Adoptive therapy with tumor-isolated and ex vivo amplified T cells is showing spectacular success in the treatment of malignant diseases supporting the overall concept that the patient's immune system can control cancer in the long-term. In particular, tumor infiltrating lymphocytes (TILs), isolated and expanded from melanoma lesions, are capable in inducing tumor regressions and long-term remissions in a substantial number of patients.1 The antigen specificity of most TILs is frequently not known, however, assumed to be redirected towards the respective tumor from which the cells were isolated. The assumption is supported by the recent report that the T cell receptor (TCR), isolated from TILs from a mammary tumor lesion and engineered on peripheral blood T cells, was capable to induce tumor regression.2
However, the number of available TCRs with known specificity for tumors is still limited and cancer cells frequently lose the capacity to present antigen, either by deficient antigen processing or by suppressed expression of the major histocompatibility complex (MHC). In this situation Zelig Eshhar and colleagues (Weizmann Institute of Science) designed a chimeric antigen receptor (CAR), previously called “immunoreceptor” or nick-named “T-body”, which consists in the extracellular moiety of an antigen binding and in the intracellular moiety of a signaling domain capable to initiate T cell activation upon antigen engagement.3 The CAR is a composite receptor which for binding frequently uses a single chain fragment of variable region (scFv) antibody; the T cell activating signal is mostly transmitted through the TCR CD3ζ signaling chain in the intracellular part with or without a linked costimulatory moiety (Fig. 1). Engagement of cognate antigen on the surface of cancer cells by the CAR engineered T cell initiates a cascade of signaling events resulting in T cell activation and an antigen-specific response towards the cognate target cells.3,4
Adoptive therapy with CAR modified T cells takes advantage of the power of T cells and other cytolytic immune cells that actively migrate through vascular endothelia and penetrate tissues, become activated and amplify upon antigen engagement, and eliminate cognate target cells by granzyme/perforin mediated killing. As a result, tumor antigens are massively released at the site of tumor recognition which are captured and cross-presented by tumor infiltrating DCs, monocytes, and tumor-associated macrophages which recruit and initiate a second wave of immune response.5
By using an antibody for target recognition, CAR T cells can recognize target cells independently of MHC presentation of antigen. Consequently, CAR T cells can recognize target cells with downregulated MHC or deficiency in the antigen processing machinery and, moreover, can recognize other targets than proteins, like carbohydrates and lipids, which broadens the panel of potential targets compared with the TCR mediated T cell recognition. CAR T cells are repetitively re-stimulated as long as the antigen is present; withdrawal from antigen turns the CAR T cell to energy or to entry into apoptosis; however, some CAR T cells can persist in the long-term and provide an antigen-specific memory as suggested by experimental models.6
After application to the cancer patient, CAR T cells amplify upon antigen engagement giving rise to increasing CAR T cell numbers of CAR T cells in the peripheral blood within the first weeks; anti-CD19 CAR T cells and concomitant B cell aplasia can persist for years.7 There is increasing evidence that CAR T cells need to persist in a substantial number and with active effector functions providing long-term control of the disease; relapse of the disease was frequently observed when the number of CAR T cells in the patient after initial amplification dropped to undetectable levels. In some cases of CD19 CAR T cell loss, relapse was heralded by recovering healthy B cells. In this situation, monitoring healthy B cell counts may identify patients with a high risk of relapse.8 In the treatment of neuroblastoma, duration of CAR T cell persistence in the peripheral blood beyond 6 weeks was associated with superior clinical outcome; long-term persistence was concordant with the percentage of CD4+ cells and central memory cells in the manufactured cell product9 implying that those cells promote the extended low-level persistence in patients. Experimental models indicate that in the presence of constant antigenic stimulation CAR T cells can provide long-term, antigen-specific tumor rejection,6 moreover indicating that there is a persistent, functional population of CAR T cells capable to re-expand and eradicate secondary tumor challenge. The endogenous CD19 antigen seems to be required to promote persistence of CD4+ CAR T cells, and to some extend of CD8+ T cells, implying repetitive antigen stimulation as driving force for CAR T cell persistence in the long-term.10 CAR T cell persistence in the long-term is thereby not equivalent to canonical T cell memory in the absence of antigen. Classical T cell memory depends on appropriate costimulation; costimulation through ICOS increased the number of CD4+ CAR T cells which in turn promote persistence of CD8+ CAR T cells redirected by a CD28 or 4–1BB signaling CAR.11 As a consequence, a third generation CAR providing ICOS and 4-1BB costimulation may be superior in promoting long-term CAR T cell persistence and anti-tumor activities. However, some patients stayed in remission although CAR T cells dropped below detection limit in the peripheral blood12 which indicates that apart from the specific CAR T cells, a recruited secondary immune response may also substantially contribute to control the disease.
Treatment of B cell malignancies with CD19 specific CAR T cells induces clinical and molecular remissions with high frequencies which can last over years.13 However, CD19 specific CAR T cells deplete also the healthy CD19+ B cell compartment resulting in hypogammaglobulinemia within the first weeks of CAR T cell infusion; most plasma cells are spared by anti-CD19 CAR T cells due to lack of CD19.14 The symptoms due to B cell aplasia are clinically manageable by immunoglobulin replacement and in some cases by prophylactic antibiotic therapy.15,16 B cell depletion rapidly reverses after CAR T cell ablation17 which, however, not necessarily indicates a pending relapse of the disease; complete lymphoma remissions can continue after the disappearance of an effective anti-CD19 T cell response.12 Recovering non-malignant B cells in peripheral blood of treated patients were CD19+ and CD20+, were polyclonal, and of naive phenotype indicated by IgD and lack of CD27.
Up to today, more than 1000 patients were treated with anti-CD19 CAR T cells in the US alone. Currently, 439 clinical trials are listed, more than 270 trials are actively exploring CAR T cells in the treatment of hematologic and solid cancer around the world with clear hotspots in frequencies in the US and in China; a minority of trials are performed in Europe (https://www.the-scientist.com/infographics/cell-and-gene-therapy-tracker-64450). Based on the considerable remission rate in patients with B cell lymphoma, the anti-CD19 CAR T cell product tisagenlecleucel (KymriahTM, Novartis) was approved on August 30th, 2017, by the U.S. Food and Drug Administration (FDA) and subsequently by the European Medicines Agency (EMA) for the treatment of relapsed and/or refractory B cell acute lymphoblastic leukemia. Shortly thereafter, the second anti-CD19 CAR T cell product axicabtagene ciloleucel (YescartaTM, Gilead/Kite) was approved for the treatment of adult patients with relapsed and/or refractory large B cell lymphoma (https://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm574154.htm). The anti-CD19 CAR T cell product lisocabtagene maraleucel (liso-cel; JCAR017, Celgene/Juno) received an FDA breakthrough therapy designation for the treatment of DLBCL.
