Annals of Oncology 30: 1740–1750, 2019 doi:10.1093/annonc/mdz285 Published online 21 August 2019 REVIEW Development of adaptive immune effector therapies in solid tumors P. Comoli1, C. Chabannon2, U. Koehl3,4, F. Lanza5, A. Urbano-Ispizua6, M. Hudecek7, A. Ruggeri8, S. Secondino9, C. Bonini10 & P. Pedrazzoli9*, on behalf of the European Society for Blood and Marrow Transplantation, Cellular Therapy & Immunobiology Working Party – Solid Tumor Sub-committee 1 Cell Factory and Pediatric Hematology Oncology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy; 2Institut Paoli-Calmettes, Aix-Marseille University, INSERM CBT 1409, Centre for Clinical Investigation in Biotherapy, Marseille, France; 3Institute of Clinical Immunology, University of Leipzig and Fraunhofer Institute for Cell Therapy and Immunology, Leipzig; 4Institute of Cellular Therapeutics, Hannover Medical School, Hannover, Germany; 5Hematology and Stem Cell Transplant, Romagna Transplant Network, Ravenna, Italy; 6Department of Hematology, IDIBAPS, Institute of Research Josep Carreras, Hospital Clı́nic, University of Barcelona, Barcelona, Spain; 7Department of Medicine II, University Hospital Würzburg, Würzburg, Germany; 8Department of Pediatric Hematology and Oncology, Bambino Gesù Children’s Hospital, Rome; 9Oncology Unit, Fondazione IRCCS Policlinico San Matteo, Department of Internal Medicine and Medical Therapy, University of Pavia, Pavia; 10Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, University Vita-Salute San Raffaele and Ospedale San Raffaele Scientific Institute, Milan, Italy *Correspondence to: Prof. Paolo Pedrazzoli, UOC Oncology, Fondazione IRCCS Policlinico S. Matteo, Piazzale Golgi, 19-27100 Pavia, Italy. Tel: þ39-0382-501659; E-mail: p.pedrazzoli@smatteo.pv.it State-of-the-art treatment strategies have drastically ameliorated the outcome of patients affected by cancer. However, resistant and recurrent solid tumors are generally nonresponsive to conventional therapies. A central factor in the sequence of events that lead to cancer is an alteration in antitumor immune surveillance, which results in failure to recognize and eliminate the transformed tumor cell. A greater understanding of the dysregulation and evasion of the immune system in the evolution and progression of cancer provides the basis for improved therapies. Targeted strategies, such as T-cell therapy, not only generally spare normal tissues, but also use alternative antineoplastic mechanisms that synergize with other therapeutics. Despite encouraging success in hematologic malignancies, adaptive cellular therapies for solid tumors face unique challenges because of the immunosuppressive tumor microenvironment, and the hurdle of T-cell trafficking within scarcely accessible tumor sites. This review provides a brief overview of current cellular therapeutic strategies for solid tumors, research carried out to increase efficacy and safety, and results from ongoing clinical trials. Key words: immunotherapy, solid tumors, T cells, CAR-T, checkpoint inhibitors Tumor cells develop multiple mechanisms to evade immune rec- Introduction ognition, including downregulation of tumor antigens, gener- Resistant, metastatic, or recurrent solid tumors represent unmet ation of an immunosuppressive microenvironment though clinical challenges, since they are seldom surgically resectable, secretion of anti-inflammatory cytokines and expression of nega- and largely nonresponsive to radiation and chemotherapy. tive immune regulators able to silence immune effectors [3]. Relapse and chemoresistance are generally due to cancer cells ATCT was introduced and tested in the field of solid tumors; endowed with stem-like features (CSC) [1], which persist in com- however, it has produced only sporadic responses. This standstill plex cellular niches that provide a unique microenvironment to is attributable to limitations in the understanding of tumor biol- protect and promote CSC survival, self-renewal, maintenance, ogy and interactions with its microenvironment. In addition, and migratory ability [2]. techniques used to identify, isolate, and amplify immune effector Modern medical innovations simultaneously try to address cells have not been amenable to manufacturing processes that tumor heterogeneity in space and over time—including cancer comply with industry standards. Considerable progress has been stem cell recognition and eradication—and to initiate, stimulate made along these two avenues: improved knowledge of cancer or amplify a clinically meaningful antitumor immune response. immune evasion mechanisms and host–tumor interactions have C The Author(s) 2019. Published