Title: New Cell Sources for T Cell Engineering and Adoptive Immunotherapy
Abstract: The promising clinical results obtained with engineered T cells, including chimeric antigen receptor (CAR) therapy, call for further advancements to facilitate and broaden their applicability. One potentially beneficial innovation is to exploit new T cell sources that reduce the need for autologous cell manufacturing and enable cell transfer across histocompatibility barriers. Here we review emerging T cell engineering approaches that utilize alternative T cell sources, which include virus-specific or T cell receptor-less allogeneic T cells, expanded lymphoid progenitors, and induced pluripotent stem cell (iPSC)-derived T lymphocytes. The latter offer the prospect for true off-the-shelf, genetically enhanced, histocompatible cell therapy products. The promising clinical results obtained with engineered T cells, including chimeric antigen receptor (CAR) therapy, call for further advancements to facilitate and broaden their applicability. One potentially beneficial innovation is to exploit new T cell sources that reduce the need for autologous cell manufacturing and enable cell transfer across histocompatibility barriers. Here we review emerging T cell engineering approaches that utilize alternative T cell sources, which include virus-specific or T cell receptor-less allogeneic T cells, expanded lymphoid progenitors, and induced pluripotent stem cell (iPSC)-derived T lymphocytes. The latter offer the prospect for true off-the-shelf, genetically enhanced, histocompatible cell therapy products. T cells are essential mediators of immune defense against infectious pathogens and cancer. Their insufficiency, which occurs in hereditary or acquired immune deficiencies, results in life threatening infections, increased cancer incidence, and disrupted immunoregulation. T cells can also be harmful and cause normal tissue destruction, as seen in autoimmune disorders, graft rejection, and graft-versus-host disease (GVHD). T cells develop from precursors that rearrange germline antigen receptor VDJ genes in the thymus, thereby generating clonotypic T cell receptors (TCRs) that undergo positive and negative thymic selection (Figure 1). The resulting T cells are self-restricted and tolerant of self tissues. The newly generated T cell clones, known as naive T cells, initially circulate throughout the body at low frequency. Upon encountering antigen, T cells expand and acquire effector and/or memory functions. This T cell priming requires TCR engagement by Human Leucocyte Antigen (HLA)-peptide complexes on the surface of antigen presenting cells (APCs) and concomitant ligation of costimulatory receptors by ligands borne by the APCs (Chen and Flies, 2013Chen L. Flies D.B. Molecular mechanisms of T cell co-stimulation and co-inhibition.Nat. Rev. Immunol. 2013; 13: 227-242Crossref PubMed Scopus (108) Google Scholar, Krogsgaard and Davis, 2005Krogsgaard M. Davis M.M. How T cells ‘see’ antigen.Nat. Immunol. 2005; 6: 239-245Crossref PubMed Scopus (110) Google Scholar). Pathogen-specific T cells can be effectively expanded through vaccination, a medical intervention that allows prevention of a number of infectious diseases. In this instance, immunization proceeds in vivo within secondary lymphoid organs where T cells engage their TCRs on professional APCs that initiate productive T cell activation and clonal expansion. Active immunization has, however, proven far less effective when infection or cancer is already established and progressing. In such circumstances, T cells, whether they are naturally activated or elicited through immunization, often fail to eradicate disease owing to their inadequate number or suboptimal function. The infusion of T cells, or adoptive transfer, has proven to overcome the limitations of active immunization in some pathologies. The therapeutic use of isolated T cells began somewhat inadvertently with allogeneic bone marrow transplantation (BMT). The use of whole marrow grafts containing donor T cells revealed the beneficial (graft-versus-tumor responses) and deleterious (GVHD) effects of adoptive T cell transfer (Ferrara and Deeg, 1991Ferrara J.L. Deeg H.J. Graft-versus-host disease.N. Engl. J. Med. 1991; 324: 667-674Crossref PubMed Google Scholar). Several forms of T cell therapy subsequently developed, including donor leukocyte infusion (Kolb et al., 2005Kolb H.J. Schmid C. Buhmann R. Tischer J. Ledderose G. DLI: where are we know?.Hematology. 