The authorization of Chimeric Antigen Receptor (CAR) cell therapies by the US Food and Drug Administration (FDA) marked a significant breakthrough for cancer treatments, particularly relapsed/refractory hematological malignancies.
By targeting surface markers on cancer cells, engineered synthetic receptors expressed on immune cells can be directed to tumor sites. This specific binding enhances immune cell targeting, reduces immune escape, and triggers a strong anti-tumor response.1
As cell therapies are increasingly integrated into conventional cancer treatment strategies, attention has turned to addressing challenges in production and quality control that hinder cell therapy development.2
CAR-T and other cell therapies present a distinct challenge as a biologics-based treatment, particularly in terms of regulations and guidelines. Due to the inherent variability, errors and safety concerns in cell production, implementing controls and quality checks to guarantee product specificity, safety and function is of paramount importance.
This necessity is reflected by the FDA’s 2022 guidance, which details the necessary analytical tests and controls for cell manufacturing. A key component of these regulations is CAR cell characterization, which involves evaluating all characteristics of the altered cells, especially CAR expression detection.8
Detecting CAR expression has been a major hurdle in this domain, necessitating a rapid, sensitive and robust assay, particularly in production settings.
Since the introduction of CARs in 1989, various methods have been suggested, including anti-fragment antigen-binding (Fab) antibodies, antigen-Fc fusion proteins, biotinylated protein L with fluorescently conjugated streptavidin and murine luciferases.4-7
Nevertheless, significant challenges persist, primarily related to indirect CAR detection, difficulties in fusion protein development and potential interference from fused or conjugated fluorophore on the target antigen.
The development of chemically labeled fluorescent proteins has addressed many obstacles associated with fusion proteins and indirect detection modalities. Two development methods exist: traditional and site-specific labeling, as shown in Figure 1.
Traditional labeling methods involve binding a fluorescent tag to a reactive group for protein conjugation, offering a rapid and easy labeling process for laboratory use. However, this method can conjugate a different number of fluorophores and varies in conjugation site, potentially covering active protein sites.
This randomness introduces variability that may impact subsequent CAR expression evaluations.
Conversely, site-specific labeling methods regulate and reduce the variation in each batch and between batches. Control of the conjugate site is also possible, focusing on regions away from active sites while maintaining the natural conformation and post-translational modifications unique to each antigen.
As with any analytical assay, reagent quality directly influences result quality. This is particularly critical for CAR expression detection, where binding reproducibility and quantifiability are essential.
The study discussed in this article examines the benefits of site-specific fluorescently labeled proteins and how they enhance CAR expression detection via flow cytometry.
Figure 1. (A) Site-specific and (B) traditional chemical labeling methods. Synthetic reagents for chemical fluorescent labeling through traditional labeling result in significant variation in a number of the conjugated fluorophores and conjugation sites. Image Credit: ACROBiosystems
Methods and materials
A detailed comparison of site-specific labeled recombinant proteins (Star Staining) and traditional chemical labeling methods was conducted using fluorescence activated cell sorting (FACS).
Experimental reagents and materials
Lyophilized proteins were reconstituted using FACS buffer into the appropriate working solution (1 ug/mL) and diluted as required.
Table 1. Materials used for FACS. Source: ACROBiosystems
| Materials |
Catalog No. |
Vendor |
FITC-Labeled Human BCMA, His Tag,
Star Staining |
BCA-HF2H3 |
ACROBiosystems |
PE Labelled Human BCMA, His Tag,
Star Staining |
BCA-HP2H7 |
ACROBiosystems |
AF-488 Labeled Human Mesothelin, His Tag,
Star Staining |
MSN-HA2H9 |
ACROBiosystems |
AF-647 Labeled Human Mesothelin, His Tag,
Star Staining |
MSN-HA2H5 |
ACROBiosystems |
| Negative Control Protein |
PSA-HF244 |
ACROBiosystems |
| Fetal Bovine Serum (FBS) |
SA212.02 |
CellMax |
| DMEM media |
SH30022.01 |
Hyclone |
Table 2. Reagents used in FACS. Source: ACROBiosystems
| Conditions |
Description |
| Anti-BCMA CAR-293 |
Used to evaluate CAR binding to BCMA. |
| Anti-MSLN CAR-293 |
Used to evaluate CAR binding to Mesothelin. |
| HEK 293 |
Used as control as non-transfected cells. |
| PBMCs |
Used to evaluate non-specific binding |
| FACS Buffer |
2% bovine serum albumin (BSA) in phosphate
buffered solution (PBS), pH 7.2-7.4 |
Table 3. Instruments for Cell Culture & FACS. Source: ACROBiosystems
| Type |
Instrument |
| Incubator |
CO2 Incubator |
| Cell Counter |
Logos Biosystems LUNA |
| Flow Cytometer |
BD Biosciences FACSLyric |
| Data Visualization Software |
FCS Express 7 |
Fluorescence Activated Cell Sorting (FACS) protocol
- Cell Culturing First, cells were cultured in DMEM medium with 10 % FBS in the CO2 incubator at 37 °C, 5 % CO2.
