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How to use non-viral methods to produce CAR T cells?
How to use non-viral methods to produce CAR T cells? Chimeric antigen receptor T cells (CAR T cells) represent a novel and promising approach in cancer immunotherapy. CAR T cell therapy has achieved extraordinary results in patients with B-cell malignancies. To date, the most common method of CAR T cell generation is the use of viral vectors.
However, due to strict regulations and high cost requirements, handling virus-derived vectors may cause obstacles to the CAR T cell manufacturing process. If CAR T cell therapy is to be routinely used in medical practice, manufacturing costs and complexity need to be as low as possible. Transposon-based vectors seem to meet these standards better than virus-based vectors.
1. The evolution of T cells with chimeric antigen receptors
1.1. CAR T cell: a brief definition
T cells are genetically engineered to express a CAR that recognizes cancer cell antigens. The CAR T cell then recognizes the cancer cell and removes it from the organism. The specificity of this tumor target antigen is crucial, because its expression in healthy tissues may cause serious side effects and even death.
1.2. History and Breakthrough
The most important CAR T cell clinical trial was conducted by Carl June’s research team in 2011 [2, 3]. They treated 3 patients with chronic lymphocytic leukemia (CLL) using CAR targeting the B-cell antigen CD19. CAR T cells expanded 1,000-fold after retransfusion, eliminated lymphoma cells, and induced complete and sustained remission.
Although the CAR T cell-based technology is still under development, the first batch of therapeutic products have entered clinical practice. Kymriah (Novartis) and Yescarta (Gilead-KitePharma) are both approved by the U.S. Food and Drug Administration (FDA) for the treatment of B-cell-related malignancies. Among the hematological carcinogenic diseases, the treatment of B-cell malignancies has proven to be the most successful. Although the treatment itself may cause B-cell aplasia, because healthy B-cells will also be affected, this state is clinically controllable.
At present, it is recognized that there are five generations of CAR T cells, and each generation enhances the characteristics of CAR constructs by modifying their domains. The second-generation CAR T cells include various costimulatory domains (CD28  or 4-1BB ), which can improve the efficiency of T cell construction .
The third generation utilizes the cooperation between multiple costimulatory domains (for example, CD3ζ+CD28+4-1BB). The activation of PI3K/AKT signaling pathway may be the reason for the enhanced characteristics of third-generation CAR T cells [7, 8].
The fourth-generation CAR T cell or TRUCK (redirected T cell for universal cytokine-mediated killing) expands the cytotoxic properties through the ability to express genetically modified cytokines (such as IL-12 ). The expression of transgenic IL-12 is related to the specific environment (the proximity of the targeted cancer cells), so it may be particularly helpful for the treatment of solid tumors .
The fifth-generation CAR T cells have added other cytokine expression domains (IL-15 ; IL-18 ), and have the ability to target multiple antigens-targeting HER2 + IL13Rα2-in animal models Successfully tested glioblastoma to prevent the escape of tumor antigens .
1.3. Viral and non-viral methods for CAR T cell preparation
One of the main aspects of CAR T cell preparation is to select the appropriate vector to bring the CAR construct into the cell. The two most commonly used options for CAR T cell generation are viral-based vectors (usually retrovirus or lentivirus) or non-viral vectors, which are mainly based on transposons to construct CAR T cells.
1.3.1. Virus-based vectors
Currently, the vast majority of CAR T cell production relies on the transfer of genetic information to T cells through viral vectors. The combination of retroviral genes (gag, pol, env) with inducible promoters can increase the transduction rate and generate a relatively large number of CAR+ T cells (see the review in ). Vector-based murine leukemia virus (MLV) is the most commonly used gamma retroviral vector (reviewed in ) and has been successfully used as a new method for T cell immunotherapy for severe combined immunodeficiency- (SCID- ) X1 disease . Although immunodeficiency diseases have been successfully treated, T cell-associated leukemia has appeared in some cases [16, 17].
