Translational Implications for Off-the-shelf Immune Cells Expressing Chimeric Antigen Receptors
Authors: Hiroki Torikai, Laurence JN Cooper
Journal: Molecular Therapy (2016)

Chimeric antigen receptor (CAR) endows specificity to T-cells independent of human leukocyte antigen (HLA). This enables one immunoreceptor to directly target the same surface antigen on different subsets of tumor cells from multiple HLA-disparate recipients. Most approaches manufacture individualized CAR+T-cells from the recipient or HLA-compatible donor, which are revealing promising clinical results. This is the impetus to broaden the number of patients eligible to benefit from adoptive immunotherapy such as to infuse third-party donor derived CAR+T-cells. This will overcome issues associated with (i) time to manufacture T-cells, (ii) cost to generate one product for one patient, (iii) inability to generate a product from lymphopenic patients or patient's immune cells fail to complete the manufacturing process, and (iv) heterogeneity of T-cell products produced for or from individual recipients. Establishing a biobank of allogeneic genetically modified immune cells from healthy third-party donors, which are cryopreserved and validated in advance of administration, will facilitate the centralizing manufacturing and widespread distribution of CAR+T-cells to multiple points-of-care in a timely manner. To achieve this, it is necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to toxicity and rejection. We review the strategies to establish "off-the-shelf" donor-derived biobanks for human application of CAR+T-cells as a drug. Chimeric antigen receptor (CAR) endows specificity to T-cells independent of human leukocyte antigen (HLA). This enables one immunoreceptor to directly target the same surface antigen on different subsets of tumor cells from multiple HLA-disparate recipients. Most approaches manufacture individualized CAR+T-cells from the recipient or HLA-compatible donor, which are revealing promising clinical results. This is the impetus to broaden the number of patients eligible to benefit from adoptive immunotherapy such as to infuse third-party donor derived CAR+T-cells. This will overcome issues associated with (i) time to manufacture T-cells, (ii) cost to generate one product for one patient, (iii) inability to generate a product from lymphopenic patients or patient's immune cells fail to complete the manufacturing process, and (iv) heterogeneity of T-cell products produced for or from individual recipients. Establishing a biobank of allogeneic genetically modified immune cells from healthy third-party donors, which are cryopreserved and validated in advance of administration, will facilitate the centralizing manufacturing and widespread distribution of CAR+T-cells to multiple points-of-care in a timely manner. To achieve this, it is necessary to engineer an effective strategy to avoid deleterious allogeneic immune responses leading to toxicity and rejection. We review the strategies to establish "off-the-shelf" donor-derived biobanks for human application of CAR+T-cells as a drug. The chimeric antigen receptor (CAR) is an artificial immune receptor to redirect T-cell specificity to tumor-associated cell-surface molecules independent of HLA. The extracellular antigen-recognition domain of the prototypical CAR uses a single chain variable fragment from monoclonal antibody (mAb); however, this can be replaced with a receptor–ligand interaction of sufficient affinity, such as modified cytokine (e.g., IL-13) to target a cytokine receptor (e.g., IL-13Rα),1Brown CE Starr R Aguilar B Shami AF Martinez C D'Apuzzo M et al.Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T cells.Clin Cancer Res. 2012; 18: 2199-2209Crossref PubMed Scopus (148) Google Scholar a cell surface molecule (e.g., CD27) for targeting its ligand (e.g., CD70),2Shaffer DR Savoldo B Yi Z Chow KK Kakarla S Spencer DM et al.T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies.Blood. 2011; 117: 4304-4314Crossref PubMed Scopus (110) Google Scholar or a pattern-recognition receptor (e.g., Dectin-1) for targeting foreign carbohydrates such as β-glucan on germinating Aspergillus.3Kumaresan PR Manuri PR Albert ND Maiti S Singh H Mi T et al.Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.Proc Natl Acad Sci USA. 2014; 111: 10660-10665Crossref PubMed Scopus (0) Google Scholar These binding motifs are typically fused to an extracellular stalk or varying lengths and composition, such as derived from the hinge with or without CH2–CH3 domains from IgG1 and IgG4 or hinge with extracellular domain of CD8α. The use of immunoglobulin regions raises the possibility that a CAR may bind to Fc receptors and unwanted elimination of infused T-cells which may be alleviated by introducing site-directed changes or eliminating CH2 region to reduce potential for such clearance.4Hudecek M Sommermeyer D Kosasih PL Silva-Benedict A Liu L Rader C et al.The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity.Cancer Immunol Res. 2015; 3: 125-135Crossref PubMed Scopus (281) Google Scholar,5Hombach A Hombach AA Abken H Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc 'spacer' domain in the extracellular moiety of chimeric antigen receptors avoids 'off-target' activation and unintended initiation of an innate immune response.Gene Ther. 2010; 17: 1206-1213Crossref PubMed Scopus (148) Google Scholar,6Jonnalagadda M Mardiros A Urak R Wang X Hoffman LJ Bernanke A et al.Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy.Mol Ther. 2015; 23: 757-768Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Recent evidence also suggests the importance of the stalk to impact the effector functioning of CAR+T-cells interrogating the binding motifs within tumor associated antigens (TAAs) proximal versus distal to the cell surface.6Jonnalagadda M Mardiros A Urak R Wang X Hoffman LJ Bernanke A et al.Chimeric antigen receptors with mutated IgG4 Fc spacer avoid fc receptor binding and improve T cell persistence and antitumor efficacy.Mol Ther. 2015; 23: 757-768Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar,7Hudecek M Lupo-Stanghellini MT Kosasih PL Sommermeyer D Jensen MC Rader C et al.Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells.Clin Cancer Res. 2013; 19: 3153-3164Crossref PubMed Scopus (311) Google Scholar,8James SE Greenberg PD Jensen MC Lin Y Wang J Till BG et al.Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.J Immunol. 2008; 180: 7028-7038Crossref PubMed Scopus (157) Google Scholar,9Almåsbak H Walseng E Kristian A Myhre MR Suso EM Munthe LA et al.Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model.Gene Ther. 2015; 22: 391-403Crossref PubMed Scopus (0) Google Scholar CAR-dependent activation is dependent on one or more intracellular signaling domains expressed in cis or trans. The signaling domain of CARs brought forward to clinical applications employed a single signaling molecule containing immunoreceptor tyrosin-based activation motifs (ITAMs) in cytoplasmic domain to mimic the T-cell receptor (TCR)/CD3 signal.10Eshhar Z Waks T Gross G Schindler DG Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.Proc Natl Acad Sci USA. 1993; 90: 720-724Crossref PubMed Scopus (994) Google Scholar These "first generation" CARs typically delivered an incomplete T-cell activation event which prompted embedding additional activation motifs within the CAR endodomain. Inclusion of costimulatory signaling, e.g., through CD28 (ref. 11Maher J Brentjens RJ Gunset G Rivière I Sadelain M Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.Nat Biotechnol. 2002; 20: 70-75Crossref PubMed Scopus (642) Google Scholar), CD137 (refs. 12Imai C Mihara K Andreansky M Nicholson IC Pui CH Geiger TL et al.Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.Leukemia. 2004; 18: 676-684Crossref PubMed Scopus (504) Google Scholar,13Milone MC Fish JD Carpenito C Carroll RG Binder GK Teachey D 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: 1453-1464Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar), OX40 (ref. 14Pulè MA Straathof KC Dotti G Heslop HE Rooney CM Brenner MK A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells.Mol Ther. 2005; 12: 933-941Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar), and CD27 domains,15Song DG Ye Q Poussin M Harms GM Figini M Powell Jr DJ CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo.Blood. 2012; 119: 696-706Crossref PubMed Scopus (228) Google Scholar improved function of CAR+T-cells manifested by sustained persistence after adoptive transfer leading to improved therapeutic effect.16Savoldo B Ramos CA Liu E Mims MP Keating MJ Carrum G et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.J Clin Invest. 2011; 121: 1822-1826Crossref PubMed Scopus (720) Google Scholar,17Carpenito C Milone MC Hassan R Simonet JC Lakhal M Suhoski MM et al.Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains.Proc Natl Acad Sci USA. 2009; 106: 3360-3365Crossref PubMed Scopus (620) Google Scholar In addition to "signal 1" delivered by phosphorylation of ITAMs and "signal 2" mediated by costimulatory molecules, T-cells typically require a third signal to achieve and perhaps sustain full activation. This third signal is mediated through the common γ-chain cytokine receptor and thus coordinated delivery or coexpression of certain cytokines can enhance CAR+T-cell functions, which may be especially useful for the application of genetically modified T-cells targeting solid tumors.18Hoyos V Savoldo B Quintarelli C Mahendravada A Zhang M Vera J et al.Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety.Leukemia. 2010; 24: 1160-1170Crossref PubMed Scopus (359) Google Scholar,19Pegram HJ Lee JC Hayman EG Imperato GH Tedder TF Sadelain M et al.Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning.Blood. 2012; 119: 4133-4141Crossref PubMed Scopus (438) Google Scholar,20Chinnasamy D Yu Z Kerkar SP Zhang L Morgan RA Restifo NP et al.Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.Clin Cancer Res. 2012; 18: 1672-1683Crossref PubMed Scopus (184) Google Scholar Human applications of CAR+T-cells have shown promise in several early phase clinical trials, such as infusing targeting CD19 on B-cell leukemias21Grupp SA Kalos M Barrett D Aplenc R Porter DL Rheingold SR et al.Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.N Engl J Med. 2013; 368: 1509-1518Crossref PubMed Scopus (2338) Google Scholar,22Porter DL Levine BL Kalos M Bagg A June CH Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med. 2011; 365: 725-733Crossref PubMed Scopus (2480) Google Scholar,23Kalos M Levine BL Porter DL Katz S Grupp SA Bagg A et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Sci Transl Med. 2011; 3: 95ra73Crossref PubMed Scopus (1696) Google Scholar,24Brentjens RJ Davila ML Riviere I Park J Wang X Cowell LG et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med. 2013; 5: 177ra38Crossref PubMed Scopus (1423) Google Scholar,25Brentjens RJ Rivière I Park JH Davila ML Wang X Stefanski J et al.Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.Blood. 2011; 118: 4817-4828Crossref PubMed Scopus (946) Google Scholar,26Cruz CR Micklethwaite KP Savoldo B Ramos CA Lam S Ku S et al.Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study.Blood. 2013; 122: 2965-2973Crossref PubMed Scopus (378) Google Scholar,27Lee DW Kochenderfer JN Stetler-Stevenson M Cui YK Delbrook C Feldman SA 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: 517-528Abstract Full Text Full Text PDF PubMed Scopus (1876) Google Scholar,28Maude SL Frey N Shaw PA Aplenc R Barrett DM Bunin NJ et al.Chimeric antigen receptor T cells for sustained remissions in leukemia.N Engl J Med. 2014; 371: 1507-1517Crossref PubMed Scopus (3235) Google Scholar,29Davila ML Riviere I Wang X Bartido S Park J Curran K et al.Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.Sci Transl Med. 2014; 6: 224ra25Crossref PubMed Scopus (1657) Google Scholar and lymphomas, 16Savoldo B Ramos CA Liu E Mims MP Keating MJ Carrum G et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.J Clin Invest. 2011; 121: 1822-1826Crossref PubMed Scopus (720) Google Scholar,30Kochenderfer JN Dudley ME Kassim SH Somerville RP Carpenter RO Stetler-Stevenson M et al.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. 2015; 33: 540-549Crossref PubMed Scopus (1132) Google Scholar,31Kochenderfer JN Yu Z Frasheri D Restifo NP Rosenberg SA Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.Blood. 2010; 116: 3875-3886Crossref PubMed Scopus (226) Google Scholar and targeting GD2 on neuroblastoma.32Louis CU Savoldo B Dotti G Pule M Yvon E Myers GD et al.Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.Blood. 2011; 118: 6050-6056Crossref PubMed Scopus (774) Google Scholar Recently, several reviews have highlighted and summarized these clinical successes.33Batlevi CL Matsuki E Brentjens RJ Younes A Novel immunotherapies in lymphoid malignancies.Nat Rev Clin Oncol. 2016; 13: 25-40Crossref PubMed Scopus (175) Google Scholar,34Sadelain M CAR therapy: the CD19 paradigm.J Clin Invest. 2015; 125: 3392-3400Crossref PubMed Scopus (162) Google Scholar,35Maus MV June CH Making better chimeric antigen receptors for adoptive T-cell therapy.Clin Cancer Res. 2016; 22: 1875-1884Crossref PubMed Scopus (175) Google Scholar,36Jackson HJ Rafiq S Brentjens RJ Driving CAR T-cells forward.Nat Rev Clin Oncol. 