the ability to interpret language (expressive aphasia), lowered alertness (obtundation), and seizures. during some clinical trials, deaths caused by neurotoxicity have occurred. the main cause of death from neurotoxicity is cerebral edema. in a study carried out by juno therapeutics, inc., five patients enrolled in the trial died as a result of cerebral edema. two of the patients were treated with cyclophosphamide alone and the remaining three were treated with a combination of cyclophosphamide and fludarabine. in another clinical trial sponsored by the fred hutchinson cancer research center, there was one reported case of irreversible and fatal neurological toxicity 122 days after the administration of car t-cells. chimeric antigen receptor structure chimeric antigen receptors combine many facets of normal t cell activation into a single protein. they link an extracellular antigen recognition domain to an intracellular signalling domain, which activates the t cell when an antigen is bound. cars are composed of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular t cell signaling domain. different components of a chimeric antigen receptor antigen recognition domain the antigen recognition domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. it interacts with potential target molecules and is responsible for targeting the car t cell to any cell expressing a matching molecule. the antigen recognition domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scfv). an scfv is a chimeric protein made up of the light (vl) and heavy (vh) chains of immunoglobins, connected with a short linker peptide. these vl and vh regions are selected in advance for their binding ability to the target antigen (such as cd19). the linker between the two chains consists of hydrophilic residues with stretches of glycine and serine in it for flexibility as well as stretches of glutamate and lysine for added solubility. single domain antibodies (e.g. vh, vhh) have been engineered and developed as antigen recognition domains in the car format due to their high transduction efficiency in t cells. in addition to antibody fragments, non‐antibody‐based approaches have also been used to direct car specificity, usually taking advantage of ligand/receptor pairs that normally bind to each other. cytokines, innate immune receptors, tnf receptors, growth factors, and structural proteins have all been successfully used as car antigen recognition domains. hinge region the hinge, also called a spacer, is a small structural domain that sits between the antigen recognition region and the cell's outer membrane. an ideal hinge enhances the flexibility of the scfv receptor head, reducing the spatial constraints between the car and its target antigen. this promotes antigen binding and synapse formation between the car t cells and target cells. hinge sequences are often based on membrane-proximal regions from other immune molecules including igg, cd8, and cd28. transmembrane domain the transmembrane domain is a structural component, consisting of a hydrophobic alpha helix that spans the cell membrane. it anchors the car to the plasma membrane, bridging the extracellular hinge and antigen recognition domains with the intracellular signaling region. this domain is essential for the stability of the receptor as a whole. generally, the transmembrane domain from the most membrane-proximal component of the endodomain is used, but different transmembrane domains result in different receptor stability. the cd28 transmembrane domain is known to result in a highly expressed, stable receptor. using the cd3-zeta transmembrane domain is not recommended, as it can result in incorporation of the artificial tcr into the native tcr. intracellular t cell signaling domain depiction of first, second, and third generation chimeric antigen receptors with the scfv segments in green and the various intracellular tcr signaling components in red, blue and yellowthe intracellular t cell signaling domain lies in the receptor's endodomain, inside the cell. after an antigen is bound to the external antigen recognition domain, car receptors cluster together and transmit an activation signal. then the internal cytoplasmic end of the receptor perpetuates signaling inside the t cell. normal t cell activation relies on the phosphorylation of immunoreceptor tyrosine-based activation motifs (itams) present in the cytoplasmic domain of cd3-zeta. to mimic this process, cd3-zeta's cytoplasmic domain is commonly used as the main car endodomain component. other itam-containing domains have also been tried, but are not as effective. t cells also require co-stimulatory molecules in addition to cd3 signaling in order to persist after activation. for this reason, the endodomains of car receptors typically also include one or more chimeric domains from co-stimulatory proteins. signaling domains from a wide variety of co-stimulatory molecules have been successfully tested, including cd28, cd27, cd134 (ox40), and cd137 (4‐1bb). the intracellular signaling domain used defines the generation of a car t cell. first generation cars include only a cd3-zeta cytoplasmic domain. second generation cars add a co‐stimulatory domain, like cd28 or 4‐1bb. the involvement of these intracellular signaling domains improve t cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence. third generation cars combine multiple co-stimulatory domains, such as cd28-41bb or cd28-ox40, to augment t cell activity. preclinical data show the third-generation cars exhibit improved effector functions and better in vivo persistence as compared to second‐generation cars. research directions antigen recognition although the initial clinical remission rates after car t cell therapy in all patients are as high as 90%, long-term survival rates are much lower. the cause is typically the emergence of leukemia cells that do not express cd19 and so evade recognition by the cd19–car t cells, a phenomenon known as antigen escape. preclinical studies developing car t cells with dual targeting of cd19 plus cd22 or cd19 plus cd20 have demonstrated promise, and trials studying bispecific targeting to circumvent cd19 