Chimeric Antigen Receptor Design Research Articles

NK Cells Equipped With a Chimeric Antigen Receptor That Overcomes Inhibition by HLA Class I for Adoptive Transfer of CAR-NK Cells.

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness, a process termed licensing or education, but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept, we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog, TMIGD2) and 2B4 (CD244, SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody, the CD28H transmembrane domain, and the fusion of CD28H, 2B4, and TCRζ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCRζ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore, such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore, engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies, such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing.

Precision killing of T cells via targeting of T cell receptors using two novel chimeric antigen receptor designs

Abstract Pathologic T cells drive T cell leukemia, solid organ transplant rejection, and many autoimmune diseases. Currently approved treatments have limited ability to target pathogenic vs non-pathogenic T cells. While pan-T cell treatments can be effective, they have significant risks such as infection and malignancy. Chimeric antigen receptors (CAR) are synthetic constructs that retarget T cells to a specific antigen of interest; we hypothesized that we could use this technology for targeted killing of T cells. We developed two novel CAR designs which target T cells via their T cell receptor (TCR). We opted to target OT1 T cells, a model system with a known antigenic epitope. The anti-Vβ5 CAR is a second-generation CAR with a traditional extracellular scFv domain that targets Vβ5 of the OT1 TCR. The H2Kb bait CAR instead has an extracellular H2Kb complex loaded with the SIINFEKL cognate epitope. Both anti-Vβ5 and H2Kb bait CAR T cells demonstrate degranulation, activation, and proliferation upon binding OT1 T cells. In vitro co-culture with OT1 T cells results in internalization of the OT1 TCR and specific killing of OT1 T cells. Interestingly, the changes induced by the H2Kb bait CAR were delayed relative to the anti-Vβ5 CAR. In vivo experiments show both CAR designs kill adoptively transferred OT1 T cells without significant effect on the other T cell populations. In conclusion, we report the development of two novel CAR designs which allow for the targeted killing of T cells via their T cell receptor. While the anti-Vβ5 CAR T cells generated a more rapid response, the H2Kb bait CAR offers the promise of more precisely targeting T cells. This work has the potential to transform the treatment of conditions where removal of pathogenic T cells is critical for cure. Supported by NIH HL152756

Multiomic dynamic single-cell profiling of CAR T cell populations associated with efficacy

Abstract T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative capacity; interferon responses; and a distinct cluster of pathways related to actin cytoskeleton and migration. We employed a marker-free sorting strategy for enriching T cells with persistent motility. RNA-seq on sorted motile T cells showed an enrichment of the core motility signature. These motile T cells also demonstrated superior in vivo anti-leukemia efficacy in comparison to unsorted T cells. Lastly, we also confirmed the association with increased persistent motility and killing of CAR T cells across diverse CARs. In aggregate, our data identified that independent of CAR design or biomanufacturing, persistent motility serves as a selectable cell-intrinsic biomarker, desired in the bioactivity of expanded CAR+ T cells.

The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity.

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.

CXCL9-modified CAR T cells improve immune cell infiltration and antitumor efficacy.

Chimeric antigen receptor (CAR) T cells remain unsatisfactory in treating solid tumors. The frequency of tumor-infiltrating T cells is closely related to the good prognosis of patients. Augmenting T cell accumulation in the tumor microenvironment is essential for tumor clearance. To overcome insufficient immune cell infiltration, innovative CAR designs need to be developed immediately. CXCL9 plays a pivotal role in regulating T cell migration and inhibiting tumor angiogenesis. Therefore, we engineered CAR T cells expressing CXCL9 (CART-CXCL9). The addition of CXCL9 enhanced cytokine secretion and cytotoxicity of CAR T cells and endowed CAR T cells with the ability to recruit activated T cells and antiangiogenic effect. In tumor-bearing mice, CART-CXCL9 cells attracted more T cell trafficking to the tumor site and inhibited angiogenesis than conventional CAR T cells. Additionally, CART-CXCL9 cell therapy slowed tumor growth and prolonged mouse survival, displaying superior antitumor activity. Briefly, modifying CAR T cells to express CXCL9 could effectively improve CAR T cell efficacy against solid tumors.

