Fluorescent-Labeled Recombinant Proteins

Evaluating CAR Expression on CAR-T Cells using Fluorescent-labeled Proteins

Evaluating CAR Expression on CAR-T Cells using Fluorescent-labeled Proteins

The ability to evaluate the expression of a chimeric antigen receptor (CAR) following T cell transduction is a critical step in the production of CAR-T cells for cancer immunotherapy. R&D Systems fluorescent-labeled proteins are designed to simplify the detection of a specific chimeric antigen receptor (CAR) on CAR-T cells. Our fluorescent-labeled recombinant proteins are amine-labeled and rigorously tested to ensure consistent labeling of each lot. The proteins are manufactured with controlled procedures to ensure a consistent F/P ratio, and quality control tested by flow cytometry to evaluate equivalent staining.

Using fluorescent-labeled proteins, target cells expressing the corresponding CAR can be directly stained and detected by flow cytometry. This method of evaluating CAR expression is highly specific, reduces processing time, and eliminates background staining that may occur by indirect detection of the CAR using an epitope-tagged target antigen and a fluorophore-labeled secondary antibody.

    
Protein Species Source Tag Fluorescent Label Catalog # Bioactivity
BCMA Human NS0 Fc Atto 488 ATJ193 Binds anti-BCMA monoclonal antibody conjugated fluorescent beads
Atto 647N ATM193
CD19 Human CHO Fc Atto 488 ATJ9269 Binds anti-CD19 monoclonal antibody conjugated fluorescent beads
Atto 647N ATM9269

As directly detecting a CAR with a labeled target antigen is currently the method of choice for evaluating CAR expression, we are diligently working to expand our selection of fluorescent-labeled proteins. As an alternative method, we also offer Avi-tag biotinylated proteins, which can be detected using fluorochrome-labeled streptavidin. Additionally, if you are unable to find either the fluorescent-labeled or biotinylated protein that you need on our website, our Custom Protein Services team can work with you to create a customized protein solution to meet your specific research needs.

Utilizing Fluorescent-labeled Proteins to Detect CAR-T Cells

Graphic showing that a chimeric antigen receptor (CAR) on CAR-T cells can bind to either its target antigen expressed on a tumor cell or to a purified fluorescent-labeled protein, which allows the percentage of T cells expressing the CAR to be evaluated

Demonstration of the Utility of a Fluorescent-labeled Protein for Evaluating CAR Expression. (A) CAR-T cell therapy is based on the principle that T cells removed from a patient or donor can be genetically engineered to express a specific chimeric antigen receptor (CAR). Once these CAR-T cells are infused back into the patient, the CAR will bind to its specific target antigen on the surface of the patient's tumor cells, activating the T cells, and allowing them to attack and destroy the tumor cells. (B) The ability to evaluate CAR expression following T cell transduction is an important step in the production of CAR-T cells. T cells expressing the CAR can be directly stained using a fluorescent-labeled protein (target antigen) and the percentage of CAR-expressing cells can be detected by flow cytometry. (C) CD4+CD8+ T cells were transduced with a hCD19-CAR (left) or not transduced (right) and then cultured for 11 days.  Cells were stained with a PE-Cy7-CD4 and Recombinant Human CD19 Fc Chimera Atto 488 Protein (Catalog # ATJ9269), and detected by flow cytometry. 

Advantages of Fluorescent-labeled Proteins for Evaluating CAR Expression:

  • Target cells expressing a specific chimeric antigen receptor (CAR) can be directly stained with the appropriate fluorescent-labeled protein
  • Direct staining with a fluorescent-labeled protein reduces processing time and eliminates background staining that may occur by indirect detection of the CAR using an epitope-tagged target antigen and a fluorophore-labeled secondary antibody

Bioactivity and Lot-to-Lot Consistency Testing of R&D Systems Fluorescent-labeled Proteins

  • Fluorescent-labeled proteins are validated to bind to beads conjugated to the corresponding monoclonal antibody
  • Some fluorescent-labeled proteins are also tested to ensure that they stain the appropriate CAR-T cells by flow cytometry
  • Fluorescent-labeled proteins display lot-to-lot consistency and retain the same high level of activity as the unlabeled Fc-tagged recombinant protein

Flow cytometry data showing staining of CD19-CAR-T cells by Recombinant Human CD19 Fc Chimera Atto 488, with CD4+CD8+ T cells that were not transduced with a hCD19-CAR serving as a negative control

Detection of Anti-Human BCMA-conjugated Fluorescent Beads with Recombinant Human BCMA/TNFRSF17 Fc Chimera Atto 488 Protein. Fluorescent beads conjugated to anti-Human BCMA Monoclonal Antibody were stained with (A) Recombinant Human BCMA/TNFRSF17 Fc Chimera Atto 488 Protein (Catalog # ATJ193) or (B) unstained, and detected by flow cytometry.