2005; 10: 115-116Crossref PubMed Scopus (5) Google Scholar) and virus-specific T cell therapy (Riddell and Greenberg, 1995Riddell S.R. Greenberg P.D. Principles for adoptive T cell therapy of human viral diseases.Annu. Rev. Immunol. 1995; 13: 545-586Crossref PubMed Google Scholar). These therapies utilize “donor-derived T cells,” which tap into the alloreactive potential of T cells harvested from a healthy donor but expose the recipient to the risk of normal tissue destruction by graft versus host (GVH) responses. In contrast, autologous T cells, harvested from the intended recipient (Rosenberg et al., 1986Rosenberg S.A. Spiess P. Lafreniere R. A new approach to the adoptive immunotherapy of cancer with tumor-infiltrating lymphocytes.Science. 1986; 233: 1318-1321Crossref PubMed Google Scholar), are devoid of such toxic potential. However, autologous T cells with therapeutic potential may be lacking or functionally impaired in patients with refractory infections or progressing cancer. Allogeneic and autologous T cells thus have their respective advantages and disadvantages. For some cancers, T cells may be isolated from surgically removed tumors, which are enriched in tumor-reactive T cells relative to peripheral blood. Tumor infiltrating lymphocytes (TILs) can be isolated at quite a high frequency from melanoma specimens, but this technique is not feasible or effective in many other tumor types (Rosenberg et al., 2008Rosenberg S.A. Restifo N.P. Yang J.C. Morgan R.A. Dudley M.E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy.Nat. Rev. Cancer. 2008; 8: 299-308Crossref PubMed Scopus (613) Google Scholar, Wu et al., 2012Wu R. Forget M.A. Chacon J. Bernatchez C. Haymaker C. Chen J.Q. Hwu P. Radvanyi L.G. Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook.Cancer J. 2012; 18: 160-175Crossref PubMed Scopus (29) Google Scholar). Thus, we and others have sought to generate tumor-targeted T cells through genetic engineering (Ho et al., 2003Ho W.Y. Blattman J.N. Dossett M.L. Yee C. Greenberg P.D. Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction.Cancer Cell. 2003; 3: 431-437Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, Sadelain et al., 2003Sadelain M. Rivière I. Brentjens R. Targeting tumours with genetically enhanced T lymphocytes.Nat. Rev. Cancer. 2003; 3: 35-45Crossref PubMed Scopus (209) Google Scholar). The rationale for T cell engineering is to rapidly generate populations of T cells specific for any antigen and, furthermore, to enhance their therapeutic (e.g., anti-tumor) functions. Peripheral blood T cells are easily accessible and are a perfectly suitable cell source for this purpose. Most current therapies utilizing engineered T cells process autologous peripheral blood T cells that are targeted to tumor antigens following retroviral transduction of a TCR or a chimeric antigen receptor (CAR). In recent years, a few clinical trials have resulted in encouraging and sometimes dramatic clinical responses (Couzin-Frankel, 2013Couzin-Frankel J. Breakthrough of the year 2013. Cancer immunotherapy.Science. 2013; 342: 1432-1433Crossref PubMed Scopus (79) Google Scholar). This Perspective article focuses on the sources of T cells for adoptive cell therapy, starting from blood, hematopoietic stem cell-derived lymphoid progenitor cells, embryonic stem cell (ESC), or induced pluripotent stem cell (iPSC)-derived T cells. The general premise for engineering T cells for cancer immunotherapy is to rapidly generate tumor-targeted T cells, bypassing the obstacles that preclude the induction and execution of effective immune responses in vivo. Two categories of antigen receptors are used to retarget T cell specificity: physiological TCRs and synthetic receptors referred to as CARs (Figure 2). The design of TCRs and CARs has steadily improved over the past 2 decades (Cohen et al., 2006Cohen C.J. Zhao Y. Zheng Z. Rosenberg S.A. Morgan R.A. Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.Cancer Res. 2006; 66: 8878-8886Crossref PubMed Scopus (151) Google Scholar, Cohen et al., 2007Cohen C.J. Li Y.F. El-Gamil M. Robbins P.F. Rosenberg S.A. Morgan R.A. Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond.Cancer Res. 2007; 67: 3898-3903Crossref PubMed Scopus (132) Google Scholar, Robbins et al., 2008Robbins P.F. Li Y.F. El-Gamil M. Zhao Y. Wargo J.A. Zheng Z. Xu H. Morgan R.A. Feldman S.A. Johnson L.A. et al.Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions.J. Immunol. 2008; 180: 6116-6131Crossref PubMed Google Scholar, Sadelain et al., 2003Sadelain M. Rivière I. Brentjens R. 