Taking Lessons from CAR-T Cells and Going Beyond: Tailoring Design and Signaling for CAR-NK Cells in Cancer Therapy.

Cancer immunotherapies utilize the capabilities of the immune system to efficiently target malignant cells. In recent years, chimeric antigen receptor (CAR) equipped T cells showed promising results against B cell lymphomas. Autologous CAR-T cells require patient-specific manufacturing and thus extensive production facilities, resulting in high priced therapies. Along with potentially severe side effects, these are the major drawbacks of CAR-T cells therapies. Natural Killer (NK) cells pose an alternative for CAR equipped immune cells. Since NK cells can be safely transferred from healthy donors to cancer patients, they present a suitable platform for an allogeneic “off-the-shelf” immunotherapy. However, administration of activated NK cells in cancer therapy has until now shown poor anti-cancer responses, especially in solid tumors. Genetic modifications such as CARs promise to enhance recognition of tumor cells, thereby increasing anti-tumor effects and improving clinical efficacy. Although the cell biology of T and NK cells deviates in many aspects, the development of CAR-NK cells frequently follows within the footsteps of CAR-T cells, meaning that T cell technologies are simply adopted to NK cells. In this review, we underline the unique properties of NK cells and their potential in CAR therapies. First, we summarize the characteristics of NK cell biology with a focus on signaling, a fine-tuned interaction of activating and inhibitory receptors. We then discuss why tailored NK cell-specific CAR designs promise superior efficacy compared to designs developed for T cells. We summarize current findings and developments in the CAR-NK landscape: different CAR formats and modifications to optimize signaling, to target a broader pool of antigens or to increase in vivo persistence. Finally, we address challenges beyond NK cell engineering, including expansion and manufacturing, that need to be addressed to pave the way for CAR-NK therapies from the bench to the clinics.

When Onco-Immunotherapy Meets Cold Atmospheric Plasma: Implications on CAR-T Therapies.

T cells engineered with chimeric antigen receptors (CAR) have demonstrated its widespread efficacy as a targeted immunotherapeutic modality. Yet, concerns on its specificity, efficacy and generalization prevented it from being established into a first-line approach against cancers. By reviewing challenges limiting its clinical application, ongoing efforts trying to resolve them, and opportunities that emerging oncotherapeutic modalities may bring to temper these challenges, we conclude that careful CAR design should be done to avoid the off-tumor effect, enhance the efficacy of solid tumor treatment, improve product comparability, and resolve problems such as differential efficacies of co-stimulatory molecules, cytokine storm, tumor lysis syndrome, myelosuppression and severe hepatotoxicity. As a promising solution, we propose potential synergies between CAR-T therapies and cold atmospheric plasma, an emerging onco-therapeutic strategy relying on reactive species, towards improved therapeutic efficacies and enhanced safety that deserve extensive investigations.

Better by design: What to expect from novel CAR-engineered cell therapies?

Chimeric antigen receptor (CAR) technology, and CAR-T cells in particular, have emerged as a new and powerful tool in cancer immunotherapy since demonstrating efficacy against several hematological malignancies. However, despite encouraging clinical results of CAR-T cell therapy products, a significant proportion of patients do not achieve satisfactory responses, or relapse. In addition, CAR-T cell applications to solid tumors is still limited due to the tumor microenvironment and lack of specifically targetable tumor antigens. All current products on the market, as well as most investigational CAR-T cell therapies, are autologous, using the patient's own peripheral blood mononuclear cells as starting material to manufacture a patient-specific batch. Alternative cell sources are, therefore, under investigation (e.g. allogeneic cells from an at least partially human leukocyte antigen (HLA)-matched healthy donor, universal “third-party” cells from a non-HLA-matched donor, cord blood-derived cells, immortalized cell lines or cells differentiated from induced pluripotent stem cells). However, genetic modifications of CAR-engineered cells, bioprocesses used to expand cells, and improved supply chains are still complex and costly. To overcome drawbacks associated with CAR-T technologies, novel CAR designs have been used to genetically engineer cells derived from alpha beta (αβ) T cells, other immune cells such as natural killer (NK) cells, gamma delta (γδ) T cells, macrophages or dendritic cells. This review endeavours to trigger ideas on the next generation of CAR-engineered cell therapies beyond CAR-T cells and, thus, will enable effective, safe and affordable therapies for clinical management of cancer. To achieve this, we present a multidisciplinary overview, addressing a wide range of critical aspects: CAR design, development and manufacturing technologies, pharmacological concepts and clinical applications of CAR-engineered cell therapies. Each of these fields employs a large number of ground-breaking scientific advances, where coordinated and complex process and product development occur at their interfaces.

