivated cells used in fluorescence-activated cell sorting (FACS) analysis were cultured on a small scale according to previously described protocols. Briefly, 1 × 10⁶ cells were cultured in a 96-well plate containing X-VIVO 15 (Lonza) supplemented with 5% human AB serum (Sigma) and 22 ng / mL of IL-2 (Cedarlane). 5 Cells were seeded. Activated but untransduced controls were set up using a similar protocol. After seeding, an equal volume of medium was added. Conditions activated with soluble anti-CD3 were supplemented with 100 ng / mL OKT3 (Biolegend) to a final concentration of 50 ng / mL. Conditions activated with anti-CD3 / anti-CD28 beads were supplemented with DynaBeads in a 1:1 ratio. Activated cultures were expanded from 96-well plates to 24-well plates on day 4, and based on their growth, transferred to T25 and T75 flasks and supplied every two days from day 4.

[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 fixed violet dead cell staining (ThermoFisher). To evaluate the efficiency of HER2 transduction, cells were stained as described above, except as a substitute for monocyte (CD14) staining, and cells were stained with BV421 CD271 (C40-1457 NGFR, BD Biosciences) and LIVE / DEAD fixed green dead cell staining (ThermoFisher). Next, the cells were fixed and washed. Over 20,000 events were acquired for each condition using a SA3800 Sony Spectral Analyzer. FACS analysis was performed using FlowJo 10.4.2. Gating was set up with a fluorescence minus 1 (FMO) control using non-transduction and inactivation conditions.

[0332]

[0355] Tumor cell lines. LOX-IMVI cells (National Cancer Institute), HER2-negative tumor cells, were derived from metastatic melanoma and expanded in RPMI (Sigma) containing 10% FBS (Sigma) as described above. SKOV-3 (ATCC) cells, HER2-positive tumor cells, were derived from ovarian serous cystadenoma and 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 passages were cryopreserved, and tumor lines were passaged 2-3 times before use in ALAMARBLUE or ICS assays.

[0333]

[0356] Cytokine secretion assay. As previously described (e.g., Atkuri, 2005; Avgoustiniatos, 2008), 50,000 LOX IMVI or SKOV-3 tumor cells were seeded in triples in round-bottom 96-well plates under each culture condition. The following day, T cells were seeded at 37°C for 4 hours with the protein transport inhibitor brefelzin A (Golgi Plug, BD Biosciences) at a ratio of 8:1 per well of tumor cell line. Cells were stored overnight at 4°C. Next, cells were pooled for staining and analysis. As described above, cells were stained with surface phenotype CD3, CD4, CD8a, NGFR, and LIVE / DEAD fixed green dead cell stain. Intracellular cytokine staining (ICS) was completed after fixation and permeabilization using the BD Cytofix / Cytoperm immobilization / permeabilization solution kit (554714, BD Biosciences). The activated cytokines tested included APC IFNγ (clone B27, BD Biosciences) and PETNFα (clone MAb11, BD Biosciences). Over 230,000 events (up to 500,000) were recovered for ICS analysis using the Sony SA3800. Differences in cytokine production between SKOV-3 and LOX-IMVI tumor lines were reported as the proportion of populations secreting TNFα or IFNγ. Non-transduction and inactivation conditions were used to set up gates along with FMO control.

[0334]

[0357] Cytotoxicity assay. Cytotoxicity was tested as described above (e.g., Atkuri, 2005; Avgoustiniatos, 2008). Adherent tumor cell lines were placed in 2 × 10⁶ wells of 96-well flat-bottom tissue culture treatment plates. 4Cells were plated overnight in cells / well (SKOV-3 or LOX-IMVI). CAR T cells from cocoon and control conditions were added to tumor cell wells in 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 non-adherent cells in both cases. 100 μL of a 10% solution of ALAMARBLUE cell viability reagent (Life Technologies) was added, and the wells were incubated at 37°C for 3 hours. ALAMARBLUE, a metabolic indicator of viable cells that fluoresces upon mitochondrial reduction, 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 the experimental well compared to untreated target cells. Each condition was tested in a triple-layered manner.

[0335] result

[0358] Using the automated platform Cocoon, we demonstrated the feasibility of achieving clinical-scale generation of CAR T cells using two different activation methods. The platform consists of single-use, disposable Cocoon cassettes (Figures 11A, 11E) and the Cocoon control system (Figure 11B). Figure 11F shows how syringe 1170 or bag 1172 is used for cassette 602 sampling. The cassette is designed with multiple reagent bags so that all reagents required for the process can be pre-filled and stored in the cassette's refrigeration zone and then processed in the culture zone. The cassette supports multiple linked unit operations as a closed system, including cell activation, transduction, expansion, real-time dissolved oxygen and pH monitoring, washing, and cell concentration. The bottom of the cassette contains multiple bags for holding various reagents and wastes required for culture. Cocoon provides a control system for the cassette, which includes control of fluid and cell movement, as well as oscillation, agitation, and remote monitoring of control sensors. Actuators enable automated valve control without contact with the fluid. Without actuator interaction, the valve remains closed, preventing uncontrolled fluid movement while allowing the cassette to be moved between rooms or to a microscope. After filling the cassette's fluid reservoir with the necessary reagents, it is snapped into the culture zone of the cassette where various unit operations are performed. For the removal or injection of sterile viral samples, the ICU Spiros connector is used. Prior to sample removal or virus injection, the operator proceeds at a specific time as defined in a pre-programmed protocol. After the operator signs in and confirms notifications, the cocoon automatically opens, allowing for sample removal or virus injection. The operator recognizes that the action is complete before the door automatically closes and environmental control is resumed. Once the cassette is filled into the cocoon (Figure 11C) and the outer shell is closed (Figure 11D), the lower part of the cassette is separated from the upper part by a thermal barrier. The lower part is maintained at refrigerated temperature, while the upper part is maintained at 37°C. The closed cocoon allows for gas and heat control. The cells are maintained at 37°C, and the reagents are kept in the cold zone to extend their stability.The opaque shell prevents photo-induced toxicity associated with the degradation of culture medium components. A preheating chamber is located in a 37°C zone to warm the medium before transferring it to cells. All culture steps, from PBMC filling to final concentration and cell harvesting, can be automated. As shown in Figure 11A, the cassette has a series of access ports that can be used to fill with virus after activation. Real-time dissolved oxygen and pH sensors are integrated into the cassette and provide feedback to the Cocoon software. Real-time data and historical graphs can be monitored to ensure that these factors are maintained within 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 culture and CAR T cell expansion (e.g., Lu, 2016). Figures 12B (cocoon) and 12C (permalife bag) demonstrate two forms of cell distribution, including cells in a cocoon cultured in the upper chamber of the cassette. Equivalent volumes of culture medium were used in both systems. The permalife cell culture bags utilized a fed-batch culture process, where the area expands as the total volume increases, as is commonly done. The cocoon cassette utilized a fixed area and employed a feeding strategy for the initial fed-batch culture, with partial medium changes on days 6 and 8 of culture.

