Editor’s note: Gene and cell therapy (GCT) is one of the most competitive emerging fields in global biotechnology. As a new strategy for treating malignant tumors and other human diseases, GCT has been extensively studied in clinical trials worldwide. Pharmaceutical companies have their GCT products approved for clinical use. For example, the biopharmaceutical company Janssen entered a collaboration and license agreement with Legend Biotech, a subsidiary company from GenScript Biotech Corporation, to develop investigational CAR-T Anti-Cancer therapy. It is thus foreseeable that with the continuous deepening of research and the constant advancement of commercialization, GCT will diversify and show more and more broad application prospects. However, as an emerging technology involving multiple mechanisms of action and a diverse array of active ingredients, GCT also faces its own share of controversy and challenges. To better understand the current situation of GCT opportunities, challenges, and future development trends, this series, “Decoding Gene and Cell Therapy” will extensively discuss popular topics in the GCT field such as CAR-T, TCR-T, CRISPR, oncolytic viruses, cancer vaccines and more. Stay Tuned to LifeScientist.com for the most important scientific research discoveries, progress, viewpoints and reveals from the most cutting-edge of scientific research.
Emily’s anticancer road
Five years of age, the halcyon days of youth. The simply joys of running in the sunset is one of the most carefree periods of a person’s life. However, for young Emily Whitehead who was diagnosed with acute lymphoblastic leukemia, every day was just a marathon against death.
In 2012, after all conventional treatments such as chemotherapy and bone marrow transplantation had failed, Emily, who experienced cancer recurrence at the age of seven, tragically found herself teetering on the edge of life. At this moment, her parents decided to make a desperate move. They took her to participate in the Phase I clinical trial of an experimental new cell therapy piloted by the Children’s Hospital of Philadelphia.
Although still in the experimental stages, the treatment was little Emily’s only chance of survival, and fortunately managed to miraculously eliminate all the cancer cells in her body. Since then, Emily has officially bid farewell to the cancer in her life, and publishes a “cancer-free” anniversary photo annually to celebrate her healthy life in this world. She is now in her ninth year of post-cancer recovery .
So what was this new cell therapy method that enabled Emily’s inspiring anti-cancer story? None other than the now famous CAR-T cell therapy that we will take our readers through in this issue.
CAR-T cells, the Special Forces in T cells
As its name implies, CAR-T cells, or Chimeric Antigen Receptor T cells, refer to artificial T cells used for immunotherapy after the genetic engineering and expression of cancer antigen receptors. Medical staff first start by drawing a patient’s blood during clinical treatment, isolating immune T cells, and using harmless viruses or CRISPR/CAS9 technology to inject genes into the T cells. A recent example of this can be seen in a medRxiv preprint, where Zhang et al. utilized CRISPR/Cas9 to successfully develop CAR-T cells for the two gene loci of AAVS1 and PD1 using gRNA synthesized by GenScript .
These isolated and modified CAR-T cells can now express cancer antigen receptors after gene editing. With these receptors able to bind to antigens on the surface of cancer cells, CAR-T cells can target and eliminate cancerous cells with solid specificity. As an example of personalized medicine, engineered CAR-T cells can be cultured and multiplied in the laboratory before be reinfused back into the patient’s body to treat their cancer.
If T cells are the “bandit army” that defends against diseases in the human body, then CAR-T cells are the “special forces” in this army, equipped with the most advanced tracking and positioning weapons (CAR), which can accurately strike specific “Strategic goals” (cancer cells). So, how exactly was this “new weapon” developed?
Five generations of vector construction technology: The past and the present of CAR-T technology.
For many years, people have known that the immune system plays an essential role in controlling tumors. Throughout the history of human medicine, starting way back from ancient Egypt to Europe in the early 18th century, there have been many records of tumor growth subsiding or disappearing after an infection or case of high fever . In the middle and late 18th century, the German physician Dr. Wilhelm Busch observed that some of his sarcoma patients had tumor regressions after surviving postoperative wound erysipelas, an infection with a bacterial species Streptococcus. Intrigued by this phenomenon, he deliberately infected a postoperative sarcoma with pus from another patient with postoperative erysipelas in an attempt to induce tumor regression. He did, but it was minimal and short-lived. Dr. Busch’s attempt at generating a spontaneous cancer-killing immune response was one of the earliest forays into cancer immunotherapy. By the mid to late 20th century, humans had progressed to trial the use of bone marrow transplantation (as bone marrow contains immune cells) to treat ailments such as acute myeloid leukemia, achieving notably significant treatment effects as a result . Further studies from the group reinforced that T cells played an essential role in this tumor mediation and control process, leading us to the present day. Scientists and researchers trial the injection of modified T cells into patients to fight the harmful effects of cancer, also known as the beginning of CAR-T technology as we know it.