by Oxford University Press on behalf of the European Society for Medical Oncology. V All rights reserved. For permissions, please email: journals.permissions@oup.com. Annals of Oncology Review led to the development of immune checkpoint inhibitors, which applications of cytokine-induced immune effector cells, gener- are lead products for immune modulation in vivo. ated from patients’ lymphocytes in the presence of high-dose Simultaneously, considerable technical refinements have opened rhIL-2. Objective clinical responses were reported [13], and al- new prospects for the development of immune cell-based medi- though these could not be dissociated from the effects of the co- cinal products [4–6], as recently exemplified by the unprecedent- infusion of high-dose rhIL-2, they provided the basis for clinical ed success with chimeric antigen receptor (CAR)-T cells targeting evaluation of tumor-specific T cells obtained by in vitro culture B-cell hematologic malignancies [7–9]. (Figure 1). Replicating these results in solid tumors is a major scientific challenge, and may not be feasible to the extent observed with CD19 positive malignancies. Hurdles include the difficulty of Tumor-infiltrating lymphocytes identifying target antigens that are homogeneously expressed by T cells directed to tumor-associated antigens (TAA) generally in- all tumor cells while absent on normal tissues, impairment of T- filtrate tumor tissue, represent a biomarker associated with cell trafficking to the tumor site, and limited T-cell persistence improved prognosis in some instances and may be extracted, and proliferation in a hostile tumor microenvironment that expanded ex vivo in the presence of homeostatic cytokines, and favors immune escape. Emerging evidence suggests that control reinfused into patients. Evidence that transferred T cells can of resistant or metastatic cancers will not be achieved with a single eradicate cancer in humans comes from the success of ATCT therapeutic agent, but rather with combinations of conventional with cultured lymphocytes isolated from tumor biopsies (TILs) and immunotherapeutic cancer treatments [10]. In this perspec- in patients with melanoma [14–17]. The first trial, conducted at tive, drugs that target immune checkpoints, such as the CTLA4- the National Cancer Institute (NCI) on 20 patients with metastat- B7 and the PD1-PDL1 pathways, have led to clinical benefits ic disease, showed that transfer of expanded TILs and rh-IL2, pre- across a number of different tumor types and may well represent ceded by a single dose of cyclophosphamide, could induce more a sort of adjuvant backbone facilitating response to ATCT [11]. than 50% objective responses including a complete response Here, we focus on the description of ATCT starting from early (CR) with minimal toxicity [13]. Since this preliminary report, experiments and extending to more recent approaches based on results in melanoma have further improved through the opti- gene modification, with the aim of overcoming immune evasion mization of TIL expansion protocols and preparative regimens, to pave the way for effective solid tumor control. combining fludarabine with cyclophosphamide chemotherapy, and testing different doses of total body irradiation, successfully doubling the CR rate to 24% [15, 18]. The importance of lym- phodepleting chemotherapy, highlighted in the melanoma set- T cells and tumor recognition ting, has also been crucial for CAR-T-cell therapy protocols [7, To understand how ATCT contributes to cancer immune surveil- 19]. Although TILs could be demonstrated in other solid tumors, lance, it is important to get insight into the basics of tumor recog- their expansion and in vivo efficacy did not reach the success of nition. Maturation of T cells occurs within the thymus, where the melanoma treatment, experienced in hundreds of patients treated T-cell receptor (TCR) repertoire is shaped by somatic gene re- at the NCI [5] and recently duplicated by other groups [20, 21], arrangement and selection processes, resulting in a T-cell pool and by a multicenter phase II study that is currently being characterized by limited reactivity to self but strong reactivity to expanded for registration purposes [22]. Recently, preliminary foreign antigens. The TCR does not bind directly to its antigen, data showing objective responses and CRs were reported for chol- but recognizes a limited number of short peptides derived from angiocarcinoma (CCA) [23] and cervical cancer [24], and a num- the antigen (epitopes), bound to major histocompatibility com- ber of clinical trials are currently ongoing to test ATCT with TILs plex class