Quality control methods for automated cell processing

Abstract

A method is provided for assessing and optimizing cell quality of cell-based therapies produced within an automated cell engineering system. [Solution] Provided is a method for evaluating and optimizing cell quality for cell-based therapy, the method comprising: (a) determining one or more molecular characteristics of a pre-modified cell culture; (b) genetically modifying the cell culture via an automated cell engineering system; (c) determining the one or more molecular characteristics of the modified cell culture in real time during and after the genetic modification; and (d) optimizing one or more parameters of the automated cell engineering system based on the determined one or more molecular characteristics to alter the one or more molecular characteristics of the modified cell culture before, during, and / or after the genetic modification.

Description

[Technical Field]

[0001] The present disclosure provides methods for assessing and optimizing cell quality of cell-based therapies produced in automated cell engineering systems. The methods preferably include monitoring molecular characteristics of cells before, during, and after the automated process to provide feedback to process parameters. In embodiments, the cells produced are chimeric antigen receptor (CAR) T-cells. [Background technology]

[0002] As the clinical adoption of advanced cell therapies is expected to accelerate, attention is turning to fundamental manufacturing strategies to ensure these therapies benefit patients worldwide. While cell therapies hold great clinical potential, high manufacturing costs relative to reimbursement present high barriers to commercialization. Therefore, the need for cost-effectiveness, process efficiency, and product consistency is driving automation efforts in numerous cell therapy areas, particularly for T-cell immunotherapy (see, for example, Wang 2016).

[0003] However, significant challenges remain that prevent widespread adoption of autologous cell therapies and result in manufacturing failures. The starting cell material often originates from the patient, which represents an additional level of complexity and source of variability. Many patients currently treated with cell therapies have progressive disease, experience multiple relapses, and have failed multiple other therapies. As a result, the therapeutic window in which patients can benefit from these cell therapies is narrow, necessitating accurate and reliable manufacturing processes.

[0004] After manufacturing a cell therapy product, there are additional delays before the product can be administered to patients, primarily due to numerous regulatory requirements and release testing that are currently slow and costly. Current cell therapy release testing provides regulators with the information they need to make patients comfortable, but is of little use in predicting clinical efficacy.

[0005] In automated processes for producing cell-based therapies, there is often a need to optimize and modify various parameters of the process based on individual patient needs and real-time changes during the process. The present invention meets these needs by providing a method for assessing and optimizing cell quality of cell-based therapies, preferably in an automated system. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Wang 2016 Summary of the Invention

[0007] In some embodiments, provided herein are methods for assessing and optimizing cell quality for a cell-based therapy, the method comprising determining one or more molecular characteristics of a pre-modification cell culture; genetically modifying the cell culture via an automated cell engineering system; determining one or more molecular characteristics of the modified cell culture during and after the genetic modification; and optimizing one or more parameters of the automated cell engineering system to alter the one or more molecular characteristics of the modified cell culture.

[0008] In further embodiments, provided herein are methods for assessing and optimizing cell quality for a cell-based therapy, comprising: determining one or more molecular characteristics of a pre-modified cell culture; optimizing one or more parameters of an automated cell engineering system to alter one or more molecular characteristics of one of the pre-modified cell cultures; activating the pre-modified cell culture with an activation reagent to produce an activated cell culture; transducing the activated immune cell culture with a vector to produce a transduced cell culture; expanding the transduced cell culture; (e) enriching the expanded cell culture; and (f) recovering the enriched cell culture to produce a genetically modified cell culture; during or after any one of steps (c)-(g), determining one or more molecular characteristics of the cell culture; and optimizing one or more parameters of any one of steps (c)-(g) to alter one or more molecular characteristics of the cell culture.

[0009] In an additional embodiment, a method is provided for assessing and optimizing the cell quality of a chimeric antigen receptor T (CAR T) cell culture, comprising determining one or more molecular characteristics of the pre-modification T-cell culture; optimizing one or more parameters of an automated cell engineering system to alter one or more molecular characteristics of one of the pre-modification T-cell cultures; activating the pre-modification T-cell culture with an activation reagent to produce an activated T-cell culture; transducing the activated T-cell culture with a vector encoding a chimeric antigen receptor to produce a CAR T-cell culture; expanding the CAR T-cell culture; (e) enriching the expanded CAR T-cell culture; and (f) recovering the enriched CAR T-cell culture; and during or after any one of steps (c)-(g), determining one or more molecular characteristics of the CAR T-cell culture; and optimizing one or more parameters of any one of steps (c)-(g) to alter one or more molecular characteristics of the CAR T-cell culture.

[0010] Also provided herein is a method for assessing and optimizing cell quality of a cell culture, the method comprising determining one or more molecular characteristics of a pre-modified cell culture, genetically modifying the cell culture via an automated cell engineering system, determining one or more molecular characteristics of the modified cell culture during and after the genetic modification, and optimizing one or more parameters of the automated cell engineering system to alter the one or more molecular characteristics of the modified cell culture. [Brief explanation of the drawings]

[0011] [Figure 1] 1 illustrates a generalized manufacturing process for producing a cell-based therapy as described herein. [Figure 2] 1 illustrates a laboratory space containing an exemplary cell engineering system, as described in embodiments herein. [Figure 3] 1 illustrates a cell-based therapy production process that may be performed within a cell engineering system, as described in embodiments herein. [Figure 4] 1 shows a process flow legend for an automated cell engineering system. [Figure 5] 1 illustrates the use of a syringe and bag to sample from a cassette of an automated cell engineering system. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure provides methods for monitoring, evaluating, and optimizing the automated production of various cell-based therapies.

[0013] Automated Cell Processing For autologous cell-based therapies and treatments, such as T-cell therapy, the need for cost-effectiveness, process efficiency, and product consistency is especially urgent because manufacturing microlot (single-patient-per-lot) batches lacks the economies of scale that homogeneous (multiple-patient-per-lot) processes can leverage (see, e.g., Jones 2012; Trainor 2014). The larger and more localized workforce and facilities required for microlots place significant demands on logistics, GMP compliance for manual production (especially regarding availability), and staff training. Additionally, the potential for variability in technique between operators can pose undesirable risks in consistently meeting release criteria and ensuring a safe and reliable product.

[0014] The installation and comprehensive validation of automated manufacturing, as described herein, provides a solution to these logistical and operational challenges. A key approach to introducing automation into a production process is to identify key modular steps, called "unit operations," in which operators apply physical or chemical modifications to the production materials. In the case of cell manufacturing, this includes steps such as cell isolation, genetic manipulation, growth, washing, concentration, and cell recovery. Manufacturers often identify focal process bottlenecks as immediate opportunities for implementing automation. This is reflected in the technical operating spectrum 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 through end-to-end automation that produces consistent cell output while improving inevitable process variability. The methods described herein also provide simplification, and the associated electronic records aid in compliance with GMP standards (see, e.g., Trainor 2014).

[0015] Automation of unit operations and key process sensitivities Recent rapid progress in the clinical development of various cell-based therapies, including engineered autologous T cells for cancer immunotherapy, has led to the planning of associated translational and scale-up / scale-out implications.

[0016] While specific protocols vary for different cell-based therapies, a generalized process is illustrated in Figure 1. Figure 1 describes the unit operations of cell-based therapy manufacturing, including, for example, autologous T-cell therapy, from the initial processing of a patient's blood sample to the formulation of the output cells.

[0017] To achieve cell manufacturing automation as described herein, the methods described herein provide for understanding the state of cells at each transition point and how they are affected by specific unit operations. Microlot production for patient-specific therapies must respect key process sensitivities that affect the feasibility of automation. The automation described herein accommodates various process steps.

[0018] Table 1 below highlights some process step challenges identified for cell-based therapy automation, including T cell automation, and illustrates the impact of sensitivities on automation strategies. Note that for all unit operations, open transfer of cells between respective pieces of equipment is a significant sensitivity due to the risk of contamination. [Table 1]

[0019] Tailoring manual processes around the sensitivities listed in Table 1 to automation can support successful translation, maintenance, or improvement of cell therapy performance.