2016; 13: 370-383Crossref PubMed Scopus (351) Google Scholar,37van der Stegen SJ Hamieh M Sadelain M The pharmacology of second-generation chimeric antigen receptors.Nat Rev Drug Discov. 2015; 14: 499-509Crossref PubMed Scopus (292) Google Scholar With these early successes, CAR+T-cell therapy is redeploying from academia to industry.38June CH Riddell SR Schumacher TN Adoptive cellular therapy: a race to the finish line.Sci Transl Med. 2015; 7: 280ps7Crossref PubMed Scopus (250) Google Scholar Broadening the clinical experience of CAR+T-cell therapy is thus emerging as an important hurdle, especially for those tumor targets already associated with an antitumor effect. This underscores the need to expand the list of qualified TAAs that can be safely targeted by T-cells though one or more engineered immunoreceptors.39Torikai H Moyes J Cooper LN Engineering T Cells to target tumor cells.in: Cai W Engineering in Translational Medicine. Springer, London2014: 71-101Crossref Google Scholar There are a variety of approaches to overcome this, such as to preidentify TAAs expressed solely on tumor cells and targeting aberrant cells based on combinatorial recognition of more than one TAA,40Kloss CC Condomines M Cartellieri M Bachmann M Sadelain M Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat Biotechnol. 2013; 31: 71-75Crossref PubMed Scopus (583) Google Scholar,41Fedorov VD Themeli M Sadelain M PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.Sci Transl Med. 2013; 5: 215ra172Crossref PubMed Scopus (440) Google Scholar or targeting intracellular proteins.42Inaguma Y Akahori Y Murayama Y Shiraishi K Tsuzuki-Iba S Endoh A et al.Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H.Gene Ther. 2014; 21: 575-584Crossref PubMed Scopus (27) Google Scholar,43Chames P Willemsen RA Rojas G Dieckmann D Rem L Schuler G et al.TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity.J Immunol. 2002; 169: 1110-1118Crossref PubMed Scopus (0) Google Scholar Another limitation to the broad implementation of CAR+T-cell therapy resides with the production process to generate clinical-grade biologics. Currently, T-cells are genetically modified and propagated infusing either patient-derived T-cells often after lymphodepleting chemotherapy or donor-derived T-cells administered in the context of hematopoietic stem-cell transplantation (HSCT). These T-cells are produced for a given recipient on a case-by-case basis. This personalized approach to manufacturing reflects the approach pioneered by a subset of academic institutions that operate cell processing facilities in compliance with current good manufacturing practice for phase 1 and 2 trials on campuses that are in close proximity to clinical facilities to administer CAR+T-cells.44Cooper LJN Moving from tinkering in the garage to assembly line production: the manufacture of genetically modified T cells expressing chimeric antigen receptors (CARs) comes on line.Cancer Gene Therapy. 2015; 22: 64-66Crossref PubMed Scopus (0) Google Scholar This leads to heterogeneity of manufacturing methods that provides an intellectually fertile and competitive environment to determine facets of production that result in therapeutically appealing T-cells. However, the physical alignment of multiple geographically diverse good manufacturing practice facilities with infusion units can lead to duplication of efforts compounding the financial costs associated with a labor-intensive approach to manipulation and testing of T-cells (Figure 1). This in turn raises the cost of therapy and curtails the availability of CAR+T-cells, as these products are produced in a limited number of production suites after the recipient has been identified. Moreover, the variability inherent to manufacturing CAR+T-cells from different patients and donors may confound an assessment of mechanisms associated with therapeutic success and complicate the implementation of combination therapies such as with other immune-based modalities. These challenges provide the impetus to develop and implement "off-the-shelf" (OTS) cell-based biologic therapies in which immune cells can be manufactured ahead of need (in advance of consent) and infused on demand as required by the recipient, rather than when the biologic is produced. The current production and lot release (quality control) of clinical-grade genetically modified T-cells requires time in culture within good manufacturing practice-compatible manufacturing facilities during which the intended recipient's condition can