CAR T cell
In biology, chimeric antigen receptors (CARs)—also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors—are receptor proteins that have been engineered to give T cells the new ability to target a specific antigen. The receptors are chimeric in that they combine both antigen-binding and T cell activating functions into a single receptor.
CAR T cell therapy uses T cells engineered with CARs to treat cancer. T cells are modified to recognize cancer cells and destroy them. The standard approach is to harvest T cells from patients, genetically alter them, then infuse the resulting CAR T cells into patients to attack their tumors.[1]
CAR T cells can be derived either
After the modified T cells are infused into a patient, they act as a "living drug" against cancer cells.
The surface of CAR T cells can bear either of two types of
History
The first chimeric receptors containing portions of an
In 1991,
A first generation CAR containing a CD4 extracellular domain and a CD3ζ intracellular domain was used in the first clinical trial of chimeric antigen receptor T cells by the biotechnology company
In the early 2000s, co-stimulatory domains such as
Production
The first step in the production of CAR T-cells is the isolation of T cells from human blood. CAR T-cells may be manufactured either from the patient's own blood, known as an autologous treatment, or from the blood of a healthy donor, known as an allogeneic treatment. The manufacturing process is the same in both cases; only the choice of initial blood donor is different.[citation needed]
First,
The expanded T cells are purified and then
The patient undergoes lymphodepletion chemotherapy prior to the introduction of the engineered CAR T-cells.[4] The depletion of the number of circulating leukocytes in the patient upregulates the number of cytokines that are produced and reduces competition for resources, which helps to promote the expansion of the engineered CAR T-cells.[23]
Clinical applications
As of March 2019, there were around 364 ongoing clinical trials happening globally involving CAR T cells.[24] The majority of those trials target blood cancers: CAR T therapies account for more than half of all trials for hematological malignancies.[24] CD19 continues to be the most popular antigen target,[25] followed by BCMA (commonly expressed in multiple myeloma).[24][26] In 2016, studies began to explore the viability of other antigens, such as CD20.[27] Trials for solid tumors are less dominated by CAR T, with about half of cell therapy-based trials involving other platforms such as NK cells.[24]
Cancer
T cells are genetically engineered to express chimeric antigen receptors specifically directed toward antigens on a patient's tumor cells, then infused into the patient where they attack and kill the cancer cells.
Early CAR T cell research has focused on
Solid tumors have presented a more difficult target.[34] Identification of good antigens has been challenging: such antigens must be highly expressed on the majority of cancer cells, but largely absent on normal tissues.[35][36][37][30] CAR T cells are also not trafficked efficiently into the center of solid tumor masses, and the hostile tumor microenvironment suppresses T cell activity.[33]
Autoimmune disease
While most CAR T cell studies focus on creating a CAR T cell that can eradicate a certain cell population (for instance, CAR T cells that target lymphoma cells), there are other potential uses for this technology. T cells can also mediate tolerance to antigens.[38] A regulatory T cell outfitted with a CAR could have the potential to confer tolerance to a specific antigen, something that could be utilized in organ transplantation or rheumatologic diseases like lupus.[39][40]
Approved therapies
The examples and perspective in this section may not represent a worldwide view of the subject. (November 2023) |
CAR T cell (Brand name) | Company | Approval Agency: Date | Target | Antigen recognition domain | Intracellular signaling domain | Indication (Targeted disease / Line of Therapy) | Agency Product Number, Drug Label |
---|---|---|---|---|---|---|---|
tisagenlecleucel
(Kymriah) |
Novartis | FDA: 08/30/2017 [41]
MHLW: 05/15/2019 [43]
|
CD19 | scFV | CD3ζ
|
B-cell precursor ALL (Third Line)[41][42][43]
Diffuse large B-cell lymphoma (Third Line)[44] [42][43] Follicular Lymphoma (Third Line)[45] [46] |
FDA:125646, Label
EMA:004090, Label |
axicabtagene ciloleucel
(Yescarta) |
Kite Pharma / Gilead | FDA: 10/18/2017 [47]
EMA: 08/27/2018 [48] MHLW: 12/22/2022 [50] |
CD19 | scFV | CD28 - CD3ζ | Diffuse large B-cell lymphoma (Second Line)[51] [52] [49][50]
Follicular lymphoma (Third Line) [53] [54] [49][50] Primary mediastinal large B-cell lymphoma (Third Line) [48][49][50] |
FDA:125643, Label
EMA:004480, Label |
brexucabtagene autoleucel
(Tecartus) |
Kite Pharma / Gilead | FDA: 07/24/2020 [55]
EMA: 12/14/2020 [56] |
CD19 | scFV | CD28 - CD3ζ | Mantle cell lymphoma (Third Line) [57][56]
B-cell precursor ALL (Third Line)[57] [56] |
FDA:125703, Label
EMA:005102, Label |
lisocabtagene maraleucel
(Breyanzi) |
Juno Therapeutics / BMS | FDA: 02/05/2021[58]
EMA: 04/04/2022 [59] MHLW: 12/20/2022 [60] |
CD19 | scFV | 41BB - CD3ζ | Diffuse large B-cell lymphoma (Second Line)[61] [59][60] | FDA: 25714, Label
EMA:004731, Label |
idecabtagene vicleucel
(Abecma) |
Bluebird Bio / BMS | FDA: 03/26/2021 [62]
EMA: 08/18/2021 [63] |
BCMA | scFV | 41BB - CD3ζ | Multiple myeloma (Fourth Line),[63] (Fifth Line)[62] | FDA:125736, Label
EMA:004662, Label |
ciltacabtagene autoleucel
(Carvykti) |
Janssen / J&J | FDA: 02/28/2022 [64]
EMA: 05/25/2022 [65] |
BCMA | VHH
|
41BB - CD3ζ | Multiple myeloma (Fourth Line),[65] (Fifth Line)[64] | FDA:125746, Label
EMA:005095, Label |