[0020] Disparate automation vs. fully integrated automation While there is strong evidence of the value of automation (see, for example, Trainor 2014; Levine 2017), subsequent analysis is needed on the value and practicality of integrating these automation steps in an end-to-end sequence using automated transport. There are differing perspectives on the benefits of individual process automation versus end-to-end integration.

[0021] The main benefit to individual automation is flexibility. 1) Maintaining unique process behavior 2) Acceleration of translation activities based on individual unit operation verification 3) the ability to modify processing steps to accommodate donor-to-donor variability.

[0022] The first point about increased flexibility is that it provides operators with greater control over the process. This is important in situations where the process has sensitive steps that can affect the final product. Switching to an all-in-one system may impose constraints that affect product outcomes. A discrete approach offers flexibility to choose how each step is performed, which can be especially important with sensitive unit operations. A discrete approach also allows for a gradual conversion from manual processing to automation, which helps demonstrate equivalence when each unit operation can be tested independently. Additionally, automating specific unit operations provides flexibility to make decisions based on cell performance. For example, if cells grow rapidly, it may be necessary to expand from one cell culture bag to two. Finally, a discrete system approach to automation also allows groups to choose the equipment to use for each unit operation.

[0023] Equipment utilization is another argument for individual automation. Some unit operations may take significantly more time than others. An end-to-end processing system would require multiple unit operations all to be performed on a single system, thus occupying the equipment for the duration of the culture process.

[0024] While there are benefits to individual automation, an end-to-end approach offers a variety of benefits, some of which are significant. First, a fully integrated system significantly reduces the risk of contamination. The increased handling required with individual approaches increases the likelihood of product variability due to operator intervention. Second, and as previously mentioned, this inevitably results in higher labor costs.

[0025] The flexibility provided by individual approaches is critical. In situations where the process is critical in defining the product, the end-to-end system must have the flexibility to integrate unique sensitivities. This may include specific nutrient delivery strategies, oxygen levels, surface treatments, and more. Such approaches require flexibility in both the software and disposable components. The system must offer the option to withdraw cell and media samples at various points within the process to ensure that specific unit operations meet product specification checkpoints. If modifications need to be made, the software must be able to implement these changes to provide ideal conditions. While easy-to-use and flexible software is extremely beneficial for translational 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, the operator's ability to change the protocol, if any, must be limited. However, to address issues with inherent donor variability, there must be the option to select from various validated protocols based on cell growth rate. For example, if cells grow rapidly, the system must be able to respond and adjust nutrient delivery or harvest time points accordingly.

[0026] The choice of end-to-end integration versus individual automation also depends on the long-term vision for the clinical process. A single, all-in-one system can offer significant space efficiency, minimizing the required footprint in expensive GMP clean rooms. For example, as shown in Figure 2, a fully integrated automation system is designed to maximize the required footprint and reduce expensive GMP clean room space. Figure 2 shows 96 patient-specific end-to-end units running within a standard laboratory space.

[0027] A single system also offers easier data tracking, whereas separate systems may not provide compliant software that links all electronic data files together. Software platforms such as VINETI (Vineti Ltd) and TRAKCEL (TrakCel Ltd) enable electronic monitoring and organization of supply chain logistics. However, a single, all-in-one culture system can go even further by incorporating into the batch record the history of both processing events and biomonitoring of culture conditions associated with each unit operation. Accordingly, the benefits of end-to-end integration provide a significant competitive advantage.

[0028] A commercial platform for unit operation integration The success of clinical trials for numerous autologous cell therapies, particularly immunotherapies for hematologic cancers, highlights the importance of enabling the translation of new clinical protocols onto robust production platforms to meet projected clinical demand (see, e.g., Levine 2017; Locke 2017). For autologous therapies, processing each patient-specific cell therapy preferably utilizes comprehensive manufacturing activities and operations management. The methods herein link unit operations within a turnkey automated system to achieve process optimization, security, and economics.

[0029] The challenge in designing an autologous process is twofold. First, unlike homogeneous manufacturing, where separate processing steps may occur in physically separate and optimized pieces of equipment, a scale-out autologous platform preferably performs all of the necessary steps in a single, closed, self-contained, automated environment. Second, unlike a homogeneous process, where every run theoretically begins with high-quality vials from a cell bank with known quality and predictable process behavior, the starting material in an autologous process is highly variable and generally derived from individuals with compromised health.

[0030] Thus, a method is provided herein that can sense culture conditions and respond accordingly as a sophisticated bioreactor by controlling factors such as physical agitation, pH, nutrient supply, and gas handling. Furthermore, technology transfer for autologous therapies, compared with allogeneic therapies, presents distinctly different challenges. Autologous products may have more constraints on the stability between the manufacturing process and patient treatment. Sites can be located globally, rather than in a single center. Locking down (e.g., completely closing) an all-in-one system significantly improves the technology transfer process between sites.

[0031] While source variability cannot be eliminated, automation helps eliminate variability in the final in-house product through standardization and reproducibility. This practice is adopted by major cell system providers to obtain cell performance reference points via biosensors that monitor the state of the active cell culture. With end-to-end integration, the output from any particular step in the process must be within acceptable parameters for the process to proceed.

[0032] As described herein, in embodiments, the provided methods utilize the COCOON platform (Octane Biotech, Kingston, ON), which integrates multiple unit operations in a single turnkey platform. Multiple cell protocols are available for very specific cell processing objectives. To provide efficient and effective automated translation, the described methods 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.

[0033] The methods described herein have been used to scale a variety of cell-based therapies, including CAR T cells (including activation, viral transduction, as well as expansion, enrichment, and washing), in a fully integrated, closed, automated system (Figure 3).

[0034] The benefits of automation Automation of unit operations in cell therapy production offers opportunities for universal benefits across allogeneic and autologous cell therapy applications. In the unique scenario of patient-specific autologous cell products, and further highlighted by the recent clinical success of these therapies, the benefits of automation are particularly compelling due to the complexities of microlots, where small-batch GMP compliance, economics, patient traceability, and early detection of process deviations are crucial. With the emergence of complex manufacturing protocols, the value of end-to-end integration of automated unit operations in microlot cell production has been highlighted, a fact that has been largely underexplored to date. However, given the anticipated demand for these soon-to-be-approved therapies, implementing a fully closed, end-to-end system could provide a necessary solution to manufacturing bottlenecks such as hands-on time and footprint.

[0035] Advanced Therapies developers are encouraged to consider automation early in their clinical translation deployment and scale-up of clinical trial protocols. Early automation can have an impact on protocol development, avoiding the need for comparison studies when switching from manual to automated processes at a later stage, and developing a better understanding of the longer-term route to commercialization.

[0036] Quality control of automated systems for producing cell-based therapies, including CAR T cells As described herein, methods are provided that allow for the monitoring, evaluation, and optimization of the automated production of various cell-based therapies. The methods described herein involve monitoring various molecular characteristics at different times before, during, and after the automated process, and making changes and adjustments to various parameters of the automated system to optimize the output. Such optimization may be based on the desired cell number, concentration, or characteristics for a particular therapy, or even for an individual patient.

[0037] Thus, in embodiments, provided herein are methods for assessing and optimizing cell quality for cell-based therapies. As used herein, "assessing" refers to the act of measuring or determining one or more characteristics of cells, including molecular characteristics of the cells, to help guide any modifications to the method. As used herein, "optimizing" refers to modifying one or more parameters of the automated cell engineering systems described herein. As used herein, "cell quality" refers to the characteristics necessary for cells to perform as desired in a cell-based therapy. This quality includes membrane integrity, nuclear integrity, a desired gene profile, a desired protein profile, a desired cell lifespan, a desired cell number or density, etc.

[0038] As used herein, "cell-based therapy" refers to a therapy in which cellular material is infused, transplanted, or implanted into a patient. Cell-based therapy preferably includes intact, living cells. Cell-based therapy includes various types of cells, such as immune cells, natural killer cells, cells for neurodegenerative therapy, and stem cells. The methods described herein can preferably be used for any cell type commonly used in cell-based therapy or cell culture, which may include cell culture methods for tissue engineering applications and biological production, such as viral vectors or protein expression.

