As chimeric antigen receptor (CAR) cell therapies for the treatment of solid tumors, hematologic malignancies, and autoimmune diseases continue to evolve, accurate detection and monitoring of CAR-engineered cells is critical for experimental validation. However, detection reagents for monitoring the expression of single-chain variable fragment (scFv) CARs that are based on either a target antigen or an anti-idiotype can be time-consuming to develop and optimize. Additionally, they have utility only for detecting CARs targeting a specific surface antigen or a specific scFv.
CST anti-CAR linker recombinant monoclonal antibodies are a powerful tool that can identify and confirm the presence of an engineered CAR, regardless of its antigen specificity. Because most scFv-based CARs contain either a repeating G4S or Whitlow/218 linker sequence, highly specific anti-G4S and anti-Whitlow/218 antibodies can be used to monitor the surface expression of virtually any scFv-based CAR.
Anti-CAR linker recombinant monoclonal antibodies bind to the linker sequence (either G4S or Whitlow/218) between the variable heavy and variable light domains of scFv-based CARs.
How are scientists using the CST anti-CAR linker antibodies in their research? In this research roundup, we highlight recent publications that have utilized the CST anti-CAR linker antibodies to detect, analyze, quantitate, and purify CAR-engineered immune cells in a variety of experimental settings. To help you identify studies of interest to you, the following CAR characterization workflow framework is used to describe how CST anti-CAR linker antibodies were leveraged by the study authors:
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検出 Quickly and easily evaluate CAR and target antigen expression |
解析 Interrogate immune cell activation, proliferation, viability, & signaling |
定量 Measure the transduction efficiency of the CAR transgene |
精製 Enrich CAR+ cells using bead-based or FACS-based sorting with high specificity |
TRANSLATE Directly interrogate the infiltration of CAR-T cells into the TME |
From screening assays to monitor CAR-positive cells to labeling CAR-T cells for purification or activation, these studies provide valuable insights into the methodology and applications of linker-based antibody detection approaches.
Explore the full portfolio of CAR-engineered cell characterization solutions from CST:
Following are summaries of how the CST CAR-Engineered Cell Characterization solution has been used in recent peer-reviewed papers. Please note that this blog was published in March 2025; the anti-CAR linker antibodies have been available for less than two years.
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EphA3-Targeted CAR T Cells in Glioma & Generate Curative Memory T Cell ResponsesLertsumitkul et al. (JITC 2024) | DOI: 10.1136/jitc-2024-009486 This study describes the assessment of a second-generation CAR-T cell therapy targeting Ephrin type-A receptor 3 (EphA3), which the authors identified in high-grade gliomas, including glioblastoma and diffuse midline gliomas. With tumor-specific targeting and the ability to penetrate the blood-brain barrier, the EphA3-targeted CAR-T cell therapy demonstrated strong in vitro and in vivo efficacy, leading to tumor clearance in mouse models. The study also found that EphA3-targeted CAR-T cells generated long-term immune memory, preventing tumor recurrence upon rechallenge. Use Case: Quantitate: To determine T cell transduction efficiency, the G4S Linker (E7O2V) Rabbit mAb clone was employed to confirm the presence and expansion of EphA3-targeted CAR-T cells. |
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Linker-specific Monoclonal Antibodies Present a Simple & Reliable Detection Method for scFv-Based CAR NK cellsSchindler et al. (Molecular Therapy 2024) | DOI: 10.1016/j.omtm.2024.101328 CAR natural killer (NK) cells are being investigated in clinical trials for their potential advantages over CAR-T cell therapies, including their use as allogeneic cell therapies. This study evaluated the use of anti-CAR linker antibodies as a universal and sensitive tool for detecting CAR NK cells, including comparing them to other commonly used detection methods. The study demonstrates that linker-specific mAbs can detect different CAR NK cells in vitro, spiked in whole blood, and within patient-derived tumor spheroids with high specificity and sensitivity, providing an effective and almost universal alternative for scFv-based CAR detection. Additionally, the authors demonstrate that linker-specific antibodies can be used for functional testing and enrichment of CAR NK cells. Use Cases: Detect: To confirm CAR expression, the Whitlow/218 linker (E3U7Q) Rabbit mAb and G4S Linker (E7O2V) Rabbit mAb clones were used for flow cytometry-based detection of various CAR constructs. |