- Harvesting and Washing The cells were then harvested and washed once with the FACS buffer.
- Cell Counting Cell number and viability was then counted. For Aliquot, this was up to 2x105 cells and for PBMCs, 5x105 were used. Cell viability was required to exceed 95% at this stage.
- Protein Dilution FITC-labelled Human BCMA, His Tag, Star staining and negative control protein was diluted using the FACS buffer in a series of concentrations. Diluted or working sample solution (100 µL) was then added to a tube with a cell pellet. This was then mixed well and incubated at 4 °C for 60 minutes while being shielded from light.
- Washing Cells were washed three times using the FACS buffer.
- Cell Resuspension The cells were then re-suspended in 200 uL of PBS per sample.
- Transferring to FACS The cell suspension was then transferred into the flow tube and detected using flow cytometry.
- Analysis The resulting data was then analyzed using FCS Express 7 software.
Results and discussion
Characterization of site-specific fluorescently labeled BCMA and mesothelin proteins
Characterization studies of site-specific fluorescent-labeled proteins were conducted through FACS analysis for binding activity. Two distinct fluorescently labeled proteins at different concentrations were mixed with their corresponding anti-BCMA or Mesothelin CAR-293 cells.
A considerable increase in fluorescent intensity was observed for site-specific labeled proteins (red) compared to traditional chemical labeling (blue). This difference arises due to site-specific conjugation of the fluorescent label.
At higher concentrations, the random nature of conjugation sites in conventional chemical labeling is more likely to obstruct the protein active sites, impacting binding activity.
Figure 2. FACS analysis of (A) FITC-labeled BCMA and (B) Alexa Fluor-647 Mesothelin protein. Fluorescent activity resulting from the binding of different proteins is shown to compare (Red) Site-specific and (Blue) traditional fluorescent chemical labeling methods. Image Credit: ACROBiosystems
Specificity of AF-488 labeled mesothelin protein to Anti-MSLN CAR-293 cells
To further analyze the advantages of site-specific fluorescent labeling, FACS specificity experiments were carried out using Star Staining Alexa Fluor 488 labeled Mesothelin, shown in Figure 3.
Non-transfected HEK293 cells were stained with AF-488 MSLN to establish a baseline for non-specific binding, revealing less than 0.01 % of HEK293 cells bound to MSLN. Additionally, a negative control protein was used to stain anti-MSLN CAR-293 cells, showing minimal binding.
Finally, when anti-MSLN CAR-293 cells were stained with AF488 human MSLN protein, over 99 % positive rate detection was observed, confirming the high specific binding of Star Staining labeled proteins to anti-MSLN CAR cells.
Figure 3. 5e5 of anti-MSLN CAR-293 cells were stained with 100 μL of 3 μg/mL of AF488-Labeled Human Mesothelin (296-580), His Tag (Cat. No. MSN-HA2H9) and negative control protein respectively (Fig. B and C), and non-transfected 293 cells were used as a control (Fig. A). AF488 signal was used to evaluate the binding activity. Image Credit: ACROBiosystems
Batch-to-batch consistency of star-staining FITC and PE-Labeled BCMA protein
Another advantage of site-specific labeling is enhanced consistency across batches, both inter- and intra-batch. Due to variations between traditional chemical-labeled proteins, in many cases, a single lot of fluorescent-labeled protein must be used throughout the whole experiment, which is not always feasible during early manufacturing stages.
Site-specific labeling ensures standardized and controlled fluorophore conjugation and conjugation site selection, theoretically eliminating variation between fluorescent-labeled proteins.
To evaluate batch-to-batch consistency of site-specific labeled proteins, the binding activity of FITC, PE and Alexa Fluor 555 BCMA to anti-BCMA CAR-293 cells was determined, as shown in Figure 4. Results revealed minimal variation across all three Star-Staining BCMA proteins, confirming their batch-to-batch consistency.