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Robbins P.F. Rosenberg S.A. Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes.J. Immunol. 2006; 177: 6548-6559Crossref PubMed Google Scholar), from humanized murine models (Cohen et al., 2005Cohen C.J. Zheng Z. Bray R. Zhao Y. Sherman L.A. Rosenberg S.A. Morgan R.A. Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR.J. Immunol. 2005; 175: 5799-5808Crossref PubMed Google Scholar, Parkhurst et al., 2009Parkhurst M.R. Joo J. Riley J.P. Yu Z. Li Y. Robbins P.F. Rosenberg S.A. Characterization of genetically modified T-cell receptors that recognize the CEA:691-699 peptide in the context of HLA-A2.1 on human colorectal cancer cells.J. Clin. Cancer Res. 2009; 15: 169-180Crossref Scopus (33) Google Scholar), or through the use of phage display technology (Li et al., 2005Li Y. 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Riviere I. Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.Nature biotechnology. 2002; 20: 70-75Crossref PubMed Scopus (248) Google Scholar, Figure 2) not only mediate antigen recognition and initiate T cell activation but also harness costimulation to enhance T cell function and prolong T cell persistence (Sadelain et al., 2009Sadelain M. Brentjens R. Rivière I. The promise and potential pitfalls of chimeric antigen receptors.Curr. Opin. Immunol. 2009; 21: 215-223Crossref PubMed Scopus (164) Google Scholar). Over a decade ago, we selected the CD19 antigen as a potential CAR target for B cell malignancies (Brentjens et al., 2003Brentjens R.J. Latouche J.B. Santos E. Marti F. Gong M.C. Lyddane C. King P.D. Larson S. Weiss M. Rivière I. Sadelain M. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15.Nat. 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Sherry R.M. et al.B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.Blood. 2012; 119: 2709-2720Crossref PubMed Scopus (235) Google Scholar), the University of Pennsylvania for chronic lymphocytic leukemia (Kalos et al., 2011Kalos M. Levine B.L. Porter D.L. Katz S. Grupp S.A. Bagg A. June C.H. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.Science translational medicine. 2011; 3: 95ra73Crossref PubMed Scopus (378) Google Scholar, Porter et al., 2011Porter D.L. Levine B.L. Kalos M. Bagg A. June C.H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.The New England journal of medicine. 2011; 365: 725-733Crossref PubMed Scopus (630) Google Scholar, Brentjens et al., 2011Brentjens R.J. Rivière I. Park J.H. Davila M.L. Wang X. Stefanski J. Taylor C. Yeh R. Bartido S. 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Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+ T cell-mediated tumor eradication.Mol. Ther. 2010; 18: 413-420Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), although their effectiveness remains to be evaluated in clinical trials. The genetic modification of autologous peripheral blood T lymphocytes to generate tumor-targeted T cells is now a well-established approach that was developed in a handful of academic centers. The power and promise of TCR and CAR therapies utilizing these manufacturing processes are best illustrated by the exciting clinical results obtained with NY-ESO-1 TCR (Robbins et al., 2011Robbins P.F. Morgan R.A. Feldman S.A. Yang J.C. Sherry R.M. Dudley M.E. Wunderlich J.R. Nahvi A.V. Helman L.J. Mackall C.L. et al.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.J. Clin. 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These cell manufacturing processes combine T cell activation and transduction steps to generate expanded, genetically targeted T cell products. For example, T cells engineered to express specific CARs or TCRs may be initiated from Ficoll-purified PBMCs, which are next activated with anti-CD3 monoclonal antibody (mAb) in the presence of irradiated allogeneic feeder cells and transduced with a vector encoding either the CAR or TCR α and β chains (Till et al., 2012Till B.G. Jensen M.C. Wang J. Qian X. Gopal A.K. Maloney D.G. Lindgren C.G. Lin Y. Pagel J.M. Budde L.E. et al.CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1BB domains: pilot clinical trial results.Blood. 