Radiol Oncol 2022; 56(4): 409-419. doi: 10.2478/raon-2022-0049 409 review Cancer immunotherapy with CAR T cells: well-trodden paths and journey along lesser-known routes Anze Smole Immunology and Cellular Immunotherapy (ICI) Group, Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia Radiol Oncol 2022; 56(4): 409-419. Received 19 October 2022 Accepted 27 October 2022 Correspondence to: Anže Smole, Ph.D., Immunology and Cellular Immunotherapy (ICI) Group, Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, SI-1000 Ljubljana, Slovenia. E-mail: anze.smole@nib.si Disclosure: A.S. is a co-inventor on PCT International Patent Applications by The Trustees of the University of Pennsylvania, which include discoveries and inventions related to cellular immunotherapies using CAR and TCR T cells. This is an open access article distributed under the terms of the CC-BY license (https://creativecommons.org/licenses/by/4.0/). Background. Chimeric antigen receptor (CAR) T cell therapy is a clinically approved cancer immunotherapy ap- proach using genetically engineered T cells. The success of CAR T cells has been met with challenges regarding effica- cy and safety. Although a broad spectrum of CAR T cell variants and applications is emerging, this review focuses on CAR T cells for the treatment of cancer. In the first part, the general principles of adoptive cell transfer, the architecture of the CAR molecule, and the effects of design on function are presented. The second part describes five conceptual challenges that hinder the success of CAR T cells; immunosuppressive tumour microenvironment, T cell intrinsic proper- ties, tumour targeting, manufacturing cellular product, and immune-related adverse events. Throughout the review, selected current approaches to address these issues are presented. Conclusions. Cancer immunotherapy with CAR T cells represents a paradigm shift in the treatment of certain blood cancers that do not respond to other available treatment options. Well-trodden paths taken by pioneers led to the first clinical approval, and now the journey continues down lesser-known paths to treat a variety of cancers and other serious diseases with CAR T cells. Key words: chimeric antigen receptor; adoptive cell therapy; cancer; cellular immunotherapy; gene-engineered immune cells Introduction It took a series of ground-breaking ideas and clever experiments to establish the role of the im- mune system in controlling cancer (reviewed in1). Current understanding of cancer immunosurveil- lence also considers the notion that the immune system not only controls tumour formation and growth, but also influences the immunogenicity of the tumour and potential outgrowth. This hy- pothesis is referred to as cancer immunoediting, in which the three phases of elimination, equilib- rium, and escape can be distinguished (reviewed in2). These foundations are important for under- standing the concepts of cancer immunotherapy, which aims to enhance the immune system’s re- sponses to tumour cells. In the landmark study in 19883, ex vivo expand- ed autologous tumour-infiltrating lymphocytes (TILs) in combination with human interleukin-2 (rhIL-2) were developed and demonstrated objec- tive responses in patients with metastatic malig- nant melanoma. In addition, this work provided the unequivocal evidence of tumour-specific T cell mediated immunity leading to cancer recognition and elimination in humans.3 The next milestone was the development of a T cell-based cancer im- munotherapy using genetically engineered T cells, Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells410 made possible by a better understanding of basic T cell biology and genetic engineering approaches.4 Currently, the two most widely used immune receptors that confer tumour specificity and func- tionality to genetically engineered T cells are a tu- mour-reactive synthetic chimeric antigen receptor (CAR) and an identified (e.g., from TILs) or further engineered T-cell receptor (TCR). To date, CD19- targeting CAR T cells emerged as the most suc- cessful cellular immunotherapy approach. Clinical trials in relapsed or refractory paediatric acute lymphoblastic leukaemia (ALL)5–7 and high-grade B-cell lymphoma in adults8–16 have demonstrated that CAR T cell immunotherapy can produce ef- fective, long-lasting, and overall unprecedented clinical responses. CD19-targeting CAR T cells received the U.S. Food and Drug Administration and European Medicines Agency approval in 2017 and 2018, respectively. To date, genetically engi- neered T cell immunotherapies have mediated un- precedented clinical responses in hematologic ma- lignancies5–16 but the efficacy of these therapies is limited in solid tumours and also in certain blood cancers due to several factors, some of which are discussed in this review. In addition, adoptive cel- lular immunotherapies can cause potentially life- threatening complications such as cytokine release syndrome (CRS) and neurological toxicities.17,18 Nowadays, cellular immunotherapies include exciting research and clinical successes with TILs and T cells genetically modified with TCRs and CARs. In addition, alternative immune cells are being engineered with CARs19,20 and CAR T cells are now being used outside of cancer treatment.21–27 This review article focuses on CAR T cells to treat cancer. First, the concepts of adoptive cellular im- munotherapy with CAR T cells are introduced. Then, the architecture of the CAR molecule is de- scribed and how design affects function. Current challenges and limitations regarding efficacy and safety are then presented, focusing on the immu- nosuppressive tumour microenvironment (TME), T cell intrinsic properties, tumour targeting, cel- lular product manufacturing and immune-related adverse events. Throughout, this paper presents selected recent next-generation approaches to the development of CAR T cells that have the potential to overcome some of these challenges. Principles of cellular immunotherapy Adoptive cell transfer In its broadest sense, adoptive T cell transfer (ACT) involves the isolation of T lymphocytes from blood and their reinfusion into patients for the treatment of disease. Advances in the understanding of ba- sic mechanisms in T cell biology, including target recognition, T cell activation, signal transduction, role of soluble factors, and co-stimulation signals, have led to a better understanding of T cell func- tion, expansion, and persistence.28 This knowl- edge has been critical for establishing optimized protocols for ex vivo culturing conditions, activa- tion, and expansion. To redirect the specificity of T cells, genetic engineering approaches had to be developed to introduce the genetic cassette en- coding TCR or CAR into primary T cells.4 These significant advances enabled the development of sophisticated T cell-based therapies such as CAR T cells that transformed oncology. Current clinical adoptive transfer of CAR T cells involves three steps (Figure 1). (1) Collection of T cells: The patient’s own T cells (in the autologous ACT setting), which are