The mentioned anti-CD19 CARs differ in their design; the KymriahTM CAR signals through 4-1BB-CD3ζ as does the JCAR017 while the YescartaTM CAR uses the CD28-ζ chain. While CD28 costimulation induces IL-2 release along with IFN-γ secretion and rapid expansion of CAR T cells, costimulation through 4-1BB does not induce IL-2, but IFN-γ secretion and a more prolonged T cell activation and amplification period. All 3 CARs use the murine FMC63 scFv for binding while the extracellular hinge and transmembrane domains differ: Kymriah uses the CD8, Yescarta the CD28 and JCAR017 use the IgG4 hinge and transmembrane domains. The Kymriah and JCAR17 CAR are transduced into T cells by lentiviruses while the Yescarta CAR by retroviruses. Given the CAR design, the functional capacities of engineered T cells and the patient eligibility differences caution needs to be taken when comparing data across studies.
Recent efforts have broadened the potential of CAR T cell therapies beyond oncology towards the treatment of auto-immune diseases. In particular, regulatory T cells (Tregs) were engineered with and activated by CARs18; in a pre-clinical model, FoxP3 induced Treg cells were redirected by an engineered CAR towards myelin oligodendrocyte glycoprotein (MOG) in order to repress murine experimental auto-immune encephalomyelitis.19 T cells were engineered with CAR redirected specificity for the auto-antigen Dsg3 to eliminate auto-immune B cells in order to treat auto-immune diseases like pemphigus vulgaris.20
CAR T cell therapy is frequently associated with toxic side effects, mostly due to on-target effects depending on the antigen specificity of the CAR and the degree and kinetics of redirected T cell activation. Most toxic effects are reversible or disappear upon CAR T cell elimination or termination of engraftment. This is in contrast to cytotoxic chemotherapy that can cause permanent genetic alterations in healthy tissues with substantial risk of secondary malignancies in the long-term. For further information on clinical aspects, we refer to a recent review by June and Sadelain.13
We here discuss some major aspects in the CAR design and review recent developments towards a safer CAR which intend to make T cell therapy applicable to a broad variety of diseases and to a growing number of patients.
The modular composition of the CAR
The prototype CAR comprises in the extracellular domain a single chain fragment of variable region (scFv) antibody for binding and a spacer of various lengths linked to a transmembrane domain. The intracellular signaling moiety is mostly derived from the CD3ζ intracellular chain with or without a linked costimulatory domain (Fig. 1A). The scFv is genetically engineered by joining the coding regions of the heavy chain (VH) and light chain (VL) variable regions of an antibody in the order VH-linker-VL or VL-linker-VH while the linker is frequently a short glycine-serine peptide sequence. The activating domain is mostly the CD3ζ intracellular chain of the TCR; the Fcε receptor-I (FcεRI) signaling γ-chain is used in some constructs. CARs of the “first generation” contain only the primary signal (signal-1) like CD3ζ or FcεRI γ while “second generation” CARs in addition harbor a costimulatory moiety (signal-2), like CD28, 4-1BB, OX40, ICOS, or CD27, mostly at the membrane proximal position followed by CD3ζ in the distal position. Both the primary signal-1 and the costimulatory signal-2 are required for inducing full T cell activation and for protecting from activation-induced cell death.21–23 Consequently and in contrast to the first-generation CARs, T cells redirected by the second-generation CAR show durable in cytokine release, amplification and anti-tumor activity making them suitable for clinical applications. “Third-generation” CARs contain a combination of costimulatory domains along with the primary signal and provide some benefit in sustaining survival of more matured T cells.24
The overall modular design of the prototype CAR has several advantages with respect to both the exchange of binding and signaling moieties on the engineering level and the combination of the antigen recognition with the T cell activating machinery on the functional level.
- (a) The scFv antibody provides targeting specificity to the CAR; exchange of scFv facilitates redirecting T cells towards a variety of targets. Antibody-mediated recognition by the CAR is independent of antigen presentation by the MHC; this is of advantage in case of recognizing cancer cells, which frequently lose MHC expression or are deficient in antigen processing. The CAR can potentially recognize any targetable epitope for which an antibody is available, including carbohydrates, lipids, or variants of an antigen.
- (b) CAR targets need to be cell surface antigens that are accessible to the CAR binding domain; intracellular antigens are not visible to CAR T cells. However, by using an antibody recognizing a specific peptide/MHC complex, CARs can provide TCR-like specificity to the host cell as shown for a CAR with specificity for HLA-A2/NY-ESO-1 peptide.25,26
- (c) The use of a scFv single chain antibody for CAR targeting moreover allows the design of a 1-polypeptide-chain receptor molecule. However, by the conversion from a native antibody, a number of scFvs lose their specificity and affinity. To circumvent the situation, we proposed an alternative CAR format that is composed of two, non-covalently linked polypeptide chains, that is, 1 chain is composed of the Ig heavy chain with the variable and constant region linked to the transmembrane and signaling moieties; the second chain is composed of the Ig light chain without a linked transmembrane domain (Fig. 1E). When co-expressed both chains spontaneously associate forming a fully functional antibody anchored to the T cell membrane by the linked intracellular signaling domain to provide T cell activation upon antigen engagement.27
- (d) Instead of antibodies, any other binding domains or ligands of natural receptors can be alternatively used for targeting. As an example, mutated IL-13 was linked to the extracellular CAR moiety for targeting the IL-13 receptor-α2 which is over-expressed by a broad variety of solid tumors but less by healthy tissues, thereby providing improved cancer selectivity.28–30 Adnectin, a protein fragment derived from fibronectin, was used for targeting the epithelial growth factor receptor (EGFR).31 Another example of an alternative binding domain is a designed ankyrin repeat protein (DARPin) that is composed of ankyrin repeats of 33 amino acids in length and forms a β-turn followed by 2 anti-parallel α-helices and a loop reaching the β-turn of the next repeat.32
- (e) The CAR-mediated T cell activation requires a distance of about 15 nm between the T cell and the target cell33 as it is the case for the physiological TCR-MHC interaction. To allow optimal interactions a spacer is inserted into the extracellular moiety between the antigen binding and the transmembrane domain; targeting a membrane distal epitope on the antigen requires a short spacer, targeting a proximal epitope requires a long spacer within the CAR. By using spacers of various lengths, the CAR-redirected T cell activation can be optimized. For empiric adjusting the distance of the binding domain to the membrane, the constant region of IgG1 is practical since it allows creating spacers of various lengths, that is, by using the CH1-CH2-CH3 or CH2-CH3 or CH3 moiety.34 Fine tuning the spacer length adjusts the distance between the T cell and the target cell and thereby improves the CAR T cell activation and finally efficacy in eliminating the target cells.35 Higher order structural requirements and CAR dimerization driven by the extracellular spacer domain may additionally impact T cell activation demanding a more thorough exploration in the context of a particular target antigen. The currently used anti-CD19 CARs harbor different spacers, that is, the KymriahTM CAR harbors the CD8 hinge domain, YescartaTM the CD28 spacer domain and the JCAR017 the IgG4 hinge domain. While there are additional differences between the individual CARs, the different spacers may create different lengths of the extracellular CAR domain and thereby different distances between the CAR T cell and the CD19+ B cell to initiate a cytolytic attack. The differences in CAR design demand a more detailed analysis to understand the clinical performance of the various anti-CD19 CAR T cells.