I (MHCI) or class II (MHCII) on the target cell surface. in gastrointestinal, gynecological, head and neck, breast and lung Most cells express MHCI, which presents epitopes to CD8þ cyto- cancers. toxic T lymphocytes (CTLs): lysis of cells expressing cognate epit- opes in the context of MHCI is considered the main mechanism responsible for antitumor immune surveillance. However, in re- Virus-specific cytotoxic T lymphocytes cent years, the role of CD4þ T cells, which recognize epitopes Among T cells targeting antigens through their native receptors, complexed with MHCII on the surface of professional antigen- CTLs directed to viral antigens, obtained by in vitro culture in the presenting cells, has gained increasing attention, not only for presence of virus-derived TAA, have also been extensively used their helper function in sustaining CD8þ T-cell responses and [25]. Epstein–Barr virus (EBV)-specific T cells expanded from activating innate immunity, but also for direct killing of tumor the hematopoietic stem cell donor have been demonstrated to cells [12]. prevent and treat virus-associated lymphoproliferative diseases [26–28]. Autologous EBV-specific CTLs, which have shown anti- tumor activity in patients with less immunogenic hematologic Adaptive cell therapy with natural T cells malignancies, such as Hodgkin or non-Hodgkin lymphoma [29, 30], have also been successfully employed in the treatment of endowed with antitumor activity EBV-associated solid tumors, such as nasopharyngeal carcinomas The modern era of ATCT began with the use of recombinant (NPC) [31–35]. More than 80 NPC patients treated with autolo- human interleukin-2 (rhIL-2) in the treatment of melanoma and gous EBV-targeted ATCT for recurrent, metastatic disease, have renal cell cancers, and the observation that this cytokine was able so far been reported, with a promising 20% objective response to favor human T-cell growth in vitro. This led to the first clinical rate, including 10% CR. Attempts to enhance T-cell survival, Volume 30 | Issue 11 | 2019 doi:10.1093/annonc/mdz285 | 1741 Review Annals of Oncology 1. TIL EXPANSION FROM TUMOR TISSUE TUMOR-SPECIFIC T CELLS IL-2 TILs are isolated from resected tumor tissue, selected and expanded to large numbers by culture in the presence of rhIL-2 2. EX VIVO EXPANSION OF TAA-SPECIFIC T CELLS ILs Peripheral blood T cells are stimulated by specific TAA recognition on antigen-presenting cells, and expanded to large numbers by culture in the presence of homeostatic cytokines 3. GENETIC MODIFICATION Peripheral blood T cells are activated, and their activity is redirected by insertion on of TCR CAR R or CA transgene through viral carrier infection or electroporation resting T cell tumor cell transgene electroporation aactivated T cell dendritic cell transgene in virus carrier natural TCRs Figure 1. ATCT for solid tumors: manufacturing strategies. T cells derived from tumor resection tissue or peripheral blood are manipulated according to the illustrated approaches, in order to manufacture tumor-directed cellular products for ATMP. IL-2, recombinant human inter- leukin-2; ILs, interleukins; TAA, tumor-associated antigens; CAR, chimeric antigen receptors. trafficking and effector function by administering a lymphode- technologies, a quest for mutational and transcriptional tumor pleting preparative regimen, based on the melanoma TIL therapy profiles was embraced, in order to identify optimal personalized experience, did not improve outcomes in this clinical setting [33, targeted therapies. While the majority of mutant gene products 34]. In line with EBV ATCT, cytomegalovirus-specific CTLs have are not targetable with the currently available pharmaceuticals, been explored in clinical trials for glioblastoma [36], and human the genetic sequencing approach has allowed the identification of papilloma virus (HPV)-directed TILs have been successfully neoantigens generated by gene rearrangements or mutations [38]. employed in HPV-associated malignancies [24]. Genomic studies suggest how the latter may be the main target antigens underlying the success of therapy with immune check- point inhibitors or TILs [39, 40]. Indeed, when compared with T cells targeting other TAA non-mutated self-antigens, the T-cell pool available for these anti- For tumors not associated with viruses, several classes of TAAs gens should not be affected by central T-cell tolerance. Shared have been explored as potential targets [37]. These include anti- hot-spot mutations of driver oncogenes are more likely to be gens that are mostly non-tumor-selective, as they are found in expressed by all cells within a tumor as well