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Problems and challenges in the production of CAR-T cells
Problems and challenges in the production of CAR-T cells. This article discusses current and novel CART cell processing methods and the quality control systems required by the growing clinical needs of this new therapy.
1. CAR T cell introduction
CAR is a synthetic protein that can be expressed on the surface of T cells after genetic engineering. It contains an extracellular binding domain (usually a single-chain variable fragment targeting tumor cell surface antigen), a hinge region, a transmembrane domain and one or more intracellular signaling structures for T cell activation Domain . For any given cell surface antigen, CAR specificity can be easily designed in the laboratory .
CART cells are “living drugs” that have the ability to migrate, proliferate and kill target cells expressing homologous antigens in the body. A series of early studies on CART cells of B cell hematological malignancies against the B cell antigen CD19 have provided unprecedented clinical responses [3-7]. There have been more than 400 clinical trials of CART cells registered in clinical trials.gov all over the world, and most of them are currently focused on the treatment of B-cell malignancies [8,9].
This field is developing rapidly, and CART cells can now be used to treat other hematological malignancies, such as multiple myeloma [10-12], Hodgkin lymphoma  and T cell lymphoma . Many groups are exploring whether there are solid tumors in CART cells [15,16]. The academic center has been able to run GMP-compliant CART manufacturing for small clinical studies with limited infrastructure and countless logistics and financial challenges. When planning service delivery, one must consider costs, regulatory requirements, personnel resources and training, equipment/storage capabilities, and requirements for blood separation equipment and stem cell laboratories [17,18].
This review focuses on the issues and challenges related to CART cell manufacturing, from the GMP capabilities of the manufacturing laboratory to the release of the final product, step by step throughout the entire process. Discussed the key quality indicators related to each stage of the CART cell manufacturing process, including the current consensus on release testing and the increasingly stringent requirements for performing CART cell efficacy testing. Ultimately, the goal is to ensure the release of standardized, safe and effective CART cell “drugs” for patients.
Table 1 lists the basic quality system components of products manufactured in GMP facilities used for early clinical trials. Optimizing the workflow of the GMP facility is necessary to meet the current clinical needs for CART cells and the multi-directional flow of personnel, materials, waste and waste. Products can help adapt to election campaigns. Reliable risk assessment and standard operating procedures (SOP) must be used to prevent cross-contamination [20,25].
2. Manufacture CAR T cells that meet GMP requirements
CART cells produced for clinical use comply with GMP standards [19,20]. GMP regulations define a system through which effective products can be safely produced in accordance with standardized methods under strict control, replicable and auditable conditions . Strict quality control systems are applied to the procurement and use of raw materials, reagents and consumables, equipment and methods in the process, and the final “drug” product [20-22].
2.1 CAR T cell manufacturing method: general principles
The ability of CART cells to expand in vivo (the “adaptability” of T cells) is related to the anti-tumor response  and may be affected by the manufacturing method used. Efforts to optimize and quality control the various elements of the manufacturing process are desirable.
Although the practices and technologies vary between centers, all CART cells are manufactured in a closed or functionally closed process to reduce the risk of contamination and share the common steps shown in Figure 1. Under GMP conditions, the average production time of CART cells is 12 days (range (7,19 days) [9,19], and in order to prevent exhausted cell phenotypes, there is a move towards shorter ex vivo processing (6 days) One step.
Figure 1. Flow chart of standard elements for CAR T cell manufacturing.
There are many problems and challenges in the manufacture of CART cells, and it is best to check the manufacturing process by dividing it into individual components. This is summarized in Table 2 and detailed in the following sections.
2.2 T cell harvesting using leukocyte separation: current practice and quality control
The key raw material for making CART cells is CD3+ T cells derived from non-mobilized leukocyte separation. In a functionally closed system, monocytes (MNC) are separated from anticoagulated whole blood according to a density gradient . Approved devices include COBESpectra, Spectra Optia (TerumoBCT Inc.) and Amicus cell separator (Fenwal Inc./Fresenius KabiAG).
