High-Affinity CD19 for CAR-T Cell Therapy

CD19 still remains the strongest contender in CAR-T cell therapy. According to ClinicalTrials.gov, over 600 CAR T cell clinical trials have been carried out to date, a majority of which aiming to treat leukemia or lymphoma patients using CD19‐specific CARs.

CAR-positive T cells are vital to the anti-tumor activity of anti-CD19 CAR T cell products. The percentage of CAR‐positive T cells within a product is often measured using flow cytometry, using anti-Fab antibodies, protein L, or CD19 antigen as detection antibodies. Among these typical choices, the CD19 antigen is widely believed to be the best option, as it offers high specificity and marginal background staining.

ACROBiosystems has produced a wide collection of CD19 antigen products to assist anti-CD19 CAR T investigations, including fluorescent-labeled CD19 and biotinylated CD19 that are uniquely suited for assessing anti-CD19 CAR expression.

Different CAR Detection Strategy and Product Design

Direct Detection

CD19 comes pre-labeled with FITC. Processing time can be decreased by the use of direct-labeled CD19 protein. A non-specific reaction of a secondary antibody is removed.

Cat.No. Product Description Structure
CD9-HF2H2 FITC-labeled Human CD19 (20-291) Protein
CD9-HF251 FITC-labeled Human CD19 (20-291) Protein, Fc Tag

 

CAR Detection by FITC-Labeled Human CD19, His Tag

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).

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).

CAR Detection by FITC-Labeled Human CD19, Fc Tag

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), Fc Tag (Cat. No. CD9-HF251, 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), Fc Tag (Cat. No. CD9-HF251).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), Fc Tag (Cat. No. CD9-HF251, 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), Fc Tag (Cat. No. CD9-HF251).

Competitive Advantage over Other Vendors

Binding activity of FITC-labeled Human CD19, His Tag from two different vendors were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The result showed that ACRO’s FITC-labeled Human CD19, His Tag has a much higher binding activity than another Vendor’s.

Binding activity of FITC-labeled Human CD19, His Tag from two different vendors were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The results show that ACRO’s FITC-labeled Human CD19, His Tag has a much higher binding activity than another vendor’s.

Binding activity of FITC-labeled Human CD19, His Tag from two different vendors were evaluated in the above ELISA analysis against FMC63 MAb. The result showed that ACRO’s FITC-labeled Human CD19, His Tag has a much higher binding activity than another Vendor’s.

Binding activity of FITC-labeled Human CD19, His Tag from two different vendors were evaluated in the above ELISA analysis against FMC63 MAb. The results show that ACRO’s FITC-labeled Human CD19, His Tag has a much higher binding activity than another Vendor’s.

Well-Controlled Lot Consistency of FITC-Labeled CD19

Binding activity of two different lots of FITC-labeled Human CD19, His Tag were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The result showed that the batch variation among the tested samples is negligible.

Binding activity of two different lots of FITC-labeled Human CD19, His Tag were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The results show that the batch variation among the tested samples is negligible.

Binding activity of three different lots of FITC-labeled Human CD19, Fc Tag were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The result showed that the batch variation among the tested samples is negligible.

Binding activity of three different lots of FITC-labeled Human CD19, Fc Tag were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The results show that the batch variation among the tested samples is negligible.

Consistent Binding Before and After FITC Conjugation

Binding activity of the Human CD19, His Tag before and after FITC labeling were evaluated in the above ELISA analysis. The result showed that the FITC-labeled and Unconjugated Human CD19, His Tag have almost the same binding activity.

Binding activity of the Human CD19, His Tag before and after FITC labeling were evaluated in the above ELISA analysis. The results show that the FITC-labeled and Unconjugated Human CD19, His Tag have almost the same binding activity.

Binding activity of the Human CD19, Fc Tag before and after FITC labeling were evaluated in the above ELISA analysis. The result showed that the FITC-labeled and Unconjugated Human CD19, Fc Tag have almost the same binding activity.

Binding activity of the Human CD19, Fc Tag before and after FITC labeling were evaluated in the above ELISA analysis. The results show that the FITC-labeled and Unconjugated Human CD19, Fc Tag have almost the same binding activity.

Biotin-Streptavidin Based Detection

CD19 comes pre-labeled with biotin and is detected by labeled streptavidin (the biotin-avidin complex). Streptavidin labeled with fluorochromes can bind biotinylated proteins with a high degree of specificity and affinity, intensifying the signal and enhancing the detection sensitivity and specificity.

Cat.No. Product Description Structure
CD9-H8259 Biotinylated Human CD19, Fc Tag, ultra sensitivity (primary amine labeling)

 

Indirect Detection by Biotinylated Human CD19, FC Tag

293 cells were transfected with FCM63-scFv and RFP tag. 2x105 of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml). C. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10 µg/ml) and FMC63(Mouse anti-CD19 antibody). FITC Streptavidin was used to analyze with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant biotinylated human CD19 (Cat. No. CD9-H8259).