as across tumor sam- normal tissues but overexpressed on tumors, or expressed only ples and histologies, and identification of TCRs targeting these during fetal development or in immune-privileged sites such as neoantigens may be used as off-the-shelf reagents for TCR gene testes. T cells targeting these TAAs may potentially attack healthy therapy. Moreover, their use is likely to limit the risk of on-target tissues expressing even low-level antigen, causing severe adverse off-tumor toxicity. A proof of principle for the potential in vivo ef- events, referred to as on-target off-tumor toxicities, when an es- ficacy of T cells that target tumor neoantigens arising from cancer sential organ is involved. With recent advances in genomic mutations has been provided in the setting of solid tumors [41]. 1742 | Comoli et al. Volume 30 | Issue 11 | 2019 Annals of Oncology Review TUMOR CELL MHC-TAA MHC-TAA TAA TAA TAA Extracellular domain Transmembrane domain CoDomain 1 CoDomain 1 CD3 CD3 CD3 (CD28 or (CD28) 4-1BB) CoDomain 2 Intracellular (4-1BB or OX40) CD3 domain CD3 endogenous engineered 1st generation 2nd generation 3rd generation TCR TCR CAR CAR CAR T-CELL Figure 2. Structure of endogenous TCR, engineered TCR and CAR receptors. In first generation CARs, the variable heavy and light single- chains (antigen-binding moiety) are linked by a spacer to the transmembrane region, usually derived from CD28. The intracellular domain includes the CD3f signaling pathway machinery, which activates the T cell in response to the specific tumor antigen binding. In second and third generation CARs, co-stimulatory domains, such as CD28 and/or 4-1BB, are added to improve antitumor potency, cytokine production, and persistence of the T cell. MHC, major histocompatibility complex; TAA, tumor-associated antigen; CoDomain, costimulatory domain. Adaptive cell therapy with engineered T retrovirally transduced with a TCR specific for NY-ESO-1 resulted in objective clinical responses in 61% of patients with synovial cell cells: the era of CARs sarcoma and 55% of patients with melanoma [44, 45], and one of ATCT with TILs/CTLs has been mostly successful in cancers three patients treated with autologous T cells engineered to ex- characterized by strong immunogenicity due to high frequency of press a TCR against human carcinoembryonic antigen (CEA) had mutational events [38]. The targeting of cancers such as pediatric an objective regression of metastatic colorectal cancer (CRC) [46]. solid malignancies or other frequent tumors, such as breast or The rate of objective responses obtained in melanoma patients did prostate cancer, for which neoantigens generated by non- not significantly differ from those obtained in clinical trials evalu- synonymous mutations are limited, is a more daunting task. ating TILs, but fewer sustained CRs were observed. These data are Indeed, TAA are mostly overexpressed self-antigens, and, as such, likely due to inherent limitations of TCR biology. In particular, subject to tolerance. In addition, TAA recognition by T cells is HLA-restricted specificity limits the fraction of potential patients MHC-restricted, and the majority of solid tumors downregulate to those expressing the relevant HLA allele recognized by T cells, MHC expression as an immune escape mechanism. In order to and clinical efficacy is dependent upon TCR affinity and the ex- overcome these hurdles, genetically engineered T cells have pression of MHC-antigen complexes on the tumor cell surface emerged as an alternative to TILs and CTLs (Figure 1). [42]. The transferred and endogenous TCRs compete for CD3 Genetic retargeting has been obtained by means of two different binding, resulting in mutual receptor dilution and lower antitu- approaches: (i) transfer of natural tumor-specific TCRs isolated mor efficacy. Moreover, the a and b chains of the endogenous from high-avidity T cells that recognize cancer antigens [42] and (ii) TCR may mispair with the respective chains of the transferred transfer of synthetic TCRs, also referred to as CARs [43] (Figure 2). TCR, forming hybrid receptors with unpredictable and potentially detrimental specificities. Furthermore, targeting of antigenic spe- cificities that are expressed at low level on normal tissues has led to T cells engineered with natural TCRs severe on-target off-tumor toxicity [47, 48]. TCR therapy trials have mostly targeted overexpressed self/tumor antigens also expressed by healthy adult cells, such as gp100 and Melan-A/MART-1, or oncofetal antigens, present on healthy cells T cells engineered with CARs exclusively during fetal development, and ectopically expressed In order