the body’s primary com- ponent for fighting infection and cancer, are first isolated from the blood in a procedure called leu- kapheresis. These cells express endogenous TCR. (2) Ex vivo reprogramming and manufacturing of the cellular product: Primary T cells are first ac- tivated using activation beads and then a genetic cassette encoding the CAR molecule is introduced into the primary T cells by viral transduction, which transforms donor T cells into CAR T cells. Introduction of these molecules reprograms T FIGURE 1. The principle of adoptive cellular immunotherapy. CAR = chimeric antigen receptor; TCR = T-cell receptor Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells 411 cells to specifically recognize, target and eliminate cancer cells, while ex vivo expansion allows man- ufacturing of sufficient numbers of CAR T cells. (3) Infusion: Patients are treated with a prepara- tory chemotherapy and then reinfused with the modified T cells. After ex-vivo expansion, the re- programmed cells are infused back to the patient where they find and eliminate the disease.29 Design of a CAR molecule The Chimera is a creature of Greek mythology that consists of parts of various animals. Based on this analogy, CAR is a molecule that combines the properties of a monoclonal antibody that enables antigen recognition with the components of the TCR that drive T cell signalling and activation. CAR is a molecule composed of different domains, each of which contributes to a specific functional- ity, and together they effectively redirect T cells to the target of interest and elicit T cell responses (Figure 2). Design of CAR molecule continues to evolve as we gain more knowledge from basic immunology and clinical trials. First-generation CARs consisted of an extracellular antigen-binding domain, usu- ally in the form of an antibody-derived single- chain variable fragment (scFv) linked to intracel- lular signalling domains, most often derived from the components of the TCR complex, for example the CD3 zeta chain (CD3ζ).30,31 This molecule was capable of recognizing antigens independent of HLA (human leukocyte antigens) presentation. First-generation CARs provided proof of principle but did not enable long-term T cell persistence and effector responses due to their limited signalling capacity.32 This section describes CAR molecule architecture and its individual domains. Antigen recognition domain The specificity of the CAR molecule is defined by the antigen-targeting ectodomain. In most current designs, this is scFv, which is a fusion between variable heavy and variable light chains of an an- tibody connected by a flexible linker. The affinity of CAR has been shown to have important effects on the functions of CAR T cells. In a clinical study, enhanced CAR T cell expansion and prolonged persistence were observed with a low affinity CD19 CAR compared to CAR T cells with FMC63, a scFv in clinically approved CD19 targeting CAR T cells.33 Interestingly, in a different study, linker length has also been shown to influence CAR clustering, antigen-independent signalling and function of CAR T cells.34 ScFv have now been de- signed to target several cell surface molecules as- sociated with cancer, most often proteins, but also glycans such as the aberrant cancer-associated Tn glycoform of MUC1, which is expressed in a varie- ty of cancers.35 Although the mechanism by which binding of CAR to its cognate antigen leads to T cell activation shares key similarities, it also differs substantially from the mechanism by which TCR binding leads to T cell activation. While CARs gen- erally exhibit higher affinity that can also be tuned, the sensitivity is higher in TCRs.36 Currently, CAR T cells that target CD19 (tisagenlecleucel, axicabta- gene ciloleucel, lisocabtagene maraleucel and brexucabtagene autoleucel) and B-cell maturation antigen (BCMA also known as TNFRSF17) (ide- cabtagene vicleucel, ciltacabtagene autoleucel) are being FDA-approved and marketed37,38 while sev- eral others are in clinical trials, including CD20, CD22, CD33, CD5, and CD7 (reviewed in39). Some of widely explored targets in solid tumours include alpha folate receptor (FOLR1), human epidermal growth factor receptor 2 (HER2), carcinoembry- onic antigen (CEA), ganglioside G2 (GD2), meso- thelin, epidermal growth factor receptor variant III (EGFRvIII), mucin1 (MUC1), interleukin-13 re- ceptor subunit alpha-2 (IL13Ra2), prostate specific membrane antigen (PSMA), B7 homolog 3 (B7-H3), epidermal growth factor receptor (EGFR), and fi- broblast activation protein (FAP) (reviewed in39,40). FIGURE 2. Schematics of the basic CAR architecture. CAR = chimeric antigen receptor; scFv = single-chain variable fragment Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells412 Hinge and transmembrane domain The scFv domain is connected via a hinge region to the transmembrane (TM) domain. The TM do- main is often derived from CD8 or CD28 molecules and functions to anchor CAR in the membrane and facilitate signal transduction. The choice or engineering of TM domain may affect the inter- actions between CAR molecules themselves41, or with other endogenous molecules such as CD28.42 Innovative designs in hinge and TM domains may provide opportunities to tune CAR signalling. Co-stimulatory domain In the clinically approved CARs, the membrane proximal intracellular domain is the co-stimula- tory domain. The need for costimulatory domain arose when limited clinical efficacy of the first gen- eration CAR T cells was observed.43 The authors concluded that genetically engineered tumour- reactive T cells are safe but do not persist and that strategies to prolong T cell persistence are needed. The first domain included in the CAR design was the CD28 costimulatory domain, initially alone44 and then in combination with CD3ζ.45,46 The CD28 domain provides robust response with an effec- tor phenotype and high levels of secreted IL-2 and tumour lysis activity.47 The other widely used co- stimulatory domain introduced into CAR design is CD137 (4-1BB). Compared to CD28, 4-1BB provides improved persistence, shift towards central mem- ory phenotype differentiation, a lower propensity to exhaustion and reduced toxicity.15,47,48 A recent comparison between the two marketed products, axicabtagene ciloleucel and tisagenlecleucel ex- amined the differences between CD28 and 41BB in relapsed or refractory diffuse large B cell lym- phoma and concluded that axicabtagene ciloleucel provides higher efficacy and also a higher toxic- ity.48 Other co-stimulatory domains are also be- ing studied including CD2749, ICOS50, and OX-4051, each of which has certain favourable properties. Finally, third generation CARs comprise a combi- nation of two costimulatory domains and some of these have already been tested in clinical trials.52 However, excessive stimulation can lead to dys- functional CAR T cells.53 The design of the second-generation CARs, which includes additional co-stimulatory domains that enhance the expansion, persistence, and effec- tor functions of CAR T cells, has been key to the