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Magnetic CAR T Cell Purification Using an Anti-G4S Linker AntibodyHarrer et al. (JIM 2024) | DOI: 10.1016/j.jim.2024.113667 This study introduced a novel CAR-T cell purification method using magnetic separation based on an anti-G4S linker antibody. The approach allowed for the enrichment of CAR-T cells to high purity levels without affecting their viability or functionality. The method was tested on CAR-T cells targeting CEA and Her2, as well as a dual-specific tandem CAR. Use Case: Purify: The G4S Linker (E7O2V) Rabbit mAb clone was used to label CAR-T cells before magnetic-activated cell sorting (MACS) using a one-step protocol (explore magnetic bead CAR enrichment assays from CST), demonstrating efficient purification without impairing cell viability. |
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Synthetic Immune Checkpoint Engagers Protect HLA-deficient iPSCs and Derivatives from Innate Immune Cell CytotoxicityGravina et al. (Cell Press 2023) | DOI: 10.1016/j.stem.2023.10.003 The authors of this study designed and tested a new class of synthetic cell surface molecules (engagers) that activate immune checkpoints in immune cells and inhibit cytotoxicity. Specifically, the authors designed and tested engagers targeting the immune checkpoints SIRPa, TIM3, and LILRB1, to prevent cytotoxic immune attacks from NK cells and macrophages. The approach aims to help protect allogeneic immune-oncology or regenerative cell therapeutics from rejection and improve their efficacy. Detect: The G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor® 647 Conjugate) #69782 was used to detect and quantify engager surface expression in a flow cytometry analysis that examined the expression dynamics on the engineered engagers before and after binding with their target molecules. |
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Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell PretreatmentGrahnert et al. (Biomedicines 2024) | DOI: 10.3390/biomedicines12081641 This study examined the use of anti-CAR-linker antibodies for monitoring CAR-T cell therapies in patients treated with either bispecific antibodies (bsAbs) or bispecific T cell engagers (BiTEs). Traditional antigen-based CAR detection methods can produce false positives when the same antigen is targeted by both the CAR-T cells and the bsAbs/BiTEs. The researchers analyzed specimens containing all of the BCMA- and CD19-targeting CAR-T cell products currently approved for clinical use to demonstrate that anti-CAR-linker mAbs offer a highly specific detection method that was unaffected by treatment with either BiTEs or bsAbs. However, the sensitivity varied across different clinically available CAR-T cell therapies. Use Case: Detect: CST anti-CAR linker antibodies were found to be highly sensitive and specific for the detection of CAR-T cells in blood samples treated with Idecel, Tisa-cel, Axi-cel, Brexu-cel, and Liso-cel, offering a reliable method to distinguish CAR-T cell staining from background staining caused by concurrent detection of immune cell engagers when using antigen-based detection methods. However, Cilta-cel (which contains a G4S-CAR linker) could not be detected by the anti-CAR linker mAb. |
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CD8α Structural Domains Enhance GUCY2C CAR-T Cell EfficacyBaybutt et al. (Cancer Biology & Therapy 2024) | DOI: 10.1080/15384047.2024.2398801 This study examined the hinge and transmembrane domains of engineered CAR cells to determine how different constructs might affect CAR performance in solid tumor immunotherapies. Specifically, the researchers compared the hinge and transmembrane domains derived from either the CD8ɑ or CD28 molecule in GUCY2C-targeted CAR designs for colorectal cancer. The researchers found that, while the structural domains did not contribute to differences in antigen-independent contexts, such as CAR expression, differentiation, and exhaustion phenotypes, the CD8ɑ structural domain CAR demonstrated a greater affinity for the target. Use Cases: Detect: The G4S Linker (E7O2V) Rabbit mAb (PE Conjugate) #38907 was used in a flow cytometry assay to detect and quantify CAR surface expression. Surface expression was also detected using Protein L, yielding equivalent results to those obtained with the linker antibody.
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