Safety
There are serious side effects that result from CAR T-cells being introduced into the body, including cytokine release syndrome and neurological toxicity.[4] Because it is a relatively new treatment, there are few data about the long-term effects of CAR T-cell therapy. There are still concerns about long-term patient survival, as well as pregnancy complications in female patients treated with CAR T-cells.[66] Anaphylaxis may be a side effect, as the CAR is made with a foreign monoclonal antibody, and as a result provokes an immune response.[citation needed]
On-target/off-tumor recognition occurs when the CAR T-cell recognizes the correct antigen, but the antigen is expressed on healthy, non-pathogenic tissue. This results in the CAR T-cells attacking non-tumor tissue, such as healthy B cells that express CD19 causing B-cell aplasia. The severity of this adverse effect can vary but the combination of prior immunosuppression, lymphodepleting chemotherapy and on-target effects causing hypogammaglobulinaemia and prolonged cytopenias places patients at increased risk of serious infections.[20][67]
There is also the unlikely possibility that the engineered CAR T-cells will themselves become transformed into cancerous cells through
Cytokine release syndrome
The most common issue after treatment with CAR T-cells is cytokine release syndrome (CRS), a condition in which the immune system is activated and releases an increased number of inflammatory cytokines. The clinical manifestation of this syndrome resembles
Immune effector cell-associated neurotoxicity
Neurological toxicity is also often associated with CAR T-cell treatment.
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.[75][76]
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.[citation needed]
The antigen recognition domain is typically derived from the variable regions of a
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.[75] Cytokines, innate immune receptors, TNF receptors, growth factors, and structural proteins have all been successfully used as CAR antigen recognition domains.[75]
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.[83] Hinge sequences are often based on membrane-proximal regions from other immune molecules including IgG, CD8, and CD28.[75][84][80][81]
Transmembrane domain
The transmembrane domain is a structural component, consisting of a
Using the CD3-zeta transmembrane domain is not recommended, as it can result in incorporation of the artificial
Intracellular T cell signaling domain
The intracellular T cell signaling domain lies in the receptor's endodomain, inside the cell.[75] 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.[77]
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.[76]
T cells also require
The intracellular signaling domain used defines the generation of a CAR T cell.[4] First generation CARs include only a CD3-zeta cytoplasmic domain.[4] 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.[4] 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.[4]
Research directions
Antigen recognition
Although the initial clinical remission rates after CAR T cell therapy in all patients are as high as 90%,[87] 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.[33] 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.[33]
In 2018, a version of CAR was developed that is referred to as SUPRA CAR, or split, universal, and programmable.[88] 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.[89]
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.[90][91]
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.[92][93]
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.[94] 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.[citation 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.[94][95][96] 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.[94][97] It is also currently unclear whether the suicide gene strategies will act quickly enough in all situations to halt dangerous off-tumor cytotoxicity.[citation needed]
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.[98] An in vivo study in mice shows that dual-receptor CAR T cells effectively eradicated prostate cancer and achieved complete long-term survival.[99]
ON-switch and OFF-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.[100] Human EGFR truncated form (hEGFRt) has been used as an OFF-switch for CAR T cells using cetuximab.[35][37][80]
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.[101] The anti-CD20/CD3 bispecific molecule shows high specificity to both malignant B cells and cancer cells in mice.[102] 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.[103]
Advances in CAR T cell manufacturing.
Due to the high costs of CAR T cell therapy,[104] a number of alternative efforts are being investigated to improve CAR T cell manufacturing and reduce costs. In vivo CAR T cell manufacturing strategies[105][106] are being tested. In addition, bioinstructive materials have been developed for CAR T cell generation.[107] Rapid CAR T cell generation is also possible through shortening or eliminating the activation and expansion steps.[108]
In situ modification
Another approach is to modify T cells and/or B cells still in the body using viral vectors.[109]
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.[104] 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.[104] In the United States, CAR T cell therapies are covered by Medicare and by many but not all private insurers.[110][111] 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.[112]
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.[113] In February 2022 Brazil approved tisagenlecleucel (Kymriah) treatment.[114]
See also
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