Scalable protocols for T cell activation

This article is based on a poster originally authored by Weiping Sun, Xing Zhang, Zhaolu Yan, Chen Yu, Spencer Chiang, and Tianfu Zhang.

Immunotherapy is an advanced and widely used treatment due to its capacity to repress tumor cells, activating the immune system to limit these cells’ potential for survival, proliferation, and invasion.

Several immune cell types have been used as part of immunotherapeutic strategies in cancer treatment, including T cells, B cells, and dendritic cells. The use of T cells, in particular, has been widely documented.¹

Tumor-reactive immune cells can be grown following patient leukapheresis. These can then be gene-edited and/or cultivated with immunomodulatory functions and reinfused to help inhibit tumor growth.

Adoptive cell transfer therapies include:

• Chimeric antigen receptor T cells (CAR-T)
• T cell receptor-modified T cell (TCR-T)
• Tumor-infiltrating lymphocytes (TILs)

The adoptive transfer of CAR-T cells is leading this new therapeutic paradigm, but there are longstanding challenges surrounding the manufacture of T cells, with critical steps, including:

• T cell isolation
• T cell activation
• T cell expansion

ACROBiosystems has developed a workflow to help address these challenges. It leverages its innovative solutions to support T cell activation and expansion and includes a potent range of activating antibodies, cytokines, and expansion medium.

The company’s GMP ActiveMax^® Human T cell Activation/Expansion CD3/CD28 Beads are suitable for the in vitro isolation, stimulation, and expansion of T cell populations, including CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and human PBMCs.

Once activated, it is necessary to expand T cells to therapeutic-relevant numbers. This can be achieved using CelThera^™ GMP T Cell Expansion Medium – an animal origin-free, serum-free, and chemically defined medium produced under GMP conditions.

Using this medium mitigates any risk of pathogenic microorganism introduction, improving batch-to-batch consistency versus conventional and xeno-free media. This medium has also been specifically designed to enable the large-scale expansion of T cells, removing the need for serum or serum replacements.

Additional cytokines, such as GMP human IL-2 protein, human IL-7, human IL-15, or human IL-21 protein, are also required for T cell expansion. Global regulatory guidelines stipulate that GMP grades of each reagent should be available throughout the whole process to eliminate contamination risks and ensure that the final therapeutic is safe for use.

Methods and materials

CelThera^™ GMP T Cell Expansion medium (ACROBiosystems, Cat. No. GMP-CM3101) was initially supplemented with:

• 500 IU/ml GMP Human IL-2 Protein (ACROBiosystems, Cat. No. GMP-L02H14)
• 10 ng/ml GMP Human IL-7 Protein (ACROBiosystems, Cat. No. GMP-L07H24)
• 10 ng/ml GMP Human IL-15 Protein (ACROBiosystems, Cat. No. GMP-L15H13)

PBMCs were isolated from the blood sample using density gradient centrifugation with a Ficoll solution.

The activation and expansion phase begins with the preparation of 3 × 10⁶ PBMCs in 3 mL of CelThera^™ GMP T Cell Expansion medium with cytokines in a 6-well tissue culture plate.

Next, pre-washed and resuspended GMP ActiveMax^® Human T cell Activation/Expansion CD3/CD28 Beads (ACROBiosystems, Cat. No. GMP-MBS001) were added to the well at a 1:1 ratio.

These were then incubated in a humidified CO₂ incubator at 37 °C, with 3 mL of fresh medium gently added on day two. Cell concentrations were counted using an AOPI-staining method on days 4, 6, 8, 10, and 13.

Cell concentrations were adjusted to 3-5 × 10⁵ / mL with fresh medium before testing for CD3, CD4, CD8, CD25, CD69, CD45RO, CD62L, and PD-1 expression using flow cytometry.