to circumvent the challenges posed by antigen escape in tumors, such as NY-ESO-1 and MAGE. Autologous T cells variants, researchers have focused on the delivery of synthetic Volume 30 | Issue 11 | 2019 doi:10.1093/annonc/mdz285 | 1743 Review Annals of Oncology CARs, constructed by fusing an antigen-binding domain that is high-risk neuroblastoma, 3 had a CR to CAR-T-cell infusion, derived from a single-chain variable fragment of a monoclonal with only local pain and slight fever as adverse events [53, 66]. antibody against a tumor surface antigen, to a flexible spacer re- The same group evaluated the safety and antitumor activity of gion, a trans-membrane domain and the TCR intracellular do- second-generation CD28f HER2-specific CAR-modified virus- main, traditionally consisting of the CD3 zeta (f) chain, capable specific T cells in patients with progressive glioblastoma of activating T cells. As the antigen-directed exodomain binds multiforme (GBM). The results showed that CAR-T cells are well directly to target cell surface epitopes, CAR recognition of tumor tolerated, with no dose-limiting toxic effects, and can produce target cells is HLA-unrestricted, thus resistant to tumor escape objective responses, as one patient showed a PR for more than mechanisms related to HLA downmodulation and altered proc- 9 months, and seven patients had SD lasting several months [67]. essing. Initial trials with CAR-T cells targeting solid and hemato- Other clinical trials have demonstrated the feasibility and safety logic tumors showed limited clinical results, with poor in vivo [68] and clinical efficacy [69] of second-generation EGFRvIII- expansion and duration [49–52]. In the setting of solid tumors, specific and IL13BBf-specific CAR-T cells, respectively, in the unique exception was CAR-T-cell therapy targeting the patients with refractory GBM. disialo-ganglioside GD2, which induced complete remission in 3/ Investigators at the University of Pennsylvania explored an ap- 11 pediatric patients with neuroblastoma [53]. proach based on mRNA-transduced CAR-T cells that target mes- othelin (CART-meso) in patients with advanced malignant CAR-T-cell therapy in hematologic malignancies. Progressive pleural mesothelioma or advanced pancreatic cancer. In the first improvements in the design of CARs have led to second and third two patients reported, CART-meso cells showed some antitumor generation molecules (Figure 2), that incorporate one or two activity in vivo, in the absence of distinct toxicities [70]. additional costimulatory domains, such as CD28, 4-1BB and Second-generation CAR-T cells specific for epidermal growth OX40, which enhance killing activity and expansion potential factor receptor (EGFR) were employed in a phase I study that (CD28), as well as in vivo persistence (4-1BB), resulting in dra- treated 11 patients with advanced non-small-cell lung cancer, matic results in hematologic malignancies [54–61], with 81% 3- obtaining 2 PR and 5 SD lasting from 2 to 8 months, with limited month remission rate and 50% event-free survival at 12 months adverse events including skin toxicity, nausea, vomiting, dyspnea in a phase II global multicenter clinical trial with CD19-CAR-T and hypotension [71]. EGFR-CAR-T cells were also applied by cells in children and young adults treated for acute lymphoblastic the same investigators in the treatment of one patient with refrac- leukemia [61]. tory, metastatic CCA, demonstrating a PR lasting more than In every trial, efficacy correlated with the expansion of CAR-T 1 year. However, the objective response was accompanied by epi- cells, and relapses were due to immune escape mechanisms, such dermal and endothelial toxicity [72]. A clinical trial of CEA CAR- as targeted antigen loss or development of immunity directed to T therapy in 10 patients with metastatic CRC achieved SD in 7 the CAR molecule mouse portion [58, 59]. The increase in CAR- patients who were in progression after previous treatments, with- T-cell clinical efficacy, however, was paralleled by the potential to out severe adverse events related to CAR-T therapy [73]. Similar induce severe adverse events, such as cytokine release syndrome to these experiences, second-generation HER2-specific CAR-T (CRS), neurological toxicity, and on-target off-tumor toxicities cells, used in a phase I clinical trial conducted on 19 patients with [57–62]. While still within the field of lymphoid malignancies, refractory HER2-positive sarcoma, could induce SD lasting from the fast-growing experience when treating multiple myeloma 12 weeks to 14 months in 4 assessable patients [74]. patients with CAR-T cells targeting BCMA