[0337]

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

[0338]

[0361] Unless otherwise instructed, the same donor cells were used for Donor 2 under all conditions. All conditions were 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 variability. 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 was over 95% under all conditions (Figure 13A). The population doubling levels (PDL) 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 while 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 times) 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 times).

[0341]

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

[0342]

[0365] High transduction efficiency was determined by the expression of the surrogate surface marker CD271 (NGFR) for T cell HER2 specificity, which was present in 62–78% of CD3+ cells in the cocoon and 42–60% of CD3+ cells in the permalife bags expressing NGFR (Figure 14A). Transduction efficiency was greater in the cocoon compared to bag culture. Due to high transduction and expansion, the total number of viable CAR T cells was 1.26–1.66 × 10⁶ in the cocoon. 9 In Permalife bags, 0.62~1.20×10 9The range was (Figure 14B). The percentage and total number 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 NGFR-expressing cocoons, which was greater than that of CD8 cells. In permalife bags, 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 DynaBeads-activated bag culture (Figure 14D). In cocoons, the range was 0.25–0.64 × 10⁻⁶. 9 Transduced CD4 cells and 0.66~1.43 × 10 9 Transduced CD8 cells were present. Under permalife bag conditions, the density was 0.09–0.41 × 10⁻⁶. 9 Transduced CD4 cells and 0.25~1.06 × 10 9 Transduced CD8 cells were present. Representative contour plots of transduction efficiency under cocoon conditions and permalife bag conditions are shown in Figures 14E and 14F, respectively.

[0343]

[0366] Functional testing of the cells was performed using intracellular cytokine release assays and the ALAMARBLUE killing assay (see Nociari, 1998) (Figure 15). In all cases, the cells demonstrated the production of TNFα and IFNγ characteristic of type 1 T helper CD4+ cells and cytotoxic CD8+ cells (Figures 15A and 15B) (see, e.g., Romagnani, 1991). A higher proportion of CD4+ cells secreted TNFα. The generation of TNFα-secreting cells was greater under cocoon conditions compared to bag cultures of the same donor cells. DynaBeads activation conditions generated TNFα and IFNγ-secreting transdermal cells at a higher rate than OKT3 activation conditions. The ALAMARBLUE killing assay demonstrated effective killing of ovarian cancer cell line SKOV-3 HER2+ tumor cells by CAR T cells (Figures 15C and 15D). The killing trend was observed following serial dilution of effector T cells, with strong responses from both permalife and cocoon-generating cells. HER2 tumor cells, LOX IMVI, were also exposed to T cells and demonstrated HER2 specificity. No killing trend was observed in HER2-negative cultures responding to CAR T cells.

[0344] Consider

[0367] Activation methods. Evaluation of CAR T cell production included activation using soluble anti-CD3 (OKT3) and bead-bound anti-CD3 / anti-CD28 DynaBeads. Cultures activated with OKT3 demonstrated 19–36% improved proliferation compared to cultures activated with DynaBeads (Figure 13A). The activation method also produced significant differences in the final phenotype (Figure 13E). Under DynaBeads activation, the average CD3+CD8+ cell ratio was 52.7%, compared to 84.5% under OKT3 activation. This indicates that the CD8+ to CD4+ ratio was approximately 1.2:1 under DynaBeads activation, compared to 9.8:1 under OKT3 activation. The increase in CD8+ cell count was observed regardless of whether the cells were cultured under bag or cocoon conditions.

[0345]

[0368] The improved yield due to OKT3 activation was an unexpected result. DynaBeads activate T cells by binding to the TCR / CD3 complex and the CD28 costimulatory receptor. Unlike DynaBeads with anti-CD28 antibodies for costimulation, activation by soluble anti-CD3 is monocyte-dependent, presenting the B7 receptor, 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, thus inhibiting T cell proliferation. The improved total cell yield based on the activation method was found regardless of whether the cells were cultured under bag or cocoon conditions. Bead-conjugated anti-CD3 / anti-CD28 antibodies may promote the expansion of helper T cells (CD4+ cells), and OKT3 may 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 dominance, may be due to the stimulation of additional receptors when activated using OKT3 and monocytes. It has been previously reported that 95% of CD4+ T cells express CD28, while only 50% of CD8 cells express CD28 (see Ledbetter, 1990). Consequently, DynaBeads can activate up to 50% of CD8+ cells. Cultures activated with OKT3 may benefit from other co-stimulatory ligands present in monocytes rather than the beads.

[0347]

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

[0348]

[0371] Since OKT3 activation is dependent on other cells, the influence of donor variability may be more important than activation by DynaBeads. The starting cell population for this study consisted of 12.0% CD14+ cells and 66.6% CD3+ cells on day 0. Dose studies were possible to investigate the effects of monocyte sensitivity on final yield and phenotype.