2012; 119: 3940-3950Crossref PubMed Scopus (65) Google Scholar, Morgan et al., 2006Morgan R.A. Dudley M.E. Wunderlich J.R. Hughes M.S. Yang J.C. Sherry R.M. Royal R.E. Topalian S.L. Kammula U.S. Restifo N.P. et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (1172) Google Scholar). We and others have established cGMP-compliant large-scale transduction and expansion processes, which are applicable to CARs or TCRs, utilizing either γ-retroviral or lentiviral T cell manufacturing (Figure 3). These processes begin with the selection and activation of T cells from patient apheresis products using materials coated with anti-CD3 and anti-CD28 mAbs. In the case of iron beads, CD3+CD28+ T cells are enriched using a magnetic particle concentrator and subsequently cultured. Activated T cells are retrovirally transduced in RetroNectin-coated cell bags and inoculated in a WAVE bioreactor where they are expanded with a continuous perfusion regimen, reaching cell densities of 10 million T cells/ml or more (Hollyman et al., 2009Hollyman D. Stefanski J. Przybylowski M. Bartido S. Borquez-Ojeda O. Taylor C. Yeh R. Capacio V. Olszewska M. Hosey J. et al.Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.J. Immunother. 2009; 32: 169-180Crossref PubMed Scopus (53) Google Scholar). At the end of the production, the beads are removed and the cells are formulated for immediate infusion or frozen for deferred use. The entire process typically takes 10–14 days, depending on the disease and the targeted T cell dose. This semi-closed large-scale manufacturing platform can be easily adapted for various vectors and for the expansion of either patient (autologous) or donor (allogeneic) T cells. It successfully supports several ongoing clinical trials in which therapeutic efficacy has been demonstrated (Brentjens et al., 2011Brentjens R.J. Rivière I. Park J.H. Davila M.L. Wang X. Stefanski J. Taylor C. Yeh R. Bartido S. Borquez-Ojeda O. 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 (215) Google Scholar, Brentjens et al., 2013aBrentjens R.J. Davila M.L. Riviere I. Park J. Wang X. Cowell L.G. Bartido S. Stefanski J. Taylor C. Olszewska M. et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci. Transl. Med. 2013; 5: 177ra138Crossref Scopus (180) Google Scholar, Davila et al., 2014bDavila M.L. Riviere I. Wang X. Bartido S. Park J. Curran K. Chung S.S. Stefanski J. Borquez-Ojeda O. Olszewska M. et al.Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.Sci. Transl. Med. 2014; 6: 224ra225Crossref Scopus (41) Google Scholar). This process starts from bulk T cells harvested from each individual subject. Several groups are currently evaluating what T cell phenotype and T cell subset or subsets account for the anti-tumor activity of these cells and what will be optimal tools to activate and expand T cells for different T cell therapies. Various means to enhance the activation and expansion of T cells for adoptive cell therapy have been reviewed elsewhere (Vacchelli et al., 2013Vacchelli E. Eggermont A. Fridman W.H. Galon J. Tartour E. Zitvogel L. Kroemer G. Galluzzi L. Trial Watch: Adoptive cell transfer for anticancer immunotherapy.Oncoimmunology. 2013; 2: e24238Crossref PubMed Scopus (20) Google Scholar). The functional, proliferative, and persistence potential of adoptively transferred T lymphocytes is determined by multiple factors. These include the TCR or CAR design, the manufacturing platform, the selected T cell subsets, and the differentiation stage of the harvested T cells. Peripheral blood T cells comprise naive (TN), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and terminal effector (TE) cells (Klebanoff et al., 2012Klebanoff C.A. Gattinoni L. Restifo N.P. Sorting through subsets: which T-cell populations mediate highly effective adoptive immunotherapy?.J. Immunother. 2012; 35: 651-660Crossref PubMed Scopus (27) Google Scholar). Several groups have investigated which of these T cell subsets are best suited for use in different adoptive therapy settings (Klebanoff et al., 2012Klebanoff C.A. Gattinoni L. Restifo N.P. Sorting through subsets: which T-cell populations mediate highly effective adoptive immunotherapy?.J. Immunother. 2012; 35: 651-660Crossref PubMed Scopus (27) Google Scholar, Riddell et al., 2014Riddell S.R. Sommermeyer D. Berger C. Liu L.S. Balakrishnan A. Salter A. Hudecek M. Maloney D.G. Turtle C.J. Adoptive therapy with chimeric antigen receptor-modified T cells of defined subset composition.Cancer J. 2014; 20: 141-144Crossref PubMed Scopus (2) Google Scholar). In non-human primates and murine NSG models, T cell transfer studies have shown that virus-specific and CAR-redirected anti-tumor CD8 TEM rapidly mature to terminal effector T cells and do not persist beyond 7–14 days, while a subset of transferred CD8+ TE/CM can acquire memory cell features and persist for months and even years (Wang et al., 2012Wang X. Naranjo A. Brown C.E. Bautista C. Wong C.W. Chang W.C. Aguilar B.