[0039] Methods for evaluating and optimizing cell quality for cell-based therapies preferably include determining one or more molecular characteristics of the pre-modified cell culture. As used herein, "molecular characteristics" include one or more of the following: a genetic profile (e.g., a gene expression profile), an amino acid or protein profile (e.g., proteins expressed within or on the cell surface), a lipid profile, etc. of the cells. As used herein, "cell culture" refers to single cells and collections of cells for use in the automated methods described herein.

[0040] The method preferably begins with determining one or more molecular characteristics of a pre-modified cell culture. A pre-modified cell culture is a cell culture that has not yet been placed in an automated cell engineering system described herein. A pre-modified cell culture can include cells taken directly from a patient (e.g., a blood draw or plasma sample) and cells removed from a patient that have undergone some filtering, sorting, or other modification to arrive at the desired cell culture population to be modified in the automated cell engineering system described herein.

[0041] The method preferably includes genetically modifying a cell culture via an automated cell engineering system. As used herein, "genetic modification" includes introducing one or more genes into a cell (e.g., via transduction) to suitably modify the genome of the cell. Genetic modification may also include transient modifications that are not integrated into the genome. Methods for genetically modifying a cell culture are described herein and preferably include activating the cell culture with an activation reagent to produce an activated immune cell culture, transducing the activated cell culture (e.g., with a vector) to produce a transduced cell culture, and expanding the transduced immune cell culture (see, e.g., Figures 1 and 3). Methods for preparing, activating, transducing, and expanding cell cultures are described herein.

[0042] In exemplary embodiments, the method for evaluating and optimizing cell quality further comprises determining one or more molecular characteristics of the modified cell culture during and after genetic modification.That is, the cell culture can be evaluated at any time during any one of activation, transduction, and / or proliferation, and preferably one or more molecular characteristics of the cell culture are determined.This sampling and evaluation provides data on the characteristics of the cell culture at various times during and after automated processing, allowing one or more molecular characteristics to be tracked at each stage of automation.

[0043] The method for assessing and optimizing cell quality preferably further comprises optimizing one or more parameters of the automated cell engineering system to alter one or more molecular characteristics of the modified cell culture. As described herein, parameters of the automated cell engineering system that may be optimized include processing parameters (e.g., pH, temperature, heat), as well as the quality and duration of activation, transduction, and growth, cell sorting, and the like.

[0044] In exemplary embodiments, the one or more molecular characteristics include one or more of gene expression, protein expression, mRNA expression, and copy number variation.

[0045] Preferably, the molecular signature is determined by one or more methods for multiplexed analysis of RNA, DNA, and / or protein targets of cell-based therapies and / or cell cultures. Exemplary multiplexed analysis tools include various arrays, barcode technologies, next-generation sequencing methods, quantitative PCR, etc.

[0046] In an exemplary embodiment, molecular characteristics can be determined using techniques such as the molecular barcoding technique NCOUNTER®, developed by NANOSTRING® (Seattle, WA). Molecular barcoding techniques utilize unique capture probes designed to bind to desired targets (e.g., nucleic acids (RNA, DNA), proteins, or peptides). A reporter probe containing a barcode (e.g., a fluorescent-, color-, or radioactive-based tag) binds to the capture probe. The sample is purified and immobilized, and the barcoded target molecules are then counted and analyzed. See, for example, Geiss, et al., “Direct multiplexed measurement of gene expression with color-coded probe pairs,” Nat. Biotechnol. 26:317-325 (2008), the disclosure of which is incorporated herein by reference in its entirety.

[0047] As described herein, cell cultures that can be utilized in the automated cell engineering systems described herein include immune cell cultures, natural killer cell cultures, and cell cultures for neurodegenerative therapy.

[0048] In an exemplary embodiment, the immune cell culture is a T-cell culture, and in embodiments, a chimeric antigen receptor T (CAR T) cell culture. Chimeric antigen receptor T cells, or "CAR T cells," are, more specifically, T cells engineered with a chimeric antigen receptor (CAR) to target cancer cells. Generally, CARs comprise three parts: an ectodomain, a transmembrane domain, and an endodomain. The ectodomain is the region of the receptor exposed to extracellular fluid and comprises three parts: a signal peptide, an antigen recognition region, and a spacer. The signal peptide directs the nascent protein to the endoplasmic reticulum. In CARs, the signal peptide is a single-chain variable fragment (scFv). An scFv is a light chain (V) of an immunoglobulin linked with a short linker peptide. L ) and heavy chain (V H In some embodiments, the linker comprises glycine and serine. In some embodiments, the linker comprises glutamic acid and lysine.

[0049] The transmembrane domain of the CAR is a membrane-spanning hydrophobic α-helix. In some embodiments, the transmembrane domain of the CAR is a 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 a CD3ζ transmembrane domain. In some embodiments, the CD3ζ transmembrane domain results in a CAR that is incorporated into a natural T cell receptor.

[0050] The endodomain of a CAR is generally considered the "functional" end of the receptor. After antigen recognition by the antigen recognition region of the ectodomain, the CAR clusters and a signal is transmitted to the cell. In some embodiments, the endodomain is a CD3ζ endodomain, which contains three immunoreceptor tyrosine-based activation motifs (ITAMs). In this case, the ITAMs transmit activation signals to T cells after antigen binding, triggering a T cell immune response.

[0051] During the generation of CAR T cells, T cells are removed from a human subject, genetically engineered, and reintroduced into the patient to attack cancer cells. CAR T cells can be derived either from the patient's own blood (autologous) or from another healthy donor (allogeneic). Generally, CAR T cells are developed to be specific for antigens expressed on tumors that are not expressed in healthy cells.

[0052] In exemplary embodiments, the one or more molecular characteristics include one or more of gene expression, protein expression, mRNA expression, and copy number variation of the CAR T cell. In additional embodiments, the one or more molecular characteristics relate to T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

[0053] Preferably, molecular characteristics are determined using a CAR T characterization panel, using the molecular barcoding method NCOUNTER®, developed by NANOSTRING® (Seattle, WA). Exemplary characteristics of a CAR T characterization panel include the ability to examine the expression of about 500-1000 genes, preferably about 700-800 genes, including about 700, about 750, about 760, about 770, about 780, about 790, about 800, or about 850 human genes. The genes include eight of the elements of CAR T cell biology, including T cell activation, metabolism, exhaustion, TCR receptor diversity, and transgene expression.

[0054] Molecular characteristics are preferably determined using a non-enzymatic, non-amplified barcoding technology (e.g., NCOUNTER®) that allows for direct digital detection of molecules with a dynamic range of up to 6 logs. With a total hands-on time of only 15 minutes and data generation time of less than 24 hours, the workflow offers a productivity boost compared to either next-generation sequencing (NGS) or quantitative PCR (qPCR) approaches. Both of these can take days to weeks and involve library preparation, DNA synthesis, and amplification (all of which require additional hands-on time), potential user error, and reproducibility challenges introduced through the use of enzymes. Data generated from barcoding technology results in direct counts of molecules, does not require specialized bioinformaticians, and is amenable to simple analytical visualization and reporting through various analysis software or other customized reports.

[0055] The nCounter CAR-T Characterization Panel, which measures gene expression on the nCounter platform, was created specifically for use in the cell therapy field, further enabling characterization, optimization, and signature development, profiling various stages in manufacturing and enabling better control of the process, which in turn addresses the challenges surrounding consistent and reproducible production of cell therapies. The nCounter CAR-T Characterization Panel was created in collaboration with eight leading centers in the field of CAR T therapy and is designed for use across the entire CAR T workflow, enabling uniform and robust profiling of CAR T cells after leukapheresis, manufacturing product, and infusion. The customizable 780-gene expression panel incorporates content to measure eight essential elements of CAR T cell biology, including T cell activation, metabolism, exhaustion, and TCR receptor diversity, with optional customization to measure transgene expression.

[0056] The following table provides exemplary information available from the CAR T characterization panel (see nanostring.com; car-t-characterization panel): [Table 2]

[0057] Genes included in the CAR T characterization panel can include (see nanostring.com; car-t-characterization panel): [Table 3]

[0058] For CAR T characterization panels, sample inputs can include sorted T-cells (e.g., before the genetic modification step), CAR-T cells (e.g., after the final step of the automated process), CAR-T manufacturing products (e.g., cells during different stages of the automated process), whole blood, or nucleic acids.