Flow cytometry data showing staining of anti-BCMA-conjugated beads with Recombinant Human BCMA/TNFRSF17 Fc Chimera Atto 488 Protein, with unstained anti-BCMA-conjugated beads serving as a negative control.

Detection of CD19-CAR-T Cells with Recombinant Human CD19 Fc Chimera Atto 647N Protein. CD4+CD8+ T cells were either (A) transduced with a hCD19-CAR or (B) not transduced, and then cultured for 11 days. Cells were stained with a PE-Cy7-CD4 and Recombinant Human CD19 Fc Chimera Atto 647N Protein (Catalog # ATM9269), and detected by flow cytometry. 

Bioactivity assay data showing binding of different concentrations of three different lots of Recombinant Human BCMA Fc Chimera Atto 647N or the unlabeled Recombinant Human BCMA Fc Chimera protein to Recombinant Human APRIL. The data demonstrates consistent bioactivity between the fluorescent-labeled and unlabeled proteins and lot-to-lot consistency in the bioactivity of the different lots of the fluorescent-labeled protein.

Bioactivity and Lot-to-Lot Consistency Testing of Recombinant Human BCMA Fc Chimera Atto 647N Protein. Recombinant Human APRIL/TNFSF13 (Catalog # 5860-AP) was immobilized at 0.1 ug/mL, 100 ul/well, and the indicated concentrations of three independent lots of Recombinant Human BCMA Fc Chimera Atto 647N (Catalog # ATM193; red, green, orange lines) or unlabeled Recombinant Human BCMA Fc Chimera (Catalog # 193-BC; blue line) were added. The data demonstrates consistent bioactivity between the fluorescent-labeled and unlabeled proteins and lot-to-lot consistency in the bioactivity of the three different lots of the fluorescent-labeled protein.

What Are CAR-T Cell Therapies?

CAR-T cell therapies are being increasingly investigated and used for treating hematologic cancers, including acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), lymphoma, and multiple myeloma (MM).1 This type of cell therapy utilizes T cells removed from a patient or a donor and genetically engineers the cells to express a chimeric antigen receptor (CAR). The CAR is a synthetic T cell receptor consisting of an extracellular antibody-derived single chain variable fragment (scFv) that binds to a specific target antigen, linked to one or more intracellular signaling sequences that promote T cell activation following scFv binding. Once the CAR-modified T cells are expanded, they are then infused back into the patient, where they bind to their specific antigen on the surface of the patient’s tumor cells and become activated, allowing them to attack and destroy the tumor cells. The ability to evaluate the expression of the CAR following retroviral or lentiviral transduction of the patient’s T cells is an important step for understanding the dose of CAR+ T cells that is being transferred back into the patient. Proteins labeled with a fluorescent dye allow target cells expressing the corresponding CAR to be directly stained and detected by flow cytometry.

CD19 and BCMA Are Commonly Targeted Antigens for CAR-T Cell Therapy

CAR-T cells expressing CARs specific for different surface molecules, including CD19 and BCMA, have been generated and are being evaluated as anti-cancer therapies. CD19 is an antigen that is widely expressed on B cell-derived cancers and is one of the most popular targets for CAR-T cell therapies.1 Two anti-CD19-CAR-T cell therapies have been approved by the U.S. Food and Drug Administration (FDA). Tisagenlecleucel (KYMRIAH) was approved in August 2017 for treating relapsed or refractory B cell precursor acute lymphoblastic leukemia (ALL), and axicabtagene ciloleucel (Yescarta) was approved in October 2017 for treating adult patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL). Although remarkable clinical remission has been observed using anti-CD19 CAR-T cells, relapses have also occurred due to either the escape and proliferation of CD19- tumor cells, or factors that may contribute to the loss of CAR-T cells or CAR-T cell function, including a loss of CAR-T cell persistence, activation-induced cell death or senescence, or an immunosuppressive tumor microenvironment.2 Dual targeting strategies are being pursued to try to overcome these obstacles.

The second most-targeted tumor antigen for CAR-T cell therapy is B-cell maturation antigen (BCMA).1 BCMA is involved in regulating B cell maturation and differentiation into plasma cells and is typically expressed at higher levels on malignant plasma cells than on normal plasma cells.3, 4 Anti-BCMA CAR-T cell therapy is currently being pursued for the treatment of multiple myeloma and has thus far shown promising results.3, 4 Other top targets being investigated in clinical trials for CAR-T cell therapies include GD2, EGF R, HER2, Mesothelin, CD20, CD22, CD30, CD33, CD123, Glypican-3, and NKG2D.1

References

  1. Yu, J.X. et al. (2019) Nat. Rev. Drug Discov. 18:821.
  2. Hay, K.A. & C.J. Turtle (2017) Drugs 77:237.
  3. Mikkilineni, L, & J.N. Kochenderfer (2017) Blood 130:2594.
  4. D’Agostino, M. & N. Raje (2020) Leukemia 34:21.

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