Chimeric antigen receptor
Chimeric antigen receptors (CARs) are genetically engineered receptors that provide specific properties to an immune effector cell (e.g., lymphocyte T-cell). These receptors gain the specificity of a monoclonal antibody targeted against specific tumor cells. The coding sequence is transferred to the receptor by viral (retroviral and lentiviral) vectors. The term “chimeric” indicates different sources of composing parts of the receptor. T cells with engineered CARs acquire potent immunological properties and by redirecting the immune system in order to eliminate malignant cells act as a living drug, expanding in the patient and ensuring long-term antitumor memory [1, 2, 3].
CAR-T cells can be either autologous or allogenic. Autologous CAR-T cells are derived via leukapheresis from the blood of the patient. Allogenic CAR-T cells are derived from the blood of a healthy donor. CARs as recombinant receptors transferred in various T-cell subsets provide in a restricted manner specific antigen binding in a non-major histocompatibility complex (non-MHC) . Theoretically, CAR-T cells can be engineered to target virtually any tumor-associated antigen and, in general, any other antigen, e.g., fungal antigen .
Four generations of CARs
First engineered T-cell with chimeric molecule was developed in 1989–1993 by Israeli immunologists Zelig Eshhar and Gideon Gross in the Department of Chemical Immunology of Weizmann Institute of Science in Rehovot, Israel (Tab. I). These first-generation CARs were not yet clinically effective [5, 6, 7].
History of development of CAR effector cells
|1989||Generation of effector T cells expressing chimeric T-cell receptor [5, 6]|
|1993||First-generation CAR-T cells, not clinically effective |
|2002||First effective CAR-T cells against prostate cancer antigen in the laboratory|
|2003||Second-generation CARs: CD19-directed CAR-T cells can kill leukemia cells in mouse|
|2009||CD19 CAR-T cells used in relapsed/refractory leukemia|
|2011||CD19 CAR-T cells in patients with chronic lymphocytic leukemia |
|2013||CD19 CAR-T cells in pediatric acute lymphoblastic leukemia |
|2013||Science magazine announced cancer immunotherapy as “Breakthrough of the Year”|
|2014||Third-generation CAR-T cells: inducible caspase-9 suicide gene system as a “safety switch” to limit on-target, off-tumor toxicities |
|2015||Fourth-generation CARs, the so-called TRUCKs (CAR redirected T cells that deliver a transgenic product to the targeted tumor tissue) or armored CARs, which produce other molecules built and being tested for ovarian cancer |
|2015||Concept of CAR-NK cells |
|2017||Clustered regularly interspaced short palindromic repeats (CRISPR) used to optimize CAR placement in T cells |
|2017||FDA approves CD19-CAR-T cells for relapsed/refractory acute lymphoblastic leukemia in children and young adults|
|2017||FDA approves CD19-CAR-T cells for relapsed/refractory DLBCL in adults|
|2018||EMA approves CD19-CAR-T cells for relapsed/refractory acute lymphoblastic leukemia in children and young adults and for relapsed/refractory DLBCL in adults|
|2019||Dual CD19/CD22 CAR-T cells in acute lymphoblastic leukemia in children and adults |
Over next thirty years, CARs were immunologically and technologically modernized and sophisticated as first, second, third, and currently fourth generation, depending on their composition. The second-generation CARs had improved antitumor activity of T cells: improved T-cell proliferation, resistance to apoptosis, cytokine secretion, and in vivo persistence. The third-generation CARs, in comparison with second-generation CARs, had improved effector functions and in vivo persistence [8, 9].
Fourth-generation CARs, the so-called TRUCKs (CAR redirected T cells that deliver a transgenic product to the targeted tumor tissue) or armored CARs, present further enhancement in antitumoral potency, cytokine activity, and costimulatory ligands and enzymes that can degrade the extracellular matrix in solid tumors [9, 10]. Further enhancement in the safety of CAR-T cell therapy can be obtained with the so-called smart T cells, which is under investigation [11, 12, 13].
Finally, new engineering modalities, based on the use of targeted nucleases like clustered regularly interspaced short palindromic repeats (CRISPR), may further enhance the efficacy and safety of CAR-T cells .
First clinical application
Clinical application of CAR-T cells became available through the dedicated team in the University of Pennsylvania and Children’s Hospital in Philadelphia. Carl June was the immunologist who led the development of CAR-T cell technology, striving with many difficulties. Together with David Porter and Stephan Grupp, he administered CAR-T cells to patients with chronic lymphocytic leukemia (CLL) in 2011  and ALL in 2012 .