[0059] Based on the information obtained from the molecular characterization, one or more optimizations can occur in the automated process. These optimizations can occur before, during, and / or after the genetic modification step of the automated process.

[0060] Optimization may include one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying the cell transduction procedure, modifying the vector for use in the transduction procedure, and modifying the cell isolation procedure.

[0061] Methods for optimizing automated processes include optimizing cell culture conditions prior to initiating the automated method, as well as using feedback from various sensors to assist in real-time modifications to growth conditions (e.g., gas concentrations, media conditions, temperature, pH, waste, and nutrient concentrations, etc.), in combination with information collected on molecular properties.

[0062] As described herein, optimizing one or more parameters of an automated cell engineering system preferably alters one or more molecular characteristics of the modified cell culture. That is, modifications made to the automated cell engineering system (e.g., changes to gas concentrations, media conditions, temperature, pH, waste, and nutrient concentrations, etc.) alter the molecular characteristics of the cell culture such that the optimized cell culture has improved properties. These improved properties may include, for example, higher antigen concentrations, more efficient or complete transduction, or higher cell density or cell count. Additional improved properties may manifest at the genetic level, including, for example, longer-lived cells or improved characteristics for specific patients.

[0063] In embodiments, the optimization process is a self-regulating process, i.e., a process that does not require input from an external (human) user and that, through various computer programs and conditions, can determine necessary modifications to the cell culture or other properties to optimize the automated process. In embodiments, the self-regulating process includes monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor. These self-regulating processes can also monitor one or more molecular properties as described herein, providing real-time feedback to the automated system either before the automated process begins, during the automated process, or after the process has finished.

[0064] As described herein, the use of these various sensors in a fully enclosed cell engineering system works in concert at various times and places within the system to provide optimization. For example, a self-regulating process can adjust (e.g., increase or decrease) 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 this monitoring.

[0065] 4 shows a process flow legend for an automated cell engineering system as described herein, including the positioning of one or more fluid pathways 440, preferably comprising various sensors (e.g., pH sensor 450, dissolved oxygen sensor 451), as well as sampling / sample ports 452, and various valves (control valve 453, bypass check valve 454), and silicone-based tubing components connecting the components. Also shown in FIG. 4 is the use of one or more hydrophobic or hydrophilic filters 455 or 456 within the flow paths of cassette 402, along with pump tubing 457 and bag / valve module 458. Also shown is the positioning of cell culture chamber 410 within the flow paths.

[0066] 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, cell age, and molecular characteristics of the cells (including CAR T cells) determined as described herein. These starting cell population characteristics can be input into a computer-controlled system before initiating the automated method, and the system will then make various initial modifications, such as oxygen and carbon dioxide concentrations, flow rates, incubation times, pH, etc., to optimize the method. In turn, monitoring of cellular processes allows for automated characterization of the progression of cell culture sequences from the starting population, allowing for case-by-case adjustments of conditions for optimized final cell culture properties.

[0067] While the optimization parameters and modifications described herein are generally applicable to the COCOON® automated cell engineering system, it should be understood that such optimization may be applied to other automated cell engineering systems. In general, controls such as medium flow rate, gas concentration, and pH are all parameters that may be controlled and optimized regardless of the type of automated cell engineering system utilized.

[0068] In an exemplary embodiment, the methods described herein generate at least about 50 million viable genetically modified immune cells. In preferred embodiments, the described methods generate at least about 100 million viable genetically modified immune cells, or at least about 200 million cells, at least about 300 million cells, at least about 400 million cells, at least about 500 million cells, at least about 600 million cells, at least about 700 million cells, at least about 800 million cells, at least about 1 billion cells, at least about 1.1 billion cells, at least about 1.2 billion cells, at least about 1.3 billion cells, at least about 1.4 billion cells, at least about 1.5 billion cells, at least about 1.6 billion cells, at least about 1.7 billion cells, at least about 1.8 billion cells, at least about 1.9 billion cells, at least about 2 billion cells, at least about 2.1 billion cells, at least about 2.2 billion cells, at least about 2.3 billion cells, at least about 2.4 billion cells, at least about 2.5 billion cells, at least about 2.6 billion cells, at least about 2.7 billion cells, at least about 2.8 billion cells, at least about 2.9 billion cells, or at least about 3 billion genetically modified immune cells. In some embodiments, the methods can be used to generate greater than about 3 billion genetically modified immune cells, such as, for example, 10 billion cells, 12 billion cells, or 15 billion cells. Suitably, but not exclusively, these genetically modified immune cells are CAR T cells.

[0069] As described herein, the genetically modified immune cell cultures produced by the present methods are preferably T cell cultures, including chimeric antigen receptor T (CAR T) cell cultures. In such embodiments, the vector utilized to generate such CAR T cells is a vector encoding a chimeric antigen receptor. Preferably, the immune cell cultures comprise peripheral blood mononuclear cells and / or purified T cells. In embodiments, the immune cell cultures comprise at least one accessory cell, preferably a monocyte or monocyte-derived cell. As described herein, in embodiments, the accessory cell comprises an antigen for the T cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0070] The methods described herein can also provide information regarding molecular characteristics of cell-based therapies before, during, or after genetic modification that are found to correlate with either low or high efficacy, or low or high toxicity or side effects. Thus, the methods will provide quality control information before, during, or after automated processes that can be used to determine whether a cell-based therapy should be administered to a patient, or alternatively, whether there is a high likelihood of problems when such a cell population is utilized.

[0071] In additional embodiments, information regarding the molecular characteristics of a cell-based therapy can also be determined after the prepared cell-based therapy has been administered to a patient. For example, the patient's blood can be drawn and the molecular characteristics of the desired cell type determined, allowing for improved monitoring of efficacy and increased insight into how clinical outcomes are linked to cell characteristics and automated cell engineering processes.

[0072] In further embodiments, provided herein is a method for assessing and optimizing cell quality for a cell-based therapy, the method comprising determining one or more molecular characteristics of a pre-modified cell culture; optimizing one or more parameters of an automated cell engineering system to alter one or more molecular characteristics of one of the pre-modified cell cultures; activating the pre-modified cell culture with an activation reagent to produce an activated cell culture; transducing the activated immune cell culture with a vector to produce a transduced cell culture; expanding the transduced cell culture; enriching the expanded cell culture; and recovering the enriched cell culture to produce a genetically modified cell culture.

[0073] As described herein, the method preferably includes determining one or more molecular characteristics of the cell culture during or after any one of the activation, transduction, expansion, enrichment, or recovery steps, and optimizing one or more parameters of any one of these steps to alter the one or more molecular characteristics of the cell culture.

[0074] As described herein, the one or more molecular characteristics preferably include gene expression, protein expression, mRNA expression, and copy number variation.

[0075] Exemplary cell cultures include immune cell cultures, such as natural killer cell cultures and cell cultures for neurodegenerative therapy. In a preferred embodiment, the immune cell culture is a T-cell culture, including a chimeric antigen receptor T (CAR T) cell culture. In such an embodiment, the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

[0076] Figure 5 illustrates a method for accessing cells in an automated cell engineering system, including methods described herein for producing various cell therapies. As shown, cassette 402, in which various processes (transduction, expansion, etc.) are performed, can be attached to syringe 502. This syringe can be used to take a sample of cells during any stage of the process (e.g., during or after one of activation, transduction, expansion, and / or harvest). This sample can then be used to analyze for various molecular properties described herein. Also shown in Figure 5 is a bag 504 that can be connected to the cassette if a larger cell sample is desired or needed.

[0077] Various optimization methods are described herein and may include one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying the cell transduction procedure, modifying the vector for use in the transduction procedure, and modifying the cell isolation procedure.

[0078] For example, if the molecular characteristics of the cell culture indicate that the culture will not achieve the growth necessary for the desired cell culture size, the cell engineering system can automatically increase 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 oxygenating component (i.e., silicone tubing).

