The Problem with Gene-Modified Cell Therapy Manufacturing

By Ryan Pawell

Gene-modified cell therapies (GMCTs) represent the most effective therapeutic platform for many patients with advanced diseases, including relapsed and refractory leukemia, non-Hodgkin lymphoma, and other blood cancers [1].

Specifically, chimeric antigen receptor T cell (CAR-T) therapies targeting CD19 have demonstrated remarkable responses and possibly cures in patients with advanced acute lymphoblastic leukemia (ALL) unresponsive to all prior therapies. Gene-modified CAR-T cells are the first cellular therapy to gain FDA approval for cancer treatment following the demonstration of an 83% remission rate in ALL [2].

CAR-T cells are generated via genetic modification of human T cells to display an extracellular antibody single-chain variable fragment (scFv) linked to a hinge region, one or more co-stimulatory domain(s), and an intracellular activating domain. 

Sounds Great, Right?

The most problematic step in GMCT manufacturing is the intracellular delivery of nucleic acids via transfection or transduction to express the CAR by the T-cell. Viral transduction using lentiviruses is used to generate CAR-T therapies for clinical trials. However, these methods require significant hands-on time during production and extensive intra- and post-production safety testing to avoid infusion of replication competent viruses at therapy administration [3,4].

There are several practical metrics when considering intracellular delivery for GMCT development and manufacturing, including:

1. Cell recovery

2. Cell viability

3. Delivery or expression efficiency

4. Throughput

5. Maintenance of normal or desired cell state and function.

Let’s Break It Down

Physical transfection methods, such as electroporation, are appealing alternatives for GMCT manufacturing [5,6]. Electroporation does not require extensive safety or release precautions. It can deliver a broad range of constructs into cells (e.g., DNA, RNA, proteins and/or various complexes) but can result in significant cell losses or alteration of normal cell function [7,8].

Low cell recovery rates are not ideal due to the large number of cells required for GMCTs. High cell viability is also essential as cells are frequently expanded after modification, and low cell viability or dead cells is known to reduce cell growth rates. Additionally, nonviable cells can induce an adverse immune response [9]. 

Regulators require a minimum percentage of viable cells for cell therapies as low cell viability at the time of administration can induce infusion toxicity and lessen therapeutic effects [10,11].

Delivery should induce a therapeutic effect without altering cell state, and the efficiency of cell modification needs to be sufficiently high to avoid additional processing steps like dead cell removal. Zhang et al. used electroporation to transfect naïve T-cells with plasmids and found recovery rates of less than 20% after 24 hours [7].

In addition to greatly reducing viability, electroporation increased the expression of T-cell surface activation markers, which requires additional post-transfection recovery time before therapy administration [7].

Intracellular Delivery Via Microfluidic Vortex Shedding

Indee Labs is the first to apply microfluidic vortex shedding (µVS) to intracellular delivery, where the fluid forces or hydrodynamic conditions created during µVS are used to gently and temporarily porate the cell membrane, allowing for gene delivery in a novel manner.

Specifically, these microfluidic devices use a tiny array of posts to induce a well-known fluid dynamics phenomenon called vortex shedding. You can observe vortex shedding in nature as clouds drift past mountains or streams flow past rocks and boulders.

Microfluidics is being used to actively improve traditional intracellular delivery methods [12,13]. However, a substantial need for a practical microfluidic intracellular delivery method remains, particularly within the scope of GMCT development and manufacturing. At Indee Labs, we have developed a hydrodynamic intracellular delivery platform based on µVS.

 We have optimized our platform for enhanced green fluorescent protein (EGFP) mRNA delivery to human pan T cells [14]. We demonstrate µVS results in

1. High cell recovery (e.g., 96.3 ± 1.1%, mean ± stdev),

2. High cell viability (e.g., 83.7 ± 0.7%) and

3. High EGFP expression efficiency (e.g., 57.4 ± 6.8%) resulting a

4. Yield of 46.3 ± 5.6% recovered, viable, and EGFP expressing pan T cells after intracellular delivery.

We also demonstrate:

1. µVS does not adversely affect T cell growth.

2. µVS results in even EGFP expression profiles amongst T cell types.

3. µVS does not change T cell activation profiles.

Introducing Hydropore

Now, we are developing Hydropore™, a user-friendly µVS instrument, for research then clinical use in collaboration with the Westmead Institute for Medical Research and funded by NSW Health.

These µVS devices can potentially eliminate the two primary barriers to widespread gene-modified cell therapy like CAR-T: cost and scalability. The device may also reduce or eliminate many of the challenges described above.

We look forward to updating you on our progress as we work towards practical and scalable gene-modified cell therapies like CAR-T.

References

1. Kochenderfer, J. N. et al. Long-Duration Complete Remissions of Diffuse Large B Cell Lymphoma after Anti-CD19 Chimeric Antigen Receptor T Cell Therapy. Mol. Ther. J. Am. Soc. Gene Ther. 25, 2245–2253 (2017).

2. Ledford, H. Engineered cell therapy for cancer gets thumbs up from FDA advisers. Nat. News 547, 270 (2017).

3. Wang, X. & Rivière, I. Clinical manufacturing of CAR T cells: foundation of a promising therapy. Mol. Ther. Oncolytics 3, 16015 (2016).

4. Levine, B. L., Miskin, J., Wonnacott, K. & Keir, C. Global Manufacturing of CAR T Cell Therapy. Mol. Ther. - Methods Clin. Dev. 4, 92–101 (2017).

5. Stewart, M. P. et al. In vitro and ex vivo strategies for intracellular delivery. Nature 538, 183–192 (2016).

6. Ramanayake, S. et al. Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials. Cytotherapy 17, 1251–1267 (2015).

7. Zhang, M. et al. The impact of Nucleofection® on the activation state of primary human CD4 T cells. J. Immunol. Methods 408, 123–131 (2014).

8. Liu, L., Johnson, C., Fujimura, S., Teque, F. & Levy, J. A. Transfection optimization for primary human CD8+ cells. J. Immunol. Methods 372, 22–29 (2011).

9. Peng, Y. et al. Innate and adaptive immune response to apoptotic cells. J. Autoimmun. 29, 303–309 (2007).

10. Morgenstern, D. A. et al. Post-thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes. Br. J. Haematol. 174, 942–951 (2016).

11. Watts, M. J. & Linch, D. C. Optimisation and quality control of cell processing for autologous stem cell transplantation. Br. J. Haematol. 175, 771–783 (2016).

12. Ding, X. et al. High-throughput Nuclear Delivery and Rapid Expression of DNA via Mechanical and Electrical Cell-Membrane Disruption. Nat. Biomed. Eng. 1, (2017).

13. Woodruff, K. & Maerkl, S. J. A High-Throughput Microfluidic Platform for Mammalian Cell Transfection and Culturing. Sci. Rep. 6, 23937 (2016).

14. Twite, A. et al. Intracellular delivery of mRNA to human primary T cells with microfluidic vortex shedding. bioRxiv (2018).