- (e) The commonly used IgG1 constant domain as a spacer in the extracellular CAR moiety can bind to Fcγ receptor (FcγR) (CD64) with the risk to initiate an unintended “off-target” activation of both T cells and myeloid cells. To avoid the situation, the FcγR binding motif within IgG1 CH2 was modified by deleting the Asn297 glycosylation site36,37 or by deleting the IgG1 CH2 domain to abrogate binding to Fc receptors.35 CARs with the CH2CH3 spacer moreover show a pronounced tonic signaling which accelerates T cell senescence which can be mitigated by removing the CH2 region.35 These specific IgG1 properties provide the rationale to use alternative spacers like the constant domain of IgG4 or the extracellular domains of CD4 and CD8.4,36
- (f) CARs of the prototype design are also functional in NK cells based on the capacity of the TCR derived signaling domain to efficiently recruit and associate with kinases to initiate a productive signaling cascade in NK cells38–44; NK cell-derived signaling chains, like DAP10 or DAP12, are also applied.45–47
The growing family of CARs
TRUCK: a CAR T cell producing and releasing a transgenic protein product
CAR T cells target the defined tissues and are activated upon antigen engagement; the mechanism can be used to release a transgenic polypeptide product “on demand” upon CAR signaling. CAR T cells are additionally engineered with an expression cassette for the transgenic protein in order to deliver the protein in a therapeutic concentration in the targeted tissue while the protein concentrations in the periphery remain low. “T cells redirected for universal cytokine-mediated killing”, so-called TRUCKs or the “fourth generation” of CAR cells, combine the redirected CAR T cell attack with the locally restricted release of a biologically active protein while avoiding its systemic toxicity (Fig. 1A).48 The protein for release can be produced in a constitutive or in an inducible fashion upon CAR signaling; the expression of the inducible protein is under control of the NFAT6-IL-2 minimal promoter that induces protein synthesis upon CAR signaling and remains silent as long as no T cell activation occurs. The TRUCK concept is universal with respect to the delivered protein; nearly every protein can be produced and released in this fashion converting CAR T cells into “living factories”. The delivery of IL-12 and IL-18 to the targeted tissue was reported to induce different biological effects49–55; CAR IL-12 T cells (IL-12 TRUCKs) recruited and activated an innate immune response in the targeted tumors,51 resisted suppression by Treg cells,52 and showed an increased cytokine release and expansion.56 IL-18 TRUCKs converted the CAR T cells into Tbethigh FoxOlow killer cells with the reduction of Treg cells and the increase in Th17 cells in the targeted tumor tissue54; TRUCKs delivering both IL-12 and IL-18 were equally potent in this respect as IL-18 TRUCKs. The transgenic release of IL-15 improved T cell amplification and anti-tumor activity.57 Since IL-15 is potentially leukemogenic,58 IL-15 TRUCKs need to be controlled by a suicide gene in case of uncontrolled T cell amplification.59 CAR T cells can be protected from oxidative stress through the release of catalase,60 and tumor penetration can be sustained by targeting the tumor vasculature by delivering the soluble HVEM ectodomain61. A variety of other examples can be envisaged, some are currently evaluated in pre-clinical models. A clinical trial (NCT02498912) is currently testing CAR T cells targeting Muc16 and secreting IL-12,62 other combinations will follow in the near future.
bispecific CAR T cells
Treatment with antigen-redirected therapies is associated with the risk of antigen/epitope loss and subsequent risk of relapse of the disease. This is the case in a substantial number of acute B cell lymphoma/leukemia patients treated with CD19 CAR T cells.63 To reduce the risk of relapse upon antigen loss, T cells are engineered with a CAR with 2 linked scFvs of 2 specificities (tandem CAR, “TanCAR”); binding to either antigen is sufficient to induce CAR T cell activation (Fig. 1B).64 TanCAR T cells with specificity for CD19 and CD20 target even those leukemic cells which lost CD19 upon a primary CAR T cell attack.65 Pediatric acute B cell lymphocytic leukemia with heterogeneity in CD19 and CD20 expression can be controlled by bispecific CD20-CD19 CAR T cells in a transplanted mouse model.66 In the same line, dual antigen targeting is explored for the treatment of B-ALL by targeting CD19 and CD123,67 CD22,68 ROR1,69 and immunoglobulin kappa light chain (Igκ).70
logic gating CAR T cells
By combining CARs of different specificities, CAR T cells can recognize an antigen pattern in a Boolean logic computation aiming at reducing toxicities towards healthy tissues. Two CARs of different specificities are co-expressed, 1 CAR providing the primary CD3ζ, the other CAR the costimulatory CD28 signal (Fig. 1B). Consequently, engagement of both antigens and thereby signaling through both signaling chains are simultaneously required to induce full T cell activation; engagement of only 1 antigen is not sufficient. Such Boolean “AND” gating was exemplarily shown for targeting ErbB2 and Muc1,71 mesothelin and folate receptor-α,72 and targeting PSCA and PSMA,73 the latter using combined CD28 and 4-1BB costimulation. The synNotch system can alternatively be used for logic gating; synNotch activation controls the transcription of a CAR (synNotch CAR) for recognizing a second antigen on cancer cells for providing a signal required for final T cell activation (Fig. 1C).74–76 Thereby a synNotch CAR integrates dual antigens through transcriptional regulation of CAR expression rather than through signaling.77 In contrast to “AND” integration, a fully signaling CAR or a bispecific CAR provides “OR” computation which initiates T cell activating also upon engagement of either cognate antigen (Fig. 1B). T cells with a second-generation activating CAR recognizing antigen 1 and an inhibitory CAR recognizing antigen 2 are only activated if no signaling by the inhibitory CAR occurs (“antigen 1 but no antigen 2”); in case of engaging both antigens, the T cell is blocked by the inhibitory CAR (Fig. 1B).
CARs with “universal” antigen recognition
Changing the specificity of a prototype CAR requires engineering of a new CAR molecule. To make the strategy more flexible, a CAR with specificity for CD16 was used to capture any antibody through binding the constant domain.78 By adding specific antibodies, CARs are loaded with defined tumor specificities; different antibodies provide different specificities to the same CAR T cell, for example, CD16 CAR T cells target Her2+ cancer cells in the presence of the anti-Her2 antibody. CARs recognizing epitopes artificially linked to the targeting antibody can likewise be used, like CARs with specificity for fluorescein isothiocyanate (FITC),79,80 biotin81 or a protein epitope.82 The strategy will have substantial potential in simultaneous targeting different antigens on tumors by the same CAR T cell loaded with various targeting antibodies. An additional benefit of the strategy is that the toxicity can be controlled by titrating the amount of targeting antibody; withdrawal from antibody administration is thought to reduce and limit potential side effects.