[0349]

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

[0350]

[0373] The improved yield of the Cocoon compared to Permalife bags may be due to increased activation. This could be attributable to the distribution across the culture area. The Cocoon utilizes a solid, non-yielding chamber, whereas the bags are flexible. After cell sedimentation, it was observed that the bending of the bags caused a heterogeneous distribution of cells. This may have caused a heterogeneous distribution of the activator and / or cells. Another possible cause may relate to the amount of agitation during the activation phase. Since the cells were transduced the day after activation, it is possible that activation was still in progress or that the activator was not taken up by the cells. During the transduction step, the bag cultures were moved from the incubator to a biosafety cabinet, and sterilization techniques were used to deliver the cells. Bag movement promotes viral distribution within the bag; however, cells are also disturbed during the transfer of bags from incubator to incubator. Since stable contact may be important for cell activation, this movement may have negatively affected the cells. Cells in the Cocoon cultures were not disturbed between the activation or transduction steps. In the Cocoon, the culture medium used for activation is removed from the culture before transduction. A small amount of medium remains in the chamber, allowing the cells to stay at the bottom and undisturbed during volume transfer. The medium removed from the chamber is used to dilute and mix the virus, and then transferred back into the cell population. The cells remain unaffected throughout this process.

[0351]

[0374] Efficient activation may correlate with more efficient transduction. That is, if cells are activated and actively dividing, lentiviruses may integrate more effectively. To assess this, samples can be taken before transduction to determine 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 culture medium and distributed uniformly to the cells. Using a flat, non-flexible container improves uniform distribution, which in turn helps with uniform exposure of the virus among the cell population.

[0352]

[0375] Another reason for the improved performance may be related to gas exchange. Increased oxygen levels may support increased growth. High oxygen levels were maintained on the automated platform by using recirculation of the culture supernatant via a silicone gas exchange line. Gas exchange is achieved in the bag state by diffusion through fluorinated ethylene propylene (FEP), the material of the bag. The permeability coefficient of silicone is significantly higher than that of FEP (see, e.g., Avgoustiniatos, 2008). The Cocoon protocol was designed to ensure sufficient oxygen concentration, which was confirmed by biosensor data generated throughout the culture period.

[0353]

[0376] Gas exchange via silicone tubing also supports pH levels. Specifically, at the start of culture, the medium maintains its target pH through gas exchange with a CO2-rich environment. As the number of cells increases during culture, the cells produce lactic acid and CO2, eliminating the need for a CO2 environment. The CO2 in the cocoon environment decreased during the culture period, helping to maintain pH. Permalife bags followed a conventional protocol, being stored in a 5% CO2 environment throughout the entire culture process.

[0354]

[0377] A further advantage of continuous recirculation, which does not disrupt cells, is the more uniform distribution of positive and negative factors. These include nutrients, waste products, released cytokines, and dissolved gases. Continuous recirculation helps reduce local effects and improve the efficiency of the culture medium by evenly distributing factors.

[0355]

[0378] Automated conversion. In this example, CAR T cells activated by either bead-conjugated antibodies or soluble OKT3 were generated using a closed, automated generation system called Cocoon. The results demonstrate that clinically relevant yields can be generated from Cocoon with high transduction efficiency using low concentrations of virus. Furthermore, the cell phenotype can be driven by the activation method.

[0356]

[0379] The results were primarily generated from a single donor to compare the effects of activation methods. Variability between conditions was very low. When the experiment was repeated with different donors, the results were similar across donors when the same activation method was used. This study demonstrates an efficient method for effectively automating CAR T cell generation in a clinically relevant, scalable, and user-friendly way.

[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. Octane's 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 nonviral methods, including primary cells, stem cells, neurons, and quiescent or non-proliferating cells. The system includes the electroporation unit, electroporation solution, electroporation cartridge, and an optimized electroporation protocol. The electroporation unit consists of a core unit and 1 to 3 additional functional add-on units to meet various needs. For example, the electroporation unit can be used to transfect various cell counts from 20 μL to 100 μL and 1 × 10⁶ cells in volumes from 1 mL to 20 mL. 7 ~1 × 10 9 Transfection can be performed.

[0359]

[0382] This specification describes automated, fully enclosed, sterile, and robust transfection and cell expansion procedures using an electroporation unit and an Octane Cocoon® system. In proof-of-concept (PoC) evaluations, the respective electroporation software and Octane Cocoon® ACTES software operate independently of each other. In other embodiments, the software is fully integrated between the systems. method

[0383] The evaluation of peripheral blood monocyte (PBMC) transfection and expansion using electroporation units and the Cocoon® system was divided into three main focal regions.

[0360]

[0384] Cell concentration in the Cocoon® cassette, cell movement between the Octane Cocoon® and the electroporation unit, expansion of transfected cells moved between the Cocoon® and the electroporation unit, and cell concentration within the Cocoon® cassette.

[0361]

[0385] The Cocoon® ACTES cassette recirculates approximately 450 mL of culture medium within its culture chamber. The cell proliferation chamber is typically 260 cm². 2 A fixed amount of culture medium, up to 180 mL, is maintained within this area. 260 cm 2 Additional culture medium volumes exceeding the 180 mL capacity of the growth chamber are supplied from various satellite reservoirs and chambers in the Cocoon® cassette. These additional culture media from satellite reservoirs are recycled within the disposable Cocoon® culture chamber to provide fresh nutrients and 260 cm³. 2 It can remove waste products from cells within the proliferation chamber.

[0362]

[0386] An exemplary volume that an electroporation unit can transfect is 20 mL. A volume of 20 mL must be properly composed of at least 90% of the appropriate electroporation solution. Therefore, 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 to 18 mL.

[0363]

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

[0388] A Nordson EFD 20-gauge, 0.024-inch / 0.036-inch inner diameter flow limiter was added to the end of the permeable line.

[0364]

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

[0365]

[0390] Dual counting was performed using a Nucleocounter NC-200 (Chemometec) on the cell cultures before dilution, the diluted cultures, and the final concentrated cell suspension. Volume was 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. The percentage of serum remaining after dilution and concentration was determined using a human serum ELISA kit (Bethyl Laboratories). FACS analysis was performed on control cells and concentrated cell suspension for CD4+ and CD8+ expression.

