Discover solutions for CAR detection 

Monitoring CAR T manufacturing and analyzing CAR T persistence and expansion during CAR T cell therapy requires precise and reproducible detection of the chimeric antigen receptor (CAR) by flow cytometry. Discover our solutions for CAR detection designed with REAfinity™ Recombinant Technology and choose a highly specific and sensitive antigen-based or antibody- based (idiotype) CAR detection reagent for CD19 CAR, BCMA CAR, CD22 CAR, or CD33 CAR evaluation.

 

Basic principle of CAR detection reagents

Mechanism of tumor cell recognition by CAR engineered cells.

CAR detection reagents for engineered cell therapies

Engineered cell therapies are a new and innovative treatment option against cancer. They are based on the principle that the patient’s immune cells (e.g., T or NK cells) can be genetically engineered to carry a chimeric antigen receptor (CAR) on their surface, which binds a specific antigen expressed on tumor cells (e.g., CD19 antigen). Upon re-infusion into the patient, the engineered immune cells are directed to the tumor cells expressing the target antigen. Recognition of the target antigen leads to activation of the engineered cell resulting in proliferation, cytokine secretion, and tumor cell lysis. CAR detection reagents allow efficient recognition of CAR engineered cells in vitro.

Detection of CAR-engineered cells using antigen-based CAR detection reagents.

Antigen-based CAR detection reagents

Our antigen-based CAR detection reagents consist of the CAR target protein (e.g., CD19) fused with a biotin-labeled mutated Fc backbone, eliminating background signal. The target protein is bound by the CAR receptor and can be readily identified using fluorochrome-labeled biotin antibodies (Clone: REA746). This approach multiplies the signal and increases assay sensitivity compared to fluorescent-labeled proteins. 

Features of antigen-based CAR detection reagents:

  • Broad specificity
  • Highly sensitive
  • Show functional CAR folding
Detection of CAR engineered cells using antibody-based (idiotype) CAR detection reagents.

Antibody-based (idiotype) CAR detection reagents

CAR receptor specificity is typically derived from the single-chain variable fragment (scFv) region of a monoclonal antibody, e.g., CD19-specific mouse monoclonal antibody FMC63. Idiotype antibodies recognize the scFv of a specific CAR receptor, meaning the variable part which includes the antigen-binding domain. Biotin and fluorochrome-labeled idiotype antibodies can be used for sensitive CAR detection.

Features of idiotype CAR detection reagents:

  • Narrow specificity
  • One-step staining possible
  • Compatible applications: flow cytometry, imaging (MICS, IF, IHC), and mass cytometry
REAfinity Recombinant Antibody model. 

REAfinity Recombinant Antibodies – the underlying technology 

A reliable CAR detection reagent is essential to achieve precise and reproducible results in flow cytometric analysis of CAR engineered cells. Labeled target proteins and anti-idiotype antibodies are widely considered to be the favorable options for CAR detection. Nevertheless, conventional fluorescent-labeled proteins and hybridoma-derived antibodies often lack the sensitivity and staining intensity required for optimal CAR+ population identification. 

To overcome this limitation, Miltenyi Biotec provides CAR detection reagents designed with REAfinity Recombinant Technology. The benefits of Miltenyi Biotec’s CAR detection reagents are:

  • No background signal due to unique mutated Fc region
  • Optimal staining results due to biotin/antibody signal amplification
  • Superior lot-to-lot consistency and purity
  • Liquid ready-to-use formulation for streamlined workflow

Moreover, all our CAR detection reagents undergo extensive testing on CAR engineered cells during development, instead of using antibody-conjugated beads, to optimize their performance in the relevant assays.

Comparison of the specificity of different commercial CAR detection reagents.

No unspecific binding

Our unique REAfinity Recombinant Technology minimizes unspecific signal in CAR analysis by flow cytometry. The significant lower background staining compared to other vendors makes Miltenyi Biotec´s CAR detection reagents the ideal tool for reliable distinction of CAR-expressing and negative cells during cell manufacturing and patient monitoring research.

“We are using CD19 CAR detection reagent for our in vitro and in vivo R&D experiments. They are easy, fast to use, and show far better affinity than other CD19-based detection reagents. I also want to thank the scientific and technical support for their useful help and availability.” 
Rachel Pacherie, PhD
R&D Director IXAKA

Available options of CAR detection reagents

Compatibility for complete workflow solution

Our CAR detection reagents are compatible with our StainExpress™ Dry Antibody Cocktail “CAR T Transduction” for standardized and accelerated CAR T cell identification by flow cytometry. Additionally, they are tested with our CAR T Cell Express mode package and panels, a unique add-on for the MACSQuantify™ Software, developed to automate data acquisition and analysis and minimize operator variation and hands-on time. 

 

Applications of CAR detection reagents

Example plots of CAR T cells stained with various CAR detection reagents.

In-process and quality control (IPC/QC) during manufacturing

Our CAR Detection reagents enable a clear distinction of CAR+ and CAR- cells, which makes them the ideal tool for the assessment of transduction efficiency and viability of CAR T cells during manufacturing. 

Example plots of CAR NK cells stained with various CAR detection reagents.

Our CAR detection reagents also allow CAR identification on other cell types of interest. This includes but is not limited to CAR NK cells

CAR monitoring in blood samples. 

Follow-up of patients undergoing CAR T cell therapy 

To model the analysis of CAR T cell persistence after adoptive transfer, CAR T cells were spiked into whole blood. Our CAR detection reagents enable a clear distinction of CAR+ and CAR- cells, even with very low CAR T cell numbers during post-infusion monitoring.  

CAR Imaging using the MACSima™ Platform.

Highly specific imaging of CAR T cells

CD19 CAR FMC63 Idiotype Antibody-PE (REA1297) is a highly specific and sensitive tool to detect FMC63-derived CD19 CAR, not only in flow cytometry, but also in a range of other applications, e.g., immunostainings for microscopy.

“Working with both antigen-based and anti-idiotype CAR detection reagents, I value the high quality of the REAfinity Technology. Consistent performance over time, and the elimination of background, allows sensitive detection of CAR+ cells in blood samples as well as tissues by immunofluorescence and ultra-high content imaging. The ready-to-use format and reduced pipetting steps support an efficient workflow.”
Dr. med. Christian Seitz
Universitätsklinikum Tübingen
 

All CAR detection reagents

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