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.
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.
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.
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
1. Maude SL, Frey N, Shaw PA, et al., Chimeric antigen receptor T cells for sustained remissions in leukemia, N Engl J Med, 2014;371:1507–17.
2. Porter D,L.Levine BL, Kalos M, Bagg A, June CH, Brief report: Chimeric antigen receptor–modified T cells in chronic lymphoid leukemia, N Engl J Med, 2011,365:725–33.
3. Brentjens RJ, Rivière I, Park JH, et al., Blood, 2011;118:4817–28.
4. Kuwana Y, et al., Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions, Biochem Biophys Res Commun, 1987;149:960–8.
5. Gross G, Waks T, Eshhar Z, Expression of immunoglobulin- T-cell receptor chimeric molecules as functional receptors with antibody–type specificity, Proc Natl Acad Sci U S A, 1989;86:10024-8.
6. Gacerez AT, Arellano B, Sentman CL, How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy, J Cell Physiol, 2016;231:2590–8.
7. Jensen MC, Riddell SR, Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells, Immunol Rev, 2014;257:127–44.
8. Sadelain M, Brentjens R, Riviere I, The basic principles of chimeric antigen receptor design, Cancer Discov, 2013;3:388–98.
9. Ikeda H, Akahori Y, Yoneyama M, Immunotherapy with Chimeric Antigen Receptor Targeting Intracellular WT1 Gene Product Complexed with HLA-a*24:02 Molecule, Blood, 2015;126:4292.
10. Dao T, Yan S, Veomett N, et al., Targeting the intracellular WT1 oncogene product with a therapeutic human antibody, Sci Transl Med, 2013;5:176ra133.
11. Smith JW, Apheresis techniques and cellular immunomodulation, Ther Apher, 1997;1:203–6.
12. Stroncek DF, Ren J, Lee DW, et al., Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells, Cytotherapy, 2016;18:893–901.
13. Stroncek DF, Fellowes V, Pham C, et al., Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies, J Transl Med, medicine, 2014;12:241.
14. Janssen WE, Ribickas A, Meyer LV, Smilee RC, Large-scale Ficoll gradient separations using a commercially available, effectively closed, system, Cytotherapy, 2010;12:418–24.
15. Westermann J, Pabst R, Distribution of lymphocyte subsets and natural killer cells in the human body, Clinical Investig, 1992;70:539–44.
16. Levine BL, Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells, Cancer Gene Ther, 2015;22:79–84.
17. Di Mascio M, Paik CH, Carrasquillo JA, et al., Noninvasive in vivo imaging of CD4 cells in simian-human immunodeficiency virus (SHIV)-infected nonhuman primates, Blood, 2009;114:328–37.
18. Lee G, Arepally GM, Anticoagulation techniques in apheresis: from heparin to citrate and beyond, J Clin Apher, 2012;27:117–25.
19. Dettke M, Buchta C, Wiesinger H, et al., Anticoagulation in largevolume leukapheresis: comparison between citrate- versus heparin-based anticoagulation on safety and CD34 (+) cell collection efficiency, Cytotherapy, 2012;14:350–8.
20. Passos-Coelho JL, Braine HG, Wright SK, et al., Large-volume leukapheresis using regional citrate anticoagulation to collect peripheral blood progenitor cells, J Hematother, 1995;4:11–9.
21. Bolan CD, Leitman SF, Management of anticoagulationassociated toxicity during large-volume leukapheresis of peripheral blood stem cell donors, Blood, 2002;99:1878.
22. Bojanic I, Dubravcic K, Batinic D, et al., Large volume leukapheresis: Efficacy and safety of processing patient’s total blood volume six times, Transfus Apher Sci, 2011;44:139–47.
23. June CH, Principles of adoptive T cell cancer therapy, J Clin Invest, 2007;117:1204–12.
24. Kochenderfer JN, Dudley ME, Kassim SH, et al., Chemotherapyrefractory 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, 2015;33:540–9.
25. Hami LS, Green C, Leshinsky N, et al., GMP production and testing of Xcellerated T Cells for the treatment of patients with CLL, Cytotherapy, 2004;6:554–62.