[0366]

[0391] The successful demonstration of volume reduction by the Cocoon™ transfection protocol is defined as follows: ≥85% recovery of cells, ≤10% reduction in cell viability, and ≤10% of the initial concentration of residual human serum.

[0367]

[0392] Cell transfer between Octane Cocoon (trademark) and electroporation unit

[0393] Transferring cells between the Cocoon® and the electroporation unit requires several disposable consumables: a Cocoon® cassette, an electroporation cartridge, two modified electroporation reservoirs, and two sets of connecting tubes (see Figure 17).

[0368]

[0394] The modified electroporation reservoir includes weldable inlet and outlet tubing with Luer lock connection ends, a cell loading port in the reservoir housing connected to an external inlet reservoir tube for sterile cell transfer to the reservoir, a Luer lock substrate attachment port on the LV reservoir loading tube, and a vent filter on the cap for releasing air during volume transfer. A Cocoon® cassette is designed with ports that can automate the transfer of fluids and cell suspensions out of the Cocoon® in a controlled manner without compromising the sterility of the culture or the health of the cells.

[0369]

[0395] The successful demonstration of sterile cell transfer between Cocoon® and the electroporation unit demonstrated the following: the supernatant of transferred and transfected cells passed sterility tests; no mycoplasma was detected in culture samples before and after transfection; after transfection, more than 90% recovery of cells / volume to transfection in the Cocoon® cassette after delivery to the Cocoon® cassette growth chamber; less than 5% change in the viability of untransfected cells during cell transfer between Cocoon® and the electroporation unit; and less than 20% change in CD3+, CD4+, and CD8+ cells compared to transfected cells with or without automated transfer between Cocoon® and the electroporation unit.

[0370]

[0396] The Cocoon® ACTES cassette has two sampling ports with BD Q-Syte female Luer lock ends, as well as inlet and outlet ports with cannulas that allow for automated transfer of cell suspensions from the Cocoon® cassette via a set of connecting tubes aseptically connected to these locations. During the PoC study, the connections between the Cocoon® cassette, electroporation reservoir, electroporation cartridge, and set of connecting tubes were aseptically connected to create a sterile loop between the Cocoon® and the electroporation system as follows:

[0371]

[0397] A set of connecting tubes equipped with ICU Medical's Spiros® male Luer lock terminal connectors was connected to the two BD Q-Syte female Luer lock sampling ports of the Cocoon® cassette. To create a sterile route from the Cocoon® cassette to the electroporation reservoir, another Spiros® male Luer lock connection (ICU Medical) from the connecting 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 lane line was attached to the Spiros® male Luer lock connection (ICU Medical) at the electroporation cartridge inlet. To collect 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 a Spiros® male Luer lock connector on a set of connecting tubes located at the second automated sampling port of the Cocoon® cassette.

[0372]

[0398] In embodiments, the Cocoon® pump transfers transfected cells to a Cocoon® growth chamber and recovers newly transfected cells before delivery of the Cocoon® cassette to the growth chamber using a second electroporation reservoir or other recovery container capable of sterile cell transfer. Instead of sterile Luer lock connections between the PVC tubing lines at the inlet and outlet of the modified electroporation reservoir, sterile welding techniques can be used, and a connecting 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 guided from the internal Cocoon® environment through a 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 losing control of key process parameters. Cell movement between the Cocoon® and the electroporation unit was controlled using peristaltic pumps and software for two independent control systems, although the software for the combined system can also be used to control the independent pump systems.

[0374]

[0400] Prior to transfection, cells / fluid were manually transferred to a sterile electroporation reservoir to mimic the pre-expansion (day 0) transfection procedure, or the post-expansion transfection procedure was mimicked by transferring from the Cocoon® cassette growth chamber to the sterile electroporation reservoir using the Cocoon® pump, software, and connecting tube set (described above). The Cocoon® pump and software then automated the transfer of cells / fluid from the Cocoon® cassette to the inlet of the electroporation reservoir. The electroporation system performed a pre-programmed pumping motion of up to 20 mL from the electroporation reservoir, through the electroporation cartridge, to a second electroporation reservoir. The Cocoon® pump then 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 collect the transfected cells and hold them until they were ready to be transferred to the Cocoon® growth chamber by the Cocoon® pump. To transfer the transfected cells from the electroporation unit to the Cocoon® growth chamber using only the electroporation unit pump, the "connection tube set clearing" program can be utilized. Furthermore, the length of the connection tube set must be consistent.

[0376]

[0402] Using the aforementioned Cocoon® cassette, connecting tube set, and modified electroporation reservoir connection (Figure 17), 11 mL of phosphate buffer (Lonza) was transferred from the Cocoon® cassette to the modified electroporation reservoir using a Cocoon® pump. Using the electroporation program, a simulated transfection of the PBS solution was performed, and an 11 mL volume was transferred to a second modified electroporation reservoir. Next, using the Cocoon® pump and software, an 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 in a single run. The actual volume was measured using a serum pipette after transfer to the first modified electroporation reservoir, the second modified electroporation reservoir, and the Cocoon® output bag. The pass criterion was established as a fluid recovery rate of ≥90% from the initial modified electroporation reservoir to the Cocoon™ output bag. Cell suspension test

[0403] 1 x 10 8 and 5×10 8The total 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 held for sterility and mycoplasma testing. The cells are diluted with 90 mL of supplemented P3 electroporation solution (Lonza). The cells are then concentrated into approximately 10 mL of cell suspension in a Cocoon® cassette 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. Using a Nucleocounter NC-200 (Chemometec), samples are taken from enriched cells in a satellite bag and kept sterile to measure duplicate cell counts using mycoplasma. Next, as previously described, the cells are transferred to a modified electroporation reservoir via a Cocoon® pump and connecting tube set. Using the electroporation unit pump and EO-210 program, T cells are transfected with pmax GFP vector (Lonza), and the transfected cells are transferred to a second modified electroporation reservoir. The Cocoon® pump then transfers the cells from the modified electroporation reservoir to the growth chamber of a Cocoon® ACTES cassette. Cell samples are taken from the ACTES cassette growth chamber for duplicate cell count, mycoplasma, and sterility testing. This procedure is repeated with a control culture in which cells are not transfected but instead pass through the electroporation unit using the simulated 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 assessed in all cultures. Flow cytometry is used to evaluate GFP, CD3+, CD4+, CD8+, and additional marker expressions.