In case of uncontrolled toxicity, CAR T cells need to be selectively and rapidly eliminated, preferentially by a co-expressed suicide gene. CAR T cell apoptosis can be induced by dimerization of the transgenic inducible caspase-9 (iCasp9) upon addition of the dimerizing agent AP1903; as a result, more than 90% of T cells undergo apoptosis within minutes.83–86 However, spontaneous dimerization of iCasp9 occurs to some extent in such engineered CAR T cells resulting in a certain baseline frequency of apoptotic cells. Alternatively, CAR T cells can be eliminated by antibody-dependent cellular cytotoxicity (ADCC) using antibodies targeting an integrated CAR domain, for instance by application of rituximab targeting a CD20 epitope integrated into the extracellular CAR domain, or a co-expressed truncated receptor molecule like truncated EGFR which is targeted by cetuximab.87–89 It remains a matter of debate whether in case of toxicity cancer patients with a dysfunctional immune system have sufficient capacities to remove all CAR T cells by ADCC in due time. Another strategy uses the “switch-on/switch-off” key to control the CAR T cell response by splitting the CAR signaling moieties onto 2 polypeptide chains which dimerize in the presence of a small dimerizer molecule (“switch-on”) while being dissociated without dimerizer and “switched-off” (Fig. 1C).90,91 Increase in the concentration of the dimerizer improves CAR-mediated T cell activation and withdrawal switches off CAR activities. The CAR design is based on inducible MyD88/CD40 (iMC) to provide the activation switch upon administration of the drug rimiducid that links the 2 signaling domains required for T cell activation.92
In the opposed application, co-expressed inhibitory CARs (iCARs) with inhibitory signals instead of activating signals are used to block CAR T cell activation, for instance, when engaging antigens on healthy cells (Fig. 1D).93
CARs targeting inhibitory ligands
T cell activation is frequently blocked by inhibitory signals present in the tumor environment. To prevent T cell repression, CARs were engineered which target the inhibitory ligands by their extracellular domain and provide a stimulatory signal to the T cell by their intracellular moiety in order to overrun the inhibitory signals (Fig. 1D). For instance, a CAR recognizing PD-L1 through its extracellular PD-1 domain provides CD28 costimulation to sustain T cell activation when engaging PD-L1+ target cells.94–96
Armored CAR T cells with recombinant cytokine receptors
In order to increase T cell amplification, CAR T cells were equipped with the transgenic IL-7 receptor-α chain with IL-2β signaling in order to stimulate T cell amplification in presence of IL-7.97–99 Similarly, an IL-4 binding/IL-7 signaling receptor improved anti-tumor activity of T cells with anti-PSCA CAR in pancreatic cancer with increased IL-4 levels.100 On the other hand, a dominant negative TGF-β receptor on CAR T cells competes with TGF-β in a melanoma model providing CAR T cell resistance in the presence of the suppressive cytokine.101
CAR T cell production for clinical application
For adoptive therapy, T cells from each individual patient are ex vivo genetically engineered with the CAR, amplified to clinically relevant numbers and re-infused to the patient after non-myeloablative pre-conditioning. In particular, the procedure includes collecting the cells by leukapheresis, genetic transduction by retro- or lentivirus infection or by RNA electroporation, T cell amplification, and quality control of the final cell product, all processes in accordance to good manufacturing procedure (GMP) guidelines.102
The viral transduction procedure has the advantage of a gentle gene transfer to the T cell by infection with low toxicity as long as the T cells are activated and replicate; lentivirus infection requires much lower activation than retroviruses. The viral vector integrates into the host genome in a “semi”-random fashion; episomal DNA vectors are gradually lost in cycling cells with the consequence of loss of transgene expression. While genotoxic and mutagenic effects elicited by vector insertion near proto-oncogenes may occur, to our best knowledge, no malignant transformation of mature T cells upon vector integration occurred in mature T cells during clinical trials so far. While genetic modification of T cells by lenti- and retroviruses is highly efficient, the virus production is extremely labor-, time- and cost-intensive including the generation and characterization of a master cell bank for virus production which makes non-viral transfection systems increasingly attractive.
Efforts are currently made to obtain permanently modified T cells by non-viral vectors; transposon-based vectors like Sleeping Beauty (SB) and PiggyBac as well as CRISPR/Cas9 mediated insertions are currently adopted to clinical CAR T cell applications.103–105 The sleeping beauty transposon system combines the benefit of an integrating viral vector with long-lasting transgene expression. Naked mini-circle DNA, which can be easily produced under GMP conditions, is transferred into T cells by electroporation; the sleeping beauty integration takes place into “safe harbor” genomic loci with low risk of unintended genetic mutations. Delivering the sleeping beauty transposase by mRNA transfer and delivering a CAR by mini-circle DNA electroporation improves the transposition efficacy and finally the number of genetically modified T cells.106 Compared with viral vectors, transposon-based vectors can transfer larger genetic elements into the target cell which makes the system convenient for co-expressing 2 or more CARs, for delivering larger transgenic “payloads” by TRUCKs, and for others.
The starting material for the manufacturing of the therapeutic T cell product is usually a leukapheresis product obtained from the patient. T cells are ex vivo isolated, stimulated, transduced by a retro- or lentivirus vector and amplified to obtain a final T cell product containing 10% to 50% CAR-modified cells. The manufacturing process from leukapheresis to the release of the final T cell product requires about 12 to 14 days.107,108 Rigorous quality control management accompanies the entire process and governs the starting material, the genetic engineering and cell amplification process and the final cell product after harvesting. Most trials are using bulk CD3+ T cell preparations engineered with the respective CAR; isolated CD4+ and CD8+ T cell subsets,109 naïve T cells,110 central memory111 or memory stem cells112 are also explored in some trials. The most suitable T cell population for CAR therapy is thought to be cells in a naïve or young central memory stage of maturation due to their prolonged persistence. In particular, CD45RO+ CD62L+ memory CAR T cells provide a more durable anti-tumor response than effector T cells.112–114 Therefore T cells are ex vivo amplified in the presence of IL-7, IL-15 or IL-21 that improve clonotypic T cell persistence and survival and induce a robust cytokine release.115–117
The manufacturing process is currently an entirely hands-on manufacturing procedure; process controlled automatic manufacturing is increasingly applied for a variety of T cell products. For a detailed description of the manufacturing processes, we refer to Köhl et al.102
Obtained from patients with leukemia, the leukapheresis product and thereby the final CAR T cell product may be contaminated with leukemic cells, although the risk is virtually low. Recently, a single contaminating leukemic B cell was unintentionally transduced with an anti-CD19 CAR during the manufacturing process which initiated a CAR T cell refractory relapse of B cell leukemia.118 The anti-CD19 CAR bound in cis to the CD19 epitope on the leukemic B cell masking it from recognition by the anti-CD19 CAR T cells. The event seems to be very rare since this is the only reported case of “epitope masking” upon unintentional transduction of leukemic cells out of currently 369 leukemia patients treated so far.