Results

Activation of PBMCs

Figure 1. Human PBMCs were activated using Human T cell Activation/Expansion CD3/CD28 Beads (ACRO, Cat. No. GMP-MBS0 01) for 24 hours with CelThera^™ GMP T Cell Expansion medium (ACROBiosystems, Cat. No. GMP- CM3101) and Company A medium, respectively. The result shows that CD3/CD28 beads activate more CD3⁺CD25 ⁺CD69⁺ T cells in our GMP medium, outperforming Company A medium. Image Credit: ACROBiosystems

T cell activation and expansion workflow

Image Credit: ACROBiosystems

Expansion and viability of the human CD3⁺ T cells

Figure 2. CelThera^™ GMP T Cell Expansion Medium supports a higher expansion fold of T cells, regardless of (A) CD3⁺ T cells, (B) CD3⁺ CD4⁺ T cells, and (C) CD3⁺CD8⁺ T cells, compared to the Company A medium. (D) The percentage of CD3⁺ cells in living cells is still above 99%, with more than 3,000-fold proliferation, at the end of the culture process. The proportion of CD3⁺ CD8⁺ T cells in CD3⁺ T cells raises rapidly along the culture process. (E,F) At the end of culture, the percent of CD3⁺ CD8⁺ T cells reaches to more than 60%, and the percent of CD3⁺ CD4⁺ T cell is less than 30%. The expansion fold of CD3⁺ CD8⁺ T cells and CD3⁺ CD4⁺ T cells is 6000-fold and 1500-fold, respectively. (G) After 8 days of culture, T cell viability was 95% in CelThera^™ GMP T Cell Expansion Medium, compared to 90% in the Company A medium. Image Credit: ACROBiosystems

Memory/ Exhaustion phenotypes of T cells

Figure 3. (A) The percentages of T_N/_SCM and T_CM in CD4⁺ and CD8⁺ parent groups cultured in CelThera^™ GMP T Cell Expansion Medium were much higher than in Company A medium throughout the culture period. Moreover, the majority memory phenotype of T cells are T_N/_SCM, followed by T_CM, which means that the final cell product likely have sustained proliferation, prolonged survival, and so on. (B) Activated T cells were expanded for up to 14 days and evaluated for PD-1 expression, a marker for cell death. Low PD -1 expression of the activated human T cells was observed with PD1⁺ T cell percentages in the CelThera^™ GMP T Cell E xpansion Medium lower than other company’s medium. Image Credit: ACROBiosystems

Conclusion

The combination of CelThera^™ T cell Expansion Medium, ActiveMax^® Human T cell Activation/Expansion CD3/CD28 Beads, and GMP-grade growth factors was essential in ensuring a successful T cell activation and expansion protocol.

Because it exclusively uses GMP-grade reagents, this workflow is highly scalable and suitable for use when transitioning into clinical trials.

This workflow demonstrates improved efficiencies at every stage, with ActiveMax^® Human T Cell Activation/Expansion CD3/CD28 Beads ensuring effective T cell activation and expansion from PBMCs.

It is simple and straightforward to incorporate this method into any workflow designed to isolate, activate, and expand human T cells or human PBMCs.

The CelThera^™ GMP T Cell Expansion Medium is a chemically defined and serum-free culture medium specifically developed to support human T cell culture, 14 days to 3,000-fold expansion.

This workflow enables the effective monitoring of different cell types throughout the culturing period, which changes daily. It is important to note that the proportion of T_N/_SCM cells can be defined from day seven – a key consideration when manufacturing cell therapies.

References and further reading

1. Dasgupta, S., et al. (2024). Potential role of immune cell therapy in gynecological cancer and future promises: a comprehensive review. Medical oncology (Northwood, London, England), (online) 41(5), p.98. https://doi.org/10.1007/s12032-024-02337-1.
2. Speiser, D.E., Ho, P.-C. and Verdeil, G. (2016). Regulatory circuits of T cell function in cancer. Nature Reviews Immunology, 16(10), pp.599–611. https://doi.org/10.1038/nri.2016.80.
3. Watanabe, N., Mo, F. and McKenna, M.K. (2022). Impact of Manufacturing Procedures on CAR T Cell Functionality. Frontiers in Immunology, (online) 13. https://doi.org/10.3389/fimmu.2022.876339.

Acknowledgments

Produced from materials originally authored by Weiping Sun, Xing Zhang, Zhaolu Yan, Chen Y, Spencer Chiang, and Tianfu Zhang from ACROBiosystems.

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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