deteriorate. Currently, the "distributed" approach to manufacturing by academic centers at multiple point-of-care has resulted in a portfolio of biologic products that differ in terms of quality and quantity (Table 1) which may impact the results of immunotherapy of CAR+T-cells. OTS immunotherapy may overcome these limitations as it lends itself to centralized manufacture of a well-characterized product.Table 1Heterogeneity in the biologic products as derived from different autologous or allogeneic donorsNumber of copies of integrated chimeric antigen receptorGenomic sites of transgene insertion(s)T-cell immunophenotypeTCR repertoireEffector function due to the phenotype, single nucleotide polymorphisms, etc. Open table in a new tab Our present definition of OTS CAR+T-cells is defined as a biologic that is pre-prepared in advance from one or more healthy unrelated donors, validated, and cryopreserved. Their manufacture can be readily undertaken in a centralized manufacturing facility for predeployment to treatment facilities and infused as needed rather than when the product is available (Figure 1). One or more biobanks of the OTS T-cell product(s) could then be administered into multiple recipients in multicenter trials powered for efficacy and to establish the maximum tolerated dose. The administration of a well-characterized product will then facilitate combination immunotherapies infusing OTS T-cells with other treatments (Table 2). To prepare the cells in advance, we propose the use of "universal" allogeneic donor-derived T-cells. These third party cells might be genetically modified, and as required genetically edited, to safely maintain CAR-mediated effector functions and sustain in vivo persistence by avoiding deleterious immune-mediated recognition by the recipient of allogeneic features on the product (Figure 2).Table 2Advantages associated with infusing off-the-shelf (OTS) chimeric antigen receptor (CAR)+T-cells manufactured from one or more third party donor(s) for administration and readministration into multiple unrelated recipientsAvoids difficulties in generating CAR+T-cells from and for a recipient due to poor quality and/or quantity of T-cellsReduces the cost, time, and resources to manufacture CAR+T-cells that are needed for infusion into a single patientOTS CAR+T-cells can be pre-prepared for infusion as needed versus when availableOTS CAR+T-cells can be predeployed at multiple points-of-careOTS CAR+T-cells have reduced heterogeneity compared with multiple patient-specific CAR+T-cellsOTS CAR+T-cells can be produced using centralized manufacturing site(s)OTS CAR+T-cells reduce the barriers to undertaking phase 2b multicenter trials powered for efficacyOTS CAR+T-cells can be infused as part of multicomponent trials administering this biologic at predefined maximally tolerated dose Open table in a new tab In the setting of an HLA-mismatch between donor and recipient, the frequency of T-cells specific for disparate HLA is estimated ~1 in 104.45Heeger PS T-cell allorecognition and transplant rejection: a summary and update.Am J Transplant. 2003; 3: 525-533Crossref PubMed Scopus (119) Google Scholar,46Felix NJ Allen PM Specificity of T-cell alloreactivity.Nat Rev Immunol. 2007; 7: 942-953Crossref PubMed Scopus (164) Google Scholar In clinical trials, the number of administered CAR+T-cells is typically between 108 and 109 which could lead to the delivery 103–105 T-cells expressing αβTCR specific for allogeneic antigens and thus likely be sufficient to induce graft-versus-host-disease (GvHD). We describe five strategies to prevent this unwanted activation through endogenous TCR (Table 3).Table 3Potential strategies to generate off-the-shelf (OTS) cell productsAvoid GvHDEliminate alloreactive T-cells CD25, CD69, CD137 depletion, photodepletionSuppress TCR mediated activation B7 specific mAb for blocking CD28 mediated costimulationDecrease the TCR repertoire Memory T-cellsT-cells expressing TCR with defined specificity Virus or tumor specific T-cells, γ9δ2 T-cellUse cell types that are not expressing alloreactive immunoreceptors γδ T-cells, NK-cells, NKT-cellsUse precursor T-cells Conditional ablation HSV1-TK, iCasp9Eliminate of endogenous TCR ZFN, TALEN, CRISPR/Cas9 and other gene editing technologiesAvoid rejectionSuppress immune system in recipients lymphodepletion (CY, Fludarabine, TBI, anti-CD52Ab)Modulation of immune system in recipients FTY720Enforced expression of immune checkpoint molecules PD-L1, CTLA4-IgHLA homozygous donor Gene editing of HLA ZFN, TALEN, CRISPR/Cas9 and other gene editing technologies Open table in a new tab T-cells displaying αβTCRs play a central role inducing GvHD and therefore several strategies have been developed to ex vivo remove alloreactive T-cells such as contaminating the coinfusion of HLA-mismatched hematopoietic stem-cells (HSCs) to restore hematopoiesis without GvHD in the context of HSCT. These include the numeric depletion of T-cells that express one or more cell-surface markers consistent with activation (e.g., CD25 (refs. 47Amrolia PJ Muccioli-Casadei G Huls H Adams S Durett A Gee A et al.Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.Blood. 2006; 108: 1797-1808Crossref PubMed Scopus (199) Google Scholar,48Amrolia PJ Muccioli-Casadei G Yvon E Huls H Sili U Wieder ED et al.Selective depletion of donor alloreactive T cells without loss of antiviral or antileukemic responses.Blood. 2003; 102: 2292-2299Crossref PubMed Scopus (125) Google Scholar,49Solomon SR Mielke S Savani BN Montero A Wisch L Childs R et al.Selective depletion of alloreactive donor lymphocytes: a novel method to reduce the severity of graft-versus-host disease in older patients undergoing matched sibling donor stem cell transplantation.Blood. 2005; 106: 1123-1129Crossref PubMed Scopus (123) Google Scholar), CD69 (ref. 50Hartwig UF Nonn M Khan S Meyer RG Huber C Herr W Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes.Bone Marrow Transplant. 2006; 37: 297-305Crossref PubMed Scopus (0) Google Scholar), and CD137 (ref. 51Wehler TC Nonn M Brandt B Britten CM Gröne M Todorova M et al.Targeting the activation-induced antigen CD137 can selectively deplete alloreactive T cells from antileukemic and antitumor donor T-cell lines.Blood. 2007; 109: 365-373Crossref PubMed Scopus (78) Google Scholar)) upon coculture with antigen-presenting cells (APCs) derived from the intended recipient. Activated T-cells can then be reduced using magnetic beads or immunotoxin conjugated to mAb.47Amrolia PJ Muccioli-Casadei G Huls H Adams S Durett A Gee A et al.Adoptive immunotherapy with allodepleted donor T-cells improves immune reconstitution after haploidentical stem cell transplantation.Blood. 2006; 108: 1797-1808Crossref PubMed Scopus (199) Google Scholar,50Hartwig UF Nonn M Khan S Meyer RG Huber C Herr W Depletion of alloreactive T cells via CD69: implications on antiviral, antileukemic and immunoregulatory T lymphocytes.Bone Marrow Transplant. 2006; 37: 297-305Crossref PubMed Scopus (0) Google Scholar Photodepletion is an alternative approach for reduction of alloreactive T-cells based on the inability of activated T-cells to efflux of a phototoxic dye.52Mielke S Nunes R Rezvani K Fellowes VS Venne A Solomon SR et al.A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique.Blood. 2008; 111: 4392-4402Crossref PubMed Scopus (86) Google Scholar Such methods have already tested in the clinic and significantly decreased the frequency of alloreactive T-cells while preserving viral- and tumor-specific T-cells which may benefit immune reconstitution in an immunocompromised recipient. However, strategies that rely on ex vivo depletion cannot completely eliminate alloreactive T-cells. Moreover, the requirement to coculture the CAR+T-cells with recipient's cells reduces the speed and convenience associated with producing this OTS biologic. We demonstrated that anergization of CAR+T-cells can be achieved in tissue culture by combining allostimulation with HLA-mismatched APC and concomitant blockade of CD28-mediated costimulation.53Davies JK Singh H Huls H Yuk D Lee DA Kebriaei P et al.Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res. 2010; 70: 3915-3924Crossref PubMed Scopus (34) Google Scholar This resulted in the reduction of recognition of disparate HLA by third-party T-cells mediated by αβTCR while preserving CAR-mediated effector function. The induction of alloanergization requires patient-derived stimulator cells that undermines its suitability for OTS CAR+T-cells. One strategy to reduce TCR diversity and thus potential of alloreactivity is to employ T-cells from memory pools as a cellular template for introduction of CAR. Injecting naive T-cells induced GvHD in a mouse model, whereas administering memory T-cells did not.54Anderson BE McNiff J Yan J Doyle H Mamula M Shlomchik MJ et al.Memory CD4+ T cells do not induce graft-versus-host disease.J Clin Invest. 2003; 112: 101-108Crossref PubMed Sc