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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,
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.
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