The optimal CD3+ T cell blood separation parameters for downstream CAR T cell production are not well described. The low yield of CD3+ T cell apheresis will make CART cell expansion difficult in vitro. It is foreseeable that low CD3+ absolute vs. cell number and/or high proportion of natural killer (NK) cells and blasts have impaired the peripheral blood cell count before plasma exchange. It is common in patients with toxic chemotherapy.
Patients with lymphopenia can benefit from prolonged blood sampling to improve T cell production. In University College London (abbreviated as UCL, the unit where the author works), the absolute lymphocyte count <0.5×109/L was upgraded from the standard 2 times the single blood collection yield to 2.5 times the volume of blood collection to increase the CD3+ obtained The number of T cells . In a CART cell center in the United States, two apheresis indicators for apheresis of 109 CD3+ T cells are defined (the lowest threshold is 0.6 109 CD3+ T cells). These goals were achieved in 77% and 97% of patients, respectively, and in most cases, the number of cells expanded sufficiently to produce a product that met the release criteria .
From a technical point of view, lymphopenia uses conventional blood separation methods to narrow the MNC layer and increase the difficulty of harvesting a pure lymphocyte population . To solve this problem, TerumoBCT developed SpectraOptia, which uses a color coding system to separate specific parts of the MNC layer with the purpose of providing a harvest that is rich in T cells [29,30]. In UCL, as a standard operation, all T cell harvesting is performed on SpectraOptia.
In a multi-site testing environment where apheresis blood component infrastructure, equipment, reagents, and trained staff differ, it is vital to demonstrate consistent collection of comparable MNC products across sites to minimize The variability of the starting material in an attempt to further quality control the manufacture of CART cells for patients [27,32].
2.3 T cell enrichment and initial processing steps after decellularization: quality aspects
From a quality control point of view, there is a great need for standardized cell materials. Contamination of platelets, plasma and residual anticoagulants can alter the responsiveness of T cells to activators ; red blood cells and platelets can impair cell counting and flow cytometry in the process , while granulocytes and monocytes It inhibits the expansion and transduction of T cells in the process of CART cell manufacturing [35 37].
The washing step can remove platelets, plasma and residual anticoagulants. Methods include manual Ficoll density gradient centrifugation, GMP-compliant closed system Ficoll separation and automated processes, such as COBE2991 cell processor, BiosafeSepax II and CaridianBCTElutra [27,38]. The use of countercurrent elutriation (CFE) can achieve the separation of monocytes and lymphocytes from the MNC harvest in compliance with GMP requirements. The commercially available CFE technology has been used in a number of CAR-T cell clinical trials (ElutraCell suspension system; TerumoBCTInc.) [32,39,40].
Immunomagnetic separation can be used to enrich T cell subpopulations from MNC harvests. MiltenyiBioTech provides GMP-compliant monoclonal antibody (mAb) conjugated paramagnetic beads for CliniMACS and Prodigy systems. The manufacturer confirms that the selected beads have been removed in multiple washing steps and do not contaminate the final patient product. In clinical trials based on CD4+/CD8+ magnetic beads, selection techniques have been used to produce CART cell products with a defined CD4+/CD8+ ratio [5,41], and magnetic beads can be used to select CD62L+ T cells, so that at the beginning of production Time enriches the central memory T cell subsets . Some groups use CD62L to choose to observe NK cell contamination, and recommend NK cell depletion first, the number of which exceeds 10% of the total harvest of leukocyte depletion .
At UCL, current practices have evolved from using total leukocyte depletion in all cases to include bead-based lymphocyte selection. This is to minimize manufacturing failures and produce more consistent CART cell products between patients. Immunomagnetic selection will affect cost and manufacturing complexity, and the impact of this step in our hands will be evaluated downstream of clinical research.
At this stage, the test includes flow cytometry analysis of samples before and after selection to quantify the efficacy of selection before the selection of activated T cells. Perform extended phenotypic analysis of all cell starting materials to determine cell composition and expression of cell maturity and failure markers.
These baseline readings allow us to track changes throughout the manufacturing process and begin to understand the effects of downstream culture steps on T cells. According to the expression of surface markers, T lymphocytes can be classified as naive T cells without antigen (TN), central memory T cells (TCM), effector memory T cells (TEM) or terminal differentiation effectors (TEFF), such as CD62L , CCR7, CD45RA, CD45RO and CD95. Although TEFF and TEM cells have strong cytotoxicity to target cells in vitro, the less differentiated TCM subtypes can migrate to lymph nodes and rapidly expand after exposure to antigens, and are known to have better proliferation potential in vivo , Durability and functionality .