Vectors derived from the subfamily of lentiviruses from another retrovirus family show better integration properties than their gamma retroviral counterparts. Lentiviral vectors can target non-dividing cells relatively easily , while in γ retroviral vectors, the transduction rate into non-dividing cells is significantly lower . The increased biosafety of lentiviral vectors is due to different integration tendencies. γ retroviral vectors prefer to integrate into gene promoters, which may be the reason for the oncogenic properties described previously (reviewed in ).
Viral vectors may be very effective in the production of CAR T cells, but several key features hinder their clinical use and instead use non-viral methods. First of all, the possibility of carcinogenic and mutagenic potential requires a more stable carrier, which will ultimately be used to prepare clinical grade CAR. Second, the use of viruses in current good manufacturing practices (cGMP) laboratories is bound by a series of strict regulations, and non-viral methods of gene transfer may be more feasible for clinical-grade production. Third, lentiviral/retroviral transduction is limited by the size of the transferred DNA . Finally, some other vector systems (for example, those that use electroporation, lipofection, ultrasound, or magnetofection for transduction) significantly reduce the overall price of CAR T cell preparation. Generally speaking, the manufacturing cost of viral vectors is often higher than that of transposon-based counterparts because the manufacturing process of such vectors is more demanding (see ).
1.3.2. Non-viral vectors
The most common alternative to viral vectors is transposons. A variety of transposon-based systems have been reported for CAR T cell production. These systems provide safe and reliable DNA transfer into T cells. The Sleeping Beauty (SB) transposon system is currently used as an alternative viral vector to prepare, for example, CD19+ CAR T cells , and is reported to have anti-tumor activity in vitro and in vivo . The main advantage of this method lies in the better integration of the entire transduced genetic material. This improved integration is due to the low promoter activity of the integrated transposon . The SB system also triggers fewer epigenomic changes near the integration site. The relatively low manufacturing cost is also an important factor to further improve the status of the SB system . The main obstacle to the use of SB transposons is their significantly lower integration rate of genetically modified materials. With the further development of the SB system, the problem of low integration is significantly reduced. SB11 is a prime example of this effort, and its transposition rate is 100 times higher than that of natural SB transposons . Through other modifications to the SB transposon system, the SB100X system improves the transposition rate by 100 times compared with the SB11 system .
The integration profile of SB transposon is close to random. The SB system is integrated for the TA site . Compared with viral vectors (), SB transposons have repeatedly demonstrated no integration bias towards coding sequences [31, 32]. Although it can be considered that the SB integration profile is biologically safe, other transposon vectors exhibit characteristics that are more similar to virus-based vectors .
The transposon Tol2 is another example of a successful transposon system suitable for the creation of CAR T cells . Compared with naïve SB transposon, Tol2 provides greater coding capacity (100-200 kb) and sufficient transposition activity . SB transposons prefer T-A bases for integration, while Tol2 seems to have a random integration preference .
The biggest competition for SB transposons is likely to be the PiggyBac (PB) transposon system. Similar to the previously mentioned transposons, PB transposons use a simple cut and paste mechanism to integrate themselves into human cells. The integration site is non-random, because PB transposons usually prefer the TTAA sequence . The mapping of the PB integration map reveals the similarity with γ-retrovirus and lentiviral vector , and has the same insertion bias of expressed genes as MLV retrovirus .
Compared with other (Tol2 and SB11) transposon systems, the PB transposon system exhibits excellent transposable activity in mammalian cells . The higher transposition activity in various mammalian cells places the PB transposon on top of potential non-viral vectors for the production of human cell transgenes and CAR T cells.
Another basic ability of plasmids is the size of the “cargo” capacity. Cancer cells have multiple mechanisms to avoid immune responses, and the best CAR structure needs to overcome most of these mechanisms to make the treatment successful. The PB transposon system has been shown to be able to transfer multiple genes into T cells, making them more effective for cancer treatment . Nakazawa’s  study also proved that the PB system can carry and transfer safety switches to human T cells in the form of suicide genes. The ability to shut down CAR T cells in patients in the case of severe cytokine release syndrome (CRS) may be the most important safety feature of this technology . Many highly active mutant variants of the PB transposon have been successfully isolated . The 7PB variant can outperform the original PB transposon and SB100X transposon in terms of transposition activity . Another example of an improved PB system is the “mouse codon optimized PB transposase gene”-mPB .
