Research Round-Up: CST^® Anti-CAR Linker Antibodies in the Literature

EphA3-Targeted CAR T Cells in Glioma & Generate Curative Memory T Cell Responses

Lertsumitkul 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.

Synthetic Immune Checkpoint Engagers Protect HLA-deficient iPSCs and Derivatives from Innate Immune Cell Cytotoxicity

Gravina 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.

Use Cases:

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. 

Detect: Biotinylated anti-G4S-linker F(ab’)₂ fragments were used in a flow cytometry assay to assess the feasibility of engineering immune evasion using B2M-knockout iPSCs expressing the synthetic transgenes (SIRPa-E and CD64t).

Evaluation of Anti-CAR Linker mAbs for CAR T Monitoring after BiTEs/bsAbs and CAR T-Cell Pretreatment

Grahnert 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.

CD8α Structural Domains Enhance GUCY2C CAR-T Cell Efficacy

Baybutt 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.

Detect: The G4S Linker (E7O2V) Rabbit mAb (Alexa Fluor^® 488 Conjugate) #50515 was used in two different flow cytometry experiments to label and identify CAR+ T-cells:

• Intracellular flow cytometry experiment where cells were permeabilized to measure cytokine production
  
• Cell surface marker analysis for T-cell phenotyping to determine memory status and detect exhaustion markers