Perspectives and future developments
The ideal “universal” CAR T cell
For most clinical applications, patient's T cells are engineered and amplified on the individual basis which demands an individualized and cost-intensive manufacturing and delivery process. To overcome the limiting situation tremendous efforts are made to produce ideally “universal” allogeneic T cells that are produced in advance for the “off-the-shelf” administration to a number of patients.119 In this line, donor T cells were disrupted in the TCR α-chain locus using transcription activator-like effector nucleases (TALENs) to obtain TCR-negative T cells which were finally engineered with the CAR.119–121 In a first clinical application, TALEN edited CAR T cells were administered to 2 pediatric B-ALL patients for whom autologous T cells could not be produced in sufficient numbers, demonstrating the feasibility of the approach.120 One patient, who was mismatched in all MHC-I alleles, developed graft-versus-host disease potentially due to the expansion of contaminating, non-edited CAR T cells and/or the elimination of edited, MHC-deficient CAR T cells by NK cells. To reduce the risk of MHC deficiency induced elimination, CAR T cells were engineered with HLA-E and deleted from HLA-A, -B, -C which prevents their recognition by host immune cells.122 Basically the same TCR-negative T cells can be achieved using virus-transmitted zinc finger nucleases123 or non-virally transmitted megaTALs.124 Recent research achieved TCR gene editing and engineering with the CAR by one CRISPR/Cas9 mediated process125; the CAR expression cassette is targeted into the endogenous TCR α-chain and β2-microglobulin locus in order to control CAR expression by the physiologic TCR promoter and to disrupt the endogenous TCR.126–128 Thereby the T cell function is aimed at being more sustained through diminished tonic signaling and delayed T cell exhaustion.128 To the end, a cell bank with “universal” CAR T cells for allogeneic cell transfer would provide much more flexibility in the application, more rapid delivery to the patient and less cost-intensive manufacturing, alltogether making the therapy more applicable to a high number of patients.
The target antigen
Since most targetable tumor-associated antigens are also expressed by healthy cells, their elimination by CAR T cells may become a severe side effect dependent on the targeted antigen and the attacked tissue. In the treatment of B cell malignancies by targeting CD19 or CD20, depletion from healthy mature B cells is clinically manageable; the same is the case for CAR T cell targeting B cell maturation antigen BCMA (CD269) in the treatment of myeloma which results in the elimination of healthy plasma cells with BCMA expression (NCT02546167).129 Current efforts are aiming at identifying truly tumor-selective antigens suitable for a more selective targeting of tumor lesions while sparing healthy tissues. Such tumor-selective antigens may be tumor-specific mutations of surface proteins, like claudin-6,130 or glycosylation variants like Muc1 or Muc16.131
Expression of the targeted antigen by healthy tissues does not exclude per se the application of CAR-redirected T cells. For instance, CAR T cell targeting carcinoembryonic antigen (CEA) proofed to be safe since CEA is expressed in a strictly luminal fashion by healthy epithelia in the gastrointestinal tract and the lung; in contrast, cancer cells show a depolarized expression pattern on the entire cell surface.132 Two trials showed that systemic anti-CEA CAR T cell administration is safe without inducing treatment related colitis; the same trials provided some clinical efficacy in the treatment of gastrointestinal adenocarcinoma (NCT01212887, NCT02349724).133,134 Local administration of anti-CEA CAR T cells by hepatic artery infusion was safe and also declined tumor progression (NCT01373047).135
The choice of target antigen is also crucial with respect to antigen loss during therapy. The situation became obvious upon CD19 CAR T cell therapy of pediatric B-ALL, which regressed into complete remissions with high frequencies, however, relapsed in about 40% of patients despite persisting CAR T cells.8,136,137 The dominant cause of relapse seems to be the expression of a CD19 isoform which lacks exon-2 and is not recognized by the CAR with the FMC63 scFv.63 Loss of CD19 occurred in 28% of patients in a recent trial for the treatment of pediatric and young adult B-ALL making the malignant cells invisible to CD19 CAR T cells138; CD19 loss was so far not observed in the treatment of CLL. The risk of relapse due to antigen loss can be reduced by co-targeting a second antigen, for example, CD20 by a bispecific CAR, by T cells with 2 CARs or by the application of 2 CAR T cell products. In the long-term, an antigen-independent anti-tumor response by innate immune cells, which is initiated by a targeted CAR T cell activation, may improve the overall therapeutic efficacy against cancer with profound antigen heterogeneity and antigen loss. TRUCK cells, that is, CAR T cells with the inducible release of transgenic cytokines like IL-12, are capable to induce an innate response against antigen-negative cancer cells in an experimental model51; clinical exploration will prove whether the combined activation of a redirected T cell and a cytokine initiated innate response is capable to control cancer with high antigen heterogeneity.
Optimization of the CAR design
While a prototype design of a CAR established during the last decade, each CAR needs optimization with respect to the target antigen and the redirected immune cell. The optimization steps include the targeted epitope of the particular antigen, the binding affinity and the extracellular spacer length to ensure proper accession to the target cell.139,140 As an example, targeting Her2 by a medium affinity CAR caused no substantial toxicity141 while a high-affinity CAR targeting a different Her2 epitope caused fatal adverse events.142 There is an optimal binding affinity by which CAR T cells recognize cancer cells with high antigen load and spare healthy cells with low antigen levels.143,144
The T cell subset
The most suitable maturation stage of T cells for adoptive cell therapy is still a matter of debate; naïve or early central memory T cells are preferred due to their enhanced capacity in cytolysis, amplification, and persistence.110 In previous trials, T cells with a CD62L+ phenotype were used for CAR engineering145; other memory T cell subsets may likewise be suitable. Since T cell maturation is modulated by costimulation and/or cytokine signals, efforts are made to shape a T cell response in a specific fashion. Costimulation through 4-1BB in young T cells initiates a central memory T cell response while CD28 mediates an acute effector cell response.146 In advanced stages of maturation, CCR7− T cells require combined CD28-OX40 costimulation to be protected from activation-induced cell death.24 IL-18 differentiates T cells towards Tbethigh FoxO1low cytolytic T cells with improved anti-tumor activities; CAR and TCR modified T cells with induced release of transgenic IL-18 showed superior activities against advanced tumors.54,55 Ibrutinib, a Bruton tyrosine kinase (BTK) inhibitor, reduces PD-1 levels on CAR T cells and finally exhaustion which increases anti-tumor activity of CAR T cells in the long-term.147
Manufacturing the CAR T cell product
Currently, each CAR T cell product is manufactured by a manual or semi-automated process by a few specialized centers. The process will come to its limits when thousands of patients in various hospitals require their individualized cell products in due time demanding a de-centralized, in hospital manufacturing process by an automated and entirely controlled procedure with a high degree of standardization. Procedures are going to be set up in this direction; major achievements towards automatization and hospital-based production are expected in the near future.