The PB system has been successfully used to generate CD19 CAR for hematological malignancies  and CAR for selected solid tumor antigens, such as CD73 , MSLN , EGFRvIII  and PSMA . The comparison between the viral vector and the two most common transposon vectors is shown in Table 1.
Transposon-derived plasmid vectors rely on the system to deliver them into the cell. Recently, the most commonly used method is cell electroporation, and its main advantage is that the procedure is relatively simple, and the overall cost of the method is low. The electroporation of plasmid DNA is currently being surpassed by the electroporation of messenger RNA (mRNA). The main barrier for plasmid DNA to enter the nucleus is the nuclear envelope. Therefore, dividing cells show a much higher transduction rate than non-dividing cells . With the transfer of mRNA, the need to overcome the nuclear membrane becomes redundant, despite other problems (mainly the reduced stability of the mRNA molecule compared to plasmid DNA). Chemical modification of RNA nucleosides (for example, the incorporation of pseudouridine ) significantly reduces the problem of insufficient mRNA stability.
The successful transformation of T cells in a mouse model by RNA electroporation was reported more than 15 years ago , and significant progress has been made in this regard since then [53, 54]. Preclinical testing of mRNA-mediated CAR has shown that this method is suitable for the treatment of solid tumors [55, 56]. In addition to electroporation, the use of lipid nanoparticles as a form of mRNA transport has recently been demonstrated in the preparation of CAR T cells . Unlike electroporation, this method is much less toxic to transduced cells.
Although both methods are suitable for cGMP quality CAR T cell production, electroporation is used much more frequently.
2. CAR practice: clinical application
Clinical trials based on CAR T cell therapy are developing rapidly, and more and more requests for approval of clinical trials are submitted every year. As of the end of 2016, there were 124 ongoing clinical trials of CAR T cell therapy for hematological malignancies and 57 clinical trial registrations for solid tumors . Most of these trials were conducted in the United States or China. Less than 10% of these trials were conducted in Europe.
Since then, the situation has changed significantly. According to data from ClinicalTrials.gov, there are more than 600 clinical trials in various stages of CAR T cell therapy. Most clinical trials still focus on hematological malignancies (of which 267 trials involve CARs that target CD19). Although the portion of research using transposon vectors is growing steadily, most ongoing clinical studies use virus-based vectors for CAR T cell manufacturing. Although concerns about possible vector-induced oncogenic activation have not been observed in clinical applications, there are still theoretical risks .
The LV system used in the clinic is mainly derived from the HIV-1 virus. Several methods have been implemented to reduce the biohazard properties of LV vectors . A popular example of such modifications that can be used in clinical practice is the four-plasmid system, which can effectively segment the HIV-1 genome, making viral gene expression dependent on different isolated transcription units and genes responsible for packaging that are expressed only in production cells (HEK293T cell line is often used) . The integration of multiple plasmids carrying part of the LV vector ultimately increases the logistical complexity of large-scale CAR T cell manufacturing; therefore, a more optimized low-pressure system is still needed . The main clinical application of virus-constructed CAR is still the treatment of blood-related malignancies [67-69].
2.1. Transposon clinical trials
Similar to virus-based CAR, the clinical application of transposon-mediated CAR T cells is mainly focused on blood cancer . CD19-specific CAR T cells transduced by SB vector have shown promising results in phase I clinical trials [31, 70, 71]. A successful method to prepare cGMP-grade CAR T cells (for phase I/II clinical trials) is to use electroporation of the SB system DNA plasmid and co-culture of T cells with inactivated aAPC (artificial antigen presenting cells). Although the effect of transposon-mediated transduction is poor, a sufficient number (1010) of CAR T cells (95% purity) were repeatedly prepared during the 28-day culture process . Analysis of the SB integration profile of the used CAR showed that there were no obvious hot spots or integration deviations in the transplanted cells.