Strategies for Improving the Efficacy of CAR T Cells in Solid Cancers.

Simple SummaryCell therapy with genetically retargeted T cells shows strong clinical efficacy against leukaemia and lymphoma. To make this therapy efficient against solid cancers, a series of hurdles must be addressed. This includes the need to enable the T cells to survive long term in patients and to overcome immunosuppressive mechanisms in the tumour. Further, it is essential to prevent tumour cells from escaping by losing the protein that is recognised by the infused cells. The present article provides an overview of the key strategies that are currently being investigated to overcome these hurdles. A series of approaches have been described in preclinical models, but these remain untested in patients. The further progress of the field will depend on evaluating more strategies in a proper clinical setting.Therapy with T cells equipped with chimeric antigen receptors (CARs) shows strong efficacy against leukaemia and lymphoma, but not yet against solid cancers. This has been attributed to insufficient T cell persistence, tumour heterogeneity and an immunosuppressive tumour microenvironment. The present article provides an overview of key strategies that are currently investigated to overcome these hurdles. Basic aspects of CAR design are revisited, relevant for tuning the stimulatory signal to the requirements of solid tumours. Novel approaches for enhancing T cell persistence are highlighted, based on epigenetic or post-translational modifications. Further, the article describes CAR T strategies that are being developed for overcoming tumour heterogeneity and the escape of cancer stem cells, as well as for countering prevalent mechanisms of immune suppression in solid cancers. In general, personalised medicine is faced with a lack of drugs matching the patient’s profile. The advances and flexibility of modern gene engineering may allow for the filling of some of these gaps with tailored CAR T approaches addressing mechanisms identified as important in the individual patient. At this point, however, CAR T cell therapy remains unproved in solid cancers. The further progress of the field will depend on bringing novel strategies into clinical evaluation, while maintaining safety.

A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T Cells.

As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti-CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically reversible chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR-T activation during cancer treatment.

A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T Cells**

AbstractAs a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR‐T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV‐NS3) between the single‐chain variable fragment (scFv) and the hinge domain, we showed that the display of anti‐CD19 scFv on CAR‐T cells was positively correlated to the presence of a clinical HCV‐NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti‐CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically reversible chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR‐T activation during cancer treatment.

CAR-Ts: new perspectives in cancer therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a promising anticancer treatment that exploits the host's immune system to fight cancer. CAR-T cell therapy relies on immune cells being modified to express an artificial receptor targeting cancer-specific markers, and infused into the patients where they will recognize and eliminate the tumour. Although CAR-T cell therapy has produced encouraging outcomes in patients with haematologic malignancies, solid tumours remain challenging to treat, mainly due to the lack of cancer-specific molecular targets and the hostile, often immunosuppressive, tumour microenvironment. CAR-T cell therapy also depends on the quality of the injected product, which is closely connected to CAR design. Here, we explain the technology of CAR-Ts, focusing on the composition of CARs, their application, and limitations in cancer therapy, as well as on the current strategies to overcome the challenges encountered. We also address potential future targets to overcome the flaws of CAR-T cell technology in the treatment of cancer, emphasizing glycan antigens, the aberrant forms of which attain high tumour-specific expression, as promising targets for CAR-T cell therapy.

Synthetic Biology in Chimeric Antigen Receptor T (CAR T) Cell Engineering.