The analysis of exhaustion and senescence markers (such as CD57, 2B4, PD-1, LAG-3, and TIM-3) can further characterize T cells after the incubation period and help predict their sensitivity to inhibitory signals.
Figure 2. (b)
An example of an extended phenotypic analysis performed in patient materials is shown in Figure 2a. In Figure 2a, we see the proportion of natural or central memory T cells based on the expression of cell surface markers CD45RA and CCR7. Among the vast majority of manufacturers in our department, we have noticed the richness of the central memory subset in the final CART cell product.
Representative images of patient A and patient B show that on the 8th day of delivery, more than 50% of the CD3+ T cells are TCM. Figure 2b shows the expression of PD1 and TIM3, which are the exhaustion markers of patients A and B at the beginning and end of CART cell production (a diagram representing most of the products in this unit). During this process, the expression of TIM3 increased, but the expression of PD1 was lower during the release test and cryopreservation on day 8. Figure 2c is a representative example of autologous CART cell products produced for patients suffering from B-cell malignancies (in this case B-ALL).
It illustrates that the final product for cryopreservation is rich in CD3+ T cells, while from other cell types (including monocytes (CD14+), NK cells (CD16+/CD56+), natural killer T (NKT) cells (CD3+/CD16+/CD56+) and B cells (CD19+). B-ALL leukocyte depletion is usually rich in CD34+ blasts, but these pronuclei can be eliminated by CD4/CD8 selection, and so far, our manufacturer has not been contaminated by CD34+ blasts detected by flow cytometry.
2.4 T cell activation methods: problems and challenges
T cell activation is critical to the efficiency of CART cell transduction. It is achieved by using antigen presentation technology to deliver concurrent T cell receptor (TCR) and costimulatory signals in vitro. Alternative methods include the use of autologous antigen presenting cells or cell lines modified to express costimulatory ligands. These are labor-intensive and produce high efficiency .
Artificial antigen presentation technologies, such as immobilized anti-CD3 and anti-CD28 mAbs adsorbed on plastic surfaces/beads, provide a cheaper and more standardized method for T cell activation . Superparamagnetic anti-CD3/CD28 antibody coated magnetic beads (such as DynalDynabeads; Human-ativator) are used in the manufacture of many CART cell products (including CTL019, which has now been licensed by the FDA for Kymriah (Novartis)) .
At UCL, DynalDynabeads are used in a number of CART cell studies. Considering the potential for polyclonal T cell activation, quality control requires validated assays to ensure that DynalDynabeads are effectively removed from the product before infusion. We use a simple, validated morphological assay to perform stringent analysis of <100 beads per 3×106 cells .
From a technical point of view, we found that removing magnetic beads in a GMP clean room can be troublesome and time-consuming (requires multiple operators), and is associated with a large amount of cell loss. This prompted the team to explore other non-bead-based activators.
An example is Expamer (JunoTherapeutics), a soluble T cell activation reagent that contains a streptomycin backbone associated with a low-affinity anti-CD3/CD28 Fab fragment. This is depleted from the T cell culture by washing steps [9,19].
At UCL, we have the experience of MACSGMPTransAct (MiltenyiBioTec), which is a biodegradable polymerizable nanomatrix and impregnated with anti-CD3/CD28mAb, thereby eliminating the requirement to remove magnetic beads .
At present, there is no measurement method that can be used to determine whether there is residual TransAct in the final CART cell product, but the product information file reports that the built-in culture washing and medium exchange steps in T cell transduction will deplete the excess TransAct (TCT) protocol. At UCL, the mathematical model that outlines the dilution of TransAct to negligible levels during the TCT process was incorporated into the relevant ATIMPD report submitted to the MHRA for approval of the CART cell study.