[0079] In another example, if the molecular signature indicates that the cell culture is growing too rapidly (e.g., possible cell overcrowding may lead to undesirable characteristics), the cell engineering system can automatically reduce the temperature of the cell culture to maintain a constant growth rate of the cells (or, if desired, an exponential growth rate). In still further embodiments, based on the analyzed molecular signature, the cell engineering system can automatically adjust a cell nutrient feeding schedule based on the molecular signature (i.e., providing fresh medium and / or nutrients to the cell culture).

[0080] In a further embodiment, provided herein is a method for assessing and optimizing the cell quality of a chimeric antigen receptor T (CAR T) cell culture. The method preferably includes determining one or more molecular characteristics of a pre-modified T-cell culture, optimizing one or more parameters of an automated cell engineering system to alter one or more molecular characteristics of one of the pre-modified T-cell cultures, activating the pre-modified T-cell culture with an activation reagent to produce an activated T-cell culture, transducing the activated T-cell culture with a vector encoding a chimeric antigen receptor to produce a CAR T-cell culture, expanding the CAR T-cell culture, enriching the expanded CAR T-cell culture, and recovering the enriched CAR T-cell culture.

[0081] Suitably, the method comprises determining one or more molecular characteristics of the CAR T-cell culture during or after any one of the activation, transduction, expansion, enrichment or harvesting steps, and optimizing one or more parameters of any one of these steps to alter one or more molecular characteristics of the CAR T-cell culture.

[0082] As described herein, preferably, the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity. In an exemplary embodiment, the one or more molecular characteristics include gene expression, protein expression, mRNA expression, and copy number variation.

[0083] As described herein with respect to CAR T characterization panels, preferably the method comprises determining the expression of at least about 500 genes, preferably at least about 700 genes, including when the expression of about 700 to 800 genes is determined (including the expression of about 750, 760, 770, 780, 790, or 800 genes).

[0084] As described herein, optimization of the automated process preferably includes one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying the cell transduction procedure, modifying the vector for use in the transduction procedure, and modifying the cell isolation procedure.

[0085] The methods described herein are preferably optimized to generate at least about 100 million viable CAR T-cells, including at least about 2 billion viable CAR T-cells.

[0086] Exemplary starting T-cell cultures as described herein preferably include peripheral blood mononuclear cells and / or purified T-cells.

[0087] In an exemplary embodiment, the T-cell culture includes at least one accessory cell, which may be a monocyte or a monocyte-derived cell.

[0088] Preferably, the accessory cells comprise antigens for the T-cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0089] As described herein, in embodiments, the activation reagent comprises an antibody or a dendritic cell. Preferably, the antibody is immobilized on a surface, including the surface of a bead. Preferably, the antibody is a soluble antibody, including at least one of an anti-CD3 antibody and an anti-CD28 antibody.

[0090] In exemplary embodiments, the transducing comprises viral infection, electroporation, membrane disruption, or a combination thereof.

[0091] In a preferred embodiment, the vector used in transduction is a lentiviral vector or a retrovirus.

[0092] Exemplary process flow for assessing and optimizing CAR T production Leukapheresis of healthy donors and patients is used to manufacture CAR-T cells in an automated cell engineering system.

[0093] At multiple points throughout cell therapy manufacturing, cell samples are taken and characterized with the CAR-T cell panel.

[0094] Data from all testing will be analyzed to generate knowledge to continually improve the detection panel (potential detection panel modifications) and understand how to translate cell characteristics into tools that enable further manufacturing process optimization.

[0095] Through collaborations with medical centers, databases will be created to gain insight into how clinical outcomes can be linked to cell characteristics, along with improved manufacturing processes aimed at improving clinical efficacy and reducing treatment adverse events.

[0096] Improved CAR-T cell panels combined with translating cell characteristics and optimizing automated manufacturing processes will provide an analytical package that can be delivered.

[0097] The following sections provide a description of the methods used in the automated cell engineering system for the generation of CAR T cells.

[0098] T cell activation. In some embodiments, the immune cell culture generated by the methods described herein is a CAR T cell culture. CAR T cell cultures 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 co-receptor that serves to activate both cytotoxic T cells (e.g., CD8+ naive T cells) and T helper cells (e.g., CD4+ naive T cells). Generally, T cell activation follows a two-signal model, requiring stimulation of the TCR / CD3 complex and a costimulatory receptor. T cell activation is further described, for example, in Kochenderfer 2015; Kalos 2011.

[0099] Without costimulatory signals, cells are prone to anergy and become unresponsive. Therefore, costimulation of T cells can be important for T cell proliferation, differentiation, and survival. Non-limiting examples of costimulatory molecules for T cells include CD28, a receptor for CD80 and CD86 on the membrane of APCs, and CD278 or ICOS (inducible T-cell costimulatory factor), a CD28 superfamily molecule expressed on activated T cells that interacts with ICOS-L. Thus, in some embodiments, the costimulatory molecule is CD28. In other embodiments, the costimulatory molecule is ICOS. In vivo, costimulatory signals can be provided by B7 molecules on APCs that bind to the CD28 receptor on T cells. B7 is a surface transmembrane protein found on activated APCs that can interact with CD28 or CD152 surface proteins on T cells to generate costimulatory signals. Thus, in some embodiments, the costimulatory molecule is B7. Costimulatory receptors are further described, for example, in Lafferty 1975; Harding 1992; Clavreul 2000; Charron 2015; Fathman 2007; Greenwald 2005. Costimulation is further described, for example, in Carpenter 2000; Andris 2004. B7 molecules are further described, for example, in Fleischer 1996; Schwartz 2003.

[0100] Various methods of activation are utilized in vitro to simulate T cell activation. In embodiments, T cell cultures are activated with an activation reagent. In further embodiments, the activation reagent is an antigen-presenting cell (APC). In yet further embodiments, the activation reagent is a dendritic cell. Dendritic cells are APCs that process antigen and present it on their cell surface to T cells. In some embodiments, the activation reagent is co-cultured with the T cell culture. Co-culturing may require separate purification and culturing of a second cell type, which can increase labor requirements and sources of variability. Therefore, in some embodiments, alternative activation methods are used.

[0101] In some embodiments, the activation reagent is an antibody. In some embodiments, the cell culture is activated with an antibody bound to a surface, including a polymeric surface, including beads. In further embodiments, one or more antibodies are anti-CD3 and / or anti-CD28 antibodies. For example, the beads can be magnetic beads, such as DYNABEADS®, coated with anti-CD3 and anti-CD28. The anti-CD3 and anti-CD28 beads can preferably provide a stimulatory signal to support T cell activation. See, e.g., Riddell 1990; Trickett 2003.

[0102] 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 immunoglobulin IgG2a isotype that targets CD3. Thus, in some embodiments, the soluble anti-CD3 antibody is OKT3. OKT3 is further described in, for example, Dudley 2003; Manger 1985; Ceuppens 1985; Van Wauwe 1980; Norman 1995.

[0103] In some embodiments, costimulatory signals for T cell activation are provided by accessory cells. Accessory cells may contain, for example, Fc receptors that allow cross-linking of CD3 antibodies with the TCR / CD3 complex on T cells. In some embodiments, the cell culture is a mixed population of peripheral blood mononuclear cells (PBMCs). PBMCs may contain accessory cells that can support T cell activation. For example, a CD28 costimulatory signal may be provided by B7 molecules present on monocytes within PBMCs. Accordingly, in some embodiments, accessory cells comprise monocytes or monocyte-derived cells (e.g., dendritic cells). In additional embodiments, accessory cells comprise 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; Austin 1987; and Tax 1983.

[0104] As described herein, the activation reagent determines the phenotype of the generated CAR T cells and can promote a desired phenotype. In some embodiments, the activation reagent determines the ratio of T cell subsets, i.e., CD4+ helper T cells to CD8+ cytotoxic T cells. Cytotoxic CD8+ T cells are typically responsible for killing cancer cells (i.e., anti-tumor response), infected cells (e.g., by a virus), or otherwise damaged cells. CD4+ T cells typically produce cytokines, help modulate the immune response, and in some cases may support cell lysis. CD4+ cells activate APCs, which then prime naive CD8+ T cells for an anti-tumor response. Accordingly, 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 can be, for example, a predetermined ratio of CD8+ cells to CD4+ cells. In some embodiments, the ratio of CD8+ cells to CD4+ cells in the population of CAR T cells is about 1: 1, about 0.25: 1, or about 0.5: 1. In other embodiments, the ratio of CD8+ cells to CD4+ cells in the population of CAR T cells is about 2: 1, about 3: 1, about 4: 1, or about 5: 1.