Figure 4. Binding activity of different lots of FITC-Labeled Human BCMA (Cat. No. BCA-HF2H3), AF555-Labeled Human BCMA (Cat. No. BCA-HA2H6) and PE-Labeled Human BCMA (Cat. No. BCA-HP2H7) against anti-BCMA CAR-293 cells was evaluated by flow cytometry with high batch-to-batch consistency throughout Star-stained FITC, AF555, and PE-labelled BCMA proteins. Image Credit: ACROBiosystems
Minimizing non-specific binding of consistency of star-staining FITC and PE-labelled BCMA protein
Non-specific binding to non-transduced cells is a major limitation when evaluating CAR expression detection. Generally, non-specific binding directly affects the evaluated CAR expression level, affecting the assay sensitivity due to elevated background noise.
Non-specific binding was tested by comparing PBMC binding to site-specific labeled protein and traditionally labeled protein, shown in Figure 5b and 5c respectively.
Traditionally labeled protein displayed a significant level of non-specific binding, with an unclean cell population distribution expected from a standard-solution FACS assay. On the other hand, site-specific labeled protein displayed almost no non-specific binding, leading to a FACS distribution mimicking the PBMC control.
As a result, background noise was minimized and sensitivity and accuracy of CAR expression evaluation increased.
Figure 5. Non-specific binding to non-transduced PBMCs between PE-Labeled Human BCMA Protein of Acro and competitor. 5e5 of non-transduced PBMCs were stained with PE -Labeled Human BCMA Protein and anti-CD3 antibody, washed and then analyzed with FACS. FITC signal was used to evaluate the expression of CD3+ T cells in non-transduced PBMCs, and PE signal was used to evaluate the non-specific binding activity to non-transduced PBMCs. Image Credit: ACROBiosystems
Conclusions
Despite advancements in next-generation of CAR cells, cell therapy remains in its early stage and is expected to expand significantly in the near future. As more cell therapies progress through clinical pipelines, ensuring high-quality manufacturing standards has become a primary concern in drug development.
Employing robust, specific and stable reagents for analytical assessments is vital to guaranteeing safe and effective final cell therapies. ACROBiosystems’ Star-staining products have been optimized to deliver highly sensitive and consistent CAR expression detection for cell manufacturing.
Acknowledgments
Produced from materials originally authored by Siqi Huang, Xiaojuan Shi, Yanke Liu, Jane Liu and Spencer Chiang from ACROBiosystems.
References and further reading
- Sterner, R.C. and Sterner, R.M. (2021). CAR-T Cell therapy: Current Limitations and Potential Strategies. Blood Cancer Journal, 11(4), pp.1–11. https://doi.org/10.1038/s41408-021-00459-7.
- Saez-Ibañez, A.R., et al. (2022). Landscape of cancer cell therapies: trends and real-world data. Nature Reviews Drug Discovery. https://doi.org/10.1038/d41573-022-00095-1.
- Chimeric antigen receptors. (2022). Nature Biotechnology, 40(5), pp.654–654. https://doi.org/10.1038/s41587-022-01320-3.
- Jena, B., et al. (2013). Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials. PLoS ONE, 8(3), p.e57838. https://doi.org/10.1371/journal.pone.0057838.
- Zheng, Z., Chinnasamy, N. and Morgan, R.A. (2012). Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry. Journal of Translational Medicine, [online] 10, p.29. https://doi.org/10.1186/1479-5876-10-29.
- De Oliveira, S.N., et al. (2013). A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors. Journal of Translational Medicine, 11(1). https://doi.org/10.1186/1479-5876-11-23.
- Gopalakrishnan, R., et al. (2019). A novel luciferase-based assay for the detection of Chimeric Antigen Receptors. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-018-38258-z.
- Research, C. for B.E. and (2022). Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products. [online] U.S. Food and Drug Administration. Available at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/considerations-development-chimeric-antigen-receptor-car-t-cell-products.
About ACROBiosystems
ACROBiosystems is a cornerstone enterprise of the pharmaceutical and biotechnology industries. Their mission is to help overcome challenges with innovative tools and solutions from discovery to the clinic. They supply life science tools designed to be used in discovery research and scalable to the clinical phase and beyond. By consistently adapting to new regulatory challenges and guidelines, ACROBiosystems delivers solutions, whether it comes through recombinant proteins, antibodies, assay kits, GMP-grade reagents, or custom services. ACROBiosystems empower scientists and engineers dedicated towards innovation to simplify and accelerate the development of new, better, and more affordable medicine.
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