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Solutions for Evaluation of CAR Expression

Specific detection of CAR expression using target antigens
 

Introduction

The chimeric antigen receptor T (CAR-T) cell therapy is a new treatment for a variety of cancers. The idea is to take out the T-cells from the patient, and genetically engineer the cells to make them express a chimeric antigen receptor (CAR) recognizing a specific tumor-associated antigen (TAAs). As a result, the CAR-expressing T cells, when reintroduced into the patient's body, will target and eliminate the TAA-expressing tumor cells.

2017 is a milestone year for CAR-T cell therapy. The FDA approved two CD19-targeting CAR-T therapies. Novartis's Kymriah is approved for B-cell precursor ALL treatment in children and young adults, while Kite Pharma's Yescarta is approved for the treatment of adult patients. This success fueled the efforts to develop / advance CAR-T treatment targeting other cancer-specific antigens. Beyond CD19, there is a growing list of targets being investigated for therapeutic intervention.

ACROBiosystems has developed an extensive collection of recombinant proteins to support CAR-T therapy development. This growing list of proteins includes many fluorescent-labeled target antigens and pre-biotinylated proteins that are uniquely suitable for evaluation of CAR expression. In addition, we also supply hard-to-make proteins such as BCMA, CD19, ROR1, and EGFRVIII.

Evaluation of CAR Expression

Evaluating CAR expression is an essential step in the production of CAR-T cells. This is often done by flow cytometry, using protein L, anti-Fab antibodies or target antigens as detection antibodies. Among these common choices, target antigens are widely considered to be the best option, because it offers high specificity and minimal background staining.

Reagents Mechanism Pros Cons
Target Antigens Specifically bind to the antigen-binding domains of CARs. High Specificity; Minimal backgound staining. Each unique CAR has to be stained with corresponding antigens
Protein L Binds to the kappa light chain of immunoglobulin. Universal. High background staining; Cannot detect the anti-lambda light chain CAR.
 Anti-Fab antibody Binds to the Fab portion of immunoglobulin. Universal. High background staining.

CAR Detection Strategy and Product Design

Currently, target antigens for CAR-T cells are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available. In an effort to fulfill these needs, we have developed an extensive collection of CAR-T target antigens includes many fluorescent-labeled proteins and pre-biotinylated proteins that are uniquely suitable for evaluation of CAR expression by flow cytometry.

Detection Methods

  • Direct detection
    Target antigens are pre-labeled with fluorescent dye. Processing time can be reduced by the use of direct-labeled proteins.
    Non-specific reaction of a secondary antibody is eliminated.

ACRO's specially designed products:

FITC-labeled proteins;
PE-labeled proteins;

 

  • Biotin-streptavidin based detection
    Target antigens are pre-labeled with biotin and detected by labeled streptavidin (the biotin-avidin complex).
    Streptavidin labeled with fluorochromes can bind biotinylated proteins with a high degree of affinity and specificity, amplifying the signal and improving the detection sensitivity and specificity.

ACRO's specially designed products: AvitagTM biotinylated proteins;
Chemically biotinylated proteins;

 

  • Indirect detection
    Target antigens are designed to carry a specific tag and  detected using a secondary antibody (anti-epitope tag antibody) labeled with a fluorophore.
    Non-specific reaction of a secondary antibody may occur.
     

ACRO's specially designed products:
Fc-tag fusion proteins;
His-tag fusion proteins;

Case Studies

Evaluation of Anti-CD19 CAR Expression with FITC-labeled CD19

  • Reagents
    FITC-labeled Human CD19 (20-291) Protein (ACROBiosystems, Cat. No. CD9-HF2H2);
  • Samples
    R1013-C6 cells (Transfected 293 cells expressing the anti-CD19 [FCM63] scFv & RFP tag).
  • Protocol
    1. Culture R1013-C6 cells in DMEM medium with 10% FBS in the CO2 incubator (at 37°C, 5%CO2 ).
    2. Harvest the cells and wash the cells once by wash buffer.
    3. Count the cells number and the viability, aliquot up 2e5 live cells (Anti-CD19-scFv positive cell is 98%) into each tube. (Note: the cell viability must be ≥ 95%.)
    4. Add 100 µl, 10 µg/ml of FITC-labeled Human CD19 (20-291) Protein or FITC-labeled Protein control into each tube, incubating at 4℃ for 1 hour.
    5. Wash the cells 3 times by wash buffer and resuspend the cells in 200 µl PBS per sample.
    6. Transfer the cells into flow tube and detect by Flow cytometry.
    7. Analyze result using FACS Celesta software and FCS Express 6 Flow software. 
  • Results
    The data showed that the expression level of anti-CD19 scFv on the surface of R1013-C6 cells was 95.37% .


293 cells were transfected with anti-CD19-scFv and RFP tag. 2e5 of the cells were stained with B. FITC-Labeled Human CD19 (20-291) (Cat. No. CD9-HF2H2, 10 µg/ml) and C. FITC-labeled protein control. A. Non-transfected 293 cells and C. FITC-labeled protein control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of FITC-labeled Human CD19 (20-291) (Cat. No. CD9-HF2H2).

