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Like many other emerging cancer treatments, the advent of CAR-T is not always smooth, and public skepticism has always existed, such as effectiveness, sustainability, safety, versatility, etc., which has aggravated the development and transformation of CAR-T therapy. Application concerns. However, it is gratifying that after decades of development and improvement, CAR-T therapy has also experienced a path of continuous improvement, maturity, optimization and improvement.
From the perspective of technical and functional characteristics, CAR-T has developed five technical generation systems to date. 
How does CAR-T develop to the fifth generation, their respective technical characteristics and pre-clinical or clinical applications? This column will give you a detailed account.
Generation I CAR-T prototype : ScFv - hinge region - transmembrane region - structure of CD3z fusion expression
Appearance: In 1989, the Proceedings of the National Academy of Sciences (PNAS) reported the first generation of CAR-T therapy
Structure: a single-chain antibody that specifically expresses a tumor cell surface antigen by T-cells, and binds to an extracellular hinge region and a transmembrane region, as well as an intracellular T cell activating receptor CD3z chain, targeting B cell malignancies. CD19 and CD20 protein molecules.
Features: low utility, easy to exhaust
Clinical application: The first generation of CAR was quickly put into clinical trials, but although some specific cytotoxicity could be seen, it was found to be unsatisfactory in clinical trials in 2006. The reason is that the first generation of CAR-T cells are quickly depleted in patients, and their persistence is so poor that CAR-T cells have become apoptotic when they have not been exposed to a large number of tumor cells. 
Generation II: Adding co-stimulatory signaling molecules necessary for T cell activation and survival
Appearance time: In 1998, facing the defects of the first generation of CAR-T, the second generation of CAR-T technology came into being
Improved structure: The co-stimulatory signaling molecule CD137 (or CD28, ICOS, OX-40) necessary for T cell activation and survival is assembled into CAR, which enhances the ability of CAR-T cells to expand and survive in vivo.
Features: significant effect, mature development
Clinical application: First successful in the mouse B lymphocytic leukemia model, and its performance in the subsequent clinical trials exceeded the expected results for relapsed or refractory acute B lymphocytic leukemia (r/r-ALL) The patient's response rate is as high as 90% or more. Based on its significant tumor killing effect, most of the current clinical trials are second-generation CAR-T.
It is worth mentioning that the CAR-T drug CTL019, which is approved by FDA experts and approved for marketing, belongs to the second-generation CAR-T category. The intra-stimulus signal is co-stimulus signal CD137 (ie 4-1BB).
In addition, JCAR014, JCAR015, JCAR017 products developed by Juno Therapeutics, KTE-C19 developed by Kite Pharma and CART19 developed by Bluebird/Celgene belong to the second generation CAR-T. 
III generation CAR-T: walking on two legs, but knocking down the foot <br> Since the integration of the costimulatory signal can significantly improve the therapeutic effect of CAR-T, and T cells have a large number of costimulatory signaling molecules, then these Co-stimulatory signal molecules are integrated into the CAR molecular structure. Isn't the therapeutic effect more ideal than the second-generation CAR-T? This idea seems to be a very good idea in many ways, so the third generation of CAR will come out immediately.
Improved structure: A common approach is to add a co-stimulatory signal molecule to the second generation of CAR, such as the simultaneous assembly of CD28-CD137 (4-1BB) molecules into the CAR. CART19 (NCT02132624), which targets the CD19 molecule developed by Uppsala University, which was previously reported, belongs to the third generation CAR, and the co-stimulatory signal in the intracellular signal region is also CD28-CD137.
Features: However, there are major defects, rapid depletion of cells, and high recurrence rate. <br> Some studies have shown that CD28 has significant defects compared with 4-1BB: CD28 is characterized by stimulating rapid expansion of CAR-T cells in vivo ( The risk of cytokine storms is greatly increased, but it also causes rapid depletion of cells and a high recurrence rate. Overall, the three-generation CAR-T with integrated CD28 molecules is no better than the second-generation CAR-T, and sometimes has catastrophic consequences (this is awkward, 囧~~).
Clinical application: JUNO's CAR-T product (CD28zCAR) JCAR015 with CD28 as a costimulatory domain is not successful in clinical trials, and five deaths have occurred. In March 2017, Juno announced the abandonment of JCAR015 and instead focused on the development of CAR-T products (4-1BBzCAR) JCAR017 and JCAR014, which are also CD19 targets and 4-1BB as costimulatory domains.
The lessons of JUNO have caused the major CAR-T R&D institutions to be shocked and worried. The three generations of CAR-T have quietly closed in people's disappointment. Even if there is still some research, the main flag of CAR-T has been transferred to it without any suspense. The second generation of CAR-T. The three generations of CAR-T have returned to this situation, but even this does not erode the confidence of CAR-T therapy. The development and improvement of CAR-T technology is still on the way. 