CARs to manipulate the immune network
Evidences are raising that successful CAR T cell therapy involves the entire host immune system for tumor rejection in the long-term. This is underlined by experimental models that showed resistance to EGFRvIII-negative tumors mediated by host immune cells after transfer of anti-EGFRvIII CAR T cells.148 A secondary innate cell response can be induced by IL-12 TRUCKs, that is, IL-12 releasing CAR T cells, which activate macrophages in the tumor tissue for eliminating those cancer cells which are invisible to CAR T cells.51 IL-18 TRUCKs increase the numbers of CD206− M1 macrophages and NKG2D+ NK cells and reduce Treg cells, suppressive CD103+ dendritic cells and M2 macrophages in the tumor tissue.54 The strategy can also be used to deliver other immune response modifiers to the tumor tissue in order to shape a broad immune response against cancer. Checkpoints are part of a regulatory network and specific checkpoints like TIM-3 are upregulated upon PD-1 blockade149; targeting PD-1/PD-1L by blocking antibodies along with CD19 specific CAR T cell therapy is currently explored in trials (NCT02926833, NCT02650999, NCT02706405).
Work in the authors’ laboratory was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Deutsche Krebshilfe, Bonn, Deutsche José Carreras-Leukämie Stiftung, München, Sander Stiftung, München, Else Kröner-Fresenius Stiftung, Bad Homburg v.d.H., the Fortune Program of the Medical Faculty of the University of Cologne and the Regensburger Center for Interventional Immunology (RCI).
1. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science.
2. Zacharakis N, Chinnasamy H, Black M, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med.
3. Eshhar Z, Waks T, Gross G, et al. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA.
4. Bridgeman JS, Hawkins RE, Hombach AA, et al. Building better chimeric antigen receptors for adoptive T cell therapy. Curr Gene Ther.
5. Broz ML, Binnewies M, Boldajipour B, et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell.
6. Chmielewski M, Hahn O, Rappl G, et al. T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice. Gastroenterology.
7. Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med.
8. Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med.
9. Louis CU, Savoldo B, Dotti G, et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood.
10. Yang Y, Kohler ME, Fry TJ. Effect of chronic endogenous antigen stimulation on CAR T cell persistence and memory formation. Blood.
11. Guedan S, Posey AD, Shaw C, et al. Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight.
12. Kochenderfer JN, Somerville RPT, Lu T, et al. Long-duration complete remissions of diffuse large B cell lymphoma after anti-CD19 chimeric antigen receptor T cell therapy. Mol Ther J Am Soc Gene Ther.
13. June CH, Sadelain M. Chimeric antigen receptor therapy. N Engl J Med.
14. Bhoj VG, Arhontoulis D, Wertheim G, et al. Persistence of long-lived plasma cells and humoral immunity in individuals responding to CD19-directed CAR T-cell therapy. Blood.
15. Ueda M, Berger M, Gale RP, et al. Immunoglobulin therapy in hematologic neoplasms and after hematopoietic cell transplantation. Blood Rev.
16. Doan A, Pulsipher MA. Hypogammaglobulinemia due to CAR T-cell therapy. Pediatr Blood Cancer.
17. Paszkiewicz PJ, Fräßle SP, Srivastava S, et al. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia. J Clin Invest.
18. Hombach AA, Kofler D, Rappl G, et al. Redirecting human CD4+CD25+ regulatory T cells from the peripheral blood with pre-defined target specificity. Gene Ther.
19. Fransson M, Piras E, Burman J, et al. CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery. J Neuroinflammation.
20. Ellebrecht CT, Bhoj VG, Nace A, et al. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science.
21. Alvarez-Vallina L, Hawkins RE. Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors. Eur J Immunol.
22. Finney HM, Lawson AD, Bebbington CR, et al. Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. J Immunol Baltim (Md 1950).
23. Hombach A, Sent D, Schneider C, et al. T-cell activation by recombinant receptors: CD28 costimulation is required for interleukin 2 secretion and receptor-mediated T-cell proliferation but does not affect receptor-mediated target cell lysis. Cancer Res.
24. Hombach AA, Chmielewski M, Rappl G, et al. Adoptive immunotherapy with redirected T cells produces CCR7- cells that are trapped in the periphery and benefit from combined CD28-OX40 costimulation. Hum Gene Ther.
25. Stewart-Jones G, Wadle A, Hombach A, et al. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci U S A.
26. Ma Q, Garber HR, Lu S, et al. A novel TCR-like CAR with specificity for PR1/HLA-A2 effectively targets myeloid leukemia in vitro when expressed in human adult peripheral blood and cord blood T cells. Cytotherapy.
27. Faitschuk E, Nagy V, Hombach AA, et al. A dual chain chimeric antigen receptor (CAR) in the native antibody format for targeting immune cells towards cancer cells without the need of an scFv. Gene Ther.
28. Kahlon KS, Brown C, Cooper LJ, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res.
29. Kong S, Sengupta S, Tyler B, et al. Suppression of human glioma xenografts with second-generation IL13R-specific chimeric antigen receptor-modified T cells. Clin Cancer Res Off J Am Assoc Cancer Res.
30. Krebs S, Chow KKH, Yi Z, et al. T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1. Cytotherapy.
31. Han X, Cinay GE, Zhao Y, et al. Adnectin-based design of chimeric antigen receptor for T cell engineering. Mol Ther J Am Soc Gene Ther.
32. Hammill JA, VanSeggelen H, Helsen CW, et al. Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors. J Immunother Cancer.
33. Grakoui A, Bromley SK, Sumen C, et al. The immunological synapse: a molecular machine controlling T cell activation. Science.
34. Srivastava S, Riddell SR. Engineering CAR-T cells: design concepts. Trends Immunol.
35. Watanabe N, Bajgain P, Sukumaran S, et al. Fine-tuning the CAR spacer improves T-cell potency. Oncoimmunology.
36. Hudecek M, Sommermeyer D, Kosasih PL, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res.
37. Hombach A, Hombach AA, Abken H. Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc “spacer” domain in the extracellular moiety of chimeric antigen receptors avoids “off-target” activation and unintended initiation of an innate immune response. Gene Ther.
38. Kruschinski A, Moosmann A, Poschke I, et al. Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas. Proc Natl Acad Sci USA.
39. Mehta RS, Rezvani K. Chimeric antigen receptor expressing natural killer cells for the immunotherapy of cancer. Front Immunol.
40. Simon B, Wiesinger M, März J, et al. The generation of CAR-transfected natural killer T cells for the immunotherapy of melanoma. Int J Mol Sci.
41. Murakami T, Nakazawa T, Natsume A, et al. Novel human NK cell line carrying CAR targeting EGFRvIII induces antitumor effects in glioblastoma cells. Anticancer Res.
42. Li Y, Hermanson DL, Moriarity BS, et al. Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity. Cell Stem Cell.
43. Tang X, Yang L, Li Z, et al. First-in-man clinical trial of CAR NK-92 cells: safety test of CD33-CAR NK-92 cells in patients with relapsed and refractory acute myeloid leukemia. Am J Cancer Res.
44. Müller N, Michen S, Tietze S, et al. Engineering NK Cells Modified With an EGFRvIII-specific chimeric antigen receptor to overexpress CXCR4 improves immunotherapy of CXCL12/SDF-1α-secreting Glioblastoma. J Immunother Hagerstown (Md 1997).
45. Töpfer K, Cartellieri M, Michen S, et al. DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy. J Immunol (Baltim Md).
