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CAR-Engineered Cell Characterization Solutions

The development of chimeric antigen receptor (CAR) -based cell therapies is revolutionizing the treatment of human cancers and autoimmune diseases. As this innovative therapeutic modality continues to improve and become more accessible, better reagents are improving our ability to detect, identify, and fully characterize CAR-expressing cells. With a growing portfolio of innovative products, CST is helping to advance researchers' work in this field.

Characterize Your CAR-Engineered Cells Easier and Faster

One of the most significant challenges facing scientists developing and characterizing CAR-engineered cells is the need for robust, sensitive, and selective research reagents for the identification of CAR-engineered cells. If you plan to use an immunoassay platform in your research, developing your own anti-idiotype antibodies to identify CAR-engineered cells can be a long and resource-intensive process.

A Workflow Solution for Characterization of CAR-Engineered Cells

There are many stages in the complex process of characterizing CAR-engineered cells. CST focuses on one critical step in the process with a suite of purpose-built research tools for CAR cell characterization.

DETECT

ANALYZE

QUANTITATE

PURIFY
Step 1 Step 2 Step 2 Step 2

Evaluate CAR and target antigen expression

Interrogate immune cell activation, proliferation, viability, and signaling

Measure the density of CAR expression on the cell surface or the transduction efficiency of the CAR transgene

Enrich CAR+ cells using either bead-based or FACS-based sorting

To streamline your characterization assays, CST scientists developed recombinant monoclonal antibodies to the ubiquitous peptide linker sequences of scFv-based CARs. By targeting these ubiquitous linker sequences, you can significantly reduce time, effort, and cost by eliminating the need for anti-idiotype antibodies to every CAR variant you’re testing. With high specificity to either the G4S or Whitlow/218 linkers, our CAR linker antibodies can detect the expression of scFv-based CARs, regardless of scFv specificity.

Video: Detecting CAR Expression with Versatile, Innovative Antibodies (2:04)

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CAR Detection

Flow cytometric analysis of live Jurkat cells (blue, negative) or Jurkat cells engineered to stably express an scFv-based Anti-CD20 CAR containing a G4S linker (green, positive) using G4S Linker (E7O2V) Rabbit mAb (PE Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines). Cell line was provided by the Lohmueller Lab, University of Pittsburgh.

Controls

Whitlow/218 Linker Posibeads™ controls were added to a mixed population of live cells containing wild-type Jurkat cells and Jurkat cells engineered to express an scFv-based Anti-CD19 CAR containing a Whitlow/218 linker. The cells and Posibeads controls were then immunostained with either Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (center) or Whitlow/218 Linker (E3U7Q) Rabbit mAb (Alexa Fluor® 488 Conjugate) #55809 (right). Cells and beads were differentiated by forward scatter (FSC) and side scatter (SSC) as shown (left), and fluorescence data (FL1) are displayed for cells (blue) and Posibeads controls (green) for both immunostaining conditions. CAR cell line was provided by the Lohmueller Lab, University of Pittsburgh.

Target Antigen Detection

Immunohistochemical analysis of paraffin-embedded human lymphoma using CD19 (Intracellular Domain) (D4V4B) XP® Rabbit mAb #90176.

Immunohistochemical analysis of paraffin-embedded human normal colon using TNFRSF17/BCMA (E6D7B) Rabbit mAb #88183.

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using Claudin-6 (E7U2O) XP® Rabbit mAb #18932 performed on the Leica BOND Rx.

T-Cell Activation

Flow cytometric analysis of mouse splenocytes, untreated (left column) or treated with Rapid-Act T-Cell Activation Kit #86772 (Mouse, Anti-CD3/CD28) (15 min; right column), using Phospho-SLP-76 (Ser376) (E3G9U) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #47876 (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (bottom row), and co-stained with CD3 (17A2) Rat mAb (APC Conjugate) #24265.

Cell proliferation

Cell division tracking in live Jurkat cells over the course of 4 days (d0-d3). Cells were labeled with the Cell Proliferation Tracer Kit (Fluorometric, Violet 450) #48444 on day 0, and analyzed by flow cytometry each day. Each successively dimmer peak represents one cell division. Unstained cells are represented by the unshaded peak.

Cytotoxicity

HeLa cells were seeded into a 96-well plate at varying densities using media containing 10% FBS. After overnight incubation, the cells were replaced with serum-free media and then treated with either Assay Buffer (Spontaneous LDH Release) or 10% Triton X-100 solution (Maximum LDH Release). After treatment, the medium was removed and placed into a new 96-well plate. The amount of LDH released into the medium was determined using the LDH Cytotoxicity Assay Kit #37291 protocol.

Immunophenotyping by Flow Cytometry

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left column) or treated with cross-linked anti-CD3 plus anti-CD28 (10 μg/ml each, 15 min; right column), using Phospho-SLP-76 (Ser376) (E3G9U) XP® Rabbit mAb (PE Conjugate) #76143 (top row) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (bottom row), and co-stained with CD3 (UCHT1) Mouse mAb (FITC Conjugate) #86774.