IV generation CAR-T: regulatory gene, performance upgrade
Improved structure: Co-expression of some key cytokines or costimulatory ligands based on the second/third generation CAR-T can significantly increase T cell expansion activity and longevity. This type of CAR-T technology is also known as "TRUCK".
Features: In the latest development, the fourth-generation CAR-T has demonstrated its ability to overcome the micro-environment of solid tumor immunosuppression, and is expected to become an important means in the treatment of solid tumors.
Taking IL-12 as an example, this cytokine is known as the third signaling molecule of T lymphocytes, which shows its importance to T cell function. Systemic injection of IL-12 causes severe inflammatory side effects, but after local expression is achieved by fourth-generation CAR-T cells co-expressing IL-12, NK cells can be recruited at the tumor site, or the depleted tumor can be directly reversed. Invasive T lymphocytes, thereby effectively overcoming the tumor immunosuppressive microenvironment. Other studies have found that co-expression of certain chemokines in CAR-T cells can also enhance the homing ability of CAR-T cells.
In addition, other cytokines with important immunomodulatory capabilities are also being tried to co-express in CAR-T cells, such as IL-7, IL18, IL-21, IL15, etc. We will introduce this in detail in the following columns. Several important cytokines and their progress with CAR-T research , please pay attention to ~ 
Of course, cytokine storms are one of the key side effects of CAR-T treatment. The integration of cytokine co-expression seems to be “fueling the fireâ€, and researchers have noticed this. In other clinical studies, Scientists are trying to set up a "molecular switch or gene switch" mechanism to try to control the efficacy of CAR-T in the body, if necessary, to stop the release of cytokines to avoid damage to the body.
Integrating cytokine expression can realize the upgrading of CAR-T cells in tumor killing function and microenvironment regulation. Its clinical effect, applicability, safety and stability are believed to be in the future. There will be many preclinical and clinical data to respond. Industry concerns, we will wait and see!
V
Generation CAR-T: universal, shiny star
Improved thinking: Although CAR-T cell therapy has many advantages, its highly personalized characteristics make it inconvenient for cGMP production management and large-scale immediate application, and the cost of treatment is high. Therefore, the development of general-purpose CAR-T cells In order to make up for the above shortcomings, therapy is gradually becoming one of the inevitable trends in the field of CAR-T cell therapy.
Improved structure : The universal type of CAR-T is essentially an allogeneic CAR-T cell therapy, which uses gene editing technology and does not require modification according to the patient, but directly from non-patient Body T cells are engineered for treatment in multiple patients. In short, it is to obtain T cells from healthy donors and knock out the TLA HLA gene and TCR gene to avoid host immune rejection (or graft-versus-host disease) infusion of CAR-T cells, thereby producing A versatile T cell product that is theoretically useful for clinical treatment in multiple patients.
Clinical application: Recently, the universal CAR-T cell therapy has brought us one surprise after another. On February 7, Cellectis announced that its universal CAR-T therapy UCART123 was approved by the US FDA's IND (Investigational New Drug) and became the first such product to be approved by the US FDA for clinical trials. On March 10th, Servier, Pfizer and Cellectis jointly announced that the US FDA has approved the UCART19 IND for universal CAR-T therapy, which can enter clinical development in the United States for the treatment of relapse/refractory Indications for acute lymphoblastic leukemia. 
We are also paying attention to and expecting the clinical treatment effect of the fifth-generation universal CAR-T therapy! At the same time, we also hope that with the joint efforts of researchers from all over the world, CAR-T cell therapy will achieve better and faster development in the future, so that more and more high-quality CAR-T therapy will come out as soon as possible, benefiting the majority of patients!
This column will come to an end. To learn more about CAR-T's latest information and "knowledge dry goods", don't forget to continue to pay attention to us, see you next time!
Reference materials:
1. Ruella M, June C H. Chimeric antigen receptor T cells for B cell neoplasms: choose the right CAR for you [J]. Current hematologic malignancy reports, 2016, 11(5): 368-384.
2. Engineered T cells: the promise and challenges of cancer immunotherapy. Nature Reviews, SEPTEMBER 2016, VOLUME 16
3. The Basic Principles of Chimeric Antigen Receptor Design. CANCER DISCOVERY, 389,2013
4. Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy. Clinical Cancer Research.2017
5. Therapeutic T cell engineering. 25 may 2017(545), Nature Review.2017
The road to improvement of CAR-T therapy
In the last issue, we have had some initial understanding of CAR-T therapy. Because of its great potential in tumor immunotherapy, it has become a star that has attracted worldwide attention. All the successes are not achieved overnight. The same is true of CAR-T. How does it become a well-known big star step by step from an unknown little person? Let us take a look at the growth of CAR-T generations today.