2.5 Gene delivery/transduction
This can be subdivided into viral and non-viral delivery systems . Retroviruses and lentiviral vectors are the most commonly used gene delivery methods in the production of CART cells, accounting for about 41% and 54% of all manufactured products . The transduction efficiency is usually very high, and T cell activation is required for gene transfer . The viral vectors are harvested into sub-batches, purified and sterile filtered, and then subjected to cryopreservation and quality control tests . The GMP viral vector production facility in the United Kingdom is responsible to MHRA, and in the United States to the FDA [50,51].
Due to labor-intensive manufacturing methods, requirements for clean room infrastructure, and extensive safety testing required to release the vector, viral vectors are very expensive. In order to reduce costs, stable packaging cell lines can be used to produce retroviral vectors . Equivalent stable lentiviral packaging cell lines are under development .
Potential risks of reverse transcription and lentiviral vectors include insertional mutagenesis and the production of replication competent viruses (RCV). To date, insertional mutagenesis has not been described in more than 100 patients receiving CART cell therapy . Strategies to minimize the risk of viral recombination and RCV production include “direct” delivery of plasmids needed for transient transfection of the production cell line (usually HEK293 cells) .
The use of “Sleeping Beauty” (SB) [54,55] or PiggyBac[56-58] transposon/transposase technology for non-viral gene delivery is much cheaper than viral vectors. Due to the randomness of gene integration, there is a risk of insertional mutagenesis , but it is believed that the efficiency is lower than that of viral vectors. Disadvantages include low gene transfer efficiency and the need to extend in vitro culture to produce clinically relevant cell numbers [59,60]. SBCAR T cell trials are ongoing [43,61].
At UCL, GMP-compliant reverse transcription and lentiviral vectors are used for CART cell production. Due to the nature of the multi-directional campaign carried out at UCL, we have adopted a closed system CART cell manufacturing method, which has been covered in the appropriate risk assessment. In order to strictly ensure the lack of carrier infectivity in the waste generated during the entire process, we perform titration analysis on the cell lines of the waste collected at intervals during the process. This confirms the lack of infectivity of viral vectors in the final stages of production and in cryopreserved products.
From a clinical risk point of view, the MHRA requires 15-year follow-up of patients to monitor the emergence of RCV and perform integrated site analysis if necessary. Long-term follow-up clinics have been established at UCL to follow these patients over time.
2.6 T cell expansion and fully automated CAR T cell manufacturing platform
After gene transfer, T cells are expanded to reach the number of cells required for clinical applications in a functionally closed culture system . A variety of methods can be used, including T bottles, plates or culture bags, and bioreactors such as G-Rex flasks (Wilson Wolf Manufacturing), WAVE bioreactors (GE Life Systems) and CliniMACS Prodigy (Miltenyi BioTec). Recent reviews indicate that 43% of CAR T cell clinical trials use rocking bioreactors [46,49], while 35% use static culture bags and 22% use T bottles .
T-flasks are not suitable for large-scale work because they are labor intensive and require multiple open processing steps in a safety cabinet [31,62]. Static culture bags can be welded together by sterile tubes and provide a semi-closed culture system, but the medium exchange is a manual process and is not easy to expand . The bioreactor has several advantages: The G-Rex flask is a closed system cylindrical vessel with optimized gas exchange and sampling options, which can rapidly expand cells in an incubator [64,65].
The GE WAVE swing bioreactor is an automated closed culture system, including a gas permeable culture bag (Cellbag; GE Healthcare Life Sciences) placed on a swing platform. The built-in mass flow, pH, air pressure and CO2 and O2 concentration sensors allow automatic perfusion and gas exchange to reduce physical labor and media consumption [49,66]. The problems of WAVE include lack of scalability (each bioreactor can only produce one product), cost and potential biological impact, for example, it is reported to be biased towards CD4+ T cell expansion .
The choice of medium varies between different studies and is moving towards the use of serum-free preparations such as X-VIVO 10 (Lonza). Interleukin 2 (IL-2) or interleukin 7 (IL-7) plus interleukin 15 (IL-15) for cytokine support is center-dependent . From a practical point of view, it is important for any CART cell manufacturing company to ensure its supply chain and be able to verify alternative GMP-compliant reagents and consumables to protect its processes.