293 cells were transfected with FCM63-scFv and RFP tag. 2x105 of the cells were first incubated with A. Biotinylated protein control. B. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10ug/ml). C. Recombinant biotinylated human CD19 (Cat. No. CD9-H8259, 10 µg/ml) and FMC63(Mouse anti-CD19 antibody). FITC Streptavidin was used to analyze with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant biotinylated human CD19 (Cat. No. CD9-H8259).

Case Study on Real CART Cells

Human T cells were lentivirally transduced with anti-CD19 CAR and cultured for 11 days. Eleven days post-transduction, 1e6 cells were stained for the expression of CD3 and anti-CD19 CAR with FITC anti-human CD3 antibody and biotinylated human CD19 (Cat. No. CD9-H8259) followed by PE-conjugated streptavidin, respectively. A. Non-transduced T cells were used as a control for gating of CAR expression. (Data are kindly provided by Beijing Bowei Huaen Medical Technology Co. Ltd.)

Human T cells were lentivirally transduced with anti-CD19 CAR and cultured for 11 days. Eleven days post-transduction, 1e6 cells were stained for the expression of CD3 and anti-CD19 CAR with FITC anti-human CD3 antibody and biotinylated human CD19 (Cat. No. CD9-H8259) followed by PE-conjugated streptavidin, respectively. A. Non-transduced T cells were used as a control for gating of CAR expression. (Data was kindly provided by Beijing Bowei Huaen Medical Technology Co. Ltd.)

Indirect Detection

CD19 is engineered to carry a specific tag and is detected using a secondary antibody (anti-epitope tag antibody) labeled with a fluorophore. A non-specific reaction of a secondary antibody may occur.

Cat.No. Product Description Structure
CD9-H52H2 Human CD19 (20-291) Protein, His Tag
CD9-H5251 Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity)

 

Indirect Detection by Human CD19, His Tag

293 cells were transfected with FMC63-scFv and RFP tag. 2e5 of the cells were first incubated with A. His Tag-protein control. B. Recombinant human CD19, His Tag (Cat. No. CD9-H52H2, 10 μg/ml). The FITC Anti-6xHis tag antibody was used to analyze with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant human CD19, His Tag (Cat. No. CD9-H52H2).

293 cells were transfected with FMC63-scFv and RFP tag. 2e5 of the cells were first incubated with A. His Tag-protein control. B. Recombinant human CD19, His Tag (Cat. No. CD9-H52H2, 10 μg/ml). The FITC Anti-6xHis tag antibody was used to analyze with FACS. RFP was used to evaluate CAR(FMC63-scFv) expression and FITC was used to evaluate the binding activity of recombinant human CD19, His Tag (Cat. No. CD9-H52H2).

Indirect Detection by Human CD19, Fc Tag

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, low endotoxin (Super affinity) (Cat. No. CD9-H5251).

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, low endotoxin (Super affinity) (Cat. No. CD9-H5251).

Higher Binding Affinity than Competitive Products

Binding activity of Human CD19, His Tag from two different vendors were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The result showed that ACRO’s Human CD19, His Tag has a higher binding activity than another Vendor’s.

Binding activity of Human CD19, His Tag from two different vendors were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The results show that ACRO’s Human CD19, His Tag has a higher binding activity than another Vendor’s.

Binding activity of Human CD19, His Tag from two different vendors were evaluated in the above ELISA analysis against FMC63 MAb. The result showed that ACRO’s Human CD19, His Tag has a higher binding activity than another Vendor’s.

Binding activity of Human CD19, His Tag from two different vendors were evaluated in the above ELISA analysis against FMC63 MAb. The results show that ACRO’s Human CD19, His Tag has a higher binding activity than another Vendor’s.

Binding activity of Human CD19, Fc Tag from two different vendors were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The result showed that ACRO’s Human CD19, Fc Tag has a higher binding activity than another vendor’s.

Binding activity of Human CD19, Fc Tag from two different vendors were evaluated in the above flow cytometry analysis against anti-CD19-CAR-293 cells. The results show that ACRO’s Human CD19, Fc Tag has a higher binding activity than another vendor’s.

Binding activity of Human CD19, Fc Tag from two different vendors were evaluated in the above ELISA analysis against FMC63 MAb. The result showed that ACRO’s Human CD19, Fc Tag has a higher binding activity than another vendor’s.

Binding activity of Human CD19, Fc Tag from two different vendors were evaluated in the above ELISA analysis against FMC63 MAb. The results show that ACRO’s Human CD19, Fc Tag has a higher binding activity than another vendor’s.