[0378]

[0405] Aseptic transfer of cell suspension between Cocoon® and the electroporation unit provides a sterile, mycoplasma-free supernatant both before and after migration, and after transfection, resulting in ≥90% recovery of pre-transfection cells in the Cocoon® cassette after delivery to the Cocoon® cassette proliferation chamber, ≤5% change in the viability of untransfected cells, and ≤20% change in the CD3+, CD4+, and CD8+ cell ratio after transfection.

[0379] Expansion of transfected cells transferred between Cocoon (trademark) and the electroporation unit.

[0406] 1 x 10 8 and 5×10 8 Total viable PBMCs are expanded and concentrated in a Cocoon® cassette, transfected via a sterile connection of an electroporation LV unit, and sterilely transferred to a Cocoon® as described in the Methods section concerning cell transfer between Octane Cocoon® and electroporation LV unit, and cell suspension testing. Transfected cells are cultured in a 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, control cultures are aseptically and manually concentrated, transfected via an electroporation LV unit EO-210 program, and returned to the original vessel for continuous 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 transfected cells transferred between the Cocoon™ and the electroporation unit, as determined by FACS, resulted in a ±10% variation in transfection efficiency, ≥80% of the final cell concentration of the control culture, a ±5% variation in final cell viability compared to the control culture, and a ±10% variation in GFP+, CD3+, CD4+, and CD8+ expression compared to the control culture, both 24 hours after transfection and on the day of collection. The supernatant of the transferred transfected cells passed sterility testing, and no mycoplasma was detected in the culture samples before and after transfection.

[0381] result

[0408] Cocoon® Cassette Cell Concentration

[0409] Cells derived from two donors were concentrated to 4.4 × 10⁶ 8 and 4.2 × 10 8 The total viable cells were precipitated in 10 mL of liquid. These two cell suspensions were then diluted and concentrated in 90 mL of supplemented electroporation solution (NFS). Cell recovery rates after concentration were 92% and 87%. Cell viability before transfection was 92% and 74%, a decrease of less than 5%. In both runs, 6% and 8% of the initial culture supernatant were 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 unconcentrated control culture.

[0383]

[0411] The results demonstrated fluid recovery from the Cocoon® satellite bag to the Cocoon® output bag. Enlarged transfected cells were transferred between the Cocoon® and the electroporation unit. Successful electroporation was 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® system. A method for enriching cells using the Cocoon® system can be used.

[0385] Example 4 - Expansion of hematopoietic stem cells

[0413] CD34+ was focused on umbilical cord blood expansion. This particular application involved CD34+ expansion from umbilical cord blood samples containing low CD34+ counts, for use in the treatment of adults using a single, well-matched umbilical cord. Therefore, the starting cell number and concentration were very low compared to some other protocols. It was expected that larger starting cell numbers and concentrations would result in smaller cell expansion.

[0386]

[0414] Selected and enlarged CD34+ cells

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

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

[0417] Cell enlargement was found to vary depending on the harvesting protocol (Figure 19).

[0387]

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

[0419] 25.3% of TNCs were CD34+ after 12 days of expansion (Figure 20).

[0388]

[0420] The differentiated cell phenotype is shown in Figure 21. Figure 22 shows that a single colony can form a multiphyletic differentiation.