success of clinical trials. A recent study revealed that CAR T cells persisted for more than ten years after infusion, with sustained remission in a pa- tient treated with CD19 targeting 4-1BB CAR T in 2010.54 Selection of the co-stimulatory domain in- fluences important parameters of CAR T cell ther- apy including effector function, response kinetics, expansion, differentiation, metabolism and toxic- ity.47 Innovative studies are attempting to address the complexities and unknowns by characterizing multiple intracellular signalling domains in a high throughput manner to identify to the CAR designs that have improved functions compared to clini- cally used CAR T cells.55 Activation domain The distal intracellular domain is CD3ζ, a signal transduction component of the TCR complex that has been repurposed to drive CAR signalling after recognition of its cognate target. Immunoreceptor tyrosine-based activation motifs (ITAMs) are key motifs in the CD3ζ domain. When the TCR recog- nises its target, ITAMs are phosphorylated through a series of molecular interactions mediated by Lck kinase (lymphocyte-specific protein tyrosine ki- nase). This leads to the recruitment and activation of ZAP-70 (Zeta-chain-associated protein kinase 70), which orchestrates a series of downstream phosphorylation events that result in the complex and highly regulated signal transduction required for T cell activation and effector functions.56 CAR signalling resamples key features of TCR signal- ling but also differs in important ways. Analogous to the “two-step” T cell activation model, CD3ζ provides signal 1 whereas the co-stimulatory do- main provides signal 2. CAR signalling is active area of research in basic T cell biology and has direct importance for the therapeutic implementa- tions. As an alternative to the CD3ζ, other domains are investigated for CAR T cell therapy including the CD3ε.57 An example of rational tuning and cal- ibration of CAR activation and signalling demon- strated that combinatorial mutation of ITAM mo- tifs directs differentiation towards memory T cell states, which translated in improved persistence and therapeutic potency in preclinical mouse models.58 Moreover, using the genome editing ap- proach, the TRAC locus was modified in primary human T cells to target cell-surface molecules via their TCR complex, which was reconfigured to use the same targeting component as a corresponding CAR. These HLA-independent TCRs, referred to by the authors as HIT receptors, have been shown to be particularly sensitive compared to CD28- based CARs.59 Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells 413 Challenges and opportunities of cellular immunotherapy The success of CAR T cells is countered by chal- lenges in efficacy in solid tumours40 and immune- related adverse events. Underlying causes of limit- ed efficacy include immunosuppressive TME and T cell and tumour intrinsic properties. In addition, the manufacturing of the cellular product and lack of tumour specific targets represent a major chal- lenge. Here, some of these aspects are outlined and selected recent publications are presented that at- tempt to meet these challenges (Figure 3). Immunosuppressive tumour microenvironment Immunosuppressive TME limits the efficacy of CAR T cells by interfering with their function. Various approaches have been developed to ad- dress these challenges, including upgrading en- gineered T cells with the expression of accessory molecules. Pioneering work has been done with tumour infiltrating-lymphocytes (TILs) engi- neered with inducible expression of the potent im- mune-enhancing molecule IL-12.60 This approach was tested in human clinical trials and clinical activity but also toxicity were observed. Similarly, CAR T cells have been equipped with accessory molecules to counteract various aspects of the hos- tile immunosuppressive TME. These molecules include IL-1861–64, PD-165, CTLA-4, or TIM366 block- ing scFvs and minibodies, CD40L67, dominant-neg- ative Fas68 or Fas-41BB switch69 receptors, pro-in- flammatory neutrophil-activating protein (NAP) from Helicobacter pylori70 and dominant-negative TGFβ Receptor.71 Recently, a pooled knock-in plat- form has been developed to screen for genetic constructs that can improve T cell functions for effective cell therapies when constitutively over- expressed.72 Additional genetic approach coupling expression of effector molecule with specific anti- gen recognition was developed using a synNotch platform.73 These approaches improve the efficacy of T cell therapy and highlight the need to develop robust and efficient gene expression systems suit- able for clinical translation. Depleting of cells that limit the efficacy of CAR T cells is a viable approach to increase CAR T cell activity in TME. One approach is the depletion of immunosuppressive M2 tumour-associated mac- rophages (TAMs) by CAR-mediated targeting of a folate receptor β (FRβ) positive subset of TAMs that exhibit an immunosuppressive M2-like pro- file. CAR T cells eliminated these FRβ+ TAMs, resulting in recruitment of endogenous tumour- specific CD8+ T cells, improved tumour control, and prolonged survival.74 Therefore, overcoming immunosuppressive TME with innovative approaches is an important pillar in improving the activity of CAR T cells. T cell intrinsic properties It is becoming increasingly clear that intrinsic T cell dysfunctions, such as T cell exhaustion limit the success of CAR T cells in solid tumours but also in hematologic malignancies that induce dysfunctional T cell states. A recent correlative study examined the determinants of response at genomic, phenotypic and functional levels and demonstrated that clinical efficacy in patients with chronic lymphocytic leukaemia (CLL) treated with CAR T cells is affected by complex intrinsic im- mune cell functions and dysfunctions.75 Chronic stimulation of T cells with an antigen, as occurs also with CAR T cells targeting solid tumours, is an important reason for the dysfunction.76 One ap- proach to overcome this problem is a temporary resting period in which the functionality of the CAR T cells is restored.77 Innovative approaches FIGURE 3. Challenges of cellular immunotherapy with chimeric antigen receptor (CAR) T cells. Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells414 have been developed to maintain functionality of CAR T cells, including overexpression of c-Jun78 or a combination of BATF and IRF4.79 Recent studies linked the heterogeneity of autologous CAR T cells in terms of cellular and molecular characteristics of the infusion products to differences in efficacy and toxicity following CD19 CAR T therapy.80 In a distinct approach, CAR T cells were designed to express interleukin IL-7 and CCL19 to mimic a favourable milieu that forms and maintains T cell zones in lymphoid organs.81 These upgraded CAR T cells demonstrated enhanced recruitment of T cells and dendritic cells into tumour and aug- mented therapeutic effects against solid tumours. Favourable effect on differentiation and persistence of CAR T cells has been demonstrated with the constitutive IL-7 receptor82, IL-1583, and synthetic receptors combining orthogonal extracellular IL-2 and intracellular IL-9 domains.84 In a recent study, overexpression of more than 10,000 barcoded hu- man open reading frames (ORFs) identified posi- tive regulators of T cell function, with the aim of developing improved cellular immunotherapies including CAR T cells.85 The intrinsic properties of T cells in the context of CAR T cell therapy require careful study from the perspective of basic immunology. This knowl- edge is important to overcome the dysfunction that limits the activity of CAR T cells. Tumour targeting CD19 is an example of a target that is also ex- pressed on normal cells (B cells), but humans can live with B cell aplasia and appropriate treatment, namely intravenous immunoglobulin (IVIG) treat- ment, which overcomes antibody deficiencies. However, a major challenge in the development of CAR T cells is to identify targets that are homoge- neously expressed at sufficient levels on the sur- face of tumour cells and are not present on healthy tissues at levels that would cause damage. A tragic example is described in a case report where CAR T cells based on the humanized monoclonal anti- body trastuzumab (Herceptin), which recognizes ERBB2, led to the patient’s death.86 The authors hypothesize that the large number of CAR T cells infiltrated in the lungs and triggered cytokine release after recognizing low levels of ERBB2 on lung epithelial cells. Acute myeloid leukemia (AML) is a candidate disease for cellular immunotherapy. However, tar- geting the myeloid marker CD33 in (AML) leads to toxicity from destroying normal myeloid cells. The authors demonstrated the artificial generation of a leukaemia-specific antigen by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells.87 In this approach, the host was genetically engineered to avoid on-target and off-tumour toxicity. Heterogeneity88 and loss of antigen expression on cancer cells under selective pressure of targeted immunotherapy can lead to evasion strategies by cancer cells.89 This has sparked the development of CARs with multiple specificities. Examples for he- matologic malignancies include a dual CD19 and CD22 CAR T cells expressing two CAR receptors90 or CAR T cells with a tandem scFv CAR molecule with dual targeting of CD19 and CD22.91,92 Another approach that allows on demand mul- tiple antigen targeting to mitigate a potential anti- gen escape in CAR T cell therapy is adapter CAR platform. One example is the universal immune receptor based on SpyCatcher-SpyTag chemistry. The SpyCatcher immune receptor redirects prima- ry human T cells upon adding SpyTag-labeled tar- geting ligands.93 Another example is the so-called SUPRA CAR, a split-CAR design that allows the development of CAR T cells with multiple features and provides the ability to switch targets without re-engineering the T cells.94 TCRs have been shown to enable targeting of neoantigens95–98 and recently CARs have also been developed that specifically target peptides derived from intracellular proteins presented by HLAs.99 These results demonstrate that CAR T cells are not limited to recognizing molecules expressed on the surface, but can now be engineered to recognize intracellular targets presented by the HLAs, which mimics recognition by TCRs. This significantly in- creases the potential pool of CAR T targets. Tumour targeting represents a challenge and an opportunity for innovative approaches and advances will be necessary to develop CAR T cell therapies for new disease indications, particularly in solid tumours. Manufacturing cellular product The manufacturing process, which involves the collection of autologous T cells and the generation of CAR T cells for each individual patient, is ex- pensive and complex from an infrastructural and logistical perspective. In addition, unexpected challenges can emerge with some of the existing pipelines. One such example is the discovery that Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells 415 the lentivirally delivered CAR gene was inadvert- ently introduced into a single leukemic B cell dur- ing T cell manufacturing. This anti-CD19 CAR molecule then bound the CD19 epitope on the sur- face of the same leukemic cells, which masked it from being recognized by the CD19-targeting CAR T cells, resulting in relapse.100 Therefore, there is great interest in optimizing the manufacturing of the cellular product to make it safer, more effective and broadly available. Recent study presented the shortened process of manufacturing of non-activated CAR T cells with improved functionality.101 Another study investi- gated the approach where CAR T cells have been manufactured from the defined CD4+ and CD8+ T cell subsets and infused in a defined CD4+: CD8+ composition.102 Recent study investigated the ef- ficacy and safety of CAR T cells generated from preselected naïve/stem memory T cells, observing a superior safety and efficacy profile compared to unselected bulk T cells.103 In addition, alterna- tive sources of donor T cells are being explored, including allogeneic off-the-shelf approaches.104,105 Recently, the first human clinical trials were re- ported with CRISPR/Cas9-engineered T cells that edited PD-1106 or even demonstrated multiplex CRISPR/Cas9 editing of the endogenous T cell re- ceptor and PD-1.107 Currently CAR T cells are produced via lentivi- ral or retroviral transduction, where integration of a gene encoding CAR is semi random and poses certain risks and challenges. Recent studies have demonstrated that genome editing technologies can be used for CRISPR/Cas9-mediated targeted integration of a CAR into an endogenous locus via homology-directed repair (HDR) and an adeno- associated virus (AAV) vector as a HDR donor template.108,109 Further, a non-viral strategy using a double stranded DNA as a HDR donor template for CRISPR/Cas9-mediated targeted integration has been demonstrated.110 CAR T cells generated with non-viral targeted integration have even been test- ed in a clinical trial.111 Finally, approaches to gener- ate CAR T cells in vivo are also being explored.112 Bringing the manufacture of cellular products to a level that enabled clinical approval required extensive efforts by pioneers and now continues to represent an area of opportunity to make CAR T cells safer, more effective, and broadly available. Immune-related adverse events Unfortunately, adoptive cancer immunotherapy carries safety risks such as cytokine release syn- drome (CRS) and neurologic toxicities113, that have led to life-threatening complications.17 Current management strategies include systemic use of the antibody tocilizumab, which blocks IL-6 re- ceptor.114 CRS and neurotoxicity are the two main toxicities associated with clinically used CD19- targeting therapies. B-cell aplasia is on-target, off- tumour adverse effect of CARs that target B-cell differentiation antigens such as CD1917 and can be effectively managed by IVIG, as mentioned earlier in the paper. Further, on-target off-tumour toxic- ity can have devastating effects86 as described in previous sections. A recent study illuminated a contributor to se- vere neurotoxicity observed in a subset of patients treated with CD19-targeting therapies. The au- thors show that brain mural cells, which surround the endothelium and are critical for the integrity of the blood-brain-barrier, express CD19, implying that on-target off-tumour toxicities may occur.115 Several approaches are being developed to mitigate toxicities, including platforms in which the activity of CAR T cells can be regulated by ge- netically encoded transient functions in a combi- nation with the small molecules116–118 or targeting ligands.93,94 Suicide switches based on inducible caspase-9119 or on expression of surface molecules, such as a truncated version of epidermal growth factor receptor (EGFRt) are being developed. In the latter case, EGFRt is expressed together with CAR on the surface of T cells, so that CAR T cells can be eliminated by addition of an antibody targeting EGFRt.120 SynNotch enabled AND-gate combinatorial tar- geting, in which the synNotch receptor first recog- nized one tumour antigen, which led to the release of a transcriptional activator domain to drive ex- pression of a CAR targeting another tumour an- tigen.121 New insights into the biology of CAR T cells, experience from clinical trials, and advances in engineering approaches now provide the basis for making CAR T cells safer while maintaining their efficacy. Conclusions This review article focuses on CAR T cells for can- cer immunotherapy. However, it is important to note that cellular immunotherapy using TILs122,123 or T cells with engineered TCRs has achieved re- markable success in clinical studies in solid tu- mours and established approaches to target intra- Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells416 cellular antigens presented in the context of ma- jor histocompatibility complex (MHC) molecules including neoantigens.95–97 The success of CAR T cells in treating cancer has led to their use outside of cancer treatment, including autoimmunity21–23, infections24,25, senescence-associated pathologies26, and cardiac fibrosis.27,112 Several cell types includ- ing Natural Killer (NK)19 cells and macrophages20 are being explored as alternatives to T cells that have certain advantages and provide new features. Cancer immunotherapy with CAR T cells repre- sents a paradigm shift in the treatment of certain blood cancers that do not respond to other avail- able treatment options. Well-trodden paths blazed by pioneers led to the first FDA and EMA approv- al, and the journey now continues on lesser-known paths to treat a variety of cancers and other serious diseases with CAR T cells. Acknowledgments A.S. thanks J. Pohar and K. Butina Ogorelec for re- viewing and providing valuable feedback on the manuscript. A.S. received funding from Slovenian Research Agency (ARRS) for Project J3-3084 and Program P1-0245 and from Research fund of the National Institute of Biology for Project 10ICIGEN (ICI). Figures created with BioRender.com References 1. Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ. Natural innate and adaptive immunity to cancer. Annu Rev Immunol 2011; 29: 235-71. doi: 10.1146/annurev-immunol-031210-101324 2. Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating im- munity’s roles in cancer suppression and promotion. Science 2011; 331: 1565-70. doi: 10.1126/science.1203486 3. Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immu- notherapy of patients with metastatic melanoma. N Engl J Med 1988; 319: 1676-80. doi: 10.1056/nejm198812223192527 4. Sadelain M, Rivière I, Riddell S. Therapeutic T cell engineering. Nature 2017; 545: 423-31. doi: 10.1038/nature22395 5. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013; 368: 1509-18. doi: 10.1056/NEJMoa1215134 6. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018; 378: 439-48. doi: 10.1056/nejmoa1709866 7. Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 2015; 385: 517-28. doi: 10.1016/S0140- 6736(14)61403-3 8. Schuster SJ, Svoboda J, Chong EA, Nasta SD, Mato AR, Anak Ö, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017; 28: 2545-54. doi: 10.1056/NEJMoa1708566 9. Sommermeyer D, Hudecek M, Kosasih PL, Gogishvili T, Maloney DG, Turtle CJ, et al. Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 2016; 30: 492-500. doi: 10.1038/leu.2015.247 10. Brentjens RJ, Rivière I, Park JH, Davila ML, Wang X, Stefanski J, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 2011; 118: 4817-28. doi: 10.1182/blood-2011-04-348540 11. Porter DL, Levine BL, Kalos M, Bagg A, June CH, Levine BL, et al. Chimeric antigen receptor–modified T cells in chronic lymphoid leukemia. N Engl J Med 2011; 365: 725-33. doi: 10.1056/nejmoa1103849 12. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lym- phoma. N Engl J Med 2017; 377: 2531-44. doi: 10.1056/nejmoa1707447 13. Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, et al. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lym- phoma. N Engl J Med 2019; 380: 45-56. doi: 10.1056/nejmoa1804980 14. Abramson JS, McGree B, Sarah Noyes N, Sean Plummer B, Curtis Wong B, Chen YB, et al. Anti-CD19 CAR T cells in CNS diffuse large-B-cell lymphoma. N Engl J Med 2017; 377: 783-4. doi: 10.1056/NEJMc1704610 15. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cells with chimeric antigen receptors have potent antitumor effects and can estab- lish memory in patients with advanced leukemia. Sci Transl Med 2011; 3: 95ra73. doi: 10.1126/scitranslmed.3002842 16. Kochenderfer JN, Wilson WH, Janik JE, Dudley ME, Stetler-Stevenson M, Feldman SA, et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically en- gineered to recognize CD19. Blood 2010; 116: 4099-102. doi: 10.1182/ blood-2010-04-281931 17. Neelapu SS, Tummala S, Kebriaei P, Wierda W, Gutierrez C, Locke FL, et al. Chimeric antigen receptor T-cell therapy – assessment and manage- ment of toxicities. Nat Rev Clin Oncol 2017; 15: 47-62. doi: 10.1038/ nrclinonc.2017.148 18. Brudno JN, Kochenderfer JN. Recent advances in CAR T-cell toxicity: mechanisms, manifestations and management. Blood Rev 2019; 34: 45-55. doi: 10.1016/j.blre.2018.11.002 19. Liu E, Marin D, Banerjee P, MacApinlac HA, Thompson P, Basar R, et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N Engl J Med 2020; 382: 545-53. doi: 10.1056/nejmoa1910607 20. Klichinsky M, Ruella M, Shestova O, Lu XM, Best A, Zeeman M, et al. Human chimeric antigen receptor macrophages for cancer immunothera- py. Nat Biotechnol 2020; 38: 947-53. doi: 10.1038/s41587-020-0462-y 21. Ellebrecht CT, Bhoj VG, Nace A, Choi EJ, Mao X, Cho MJ, et al. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune dis- ease. Science 2016; 353: 179-84. doi: 10.1126/science.aaf6756 22. Mackensen A, Müller F, Mougiakakos D, Böltz S, Wilhelm A, Aigner M, et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat Med 2022; 28: 2124-32. doi: 10.1038/s41591-022-02017-5 23. Mougiakakos D, Krönke G, Völkl S, Kretschmann S, Aigner M, Kharboutli S, et al. CD19-Targeted CAR T cells in refractory systemic lupus erythemato- sus. N Engl J Med 2021; 385: 567-9. doi: 10.1056/nejmc2107725 24. Maldini CR, Gayout K, Leibman RS, Dopkin DL, Mills JP, Shan X, et al. HIV- resistant and HIV-specific CAR-modified CD4+ T cells mitigate HIV disease progression and confer CD4+ T cell help in vivo. Mol Ther 2020; 28: 1-15. doi: 10.1016/j.ymthe.2020.05.012 25. Maldini CR, Claiborne DT, Okawa K, Chen T, Dopkin DL, Shan X, et al. Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis in vivo. Nat Med 2020; 26: 1776-87. doi: 10.1038/s41591- 020-1039-5 26. Amor C, Feucht J, Leibold J, Ho YJ, Zhu C, Alonso-Curbelo D, et al. Senolytic CAR T cells reverse senescence-associated pathologies. Nature 2020; 583: 127-32. doi: 10.1038/s41586-020-2403-9 27. Aghajanian H, Kimura T, Rurik JG, Hancock AS, Leibowitz MS, Li L, et al. Targeting cardiac fibrosis with engineered T cells. Nature 2019; 573: 430-3. doi: 10.1038/s41586-019-1546-z 28. Kalos M, June CH. Adoptive T Cell Transfer for cancer immunotherapy in the era of synthetic biology. Immunity 2013; 39: 49-60. doi: 10.1016/j. immuni.2013.07.002 Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells 417 29. June CH, O ’connor RS, Kawalekar OU, Ghassemi S, Milone MC, O’Connor RS, et al. CAR T cell immunotherapy for human cancer. Science 2018; 1365: 1361-5. doi: 10.1126/science.aar6711 30. Kuwana Y, Asakura Y, Utsunomiya N, Nakanishi M, Arata Y, Itoh S, et al. Expression of chimeric receptor composed of immunoglobulin-derived V resions and T-cell receptor-derived C regions. Biochem Biophys Res Commun 1987; 149: 960-8. doi: 10.1016/0006-291X(87)90502-X 31. Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the γ or ζ subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci U S A 1993; 90: 720-4. doi: 10.1073/ pnas.90.2.720 32. Brocker T, Karjalainen K. Signals through T cell receptor-ζ chain alone are insufficient to prime resting T lymphocytes. J Exp Med 1995; 181: 1653-9. doi: 10.1084/jem.181.5.1653 33. Ghorashian S, Kramer AM, Onuoha S, Wright G, Bartram J, Richardson R, et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med 2019; 25: 1408-14. doi: 10.1038/s41591-019-0549-5 34. Singh N, Frey N V., Engels B, Barrett DM, Shestova O, Ravikumar P, et al. Antigen-independent activation enhances the efficacy of 4-1BB- costimulated CD22 CAR T cells. Nat Med 2021; 27: 842-50. doi: 10.1038/ s41591-021-01326-5 35. Posey AD, Schwab RD, Boesteanu AC, Steentoft C, Mandel U, Engels B, et al. Engineered CAR T cells targeting the cancer-associated Tn-glycoform of the membrane mucin MUC1 control adenocarcinoma. Immunity 2016; 44: 1444-54. doi: 10.1016/j.immuni.2016.05.014 36. Harris DT, Kranz DM. Adoptive T Cell therapies: a comparison of T cell receptors and chimeric antigen receptors. Trends Pharmacol Sci 2015; 37: 220-30. doi: 10.1016/j.tips.2015.11.004 37. Bashor CJ, Hilton IB, Bandukwala H, Smith DM, Veiseh O. Engineering the next generation of cell-based therapeutics. Nat Rev Drug Discov 2022; 21: 655-75. doi: 10.1038/s41573-022-00476-6 38. Mullard A. FDA approves second BCMA-targeted CAR-T cell therapy. Nat Rev Drug Discov 2022; 21: 249. doi: 10.1038/d41573-022-00048-8 39. MacKay M, Afshinnekoo E, Rub J, Hassan C, Khunte M, Baskaran N, et al. The therapeutic landscape for cells engineered with chimeric antigen receptors. Nat Biotechnol 2020; 38: 233-44. doi: 10.1038/s41587-019- 0329-2 40. Johnson LA, June CH. Driving gene-engineered T cell immunotherapy of cancer. Cell Res 2016; 27: 38-58. doi: 10.1038/cr.2016.154 41. Elazar A, Chandler NJ, Davey AS, Weinstein JY, Nguyen J V., Trenker R, et al. De novo-designed transmembrane domains tune engineered receptor functions. Elife 2022; 11: 1-29. doi: 10.7554/eLife.75660 42. Muller YD, Nguyen DP, Ferreira LMR, Ho P, Raffin C, Valencia RVB, et al. The CD28-transmembrane domain mediates chimeric antigen receptor heter- odimerization with CD28. Front Immunol 2021; 12: 639818. doi: 10.3389/ fimmu.2021.639818 43. Kershaw MH, Westwood J a, Parker LL, Wang G, Eshhar Z, Mavroukakis S a, et al. A phase I study on adoptive immunotherapy using gene- modified T cells for ovarian cancer. Clin Cancer Res 2006; 12: 6106-15. doi: 10.1158/1078-0432.CCR-06-1183 44. Krause A, Guo HF, Latouche JB, Tan C, Cheung NK V., Sadelain M. Antigen- dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes. J Exp Med 1998; 188: 619-26. doi: 10.1084/jem.188.4.619 45. Maher J, Brentjens RJ, Gunset G, Rivière I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ/CD28 re- ceptor. Nat Biotechnol 2002; 20: 70-5. doi: 10.1038/nbt0102-70 46. Hombach A, Wieczarkowiecz A, Marquardt T, Heuser C, Usai L, Pohl C, et al. Tumor-specific T cell activation by recombinant immunoreceptors: CD3ζ signaling and CD28 costimulation are simultaneously required for ef- ficient IL-2 secretion and can be integrated into one combined CD28/CD3ζ signaling receptor molecule. J Immunol 2001; 167: 6123-31. doi: 10.4049/ jimmunol.167.11.6123 47. Kawalekar OU, O’Connor RS, Fraietta JA, Guo L, McGettigan SE, Posey AD, et al. Distinct signaling of coreceptors regulates specific metabolism path- ways and impacts memory development in CAR T Cells. Immunity 2016; 44: 380-90. doi: 10.1016/j.immuni.2016.01.021 48. Bachy E, Le Gouill S, Di Blasi R, Sesques P, Manson G, Cartron G, et al. A real-world comparison of tisagenlecleucel and axicabtagene ciloleucel CAR T cells in relapsed or refractory diffuse large B cell lymphoma. Nat Med 2022; 28: 2145-54. doi: 10.1038/s41591-022-01969-y 49. Song DG, Ye Q, Poussin M, Harms GM, Figini M, Powell DJ. CD27 costimulation augments the survival and antitumor activity of redi- rected human T cells in vivo. Blood 2012; 119: 696-706. doi: 10.1182/ blood-2011-03-344275 50. Guedan S, Chen X, Madar A, Carpenito C, McGettigan SE, Frigault MJ, et al. ICOS-based chimeric antigen receptors program bipolar TH17/ TH1 cells. Blood 2014; 124: 1070-80. doi: 10.1182/blood-2013-10-535245 51. Hombach AA, Heiders J, Foppe M, Chmielewski M, Abken H. OX40 costim- ulation by a chimeric antigen receptor abrogates CD28 and IL-2 induced IL- 10 secretion by redirected CD4+ T cells. Oncoimmunology 2012; 1: 458-66. doi: 10.4161/onci.19855 52. Ramos CA, Rouce R, Robertson CS, Reyna A, Narala N, Vyas G, et al. In vivo fate and activity of second- versus third-generation CD19-specific CAR-T cells in B cell Non-Hodgkin’s lymphomas. Mol Ther 2018; 26: 2727-37. doi: 10.1016/j.ymthe.2018.09.009 53. Wijewarnasuriya D, Bebernitz C, Lopez AV, Rafiq S, Brentjens RJ. Excessive costimulation leads to dysfunction of adoptively transferred T cells. Cancer Immunol Res 2020; 18: 732-42. doi: 10.1158/2326-6066.CIR-19-0908 54. Melenhorst JJ, Chen GM, Wang M, Porter DL, Chen C, Collins MKA, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 2022; 602: 503-9. doi: 10.1038/s41586-021-04390-6 55. Gordon KS, Kyung T, Perez CR, Holec P V., Ramos A, Zhang AQ, et al. Screening for CD19-specific chimaeric antigen receptors with enhanced signalling via a barcoded library of intracellular domains. Nat Biomed Eng 2022; 6: 855-66. doi: 10.1038/s41551-022-00896-0 56. Love PE, Hayes SM. ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb Perspect Biol 2010; 2: 1-11. doi: 10.1101/cshperspect. a002485 57. Wu W, Zhou Q, Masubuchi T, Shi X, Li H, Xu X, et al. Multiple signaling roles of CD3ε and its application in CAR-T cell therapy. Cell 2020; 182: 855-871. e23. doi: 10.1016/j.cell.2020.07.018 58. Feucht J, Sun J, Eyquem J, Ho YJ, Zhao Z, Leibold J, et al. Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency. Nat Med 2019; 25: 82-8. doi: 10.1038/s41591-018-0290-5 59. Mansilla-Soto J, Eyquem J, Haubner S, Hamieh M, Feucht J, Paillon N, et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 2022; 28: 345-52. doi: 10.1038/s41591-021-01621-1 60. Zhang L, Morgan RA, Beane JD, Zheng Z, Dudley ME, Kassim SH, et al. Tumor-infiltrating lymphocytes genetically engineered with an induc- ible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res 2015; 21: 2278-88. doi: 10.1158/1078-0432. CCR-14-2085 61. Hu B, Ren J, Luo Y, Scholler J, Zhao Y, June CH, et al. CAR T cells secreting IL18 augment antitumor immunity and increase T cell proliferation and costim- ulation. Cell Rep 2017; 20: 3025-33. doi: 10.1016/j.celrep.2017.09.002 62. Kunert A, Chmielewski M, Wijers R, Berrevoets C, Abken H, Debets R. Intra- tumoral production of IL18, but not IL12, by TCR-engineered T cells is non- toxic and counteracts immune evasion of solid tumors. Oncoimmunology 2017; 7: 1-12. doi: 10.1080/2162402X.2017.1378842 63. Chmielewski M, Abken H. CAR T cells releasing IL-18 convert to T-Bet high FoxO1 low effectors that exhibit augmented activity against advanced solid tumors. Cell Rep 2017; 21: 3205-19. doi: 10.1016/j.celrep.2017.11.063 64. Zimmermann K, Kuehle J, Dragon AC, Galla M, Kloth C, Rudek LS, et al. Design and characterization of an “all-in-one” lentiviral vector system combining constitutive anti-gd2 car expression and inducible cytokines. Cancers 2020; 12: 1-22. doi: 10.3390/cancers12020375 65. Rafiq S, Yeku OO, Jackson HJ, Purdon TJ, van Leeuwen DG, Drakes DJ, et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nat Biotechnol 2018; 36: 847-56. doi: 10.1038/ nbt.4195 66. Yin Y, Boesteanu AC, Binder ZA, Xu C, Reid RA, Rodriguez JL, et al. Checkpoint blockade reverses anergy in IL-13Rα2 humanized scFv-based CAR T cells to treat murine and canine gliomas. Mol Ther Oncolytics 2018; 11: 20-38. doi: 10.1016/j.omto.2018.08.002 Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells418 67. Kuhn NF, Purdon TJ, van Leeuwen DG, Lopez A V., Curran KJ, Daniyan AF, et al. CD40 ligand-modified chimeric antigen receptor T cells enhance antitu- mor function by eliciting an endogenous antitumor response. Cancer Cell 2019; 35: 473-88. doi: 10.1016/j.ccell.2019.02.006 68. Yamamoto TN, Lee P-HH, Vodnala SK, Gurusamy D, Kishton RJ, Yu Z, et al. T cells genetically engineered to overcome death signaling enhance adop- tive cancer immunotherapy. J Clin Invest 2019; 129: 1551-65. doi: 10.1172/ JCI121491 69. Oda SK, Anderson KG, Ravikumar P, Bonson P, Garcia NM, Jenkins CM, et al. A Fas-4-1BB fusion protein converts a death to a pro-survival signal and enhances T cell therapy. J Exp Med 2020; 217: 1-16. doi: 10.1084/ jem.20191166 70. Jin C, Ma J, Ramachandran M, Yu D, Essand M. CAR T cells expressing a bacterial virulence factor trigger potent bystander antitumour responses in solid cancers. Nat Biomed Eng 2022; 6: 830-41. doi: 10.1038/s41551-022- 00875-5 71. Narayan V, Barber-Rotenberg JS, Jung IY, Lacey SF, Rech AJ, Davis MM, et al. PSMA-targeting TGFβ-insensitive armored CAR T cells in metastatic castration-resistant prostate cancer: a phase 1 trial. Nat Med 2022; 28: 724-34. doi: 10.1038/s41591-022-01726-1 72. Roth TL, Li PJ, Blaeschke F, Nies JF, Apathy R, Mowery C, et al. Pooled knockin targeting for genome engineering of cellular immunotherapies. Cell 2020; 181: 728-744.e21. doi: 10.1016/j.cell.2020.03.039 73. Roybal KT, Williams JZ, Morsut L, Walker WJ, Mcnally KA, Lim WA. Engineering T cells with customized therapeutic article engineering T cells with customized therapeutic response programs using synthetic notch receptors. Cell 2016; 167: 419-32. doi: 10.1016/j.cell.2016.09.011 74. Rodriguez-garcia A, Lynn RC, Poussin M, Eiva MA, Shaw LC, Connor RSO, et al. CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy. Nat Commun 2021; 12: 877. doi: 10.1038/ s41467-021-20893-2 75. Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med 2018; 24: 563-71. doi: 10.1038/s41591-018-0010-1 76. Good CR, Aznar MA, Kuramitsu S, Samareh P, Agarwal S, Donahue G, et al. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell 2021; 184: 6081-6100.e26. doi: 10.1016/j.cell.2021.11.016 77. Weber EW, Parker KR, Sotillo E, Lynn RC, Anbunathan H, Lattin J, et al. Transient rest restores functionality in exhausted CAR-T cells through epi- genetic remodeling. Science 2021; 372: eaba1786. doi: 10.1126/science. aba1786 78. Lynn RC, Weber EW, Sotillo E, Gennert D, Xu P, Good Z, et al. c-Jun overex- pression in CAR T cells induces exhaustion resistance. Nature 2019; 576: 293-300. doi: 10.1038/s41586-019-1805-z 79. Seo H, González-Avalos E, Zhang W, Ramchandani P, Yang C, Lio CWJ, et al. BATF and IRF4 cooperate to counter exhaustion in tumor-infiltrating CAR T cells. Nat Immunol 2021; 22: 983-95. doi: 10.1038/s41590-021-00964-8 80. Deng Q, Han G, Puebla-Osorio N, Ma MCJ, Strati P, Chasen B, et al. Characteristics of anti-CD19 CAR T cell infusion products associated with efficacy and toxicity in patients with large B cell lymphomas. Nat Med 2020; 26: 1878-87. doi: 10.1038/s41591-020-1061-7 81. Adachi K, Kano Y, Nagai T, Okuyama N, Sakoda Y, Tamada K. IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor. Nat Biotechnol 2018; 36: 346-51. doi: 10.1038/ nbt.4086 82. Shum T, Omer B, Tashiro H, Kruse RL, Wagner DL, Parikh K, et al. Constitutive signaling from an engineered IL7 receptor promotes durable tumor elimination by tumor-redirected T cells. Cancer Discov 2017; 7: 1238-47. doi: 10.1158/2159-8290.CD-17-0538 83. Alizadeh D, Wong RA, Yang X, Wang D, Pecoraro JR, Kuo CF, et al. IL15 enhances CAR-T cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype. Cancer Immunol Res 2019; 7: 759-72. doi: 10.1158/2326-6066.CIR-18-0466 84. Kalbasi A, Siurala M, Su LL, Tariveranmoshabad M, Picton LK, Ravikumar P, et al. Potentiating adoptive cell therapy using synthetic IL-9 receptors. Nature 2022; 607: 360-5. doi: 10.1038/s41586-022-04801-2 85. Legut M, Gajic Z, Guarino M, Daniloski Z, Rahman JA, Xue X, et al. A genome-scale screen for synthetic drivers of T cell proliferation. Nature 2022; 603: 728-35. doi: 10.1038/s41586-022-04494-7 86. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of t cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 2010; 18: 843-51. doi: 10.1038/mt.2010.24 87. Kim MY, Yu KR, Kenderian SS, Ruella M, Chen S, Shin TH, et al. Genetic inactivation of CD33 in hematopoietic stem cells to enable CAR T cell im- munotherapy for acute myeloid leukemia. Cell 2018; 173: 1439-1453.e19. doi: 10.1016/j.cell.2018.05.013 88. Qazi MA, Vora P, Venugopal C, Sidhu SS, Moffat J, Swanton C, et al. Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma. Ann Oncol 2017; 28: 1448-56. doi: 10.1093/an- nonc/mdx169 89. Ruella M, Maus M V. Catch me if you can: leukemia escape after CD19- directed T cell immunotherapies. Comput Struct Biotechnol J 2016; 14: 357-62. doi: 10.1016/j.csbj.2016.09.003 90. Cordoba S, Onuoha S, Thomas S, Pignataro DS, Hough R, Ghorashian S, et al. CAR T cells with dual targeting of CD19 and CD22 in pediatric and young adult patients with relapsed or refractory B cell acute lymphoblastic leukemia: a phase 1 trial. Nat Med 2021; 27: 1797-805. doi: 10.1038/ s41591-021-01497-1 91. Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med 2018; 24: 20-8. doi: 10.1038/nm.4441 92. Spiegel JY, Patel S, Muffly L, Hossain NM, Oak J, Baird JH, et al. CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nat Med 2021; 27: 1419-31. doi: 10.1038/s41591-021-01436-0 93. Minutolo NG, Sharma P, Poussin M, Shaw LC, Brown DP, Hollander EE, et al. Quantitative control of gene-engineered T-cell activity through the covalent attachment of targeting ligands to a universal immune receptor. J Am Chem Soc 2020; 142: 6554-68. doi: 10.1021/jacs.9b11622 94. Cho JH, Collins JJ, Wong WW. Universal chimeric antigen receptors for mul- tiplexed and logical control of T cell responses. Cell 2018; 173: 1426-1438. e11. doi: 10.1016/j.cell.2018.03.038 95. Tran E, Robbins PF, Lu Y-C, Prickett TD, Gartner JJ, Jia L, et al. T-cell transfer therapy targeting mutant KRAS in cancer. N Engl J Med 2016; 375: 2255-62. doi: 10.1056/NEJMoa1609279 96. Chheda ZS, Kohanbash G, Okada K, Jahan N, Sidney J, Pecoraro M, et al. Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy. J Exp Med 2018; 215: 141-57. doi: 10.1084/jem.20171046 97. Leidner R, Sanjuan Silva N, Huang H, Sprott D, Zheng C, Shih Y-P, et al. Neoantigen T-cell receptor gene therapy in pancreatic cancer. N Engl J Med 2022; 386: 2112-9. doi: 10.1056/nejmoa2119662 98. Bear AS, Blanchard T, Cesare J, Ford MJ, Richman LP, Xu C, et al. Biochemical and functional characterization of mutant KRAS epitopes validates this on- coprotein for immunological targeting. Nat Commun 2021; 12: 1-16. doi: 10.1038/s41467-021-24562-2 99. Yarmarkovich M, Marshall QF, Warrington JM, Premaratne R, Farrel A, Groff D, et al. Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs. Nature 2021; 599: 477-84. doi: 10.1038/s41586- 021-04061-6 100. Ruella M, Xu J, Barrett DM, Fraietta JA, Reich TJ, Ambrose DE, et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat Med 2018; 24: 1499-503. doi: 10.1038/s41591-018-0201-9 101. Ghassemi S, Durgin JS, Nunez-Cruz S, Patel J, Leferovich J, Pinzone M, et al. Rapid manufacturing of non-activated potent CAR T cells. Nat Biomed Eng 2022; 6: 118-28. doi: 10.1038/s41551-021-00842-6 102. Turtle CJ, Hanafi LA, Berger C, Gooley TA, Cherian S, Hudecek M, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest 2016; 126: 2123-38. doi: 10.1172/JCI85309 103. Arcangeli S, Bove C, Mezzanotte C, Camisa B, Falcone L, Manfredi F, et al. CAR T cell manufacturing from naive/stem memory T lymphocytes en- hances antitumor responses while curtailing cytokine release syndrome. J Clin Invest 2022; 132: e150807. doi: 10.1172/JCI150807 Radiol Oncol 2022; 56(4): 409-419. Smole A / Cancer immunotherapy with CAR T cells 419 104. Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov 2020; 19: 185-99. doi: 10.1038/s41573-019-0051-2 105. Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Stafford S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene- edited CAR T cells. Sci Transl Med 2017; 9: 1-9. doi: 10.1126/scitranslmed. aaj2013 106. Lu Y, Xue J, Deng T, Zhou X, Yu K, Deng L, et al. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med 2020; 26: 732-40. doi: 10.1038/s41591-020-0840-5 107. Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, et al. CRISPR-engineered T cells in patients with refractory cancer. Science 2020; 367: 1-12. doi: 10.1126/science.aba7365 108. Eyquem J, Mansilla-Soto J, Giavridis T, Van Der Stegen SJC, Hamieh M, Cunanan KM, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 2017; 543: 113-7. doi: 10.1038/na- ture21405 109. Dai X, Park JJ, Du Y, Kim HR, Wang G, Errami Y, et al. One-step generation of modular CAR-T cells with AAV-Cpf1. Nat Methods 2019; 16: 247-54. doi: 10.1038/s41592-019-0329-7 110. Roth TL, Puig-Saus C, Yu R, Shifrut E, Carnevale J, Li PJ, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 2018; 559: 405-9. doi: 10.1038/s41586-018-0326-5 111. Zhang J, Hu Y, Yang J, Li W, Zhang M, Wang Q, et al. Non-viral, specifi- cally targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature 2022; 609: 369-74. doi: 10.1038/s41586-022-05140-y 112. Rurik JG, Tombácz I, Yadegari A, Fernández POM, Shewale S V, Li L, et al. CAR T cells produced in vivo to treat cardiac injury. Science 2022; 96: 91-6. doi: 10.1126/science.abm0594 113. June CH, Warshauer JT, Bluestone JA. Is autoimmunity the Achilles’ heel of cancer immunotherapy? Nat Med 2017; 23: 540-7. doi: 10.1038/nm.4321 114. Singh N, Hofmann TJ, Gershenson Z, Levine BL, Grupp SA, Teachey DT, et al. Monocyte lineage-derived IL-6 does not affect chimeric antigen receptor T-cell function. Cytotherapy 2017; 19: 867-80. doi: 10.1016/j. jcyt.2017.04.001 115. Parker KR, Migliorini D, Perkey E, Yost KE, Bhaduri A, Bagga P, et al. Single- cell analyses identify brain mural cells expressing CD19 as potential off- tumor targets for CAR-T immunotherapies. Cell 2020; 183: 126-142.e17. doi: 10.1016/j.cell.2020.08.022 116. Wu C-Y, Roybal KT, Puchner EM, Onuffer J, Lim WA. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science 2015; 350: aab4077. doi: 10.1126/science.aab4077 117. Juillerat A, Marechal A, Filhol JM, Valton J, Duclert A, Poirot L, et al. Design of chimeric antigen receptors with integrated controllable transient func- tions. Sci Rep 2016; 6: 1-7. doi: 10.1038/srep18950 118. Giordano-Attianese G, Gainza P, Gray-Gaillard E, Cribioli E, Shui S, Kim S, et al. A computationally designed chimeric antigen receptor provides a small- molecule safety switch for T-cell therapy. Nat Biotechnol 2020; 38: 426-32. doi: 10.1038/s41587-019-0403-9 119. Hoyos V, Savoldo B, Quintarelli C, Mahendravada A, Zhang M, Vera J, et al. Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 2010; 24: 1160-70. doi: 10.1038/leu.2010.75 120. Paszkiewicz PJ, Fräßle SP, Srivastava S, Sommermeyer D, Hudecek M, Drexler I, et al. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. J Clin Invest 2016; 126: 4262-72. doi: 10.1172/JCI84813 121. Roybal KT, Rupp LJ, Morsut L, Walker WJ, McNally KA, Park JS, et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell 2016; 164: 1-10. doi: 10.1016/j.cell.2016.01.011 122. Tran E, Ahmadzadeh M, Lu YC, Gros A, Turcotte S, Robbins PF, et al. Immunogenicity of somatic mutations in human gastrointestinal cancers. Science 2015; 350: 1387-90. doi: 10.1126/science.aad1253 123. Zacharakis N, Chinnasamy H, Black M, Xu H, Lu YC, Zheng Z, et al. Immune recognition of somatic mutations leading to complete durable regres- sion in metastatic breast cancer. Nat Med 2018; 1: 724-30. doi: 10.1038/ s41591-018-0040-8