Higher Purity than Competitive Products

The purity of Human CD19, Fc Tag from two different vendors were determined in HPLC analysis. The result showed that the purity of ACRO’s Human CD19, Fc Tag is greater than 87%, while the purity of another vendor’s Human CD19, Fc Tag is greater than 57%.

The purity of Human CD19, Fc Tag from two different vendors were determined in HPLC analysis. The results show that the purity of ACRO’s Human CD19, Fc Tag is greater than 87%, while the purity of another vendor’s Human CD19, Fc Tag is greater than 57%.

Better Performance in SPR Studies than Competitive Products

Comparative Analysis of rhCD19-His from ACRO and Another Vendor for Affinity to FMC63 by SPR

  • Experimental Design

  • Brief Protocol
    • Chip Preparation: The sensor chip CM5 was triggered using EDC/NHS. Immobilization of the anti-mouse antibody took place on the CM5 sensor chip.
    • Ligand Capturing: Capture FMC63 mAb to the CM5 chip with immobilized anti-mouse antibody
    • Analysis: A series of Human CD19, His Tag concentrations including 0 nM, 5.86 nM, 11.72 nM, 23.44 nM, 46.88 nM, 93.75 nM, 187.5 nM, and 375 nM were injected consecutively, each with a dissociation time of 480 seconds and a contact time of 120 seconds.
    • Regeneration: The chip was regenerated with the help of 10 mM Glycine-HCl buffer at PH1.7.
  • Experimental Results

Product Vendor Method Ligand Capture
Level
Analyte
Conc.
Ka
(1/Ms)
Kd
(1/s)
KD(M) Rmax
(RU)
% Ligand
Bound
RU
(375nM)
Human CD19,
His Tag
(Cat.No. CD9-H52H2)
ACRO Mouse Antibody Capture FMC63 477 RU hCD19 his
12-0.375ug/ml
(240-7.5nM)
1.02
E + 05
3.00
E-04
2.95
E-09
91.2 28.7% 85
Human CD19,
His Tag
Another Vendor N.A. N.A. N.A. 29.6 9.3% 6

 

  • Conclusions

ACRO’s rhCD19-His displayed better performance in SPR assay compared to that of the equivalent from another vendor under the same conditions. The affinity constant (KD value) of ACRO’s rhCD19-His to FMC63 was 2.95 nM, which was consistent with the published data (Nicholson, Ian C. et al.).

Comparative Analysis of rhCD19-Fc from ACRO and Another Vendor for Affinity to FMC63 by SPR

  • Experimental Design

  • Brief Protocol
    • Chip Preparation: The sensor chip CM5 was triggered using EDC/NHS. Immobilization of the anti-human IgG (Fc) antibody was done on the CM5 sensor chip.
    • Ligand Capturing: Capture Human CD19, Fc Tag to the CM5 chip with immobilized anti-human IgG (Fc) antibody.
    • Analysis: A series of FMC63 mAb concentrations including 0 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, and 40 nM were injected consecutively, each with a dissociation time of 480 seconds and a contact time of 120 seconds.
    • Regeneration: Using 3 M magnesium chloride, the chip was regenerated.
  • Experimental Results

Product Vendor Method Ligand Capture
Level
Analyte
Conc.
Ka
(1/Ms)
Kd
(1/s)
KD(M) Rmax
(RU)
% Bound
Human CD19,
His Tag
(Cat.No. CD9-H5251)
ACRO Anti-human lgG capture Fc-CD19 220 RU FMC63
6-0.19ug/ml
(40-1.25nM)
950378.1 0.00016 1.69
E-10
50.84 17.6%
Human CD19,
Fc Tag
Another Vendor Fc-CD19 2221 RU 815157.7 0.000281 3.44
E-10
19.20 6.6%

 

  • Conclusions

ACRO’s rhCD19-Fc displayed improved performance in SPR assay compared to that of the counterpart from another vendor under the same conditions. The affinity constant (KD value) of ACRO’s rhCD19-Fc to FMC63 was 0.17 nM, which was twice as high as the counterpart from another vendor.

Reference

Nicholson, Ian C. et al. “Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma.” Molecular Immunology, vol. 34, No. 16-17, 1157-1165, 1997.

ACROBiosystems

ACROBiosystems is an internationally recognized manufacturer of recombinant proteins committed to supporting cancer immunotherapy. We specialize in mammalian cell-based recombinant protein production and process development.

Our goal is to support professionals from pharmaceutical companies, CROs and research institutes who are working on the cancer immunotherapy area by providing high-quality proteins, antibodies and assay kits.

We have multiple offices and branches in North America, Europe, and Asia, and we are proud of serving customers from over 50 countries.


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Last updated: Nov 21, 2018 at 9:38 AM

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