down-regulation are ongoing. in 2018, a version of car was developed that is referred to as supra car, or split, universal, and programmable. multiple mechanisms can be deployed to finely regulate the activity of supra car, which limits overactivation. in contrast to the traditional car design, supra car allows targeting of multiple antigens without further genetic modification of a person's immune cells. smdcs (small molecule drug conjugates) platform in immuno-oncology is an experimental approach that makes possible the engineering of a single universal car t cell, which binds with extraordinarily high affinity to a benign molecule designated as fluorescein isothiocyanate (fitc). these cells are then used to treat various cancer types when co-administered with bispecific smdc adaptor molecules. these unique bispecific adaptors are constructed with a fitc molecule and a tumor-homing molecule to precisely bridge the universal car t cell with the cancer cells, which causes localized t cell activation. anti-tumor activity in mice is induced only when both the universal car t cells plus the correct antigen-specific adaptor molecules are present. anti-tumor activity and toxicity can be controlled by adjusting the administered adaptor molecule dosing. treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture of the desired antigen-specific adaptors. car t function fourth generation cars (also known as trucks or armored cars) further add factors that enhance t cell expansion, persistence, and anti‐tumoral activity. this can include cytokines, such is il-2, il-5, il-12 and co‐stimulatory ligands. control mechanisms adding a synthetic control mechanism to engineered t cells allows doctors to precisely control the persistence or activity of the t cells in the patient's body, with the goal of reducing toxic side effects. the major control techniques trigger t cell death or limit t cell activation, and often regulate the t cells via a separate drug that can be introduced or withheld as needed. suicide genes: genetically modified t cells are engineered to include one or more genes that can induce apoptosis when activated by an extracellular molecule. herpes simplex virus thymidine kinase (hsv-tk) and inducible caspase 9 (icasp9) are two types of suicide genes that have been integrated into car t cells. in the icasp9 system, the suicide gene complex has two elements: a mutated fk506-binding protein with high specificity to the small molecule rimiducid/ap1903, and a gene encoding a pro-domain-deleted human caspase 9. dosing the patient with rimiducid activates the suicide system, leading to rapid apoptosis of the genetically modified t cells. although both the hsv-tk and icasp9 systems demonstrate a noticeable function as a safety switch in clinical trials, some defects limit their application. hsv-tk is virus-derived and may be immunogenic to humans. it is also currently unclear whether the suicide gene strategies will act quickly enough in all situations to halt dangerous off-tumor cytotoxicity. dual-antigen receptor: car t cells are engineered to express two tumor-associated antigen receptors at the same time, reducing the likelihood that the t cells will attack non-tumor cells. dual-antigen receptor car t cells have been reported to have less intense side effects. an in vivo study in mice shows that dual-receptor car t cells effectively eradicated prostate cancer and achieved complete long-term survival. on-switch: in this system, car t cells can only function in the presence of both tumor antigen and a benign exogenous molecule. to achieve this, the car t cell's engineered chimeric antigen receptor is split into two separate proteins that must come together in order to function. the first receptor protein typically contains the extracellular antigen binding domain, while the second protein contains the downstream signaling elements and co-stimulatory molecules (such as cd3ζ and 4-1bb). in the presence of an exogenous molecule (such as a rapamycin analog), the binding and signaling proteins dimerize together, allowing the car t cells to attack the tumor. bispecific molecules as switches: bispecific molecules target both a tumor-associated antigen and the cd3 molecule on the surface of t cells. this ensures that the t cells cannot become activated unless they are in close physical proximity to a tumor cell. the anti-cd20/cd3 bispecific molecule shows high specificity to both malignant b cells and cancer cells in mice. fitc is another bifunctional molecule used in this strategy. fitc can redirect and regulate the activity of the fitc-specific car t cells toward tumor cells with folate receptors. advances in car t cell manufacturing. due to the high costs of car t cell therapy, a number of alternative efforts are being investigated to improve car t cell manufacturing and reduce costs. in vivo car t cell manufacturing strategies are being tested. in addition, bioinstructive materials have been developed for car t cell generation. rapid car t cell generation is also possible through shortnening or eliminating the activation and expansion steps. economics the cost of car t cell therapies has been criticized, with the initial costs of tisagenlecleucel (kymriah) and axicabtagene ciloleucel (yescarta) being $375,000 and $475,000 respectively. the high cost of car t therapies is due to complex cellular manufacturing in specialized good manufacturing practice (gmp) facilities as well as the high level of hospital care necessary after car t cells are administered due to risks such as cytokine release syndrome. in the united states, car t cell therapies are covered by medicare and by many but not all private insurers. manufacturers of car t cells have developed alternative payment programs due to the high cost of car t therapy, such as by requiring payment only if the car t therapy induces a complete remission by a certain time point after treatment. additionally, car t cell therapies are not available worldwide yet. car t cell therapies have been approved in china, australia, singapore, the united kingdom, and some european countries. in february 2022 brazil approved tisagenlecleucel (kymriah) treatment.