Adoptive 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.
Leukapheresis, chimeric antigen receptor (CAR) T cells, tumour antigens, harvesting, manufacture, toxicity
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.
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.
November 29, 2016 Accepted
January 04, 2017
Andrew Fesnak, 3 White Building, Hospital of the University of Pennsylvania, Philadephia, PA 19104, US. E: firstname.lastname@example.org
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.
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.
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 thirdgeneration 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.
Generation of CAR T cells
CAR T cell manufacture begins with collection of mononuclear cells (MNCs) from the patient’s blood via leukapheresis (Figure 2). CD3+ T cells may be further enriched ex vivo and then modified to express a transgene encoding a tumour-specific CAR. The CAR-modified T cells undergo ex vivo expansion in optimised T cell culture conditions. The final product is harvested and formulated at a specified dose. Finally, quality and release testing are performed on the product to ensure safety of the infusible product.
Harvesting and isolating T cells for CAR T cell manufacturing
To date, leukapheresis is the most efficient way to obtain significant numbers of T lymphocytes to initiate CAR T cell culture. Apheresis, which is derived from an ancient Greek word meaning “to take away”, describes the process by which whole blood is removed from an individual to be separated into components by centrifugation; subsequently, one or more components are selectively removed and the remainder of the blood is returned to the circulation.11 For the purpose of CAR T cell manufacture, the MNC layer containing lymphocytes and monocytes is the target cell layer to be collected by the apheresis device. Leukapheresis remains the method of choice for T cell collection given that semi-continuous or continuous flow of the blood allows for processing of large blood volumes providing large T cell yields.
It may be challenging to collect MNC products with adequate purity and/ or yield. Monocytes and non-T lymphocytes present in the MNC product can impede downstream CAR T cell manufacturing, and therefore additional purification may be required.12 The MNC layer itself also contains monocytes and non-T lymphocytes including B cells and NK cells in addition to T lymphocytes. Accordingly, MNC products include these cell types to varying degrees. In addition, MNC products may be contaminated with red blood cells, granulocytes and circulating tumour cells or leukemic cells, further complicating downstream manufacturing. Using leukapheresis, it is possible to enrich MNCs from several non-target cells (red blood cells, platelets and granulocytes). In addition, leukapheresis devices such as the Spectra Optia Apheresis® System (Terumo BCT, Lakewood, Colorado, US) offer the ability to further optimise collection technique. Ex vivo, a variety of techniques and devices can be used to further separate or enrich a cellular population from the MNC product. For example, counter-flow centrifugal elutriation (Elutra® Terumo BCT, Lakewood, Colorado, US) is used to separate cells according to size and density based on the cell’s sedimentation velocity. In the setting of excess monocytes, elutriation allows for separating lymphocyte and monocyte cell populations with relatively high purity, good recovery and cell viability.13 Magnetic beads bearing antibodies specific for T cell surface markers can be used to isolate the T cells from the lymphocyte fraction. MNC products with low T cell percentages may benefit from bead-based selection of the target CD3+ T cell population prior to initiation of culture. In addition, beads may be used to activate and expand the T cell population. Finally, traditional density gradients, such as Ficoll, may be used to remove contaminating granulocytes and/or red blood cells. While classically challenging to perform in a good manufacturing practice (GMP)-compliant manner, modern adaptation of Ficoll gradient separation has incorporated closed systems possibly increasing utility in CAR T cell manufacturing.14
Typically, peripheral blood comprises around 20–40% lymphocytes, although circulating lymphocytes can be suppressed in patients being treated for underlying malignancies.15 In addition, most collections occur at steady state, i.e., in the absence of haematopoietic stem cell mobilising agents.16,17 Therefore, processing large blood volumes may be necessary to obtain adequate T cell yield. In this respect, anticoagulant infusion is an important consideration. We opt for apheresis devices where anticoagulant infusion is managed carefully, for instance as with the Optia apheresis device. To pre vent the blood clotting in the apheresis device, an anti-coagulant such as Acid Citrate Dextrose Formula A (ACD-A) is mixed with the blood as it is circulating through the machine.18 As it mixes with the whole blood, citrate binds divalent cations, including calcium, a necessary component in coagulation. The patient is exposed to citrate as the blood components are returned to his/her body and may experience transient hypocalcemia.15 Heparin may also be considered as an anticoagulant; however, this drug is not without its own risk of adverse reaction.19 Several studies have provided strategies to allow larger volume apheresis collections to enhance yield and manage citrate toxicity.20–22
Activating and modifying CAR T cells
Once T cells have been collected, they must be activated, transduced and expanded before being reinfused into the patient. In vivo, endogenous antigen presenting cells (APCs), such as dendritic cells or B cells, may activate cognate T cells. Ex vivo, however, these endogenous APCs display inherent variability making their use impractical in a GMP setting.23 Robust T cell activation for CAR T cell manufacture can be achieved using soluble anti-CD3 monoclonal anitbodies (mAbs),24 anti-CD3/anti-CD28 mAb coated paramagnetic beads25 or cell-based engineered artificial APCs.26,27 Bead-based methods concurrently provide a method to positively select T cells, whereas synthetic cell based artificial APCs allow for customisation of stimulatory conditions. Commercial and technical limitations of bead and APCs respective may make widely available GMP-grade, soluble monoclonal antibody stimulation preferable in some situations. After stimulation, T cells are then genetically modified to express the CAR.
Currently, viral transduction with either gamma retroviral or lentiviral vectors are the most common method of gene transfer owing to the high efficiency of gene delivery and the persistence of integrating vectors in the modified T cells.28 Lentiviral vectors may be preferable
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Leukapheresis, chimeric antigen receptor (CAR) T cells, tumour antigens, harvesting, manufacture, toxicity