26. Suhoski MM, Golovina TN, Aqui NA, et al., Engineering artificial antigen-presenting cells to express a diverse array of costimulatory molecules, Mol Ther, 2007;15:981–8.
27. Maus MV, Thomas AK, Leonard DG, et al., Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB, Nat Biotechnol, 2002;20:143–8.
28. Kay MA, State-of-the-art gene-based therapies: the road ahead, Nat Rev Genet, 2011;12:316–28.
29. Durand S, Cimarelli A, The inside out of lentiviral vectors, Viruses, 2011;3:132–59.
30. Dropulic B, Genetic modification of hematopoietic cells using retroviral and lentiviral vectors: safety considerations for vector design and delivery into target cells, Curr Hematol Rep, 2005;4:300–4.
31. Biffi A, Bartolomae CC, Cesana D, et al., Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection, Blood, 2011;117:5332–9.
32. Cesana D, Sgualdino J, Rudilosso L, et al., Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations, J Clin Invest, 2012;122:1667–76.
33. Babaei S, Akhtar W, de Jong J, et al., J. 3D hotspots of recurrent retroviral insertions reveal long-range interactions with cancer genes, Nat Commun, 2015;6:6381.
34. Howe SJ, Mansour MR, Schwarzwaelder K, et al., Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients, J Clin Invest, 2008;118:3143–50.
35. June CH, Blazar BR, Riley JL, Engineering lymphocyte subsets: tools, trials and tribulations. Nature reviews, Immunology, 2009;9:704–16.
36. Montini E, Cesana D, Schmidt M, et al., Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration, Nat Biotechnol, 2006;24:687–96.
37. Beatty GL, Haas AR, Maus MV, et al., Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies, Cancer Immunol Res, 2014;2:112–20.
38. Simonetti FR, Sobolewski MD, Fyne E, et al., Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo, Proc Natl Acad Sci U S A, 2016;113:1883–8.
39. Scholler J, Brady TL, Binder-Scholl G, et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells, Sci Transl Med, 2012;4:132ra153.
40. Kochenderfer JN, Wilson WH, Janik JE, et al., Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19, Blood, 2010;116:4099–102.
41. Gattinoni L, Finkelstein SE, Klebanoff CA, et al., Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells, J Exp Med, 2005;202:907–12.
42. Zhao Y, Moon E, Carpenito C, et al., Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor, Cancer Res, 2010;70:9053–61.
43. Maus MV, Haas AR, Beatty GL, et al., T cells expressing chimeric antigen receptors can cause anaphylaxis in humans, Cancer Immunol Res, 2013;1:26–31.
44. Wang X, Riviere I, Clinical manufacturing of CAR T cells: foundation of a promising therapy, Mol Ther Oncolytics, 2016;3:16015.
45. Verhoeyen E, Dardalhon V, Ducrey-Rundquist O, et al., IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes. Blood, 2003;101:2167–74.
46. Saule P, Trauet J, Dutriez V, et al., Accumulation of memory T cells from childhood to old age: Central and effector memory cells in CD4+ versus effector memory and terminally differentiated memory cells in CD8+ compartment, Mech Ageing Dev, 2016;127:274–281.
47. Dai H, Wang Y, Lu X, Han W, Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy, J Natl Cancer Inst, 2016;108.
48. Milone MC, Fish JD, Carpenito C, et al., Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo, Mol Ther, 2009;17:1453–64.
49. Kawalekar OU, O’Connor RS, Fraietta JA, et al., Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells, Immunity, 2016;44:380–90.
50. Madu A, Ocheni S, Ibegbulam O, et al., Pattern of CD4 T-lymphocyte Values in Cancer Patients on Cytotoxic Therapy, Ann Med Health Sci Res, 2013;3:498–503.
51. Kean LS, Sen S, Onabajo O, et al., Significant mobilization of both conventional and regulatory T cells with AMD3100, Blood, 2011;118:6580–90.
52. Donahue RE, Jin P, Bonifacino AC, et al., Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize different CD34+ cell populations based on global gene and microRNA expression signatures, Blood, 2009;114:2530–41.
