CAR-T: from Design to Manufacturing

By: Mohamad Toutounji, Lucas Klemm, Kawthar Braysh, Alejandro Barquero, Alvaro Eguileor Giné and Alex Prokopienko

Bioprocessing of CAR-T Cell Therapy

After the successful research and development of cellular cancer therapies based on T cell activation, CAR-T therapies are being manufactured and applied on larger cohorts of patients around the globe. However, the manufacturing and regulatory challenges are complex processes to maintain high quality of the therapy. The following scheme (L. Levine et. al. 2017) shows the main steps of the ex vivo therapy manufacturing.

Culture Strategies for Cell Therapies

There are two main strategies for manufacturing of cell therapy.

1.     Planar Culture System: MSCs and hPSCs are grown as adherent colonies, anchored to tissue culture plastic, feeder layers, or synthetic substrates in vessels such as T-flasks. T-cells for CAR T are cultured in suspension in T-flasks or static cell culture bags. This strategy has restrictions in the physical depth of the media in T-flasks and stacked planar vessels mean that the volume of media per surface area, and thus achievable cell population sizes, of such vessels is limited. T-cell populations grown in T-flasks must therefore be passaged more frequently even that if a static culture bag was employed as the cell culture platform.

2.     Three-Dimensional Culture System: Online process monitoring via bioreactors provides scalability potential, reduces facility size requirements and importantly it permits strict control of environmental conditions during cell expansion and manufacturing. There are many 3D cell culture platforms for cell therapies manufacturing, these are displayed in the following table (Farid et. al. 2018).

Strategies for hPSC Differentiation and Transfection

CAR-T cells can be manufactured as either autologous therapies using patients’ own cells or as allogeneic therapies using induced pluripotent stem cells (iPSCs). Autologous CAR-T cells can only be engineered as differentiated cells and have variable editing efficiencies, though technology is improving rapidly to address this. On the other hand, allogeneic iPSCs can be engineered either before or after differentiation into effector cells, such as T cells in this case. The advantage to engineering iPSCs prior to differentiation is a homogeneous CAR-T cell product versus a heterogeneous population that will result from transfection post-differentiation. Multiple cell line clones can be characterized to determine the best combination of on-target efficacy and lowest off-cancer toxicities to patients. 

Genetic Engineering of Cells for Cell Therapies

CAR-T cells, (Chimeric antigen receptor T cells), are T lymphocytes, collected from the patient’s blood and then genetically modified in vitro to express an artificial receptor, known as a chimeric receptor. Traditionally T cells have been modified to express CARs using viral vectors, which typically work by inserting CAR-encoding DNA randomly over the cell’s genome. This arbitrary pasting of DNA can lead to disruption of endogenous genes (a potentially oncogenic process in itself) and limits the control over CAR expression levels. If the CAR is expressed constitutively, the cells often become dysfunctional and enter a state called exhaustion. To sort out this problem and expand possibilities for CAR expression, applications of gene editing technologies have been developed in the last few years to enable targeted genetic manipulation such as CRISPR-Cas9. A tighter control over CAR insertion and expression has great potential of generating tumour-specific T cells.

Delivery Systems for CRISPR/Cas9-based CAR-T Cell Therapy

A, Viral delivery of CAR or Cas9/sgRNA into T cells. CAR genes are delivered by lentivirus or retrovirus and integrate into the genome for stable expression, while cas9 and sgRNA are often transiently expressed by AdV or AAV. B, Transposition mechanisms of DNA transposon. DNA transposon contains a target gene in the middle, flanked by TIRs. Transposase binds to the TIRs, and mobilizes the transposon for integration into the target genome via a cut‐and‐paste mechanism. TIR: terminal inverted repeats. C, Cas9 and sgRNA are delivered by electroporation in the form of plasmid, RNA, chem‐RNA, or RNP. D, Delivery of DNA, RNA, or protein into target cells via nanoparticles. E, Five main steps of delivery via CellSqueeze technology. scFv, single‐chain variable fragment; Cas9, CRISPR associated protein 9; AAV, adenovirus associated virus; RNP, ribonucleoprotein

Generation of CAR-T Cell Therapy

Schematic diagram of the CAR-T cell structure. In the first generation of CARs, there was only one intracellular signal component CD3ζ. The second generation of CAR added one costimulatory molecule on the basis of the first generation. Based on the second generation of CARs, the third generation of CAR added another costimulatory molecule. Fourth-generation of CAR-T cells can activate the downstream transcription factor to induce cytokine production after the CAR recognizes the target antigens. The fifth-generation of CARs, based on the second generation, uses gene editing to inactivate the TRAC gene, leading to the removal of the TCR alpha and beta chains.

The possibilities of CRISPR-Cas9 for therapeutic T cells are broad: knock-in of functional genes, such as interleukins and suicide genes, to product next-generation CAR-T cells, other strategies comprises knock-out of endogenous genes, such as TCRs and MHCs, to develop ‘off-the-shelf’ universal CAR-T cells.

CAR-T cells Harvesting, Concentration, and Purification

CAR-T cells are easily collected as they are grown in single-cell suspension. For autologous CAR-T cell therapy products that are likely to result in small media volumes (5–10L), small scale systems such as the Haemonetics Cell Saver and COBE system can be utilized for washing and concentration steps. However, for large-scale production of allogeneic cell therapies, scalable systems such as the kSep that handle media large volumes (up to 1000L in a single run) are employed. The purification of allogenic CAR-T cells is then required to remove cells that still express unwanted TCRs and cause Graft versus host disease. This can be performed using affinity-based cell purification techniques such as Magnetic activated cell sorting (MACS) and fluorescent activated cell sorting (FACS). Given its ease of use and high throughput, CliniMACS platform is the most commonly used MACS technology where large lot sizes are entailed.

See our previous articles on CRISPR as an Ex-vivo Therapeutic Approach

We gratefully acknowledge @Mohammed Shadid for organizing this educational initiative.