[0105] The method by which a given phenotype is generated preferably involves determining the levels of one or more molecular characteristics, i.e., CD8 and CD4, and optimizing by either selecting such cells or modifying production parameters to drive the generation of such cells.

[0106] In embodiments, the activation reagent is removed from the activated T cell culture after the activation step. The activation reagent, e.g., anti-CD3 antibody and / or anti-CD28 antibody, may be present in the cell culture medium. Thus, in some embodiments, the cell culture medium containing the activation reagent, e.g., anti-CD3 antibody and / or anti-CD28 antibody, is removed from the activated T cell culture after the activation step. In some embodiments, removing the activation reagent includes removing soluble antibodies. For example, soluble antibodies can be removed by replacing the cell culture medium. Soluble antibodies can also be removed by affinity methods specific for the soluble antibodies. In other embodiments, removing the activation reagent includes removing beads containing the antibodies. Bead removal can include, for example, filtering the beads or removing them with a magnet.

[0107] Transduction of activated T cells. In some embodiments, the genetically modified immune cell culture is an activated T cell culture transduced with a vector encoding a chimeric antigen receptor to generate a transduced T cell culture. In some embodiments, the transduction comprises viral infection, transposon, mRNA transfection, electroporation, or a combination thereof. In some embodiments, the transduction comprises electroporation. Accordingly, in embodiments, the cell engineering system comprises an electroporation system or electroporation unit. In additional embodiments, the transduction comprises viral infection. The vector can be, for example, a viral vector, such as a lentiviral vector, a gammaretroviral vector, an adeno-associated viral vector, or an adenoviral vector. In embodiments, the transduction comprises introducing the viral vector into activated T cells of the cell culture. In additional embodiments, the vector is delivered as a viral particle.

[0108] In some embodiments, the transduction step comprises transducing activated T cells with a lentiviral vector, wherein the lentiviral vector is introduced at a multiplicity of infection (MOI) of about 0.5 to about 50, about 0.5 to about 30, or about 0.5 to about 20. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 8. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 6. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 4. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5 to about 2. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.6 to about 1.5. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.7 to about 1.3. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.8 to about 1.1. In some embodiments, the lentiviral vector is introduced at an MOI of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.

[0109] In some embodiments, after the activation step, cell culture medium from the T cell culture is removed, and then the medium is mixed with the vector (e.g., lentiviral vector) and evenly distributed to the cells. In some embodiments, the removed cell culture medium is used to dilute and evenly deliver the vector to the activated T cell culture. The even distribution and resulting uniform exposure of the vector (e.g., lentiviral vector) in the T cell culture improves transduction efficiency. In some embodiments, the volume of the cell culture is reduced after activation and before the addition of the vector. The reduced volume may 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, meaning that the cells generally remain in the same region of the chamber (e.g., the bottom of the cell culture chamber) while the activation reagent or vector is provided to the cells. This can facilitate even distribution and uniform exposure of the activation reagent and / or vector to the cells, thus improving activation and / or transduction efficiency.

[0110] Expansion of transduced T cells. In some embodiments, transduced T cell cultures (or other cell therapy or immune cell cultures) are expanded to a predetermined culture size (i.e., cell number). The predetermined culture size may contain a sufficient number of cells suitable for clinical use, i.e., transfusion to patients, research and development work, etc. In some embodiments, a clinical or therapeutic dose of CAR T cells for administration to patients is about 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, approximately 10 8 cells, approximately 10 9 cells, or approximately 10 10cells. 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 about 0.1 L to about 5 L, about 0.1 L to about 2 L, or about 0.2 L to about 2 L. In some embodiments, the transduced T cell culture is expanded to a total volume of about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L, or about 1.0 L. Volumes can also be varied throughout the process, as needed, based on the stage of the cell generation process. In some embodiments, the predetermined culture size is input by a user of the cell engineering system. The user can input the predetermined culture size as the desired cell count to be generated (e.g., 10 10 CAR T cells), or a predetermined culture size can be input as the 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 methods described herein is at least about 100 million (i.e., 1*10 6 cells), or at least about 300 million, at least about 500 million, at least about 600 million, at least about 700 million, at least about 800 million, at least about 900 million, at least about 1 billion (i.e., 1*10 9 ), at least about 1.1 billion, at least about 1.2 billion, at least about 1.3 billion, at least about 1.4 billion, at least about 1.5 billion, at least about 1.6 billion, at least about 1.7 billion, at least about 1.8 billion, at least about 1.9 billion, at least about 2 billion (i.e., 2*10 9cells), including 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.

[0111] In some embodiments, expanding the transduced T cell culture comprises at least one round of feeding, washing, monitoring, and sorting the transduced T cell culture. Feeding the cell culture may comprise supplementing the cell culture with medium and / or additional nutrients. Washing the cell culture may comprise removing spent medium (i.e., medium that is depleted of nutrients and / or contains cellular waste products) and replenishing the cell culture with fresh medium. Monitoring the cell culture may comprise monitoring the temperature, pH, glucose, oxygen level, carbon dioxide level, and / or optical density of the cell culture. Sorting the cell culture may comprise selecting cells with desired characteristics, such as, for example, 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 several rounds of feeding, washing, monitoring, and / or sorting the transduced T cell culture to achieve a predetermined culture size. In some embodiments, the cell engineering system performs at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or at least 100 rounds of feeding, washing, monitoring, and / or sorting the transduced T cell cultures to achieve a predetermined culture size.

[0112] In embodiments, one or more of nutritional supply, washing, and monitoring can be removed, or the order of events can be altered depending on the desired cell phenotype or cell number, etc.

[0113] In embodiments, monitoring includes monitoring with a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor. Accordingly, in some embodiments, the cell engineering system includes one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and / or an optical density sensor. In additional embodiments, the cell engineering system is configured to adjust the temperature, pH, glucose, oxygen level, carbon dioxide level, and / or optical density of the cell culture based on a predetermined culture size. For example, if the cell engineering system detects that the current oxygen level of the cell culture is too low to achieve the growth required for the desired cell culture size, the cell engineering system will automatically increase the oxygen level of the cell culture, for example, by introducing oxygenated cell culture medium, replacing the cell culture medium with oxygenated cell culture medium, or flowing the cell culture medium through an oxygenation component (i.e., silicone tubing). In another example, 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., possible cell overcrowding may lead to undesirable characteristics), the cell engineering system will automatically lower the temperature of the cell culture to maintain a constant growth rate of the cells (or, if desired, an exponential growth rate). In still further embodiments, the cell engineering system automatically adjusts the schedule of cell nutrient supply (i.e., providing fresh medium and / or nutrients to the cell culture) based on cell growth rate and / or cell number, or other monitored factors such as pH, oxygen, glucose, etc. The cell engineering system may be configured to store medium (and other reagents, such as wash solutions) in a low-temperature chamber (e.g., 4°C or -20°C) and 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.

[0114] In embodiments, washing includes washing the cells by filtration or sedimentation. In embodiments, sorting includes mixing the cell culture with one or more sorting reagents. The sorting reagents can be beads, e.g., magnetic beads, specific to the desired cell type, and the cells bound to the beads are then separated from unbound cells, e.g., by passing through a magnetic chamber. For example, the sorting beads contain antibodies specific to the desired cell type, e.g., anti-CD8 or anti-CD4 antibodies. Sorting can also be performed by filtration to remove or select certain cell types based on size. Cell sorting by plastic adhesion (i.e., starting with cells in one chamber, unwanted cells stick to the surface, and then the desired cells, still in suspension, are moved to another chamber) can also be used.