suggests that the in- tensity of side-effects may vary depending on the targeted tumor Toxicity of CAR-T-cell therapy. Various toxicities were observed antigen, and that side-effects may be manageable even in an older after CAR-T-cell infusion in solid tumors. In the setting of hema- population [63–65]. To date, two autologous CAR-T cells target- tologic malignancies, CRS is a frequent, potentially severe adverse ing CD19 have been approved by FDA and EMA for the treat- event of CAR-T-cell therapy. The release of pro-inflammatory ment of patients with refractory/relapsed ALL or high-grade cytokines by the infused cells induces monocyte and macrophage B-NHL. Since the two medicinal products benefited from the fast activation that can lead to multiple organ failure. Currently, this track procedure, the number of patients that contributed data complication is managed by cytokine level assessment and early were relatively small; thus, additional data obtained in ‘real-life’ administration of the anti-IL6R monoclonal antibody tocilizu- practice after long-term follow up are needed to fully appraise mab. However, CRS, as well as the neurological toxicity some- their clinical utility. times observed in the hematologic setting, is not a common event after CAR-T-cell therapy for solid tumors, possibly since the CAR-T-cell therapy in solid tumors. On the basis of results tumor load is lower. Conversely, trials conducted in solid tumor obtained in hematologic malignancies, a number of trials have cohorts with CAR-T cells produced critical, unexpected on-target been started to test the efficacy of CAR-T cells in solid tumors off-tumor toxicities, resulting from the recognition by CAR-T (supplementary Table S1, available at Annals of Oncology online). cells of TAA expressed on healthy tissues [75, 76]. Some antigens To date, however, clinical results in this setting have been much specific to tumors have been identified that result in more limited less encouraging, with a general lack of therapeutic response and off-tumor effects, but many of these targets have mediated poor presence of on-target off-tumor toxicity. However, some studies clinical efficacy. Moreover, as for hematologic malignancies, solid have achieved promising outcomes that justify further explor- tumors undergo antigen escape due to selection pressure favoring ation of this approach in solid tumors. The group at Baylor tumor cells lacking the targeted antigen. Strategies to increase College of Medicine pioneered the use of GD2-specific CAR-T tumor selectivity while sparing healthy tissues are being evaluated cells for neuroblastoma [53, 66]. In a trial of 19 patients with to control on-target off-tumor toxicity. 1744 | Comoli et al. Volume 30 | Issue 11 | 2019 Annals of Oncology Review Improving safety and efficacy of T-cell factor beta, vascular endothelial growth factor, and prosta- glandin E2. Additionally, negative feedback is provided by sig- therapy for solid tumors naling through the programmed death-1 (PD-1) and CTLA-1 A significant amount of research has focused on enhancing pathways (Figure 3A). the activated T-cell response against tumor cells, promoting their in vivo expansion, and improving their persistence in T-cell arming. To repolarize the tumor microenvironment and the host. Interventions encompass genetic modifications to in- fully restore the effector function of transferred T cells, preclinical crease T-cell affinity or avidity [77, 78] or to induce cytokine studies have tested the feasibility of conferring resistance to im- production [79–81], and selection of specific T-cell subsets munosuppressive molecules [92], or the ability to deliver cyto- [82, 83]. Regarding the choice of genetic modification vehicle, kines to either activate host effectors or hinder host suppressors so far retroviral gene transfer vectors have been mostly used [79–81, 93], or coupling CAR-T cells with switch receptors inter- in CAR-T-cell trials, as they have the advantage of inducing fering with the inhibitory signal provided by immune check- high-level stable transduction in stimulated human T cells. points [94] (Figure 3B). The cytokine-producing ‘TRUCKs’ (T However, immunogenicity and insertional mutagenesis, to- cells Redirected for Universal Cytokine Killing) [95] significantly gether with the costs due to viral vector production and use, enhanced efficacy of MUC-16ecto CAR against a preclinical model has led to explore alternative methods, such as lentiviral vec- of ovarian carcinoma [96], and are currently being tested in phase tors, that do not require cell cycling induction and cause very I clinical trials. limited immune and