Elsevier

Cytotherapy

Volume 21, Issue 3, March 2019, Pages 327-340
Cytotherapy

Manufacturing chimeric antigen receptor T cells: issues and challenges

https://doi.org/10.1016/j.jcyt.2018.11.009Get rights and content

Abstract

Clinical trials of adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have delivered unprecedented responses in patients with relapsed refractory B-cell malignancy. These results have prompted Food and Drug Administration (FDA) approval of two CAR T-cell products in this high-risk patient population. The widening range of indications for CAR T-cell therapy and increasing patient numbers present a significant logistical challenge to manufacturers aiming for reproducible delivery systems for high-quality clinical CAR T-cell products. This review discusses current and novel CAR T-cell processing methodologies and the quality control systems needed to meet the increasing clinical demand for these exciting new therapies.

Section snippets

Introduction to CAR T cells

Following years of pre-clinical development, immunotherapy is now recognized as a compelling alternative/adjuvant to conventional anti-cancer therapies. Adoptive transfer of chimeric antigen receptor (CAR) T cells is at the forefront of this new therapeutic paradigm.

A CAR is a synthetic protein, genetically engineered to be expressed on the T-cell surface. It comprises an extracellular binding domain (typically a single-chain variable fragment targeting a tumor cell surface antigen), a hinge

Manufacturing CAR T cells to GMP compliance

CAR T cells for clinical application are manufactured to GMP compliance [19], [20]. GMP regulations define a system by which potent products are manufactured safely according to standardized methods under closely controlled, reproducible and auditable conditions [20]. Rigorous quality control systems are applied to the procurement and use of starting materials, reagents and consumables, to in-process equipment and methods and to the final ‘drug’ product [20], [21], [22].

In the United States

CAR T-cell manufacture methodology: general principles

The ability of CAR T cells to expand in vivo (T-cell ‘fitness’) is associated with anti-tumor responses [26] and can be impacted by the manufacture method used. Efforts to optimize and quality control individual elements of the manufacture process are desirable.

Despite variation in practices and technology between centers, all CAR T cells are manufactured in closed or functionally closed processes to reduce contamination risks and share the common steps illustrated in Figure1. The average

T-cell harvesting using leukapheresis: current practices and quality control

A critical starting material for CAR T-cell manufacture is CD3+ T cells derived from non-mobilized leukapheresis. Mononuclear cells (MNCs) are separated from anticoagulated whole blood according to a density gradient in a functionally closed system [27]. Approved equipment include the COBE Spectra, the Spectra Optia (TerumoBCT Inc.) and the Amicus Cell Separator (Fenwal Inc./Fresenius Kabi AG).

Optimal CD3+ T-cell apheresis collection parameters for downstream CAR T-cell manufacture are not well

T-cell enrichment post-apheresis and initial processing steps: quality aspects

From a quality control perspective, standardized cellular starting material is highly desirable. Contamination with platelets, plasma and residual anticoagulant can alter T-cell responsiveness to activation reagents [33]; red blood cells and platelets can compromise in-process cell counting and flow cytometry [34] and granulocytes and monocytes can inhibit T-cell expansion and transduction during the CAR T-cell manufacture process [35], [36], [37].

A washing step can remove platelets, plasma and

T-cell activation methods: issues and challenges

T-cell activation is critical to CAR T-cell transduction efficiency. It is achieved in vitro using antigen-presenting technologies to deliver concurrent T cell receptor (TCR) and co-stimulatory signals. Superseded approaches include the use of autologous antigen-presenting cells or cell lines modified to express co-stimulatory ligands. These are labor intensive to generate and of variable efficiency [45].

Artificial antigen-presenting technologies such as immobilized anti-CD3 and anti-CD28 mAbs

Gene delivery/transduction

This can be subdivided into viral and non-viral delivery systems [48]. Retroviral and lentiviral vectors are the most common gene delivery methods used in CAR T-cell manufacture, accounting for 41% and 54%, respectively, of all manufactured products [9]. Transduction efficiency is generally high and requires T-cell activation for gene transfer [49]. Viral vector is harvested into sub-batches and undergoes purification and sterile filtration prior to cryopreservation and quality control testing

T-cell expansion and fully automated CAR T-cell manufacture platforms

T cells are expanded following gene transfer to reach cell numbers required for clinical application in functionally closed culture systems [20]. A variety of methods can be used including T-flasks, plates or culture bags and bioreactors such as the G-Rex flask (Wilson Wolf Manufacturing), the WAVE Bioreactor (GE Life Systems) and the CliniMACS Prodigy (Miltenyi BioTec). A recent review indicates that 43% of CAR T-cell clinical trials use rocking bioreactors [46], [49] compared with 35% that

T-cell cryopreservation

Cryopreservation of CAR T-cell products prior to infusion is standard practice. Advantages include flexible scheduling of patient infusions and time to complete extended quality control tests required for QP review and release. Validated cryopreservation is essential for large-scale centralized manufacture.

From a quality perspective, this step is critical, because suboptimal cryopreservation can lead to reduced cell numbers, impaired viability and altered cell phenotype and function [71]. Most

CAR T-cell therapy quality assurance: general principles

GMP regulation mandates assessment of CAR T-cell safety, purity and potency [25]. These metrics are used as quality release criteria [20].

Safety stipulates absolute sterility from bacteria and fungi and where viral vector is used, the absence of RCV. Culture-based sterility methods, such as the BD BacTEC system, involve inoculation of test material into culture media followed by observation for the growth of microorganisms over a set follow-up period [25]. Culture-based sterility assessment of

CAR T-cell release testing

CAR T-cells products should be released under a CoA following receipt of a physician's prescription. The CoA must detail the release assay methodology (e.g., product identity, purity, viability and potency), the testing site and the minimum specification for release alongside the actual results obtained. Release testing should be performed according to regulatory authority–approved assays. Where this is not possible, in-house assay validation will be required to prove assay integrity. According

Conclusions

CAR T cells herald an exciting era in cancer treatment and widespread adoption is expected. There remain many challenges to standardized and economical manufacture of these therapies. Furthermore, widely available validated assays to qualify potency of CAR T-cell products do not yet exist, making it challenging to compare CAR T-cell products prior to infusion to patients.

Promising engineering solutions, standardized biomaterials and in-process controls [9], [86] and huge strides in automation

Acknowledgments

We acknowledge Dr Martin Pule (Director of the UCL CAR T-cell program), Professor Karl Peggs (Principle Investigator on the CARD study) and Ms Paulina Nowosiad, Ms Mahnaz Abbassian, Ms Leticia Bosshard-Carter and Dr Rita Rego for GMP manufacture on the UCL CAR T-cell program. The CARD study has received funding from the European Union Seventh Framework Programmeunder grant agreement number 602239 ATECT.

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