Flow cytometric analysis of live human peripheral blood mononuclear cells using CD3 (UCHT1) Mouse mAb (violetFluor 450 Conjugate) #61347 (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (violetFluor 450 Conjugate) #40282 (dashed line).

Downstream Signaling Readouts for CAR T-Cell Activation
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Monitor CAR Transduction Efficiency

Flow cytometric analysis of live human peripheral blood mononuclear cells using CD4 (RPA-T4) Mouse mAb (FITC Conjugate) (solid line) compared to concentration-matched Mouse (MOPC-21) mAb IgG1 Isotype Control (FITC Conjugate) #97146 (dashed line).

Bead-Based Sorting of CAR+ Cells

Flow cytometric analysis of live cells in the input (left) and post-enrichment (right) for enrichment performed using biotinylated G4S Linker (E7O2V) Rabbit mAb. Input consists of CD4+/CD8+ human T cells containing a mixture of non-transduced cells and cells transduced with an scFv-based Anti-CD20 (Leu16) CAR containing a G4S linker. The post-enrichment sample shows a nearly pure population of cells expressing the CAR on the cell surface. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody to detect the biotinylated antibody.

FACS-based Sorting of CAR+ Cells

Flow cytometric analysis of live pan-CD3+ T cells isolated from human PBMCs and engineered to express an scFv-based Anti-CD19 CAR containing a Whitlow/218 linker, using Whitlow/218 Linker (E3U7Q) Rabbit mAb (PE Conjugate) #62405 (right) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (left). Tag Blue fluorescent protein (TagBFP) is co-expressed with the CAR. Data courtesy of Michael Kvorjak, Lohmueller lab (University of Pittsburgh).

Histology Tools for Translational Research

One of the most important questions scientists want to answer when challenging solid tumors with CAR-engineered cells is whether their therapeutic cells entered the tumor microenvironment. The most common methods include using either custom made anti-idiotype antibodies or RNA ISH, both of which have significant drawbacks. Direct interrogation of CAR expression in solid tumors using commercially available antibodies is now possible.

Study CAR Expression in Tissue

Immunohistochemical analysis of Raji B-Cell lymphoma xenograft in NSG mouse spleen transfused with primary human anti-CD19 CAR-T cells (left) or PBS control (right) using Whitlow/218 Linker (F2G3S) Rabbit mAb #47414.

Interrogate the Tumor Microenvironment

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using FAP (F1A4G) Rabbit mAb #52818.

CAR-Engineered Cell Characterization Resources

Explore a growing library of Webinars, Scientific Posters, Application Notes, and other resources focused on CAR cell characterization.

Webinars and Videos
Novel Antibodies for the Characterization of CAR T-Cells (31:47)

Learn how CAR linker antibodies can interrogate the expression of an entire panel of CARs.

Analytical Tools to Evaluate CAR T-Cell Signaling & Activation (45:23)

Learn about a streamlined workflow for generation of CD19 CAR-T cells using Bio-Techne analytical instrumentation and anti-CAR linker antibodies from CST.

Detecting CAR Expression with Versatile, Innovative Antibodies (2:04)

Innovative anti-linker monoclonal antibodies from CST eliminate the need to develop unique detection reagents for every new chimeric antigen receptor (CAR) molecule developed, speeding detection in flow cytometry panels.

 

Novel Antibodies for the Characterization of CAR T-Cells

 

Analytical Tools to Evaluate CAR T-Cell Signaling & Activation

Frequently Asked Questions

Does G4S Linker (E7O2V) Rabbit mAb detect scFv-based CARs with a single G4S repeat?

No. Two or more repeats of the G4S sequence are needed for optimal binding of E7O2V to scFv-based CARs.

Is the Kd for G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb known?

Yes. The Kd for G4S Linker (E7O2V) Rabbit mAb is ~1–2 nM and ~2.5 nM for Whitlow/218 Linker (E3U7Q) Rabbit mAb.

Have you tested whether the G4S Linker (E7O2V) Rabbit mAb and the Whitlow/218 Linker (E3U7Q) Rabbit mAb will bind to scFvs with varying Ag specificity?

Yes. We have verified G4S Linker (E7O2V) Rabbit mAb binding to 4 different scFv's and Whitlow/218 linker binding to 4 different scFvs.

Have you tested whether G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb will detect CAR-engineered cells in FFPE tissue?

Yes. Unfortunately, both G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb are unsuitable for detection of CAR-engineered cells in FFPE tissue. However, we introduced a Whitlow/218 Linker (F2G3S) Rabbit mAb that has been approved for IHC in FFPE.

Have you tested whether G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb will detect CARs in cell extracts using western blotting?

Yes. Unfortunately, both G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb are unsuitable for detection of CAR protein in cell extracts using conventional immunoblotting techniques. However, the Whitlow/218 Linker (E3U7Q) Rabbit mAb has been validated for use on the Jess Simple Western platform, and the Whitlow/218 Linker (F2G3S) Rabbit mAb has been validated for western blotting.

Have you tested whether G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb will detect CARs in either fixed cells or fixed and permeabilized cells using flow cytometry?