inflammatory responses, or the use of gene transfer forms that do not depend on costly viral vector production, such as transposons, or the non-integrating sys- Combination of T-cell therapy with checkpoint inhibitors. In add- tems like designer nucleases, DNA plasmids or RNA transfer ition to T-cell arming, an approach that combines ATCT with [52, 57, 84, 85]. Strategies to enhance affinity to self-antigens immune checkpoint inhibitors, antitumor agents that act by have increased efficacy, but also led to unexpected severe off- releasing the ‘brakes’ on pre-existing tumor-reactive T cells and target toxicity, when previously unknown cross-reactivity was facilitate generation of new T-cell responses, may represent a observed with epitopes derived from unrelated proteins strategy to improve the activity and tumor killing by T-cell gener- expressed by normal tissues [48]. ated ex vivo [97–99] (Figure 3B). Recent positive experiences have been observed in refractory hematologic malignancies with T-cell migration and intra-tumor trafficking CD19 CAR-T cells combined with the PD-1 blocking agent pem- brolizumab [100, 101]; similar strategies are being investigated in The first hurdle encountered by T cells when targeting solid solid tumors [102]. malignancies is the difficulty in migrating to and adequately pen- etrating the tumor. Solid cancers may evade immune surveillance by secreting chemokines which inhibit T-cell migration into the Increasing T-cell therapy safety tumor [86]. Several different chemotherapeutics have been Solid tumors typically exhibit a heterogeneous pattern of TAA, shown to induce chemokines and chemokine ligands [87], and promoting antigen escape. Thus, identifying patient-specific, their incorporation into a combined treatment approach with T tumor-specific mutated antigens [41, 103], potentially present cells may enhance CD8þ T-cell recruitment and reduce tumor also in cancer types that exhibit low-level immunogenicity, may growth. Alternatively, CAR-T cells may be modified to express help develop treatments for multiple cancer types so far not chemokines that enhance their intra-tumor trafficking [88]. It amenable to ATCT, and limit toxicity. Despite these advances in has been shown that the forced expression of chemokine CCR2 targeting, toxicity may still ensue. In order to limit severe conse- on CAR-T cells targeting GD2 in neuroblastoma enhances T-cell quences, strategies aimed at hampering an unwanted off-target infiltration and augments the antitumor activity of the trans- T-cell response are being investigated. Recently, a pharmacologic ferred cells [89]. In addition, T cells migrating to the tumor may on/off switch based on the use of the tyrosine kinase inhibitor be hindered by a physical barrier of fibrotic extracellular matrix dasatinib has been investigated [104]. produced by tumor fibroblasts and myeloid cells. As loss of hep- aranase, an enzyme that contributes to degradation of extracellu- Suicide gene insertion. The introduction of suicide genes may lar matrix, has been observed in T cells after in vitro culture, its help remove undesirable toxicity [105, 106]. The first attempt at overexpression in CAR-T cells can enhance T-cell infiltration introducing suicide genes into T cells involved the use of HSV- [90]. Alternatively, targeting the surrounding non-malignant thymidine kinase, which converts ganciclovir into a toxic metab- stroma using CAR-T cells may be an option [91]. olite, and proved effective in controlling graft-versus-host disease (GvHD) in several trials [105, 107]. However, the lytic response Tumor microenvironment repolarization observed was slow, and the viral proteins may stimulate immuno- The complex immunosuppressive tumor microenvironment, genicity. More recently, the inducible-caspase 9 system was and the segregation of tumor cells in areas that may be diffi- designed and tested. It is based on the expression of a monomeric cult to access, are other major hurdles to the efficacy of iCasp9 domain that dimerizes upon administration of a small ATCT. Factors that negatively regulate T-cell activity and fa- molecule, leading to cleavage of caspase 3 and apoptosis of T cells cilitate tumor immune evasion include tumor-resident regula- [106]. Alternatively, in the case of CAR-T cells, incorporating tory T cells and myeloid-derived suppressor cells, as well as epitopes like RQR8/CD20 into the CAR construct provides a tar- the presence of cytokines such as IL-10, transforming growth get for their depletion with antibodies such as rituximab [108]. Volume 30 | Issue 11 | 2019 doi:10.1093/annonc/mdz285 | 1745 Review Annals of Oncology A