53. Porter DL, Hwang WT, Frey NV, et al., Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia, Sci Transl Med, 2015;7:303ra139.
54. Sharpe M, Mount N, Genetically modified T cells in cancer therapy: opportunities and challenges, Dis Model Mech, 2015;8:337–50.
55. Maude SL, Barrett D, Teachey DT, Grupp SA, Managing cytokine release syndrome associated with novel T cell-engaging therapies, Cancer J, 2014;20:119–22.
56. Lamers CH, Sleijfer S, Vulto AG, et al., Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience, J Clin Oncol, 2006;24:e20–2.
57. Lamers CH, Willemsen R, van Elzakker P, et al., Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells, Blood, 2011;117:72–82.
58. Morgan RA, Yang JC, Kitano M, et al., Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2, Mol Ther, 2010;18:843–51.
59. Maus MV, Haas AR, Beatty GL, et al., T cells expressing chimeric antigen receptors can cause anaphylaxis in humans, Cancer Immunol Res, 2013;1:26–31.
60. Cameron BJ, Gerry AB, Dukes J, et al., Identification of a Titinderived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells, Sci Transl Med, 2013;5:197ra103.
61. Linette GP, Stadtmauer EA, Maus MV, et al., Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma, Blood, 2013;122:863–71.
62. Gargett T, Brown MP, The inducible caspase-9 suicide gene system as a “safety switch” to limit on-target, off-tumor toxicities of chimeric antigen receptor T cells, Front Pharmacol, 2014;5:235.
63. Di Stasi A, Tey SK, Dotti G, et al., Inducible apoptosis as a safety switch for adoptive cell therapy, N Engl J Med, 2011;365:1673–83.
64. Kim MS, Ma JS, Yun H, et al., Redirection of genetically engineered CAR-T cells using bifunctional small molecules, J Am Chem Soc, 2015;137:2832–5.
65. Wu CY, Roybal KT, Puchner EM, et al., Remote control of therapeutic T cells through a small molecule-gated chimeric receptor, Science, 2015;350:aab4077.
66. Kim MG, Kim D, Suh SK, et al., Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics, Arch Pharm Res, 2016;39:437–52.
67. Jackson HJ, Rafiq S, Brentjens RJ, Driving CAR T-cells forward, Nat Rev Clin Oncol, 2016;13:370–83.
68. Davila ML, Riviere I, Wang X, et al., Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia, Sci Transl Med, 2014;6:224ra225.
69. Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al., T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial, Lancet, 2015;385:517–28.
70. Steininger PA, Smith R, Geier C, et al., Leukapheresis in noncytokine- stimulated donors with a new apheresis system: first-time collection results and evaluation of subsequent cryopreservation, Transfusion, 2013;53:747–56.
71. Steininger P, Zimmermann R, Eckstein R, Strasser E, Possible reasons for variable leukapheresis collection outcomes with automated apheresis systems, Transfusion, 2014;54:2584–5.
72. Schulz M, Bialleck H, Thorausch K, et al., Unstimulated leukapheresis in patients and donors: comparison of two apheresis systems, Transfusion, 2014;54:1622–9.
73. Fischer JC, Robitzsch T, Rox JM, Composition of pbpc allografts: content of alloreactive cells and hematopoietic progenitor cells is dependent on the apheresis device. Presented at: 40th Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT), Milan, Italy, 30 March-2 April, 2014,
74. Steininger PA, Strasser EF, Weiss D, et al., First comparative evaluation of a new leukapheresis technology in non-cytokinestimulated donors, Vox Sang, 2014;106:248–55.
75. Punzel M, Kozlova A, Schmidt H, et al., Feasibility and advantages of a novel continuous Spectra-Optia apheresis system (cMNC-system) to collect nonstimulated mononuclear cells (MNC) for cellular therapy. Presented at: 41st Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT), Istanbul, Turkey, 22–25 March, 2015.
76. Robitzsch JT, Rox JM, Fischer JC, Comparison of four apheresis systems (Cobe Spectra, Spectra Optia MNC, Spectra Optia cMNC and Amicus) for non-stimulated mononuclear cell collections, Presented at: 41st Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT), Istanbul, Turkey, 22–25 March, 2015.
