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CAR-T Therapy Research Tools: Discover Efficacious Therapies Faster

Chimeric antigen receptor (CAR) T therapy is a revolutionary type of adoptive cell immunotherapy, which employs genetic engineering to redirect the specificity of cytotoxic T lymphocytes to a tumor antigen(s) expressed on the surface of tumor cells. CAR-T therapy has shown remarkable success for the treatment of hematologic malignancies and could potentially be leveraged to safely treat solid tumors and diseases of senescence, such as fibrotic liver disease and diabetes.1-3 CAR-T immunotherapies have been described as living therapies that may be efficacious decades after a single infusion.

The CAR-T development workflow is a multi-step process that ensures the development of safe, effective treatments. Cell Signaling Technology® (CST) offers product solutions to aid in preclinical research for target identification and selection, CAR-T cell characterization, and interrogation of patient response to therapy.

The CAR-T Development Workflow

 

Step 1 Step 2 Step 3 Step 4

Step 1:

Tumor Antigen Discovery and Validation

Step 2:

scFv Screening and Optimization

Step 3:

CAR Transgene Construction

Step 4:

CAR Transgene Delivery

Step 5 Step 6 Step 7  

Step 5:

Assays to
Characterize CAR-T Cells

Step 6:

Preclinical In Vivo Assays

Step 7:

Patient Selection and Monitoring

 

 

The ultimate goal of tumor antigen discovery and validation is to find tumor surface antigens that are expressed on the majority of tumor cells; display an expression pattern that minimizes risk of on-target, off-tumor toxicity; and are expressed stably so target loss will not render the treatment ineffective. Identifying a new tumor antigen is challenging enough without having to worry about whether the antibodies you’re using are specific or are of sufficient sensitivity to detect potential tumor antigens. You need to use reliable antibodies that you can trust to detect the tumor target protein accurately every time. CST antibodies undergo rigorous in-house application-specific validation, giving you confidence that your detection reagents are specific, reliable, and high-affinity.

Start with highly specific antibodies to detect known tumor antigens in your sample:

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.

IHC-validated antibodies or flow cytometry-validated antibody conjugates:

Flow cytometric analysis of HeLa cells (blue) and KARPAS-299 cells (green) using TNFRSF8/CD30 (E7E4D) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (dashed lines). KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.

Immunohistochemical analysis of paraffin-embedded human glioblastoma multiforme using IL-13RA2/CD213a2 (E7U7B) Rabbit mAb #85677.

After engineering your CAR-T cells you'll need to verify that the engineered cells are functional by monitoring CAR surface expression, cytotoxicity, and proliferation. The following validated, ready-to-use kits and reagents can help you characterize the functionality of your engineered CAR-T cells:

 

Functional Assays

Proliferation Assays

The ability of a CAR-T cell to proliferate in response to engaging a tumor antigen is an important readout of CAR-T cell function.4 Evaluate your CAR-T cells' ability to propagate with a CST proliferation assay. The Cell Proliferation Tracer Kits use flow cytometry to monitor a fluorescent tracer dye that gets diluted with each cell division.

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.

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, Blue 520) #53452 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 Assays

Cytolytic assays are routinely used to determine immune effector cell function. Interrogate cell-mediated cytotoxicity with a sensitive kit that does not employ radioisotopes.

Determination of Optimum Cell Number for LDH Cytotoxicity Assay: 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 protocol.

Phenotyping & Function Marker Antibodies

Explore the role of the immune system in oncology using immunophenotyping antibodies. Applications like immunohistochemistry (IHC) and flow cytometry allow for multiplex monitoring of cell populations and both require stringent antibody validation principles.

Phenotype T cell subsets to understand which will be most efficacious for engineering or phenotype the CAR-T cells to understand what effect the CAR has on phenotype and function.

Explore a comprehensive catalog of markers for immune phenotype and function optimized for tissue or liquid biopsy sample analysis.

Flow cytometric analysis of human peripheral blood mononuclear cells, untreated (left) or treated with cross-linked anti-CD3 plus anti-CD28 (10 μg/ml each, 15 min; right), using Phospho-SLP-76 (Ser376) (E3G9U) XP® Rabbit mAb (PE Conjugate) #76143 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).

Confirming CAR Expression

Along with validated antibodies to detect your CAR target protein of interest, CST also offers reagents to verify CAR surface expression. Monitor CAR surface expression with Protein L if you've engineered a scFv containing kappa light chain sequences.

Protein L (PE Conjugate) #58036

Analysis of CAR Signaling

Monitor the activation state of multiple proteins related to T-cell behavior to optimize CAR functionality. CST offers a diverse array of antibodies against targets in relevant signaling pathways, enabling you to evaluate downstream outcomes and better understand CAR-T cell behavior. Check out the CAR Signaling Network pathway and other pathways by research area.

To globally interrogate the phospho-proteome that is engaged by CARs, CST offers Proteomics Analytical Services for qualitative and quantitative proteomic profiling. Our experts are ready to partner with you from project planning to the delivery of a comprehensive data package. Explore our Proteomics Analytical Services offerings.

Customized CST Reagent to Fit Your Needs

Custom Conjugation Services

Don’t see the flow cytometry antibody conjugate you need? Not a problem! Take advantage of our expertise in conjugating antibodies. We’ll conjugate the antibody you need to your fluorochrome of choice so you have the flexibility to design the assay you want. Custom antibody conjugations are validated, optimized, and tested for stability using the same standards we apply to all CST antibodies.

Order your custom antibody conjugate here!

Custom Antibody Formulations

Carrier-free and customized formulations of your favorite antibody are available so you can perform CAR-T characterization assays free of BSA, glycerol, or azide. Your antibody of choice is offered in PBS for optimal use in applications such as:

  • ELISA-like assays like AlphaLISA®, HTRF®, and MSD assays
  • Metal labeling for CyTOF® mass cytometry, Imaging Mass Cytometry (IMC), and MIBI
  • Oligonucleotide labeling
  • Biotin conjugations
  • Fluorochromes for flow and immunofluorescence
  • In vivo and functional assays

Explore our list of ready-to-use, carrier-free antibodies.

Or request a custom formulation of CST catalog antibodies.

In order to determine patient eligibility for a specific CAR-T therapy, confirmation of tumor antigen expression is often one of the criteria for clinical trial selection. Furthermore, monitoring the expression of the target antigen throughout the course of a treatment is a key parameter in assessing the efficacy of the treatment.

CST offers these and many more highly validated antibodies to help detect tumor antigens in a reproducible manner:

  1. CAR T-cell Therapy: A New Era for Cancer Treatment. Mohanty et al. Oncol Rep. 2019, 42(6):2183-2195.
  2. A Fresh Approach to Targeting Aging Cells: CAR-T Cells Enhance Senolytic Specificity. Li JH and Chen YY. Cell Stem Cell. 2020, 27(2): 192–194.
  3. Regulatory T Cells Engineered with a Novel Insulin-Specific Chimeric Antigen Receptor as a Candidate Immunotherapy for Type 1 Diabetes. Tenspolde M, et al. J Autoimmun. 2019, 103:102289
  4. In vitro Tumor Cell Rechallenge for Predictive Evaluation of Chimeric Antigen T Cell Antitumor Function. Wang D, et al. J Vis Exp. 2019, (144):10.3791/59275.

For Research Use Only. Not For Any Diagnostic of Therapeutic Use.
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