In order to truly realize the scalability of autologous CART cell production, there is a great need for a standardized automated fully enclosed system that meets the requirements of GMP. Miltenyi’s CliniMACS Prodigy is currently the most widely used automated cell manufacturing platform . Prodigy allows selection, activation, transduction, expansion, sampling and harvesting of T cells on a disposable tubing set (TS520) according to a programmable activity matrix , and provides CART cells with similar phenotypes and functions. Other methods  are used for the number of cells required for clinical trials. Preselected T cell subpopulations (such as CD62L or CD4 and CD8) or unselected total leukocyte separation [47,69,70] can be used as starting materials, and the expansion of T cells may exceed 30 times after 14 days of culture.
From a quality control point of view, Clini-MACS Prodigy is an attractive choice, because the closed system eliminates the requirement for a high-grade clean room, reduces the risk of product contamination through a minimum of operator steps, and reduces Improve the labor intensity of employees. The efficacy and toxicity of CART cells produced in this way are being evaluated in clinical studies. In UCL, CAR19 donor lymphocytes (NCT02893189) for recurring CD19+ malignant tumors after allogeneic stem cell transplantation (allo-SCT) recurrence CD19CART cells allotransplantation (CARD study) are the first CART cells produced The clinical study was completed by Miltenyi’s prodigy TCT program. Other biotech companies are developing proprietary automated cell manufacturing platforms to meet demand for products. In the future, automation may allow the delivery of CART cells in a hub-and-spoke model, whereby the satellite academic center represents commercial companies that provide central coordination and quality control to generate CAR T cells .
At UCL, we use static culture bags, GE WAVE swing bioreactors and CliniMACS Prodigy to produce CART cells for patients. All equipment has passed the GMP facility installation and operation certification (IQ/OQ), and abides by the service contract. The medium is filled with non-essential materials (broth or pure medium) every six months to ensure that the process remains sterile in accordance with the requirements of the British regulatory agency.
2.7 Cryopreservation of T cells
Cryopreservation of CAR T cell products prior to infusion is standard practice. Advantages include flexible scheduling of patient infusion time and time to complete the extended quality control tests required for QP review and release. Proven cryopreservation is essential for large-scale centralized manufacturing.
From a quality point of view, this step is critical, because poor cryopreservation can lead to reduced cell numbers, impaired viability, and changes in cell phenotype and function . Most CAR T cell manufacturing plants use the cryopreservation method developed for hematopoietic cells, that is, resuspend in an isotonic buffer containing 10% dimethyl sulfoxide (DMSO) and transfer to a controlled rate freezer. And long-term storage in gas phase liquid nitrogen . CD3+ T cells cryopreserved in this way maintain viability (the recovery rate after thawing is between 50% and 90%) , and maintain the phenotype and function of CART cells . Lower DMSO concentrations (5% to 10%) can be used with extracellular protective agents, such as human serum albumin (HSA), plasma, serum, and hydroxyethyl starch solution (HES). Commercial formulas that comply with GMP are also provided, including CryoStor, the final concentration of which is 2%, 5% and 10% DMSO has been pre-formulated. For clinical use, according to the consensus of the European Association for Bone Marrow Transplantation (EBMT) and the American Association of Blood Banks (AABB), the DMSO content of the product should not exceed 1g/kg.
Some groups passively freeze CAR T cell products in a mechanical freezer at -80°C [75-77]. The cryopreservation of CAR T cells is increasingly performed in a programmable rate controlled freezer (CRF), which reduces the temperature by 1°C every minute to avoid the formation of intracellular ice crystals. Once fully dehydrated, the cells can be quickly cooled to the final storage temperature and transferred to gas phase liquid nitrogen (LN) for storage at temperatures below -150°C . From a quality point of view, the advantages of CRF include freezing consistency, programming versatility, and data traceability for a single product. Disadvantages include the high cost associated with the large amount of liquid nitrogen required to operate the equipment. New mechanical CRFs (such as asymptotes) have lower operating costs and can overcome some of the safety issues associated with the use of LN in enclosed spaces.