Evaluation of Anti-BCMA CAR Expression with Biotinylated BCMA

  • Reagents
    Biotinylated human BCMA protein, Fc & Avi Tag (ACROBiosystems, Cat. No. BC7-H82F0)
  • Samples
    Anti-BCMA CAR-transduced human primary T-cells.
    Brief Protocol
  • 1. Human T cells transfected with anti-BCMA CAR were harvested 3 days after the transfection.
    2. Aliquot up to 1e6 cells into centrifuge tube and wash the cells twice with FACS buffer.
    3. Resuspend cells in 50 µl of diluted biotinylated human BCMA  (ACROBiosystems, Cat. No. BC7-H82F0) (prepared in FACS buffer at 8 µg/ml) and incubate at 4℃ for 30 minutes.
    4. Wash cells twice with FACS buffer.
    5.Resuspend cells in 50 µl of diluted PE Streptavidin (Biolegend,  Cat. No. 405204) (prepared in FACS buffer at 1:50 dilution) and incubate at 4℃ for 30 minutes in the dark.
    6. Wash cells twice with FACS buffer and resuspend the cells in 400 µl PBS.
    7. Transfer the cells into flow tube and analyze on BD FACSCaliburTM flow cytometer using FCS Express 6 Plus software.
  • Results
    The data showed that the expression of anti-BCMA CARs on transduced T cell surface from donor 1 and donor 2 were 52.72% and 73.49%, respectively.

    Human T cells were transfected with anti-BCMA CAR and cultured for 3 days. Three days post-transfection, 1e6 cells were first incubated with 50 µl biotinylated human BCMA protein (Cat. No. BC7-H82F0, 8 µg/ml ), washed and then stained with PE Streptavidin. (Data are kindly provided by PREGENE Biopharma)

Evaluation of Anti-CD19 CAR Expression with Fc-fusion CD19

  • Reagents
    Human CD19 protein, Fc Tag (Acrobiosystems, Cat. No. CD9-H5251);
    FITC anti-human IgG Fc antibody (Biolegend, Cat. No. 409310).
  • Samples
    R1013-C6 cells (Transfected 293 cells expressing the anti-CD19 [FCM63] scFv & RFP tag).
  • Brief Protocol
    1. Culture R1013-C6 cells in DMEM medium with 10% FBS in the CO2 incubator (at 37°C, 5%CO2 ).
    2. Harvest the cells and wash the cells once by wash buffer.
    3. Count the cells number and the viability, aliquot up 2e5 live cells (Anti-CD19-scFv positive cell is 98%) into each tube. (Note: the cell viability must be ≥ 95%.)
    4. Add 100 µl, 3 µg/ml of Recombinant Human CD19 (20-291) Protein, Fc Tag or Recombinant Human Fc Tag Protein control into each tube, incubating at 4℃ for 1 hour.
    5. Wash the cells once by wash buffer.
    6. Add FITC anti-human IgG Fc (Biolegend, Cat. No. 409310) by dilution of 1:100 (100 µl), incubating at 4℃ for 1 hour.
    7. Wash the cells 3 times by wash buffer and resuspend the cells in 200 µl PBS per sample.
    8. Transfer the cells into flow tube and detect by Flow cytometry.
    9. Analyze result using FACS Celesta software and FCS Express 6 Flow software.
  • Results
    The data showed that the expression level of anti-CD19 scFv on the surface of R1013-C6 cells was 96.29% .

    293 cells were transfected with FMC63-scFv and RFP tag. 2e5 of the cells were first stained with B. Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Cat. No. CD9-H5251, 3 µg/ml) and C. Human Fc Tag Protein Control, followed by FITC-conjugated Anti-human IgG Fc Antibody. A. Non-transfected 293 cells and C. Human Fc Tag Protein Control were used as negative control. RFP was used to evaluate CAR (anti-CD19-scFv) expression and FITC was used to evaluate the binding activity of Human CD19 (20-291) Protein, Fc Tag.

Evaluation of Anti-MSLN CAR Expression with PE-labeled MSLN

  • Reagents
    PE-labeled Human Mesothelin / MSLN (296-580) Protein (ACROBiosystems, Cat. No. MSN-HP223);
  • Samples
    CAR-RC218 cells (Transfected 293 cells expressing the anti-MSLN scFv).
  • Protocol
    1. Culture CAR-RC218 cells in DMEM medium with 10% FBS in the CO2 incubator (at 37°C, 5%CO2 ).
    2. Harvest the cells and wash the cells once by wash buffer.
    3. Count the cells number and the viability, aliquot up 1e6 live cells into each tube.
    4. Add 100 µl of diluted PE-labeled Human Mesothelin (296-580) Protein (Cat. No. MSN-HP223) (prepared in dilution buffer at 1:25 dilution) into each tube, incubating at 4℃ for 1 hour.
    5. Wash the cells 3 times by wash buffer and resuspend the cells in 200 µl PBS per sample.
    6. Transfer the cells into flow tube and detect by Flow cytometry.
    7. Analyze result using FACS Celesta software and FCS Express 6 Flow software.
  • Results
    The data showed that the expression level of anti-MSLN scFv on the surface of R1013-C6 cells was 100% .
    1e6 of the MSLN-CAR-293 cells were stained with 100 µl of 1:25 dilution (4 µl stock solution in 100 µl dilution buffer) of PE- labeled Human Mesothelin / MSLN (296-580) Protein (Cat. No. MSN-HP223). PE Streptavidin was used as negative control (QC tested).