Synthetic biology is a novel interdisciplinary research area following engineering principles to redesign and construct biological systems for useful purposes. As one of the most notable clinically relevant application of synthetic biology, chimeric antigen receptor (CAR) T cells have demonstrated tremendous success for the treatment of advanced hematological malignancies in recent years. However, various unsolved obstacles limit the widespread application of CAR T cell therapies, including treatment-associated toxicities, antigen heterogeneity, antigen escape, poor CAR T cell persistence and expansion, and particularly inefficient homing, infiltrating into, and surviving within solid tumors. Accordingly, to improve therapeutic efficacy and minimize side effects, innovative CAR design becomes urgently necessary, and researchers are developing numerous methods to overcome the limitations. Here we summarize currently available bioengineering strategies and discuss the future development from a viewpoint of synthetic biology.

Combination of genetically engineered T cells and immune checkpoint blockade for the treatment of cancer

Immune checkpoint (IC) blockade using monoclonal antibodies is currently one of the most successful immunotherapeutic interventions to treat cancer. By reinvigorating antitumor exhausted T cells, this approach can lead to durable clinical responses. However, the majority of patients either do not respond or present a short-lived response to IC blockade, in part due to a scarcity of tumor-specific T cells within the tumor microenvironment. Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) provide the necessary tumor-specific immune cell population to target cancer cells. However, this therapy has been considerably ineffective against solid tumors in part due to IC-mediated immunosuppressive effects within the tumor microenvironment. These limitations could be overcome by associating adoptive cell transfer of genetically engineered T cells and IC blockade. In this comprehensive review, we highlight the strategies and outcomes of preclinical and clinical attempts to disrupt IC signaling in adoptive T-cell transfer against cancer. These strategies include combined administration of genetically engineered T cells and IC inhibitors, engineered T cells with intrinsic modifications to disrupt IC signaling, and the design of CARs against IC molecules. The current landscape indicates that the synergy of the fast-paced refinements of gene-editing technologies and synthetic biology and the increased comprehension of IC signaling will certainly translate into a novel and more effective immunotherapeutic approaches to treat patients with cancer.

Chimeric antigen receptor engineered T cells and their application in the immunotherapy of solid tumours.

In this article, we reviewed the current literature studies and our understanding of the parameters that affect the chimeric antigen receptor T cells (CAR-T's) activation, effector function, in vivo persistence, and antitumour effects. These factors include T cell subsets and their differentiation stages, the components of chimeric antigen receptors (CAR) design, the expression promoters and delivery vectors, and the CAR-T production process. The CAR signalling and CAR-T activation were also studied in comparison to TCR. The last section of the review gave special consideration of CAR design for solid tumours, focusing on strategies to improve CAR-T tumour infiltration and survival in the hostile tumour microenvironment. With several hundred clinical trials undergoing worldwide, the pace of CAR-T immunotherapy moves from bench to bedside is unprecedented. We hope that the article will provide readers a clear and comprehensive view of this rapidly evolving field and will help scientists and physician to design effective CAR-Ts immunotherapy for solid tumours.

GPC2-CAR Tcells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity.

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed Tcells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6× 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR Tcell therapy of solid tumors.

Generation of Redirected Engineered Human Chimeric Antigen Receptor (CAR) T Cells.

Chimeric antigen receptor (CAR) T cell therapy that involves genetic engineering a patient's own immune cells with antigen-specific receptors has shown remarkable efficacy in blood cancer treatment. Numerous clinical studies with CAR T cells targeting the blood cell surface protein CD19 led to the FDA 's first approval of a genetically engineered cell therapy. The process of generating potent CAR T cells involves several carefully performed manufacturing steps. Here, we describe the generation of redirected engineered human CAR T cells for preclinical studies starting with the CAR design, retroviral gene transfer, detection of CAR expression, and expansion of transduced T cells.

About the Rising Star.