At UCL, we cryopreserve CAR T cells in 7.5% DMSO in CTRF, and then store them in gas-phase LN for a long time. We stipulate the maximum CAR T cell volume for infusion of 12 mL/kg patient body weight to prevent the DMSO dose from exceeding the legal limit of 1 g/kg per day. In addition, we will conduct routine stability tests on individual products according to internally validated testing methods to ensure the viability of storage over time, the expression and functional capabilities of CAR transgenes (cytotoxicity or cytokine release data). Before infusion, the cryopreserved products should be extracted from the gas phase LN, and transported to the contracted express company’s temperature-recorded, verified LN dry shipper’s hospital in accordance with the Good Workshop (GDP) principle stipulated in the contract.
3. Quality Assurance of CAR T Cell Therapy: General Principles
GMP regulations require assessment of the safety, purity and efficacy of CAR T cells . These indicators are used as quality release standards .
Safety stipulates the absolute sterility of bacteria and fungi, and in the case of using viral vectors, there is no RCV. Culture-based sterile methods, such as the BD BacTEC system, involve test inoculation, placing it in a culture medium, and then observing the growth of microorganisms during a set follow-up period . The standard practice is to conduct a culture-based sterility assessment of the process starting materials, the culture medium manufacturing process, and the final product. When fast results are required, Gram staining can be used as a non-culture method in process testing, but it is generally considered to be less sensitive than culture-based methods . Cell-based or quantitative polymerase chain reaction (qPCR) tests must be used to prove the absence of RCV . After the product was produced, samples for RCV testing were collected and stored from patients during the production process and through clinical research for 15 years . The samples can also be screened for mycoplasma through a validated testing method in an externally accredited laboratory or internally.
Purity refers to the cellular composition of the final product and refers to the absence of contaminants (such as endotoxin and other impurities related to the manufacturing process). The cell composition and purity of the final product can be quantified using flow cytometry [20,45]. In an externally accredited laboratory or internally, in a validated assay, contaminants such as endotoxins are screened out of the final product samples. In the case of using DynalDynabeads, verification tests should be carried out to quantify the residual magnetic beads in the final product . These tests should be part of the published standards for finished products.
Efficacy measurement is an in vitro test designed to reflect/predict biological performance in vivo . The determination of the potency of the release test should be based on the requirements of the International Coordination of Technical Requirements for Human Drugs (ICH) for methodological verification, including the requirements for accuracy, precision, repeatability, specificity, linearity and durability , and should Including functional immunological determination of cytotoxicity, proliferation and cytokine release. When trying to compare different products, the lack of standardized methods to test the potential of CAR T cells poses a major challenge. Recognizing this, the FDA has issued a guide book “Efficacy Testing of Cell and Gene Therapy Products”  for researchers, and now recommends (but not mandatory) to conduct efficacy testing for Phase 3 clinical studies. Current CAR T cell research relies on flow cytometry quantification of transduction efficiency (cell surface expression of CAR or marker genes/proteins) as a substitute for CAR T cell functional activity [46,84]. Vector copy number assessment does not always reflect CAR expression on the cell surface .
CAR T cell release test
After receiving the doctor’s prescription, CAR T cell products should be released according to COA. The COA must specify the release analysis method (for example, product identification, purity, viability, and potency), test location and minimum release specifications, and the actual results obtained. The release test shall be carried out according to the analytical method approved by the regulatory agency. Where it is not possible, internal analysis verification will be required to prove the completeness of the analysis. According to published research, 2-14% of all manufacturers do not meet the published specifications .
Table 3 summarizes the square assays commonly used in CAR T cell production and reflects the current practice of UCL. For the release of a single product, performance testing is not routinely performed, but stability testing is performed after melting. UCL has performed performance testing on most products.
CAR T cells herald an exciting era of cancer treatment and are expected to be widely adopted. There are still many challenges to the standardization and economic production of these therapies. In addition, there is no widely available validated assay that can be used to verify the efficacy of CAR T cell products, which makes it challenging to compare CAR T cell products before infusing patients.
Promising engineering solutions, standardized biomaterials and process control [9,86], as well as huge advancements in automation and closed manufacturing solutions. Coupled with a deeper understanding of the knowledge that constitutes the best CAR T cell biology, and the development of standardized detection methods for more complex CAR T cell function tests, the likelihood of patients getting better-characterized products may increase.
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