As of now, eight CAR-T drugs have been launched globally and have shown significant efficacy in hematological malignancies. However, due to factors such as tumor microenvironment and T cell exhaustion, the application of CAR-T therapy in solid tumors is still under exploration.
Recent research has found that most solid tumor CAR receptors still produce a persistent basal signal when not stimulated by tumor antigens. This basal signal continuously stimulates CAR-T cells, leading to T cell exhaustion. At the same time, a clinical study led by Professor Carl June from the University of Pennsylvania has shown that a deficiency in basal signals can also affect the clinical efficacy of CAR-T cells. Therefore, understanding the mechanisms by which basal signals are formed and maintained can help design highly persistent and anti-exhaustive CAR-T cells, thereby improving the efficacy of CAR-T therapy.
School of Life Sciences and Technology, ShanghaiTech University, March 8, 2023Wang PengResearch Team and Eye, ENT & Head and Neck Hospital, Fudan UniversityWu HaitaoTeam, Center for Excellence in Molecular and Cellular Sciences, Chinese Academy of SciencesXu ChenqiLaboratory, and Shanghai First People's HospitalSong XianminCooperation within the research groupThe mechanism of basal signal formation in CAR-T cells was successfully analyzed, and a rational CAR design system based on basal signal regulation was proposed. This enables CAR-T cells to significantly enhance their survival advantage and anti-tumor effect, providing a new strategy for advancing CAR-T cell therapy against solid tumors.The study is based on “Tuning the charge density of chimeric antigen receptors optimizes tonic signaling and CAR-T cell fitness"The optimization of substrate signal and CAR-T anti-tumor ability by regulating CAR charge density" was published in the journalCell Research（Cell Research, impact factor: 46) and was listed as a selected paper by the magazineFeatured Article).
I. Characterization of Surface Charge Distribution of 10 Commonly Clinically Used CARs
CAR-targeted T lymphocytes can specifically attack tumor cells, consisting of an extracellular domain that recognizes tumor antigens and an intracellular domain that triggers T cell activation. The variable regions of the heavy and light chains are connected by flexible linkages to formsingle-stranded variable fragment(scFv) is the region in CAR that specifically recognizes tumors.
In this study, researchers first constructed and expressed ten clinically common CARs in Jurkat cells (a cell line used for studying acute T-cell leukemia), calculated the 'basal signal index', and then expressed these ten CARs on the surface of primary T cells to obtain the 'depletion fraction' of CAR-T cells. The results showed that CAR-T cells with high basal signals had a greater degree of depletion, among which GD2.CAR had the strongest basal signal, and at the same time, GD2.CAR-T cells had the highest depletion fraction; CD19.CAR had the weakest basal signal, and CD19.CAR-T cells had the lowest depletion fraction.In other words, there is a significant positive correlation between exhaustion scores and basal signal index, indicating that persistent antigen-independent basal signals can induce CAR-T cell exhaustion.
Figure 1: Ten common CAR base signal indices, corresponding T cell exhaustion scores, and a significant positive relationship between them
Based on this discovery, researchers further focused their attention on the antigen-binding domain scFv, as the structures of the other nine CARs are almost identical. Further research found thatThe positive charge patch (PCP) on the CAR surface has a certain positive correlation with basal signals. The more positively charged residues on the scFv structure surface aggregate, the higher the PCP fraction. High PCP leads to autonomous aggregation of CAR, generating corresponding basal signals.This has overturned the previous conclusion that the basal signal is due to unstable CAR proteins.
Figure 2: Schematic diagram of the PCP for ten CAR scFVs, showing the regions with continuous positive charge residues (the top three major PCPs are dark blue, blue, and light blue in sequence) and their corresponding PCP scores (the sum of all residues within the top three major PCPs); correlation between basal signal and PCP score
II. Optimizing CAR base signal by adjusting ion strength during in vitro amplification
To prove the hypothesis that the electrostatic interaction mediated by PCP is a mechanism for generating basal signals in CAR-T cells, researchers weakened the electrostatic interaction between PCPs by increasing the concentration of salt ions to enhance electrostatic shielding. The results showed that the two types with the strongest basal signalsanti-solid tumor CAR-T cell(GD2.CAR targeting glioma and CSPG4.CAR targeting head and neck tumors)In a high-salt environment, basal signals are weakened, CAR autophagy is significantly reduced, T cell exhaustion is alleviated, and the ability to kill tumor cells is significantly enhanced.However, CD19.CAR-T against hematological malignancies is almost unaffected by the same treatment, suggesting that adjusting ion strength is a convenient and direct method for optimizing CAR-based signals against solid tumors.
Figure 3: High salt treatment significantly enhances the killing efficiency of anti-solid tumor CAR-T cells, but has limited effects on anti-hematological tumor CAR-T cells
III. Adjusting the PCP score size through point mutations
Based on this, the team made point mutations in the FRs region of the scFv backbone far from the antigenic determinant region to adjust the strength of PCP.CAR-T cells with high PCP(GD2.CAR-T, CSPG4.CAR-T, etc.)After lowering the temperature, both the basal signal and the exhaustion index were weakened. Not only did affinity and specificity remain unchanged, but the tumor-killing ability was also demonstrated to be better.On the contrary,Low PCP CAR-T cells (such as CD19.CAR-T) showed enhanced basal signaling after appropriate increase in PCP, but demonstrated better persistence and anti-tumor effects.This is consistent with research findings that enhancing the basal signal of CD22.CAR-T cells improves their efficacy, indicating that a certain intensity of basal signal allows CAR-T cells to survive better in vivo. The team has preliminarily determined through experiments thatBy adjusting the PCP score to 45-56, CAR-T cells can be kept from exhausting under appropriate basal signal stimulation, achieving a win-win situation in terms of lethality and persistence.