[0389] References cited FDA, Regenerative Medicine Advanced TherapyDesignation. (2017). Available at:https: / / www.fda.gov / BiologicsBloodVaccines / CellularGeneTherapyProducts / ucm537670.htm.(Accessed:8th August 2017) Wang, X. & Riviere, I. Clinical manufacturingof CAR T cells: foundation of a promising therapy. Mol. Ther.-Oncolytics 3,16015 (2016). Jones, S. D., McKee, S. & Levine, H. L.Emerging challenges in cell therapy manufacturing. BioProcess Int 10,S4--S7(2012). Trainor, N., Pietak, A. & Smith, T. Rethinkingclinical delivery of adult stem cell therapies. Nat Biotech 32, 729-735(2014). Nilsson, C. et al. Optimal Blood MononuclearCell Isolation Procedures for Gamma Interferon Enzyme-Linked Immunospot Testingof Healthy Swedish and Tanzanian Subjects. Clin. VaccineImmunol. 15, 585-589(2008). Bohnenkamp, H., Hilbert, U. & Noll, T. Bioprocessdevelopment for the cultivation of human T-lymphocytes in a clinical scale.Cytotechnology 38, 135-145 (2002). Lu, F. et al. Automated dynamic fed-batch processand media optimization for high productivity cell culture process development.Biotechnol. Bioeng. 110, 191-205 (2013). Hollyman, D. et al. Manufacturingvalidation of biologically functional T cells targeted to CD19 antigen forautologous adoptive cell therapy. J. Immunother. 32, 169-180 (2009). FDA, Sepax Cell Separation System and singleuse kits. (2011). Available at: https: / / www.fda.gov / downloads / BiologicsBloodVaccines / BloodBloodProducts / ApprovedProducts / SubstantiallyEquivalent510kDeviceInformation / UCM278385.pdf. (Accessed: 8th November 2017) Wegener, C. Cell Washing with the LOVO CellProcessing System. BioProcess Int Industry Y, p78 (2014). Trickett, A. & Kwan, Y. L. T cell stimulationand expansion using anti-CD3 / CD28 beads. J. Immunol. Methods 275,251-255(2003). Hasegawa, K. et al. In vitro stimulationofCD8 and CD4 T cells by dendritic cells loaded with a complex of cholesterol-bearinghydrophobized pullulan and NY-ESO-1 protein: Identification of a newHLA-DR15-binding CD4 T-cell epitope. Clin. Cancer Res. 12, 1921-1927(2006). Odeleye, A. O. O., Marsh, D. T. J., Osborne,M. D., Lye, G. J. & Micheletti, M. On the fluid dynamics of alaboratoryscale single-use stirred bioreactor. Chem. Eng. Sci. 111, 299-312(2014). Grishagin, I. V. Automatic cell counting withImageJ. Anal. Biochem. 473, 63-65 (2015). Levine, B. L., Miskin, J., Wonnacott,K.& Keir, C. Global Manufacturing of CAR T Cell Therapy. Mol. Ther.MethodsClin. Dev. 4, 92-101 (2017). Locke, F. L. et al. Abstract CT019: Primaryresults from ZUMA-1: a pivotal trial of axicabtagene ciloleucel (axicel; KTE-C19)in patients with refractory aggressive non-Hodgkin lymphoma (NHL). Cancer Res.77, CT019 LP-CT019 (2017). Lu YC, Parker LL, Lu T, Zheng Z, Toomey MA,White DE, Yao X, Li YF, Robbins PF, Feldman SA, van der Bruggen P, KlebanoffCA, Goff SL, Sherry MS, Kammula US, Yang JC, Rosenberg SA. Treatment ofpatients with metastatic cancer using a major histocompatibility complex classII-restricted T-cell receptor targeting the cancer germline antigenMAGE-A3.Journal of Clinical Oncology (2017) 35: 29, 3322-3329. FDA,Available online at: https: / / www.fda.gov / downloads / AdvisoryCommittees / CommitteesMeetingMaterials / Drugs / OncologicDrugsAdvisoryCommittee / UCM566166.pdf Berdeja JG, Lin Y, Raje NS, Siegel DSD, MunshiNC, Liedtke M, Jagannath S, Maus MV, Turka A, Lam LP, Hege K, Morgan R, QuigleyMT, Kochenderfer J. First-in-human multicenter study of bb2121 anti-BCMACART-cell therapy for relapsed / refractory multiple myeloma: Updated results. Journalof Clinical Oncology 2017 35:15_suppl, 3010-3010 Kebriaei P, Singh H, Huls MH, Figiola MJ, BassettR, Olivares S, Jena B, Dawson MJ, Kumaresan PR, Su S, Maiti S, Dai J, MoriarityB, Forget MA, Senyukov V, Orozco A, Liu T, McCarty J, Jackson RN, Moyes JS,Rondon G, Qazilbash M, Ciurea S, Alousi A, Nieto Y, Rezvani K, MarinD, Popat U,Hosing C, Shpall EJ, Kantarjian H, Keating M, Wierda W, Do KA, Longsword DA,Lee DA, Hackett PB, Champlin RE, Cooper LJN. Phase I trials using SleepingBeauty to generate CD19-specific CAR T cells. J Clin Invest.2016 Sep 1; 126(9):3363–3376. Morrissey JB, Shi Y, Trainor N. End-to-end cell therapy automation: an immunotherapy case study. BioProcess International (2017)10-18. [ Abstract ] Lafferty KJ, Cunningham AJA. New analysis ofallogeneic interactions. J. Immunol. (1975) 112: 436–437. [ PubMed ] [ Cross Ref ] Harding F, McArthur J, Gross J, Raulet D, AllisonJ. CD28-mediated signaling co-stimulates murine T cells and prevents inductionof anergy in T-cell clones (1992) Nature 356:607–609. Clavreul A, Fisson S, D’ hellencourt CL, CouezD. Interelationship between CD3 and CD28 pathways in a murine T cell thymoma.Mol Immunol. (2000) 37(10): 571-7. Charron L, Doctrinal A, Choileain SN, AstierAL. Monocyte: T cell interaction regulates human T cell activation through aCD28 / CD46 crosstalk. Immunol Cell Biol. (2015) 93(9): 796-803. Fathman CG1, Lineberry NB. Molecular mechanismsof CD4+ T-cell anergy. Nat Rev Immunol. (2007) 7(8): 599-609. Greenwald RJ, Freeman GJ, Sharpe AH. TheB7family revisited. Annual Review of Immunology (2005) 23(1): 515-548. Kochenderfer JN, Dudley ME, Kassim SH, et al.Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cellmalignancies can be effectively treated with autologous T cells expressing ananti-CD19chimeric antigen receptor. Journal of Clinical Oncology (2015); 33(6):540-549. Kalos M, Levine BL, Porter DL, et al. T Cellswith Chimeric Antigen Receptors Have Potent Antitumor Effects and Can EstablishMemory in Patients with Advanced Leukemia. Science Translational Medicine(2011) 3(95): 95ra73. Riddell SR, Greenberg PD. The useofanti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specificT cells. J Immunol Methods (1990) Apr 17; 128(2): 189-201. Trickett A, Kwan YL. T cell stimulation andexpansion using anti-CD3 / CD28 beads. Journal of Immunological Methods.275(2003) 251-255. Dudley ME, Wunderlich JR, Shelton TE, Even J,Rosenberg SA. Generation of Tumor-Infiltrating Lymphocyte Cultures for Use inAdoptive Transfer Therapy for Melanoma Patients. Journal of immunotherapy (2003)26(4): 332-342. Dudley ME, Wunderlich JR, Shelton TE, Even J,Rosenberg SA. Generation of tumor-infiltrating lymphocyte cultures for use inadoptive transfer therapy for melanoma patients. J Immunother. (2003)26(4):332-342. Manger B, Weiss A, Wey and C, Goronzy J, StoboJD. T cell activation: differences in the signals required for IL2production by nonactivated and activated T cells. J Immunother. (1985) 135(6)3669-3673. Ceuppens J, Bloemmen FJ, Van Wauwe JP. T cellunresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiencyof monocyte Fc gamma receptors for murine IgG2a and inability to cross-link theT3-Ti complex. J Immunol (1985) 135 (6) 3882-3886. Van Wauwe JP, De Mey JR, Goossens JG. OKT3:a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties.J Immunol. (1980)124(6): 2708-13. Carpenter PA, Pavlovic S, Tso JY, Press OW,Gooley T, Yu XZ, Anasetti C. Non-Fc receptor-binding humanized anti-CD3antibodiesinduce apoptosis of activated human T cells. J Immunol (2000) 165(11)6205-6213. Andris F, Denanglaire S, de Mattia F, UrbainJ, Leo