The real progress was achieved with the first therapy in child with ALL: a 7-year-old Emily Whitehead with relapsed and refractory ALL, who was recommended to hospice, became the first child to receive CART-19 in 2012. She experienced all severe complications of this therapy, but she was finally cured. As a result, her survival helped reenergize a line of research that was near the failure .
The US Food and Drug Administration Agency (FDA) and the European Medicines Agency (EMA) have licensed two products of CAR-T cells: tisagenlecleucel for the use in children and young adults up to 25 years of age with B-cell ALL that does not respond to treatment or has relapsed two or more times, and tisagenlecleucel and axicabtagene ciloleucel for the use in adult patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) (Tab. II). Several other products are at different stages of research and progress toward registration (Tab. III).
Approval of CAR-T cell technology for clinical use
|August 30, 2017||US Food and Drug Administration||Tisagenlecleucel (Kymriah, Novartis)||ELIANA, Maude et al. ||Children and young adults up to 25 years of age with B-cell ALL who do not respond to treatment or have relapsed two or more times|
|October 18, 2017||US Food and Drug Administration||Axicabtagene ciloleucel (Yescarta, Gilead-Kite)||ZUMA-1, Neelapu et al. ||Adult patients with relapsed or refractory DLBCL|
|May 1, 2018||US Food and Drug Administration||Tisagenlecleucel (Kymriah, Novartis)||JULIET, Schuster et al. ||Adult patients with relapsed or refractory DLBCL, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma|
|June 28, 2018||European Medicines Agency||Tisagenlecleucel (Kymriah, Novartis)||ELIANA, Maude et al. ||Children and young adults up to 25 years of age with B-cell ALL who do not respond to treatment or have relapsed two or more times|
|June 28, 2018||European Medicines Agency||Axicabtagene ciloleucel (Yescarta, Gilead-Kite)||ZUMA-1, Neelapu et al. ||Adult patients with relapsed or refractory DLBCL|
Advanced research in CAR-T cell application
|Lisocabtagene maraleucel (Lisocel, Bristol-Myers Squibb/Celgene Company)||TRANSCEND NHL 001 trial, Abramson et al. ||Adult patients with relapsed or refractory (R/R) DLBCL after at least two prior therapies||Application to FDA (December 18, 2019)|
|Idecabtagene Vicleucel (CT053, CARsgen Therapeutics)||KarMMa||Multiple myeloma||The FDA has given (September 5, 2019) orphan drug status to CT053 (anti-B-cell maturation antigen (anti-BCMA)), an investigational chimeric antigen receptor T-cell therapy for the treatment of multiple myeloma|
|Anti-BCMA CAR-T cell bb2121 therapy (Bluebird Bio and Celgene)||CRB-401 trial, Raje et al. ||Relapsed or refractory multiple myeloma||The product was granted breakthrough therapy designation by the FDA in November 2017 and will thus receive expedited review by the agency. It has also been fast-tracked in Europe|
|Axicabtagene ciloleucel (Yescarta, Gilead-Kite)||ZUMA-3 (adult) and ZUMA-4 (pediatric and adolescents 2–21 years)
||ALL in children and adults||Registration in progress|
Further progress and worldwide distribution
Owing to emergence of relapse of ALL with cells that do not express CD19 in mechanisms of a phenomenon known as antigen escape, the Phase I study with the use of CAR-T cells with dual targeting of CD19/CD22 has demonstrated good efficacy, safety, and tolerability, thus opening possibility to trials studying bispecific targeting to circumvent CD19 down-regulation [17, 23, 24].
An option to increase the potential of CAR-T cells is its enhancement by combining CAR-T cells with a monoclonal antibody targeting the human programmed death receptor 1 (anti-PD1) performed so far in the prostate cancer model .
As of December 2019, there were 284 ongoing clinical trials (www.clinicaltrials.gov) happening globally involving CAR-T cells, mostly in the USA (130) and China (124), most of them in hematological malignancies (ALL, non-Hodgkin lymphoma, multiple myeloma), targeting mostly CD19 and also CD20, CD22, and BCMA, almost exclusively CAR-T cells, but in 10 cases also CAR-NK  (majority in China). Currently, at least 24 countries use CAR-T cell technology in therapy .
The author thanks Prof. Lidia Gil for critical review of the paper.
Conflict of interest
The author was the participant of Novartis Advisory Board, has received lecture fee, and participated in meetings organized by Gilead.
The work described in this article has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform requirements for manuscripts submitted to biomedical journals.
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