[0115] Concentration of Expanded Cultures. In some embodiments, expanded T cell cultures (or other cell therapies, including other immune cell cultures) are concentrated to a predetermined concentration. The predetermined concentration is a volume that can be suitably infused into a patient. For example, expanded T cell cultures can be concentrated to about 1 ml, about 2 ml, about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, or about 100 ml. In some embodiments, concentration is achieved by centrifugation. In some embodiments, concentration is achieved by filtration. In some embodiments, filtration is ultrafiltration and / or diafiltration. In some embodiments, the predetermined concentration is input by a user of the cytotechnology system. In other embodiments, the predetermined concentration is determined by the cell engineering system based on different parameter input by a user, such as the number or volume of clinical or therapeutic doses to be produced, or the number of cells to be produced. In some embodiments, the cell engineering system automatically adjusts the volume or number of clinical or therapeutic doses produced based on the input parameters. In some embodiments, the cell engineering system automatically adjusts centrifugation (e.g., centrifugation speed, duration) or filtration (e.g., filter size, volume, duration) parameters based on the predetermined concentration.

[0116] Sedimentation based on the port location and design of the chamber can also be utilized, i.e., the fluid volume within the chamber can be reduced to approximately 0.5 mL without removing cells.

[0117] Harvesting CAR T cell cultures. In some embodiments, enriched T cell cultures (or other cell therapies involving other immune cell cultures) are harvested, suitably to generate chimeric antigen receptor (CAR) T cell cultures. In some embodiments, harvesting involves agitation, fluid flow, and washing of the CAR T cells. In some embodiments, harvesting involves separating cells from unwanted products, including, for example, waste products of cells, sorting reagents such as beads (e.g., antibody-containing beads and / or beads used to separate cells), or excess viral vectors. In some embodiments, harvesting involves even distribution of the CAR T cells in one or more flasks, vials, or vessels. In some embodiments, harvesting involves resuspending the CAR T cells in a formulation reagent, for example, a solution that stabilizes the CAR T cells for long-term storage. In some embodiments, harvesting involves cryopreservation of the CAR T cells.

[0118] Further Downstream Steps. In some embodiments, the CAR T cells (or other cell therapies comprising other immune cells) undergo further downstream processing before therapeutic use in a patient. For example, the CAR T cells may be filtered by sterile filtration to remove potential viral particle remnants. After sterile filtration, the CAR T cells may undergo at least one or more concentration steps before being packaged into one or more vials, flasks, tanks, or containers. The packaged CAR T cells may be subjected to quality assessment and / or quality control testing as described herein. In some embodiments, the CAR T cells undergo minimal downstream processing before administration to a patient. For example, in some embodiments, the recovered CAR T cells are not cryopreserved, but are transferred to a patient within a short period of time after collection. Avoiding a cryopreservation step may increase cell viability.

[0119] Additional Exemplary Embodiments Embodiment 1 is a method for assessing and optimizing cell quality for a cell-based therapy, comprising determining one or more molecular characteristics of a pre-modified cell culture; genetically modifying the cell culture via an automated cell engineering system; determining one or more molecular characteristics of the modified cell culture during and after the genetic modification; and optimizing one or more parameters of the automated cell engineering system to alter the one or more molecular characteristics of the modified cell culture.

[0120] Embodiment 2 includes the method of embodiment 1, wherein the one or more molecular characteristics are selected from the group consisting of gene expression, protein expression, mRNA expression, and copy number variation.

[0121] Embodiment 3 includes the method of embodiment 1 or embodiment 2, wherein the cell culture is an immune cell culture, a natural killer cell culture, or a cell culture for neurodegenerative therapy.

[0122] Embodiment 4 includes the method of embodiment 3, wherein the immune cell culture is a T-cell culture.

[0123] Embodiment 5 includes the method of embodiment 4, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0124] Embodiment 6 includes the method of embodiment 5, wherein the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

[0125] Embodiment 7 includes the method of any one of embodiments 1 to 6, wherein the optimizing in (d) occurs before, during, and / or after the genetic modification.

[0126] Embodiment 8 includes the method of any one of embodiments 1-7, wherein optimizing includes one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying the cell transduction procedure, modifying the vector for use in the transduction procedure, and modifying the cell isolation procedure.

[0127]

[0013] Embodiment 9 is a method for evaluating and optimizing cell quality for a cell-based therapy, comprising: determining one or more molecular characteristics of a pre-modified cell culture; optimizing one or more parameters of an automated cell engineering system to alter one or more molecular characteristics of one of the pre-modified cell cultures; activating the pre-modified cell culture with an activation reagent to produce an activated cell culture; transducing the activated immune cell culture with a vector to produce a transduced cell culture; expanding the transduced cell culture; (e) enriching the expanded cell culture; and (f) recovering the enriched cell culture to produce a genetically modified cell culture; during or after any one of steps (c)-(g), determining one or more molecular characteristics of the cell culture; and optimizing one or more parameters of any one of steps (c)-(g) to alter one or more molecular characteristics of the cell culture.

[0128] Embodiment 10 includes the method of embodiment 9, wherein the one or more molecular characteristics are selected from the group consisting of gene expression, protein expression, mRNA expression, and copy number variation.

[0129] Embodiment 11 includes the method of embodiment 9 or embodiment 10, wherein the cell culture is an immune cell culture, a natural killer cell culture, or a cell culture for neurodegenerative therapy.

[0130] Embodiment 12 includes the method of embodiment 11, wherein the immune cell culture is a T-cell culture.

[0131] Embodiment 13 includes the method of embodiment 12, wherein the T-cell culture is a chimeric antigen receptor T (CAR T) cell culture.

[0132] Embodiment 14 includes the method of embodiment 13, wherein the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

[0133] Embodiment 15 includes the method of any one of embodiments 9-14, wherein optimizing includes one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying the cell transduction procedure, modifying the vector for use in the transduction procedure, and modifying the cell isolation procedure.

[0134] Embodiment 16 is a method for assessing and optimizing cell quality of a chimeric antigen receptor T (CAR T) cell culture, comprising determining one or more molecular characteristics of the pre-modification T-cell culture; optimizing one or more parameters of an automated cell engineering system to alter one or more molecular characteristics of one of the pre-modification T-cell cultures; activating the pre-modification T-cell culture with an activation reagent to produce an activated T-cell culture; transducing the activated T-cell culture with a vector encoding a chimeric antigen receptor to produce a CAR T-cell culture; expanding the CAR T-cell culture; (e) enriching the expanded CAR T-cell culture; and (f) recovering the enriched CAR T-cell culture; and during or after any one of steps (c)-(g), determining one or more molecular characteristics of the CAR T-cell culture; and optimizing one or more parameters of any one of steps (c)-(g) to alter one or more molecular characteristics of the CAR T-cell culture.

[0135] Embodiment 17 includes the method of embodiment 16, wherein the method generates at least about 100 million viable CAR T-cells.

[0136] Embodiment 18 includes the method of embodiment 16, wherein the method generates at least about 2 billion viable CAR T-cells.

[0137] Embodiment 19 includes the method of any one of embodiments 16 to 19, wherein the T-cell culture comprises peripheral blood mononuclear cells and / or purified T-cells.

[0138] Embodiment 20 includes the method of any one of embodiments 16 to 19, wherein the T-cell culture comprises at least one accessory cell.

[0139] Embodiment 21 includes the method of embodiment 20, wherein the accessory cells comprise monocytes or monocyte-derived cells.

[0140] Embodiment 22 includes the method of embodiment 20, wherein the accessory cells comprise an antigen for a T-cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

[0141] Embodiment 23 includes the method of any one of embodiments 16 to 22, wherein the activating reagent comprises an antibody or a dendritic cell.

[0142] Embodiment 24 includes the method of embodiment 23, wherein the antibody is immobilized on a surface.

[0143] Embodiment 25 includes the method of embodiment 24, wherein the surface is the surface of a bead.

[0144] Embodiment 26 includes the method of embodiment 23, wherein the antibody is a soluble antibody.

[0145] Embodiment 27 includes the method of embodiment 23, wherein the antibody comprises at least one of an anti-CD3 antibody and an anti-CD28 antibody.

[0146] Embodiment 28 includes the method of any one of embodiments 16-27, wherein transducing includes viral infection, electroporation, membrane disruption, or a combination thereof.