O. Naive T cells are resistant to anergy induction byanti-CD3antibodies. J of Immunology (2004) 173 (5) 3201-3208. Wolf H, Muller Y, Salmen S, Wilmanns W, JungG. Induction of anergy in resting human T lymphocytes by immobilizedanti-CD3antibodies. Eur J Immunol. (1994) 24(6): 1410-1417. Chai JG, Lechler RI. Immobilizedanti-CD3mAb induces anergy in murine naive and memory CD4+ T cells in vitro.IntImmunol. (1997) 9(7): 935-944. Verwilghen J, Baroja ML, Van Vaeck F,VanDamme J, Ceuppens JL. Differences in the stimulating capacity ofimmobilizedanti-CD3 monoclonal antibodies: variable dependence on interleukin-1as a helper signal for T-cell activation. Immunology (1991) 72(2): 269-276. Schwartz RH. A cell-culture model for lymphocyte-Tclonal anergy. Science (1990) 248: 1349-1356. Ju SW, Ju SG, Wang FM, Gu ZJ, Qiu YH, YuGH,Ma HB, Zhang XG. A functional anti-human 4-1BB ligand monoclonal antibody thatenhances proliferation of monocytes by reverse signaling of 4-1BBL.Hybridomaand Hybridomics. (2003) 22: 333-338. Baroja ML, Lorre K, Van Vaeck F,CeuppensJL. The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helpersignal and bypasses the need for accessory cells in T cell activation with immobilizedanti-CD3 and mitogens. Cell Immunol. (1989) 120(1): 205-217. Austyn JM, Smith KG, Morris PJ. T cell activationby anti-CD3 antibodies: function of Fc receptors on B cell blasts, but notresting B cells, and CD18 on the responding T cells. Eur J Immunol.1987 17(9):1329-35. Tax WJM, Willems HW, Reekers PPM, CapelPJA,Koene RAP. Polymorphism in mitogenic effect of IgG1 monoclonal antibodies againstT3 antigen on human T cells. Nature (1983) 304: 445-447. Fleischer J, Soeth E, Reiling N, Grage-GriebenowE, Flad HD, Ernst M. Differential expression and function of CD80(B7-1) andCD86 (B7-2) on human peripheral blood monocytes. Immunology (1996)89(4):592-598. Schwartz RH. T cell anergy. Annual Review Immunology(2003) 21: 305-34. Feldmann A, Arndt C, Topfer K, StamovaS,Krone F, Cartellieri M, Koristka S, Michalk I, Lindemann D, Schmitz M, TemmeA, Bornhauser M, Ehninger G, Bachmann M. Novel humanized and highly efficient bispecificantibodies mediate killing of prostate stem cell antigen-expressing tumor cellsby CD8+ and CD4+ T cells. J Immunol. (2012) 189(6): 3249-3259. Reusch U, Duell J, Ellwanger K, HerbrechtC,Knackmuss SH, Fucek I, Eser M, McAleese F, Molkenthin V, Gall FL, et al.Atetravalent bispecific TandAb (CD19 / CD3), AFM11, efficiently recruits T cells forthe potent lysis of CD19(+) tumor cells. MAbs. (2015) 7: 584-604. Church SE, Jensen SM, Antony PA, RestifoNP,Fox BA. Tumor-specific CD4+ T cells maintain effector and memory tumor-specificCD8+ T cells. Eur J Immunol. (2014) 44(1): 69-79. Feldmann A, Arndt C, Topfer K, Stamova S, KroneF, Cartellieri M, Koristka S, Michalk I, Lindemann D, Schmitz M, Temme A, BornhauserM, Ehninger G, Bachmann M. Novel humanized and highly efficient bispecificantibodies mediate killing of prostate stem cell antigen-expressing tumor cellsby CD8+ and CD4+ T cells. J Immunol. (2012) 189: 3249-3259. Reusch U, Duell J, Ellwanger K, HerbrechtC,Knackmuss SH, Fucek I, Eser M, McAleese F, Molkenthin V, Gall FL, Topp M, LittleM, Zhukovsky EA. A tetravalent bispecific TandAb (CD19 / CD3), AFM11, efficientlyrecruits T cells for the potent lysis of CD19(+) tumor cells. (2015) 7:584-604. Riddell SR, Sommermeyer D, Berger C, et al.Adoptive therapy with chimeric antigen receptor-modified T cells of defined subsetcomposition. Cancer J. (2014) 20(2): 141-144. Turtle CJ, Hanafi L-A, Berger C, et al.CD19CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. TheJournal of Clinical Investigation. (2016) 126(6): 2123-2138. Locke FL, Neelapu SS, Bartlett NL,SiddiqiT, Siddiqi T, Chavez JC, Hosing CM, Ghobadi A, Budde LE, Bot A, RossiJM, JiangY, Xue AX, Elias M, Aycock J, Wiezorek J, Go WY. Phase 1 Results ofZUMA-1: A multicenter study of KTE-C19 anti-CD19 CAR T cell therapy inrefractory aggressive lymphoma. Molecular Therapy (2017) 25(1): 285-295. Trainor N, Pietak A, Smith T. Rethinking clinicaldelivery of adult stem cell therapies. Nature Biotech (2014) 729-735. Mahdavi B, Gottschalk U, Trainor N, Smith T.The hype, hope and reality of personalization. The Medicine Maker (2015)38-41. Yan M, Schwaederle M, Arguello D, MillisS Z,Gatalica Z, Kurzrock R. HER2 expression status in diverse cancers: review ofresults from 37,992 patients. Cancer Metastasis Review (2015) 34(1):157-164. Tuefferd M, Couturier J, Penault-Llorca F, Vincent-SalomonA, Broet P, Guastalla JP, Allouache D, Combe M, Weber B, Pujade-Lauraine E,Camilleri-Broet S. HER2 Status in Ovarian Carcinomas: A Multicenter GINECOStudy of 320 Patients (2007) 2(11): e1138. Lu TL, Pugach M, Somerville R, Rosenberg SA,Kochendefer JN, Better M, Feldman SA. A rapid cell expansion process for productionof engineered autologous CAR-T cell therapies. Human Gene Therapy (2016) 27:209-218. Nociari MM, Shalev A, Benias P, Russo C. A novelone-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity.J Immunol Methods (1998) 213(2): 157-167. Romagnani S. Type 1 T helper and type 2 T helpercells: functions, regulation and role in protection and disease. Int J Clin LabRes (1991) 21(2): 152-158. Fleischer J, Soeth E, Reiling N, Grage-GriebenowE, Flad HD, Ernst M. Differential expression and function ofCD80 (B7-1) andCD86 (B7-2) on human peripheral blood monocytes. Immunology (1996) 89(4):592-598. Laux I, Khoshnan A, Tindell C, Bae D, Zhu XM,June CH, Effros RB, Nel A. Response differences between human CD4+ andCD8+T-cells during CD28 costimulation: Implications for immune cell-basedtherapies and studies related to the expansion of double-positive T-cellsduring aging. Clin Immunol. (2000) 96: 187-197. Li Y, Kurlander RJ. Comparison ofanti-CD3and anti-CD28-coated beads with soluble anti-CD3 for expanding human Tcells: Differing impact on CD8 T cell phenotype and responsiveness torestimulation. J Transl Med. (2010) 8: 104. Zhu YW, Zhu GF, Luo LQ, Flies AS, ChenLP.CD137 stimulation delivers an antigen-independent growth signal for T lymphocyteswith memory phenotype. Blood (2007) 109: 4882-4889. Ledbetter JA, Imboden JB, Schieven GL, GrosmaireLS, Rabinovitch PS, Lindsten T, Thompson CB, June CH. CD28 Ligationin T-cellActivation: Evidence for Two Signal Transduction Pathways. Blood (1990) 75(7):1531-1539. Atkuri KR, Herzenberg LA, Herzenberg LA. Culturingat atmospheric oxygen levels impacts lymphocyte function. Proceedings of theNational Academy of Sciences of the United States of America (2005)102(10):3756-3759. Avgoustiniatos ES, Hering BJ, Rozak PR,etal. Commercially Available Gas-Permeable Cell Culture Bags May NotPreventAnoxia in Cultured or Shipped Islets. Transplantation proceedings.2008;40(2):395-400. Hammill JA, VanSeggelen H, Helsen CW, DenisovaGF, Evelegh C, Tantalo DGM, Bassett JD, Bramson JL. Designed ankyrinrepeat proteins are effective targeting elements for chimeric antigen receptors.Journal for ImmunoTherapy of Cancer (2015) 3:55. VanSeggelen H, Tantalo DGM, Afsahi A, HammillJA, Bramson JL. Chimeric antigen receptor-engineered T cells a soncolytic virus carriers. Molecular Therapy-Oncolytics (2015) 2, 150014.