Recently, preliminary data from phase I/II clinical trials indicate the biological safety of donor-derived CD19+ CAR T cells, which provides opportunities for other allogeneic applications of transposon-based CAR T cells . However, the optimal dose of CAR T cells is essential to ensure the safety of treatment, because higher doses of CAR T cells can cause phase I and phase II CRS . Another factor that complicates the severity of CRS is the severity of the disease itself. The use of CAR T cell therapy (for blood cancer) within a short period of time after the patient receives hematopoietic stem cell transplantation may be beneficial to reduce the risk of CRS and ultimately eliminate the patient’s residual disease . The above study also recorded a direct comparison of SB-mediated CAR T cells in autologous and allogeneic environments . Both methods produce quite pure CAR T cells, but autologous CAR T cells can be detected in patients for a longer time. The overall survival rate and 30-month progression-free rate of autologous test patients are higher, but both methods have significantly improved compared to standard treatment patients .
Although not as extensive as SB-CAR, PB-based CAR T cells have also successfully constructed CAR T cells against CD19 antigen , and a phase I clinical trial is ongoing (ClinicalTrials.gov identifier: NCT04289220).
3. CAR T cell manufacturing
All the CAR T cell preparation methods mentioned above have their advantages, but the ultimate success of a certain method depends on its reproducibility under strictly regulated cGMP conditions. These conditions determine the complex rule set of current cell therapy production. Every aspect of CAR T cell preparation needs to be recorded in detail and comply with cGMP guidelines, starting from the clean room facility specifications, to the place where manufacturing will take place, to emphasizing aseptic laboratory technology, the content of culture media, and direct participation in product culture and Other chemicals for final product processing (cryopreservation, quality control testing, etc.) (reviewed in ).
3.1. CAR T cell culture
The separate culture process is considered to be a key part of the development of CAR T cell-based therapies. In order to avoid contamination during the culture process, closed or semi-closed culture systems and bioreactors are considered superior to simple culture in culture flasks.
G-Rex (Wilson Wolf Manufacturing) represents an example of a widely used semi-closed cell production system that utilizes culturing in a flask with a gas-permeable membrane to achieve better gas exchange and significantly enhance cell proliferation. Although the system has been upgraded on the basis of the use of ordinary culture flasks, it is not fully enclosed, and the potential contamination risk may be higher than that of a fully enclosed system.
Completely closed systems (such as CliniMACS Prodigy or Quantum Cell Expansion System) can perform the entire process (from cell transduction to expansion) in a single pipeline group. The CliniMACS Prodigy tube group mainly focuses on the preparation of CD19 CAR by lentiviral vector transduction and subsequent activation by anti-CD3/CD28 antibody . Compared with other established and conventional culture technology products, the final CAR T cell product exhibits similar characteristics . The Quantum Cell Expansion System is commonly used to produce adherent cells, such as mesenchymal stem cells (MSC) , but it also proves that the expansion of CAR T cells is possible .
Although a completely closed system minimizes the possibility of contamination, the limitations of virus-based vectors and their overall high cost greatly reduce their potential applications in clinical practice. Current estimates of the cost of CAR T cell therapy for a single patient range from 150 000 to 300 000 US dollars [63, 80].
The production of cGMP-quality CAR by plasmid/transposon is usually limited to culture in an open or semi-open culture system, because the electroporation (or lipofection) process has not been automated in a closed culture system. CAR T cells transduced by electroporation can be successfully prepared under cGMP conditions , although the requirements for aseptic skills of personnel are significantly higher.