[0147] Embodiment 29 includes the method of any one of embodiments 16 to 28, wherein the vector is a lentiviral vector or a retrovirus.

[0148] Embodiment 30 includes the method of any one of embodiments 16 to 29, wherein the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

[0149] Embodiment 31 includes the method of any one of embodiments 16 to 30, wherein the one or more molecular characteristics are selected from the group consisting of gene expression, protein expression, mRNA expression, and copy number variation.

[0150] Embodiment 32 includes the method of embodiment 31, wherein the expression of at least about 500 genes is determined.

[0151] Embodiment 33 includes the method of embodiment 31, wherein the expression of at least about 700 genes is determined.

[0152] Embodiment 34 includes the method of embodiment 31, wherein the expression of about 780 genes is determined.

[0153] Embodiment 35 includes the method of any one of embodiments 16-34, wherein optimizing includes one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying the cell transduction procedure, modifying the vector for use in the transduction procedure, and modifying the cell isolation procedure.

[0154] Embodiment 36 is a method for assessing and optimizing cell quality of a cell culture, comprising determining one or more molecular characteristics of a pre-modified cell culture; genetically modifying the cell culture via an automated cell engineering system; determining one or more molecular characteristics of the modified cell culture during and after the genetic modification; and optimizing one or more parameters of the automated cell engineering system to alter the one or more molecular characteristics of the modified cell culture.

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[0156] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.

[0157] Although particular embodiments have been illustrated and described herein, it is to be understood that the claims should not be limited to the specific forms or arrangements of parts described and shown. Although exemplary embodiments are disclosed and specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0158] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A method for assessing and optimizing cell quality for a cell-based therapy, comprising: (a) determining one or more molecular characteristics of the pre-modified cell culture; (b) genetically modifying the cell culture via an automated cell engineering system; (c) determining the one or more molecular characteristics of the modified cell culture during and after the genetic modification; (d) optimizing one or more parameters of the automated cell engineering system to alter the one or more molecular characteristics of the modified cell culture.

2. 2. The method of claim 1, wherein the one or more molecular features are selected from the group consisting of gene expression, protein expression, mRNA expression, and copy number variation.

3. 3. The method of claim 1 or 2, wherein the cell culture is an immune cell culture, a natural killer cell culture, or a cell culture for neurodegenerative therapy.

4. The method of claim 3, wherein the immune cell culture is a T-cell culture.

5. 5. The method of claim 4, wherein the T cell culture is a chimeric antigen receptor T (CAR T) cell culture.

6. The method of claim 5, wherein the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

7. 7. The method of any one of claims 1 to 6, wherein the optimizing in (d) occurs before, during, and / or after the genetic modification.

8. 8. The method of any one of claims 1-7, wherein the optimizing comprises one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying a cell transduction procedure, modifying a vector for use in the transduction procedure, and modifying a cell isolation procedure.

9. 1. A method for assessing and optimizing cell quality for cell-based therapy, comprising: (a) determining one or more molecular characteristics of the pre-modified cell culture; (b) optimizing one or more parameters of an automated cell engineering system to alter the one or more molecular characteristics of one of the pre-modified cell cultures; (c) activating the pre-modified cell culture with an activation reagent to produce an activated cell culture; (d) transducing the activated immune cell culture with a vector to generate a transduced cell culture; (e) growing the transduced cell culture; (f) concentrating the expanded cell culture of (e); (g) recovering the enriched cell culture of (f) to produce a genetically modified cell culture; (h) determining one or more molecular characteristics of said cell culture during or after any one of steps (c)-(g); and (i) optimizing one or more parameters of any one of steps (c) to (g) to alter the one or more molecular properties of the cell culture.

10. 10. The method of claim 9, wherein the one or more molecular characteristics are selected from the group consisting of gene expression, protein expression, mRNA expression, and copy number variation.

11. 11. The method of claim 9 or 10, wherein the cell culture is an immune cell culture, a natural killer cell culture, and a cell culture for neurodegenerative therapy.

12. The method of claim 11, wherein the immune cell culture is a T-cell culture.

13. 13. The method of claim 12, wherein the T-cell culture is a chimeric antigen receptor T (CAR T) cell culture.

14. 14. The method of claim 13, wherein the one or more molecular characteristics include T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

15. 15. The method of any one of claims 9 to 14, wherein the optimizing comprises one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying a cell transduction procedure, modifying a vector for use in the transduction procedure, and modifying a cell isolation procedure.

16. 1. A method for assessing and optimizing cell quality of a chimeric antigen receptor T (CAR T) cell culture, comprising: (a) determining one or more molecular characteristics of the pre-modified T-cell culture; (b) optimizing one or more parameters of an automated cell engineering system to alter said one or more molecular characteristics of one of said pre-modified T-cell cultures; (c) activating the pre-modified T-cell culture with an activation reagent to produce an activated T-cell culture; (d) transducing the activated T-cell culture with a vector encoding a chimeric antigen receptor to generate a CAR T-cell culture; (e) expanding said CAR T-cell culture; and (f) enriching the expanded CAR T-cell culture of (e); and (g) harvesting the enriched CAR-T cell culture of (f); and (h) during or after any one of steps (c)-(g), determining one or more molecular characteristics of said CAR T-cell culture; (i) optimizing one or more parameters of any one of steps (c)-(g) to alter said one or more molecular characteristics of said CAR T-cell culture.

17. 17. The method of claim 16, wherein the method generates at least about 100 million viable CAR T-cells.

18. 17. The method of claim 16, wherein the method generates at least about 2 billion viable CAR T-cells.

19. The method of any one of claims 16 to 19, wherein the T-cell culture comprises peripheral blood mononuclear cells and / or purified T-cells.

20. The method of any one of claims 16 to 19, wherein the T-cell culture comprises at least one accessory cell.

21. 21. The method of claim 20, wherein the accessory cells comprise monocytes or monocyte-derived cells.

22. 21. The method of claim 20, wherein the accessory cells comprise an antigen for a T-cell receptor, including CD28, CD40, CD2, CD40L, and / or ICOS.

23. The method of any one of claims 16 to 22, wherein the activating reagent comprises an antibody or a dendritic cell.

24. 24. The method of claim 23, wherein the antibody is immobilized on a surface.

25. 25. The method of claim 24, wherein the surface is the surface of a bead.

26. 24. The method of claim 23, wherein the antibody is a soluble antibody.

27. 24. The method of claim 23, wherein the antibody comprises at least one of an anti-CD3 antibody and an anti-CD28 antibody.

28. 28. The method of any one of claims 16 to 27, wherein the transducing comprises viral infection, electroporation, membrane disruption, or a combination thereof.

29. The method of any one of claims 16 to 28, wherein the vector is a lentiviral vector or a retrovirus.

30. 30. The method of any one of claims 16 to 29, wherein the one or more molecular characteristics comprise T-cell activation, metabolism, exhaustion, and T-cell receptor diversity.

31. 31. The method of any one of claims 16 to 30, wherein the one or more molecular characteristics are selected from the group consisting of gene expression, protein expression, mRNA expression, and copy number variation.

32. 32. The method of claim 31, wherein the expression of at least about 500 genes is determined.

33. 32. The method of claim 31, wherein the expression of at least about 700 genes is determined.

34. 32. The method of claim 31, wherein the expression of about 780 genes is determined.

35. 35. The method of any one of claims 16-34, wherein the optimizing comprises one or more of increasing or decreasing the flow rate of the cell culture medium, increasing or decreasing the oxygen concentration, increasing or decreasing the carbon dioxide concentration, increasing or decreasing the glucose level, increasing or decreasing the temperature of cell growth, increasing or decreasing the pH of the cell culture medium, modifying a cell transduction procedure, modifying a vector for use in the transduction procedure, and modifying a cell isolation procedure.

36. 1. A method for assessing and optimizing cell quality of a cell culture, comprising: (a) determining one or more molecular characteristics of the pre-modified cell culture; (b) genetically modifying the cell culture via an automated cell engineering system; (c) determining the one or more molecular characteristics of the modified cell culture during and after the genetic modification; (d) optimizing one or more parameters of the automated cell engineering system to alter the one or more molecular characteristics of the modified cell culture.

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