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Review Immunotherapy Clinical Development and Manufacture of Chimeric Antigen Receptor T cells and the Role of Leukapheresis Andrew Fesnak and Una O’Doherty University of Pennsylvania, Philadelphia, Pennsylvania, US A doptive transfer of chimeric antigen receptor (CAR) T cells is a powerful targeted immunotherapeutic technique. CAR T cells are manufactured by harvesting mononuclear cells, typically via leukapheresis from a patient’s blood, then activating, modifying the T cells to express a transgene encoding a tumour-specific CAR, and infusing the CAR T cells into the patient. Gene transfer is achieved through the use of retroviral or lentiviral vectors, although non-viral delivery systems are being investigated. This article discusses the challenges associated with each stage of this process. Despite the need for a consistent end product, there is inherent variability in cellular material obtained from critically ill patients who have been exposed to cytotoxic therapy. It is important to carefully select target antigens to maximise effect and minimise toxicity. Various types of CAR T cell toxicity have been documented: this includes “on target, on tumour”, “on target, off tumour” and “off target” toxicity. A growing body of clinical evidence supports the efficacy and safety of CAR T cell therapy; CAR T cells targeting CD19 in B cell leukemias are the best-studied therapy to date. However, providing personalised therapy on a large scale remains challenging; a future aim is to produce a universal “off the shelf” CAR T cell. Keywords Leukapheresis, chimeric antigen receptor (CAR) T cells, tumour antigens, harvesting, manufacture, toxicity Disclosure: Andrew Fesnak and Una O’Doherty have nothing to disclose in relation to this article. This study involves a review of the literature and did not involve any studies with human or animal subjects performed by any of the authors. Acknowledgements: The contents of the paper and the opinions expressed within are those of the authors, and it was the decision of the authors to submit the manuscript for publication. The authors took responsibility for the writing of this manuscript, including critical review and editing of each draft, and approval of the submitted version. The authors received writing/ editorial support in the preparation of this manuscript provided by Catherine Amey and Katrina Mountfort from Touch Medical Media, which was funded by Terumo BCT. Authorship: All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship of this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval to the version to be published. Open Access: This article is published under the Creative Commons Attribution Noncommercial License, which permits any non-commercial use, distribution, adaptation and reproduction provided the original author(s) and source are given appropriate credit. Received: 29 November 2016 Accepted: 4 January 2017 Citation: European Oncology & Haematology, 2017;13(1):28–34 Corresponding Author: Andrew Fesnak, 3 White Building, Hospital of the University of Pennsylvania, Philadephia, PA 19104, US. E: Support: The publication of this article was supported by Terumo BCT. The views and opinions expressed are those of the authors and not necessarily those of Terumo BCT. 28 Advances have been made in the use of genetically enhanced T cell therapy, in particular, chimeric antigen receptor (CAR) T cells. Such CAR T cells have been shown to be efficacious in erradicating a number of haematologic malignancies. 1–3 CARs are tailored fusion receptors that can combine the specificity of an antigen-specific antibody with numerous downstream signalling domains, the most well-described being T cell activating domains. First developed in the mid-1980s, CARs initially consisted of a variable antigen-binding region of a monoclonal antibody and the constant regions of a T cell receptor (TCR) α and β chains. 4,5 Subsequent CARs have been modified to include several functional domains (Rev. 6,7 , Figure 1) including : • an ectodomain from a single chain variable fragment (scFv) derived from the antigen binding regions of both heavy and light chains of a monoclonal antibody; • the hinge region that connects the ectodomain to the transmembrane domain, producing variations in the length and variability of the resulting CAR and affecting its function; a transmembrane domain that is usually derived from CD3-zeta (ζ), CD4, CD8 or CD28 molecules and also has an impact on CAR function; • an in cis costimulatory domain, a critical component without which the CAR T cell will become anergic upon target encounter. Adding a costimulatory domain activates different signalling pathways and prolongs T cell persistence: two costimulatory domains are included in third- generation CARs to fine-tune the T cell response; • an endodomain with a signalling domain typically derived from the T cell CD3-ζ chain. CAR T cells can recognise not only protein, but also carbohydrate and glycolipid structures that are expressed on the tumour cell surface. 8 Engineered and endogenous TCRs recognise portions of peptide presented in the context of the major histocompatibility complex (MHC). MHC-independent binding of a CAR to target allows for use of these cells regardless of host MHC polymorphisms. Unlike engineered TCR expressing cells, however, CAR T cells have long been thought to be unable to target intracellular tumour markers. Recently, CARs targeting the extracellular presentation of intracellular tumour markers in the context of a specific MHC have been developed. 9,10 This new approach may vastly expand the repertoire of potential CAR targets. This article aims to describe the process involved in the manufacture of CAR T cells, from the first critical step, harvesting via leukapheresis, to reinfusion, as well as reviewing the clinical evidence in support of the use of CAR T cells. TOU C H ME D ICA L ME D IA