Domestic Launch | New Tool for Detecting CAR Positive Rates—Easily Achieves Positive Detection with a Single Primary Antibody
Release Date:
2023-04-03 13:11
Introduction:
Still worrying about In CAR-T research, both in vitro and in vivo assays for determining CAR positivity exhibit substantial data bias, with non-specific staining observed even in negative controls. Are you plagued by such problems? Are you still overwhelmed and bewildered by Different CAR structures, direct and indirect antibodies, and antigen proteins In, because of various Protein testing is expensive. Are you hesitating again?
Even when detecting CAR-T/NK cells in the patient’s body, due to the complex Serum conditions, antigen shedding, and antigen blocking can lead to false-positive or false-negative results. As a result, is it unclear whether CAR cells have expanded, which could hinder clinicians from making accurate judgments and responses, and even lead to self-blame for misleading the experimental process?
Today, we are introducing a new tool for detecting CAR positivity rates. Shanghai Hycells Biotechnology Co., Ltd. is the first in China to launch the groundbreaking Anti-(G4S)n (B02H1) mAb CAR-positive-rate detection reagent, revolutionizing conventional testing methods and making it easy for you to detect CAR positivity with a single antibody.
Part 01
Cell therapy development holds limitless possibilities.
As one of the major breakthroughs in cancer therapy in recent years, CAR-T cell therapy has shone brightly across multiple cancer treatment domains. In addition to the robust development of CAR-T, discussions are now shifting toward the more imaginative “CAR-X” class of novel cell therapies, including CAR-macrophage (CAR-M) therapy, CAR-natural killer (NK) therapy, CAR-Treg cell therapy, and CAR γδ-T cell therapy, among others.
Part 02
Existing detection methods
CARs are engineered receptors designed to modulate immune cell responses and redirect them toward tumor-associated antigens of the patient’s own origin. This receptor is a chimeric protein that can be broken down into four major components: Extracellular antigen-binding domain (e.g., single-chain variable fragment or scFv), spacer or hinge domain, transmembrane domain, and at least one intracellular signaling domain The successful integration of these components enables CAR-immune cells to be redirected to recognize specific antigens and kill tumor cells.
During the discovery and preclinical research phases, when engineering CAR-X cells, scientists must verify cellular functionality by monitoring the surface expression of the CAR. Most existing detection methods rely on reagents that bind to the extracellular domain of CARs and exploit specific sequences within the scFv, the variable heavy domain of the hinge region, or the variable light domain. Depending on the category of CAR-X assay reagents used, the available solutions often lack specificity, are difficult to integrate into flow cytometry panels, or are limited to detecting only a single CAR.
Current Analysis of the Pros and Cons of CAR Testing Methods
Currently, common methods for detecting CAR expression include using Protein L, target antigens, tag-specific antibodies, anti-idiotype antibodies (ADA), and co-expression of GFP or tEGFR; however, each of these approaches has its own advantages and limitations, and some are not suitable for practical applications.
Protein L: Due to its low cost and broad applicability, it is currently a commonly used reagent for CAR detection and is highly favored by beginners. However, it is important to note that, despite Protein L’s wide binding spectrum, it is not a universal antibody-binding protein. Protein L specifically binds antibodies containing κ light chains, but only human κ I, III, and IV subtypes (it does not bind VκII subtype or λ light-chain antibodies) and the mouse κ I light-chain subtype; it is ineffective against other subtypes. Moreover, the affinity of antibodies for each subtype varies. Therefore, using antibodies of different subclasses in CAR constructs can lead to bias in the positive detection rate.
Target antigen protein: This is also a detection method that offers a wide range of options. It can directly bind to the CAR’s single-chain variable fragment (ScFv) or to the antigen ligand, thereby enabling direct assessment of the CAR-expressing cells’ antigen-binding capacity. However, each target antigen requires a specific detection antigen, making this approach less suitable for multi-transmembrane proteins and proteins that are difficult to express. The activity and concentration of the antigen used directly affect the accuracy of the assay; therefore, it is recommended to compare reagents from multiple brands and test a range of concentrations to optimize the assay conditions. It is worth noting that when detecting CAR-expressing cells in patients, Due to the process of phagocytosis and the blocking effect of free antigens on CAR-scFv , rendering the antigen protein highly unsuitable for in vivo CAR cell detection.
Integrated expression: It is common for many beginners and researchers to incorporate expression-tag proteins (such as His, Flag, or c-myc) and co-expression reporter proteins (such as GFP, tEGFR, or CD34) into the CAR construct to facilitate detection. It is important to note, however, that the expression of additional proteins can potentially interfere with the folding and overall expression of the CAR molecule. Molecules with 2A linkers and dual promoters that enable indirect expression often fail to accurately reflect the actual expression of CAR molecules, leading to bias in the reported CAR-positive rate.
Anti-drug antibodies (ADA): By screening anti-idiotype antibodies (ADA) that specifically recognize scFv, the CAR-positive rate can be identified and detected, demonstrating clear advantages in both in vivo and in vitro assays and making ADA a relatively ideal tool for CAR detection. However, obtaining such antibodies is challenging. Each scFv requires targeted screening, resulting in limited general applicability. Currently, only the anti-FMC63 antibody is commercially available.
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Detection |
For example |
Advantages |
Disadvantages |
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Protein L |
Protein L |
Low price, wide applicability |
Certain antibody subtypes are not well suited, resulting in a low detection rate. |
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