See related article on p. 6.Photo courtesy of Reed Hutchinson for the Broad Stem Cell Research Center at UCLAYvonne Y. Chen, PhD, is an Associate Professor of Microbiology, Immunology, and Molecular Genetics at the University of California, Los Angeles (UCLA). She is also a Member Researcher in the Parker Institute for Cancer Immunotherapy.Dr. Chen received her BS in Chemical Engineering from Stanford University and her PhD in Chemical Engineering from the California Institute of Technology. She performed postdoctoral research at the Seattle Children's Research Institute and the Department of Systems Biology at Harvard Medical School. Prior to joining UCLA in 2013, Dr. Chen was a Junior Fellow in the Harvard Society of Fellows.The focus of Dr. Chen's research is applying synthetic biology and biomolecular engineering techniques to develop robust cell-based therapies for otherwise intractable diseases. Her work has been recognized by many early career investigator awards, including the Cancer Research Institute (CRI) Lloyd J. Old STAR Award.As an inaugural CRI Lloyd J. Old STAR, Dr. Chen is exploring several strategies to improve the effectiveness of chimeric antigen receptor (CAR) T-cell therapy, which has shown remarkable success against B-cell cancers like leukemia and lymphoma, but limited efficacy and durability of benefits against other types of cancers. The approaches she is taking include the design of CARs that can recognize multiple targets, those that target soluble proteins and resist the immune-suppressing capabilities of the tumor environment, and those that can better discriminate between cancerous and healthy cells to avoid off-target effects.Overall, as a CRI Lloyd J. Old STAR, Dr. Chen is working to further improve T-cell therapy by: With these technologies that are grounded in mechanistic understanding, Dr. Chen hopes that it will enable the development of innovative technologies that can benefit more patients with hard-to-treat cancers.

Chimeric Antigen Receptor Structure and Manufacturing of Clinical Grade CAR Engineered Cells using Different Bioreactors.

Increasing success of adaptive cell therapy (ACT), such as genetically engineered T cells to express chimeric antigen receptors (CARs) proven to be highly significant technological advancements and impressive clinical outcomes in selected haematological malignancies, with promising efficacy. The evolution of CAR designs beyond the conventional structures is necessary to address some of the limitations of conventional CAR therapy and to expand the use of CAR T cells to a wider range of malignancies. There are various obstacles with a wide range of engineering strategies in order to improve the safety, efficacy and applicability of this therapeutic modality. Here we describe details of modular CAR structure with all the necessary domains and what is known about proximal CAR signalling in T cells. Furthermore, the global need for adoptive cell therapy is expanding very rapidly, and there is an urgent increasing demand for fully automated manufacturing methods that can produce large scale clinical grade high quality CAR engineered immune cells. Despite the advances in automation for the production of clinical grade CAR engineered cells, the manufacturing process is costly, consistent and involves multiple steps, including selection, activation, transduction, and Ex-Vivo expansion. Among these complex manufacturing phases, the choice of culture system to generate a high number of functional cells needs to be evaluated and optimized. Here we list the most advance fully automated to semi-automated bioreactor platforms can be used for the production of clinical grade CAR engineered cells for clinical trials but are far from being standardized. New processing options are available and a systematic effort seeking automation, standardization and the increase of production scale, would certainly help to bring the costs down and ultimately democratise this personalized therapy. In this review, we describe in detail different CAR engineered T cell platforms available and can be used in future for clinical-grade CAR engineered ATMP production.

Tuning the ignition of CAR: optimizing the affinity of scFv to improve CAR-T therapy.

How single-chain variable fragments (scFvs) affect the functions of chimeric antigen receptors (CARs) has not been well studied. Here, the components of CAR with an emphasis on scFv were described, and then several methods to measure scFv affinity were discussed. Next, scFv optimization studies for CD19, CD38, HER2, GD2 or EGFR were overviewed, showing that tuning the affinity of scFv could alleviate the on-target/off-tumor toxicity. The affinities of scFvs for different antigens were also summarized to designate a relatively optimal working range for CAR design. Last, a synthetic biology approach utilizing a low-affinity synthetic Notch (synNotch) receptor to achieve ultrasensitivity of antigen-density discrimination and murine models to assay the on-target/off-tumor toxicity of CARs were highlighted. Thus, this review provides preliminary guidelines of choosing the right scFvs for CARs.