77. Tumaini B, Lee DW, Lin T, et al., Simplified process for the production of anti-CD19-CAR-engineered T cells, Cytotherapy, 2013;15:1406–15.
78. Wilkie S, Burbridge SE, Chiapero-Stanke L, et al., Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4, J Biol Chem, 2010;285:25538–44.
79. van Schalkwyk MC, Papa SE, Jeannon JP, et al., Design of a phase I clinical trial to evaluate intratumoral delivery of ErbBtargeted chimeric antigen receptor T-cells in locally advanced or recurrent head and neck cancer, Hum Gene Ther Clin Dev, 2013;24:134–42.
80. Pardoll DM, The blockade of immune checkpoints in cancer immunotherapy, Nat Rev Cancer, 2012;12:252–64.
81. John LB, Devaud C, Duong CP, et al., Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells, Clin Cancer Res, 2013;19:5636–46.
82. Chmielewski M, Hombach AA, Abken H, Of CARs and TRUCKs: chimeric antigen receptor (CAR) T cells engineered with an inducible cytokine to modulate the tumor stroma, Immunol Rev, 2014;257:83–90.
83. Zhang L, Morgan RA, Beane JD, et al., Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma, Clin Cancer Res, 2015;21:2278–88.
84. Schmidt P, Kopecky C, Hombach A, et al., Eradication of melanomas by targeted elimination of a minor subset of tumor cells, Proc Natl Acad Sci U S A, 2011;108:2474–9.
85. Rainusso N, Brawley VS, Ghazi A, et al., Immunotherapy targeting HER2 with genetically modified T cells eliminates tumor-initiating cells in osteosarcoma, Cancer Gene Ther, 2012;19:212–7.
86. Alkins R, Burgess A, Ganguly M, et al., Focused ultrasound delivers targeted immune cells to metastatic brain tumors, Cancer Res, 2013;73:1892–9.
87. Cherkassky L, et al. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition, J Clin Invest, 2016;126:3130–44.
88. Legler DF, Johnson-Leger C, Wiedle G, et al., The alpha v beta 3 integrin as a tumor homing ligand for lymphocytes, Eur J Immunol, 2004;34:1608–16.
89. Kershaw MH, Westwood JA, Darcy PK, Gene-engineered T cells for cancer therapy, Nat Rev Cancer, 2013;13:525–41.
90. Moon EK, Carpenito C, Sun J, et al., Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor, Clin Cancer Res, 2011;17:4719–30.
91. Craddock JA, Lu A, Bear A, et al., Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b, J Immunother, 2010;33:780–8.
92. Magnani CF, Biondi A, Biagi E, Donor-derived CD19-targeted T cells in allogeneic transplants, Curr Opin Hematol, 2015;22:497–502.
93. Deniger DC, Yu J, Huls MH, et al., Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1) into Diverse Memory T-Cell Populations, PloS One, 2015;10:e0128151.
94. Yoon JH, Schmidt A, Kim YC, et al., 291 Immunosuppressive FVIII-Specific Human CAR Tregs in Hemophilia A . Presented at: 57th Annual Meeting and Exposition American Society of Hematology (ASH), Orlando, FL, USA, 5–8 December 2015.
95. Fransson M, Piras E, Burman J, et al., CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery, J Neuroinflammation, 2012;9:112.
96. Leibman RS, Riley JL, Engineering T Cells to Functionally Cure HIV-1 Infection, Mol Ther, 2015;23:1149–59.
97. Dey B, Berger EA, Towards an HIV cure based on targeted killing of infected cells: different approaches against acute versus chronic infection, Curr Opin HIV AIDS, 2015;10:207–13.
98. Dotti G, Gottschalk S, Savoldo B, Brenner MK, Design and development of therapies using chimeric antigen receptorexpressing T cells, Immunol Rev, 2014;257:107–26.
Leukapheresis, chimeric antigen receptor (CAR) T cells, tumour antigens, harvesting, manufacture, toxicity