Design principles and unique clinical treatment characteristics of the five generations of CAR-T
The first-generation CAR-T
The first-generation of CAR molecules contained three domains: an extracellular antigen recognition domain of a single-chain fragment variant (scFv) extracted from an antibody; a transmembrane domain; and an intracellular CD3ζ (CD3-Zeta) T cell activation domain.
In order for T cells to better target and kill cancer cells, in addition to allowing the receptors on T cells to bind to tumor cell antigens, a synergy of co-stimulatory receptors/ligands is also required. In the structural design of the first-generation CAR-T CAR molecule, in addition to the antigen recognition scFv, only CD3ζ or FcεRIγ (Fc-receptor common gamma chain) was introduced as the only intracellular signal transduction domain. This design unfortunately resulted in CAR molecules with limited proliferation ability and a short survival time, with no way to fully activate the cells after recognizing target tumor cells . Therefore, the first-generation CAR-T has only shown very limited therapeutic effects in clinical practice.
The second-generation CAR-T
Based on the first-generation, the second-generation of CAR-T added in the costimulatory domain of the T cell receptor, which is typically represented by the intracellular signaling region of CD28 or 4-1BB molecules . These modified T cells displayed greatly improved survival times, proliferation and tumor killing ability of cancer cells in the human body. At present, most of the currently approved CAR-T products utilize this second-generation design of CAR-T.
The third-generation CAR-T
The third-generation of CAR-T adds even more co-stimulatory domains (such as CD27 or OX40, etc.) to the basic structure of the second-generation CAR molecule, further enhancing the CAR-T’s vitality, endurance and killing effectiveness .
The second- and third-generation CAR-T have achieved great success in treating hematological tumors, however many studies have reported that it cannot cross into the tumor environments of solid tumors, resulting in poor efficacy on that front. In other words, a “super CAR-T cell” is needed to break through the immunosuppressive microenvironment of advanced solid tumors, with this “super CAR-T” being fulfilled by the fourth-generation design of CAR-T.
The fourth-generation CAR-T
The design principle guiding the fourth-generation of CAR-T focuses on allowing T cells to express CAR molecules while expressing another secretable protein, such as common pro-inflammatory cytokines like IL-12, PD-1/PD-L1 Antibodies, etc., whose main purpose is to enhance the infiltration ability of CAR-T cells into advanced solid tumors and resist the inhibitory effects of these solid tumors microenvironments . The fourth-generation CAR-T technology enables many combination therapy ideas to be realized on one CAR-T cell, with its range of applicable fields of research gradually expanding to other diseases besides cancer, such as viral infections, metabolic disorders, autoimmunity, sexually transmitted infections, etc.
The fifth-generation CAR-T
The fifth-generation of CAR-T, breaks the trend and is based on the second-generation design of CAR-T, utilizing gene editing to inactivate the TRAC gene and leading to the removal of the TCR alpha and beta chains. This is to avoid the complications of host immune rejection or graft-vs.-host disease against transplanted CAR-T cells, as scientists proposed to knock out the human leukocyte antigen (HLA) and TCR genes of T cells obtained from healthy donors. Uniquely the fifth-generation of CAR-T does not need personalized modification tailored to each individual patient, thus having the potential to be used as a strategy for treating multiple patients afflicted with similar ailments .
So, does this mean that current CAR-T technology has the potential to become an immunotherapy panacea for all tumors? How many types of cancer can CAR-T therapy cure? Regarding these questions and types of indications for CAR-T cell therapy, please look forward to the next article installment in our “Decoding Gene and Cell Therapy” series!
 Emily Whitehead Foundation, “our Journey”, achieved at https://emilywhiteheadfoundation.org/our-journey/
 Zhang, Jiqin, et al. “Development and clinical evaluation of non-viral genome specific targeted CAR T cells in relapsed/refractory B-cell non-Hodgkin lymphoma.” medRxiv (2020).
 Oiseth, Stanley J., and Mohamed S. Aziz. “Cancer immunotherapy: a brief review of the history, possibilities, and challenges ahead.” Journal of cancer metastasis and treatment 3 (2017): 250-261.
 Thomas, E. Donnall. “Bone marrow transplantation: a historical review.” Medicina 33.3 (2000): 209-218.
 Eshhar, Zelig, 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.” Proceedings of the National Academy of Sciences 90.2 (1993): 720-724.
 Porter, David L., et al. “Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.” Science translational medicine 7.303 (2015): 303ra139-303ra139.
 Zhang, Cheng, et al. “Engineering car-t cells.” Biomarker research 5.1 (2017): 1-6.
 Chmielewski M, Abken H. TRUCKs: the fourth generation of CARs. Expert Opin Biol Ther. 2015;15(8):1145–54.
 Lijun Zhao and Yu J. Cao. Engineered T cell Therapy for Cancer in the Clinic. Frontiers in Immunology. 2019;Vol.10:1–20.
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