Research paper

Single-pass, closed-system rapid expansion of lymphocyte cultures for adoptive cell therapy

Cell therapies based on T cell have gathered interest over the last decades for treatment of cancers, becoming recently the most investigated lineage for clinical trials. Although results of adoptive cell therapies are very promising, obtaining large batches of T cell at clinical scale is still challenging nowadays. We propose here a review study focusing on how bioreactor systems could increase expansion rates of T cell culture specifically towards efficient, reliable and reproducible cell therapies.

After describing the specificities of T cell culture, in particular activation, phenotypical characterization and cell density considerations, we detail the main objectives of bioreactors in this context, namely scale-up, GMP-compliance and reduced time and costs. Then, we report recent advances on the different classes of bioreactor systems commonly investigated for non-adherent cell expansion, in comparison with the current “gold standard” of T cell culture (flasks and culture bag). Results obtained with hollow fibres, G-Rex® flasks, Wave bioreactor, multiple-step bioreactors, spinner flasks as well as original homemade designs are discussed to highlight advantages and drawbacks in regards to T cells' specificities.

Although there is currently no consensus on an optimal bioreactor, overall, most systems reviewed here can improve T cell culture towards faster, easier and/or cheaper protocols. They also offer strong outlooks towards automation, process control and complete closed systems, which could be mandatory developments for a massive clinical breakthrough. However, proper controls are sometimes lacking to conclude clearly on the features leading to the progresses regarding cell expansion, and the field could benefit from process engineering methods, such as quality by design, to perform multi parameters studies and face these challenges.

Candidate cell and gene therapy products are often identified in research laboratories typically undergo extensive process development and testing prior to use in clinical trials. Methods for production and analysis of these products must adhere to strict standards for safety and demonstrate the identity, purity and potency of each product is fit for its intended use. Identifying the right partners and resources, developing robust procedures and compliance with clinical regulatory requirements all necessary for successful translation of these products to the clinic. The general considerations and examples of methods and regulations are considered in this chapter.

Candidate cell and gene therapy products are often identified in research laboratories typically undergo extensive process development and testing prior to use in clinical trials. Methods for production and analysis of these products must adhere to strict standards for safety and demonstrate the identity, purity and potency of each product is fit for its intended use. Identifying the right partners and resources, developing robust procedures and compliance with clinical regulatory requirements all necessary for successful translation of these products to the clinic. The general considerations and examples of methods and regulations are considered in this chapter.

T cells genetically targeted with a chimeric antigen receptor (CAR) to B-cell malignancies have demonstrated tremendous clinical outcomes. With the proof in principle for CAR T cells as a therapy for B-cell malignancies being established, current and future research is being focused on adapting CAR technology to other cancers, as well as enhancing its efficacy and/or safety. The modular nature of the CAR, extracellular antigen-binding domain fused to a transmembrane domain and intracellular T-cell signaling domains, allows for optimization by replacement of the various components. These modifications are creating a whole new class of therapeutic CARs. In this review, we discuss the recent major advances in CAR design and how these modifications will impact its clinical application.

The success of promising anti-cancer adoptive cell therapies relies on the abilities of the perfused CD8⁺ T lymphocytes to gain access to and persist within the tumor microenvironment to carry out their cytotoxic functions. We propose a new method for their local delivery as a living concentrate, which may not only reduce the numbers of cells required for treatment but also enhance their site-specific mobilization. Using combinations of sodium hydrogen carbonate and phosphate buffer as gelling agents, novel injectable chitosan-based biocompatible thermogels (CTGels) having excellent mechanical properties and cytocompatibility have been developed. Three thermogel formulations with acceptable physicochemical properties, such as physiological pH and osmolality, macroporosity, and gelation rates were compared. The CTGel2 formulation outperformed the others by providing an environment suitable for the encapsulation of viable CD8⁺ T lymphocytes, supporting their proliferation and gradual release. In addition, the encapsulated T cell phenotypes were influenced by surrounding conditions and by tumor cells, while maintaining their capacity to kill tumor cells. This strongly suggests that cells encapsulated in this formulation retain their anti-cancer functions, and that this locally injectable hydrogel may be further developed to complement a wide variety of existing immunotherapies.

The immune system plays a critical role in the elimination and suppression of pathogens. Although the endogenous immune system is capable of immune surveillance resulting in the elimination of cancer cells, tumor cells have developed a variety of mechanisms to escape immune recognition often resulting in tumor outgrowth. The presence of immune infiltrate in tumors has been correlated with a good prognosis following treatment (Sato et al., 2005, Loi et al., 2013, Clemente et al., 1996, Galon et al., 2006). As such, immune cells such as T cells, have been harnessed in order to target cancer. Tumor reactive lymphocytes, called tumor-infiltrating lymphocytes (TILs) have been isolated and expanded from the tumor and reinfused back into patients for the treatment of melanoma. The promise of adoptive immunotherapy utilizing TILs as a robust treatment for cancer has been highlighted in patients with advanced melanoma with greater than 50% of patients responding to treatment (Dudley et al., 2005). Although TIL therapy has shown promising results in melanoma patients, it has proved difficult to translate this approach to other cancers, given that the numbers of TILs that can be isolated are generally low. To broaden this therapy for other cancers, T cells have been genetically modified to endow them with tumor reactivity using either a T cell receptor (TCR) (Parkhurst et al., 2009, Parkhurst et al., 2011, Chinnasamy et al., 2011) or a chimeric antigen receptor (CAR) (Grupp et al., 2013, Park et al., 2007). This review will outline the origins and development of adoptive immunotherapy utilizing TILs leading to genetic modification strategies to redirect T cells to cancer. Potential hurdles and novel strategies will be discussed for realizing the full potential of adoptive immunotherapy becoming a standard of care treatment for cancer.