Figure 4: CAR-T cells exhibit higher tumor killing capacity after PCP score adjustment
In summary, the collaborative research led by Professor Wang Peng from ShanghaiTech has successfully revealed that the electrostatic interaction between CAR receptors is an important mechanism for CAR-T cells to generate basal signals, and has innovatively proposedThe 'peak tip' theory of basal signal regulation of CAR-T cell functionpoint outTo achieve the optimal (Peak) anti-tumor efficacy, the basal signal of CAR needs to be reasonably optimized. It is necessary to avoid weak basal signals that cannot maintain stable CAR-T proliferation and also prevent overly strong basal signals from leading to CAR-T functional exhaustion.This is achieved by optimizing the design of the base signal to maximize the persistence of CAR-T cells within the body without causing exhaustion.
Figure 5: 'Peak Tip' theoretical model and innovative peak tip theory diagram
IV. Breakthrough research findings help fill the gap in CAR-T treatment for solid tumors
Looking at the overall picture, from the global to China, and from regulation to the market, the cell gene therapy industry is in a period of fervor. As the application prospects of CAR-T therapies gradually become clearer, their popularity continues to grow, and it is expected that CAR-T therapies will remain a 'goldmine' in the field of innovative drugs for the next few years. According to Frost & Sullivan data, the global CAR-T therapy market size reached $1.7 billion in 2021. With the gradual advancement of research and development pipelines and breakthroughs in solid tumor application scenarios, it is estimated that the market will grow to $211 billion by 2030 at a compound annual growth rate of 32.1%.
In addition, according to Frost & Sullivan data, in 2021, the number of new cancer cases in China was nearly 5 million. Existing cancer drugs have deficiencies such as high recurrence rates. The large patient base and clinical needs have promoted the clinical research and development of CAR-T drugs for cancer. However, in terms of the number of clinical pipelines, there is still significant room for development in the field of solid tumors. As of November 2022, among the CAR-T drug pipelines under research in China, hematological malignancies accounted for 74% as the main research direction, while solid tumors only accounted for 23%. The research and development progress of CAR-T in the field of solid tumors is slower than that in hematological malignancies. Currently, the fastest clinical progress isKoji PharmaceuticalThe CT041 has taken the lead in entering Phase II. It is targeted at advanced gastric/esophageal junction adenocarcinoma that has failed at least two lines of treatment and advanced pancreatic cancer that has failed at least one line of treatment. The remaining clinical trials are still in the earlier stages of clinical research.
Figure 6: Breakdown of China's clinical CAR-T treatment pipeline by therapeutic area
Data source: 'CAR T-cell therapies in China: rapid evolution and a bright future', Frost & Sullivan analysis
Overcoming the problem of T cell exhaustion through technological innovation will undoubtedly further drive the development of cell therapies, especially the CAR-T therapy market.Professor Wang Haopeng's Research TeamA series of breakthrough achievements based on the base signalOn one hand, it provides an innovative solution to overcome the treatment barriers of solid tumors for CAR-T therapy, promising to benefit over 90% of solid tumor patients; on the other hand, it also provides CAR-T designers with a 'practical guide manual' for CAR structure design, which is of great help in accelerating the efficiency of CAR-T research and development and continuously optimizing it.
It is reported that Professor Wang Peng, the corresponding author of the article, has long focused on research in T cell immunotherapy. He graduated from Kuang Yaming College at Nanjing University with a bachelor's degree; his doctoral supervisor, Professor Vignali, is a leading figure in the field of regulatory T cells (TREG), currently serving as the director of the Tumor Immunology Center at the University of Pittsburgh; his postdoctoral supervisor is Academician Arthur Weiss from the School of Medicine at the University of California, San Francisco. Professor Weiss is one of the founders of CAR-T cell therapy. In 2019,weissProfessor Wang Hepeng, along with three other scientists, was awarded the William Coley Prize by the American Cancer Institute for his invention of chimeric antigen receptor CAR. In July 2015, Professor Wang Hepeng joined the School of Life Sciences and Technology at ShanghaiTech University through the National High-Level Talent Program to form a research team. In 2020, Professor Wang Hepeng's research team successfully developed a recyclable CAR-T design with independent intellectual property rights. This design increased the sustained ability of CAR-T by more than 100 times and effectively inhibited tumor recurrence. This breakthrough study was published as a cover article in the top journal in the field of immunology, "Immunity." In the same year, the "recyclable CAR-T" design was licensed to a leading domestic CAR-T treatment company in the form of technology transfer, completing the transformation of scientific and technological achievements with a milestone payment of up to 45 million yuan. The research described in this article received funding support from the National Key Research and Development Program, the ShanghaiTech University "Double First-Class" Venture Capital Fund, the ShanghaiTech University Center for Biomolecular and Precision Medicine Sciences, the Shanghai Local Universities Capacity Building Project, the National Natural Science Foundation of China, and the Shanghai Natural Science Foundation of China.