Yes. Both G4S Linker (E7O2V) Rabbit mAb and Whitlow/218 Linker (E3U7Q) Rabbit mAb will detect surface expressed CARs in a flow cytometry assay using live cells. The use of either fixed cells or fixed & permeabilized cells prior to staining is not recommended.

Are you able to provide the identity of the peptide linkers used in FDA-approved CAR constructs?

Yes. Yescarta, Tecartus, Breyanzi and Abecma all contain a Whitlow linker. Kymriah contains a 3X-G4S linker and Carvykti contains a 1X-G4S linker.

Magnetic CAR T cell purification using an anti-G4S linker antibody
The commercially available G4S Linker (E7O2V) Rabbit mAb (PE Conjugate) #38907 from CST enabled development of this new method of purification of CAR T cells. Authors observed high-grade enrichment of CAR T cells in a one-step procedure without impacting cell viability, which suggests that the specificity and performance of the CST antibody enabled the success of the method.
Harrer DC, Li SS, Kaljanac M, et al. Magnetic CAR T cell purification using an anti-G4S linker antibody. J Immunol Methods. 2024;528:113667. doi:10.1016/j.jim.2024.113667

Linker-specific monoclonal antibodies present a simple and reliable detection method for scFv-based CAR NK cells
The paper demonstrates the utility of the CST Whitlow/218 and G4S linker-specific monoclonal antibodies in detecting and analyzing scFv-based CAR NK cells. These antibodies were used to reliably identify CAR NK cells through flow cytometry with high specificity and sensitivity across various experimental setups, including in vitro cultures, whole-blood samples, and tumor spheroids. Additionally, the study highlights the antibodies’ role in functional assays and the positive selection of CAR NK cells, showcasing their effectiveness as versatile tools in advancing CAR NK cell research and clinical applications.
Schindler K, Ruppel KE, Müller C, Koehl U, Fricke S, Schmiedel D. Linker-specific monoclonal antibodies present a simple and reliable detection method for scFv-based CAR NK cells. Mol Ther Methods Clin Dev. 2024;32(3):101328. 2024 Aug 22. doi:10.1016/j.omtm.2024.101328

Synthetic immune checkpoint engagers protect HLA-deficient iPSCs and derivatives from innate immune cell cytotoxicity
The article explores the engineering of synthetic immune checkpoint engagers to enhance the immune evasion of HLA-deficient induced pluripotent stem cells (iPSCs) and their derivatives. A key experiment in the study involves using CST biotinylated G4S Linker F(ab')2 fragments to evaluate surface expression and potential regulatory effects. The antibody fragments enable detailed flow cytometric analysis of expression levels and interactions, highlighting their utility in optimizing immune evasion strategies.
Gravina A, Tediashvili G, Zheng Y, et al. Synthetic immune checkpoint engagers protect HLA-deficient iPSCs and derivatives from innate immune cell cytotoxicity. Cell Stem Cell. 2023;30(11):1538-1548.e4. doi:10.1016/j.stem.2023.10.003

CAR Protein Detection Tools Features

G4S monoclonal antibodies
  • Detect surface-expressed scFv-based CARs with a linker that has two or more G4S repeats, as well as (G3S)2 (*does not detect scFv-based CARs with a single G4S sequence)
  • Can detect a broad range of scFv-based CARs, independent of the antigen specificity of the scFv
  • Work well in multiparametric flow panels

Whitlow/218 monoclonal antibodies
  • Detect surface-expressed scFv-based CARs with the Whitlow/218 linker sequence
  • Can detect a broad range of scFv-based CARs, independent of the antigen specificity of the scFv
  • Work well in multiparametric flow panels

Protein L
  • Binds to human and murine kappa light chain sequences found within scFvs, but with varying affinities
  • Kappa light chain binding with varying affinities
  • Does not bind to lambda light chain sequences
  • Challenging to incorporate into multiparametric flow panels

Epitope tag antibodies (Myc, FLAG, and HA)
  • Research-grade CARs may contain epitope tags
  • Epitope tag antibodies generally display high specificity
  • Epitope tags on a CAR may interfere with proper folding and surface expression
  • Epitope tags need to be removed from clinical-grade CAR constructs

Surrogate markers of CAR expression (GFP, TagBFP, LNGFR)
  • CAR constructs often co-express surrogate markers of CAR expression
  • Co-expression of surrogate markers may not always correlate with CAR protein expression

Anti-idiotype monoclonal antibodies
  • Highly sensitive and specific
  • Challenging and time-consuming to develop and validate
  • Anti-Idiotype monoclonal antibodies need to be generated for each unique scFv, whether they are targeting the same antigen or different antigen.

Recombinant CAR target antigen
  • Can be used to detect CAR surface expression and assess whether the scFv is capable of binding to its target antigen.
  • Recombinant target antigen needs to be generated for each CAR targeting a different antigen
  • Can be challenging to express and purify
  • Stability is inconsistent
  • Not ideal for detecting CAR surface expression when a CAR is already bound to its native target antigen

Resources