Manufacturing chimeric antigen receptor T cells: issues and challenges
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.
References (86)
- et al.
Artificial T-cell receptors
Cytotherapy
(2003) - et al.
A guide to manufacturing CAR T cell therapies
Curr Opin Biotechnol
(2018 Feb 17) - et al.
T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma
Blood
(2016 29) - et al.
An APRIL-based chimeric antigen receptor for dual targeting of BCMA and TACI in multiple myeloma
Blood
(2018 Feb 15) GMP CAR-T cell production
Best Pract Res Clin Haematol
(2018 Jun)- et al.
CAR-T Cell Therapies From the Transfusion Medicine Perspective
Transfus Med Rev
(2016) - et al.
Donor lymphocyte collections using the spectra Optia MNC version 5
Transfus Apher Sci Off J World Apher Assoc Off J Eur Soc Haemapheresis
(2013 Apr) - et al.
Optimization of leukocyte collection and monocyte isolation for dendritic cell culture
Transfus Med Rev
(2010 Apr) - et al.
Activation-induced T cell apoptosis by monocytes from stem cell products
Int Immunopharmacol
(2001 Jul) - et al.
Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells
Cytotherapy
(2016)
Large-scale Ficoll gradient separations using a commercially available, effectively closed, system
Cytotherapy
Clinical manufacturing of CAR T cells: foundation of a promising therapy
Mol Ther Oncolytics
Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy
Cytotherapy
Artificial antigen-presenting cells expressing CD80, CD70, and 4-1BB ligand efficiently expand functional T cells specific to tumor-associated antigens
Immunobiology
AUtomated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy
Cytotherapy
Gene Therapy with the Sleeping Beauty Transposon System
Trends Genet TIG
Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells
Cell
Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4(+) T Cells
Mol Ther J Am Soc Gene Ther
Anti-leukemic potency of piggyBac-mediated CD19-specific T cells against refractory Philadelphia chromosome-positive acute lymphoblastic leukemia
Cytotherapy
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
Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells
Blood
Simplified process for the production of anti-CD19-CAR-engineered T cells
Cytotherapy
Optimizing the production of suspension cells using the G-Rex “M” series
Mol Ther Methods Clin Dev
IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes
Blood
Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center
Cytotherapy
Temperature fluctuations during deep temperature cryopreservation reduce PBMC recovery, viability and T-cell function
Cryobiology
Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy
Cytotherapy
CD19 chimeric antigen receptor T cell therapy for haematological malignancies
Br J Haematol
Chimeric antigen receptor T cells for sustained remissions in leukemia
N Engl J Med
Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia
N Engl J Med
CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients
J Clin Invest
CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia
Sci Transl Med
Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor
J Clin Oncol
The growing world of CAR T cell trials: a systematic review
Cancer Immunol Immunother CII
Bluebird's BCMA CAR-T impresses at ASH
Nat Biotechnol
Clinical and immunological responses after CD30-specific chimeric antigen receptor-redirected lymphocytes
J Clin Invest
Targeting the T cell receptor β-chain constant region for immunotherapy of T cell malignancies
Nat Med
CAR T Cell Therapy for Glioblastoma: Recent Clinical Advances and Future Challenges
Neuro-Oncol
Safety and Efficacy of Intratumoral Injections of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer
Cancer Immunol Res
The expanding role of the clinical haematologist in the new world of advanced therapy medicinal products
Br J Haematol
CAR T Cells in Trials: Recent Achievements and Challenges that Remain in the Production of Modified T Cells for Clinical Applications
Hum Gene Ther
Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells
Immunol Rev
Cited by (80)
Pericyte derivation and transplantation for blood-CNS barrier reconstitution in CNS disorders
2024, IBRO Neuroscience ReportsBasics of advanced therapy medicinal product development in academic pharma and the role of a GMP simulation unit
2023, Immuno-Oncology and TechnologyBeyond youth: Understanding CAR T cell fitness in the context of immunological aging
2023, Seminars in ImmunologyCAR-T cells, the first pharmaceutical cell therapy
2023, Transfusion and Apheresis ScienceAdvancing cell-based cancer immunotherapy through stem cell engineering
2023, Cell Stem CellAntigen receptor structure and signaling
2023, Advances in Immunology