The Complete Guide to CAR Design & Construction

CAR-T refers to Chimeric Antigen Receptor T-Cell Immunotherapy, which is a modification of the conventional T cell receptor TCR via a chimeric antigen receptor and is generally engineered into a monoclonal antibody antigen-binding domain. In the scFV segment, the modified CAR-T cells can specifically recognize tumor-associated antigens and are not limited by MHC, so that the targeting, killing activity, and persistence of effector T cells are improved compared with conventional immune cells. CAR-T technology generally selects cytotoxic T lymphocytes (CTLs) for modification because CTLs recognize tumor antigens and release granzymes and perforin to kill tumors.

In 1989, the first CAR T generation was reported on the PNAS. The basic design of the first-generation CAR included a tumor-associated antigen-binding region (usually derived from the scFV region of the monoclonal antibody antigen-binding region), an extracellular hinge region, a transmembrane region, and a steroid chain in the TCR complex as the intracellular signal area. The first generation of CAR can only cause transient T cell proliferation and lower cytokine secretion, but it can not provide long time T cell expansion signals and sustained in vivo anti-tumor effects. Due to the lack of co-stimulatory signals and the poor proliferating ability, CAR-T cells have eady become apoptotic before it meets a large number of tumor cells. The second-generation CAR assembles the necessary second signal molecule (usually CD28, CD134 or CD137 molecule) for the complete activation and survival of T cells, which can increase the immune memory and killing ability of CAR-T cells.

As the expectation of more antitumor efficacy, the 3rd-generation of CARs combined multiple signaling domains (eg, CD3 ζ-CD28-41BB, CD3 ζ-CD28-OX40) to acquire further enhanced activation signals, proliferation, production of cytokines and effective function. For instance, the α-CD19-CD3 ζ-4-1BB CAR-Ts for chronic lymphocyte leukemia showed complete remission to infiltrate and lyse cancer tissue. Even better, a fraction of CAR-Ts functioned as a memory phenotype for preventing tumor relapses. Despite the significant therapeutic effect, the emerging uncontrollable activity with more antitumor efficacy caused life-threatening lysis activity as the most critical adverse effect or fluency clinically significant exit of pro-inflammatory cytokines, pulmonary toxicity, multi-organ failure, and eventual Death.

The previous CAR strategies are highly specific and useful in redirecting T cells targeting malicious cancer cells. However, the major limitation on solid tumors with a tremendous phenotypic heterogeneity and relapse due to antigen-negative cancer cells is the huge challenge to trigger a novel CAR strategy. The 4th-generation CAR-T is designed to shape the tumor environment by the inducible release of additive immune modifiers, such as IL-12, which augments T-cell activation, attracts and activates innate immune cells to eliminate antigen-negative cancer cells In the targeted lesion.

At the 2015 annual meeting of the American Society of Clinical Oncology (ASCO), the first successful clinical trial was demonstrated to use CAR-T therapy for solid tumors of pancreatic cancer. Six patients with refractory pancreatic cancer were treated: 4 patients developed disease progression and 2 patients were stable (3.7 months and 5.3 months) including one patient without metastatic disease. The researchers, from the University of Pennsylvania in Philadelphia, used a special CAR-T (mesothelin + costimulatory molecule 4-1BB) cell, which was specifically infiltrated into the patient's tumor site. Mesothelin (MSLN) is a membrane-anchored protein that is commonly seen in mesothelial cells but is overexpressed in all pancreatic cancer tissues.

However, solid tumors of pancreatic cancer remain particularly challenging because many different types of markers have not been yet discovered. The researchers found a new marker in the tumor of one patient, not one of the common markers, but there were special changes in the glycosylation of the protein. Working with other researchers, they developed a novel CAR-T-cell to express a monoclonal antibody called 5E5 that specifically recognizes Tn polysaccharides on MUC1. This marker is not found in normal cells, but is abundantly expressed in different types of cancer cells. 5E5-modified car-t-cells were injected into mice with leukemia and pancreatic cancer. The tumor growth was slowed and the survival time was prolonged. At the end of the experiment, all six pancreatic cancer mice survived.

About Creative Biolabs

As a global company, Creative Biolabs has more than 200 talented and well-trained scientists located in different continents working closely with partners from the entire world to develop and produce medicines of tomorrow. Specifically, we are the established leading expert in TCR and CAR T&NK cell immune therapy development, as we offer the one-stop custom services that cover the entire new drug development pipeline. Additionally, we also offer an exclusive line of ready-to-use TCR and CAR T&NK cell construction products, such as virus packaging, purification, expansion and titer determination kits. Furthermore, we have built up a unique unparalleled CAR construction and production platform for all four CAR generations