MDSC TUMOR PD-L1 CELL PD-L1 APC PD-1 PD-1 PD-L1 TAA PD-1 MHC-TTA TGF-β CAR CD80/86 CAR CTLA-4 CAR-T CELL C LL L X IFNγ CD28 EXHAUSTED T-REG IL-4 TGF-β IL-10 B MDSC TUMOR PD CPI PD-L11 PD-L1 L1 CPI CELL PD-L1 P APC PD-1 TAA PD-1 PD-1 MHC-TTA MHC T CPI X X TGF-β CAR X CAR CD80/86 CTLA-4 IFNγ CAR-T CELL C L CD28 dnTGF-bR T-REG IL-4 IL-7 endodomain TGF-β dnTGF-bR X X IL-4 exodomain IL-10 SWITCH IL-18 CAR TRUCK IL-12 Figure 3. Summary of interventions to overcome the immunosuppressive tumor microenvironment and enhance T-cell efficacy. Tumor cells and suppressor cells (i.e. T-REGs; MDSCs) up-regulate inhibitory ligands, such as PD-L1 or CTLA-4, and secrete suppressive cytokines and fac- tors (A). The use of checkpoint inhibitor agents, or modification of CAR T cells to induce cytokine secretion (TRUCKs), expression of a domin- ant negative (dn) TGFb-receptor or conversion of a negative IL-4 signal to a positive IL-7 signal (SWITCH CARs) are examples of strategies able to help overcome tumor tolerance and restore tumor immunogenicity (B). CAR, chimeric antigen receptor; MDSC, myeloid-derived suppres- sor cells; APC, antigen-presenting cells; T-REG: regulatory T cell; MHC, major histocompatibility complex; TAA, tumor-associated antigen; CPI, checkpoint inhibitor; IL, interleukin; TGF-b, tumor growth factor-b; IFNc, interferon-c; dnTGF-bR, dominant negative TGFb-receptor; SWITCH CAR, chimeric switch receptors; TRUCK, T cells redirected for universal cytokine killing. Specificity enhancement. Other strategies have been devised to in- cells that activate T cells based on the density of target antigen ex- crease CAR-T-cell specificity, and, therefore, limit toxicity. One pression. Two independent studies demonstrated that a CAR-T approach is based on T-cell modification with two different cell with reduced affinity rendered T cells preferentially activated CARs, one containing the CD3f signaling domain to activate T- by a high, but not low, density of target antigen [112, 113]. cell function, and the other providing the co-stimulation signal by CD28 and/or CD137 [109–111]. Full CAR-T-cell activation and function are only achieved in the context of the presence of Widening T-cell therapy access both antigens, an unlikely occurrence in the case of cells from Despite the therapeutic potential of ATCT, logistics and regula- normal tissue. Another strategy to obtain differential recognition tory hurdles have limited translation into commercially available of malignant and normal cells is based on affinity-tuned CAR-T therapies. Regulatory demands for marketing authorization are 1746 | Comoli et al. Volume 30 | Issue 11 | 2019 Annals of Oncology Review burdensome for academic institutions, although the new and AIRC 5 per Mille Rif. 22737, to CB), and by Fondazione European clinical trial regulation favors accelerated novel drug IRCCS Policlinico San Matteo (grant Ricerca Corrente number evaluation and approval schemes to ensure early access to in- 08067617 to PP, 08069113 and 08069119 to PC). novative therapies [114]. The majority of subjects treated to date with ATCT have received autologous or allogeneic dedicated T cells, but this approach may not be best suited for widespread Disclosure cost-effective delivery of cellular therapy, since these are person- alized medicines that are produced on-demand through a com- PC: Atara Therapeutics: advisory board; Bellicum plex and costly supply chain, thus implying some delay in Pharmaceuticals: advisory board. CC: Gilead: co-investigator, manufacturing of the final product and the risk that the disease speaker, advisory board, hospitalities; Celgene: co-investigator, will evolve and be fatal for candidate patients. Allogeneic ATCT, speaker. Novartis: co-investigator, speaker. Sanofi SA: research including CAR-T cells, represent potential off-the-shelf products, support, speaker, advisory board, hospitalities; Miltenyi: re- that could possibly be manufactured in small batches without the search support; Terumo BCT: speaker, advisory board; need for tailored products. However, they carry the risk of im- Fresenius: research support. CB: received a Research contract mune rejection by the host, and their short persistence may re- from Intellia Therapeutics and participated to the advisory quire additional therapies to consolidate responses, and could boards of Molmed, Intellia Therapeutics, TxCell, Novartis, GSK, potentially cause GVHD, although in preliminary clinical trials Allogene. She is inventor of patents in the field of adoptive de novo GVHD was not observed [115]. Gene editing offers the T-cell therapy. 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