In order to improve the expansion and efficiency of T cells after transduction, various cytokines are added to the medium. The most commonly used cytokine to promote T cell expansion may be IL-2 . Although more and more studies have shown that higher concentrations of IL-2 can drive the CD8+ part of T cells into terminal differentiation, this higher concentration of IL-2 does not promote the formation of memory T cells[83, 84] . Therefore, different interleukin combinations are sought to improve the characteristics of T cell culture. In order to prevent terminal differentiation of T cells, a combination of IL-7, IL-15 and IL-21 is often used . IL-21 is similar to IL-2 in promoting CD8+ cytotoxicity, but in the opposite way. The addition of IL-21 can inhibit the terminal differentiation of CD8+ and can be regarded as an IL-2 antagonist . IL-7 and IL-15 promote the formation of memory phenotypes in cultured T cells . At the same time, the proliferation of T cells in the presence of the combination of IL-7 and IL-15 leads to a more effective anti-tumor CAR than the proliferation of T cells in the presence of IL-2 .
Compared with CAR T cells targeting hematological malignancies, the viability and efficacy of CAR T cells targeting solid tumor cells are affected near the aggressive tumor microenvironment. To overcome this inhibition, CAR can be modified with so-called reverse cytokine receptors (ICR). Within the ICR, the interleukin 4 (IL-4) receptor is fused to the IL-7 receptor. This fusion receptor can convert regulatory IL-4 signals into IL-7 signals, and ultimately enhance the characteristics of CAR in the tumor microenvironment . Medium supplemented with IL-4 can improve the tumor-killing ability of CAR T cells .
The current trend of cGMP tends to avoid the use of any animal-derived ingredients in the cultivation process. The emphasis on heterogeneous culture has drawn attention to the composition of the medium. Commonly used animal-derived ingredients, such as fetal bovine serum (FBS), are preferably replaced by non-exogenous substitutes. It has recently been reported that replacing FBS with human platelet lysate has a positive effect on CAR T cell status .
3.2. T cell characterization
In addition to the transduction methods and details of different CAR structures, the characteristics of modified T cells should also be addressed. Most studies only use the CD3+ portion of peripheral blood mononuclear cells (PBMC) isolated from peripheral blood or leukocyte removal. In most cases and studies, the composition of CD4+/CD8+ cells and the phenotype of a given cell are variable, which may result in failure to reproduce results in different environments. Using advanced enrichment and cell sorting techniques can solve this obstacle relatively easily .
The CD4+/CD8+ ratio, which is the ratio of T helper cells to T cytotoxic cells, should also be monitored. The physiological value of the CD4+/CD8+ ratio is considered to be between 1.5 and 2.5, and there are some differences between different races, age categories, etc. (reviewed in ). Patients undergoing chemotherapy usually have a significantly higher proportion of CD8+ cells .
CD4+ and CD8+ cells can be divided into several phenotypic subtypes-naive (Tn), effector (Teff) and memory (Tm) T cells present three main T cell phenotype subtypes. Memory T cells can be further divided into long-lived central memory (Tcm) T cells and short-lived effector memory (Tem) T cell subtypes [95-97].
CAR T cells derived entirely from CD8+ cells show higher cytolytic activity in vitro. The Tcm subtype of CD8+ CAR showed the best survival rate in tumor-inducing mice .
Similarly, CD4+ cells produce more different cytokines (IFN-γ, TNF-α, IL-2) and CD8+ cells, and each CD4+ phenotype subtype generally improves the survival rate of the mouse model  . The synergy between CD4+ and CD8+ T cells has previously been demonstrated in mouse models , and the use of optimal CAR T cell composition may be beneficial to patients.
4. Conclusion and future outlook
CAR T cell-based therapies are currently used to treat hematological malignancies, and two drugs have been approved by the US FDA. The number of drugs with similar characteristics will almost certainly increase in the future. The main challenges for the wider use of CAR T cell technology in clinical practice may be:
(i) reducing manufacturing costs,
(ii) overcoming major safety hazards,
(iii) by introducing more advanced structures.
Although viral and non-viral methods have their own advantages and disadvantages, non-viral methods have great potential in addressing these challenges and bring CAR technology beyond the field of blood diseases.
(source:internet, reference only)