 

 

Product List of CAR-T Target

Fluorescent-labeled Proteins

Targets  Cat. No. Product Description Diseases
BCMA BCA-HF2H1 FITC-labelled Human BCMA, His Tag Multiple myeloma, leukemia, B-Cell lympoma
BCMA BCA-HF254 FITC-labelled Human BCMA, Fc Tag Multiple myeloma, leukemia, B-Cell lympoma
CD19 CD9-HF2H2 FITC-labelled Human CD19, His Tag Acute leukemia, B-Cell lymphoma
CD19 CD9-HF251 FITC-labelled Human CD19, Fc Tag Acute leukemia, B-Cell lymphoma
CD22 SI2-HF2H6 FITC-labelled Human  Siglec-2 / CD22, His Tag Leukemia, B-Cell lympoma
Glypican 3 GP3-HF258 FITC-labelled Human GPC3, Fc Tag Hepatocellular carcinoma 
Mesothelin MSN-HF223 FITC-labelled Human MSLN, His Tag Mesothelioma, ovarian cancer 
Mesothelin MSN-HF253 FITC-labelled Human MSLN, Fc Tag Mesothelioma, ovarian cancer 
Mesothelin MSN-HP223  PE-labelled Human MSLN, His Tag Mesothelioma, ovarian cancer 

Biotinylated Proteins

Targets Cat. No. Product Description Diseases
BCMA BCA-H82E4 Biotinylated Human BCMA, His & Avi Tag Multiple myeloma, leukemia, B-Cell lymphoma
BCMA BC7-H82F0 Biotinylated Human BCMA, Fc & Avi Tag Multiple myeloma, leukemia, B-Cell lymphoma
CD19 CD9-H8259 Biotinylated Human CD19, Fc Tag Acute leukemia, B-Cell lymphoma
CD22 SI2-H82F8 Biotinylated Human CD22, Fc & Avi Tag Leukemia, B-Cell lymphoma
EGFRvIII EGR-H82E0 Biotinylated Human EGFRvIII, Avi & His Tag Glioblastoma
Glypican 3 GP3-H82E5 Biotinylated Human GPC3, His & Avi Tag  Hepatocellular carcinoma
Mesothelin MSN-H82E9 Biotinylated Human MSLN, His & Avi Tag Mesothelioma, ovarian cancer
Mesothelin MSN-H8223 Biotinylated Human MSLN, His Tag Mesothelioma, ovarian cancer

Unconjugated Proteins

Targets Cat. No. Product Description Diseases
BCMA BCA-H522y Human BCMA, His Tag Multiple myeloma, leukemia, B-Cell lymphoma
BCMA BC7-H5254 Human BCMA, Fc Tag Multiple myeloma, leukemia, B-Cell lymphoma
BCMA BCA-H5253 Human BCMA, Mouse IgG2a Fc Tag, low endotoxin Multiple myeloma, leukemia, B-Cell lymphoma
BCMA BCA-H5259 Human BCMA, Llama IgG2b Fc Tag, low endotoxin Multiple myeloma, leukemia, B-Cell lymphoma
CD19 CD9-H52H2 Human CD19, His Tag Acute leukemia, B-Cell lymphoma
CD19 CD9-H5251 Human CD19, Fc Tag, low endotoxin (Super affinity) Acute leukemia, B-Cell lymphoma
CD19 CD9-H5250 Human CD19, Llama IgG2b Fc Tag, low endotoxin Acute leukemia, B-Cell lymphoma
CD123 ILA-H52H6 Human IL-3 R alpha / CD123, His Tag Acute Myeloid Leukemia
CD123 ILA-H5255 Human IL-3 R alpha / CD123, Llama IgG2b Fc Tag, low endotoxin  Acute Myeloid Leukemia
CD138 SD1-H5228 Human Syndecan-1 / CD138, His Tag Multiple myeloma
CD22 SI2-H5228 Human Siglec-2 / CD22 (176-687), His Tag Leukemia, B-Cell lymphoma
CD22 CD2-H52H8 Human Siglec-2 / CD22 (20-687), His Tag Leukemia, B-Cell lymphoma
CD22 SI2-H525a Human Siglec-2 / CD22 (20-687), Llama IgG2b Fc Tag, low endotoxin  Leukemia, B-Cell lymphoma

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