[0390]

[0421] It will be readily apparent to those skilled in the 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] While 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 described and illustrated. Exemplary embodiments are disclosed herein, and specific terms are used, but they are general and descriptive in nature only, and not for limiting purposes. In light of the above teachings, modifications and variations of embodiments are possible. Therefore, it should be understood that embodiments may be implemented in ways other than those specifically described.

[0392]

[0423] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically incorporated by reference and is shown separately.

Claims

1. A method for the automated production of genetically modified T cell cultures, a) Mix the T cell culture with a magnetic selection reagent. b) Subjecting the T cell culture to magnetic separation. c) Activating T cell cultures using an activation reagent selected from antibodies and dendritic cells to produce activated T cell cultures. d) Transducing activated T cell cultures in a cell culture chamber using a viral vector encoding an extracellular domain, a transmembrane domain, and an intracellular domain to produce transduced T cell cultures. e) Expanding the transduced T cell culture in a cell culture chamber. f) Centrifuging the expanded T cell culture, and, g) Recovering T cell cultures and generating genetically modified T cell cultures. Includes, (a) to (f) are performed within a completely sealed automated cell engineering system, where the cell culture chamber has a fixed area. (e) The expansion of the transduced T cell culture generates at least 20% more genetically modified T cells than expansion using manual cell culture with a flexible, gas-permeable bag, and the transduction efficiency of the method is at least 20% higher than the transduction efficiency using manual cell culture with a flexible, gas-permeable bag.

2. A method for the automated production of T cell cultures in a completely sealed automated cell engineering system, To place T cell cultures in the first chamber of a completely sealed automated cell engineering system, To supply, wash, and / or oxygenate T cell cultures, the culture medium stored in the second chamber of a fully sealed automated cell engineering system is transferred to the first chamber. The system monitors one or more parameters of the first chamber via a sensor, and the one or more parameters include one or more of temperature, pH level, glucose level, oxygen level, carbon dioxide level, and optical density level, and Based on monitoring, automatically adjust one or more of the aforementioned parameters. A method comprising, including, automatically adjusting one or more of the parameters, based on monitoring, operating a pump and delivering culture medium from a second chamber to a first chamber through one or more fluid pathways.

3. An automated cell engineering system, a) A shell comprising a plurality of separate chambers configured for activating, expanding, concentrating and recovering cell cultures, wherein one of the chambers comprises an electroporation unit and another of the chambers comprises a magnetic separation unit; b) A self-contained, removable, and replaceable cassette containing a substantially non-deformable cell culture chamber; c) At least two of the following sensors configured to monitor the temperature, pH, glucose concentration, oxygen level, carbon dioxide level, and / or optical density of a cell culture in a self-contained, removable and replaceable cassette: a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor. Includes, The automated cell engineering system is configured to be pre-filled with activation reagents, vectors, cell culture medium, nutrients, and selection reagents. An automated cell engineering system in which, of the aforementioned separate chambers, the first chamber is maintained at a temperature of approximately 25°C to approximately 37°C, and the second chamber is maintained at a temperature of approximately 4°C to approximately 8°C.

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