46. Lynch A, Hawk W, Nylen E, et al. Adoptive transfer of murine T cells expressing a chimeric-PD1-Dap10 receptor as an immunotherapy for lymphoma. Immunology.
47. Wang E, Wang L-C, Tsai C-Y, et al. Generation of Potent T-cell immunotherapy for cancer using DAP12-based, multichain, chimeric immunoreceptors. Cancer Immunol Res.
48. Chmielewski M, Hombach AA, Abken H. Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma. Immunol Rev.
49. Chmielewski M, Abken H. TRUCKs: the fourth generation of CARs. Expert Opin Biol Ther.
50. Pegram HJ, Park JH, Brentjens RJ. CD28z CARs and armored CARs. Cancer J Sudbury Mass.
51. Chmielewski M, Kopecky C, Hombach AA, et al. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Res.
52. Pegram HJ, Lee JC, Hayman EG, et al. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood.
53. Hu B, Ren J, Luo Y, et al. Augmentation of antitumor immunity by human and mouse CAR T cells secreting IL-18. Cell Rep.
54. Chmielewski M, Abken H, Cells CART. Releasing IL-18 convert to T-bethigh foxo1low effectors that exhibit augmented activity against advanced solid tumors. Cell Rep.
55. Kunert A, Chmielewski M, Wijers R, et al. Intra-tumoral production of IL18, but not IL12, by TCR-engineered T cells is non-toxic and counteracts immune evasion of solid tumors. Oncoimmunology.
56. Koneru M, Purdon TJ, Spriggs D, et al. IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo. Oncoimmunology.
57. Xu A, Bhanumathy KK, Wu J, et al. IL-15 signaling promotes adoptive effector T-cell survival and memory formation in irradiation-induced lymphopenia. Cell Biosci.
58. Hsu C, Jones SA, Cohen CJ, et al. Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene. Blood.
59. Hoyos V, Savoldo B, Quintarelli C, et al. Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia.
60. Ligtenberg MA, Mougiakakos D, Mukhopadhyay M, et al. Coexpressed catalase protects chimeric antigen receptor-redirected T cells as well as bystander cells from oxidative stress-induced loss of antitumor activity. J Immunol Baltim (Md 1950).
61. Boice M, Salloum D, Mourcin F, et al. Loss of the HVEM tumor suppressor in lymphoma and restoration by modified CAR-T Cells. Cell.
62. Koneru M, O’Cearbhaill R, Pendharkar S, et al. A phase I clinical trial of adoptive T cell therapy using IL-12 secreting MUC-16(ecto) directed chimeric antigen receptors for recurrent ovarian cancer. J Transl Med.
63. Sotillo E, Barrett DM, Black KL, et al. Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov.
64. Grada Z, Hegde M, Byrd T, et al. TanCAR: a novel bispecific chimeric antigen receptor for cancer Immunotherapy. Mol Ther Nucleic Acids.
65. Zah E, Lin M-Y, Silva-Benedict A, et al. T cells expressing CD19/CD20 bispecific chimeric antigen receptors prevent antigen escape by malignant B cells. Cancer Immunol Res.
66. Martyniszyn A, Krahl A-C, André MC, et al. CD20-CD19 Bispecific CAR T cells for the treatment of B-cell malignancies. Hum Gene Ther.
67. Ruella M, Barrett DM, Kenderian SS, et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. J Clin Invest.
68. Haso W, Lee DW, Shah NN, et al. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood.
69. Hudecek M, Schmitt TM, Baskar S, et al. The B-cell tumor-associated antigen ROR1 can be targeted with T cells modified to express a ROR1-specific chimeric antigen receptor. Blood.
70. Vera J, Savoldo B, Vigouroux S, et al. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood.
71. Wilkie S, van Schalkwyk MCI, Hobbs S, et al. Dual targeting of ErbB2 and MUC1 in breast cancer using chimeric antigen receptors engineered to provide complementary signaling. J Clin Immunol.
72. Lanitis E, Poussin M, Klattenhoff AW, et al. Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo. Cancer Immunol Res.
73. Kloss CC, Condomines M, Cartellieri M, et al. Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol.
74. Roybal KT, Rupp LJ, Morsut L, et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell.
75. Roybal KT, Williams JZ, Morsut L, et al. Engineering T cells with customized therapeutic response programs using synthetic notch receptors. Cell.
76. Morsut L, Roybal KT, Xiong X, et al. Engineering customized cell sensing and response behaviors using synthetic notch receptors. Cell.
77. Cho JH, Okuma A, Al-Rubaye D, et al. Engineering Axl specific CAR and SynNotch receptor for cancer therapy. Sci Rep.
78. Kudo K, Imai C, Lorenzini P, et al. T lymphocytes expressing a CD16 signaling receptor exert antibody-dependent cancer cell killing. Cancer Res.
79. Tamada K, Geng D, Sakoda Y, et al. Redirecting gene-modified T cells toward various cancer types using tagged antibodies. Clin Cancer Res Off J Am Assoc Cancer Res.
80. Kim MS, Ma JSY, Yun H, et al. Redirection of genetically engineered CAR-T cells using bifunctional small molecules. J Am Chem Soc.
81. Urbanska K, Lanitis E, Poussin M, et al. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor. Cancer Res.
82. Cartellieri M, Feldmann A, Koristka S, et al. Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood Cancer J.
83. Thomis DC, Marktel S, Bonini C, et al. A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease. Blood.
84. Tey S-K, Dotti G, Rooney CM, et al. Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation. Biol Blood Marrow Transplant: J Am Soc Blood Marrow Transplant.
85. Di Stasi A, Tey S-K, Dotti G, et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med.
86. Zhou X, Di Stasi A, Tey S-K, et al. Long-term outcome after haploidentical stem cell transplant and infusion of T cells expressing the inducible caspase 9 safety transgene. Blood.
87. Philip B, Kokalaki E, Mekkaoui L, et al. A highly compact epitope-based marker/suicide gene for easier and safer T-cell therapy. Blood.
88. Wang X, Chang W-C, Wong CW, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood.
89. Serafini M, Manganini M, Borleri G, et al. Characterization of CD20-transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of graft-versus-host disease. Hum Gene Ther.
90. Rodgers DT, Mazagova M, Hampton EN, et al. Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies. Proc Natl Acad Sci USA.
91. Wu C-Y, Roybal KT, Puchner EM, et al. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor. Science.
92. Foster AE, Mahendravada A, Shinners NP, et al. Regulated expansion and survival of chimeric antigen receptor-modified T cells using small molecule-dependent inducible MyD88/CD40. Mol Ther J Am Soc Gene Ther.
93. Fedorov VD, Themeli M, Sadelain M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med.
94. Kobold S, Grassmann S, Chaloupka M, et al. Impact of a New Fusion Receptor on PD-1-Mediated Immunosuppression in Adoptive T Cell Therapy. J Natl Cancer Inst.
95. Liu X, Ranganathan R, Jiang S, et al. A chimeric switch-receptor targeting PD1 augments the efficacy of second-generation CAR T cells in advanced solid tumors. Cancer Res.
96. Prosser ME, Brown CE, Shami AF, et al. Tumor PD-L1 co-stimulates primary human CD8(+) cytotoxic T cells modified to express a PD1:CD28 chimeric receptor. Mol Immunol.
97. Vera JF, Hoyos V, Savoldo B, et al. Genetic manipulation of tumor-specific cytotoxic T lymphocytes to restore responsiveness to IL-7. Mol Ther J Am Soc Gene Ther.
98. Perna SK, Pagliara D, Mahendravada A, et al. Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition. Clin Cancer Res Off J Am Assoc Cancer Res.
99. Golumba-Nagy V, Kuehle J, Hombach AA, et al. T cells with CD28-ζ CAR resist TGF-β repression through IL-2 signaling which can be mimicked by an engineered IL-7/IL-2Rβ autocrine loop. Mol Ther.
100. Mohammed S, Sukumaran S, Bajgain P, et al. Improving chimeric antigen receptor-modified T cell function by reversing the immunosuppressive tumor microenvironment of pancreatic cancer. Mol Ther.
101. Zhang L, Yu Z, Muranski P, et al. Inhibition of TGF-β signaling in genetically engineered tumor antigen-reactive T cells significantly enhances tumor treatment efficacy. Gene Ther.
102. Köhl U, Arsenieva S, Holzinger A, et al. CAR T cells in trials: recent achievements and challenges that remain in the production of modified T cells for clinical applications. Hum Gene Ther.
103. Singh H, Figliola MJ, Dawson MJ, et al. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells. PloS One.
104. Singh H, Moyes JSE, Huls MH, et al. Manufacture of T cells using the sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor. Cancer Gene Ther.
105. Manuri PVR, Wilson MH, Maiti SN, et al. piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum Gene Ther.
106. Hudecek M, Ivics Z. Non-viral therapeutic cell engineering with the sleeping beauty transposon system. Curr Opin Genet Dev.
107. Kochenderfer JN, Dudley ME, Kassim SH, et al. Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol Off J Am Soc Clin Oncol.
108. Jensen MC, Riddell SR. Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev.
109. Turtle CJ, Hanafi L-A, Berger C, et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J Clin Invest.
110. Hinrichs CS, Borman ZA, Gattinoni L, et al. Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy. Blood.
111. Berger C, Jensen MC, Lansdorp PM, et al. Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates. J Clin Invest.
112. Gattinoni L, Lugli E, Ji Y, et al. A human memory T cell subset with stem cell-like properties. Nat Med.
113. Klebanoff CA, Gattinoni L, Restifo NP. Sorting through subsets: which T-cell populations mediate highly effective adoptive immunotherapy? J Immunother Hagerstown Md.
114. Singh N, Perazzelli J, Grupp SA, et al. Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies. Sci Transl Med.
115. Kaneko S, Mastaglio S, Bondanza A, et al. IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes. Blood.
116. Li Y, Bleakley M, Yee C. IL-21 influences the frequency, phenotype, and affinity of the antigen-specific CD8 T cell response. J Immunol Baltim (Md 1950).
117. Hinrichs CS, Spolski R, Paulos CM, et al. IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy. Blood.
118. Ruella M, Xu J, Barrett DM, et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat Med.
119. Poirot L, Philip B, Schiffer-Mannioui C, et al. Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies. Cancer Res.
120. Qasim W, Zhan H, Samarasinghe S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med.
121. Torikai H, Reik A, Liu P-Q, et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood.
122. Gornalusse GG, Hirata RK, Funk SE, et al. HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells. Nat Biotechnol.
123. Provasi E, Genovese P, Lombardo A, et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med.
124. Osborn MJ, Webber BR, Knipping F, et al. Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases. Mol Ther J Am Soc Gene Ther.
125. Shalem O, Sanjana NE, Hartenian E, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science.
126. Ren J, Zhang X, Liu X, et al. A versatile system for rapid multiplex genome-edited CAR T cell generation. Oncotarget.
127. Ren J, Liu X, Fang C, et al. Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition. Clin Cancer Res Off J Am Assoc Cancer Res.
128. Eyquem J, Mansilla-Soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature.
129. Ali SA, Shi V, Maric I, et al. T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood.
130. Hutzler S, Erbar S, Jabulowsky RA, et al. Antigen-specific oncolytic MV-based tumor vaccines through presentation of selected tumor-associated antigens on infected cells or virus-like particles. Sci Rep.
131. Posey AD, Schwab RD, Boesteanu AC, et al. Engineered CAR T cells targeting the cancer-associated Tn-Glycoform of the membrane mucin MUC1 control adenocarcinoma. Immunity.
132. Holzinger A, Abken H. CAR T cells targeting solid tumors: carcinoembryonic antigen (CEA) proves to be a safe target. Cancer Immunol Immunother CII.
133. Thistlethwaite FC, Gilham DE, Guest RD, et al. The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother CII.
134. Zhang C, Wang Z, Yang Z, et al. Phase I escalating-dose trial of CAR-T therapy targeting CEA+ metastatic colorectal cancers. Mol Ther J Am Soc Gene Ther.
135. Katz SC, Burga RA, McCormack E, et al. Phase I hepatic immunotherapy for metastases study of intra-arterial chimeric antigen receptor-modified T-cell therapy for CEA+ liver metastases. Clin Cancer Res Off J Am Assoc Cancer Res.
136. Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med.
137. Lee DW, Kochenderfer JN, Stetler-Stevenson M, 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 Lond Engl.
138. Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med.
139. Chmielewski M, Hombach A, Heuser C, et al. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity. J Immunol Baltim (Md 1950).
140. Hombach AA, Schildgen V, Heuser C, et al. T cell activation by antibody-like immunoreceptors: the position of the binding epitope within the target molecule determines the efficiency of activation of redirected T cells. J Immunol Baltim (Md 1950).
141. Ahmed N, Brawley VS, Hegde M, et al. Human epidermal growth factor receptor 2 (HER2) -specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma. J Clin Oncol Off J Am Soc Clin Oncol.
142. Morgan RA, Yang JC, Kitano M, et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther J Am Soc Gene Ther.
143. Liu X, Jiang S, Fang C, et al. Affinity-Tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res.
144. Caruso HG, Hurton LV, Najjar A, et al. Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent antitumor activity. Cancer Res.
145. Sabatino M, Hu J, Sommariva M, et al. Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies. Blood.
146. Kawalekar OU, O’Connor RS, Fraietta JA, et al. Distinct signaling of coreceptors regulates specific metabolism pathways and impacts memory development in CAR T cells. Immunity.
147. Ruella M, Kenderian SS, Shestova O, et al. The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 chimeric antigen receptor T Cells (CART19) improves responses against mantle cell lymphoma. Clin Cancer Res Off J Am Assoc Cancer Res.
148. Sampson JH, Choi BD, Sanchez-Perez L, et al. EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss. Clin Cancer Res Off J Am Assoc Cancer Res.
149. Koyama S, Akbay EA, Li YY, et al. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun.