Review Special Focus Issue: Adoptive cell immunotherapy for cancer For reprint orders, please contact: [email protected]
Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
Recent spectacular success in the adoptive cell therapy of leukemia and lymphoma with chimeric antigen receptor (CAR)-modified T cells raised the expectations that this therapy may be efficacious in a wide range of cancer entities. The expectations are based on the predefined specificity of CAR T cells by an antibody-derived binding domain that acts independently of the natural T-cell receptor, recognizes targets independently of presentation by the major histocompatibility complex and allows targeting toward virtually any cell surface antigen. We here discuss that targeting CAR T cells toward solid tumors faces certain circumstances critical for the therapeutic success. Targeting tumor stroma and taking advantage of TRUCK cells, in other words, CAR T cells with inducible release of a transgenic payload, are some strategies envisaged to overcome current limitations in the near future.
Hinrich Abken Clinic I Internal Medicine, Tumor Genetics, University Hospital Cologne, Cologne, Germany and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Robert-Koch-Str. 21, D-50931 Cologne, Germany hinrich.abken@ uk-koeln.de
Keywords: adoptive cell therapy • cancer • CAR • chimeric antigen receptor • gene transfer • T cell
Concept of CAR & T cells directed for universal cytokine-mediated killing modified T cells The concept of adoptive cell therapy with chimeric antigen receptor (CAR, immunoreceptor) modified cells is based on patient’s T cells that are redirected with predefined specificity toward autologous cancer cells to execute their immune response. T cells are ex vivo modified with a recombinant receptor molecule which by the extracellular part recognizes a pre-defined target by an antibody derived binding domain and by the intra cellular T-cell receptor (TCR) derived signaling part initiates T-cell activation upon target engagement (Figure 1) [1] . Such CAR T cells (also nick-named ‘T-bodies’) recognize their target independently of presentation through the major histocompatibility complex (MHC) and are thus not compromised by alterations in the antigen processing and presentation machinery as often observed during tumor progression [2,3] . CAR T cells can furthermore be modified to act as a ‘factory’ to produce a transgenic protein upon
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CAR engagement of target. Such T cell redirected for universal cytokine-mediated killing (TRUCKs) deliver in a coordinated and locally restricted fashion a ‘payload’ which may be a cytokine like IL-12 which in turn activates an innate immune response toward tumors [4,5] . For further details we refer to recent reviews [5–7] . Compared with physiological TCR recognition, the CAR mediated T-cell activation provides several advantages in the context of adoptive cell therapy. First, CARs can redirect T cells toward a broad variety of targets as long as a binding domain with suitable specificity is available. Potential CAR targets in addition to classical MHC bound peptides also include nonclassical TCR targets such as carbohydrates, lipids and conformational epitopes. While most CARs use an antibody derived scFv for targeting, some CARs were reported, which consist of receptor ligands as binding domain fused to the transmembrane and intracellular signaling moiety. Examples are heregulin and IL-13 mutein to target Her3/4 receptor and IL-13 receptor-α,
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scFv
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scFv
scFv
scFv
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Figure 1. Modular composition of the chimeric antigen receptor. The prototype CAR is composed of one polypeptide chain; the extracellular scFv antibody domain mediates binding to the target antigen in an MHC independent fashion. Upon CAR clustering, the intracellular TCR-derived CD3ζ chain, with or without costimulation through members of the CD28 family, initiates the downstream signaling for T-cell activation. Coreceptors may modulate CAR activity. In contrast to a first-generation (1°) CAR, second- (2°) and thirdgeneration (3°) CARs harbor one or two costimulatory moieties in their intracellular part. TRUCK cells are CAR T cells that are additionally modified with a CAR inducible expression cassette for a transgenic polypeptide product that is produced and released upon CAR signaling. Such TRUCK cells can be used as CAR targeted ‘factories’ that deposit a defined product in the targeted tissue. CAR: Chimeric antigen receptor; scFv: Single chain fragment of variable region; TCR: T-cell receptor.
respectively [8,9] . To be recognized by CAR T cells, the antigen needs to be present on the cell surface of the targeted cell; however, TCR-like CARs were reported, which recognize intracellular antigens presented by the MHC [10,11] . In contrast to cell-bound antigen, soluble antigen, which frequently accumulates in the serum of cancer patients, does not sufficiently activate CAR T cells [12] . CAR redirected T-cell activation initiates a plethora of T-cell effector functions including cytokine release, lytic degranulation, T-cell amplification, maturation and migration. Clinical efficacy requires lasting persistence of CAR T cells which need to be protected from activation induced cell death through appropriate costimulation, for example, CD28 or 4-1BB. This is underscored by the clinical observation that patients with achieved lasting remission showed long-term CAR T-cell persistence [13] . CAR T cells can moreover provide target-specific memory which may help to prevent tumor relapse [14] . CAR signaling blocks: combined primary & costimulatory signals are superior The intracellular signaling moiety is commonly derived from the CD3ζ chain; other signaling domains including the FcɛRI γ chain are used as well. Since full T-cell activation and fine-tuning requires appropriate costimulation, the primary signaling moiety was fused to a costimulatory signaling chain, a prototype of which is CD28, giving rise to a so-called second-generation CAR [15] . Each costimulatory endodomain in the context of a CAR orchestrates a
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distinct pattern of effector functions as revealed by a side-by-side comparison [16] . To combine the benefits, third-generation CARs were engineered with two costimulatory domains in addition to the primary signal. This is of particular interest for redirecting T-cell subsets in more progressed maturation stages which need different costimuli for activation than young T cells [17] . Repetitive stimulation by antigen forces T cells to progress in terminal differentiation which is also the case when the CAR is engaging the cognate target. Consequently, more matured CAR T cells which lack CCR7 accumulate at the tumor lesion, at least in experimental models, while both CCR7+ and CCR7- T cells are equally efficient in targeting to the tumor lesion. Since CCR7- T cells are prone to spontaneous and activation induced cell death, which is not prevented by CD28 costimulation, the antitumor response of those cells is less efficient than that of CCR7+ T cells. Similar observations were reported for CD57+ T cells [18] . Simultaneous CD28 and OX40 costimulation reduces spontaneous and induced apoptosis of CCR7- T cells. Consequently a third-generation CD28-OX40 CAR improved the CAR-mediated antitumor activity of CCR7- T cells in the long term [17] . Since CAR T cells will progress in maturation during an antitumor attack, redirecting T cells by a CD28-OX40 CAR will improve the efficacy of adoptive cell therapy when terminal T-cell differentiation occurs. It is the particular combination of costimulatory signals to achieve the effect; the optimal combination for each T-cell subset is likely different and needs to be determined in detail.
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Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
CAR T-cell production for clinical application Genetic equipment of T cells with CARs requires efficient gene transfer procedures and amplification of modified T cells ex vivo. While a variety of vector systems have alternatively been explored in preclinical settings, most clinical trials are currently performed using γ-retro- or lentiviral vectors. Both vectors produce stable integration of the transgene into the host genome, however, with the risk of insertional mutagenesis by provirus integration [19,20] . The retrovirus vector system has the advantage of scalable production from stable producer cells; lentivirus production uses transient systems with limited production scales, however, at high virus titers [21] . Compared to retrovirus, lentivirus vectors have a less mutagenic insertion profile and silencing is less observed. Stable integration of the transgene can also be achieved by plasmid or transposon mediated DNA transfer, both producing random integrations of plasmid DNA as concatamers [22,23] . In case of transposon-mediated DNA transfer the production procedure requires two reagents in good manufacturing practice (GMP) quality, transposase and transposon, or the in vitro selection for stable integrates in the case of plasmid transfection. In contrast to stable modifications, cells can be transiently modified with a CAR by RNA transfection that does not result in genomic integration and thereby has a high safety profile [24,25] . A clear disadvantage of RNA-modified T cells is the short time of CAR expression of some days which is even shorter after CAR ligand interaction and internalization. Consequently, multiple doses of RNA-modified T cells are needed in clinical applications [26] . The procedure to amplify CAR T cells ex vivo to clinically relevant numbers is still a matter of optimization. Most protocols are using magnetic beads coated with agonistic anti-CD3 and anti-CD28 antibodies in addition to IL-2 [20,21] . Traditionally, T cells are amplified in static culture systems which, however, require large volumes to obtain significant cell numbers. More advanced dynamic systems produce T cells in higher densities which facilitates their application in high-dose clinical trials. Most extended protocols for CAR T-cell amplification produce cells of the effector memory phenotype. Since naive and central memory T cells persist to a greater extent than more matured T cells [27–29] , current strategies aim at shortterm amplification or enrichment of naive or central memory cells prior or after genetic engineering. Therefore common γ-chain cytokines such as IL-7, IL-15 and IL-21 are added to favor a less differentiated T-cell type [29–31] . While these cytokines are provided in GMP quality, cell lines engineered with the costimulatory molecules represent a more physiological situa-
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tion (artificial APCs) [32] , however, have some GMP risks and are more difficult to handle in large-scale approaches. General problems in CAR T-cell therapy CAR immunogenicity
Most scFv’s of CARs in recent trials are of mouse origin; humoral and cellular responses against the murine domain occurred as recorded in detail in the CAR trial targeting carboanhydrase [33] . Blocking antibodies were recorded in sera of those patients; whether these antibodies substantially reduced therapeutic efficacy remains a matter of debate. The use of entirely human domains is one strategy to avoid an antibody response, however, the immunogenicity of the fusion sites of the individual CAR domains remains. The situation is even more complex since such early trials did not perform preconditioning of patients as it is generally incorporated into current trial protocols. As a consequence there is only a limited immune response shortly after adoptive cell therapy. The assumption is sustained by the observation that recent trials with preconditioning by nonmyeloablative chemotherapy did not produce a substantial humoral or cellular immune response against the CAR [34] . However, anaphylaxis resulting from RNA-modified CAR T cells was observed after third T-cell infusion, most likely through IgE antiCAR antibodies [26] , while human anti-mouse IgG antibodies, known to develop with CAR T cells, are thought to have no adverse effect. The current assumption is that anti-CAR immunogenicity may become more relevant after multiple infusions; long-term immunogenicity needs further exploration. CAR T-cell therapy-associated toxicity
While highly effective toward leukemia/lymphoma, CAR cell therapy can pose substantial risks of autoaggression against healthy tissues with cognate target antigen. While B-cell depletion after treatment with anti-CD19 CAR T cells is clinically manageable, activation of anti-ErbB2 (Her2/neu) CAR T cells against normal epithelial tissues including lung and heart resulted in patient’s death soon after adoptive transfer [35] . The so-called ‘on-target off-organ’ activation of modified T cells by CAR engagement of target on healthy tissues is a major issue in the treatment of solid cancer and may substantially limit clinical application. In contrast to ‘off-organ’ activation, cytokine- associated toxicity, also known as cytokine release syndrome (CRS, ‘cytokine storm’), is not target specific and due to an extensive release of toxic levels of proinflammatory cytokines by CAR T cells which are much higher than physiologic T-cell activation would produce. The clinical reactions are common with
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Review Abken antibody therapeutics such as nausea, fever, hypotension, vascular leakage and life-threatening multiple organ failure [36–38] . The severity of CRS seems to correlate with tumor burden, potency and dose of CAR T cells and goes along with high serum levels of IFN-γ and IL-6. The situation can be clinically reversed by application of an anti-IL6 receptor antibody, tocilizumab [38] , which is approved for several autoimmune indications, with or without co-application of nonspecific corticosteroids. Such aggressive immunosuppressive treatment, however, likely limits the therapeutic efficacy of CAR T cells. In order to minimize the risk of life threatening CRS, a system to grade the severity of CRS in individual patients and a treatment scheme based on patient characteristics, in other words, tumor burden, age, comorbidities and others, was recently set up [39] . It is currently a matter of speculation whether CRS is needed for maximum therapeutic efficacy of CAR T cells and merits further exploration. Taken together, CRS and off-tumor toxicity are severe adverse effects which could derail CAR T-cell therapy making strategies to eliminate CAR T cells necessary. T-cell depletion in case of toxicity
Autoreactivity seen in some patients underlines the necessity to specifically, efficiently and permanently eliminate CAR T cells from circulation. This is mostly done by expression of a ‘suicide gene’ encoding a molecule that selectively ablates genetically modified T cells. Currently, the most intensively used suicide genes are herpes simplex thymidine kinase (HSV TK) and inducible caspase 9 (iCasp9). HSV-TK has a higher affinity than the mammalian TK to specific nucleoside analogs including ganciclovir and acyclovir which upon phosphorylation incorporate into host DNA resulting in chain termination and cell death [40] . The strategy showed safe in trials and allows a gradual onset in the elimination of reactive cells [41] . A disadvantage, however, is the immunogenicity resulting in unwanted elimination of the modified T cells. The use of iCasp9 is also clinically validated and safe, and is based on dimerization of transgenic caspase 9 by a small molecule dimerizer [42] . Upon dimerization over 90% of cells are rapidly eliminated which is probably not sufficiently efficient for clinical use. Ongoing Phase I trials with CAR T cells targeting GD2 currently explore the use of iCasp9 in CAR cell therapy (NCT01822652, NCT01953900). As an alternative strategy, cells are genetically marked by a cell membrane protein which is targeted by a depleting antibody, for example, CD20 modified T cells are eliminated by anti-CD20 antibody treatment [43] . A CD34-CD20 fusion protein [44] , which allows cell enrichment and depletion,
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and a truncated EGFR [45] can likewise be used. The CAR itself can be marked by a tag, for example, a myc tag, to allow elimination of modified T cells with an antitag antibody as experimentally shown for a transgenic TCR [46] . The depleting antimyc antibody, however, is so far not available for clinical use. In contrast to stable modified cells, transient CAR expression by T cells after RNA transfer will temporally limit adverse events in case of toxicity; CAR T cells disappear upon engagement of target within a few days [47] . Specific problems in targeting CAR T cells toward solid cancer While currently ongoing CAR T-cell trials for the treatment of leukemia and lymphoma offer much promise to achieve durable remission of the disease or even cure [21,37,48–50] , some trials targeting solid cancer, however, are still in an infant stage (Table 1) . The application of the CAR T-cell strategy to nonhematopoietic cancer requires the consideration of additional qualities including the disease status and tumor burden, patient preconditioning and cytokine support, CAR T-cell sensitivity to tumor mediated repression, recruitment of other arms of the patient’s immune system and stroma targeting. CAR T-cell target
The ideal target for CAR T-cell therapy is exclusively expressed on cancer cells, not on healthy cells and will produce the greatest therapeutic effect if it is critical for survival and amplification of cancer cells. These targets may be viral or mutated cell surface antigens that have mutations large enough to produce new epitopes to be recognized by an antibody. However, such target antigens are rare; most targets are also expressed by healthy cells, in some cases at lower levels, still bearing the risk of ‘on-target off-tumor’ toxicity. Such autoimmune reaction became apparent in an early phase trial targeting carboanhydrase IX to treat renal cell carcinoma, patients suffered from reversible yet discrete cholangitis and damage to bile duct epithelia due to targeting cells with physiologic carboanhydrase IX expression [51] . A fatal event occurred when a patient treated with anti-ErbB2 CAR T cells suffered from pulmonary and cardiac failure soon after T-cell administration, probably as a consequence of targeting healthy ErbB2 positive lung and heart epithelia [35] . Increasing CAR binding affinity is thought to improve targeting selectivity; experimental evidence, however, revealed that maximum T-cell activation is independent of the affinity above threshold [52] . In contrast, low-affinity CARs can more efficiently discriminate between cells with low and high antigen load than high-affinity CARs.
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Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
Review
Table 1. Current chimeric antigen receptor T-cell trials to treat nonhematopoietic malignancies by chimeric antigen receptor modified T cells. Target
CAR
Cancer
Sponsor
Status
Clinicaltrials. gov identifier
CEA
Zeta
CEA + cancer
Cancer Research UK
Terminated
NCT01212887
CEA
CD28-zeta
CEA cancer
Roger Williams MC
Ongoing
NCT01723306
EGFR-III
Glioblastoma
Abramson Cancer Center
Ongoing
NCT02209376
EGFR-III
Zeta-4-1BB
EGFR + cancer
Chinese PLA General Hospital
Recruiting
NCT01869166
EGFR-III
CD28-4-1BB-zeta
Glioma
National Cancer Inst.
Recruiting
NCT01454596
ErbB2
ErbB2+ cancer
Chinese PLA General Hospital
Recruiting
NCT01935843
ErbB2
CD28-zeta
ErbB2+ cancer
Baylor
Recruiting
NCT00889954
ErbB2
CD28-zeta
ErbB2 glioblastoma
Baylor
Recruiting
NCT01109095
ErbB2
CD28-zeta
Sarcoma
Baylor
Recruiting
NCT00902044
ErbB2
CD28-zeta
Head and neck squamous cell cancer
King’s College London
Not yet open NCT01818323
FAP
CD28-zeta
Mesothelioma
University of Zurich
Recruiting
NCT01722149
GD2
OX40-CD28-zeta
Sarcoma, melanoma
National Cancer Inst.
Recruiting
NCT02107963
GD2
OX40-CD28-zeta
Neuroblastoma Baylor College Med.
Recruiting
NCT01822652
GD2
Neuroblastoma Children’s Mercy Hospital Kansas City
Recruiting
NCT01460901
GD2
OX40-CD28-zeta
Sarcoma
Baylor College Med.
Recruiting
NCT01953900
IL-13Ra2
4-1BB-zeta
Glioma
City of Hope MC
Recruiting
NCT02208362
Mesothelin
4-1BB-zeta
Mesothelin cancer
Abramson Cancer Center
Recruiting
NCT02159716
+
+
+
‘On-target off-organ’ activation may not always be harmful but also sustain the long-term therapeutic effect of CAR T cells. In the case of targeting CD19 in the treatment of leukemia, patients suffer from a lasting depletion of the B cell compartment [38,48] . Those CD19 + healthy B cells restimulate by CAR engagement the anti-CD19 CAR T cells and provide costimulatory ligands to boost the antitumor response. Since the targeted tumor and healthy cells reside in the same compartment repetitive restimulation and migration into the same niche come together to sustain activation and persistence of the anti-CD19 CAR T cells. This particular situation is not the case for the majority of solid tumors. Beside a target dependent cancer cell attack, antigen independent destruction of tumor stroma is required to control solid cancer lesions. The stroma consists of a variety of host cells including connective tissue cells, tumor fibroblasts, vascular endothelial cells and oth-
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ers. Targeting connective tissue cells in solid tumors by targeting fibroblast activation protein in addition to cancer cell targeting showed superior in an experimental model [53] . A second experimental model also points to the relevance of stroma targeting for the treatment of solid cancer lesions. Destruction of large established tumors relies upon targeting of stroma cells by IFN-γ and requires strong CD28 costimulation [54] . CAR T cells thereby act by both target-dependent killing of cancer cells and by indirect mechanisms which require the action of T-cell produced IFN-γ on the tumor stroma. This is in accordance to the observation that IFN-γ is crucial in preventing tumor relapse in a syngeneic model [55] . T-cell trafficking, infiltration & repression
As a fundamental prerequisite for therapeutic efficacy, adoptively transferred T cells need to traffic to the tumor lesion. While trafficking to the sites of disease
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Review Abken was shown in patients with leukemia [48,49,56] , only preclinical mouse data are available for T-cell targeting toward solid cancer lesions. Simultaneous bioluminescence imaging of cancer and T cells revealed the accumulation and persistence of systemically applied CAR T cells at the site of transplanted cancer [57] demonstrating that CAR T cells can traffic to solid cancer lesions. Trafficking is the result of a wide range of molecular interactions and depends on a number of soluble and cell bound factors, in particular chemokines. However, extensive amplification of modified T cells ex vivo may alter the panel of expressed chemokine receptors which are required for trafficking. Transgenic co-expression of CXCR2 (CXCL1 receptor) improves trafficking to melanoma [58] and co-expression of CCR2b the migration of CAR T cells to neuroblastoma [59] . On the other hand, the panel of chemokines produced by a variety of solid tumors does not favor T-cell infiltration into the tumor bed. In contrast, specific receptors like the endothelin B receptor prevent T-cell infiltration as shown for ovarian tumors [60] . Blocking those receptors is assumed to improve T-cell infiltration into the tumor lesion. Once cancer cell-specific T cells accumulate in the vicinity, they do not efficiently infiltrate into the tumor lesion. This became obvious when anti-ErbB2 CAR T cells did not control progression of metastases of a breast cancer patient although disseminated cancer cells were efficiently eliminated [61] . One conclusion is that CAR T cells did not sufficiently evade vasculature to penetrate the tumor tissue. This situation is aimed to be improved by targeting VEGF receptor-2, which is overexpressed by tumor-associated endothelial cells; indeed, anti-VEGF-R CAR T cells produced repression of vascularized tumors in an experimental model [62] . Normalization of vasculature by low-dose angiogenesis inhibitors, rather than vascular destruction, also improves the antitumor efficacy [63] and may be more efficacious in the long term. The antitumor activity of CAR T cells correlates, at least to a large extend, with the persistence and sustained activation of modified T cells [37,38,48] . Conditioning chemotherapy prior adoptive cell transfer improves CAR T-cell persistence [49] . Administration of low dose IL-2 helps to improve T-cell persistence as seen in early trials using first-generation CAR T cells [64] . In addition, costimulation improves persistence of modified T cells; in particular, CD28-ζ CAR T cells persisted longer in patients than ζ CAR T cells [56] . T cells with a 4-1BB-ζ CAR persisted more than 9 months after adoptive transfer producing a significant therapeutic effect [48] . CAR T-cell persistence can also be improved by using virus specific T cells which engage viral antigens through
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their TCR and thereby obtain necessary costimulatory signals to survive and amplify. By means of this strategy, Epstein-Barr virus (EBV) specific, autologous T cells engineered with a CAR persisted longer in a 6-week period after infusion than T cells from the same patient and engineered with the same CAR [65] . The stage of maturation of adoptively transferred T cells also impacts their persistence in the tumor tissue. Since the frequency of CD4 + CD45RO + CD62L + memory T cells in the transferred T-cell product goes along with improved T-cell persistence [13] forthcoming trials aim at using CD62L enriched T cells for CAR modification. Once migrated into the solid tumor lesion, CAR T cells face a highly suppressive environment which is a strong barrier preventing immune surveillance [66,67] . Various cellular and soluble compartments are involved, including Treg cells, myeloid derived suppressor cells, M2-type macrophages and ligands to repressive receptors on T cells such as PD-L1, CTLA-4, B7-H family members or FasL. In this situation strategies are required to make CAR T cells more resistant to repression. For instance, the anti-tumor activity of CAR T cells in presence of Treg cells is improved in an experimental model by abrogation of IL-2 release through mutation of the CD28 CAR signaling domain [68] . Blocking the PD-1/PD-L1 may furthermore improve the antitumor activity of redirected T cells. Contact to tumor cells with PD-L1 increases PD-1 on CAR T cells [69] ; a blocking anti-PD-1 antibody improves CAR T-cell activity in a preclinical model. The recent approval of the anti-PD-1 antibody pembrolizumab by the Food and Drug Administration will move the field toward testing combination therapy of CAR T cells together with PD-1 blockade. Immune cell suppression is also mediated by cytokines like TGF-β which suppresses T-cell amplification, to a lower degree cytokine release and cytolytic activity, resulting in a reduced antitumor response in the long term. CD28 costimulation provided by a second-generation CAR overcomes TGF-β repression resulting in a more pronounced tumor cell killing than by CD3ζ CAR T cells [70] . However, the best costimulation to tackle the complex network of immune repression still needs to be explored in relevant models and validated in clinical trials. Conclusion & future perspective So far, strategies to overcome the major hurdles for the success in the treatment of solid cancer lesions need to be empirically defined in forthcoming trials with respect to both safety and efficacy. Some modifications of the current concept are outlined below. A major goal of clinical activity in the field needs therefore to be testing CAR T cells against a broad
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Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
range of tumors in order to optimize protocols and treatment schedules, to avoid autoimmunity and to improve persistence and function of anti-tumor T cells in the repressive tumor environment. In this context, it needs to be addressed whether one prototype CAR design can be used to drive optimal clinical responses toward the broad variety of solid tumor entities. Targeting one antigen may select for escape variants of cancer cells which give rise to relapse; simultaneous targeting of two antigens or ideally of a tumor-driver antigen will be required. Combinatorial antigen recognition is thought to improve targeting selectivity and to reduce tumor relapse and autoimmunity. T cells are engineered with two CARs recognizing two different targets on cancer cells, one CAR providing the primary signal, the other costimulation; both signals complement when both targets on cancer cells are simultaneously engaged [71] . Currently, the strategy is merely empiric and a rationale design of the co-operating CARs needs to be explored in more detail. On the other hand, outgrowing cancer cells which lost target expression makes the tumor invisible to CAR T cells. This situation is frequently seen in solid tumors; the recruitment of the innate immune system to attack those cancer cells in an antigen-independent fashion is thought to be a strategy to prevent tumor
Review
relapse after initial tumor reduction. T cells are modified with a CAR alongside with an inducible expression vector for transgenic IL-12 to release high IL-12 levels when the CAR T cells engage their cognate antigen [4,72] . As a consequence deposited IL-12 produces a locally restricted proinflammatory response by innate cells which results in the elimination of those cancer cells which are invisible to CAR T cells [73] . Such TRUCK cells, in other words, CAR T cells engineered with an additional payload, may strengthen and fine-tune the overall immune response against solid tumors; other immune-modulating molecules need to be explored in detail to optimize the efficacy of CAR T cells toward solid tumors. Improving therapeutic efficacy of CAR T cells may require lower pretreatment intensities and lower numbers of T cells to be applied thereby potentially reducing the overall toxicities. T cells with stem celllike properties [74] may be ideal with this respect due to their strong engraftment potential after application. The general assumption is that fewer CAR T cells with a less differentiated phenotype will show similar therapeutic efficacy as high-doses of T cells with a more matured phenotype. Potentially, preconditioning may be reduced when applying those cells compared with the currently used matured central memory T cells.
Executive summary Concept of CAR-& TRUCK-modified T cells • Chimeric antigen receptor (CAR) T cells show redirected specificity toward predefined targets independently of MHC presentation; TRUCK T cells release a transgenic product in addition upon CAR engagement.
CAR signaling blocks: combined primary & costimulatory signals are superior • The costimulatory domain together with the primary T-cell receptor (TCR)-derived signal orchestrates a distinct pattern of effector functions. • The optimal combination of signals for each T-cell subset is different and needs to be determined in detail.
CAR T-cell production for clinical application • Retro- and lenti-viral vectors are commonly used to modify ex vivo patient’s T cells with CARs. • A fully automated, closed system to amplify T cells under good manufacturing practice conditions is required to establish CAR T-cell therapy for a broad panel of applications.
General problems with CAR T-cell therapy • CARs are immunogenic and provoke a humoral and cellular immune response. • CAR T-cell therapy produces some autoimmunity (on-target off-organ) which is a major challenge when targeting solid cancer. • Cytokine associated toxicity after CAR T-cell activation (cytokine storm) can be severe and life-threatening and may require immune repression and CAR T-cell elimination.
Specific problems in targeting CAR T cells toward solid cancer • A number of early phase trials are initiated to target solid cancer by CAR modified T cells. • Infiltration of solid tumor lesions and persistence of CAR T cells is insufficient and requires additional attention in the near future. • Targeting checkpoint blockade and immune repression may improve efficacy of CAR T-cell therapy in the treatment of solid cancer. • In addition to cancer cells, the tumor stroma needs to be targeted in order to reduce established large tumors.
Future perspective • Strategies to overcome the major hurdles for the success in the treatment of solid cancer lesions need to be empirically defined in forthcoming trials using a more standardized production and clinical trial protocol.
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Review Abken Based on clinical trial data, the current situation is problematic to identify the crucial steps for therapeutic effectiveness since the design of ongoing trials is highly diverse and employs CARs of different modular composition, different T-cell populations and amplification protocols, and applies different preconditioning regimes to the patient. Head-to-head testing in multiple single parameter trials against the same target in the same disease is needed to address the issue [75] . In addition, multicenter trials with a large number of patients are required to address whether CAR T-cell therapy provides benefit with respect to the overall survival and progression-free survival compared with the standard of care. The logistic of such a trial, however, is challenging and requires the optimization of a number of issues, including the production of CAR T cells which is currently performed only in a small number of trial centers and is extremely labor and cost intensive.
Standardization and automation of these processes will help to ensure that many more centers will be able to participate in such evaluation in the near future.
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Financial & competing interests disclosure Work in the author’s laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Deutsche Krebshilfe, Bonn, Else Kröner-Fresenius Stiftung, Bad Homburg v.d.H., Wilhelm Sander-Stiftung, Munich, EU (European Regional Development Fund – Investing in your future), German Federal State North Rhine-Westphalia (NRW) and the Fortune Program of the Medical Faculty of the University of Cologne. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
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Immunotherapy (2015) 7(5)
future science group
Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
Recent spectacular success in the adoptive cell therapy of leukemia and lymphoma with chimeric antigen receptor (CAR)-modified T cells raised the expectations that this therapy may be efficacious in a wide range of cancer entities. The expectations are based on the predefined specificity of CAR T cells by an antibody-derived binding domain that acts independently of the natural T-cell receptor, recognizes targets independently of presentation by the major histocompatibility complex and allows targeting toward virtually any cell surface antigen. We here discuss that targeting CAR T cells toward solid tumors faces certain circumstances critical for the therapeutic success. Targeting tumor stroma and taking advantage of TRUCK cells, in other words, CAR T cells with inducible release of a transgenic payload, are some strategies envisaged to overcome current limitations in the near future.
Hinrich Abken Clinic I Internal Medicine, Tumor Genetics, University Hospital Cologne, Cologne, Germany and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Robert-Koch-Str. 21, D-50931 Cologne, Germany hinrich.abken@ uk-koeln.de
Keywords: adoptive cell therapy • cancer • CAR • chimeric antigen receptor • gene transfer • T cell
Concept of CAR & T cells directed for universal cytokine-mediated killing modified T cells The concept of adoptive cell therapy with chimeric antigen receptor (CAR, immunoreceptor) modified cells is based on patient’s T cells that are redirected with predefined specificity toward autologous cancer cells to execute their immune response. T cells are ex vivo modified with a recombinant receptor molecule which by the extracellular part recognizes a pre-defined target by an antibody derived binding domain and by the intra cellular T-cell receptor (TCR) derived signaling part initiates T-cell activation upon target engagement (Figure 1) [1] . Such CAR T cells (also nick-named ‘T-bodies’) recognize their target independently of presentation through the major histocompatibility complex (MHC) and are thus not compromised by alterations in the antigen processing and presentation machinery as often observed during tumor progression [2,3] . CAR T cells can furthermore be modified to act as a ‘factory’ to produce a transgenic protein upon
10.2217/IMT.15.15 © 2015 Future Medicine Ltd
CAR engagement of target. Such T cell redirected for universal cytokine-mediated killing (TRUCKs) deliver in a coordinated and locally restricted fashion a ‘payload’ which may be a cytokine like IL-12 which in turn activates an innate immune response toward tumors [4,5] . For further details we refer to recent reviews [5–7] . Compared with physiological TCR recognition, the CAR mediated T-cell activation provides several advantages in the context of adoptive cell therapy. First, CARs can redirect T cells toward a broad variety of targets as long as a binding domain with suitable specificity is available. Potential CAR targets in addition to classical MHC bound peptides also include nonclassical TCR targets such as carbohydrates, lipids and conformational epitopes. While most CARs use an antibody derived scFv for targeting, some CARs were reported, which consist of receptor ligands as binding domain fused to the transmembrane and intracellular signaling moiety. Examples are heregulin and IL-13 mutein to target Her3/4 receptor and IL-13 receptor-α,
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scFv
ζ
scFv
scFv
scFv
CD28
CD28
CD28
ζ
ζ
ζ
OX40/ 4-1BB 1o
2o
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Figure 1. Modular composition of the chimeric antigen receptor. The prototype CAR is composed of one polypeptide chain; the extracellular scFv antibody domain mediates binding to the target antigen in an MHC independent fashion. Upon CAR clustering, the intracellular TCR-derived CD3ζ chain, with or without costimulation through members of the CD28 family, initiates the downstream signaling for T-cell activation. Coreceptors may modulate CAR activity. In contrast to a first-generation (1°) CAR, second- (2°) and thirdgeneration (3°) CARs harbor one or two costimulatory moieties in their intracellular part. TRUCK cells are CAR T cells that are additionally modified with a CAR inducible expression cassette for a transgenic polypeptide product that is produced and released upon CAR signaling. Such TRUCK cells can be used as CAR targeted ‘factories’ that deposit a defined product in the targeted tissue. CAR: Chimeric antigen receptor; scFv: Single chain fragment of variable region; TCR: T-cell receptor.
respectively [8,9] . To be recognized by CAR T cells, the antigen needs to be present on the cell surface of the targeted cell; however, TCR-like CARs were reported, which recognize intracellular antigens presented by the MHC [10,11] . In contrast to cell-bound antigen, soluble antigen, which frequently accumulates in the serum of cancer patients, does not sufficiently activate CAR T cells [12] . CAR redirected T-cell activation initiates a plethora of T-cell effector functions including cytokine release, lytic degranulation, T-cell amplification, maturation and migration. Clinical efficacy requires lasting persistence of CAR T cells which need to be protected from activation induced cell death through appropriate costimulation, for example, CD28 or 4-1BB. This is underscored by the clinical observation that patients with achieved lasting remission showed long-term CAR T-cell persistence [13] . CAR T cells can moreover provide target-specific memory which may help to prevent tumor relapse [14] . CAR signaling blocks: combined primary & costimulatory signals are superior The intracellular signaling moiety is commonly derived from the CD3ζ chain; other signaling domains including the FcɛRI γ chain are used as well. Since full T-cell activation and fine-tuning requires appropriate costimulation, the primary signaling moiety was fused to a costimulatory signaling chain, a prototype of which is CD28, giving rise to a so-called second-generation CAR [15] . Each costimulatory endodomain in the context of a CAR orchestrates a
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distinct pattern of effector functions as revealed by a side-by-side comparison [16] . To combine the benefits, third-generation CARs were engineered with two costimulatory domains in addition to the primary signal. This is of particular interest for redirecting T-cell subsets in more progressed maturation stages which need different costimuli for activation than young T cells [17] . Repetitive stimulation by antigen forces T cells to progress in terminal differentiation which is also the case when the CAR is engaging the cognate target. Consequently, more matured CAR T cells which lack CCR7 accumulate at the tumor lesion, at least in experimental models, while both CCR7+ and CCR7- T cells are equally efficient in targeting to the tumor lesion. Since CCR7- T cells are prone to spontaneous and activation induced cell death, which is not prevented by CD28 costimulation, the antitumor response of those cells is less efficient than that of CCR7+ T cells. Similar observations were reported for CD57+ T cells [18] . Simultaneous CD28 and OX40 costimulation reduces spontaneous and induced apoptosis of CCR7- T cells. Consequently a third-generation CD28-OX40 CAR improved the CAR-mediated antitumor activity of CCR7- T cells in the long term [17] . Since CAR T cells will progress in maturation during an antitumor attack, redirecting T cells by a CD28-OX40 CAR will improve the efficacy of adoptive cell therapy when terminal T-cell differentiation occurs. It is the particular combination of costimulatory signals to achieve the effect; the optimal combination for each T-cell subset is likely different and needs to be determined in detail.
future science group
Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
CAR T-cell production for clinical application Genetic equipment of T cells with CARs requires efficient gene transfer procedures and amplification of modified T cells ex vivo. While a variety of vector systems have alternatively been explored in preclinical settings, most clinical trials are currently performed using γ-retro- or lentiviral vectors. Both vectors produce stable integration of the transgene into the host genome, however, with the risk of insertional mutagenesis by provirus integration [19,20] . The retrovirus vector system has the advantage of scalable production from stable producer cells; lentivirus production uses transient systems with limited production scales, however, at high virus titers [21] . Compared to retrovirus, lentivirus vectors have a less mutagenic insertion profile and silencing is less observed. Stable integration of the transgene can also be achieved by plasmid or transposon mediated DNA transfer, both producing random integrations of plasmid DNA as concatamers [22,23] . In case of transposon-mediated DNA transfer the production procedure requires two reagents in good manufacturing practice (GMP) quality, transposase and transposon, or the in vitro selection for stable integrates in the case of plasmid transfection. In contrast to stable modifications, cells can be transiently modified with a CAR by RNA transfection that does not result in genomic integration and thereby has a high safety profile [24,25] . A clear disadvantage of RNA-modified T cells is the short time of CAR expression of some days which is even shorter after CAR ligand interaction and internalization. Consequently, multiple doses of RNA-modified T cells are needed in clinical applications [26] . The procedure to amplify CAR T cells ex vivo to clinically relevant numbers is still a matter of optimization. Most protocols are using magnetic beads coated with agonistic anti-CD3 and anti-CD28 antibodies in addition to IL-2 [20,21] . Traditionally, T cells are amplified in static culture systems which, however, require large volumes to obtain significant cell numbers. More advanced dynamic systems produce T cells in higher densities which facilitates their application in high-dose clinical trials. Most extended protocols for CAR T-cell amplification produce cells of the effector memory phenotype. Since naive and central memory T cells persist to a greater extent than more matured T cells [27–29] , current strategies aim at shortterm amplification or enrichment of naive or central memory cells prior or after genetic engineering. Therefore common γ-chain cytokines such as IL-7, IL-15 and IL-21 are added to favor a less differentiated T-cell type [29–31] . While these cytokines are provided in GMP quality, cell lines engineered with the costimulatory molecules represent a more physiological situa-
future science group
Review
tion (artificial APCs) [32] , however, have some GMP risks and are more difficult to handle in large-scale approaches. General problems in CAR T-cell therapy CAR immunogenicity
Most scFv’s of CARs in recent trials are of mouse origin; humoral and cellular responses against the murine domain occurred as recorded in detail in the CAR trial targeting carboanhydrase [33] . Blocking antibodies were recorded in sera of those patients; whether these antibodies substantially reduced therapeutic efficacy remains a matter of debate. The use of entirely human domains is one strategy to avoid an antibody response, however, the immunogenicity of the fusion sites of the individual CAR domains remains. The situation is even more complex since such early trials did not perform preconditioning of patients as it is generally incorporated into current trial protocols. As a consequence there is only a limited immune response shortly after adoptive cell therapy. The assumption is sustained by the observation that recent trials with preconditioning by nonmyeloablative chemotherapy did not produce a substantial humoral or cellular immune response against the CAR [34] . However, anaphylaxis resulting from RNA-modified CAR T cells was observed after third T-cell infusion, most likely through IgE antiCAR antibodies [26] , while human anti-mouse IgG antibodies, known to develop with CAR T cells, are thought to have no adverse effect. The current assumption is that anti-CAR immunogenicity may become more relevant after multiple infusions; long-term immunogenicity needs further exploration. CAR T-cell therapy-associated toxicity
While highly effective toward leukemia/lymphoma, CAR cell therapy can pose substantial risks of autoaggression against healthy tissues with cognate target antigen. While B-cell depletion after treatment with anti-CD19 CAR T cells is clinically manageable, activation of anti-ErbB2 (Her2/neu) CAR T cells against normal epithelial tissues including lung and heart resulted in patient’s death soon after adoptive transfer [35] . The so-called ‘on-target off-organ’ activation of modified T cells by CAR engagement of target on healthy tissues is a major issue in the treatment of solid cancer and may substantially limit clinical application. In contrast to ‘off-organ’ activation, cytokine- associated toxicity, also known as cytokine release syndrome (CRS, ‘cytokine storm’), is not target specific and due to an extensive release of toxic levels of proinflammatory cytokines by CAR T cells which are much higher than physiologic T-cell activation would produce. The clinical reactions are common with
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Review Abken antibody therapeutics such as nausea, fever, hypotension, vascular leakage and life-threatening multiple organ failure [36–38] . The severity of CRS seems to correlate with tumor burden, potency and dose of CAR T cells and goes along with high serum levels of IFN-γ and IL-6. The situation can be clinically reversed by application of an anti-IL6 receptor antibody, tocilizumab [38] , which is approved for several autoimmune indications, with or without co-application of nonspecific corticosteroids. Such aggressive immunosuppressive treatment, however, likely limits the therapeutic efficacy of CAR T cells. In order to minimize the risk of life threatening CRS, a system to grade the severity of CRS in individual patients and a treatment scheme based on patient characteristics, in other words, tumor burden, age, comorbidities and others, was recently set up [39] . It is currently a matter of speculation whether CRS is needed for maximum therapeutic efficacy of CAR T cells and merits further exploration. Taken together, CRS and off-tumor toxicity are severe adverse effects which could derail CAR T-cell therapy making strategies to eliminate CAR T cells necessary. T-cell depletion in case of toxicity
Autoreactivity seen in some patients underlines the necessity to specifically, efficiently and permanently eliminate CAR T cells from circulation. This is mostly done by expression of a ‘suicide gene’ encoding a molecule that selectively ablates genetically modified T cells. Currently, the most intensively used suicide genes are herpes simplex thymidine kinase (HSV TK) and inducible caspase 9 (iCasp9). HSV-TK has a higher affinity than the mammalian TK to specific nucleoside analogs including ganciclovir and acyclovir which upon phosphorylation incorporate into host DNA resulting in chain termination and cell death [40] . The strategy showed safe in trials and allows a gradual onset in the elimination of reactive cells [41] . A disadvantage, however, is the immunogenicity resulting in unwanted elimination of the modified T cells. The use of iCasp9 is also clinically validated and safe, and is based on dimerization of transgenic caspase 9 by a small molecule dimerizer [42] . Upon dimerization over 90% of cells are rapidly eliminated which is probably not sufficiently efficient for clinical use. Ongoing Phase I trials with CAR T cells targeting GD2 currently explore the use of iCasp9 in CAR cell therapy (NCT01822652, NCT01953900). As an alternative strategy, cells are genetically marked by a cell membrane protein which is targeted by a depleting antibody, for example, CD20 modified T cells are eliminated by anti-CD20 antibody treatment [43] . A CD34-CD20 fusion protein [44] , which allows cell enrichment and depletion,
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and a truncated EGFR [45] can likewise be used. The CAR itself can be marked by a tag, for example, a myc tag, to allow elimination of modified T cells with an antitag antibody as experimentally shown for a transgenic TCR [46] . The depleting antimyc antibody, however, is so far not available for clinical use. In contrast to stable modified cells, transient CAR expression by T cells after RNA transfer will temporally limit adverse events in case of toxicity; CAR T cells disappear upon engagement of target within a few days [47] . Specific problems in targeting CAR T cells toward solid cancer While currently ongoing CAR T-cell trials for the treatment of leukemia and lymphoma offer much promise to achieve durable remission of the disease or even cure [21,37,48–50] , some trials targeting solid cancer, however, are still in an infant stage (Table 1) . The application of the CAR T-cell strategy to nonhematopoietic cancer requires the consideration of additional qualities including the disease status and tumor burden, patient preconditioning and cytokine support, CAR T-cell sensitivity to tumor mediated repression, recruitment of other arms of the patient’s immune system and stroma targeting. CAR T-cell target
The ideal target for CAR T-cell therapy is exclusively expressed on cancer cells, not on healthy cells and will produce the greatest therapeutic effect if it is critical for survival and amplification of cancer cells. These targets may be viral or mutated cell surface antigens that have mutations large enough to produce new epitopes to be recognized by an antibody. However, such target antigens are rare; most targets are also expressed by healthy cells, in some cases at lower levels, still bearing the risk of ‘on-target off-tumor’ toxicity. Such autoimmune reaction became apparent in an early phase trial targeting carboanhydrase IX to treat renal cell carcinoma, patients suffered from reversible yet discrete cholangitis and damage to bile duct epithelia due to targeting cells with physiologic carboanhydrase IX expression [51] . A fatal event occurred when a patient treated with anti-ErbB2 CAR T cells suffered from pulmonary and cardiac failure soon after T-cell administration, probably as a consequence of targeting healthy ErbB2 positive lung and heart epithelia [35] . Increasing CAR binding affinity is thought to improve targeting selectivity; experimental evidence, however, revealed that maximum T-cell activation is independent of the affinity above threshold [52] . In contrast, low-affinity CARs can more efficiently discriminate between cells with low and high antigen load than high-affinity CARs.
future science group
Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
Review
Table 1. Current chimeric antigen receptor T-cell trials to treat nonhematopoietic malignancies by chimeric antigen receptor modified T cells. Target
CAR
Cancer
Sponsor
Status
Clinicaltrials. gov identifier
CEA
Zeta
CEA + cancer
Cancer Research UK
Terminated
NCT01212887
CEA
CD28-zeta
CEA cancer
Roger Williams MC
Ongoing
NCT01723306
EGFR-III
Glioblastoma
Abramson Cancer Center
Ongoing
NCT02209376
EGFR-III
Zeta-4-1BB
EGFR + cancer
Chinese PLA General Hospital
Recruiting
NCT01869166
EGFR-III
CD28-4-1BB-zeta
Glioma
National Cancer Inst.
Recruiting
NCT01454596
ErbB2
ErbB2+ cancer
Chinese PLA General Hospital
Recruiting
NCT01935843
ErbB2
CD28-zeta
ErbB2+ cancer
Baylor
Recruiting
NCT00889954
ErbB2
CD28-zeta
ErbB2 glioblastoma
Baylor
Recruiting
NCT01109095
ErbB2
CD28-zeta
Sarcoma
Baylor
Recruiting
NCT00902044
ErbB2
CD28-zeta
Head and neck squamous cell cancer
King’s College London
Not yet open NCT01818323
FAP
CD28-zeta
Mesothelioma
University of Zurich
Recruiting
NCT01722149
GD2
OX40-CD28-zeta
Sarcoma, melanoma
National Cancer Inst.
Recruiting
NCT02107963
GD2
OX40-CD28-zeta
Neuroblastoma Baylor College Med.
Recruiting
NCT01822652
GD2
Neuroblastoma Children’s Mercy Hospital Kansas City
Recruiting
NCT01460901
GD2
OX40-CD28-zeta
Sarcoma
Baylor College Med.
Recruiting
NCT01953900
IL-13Ra2
4-1BB-zeta
Glioma
City of Hope MC
Recruiting
NCT02208362
Mesothelin
4-1BB-zeta
Mesothelin cancer
Abramson Cancer Center
Recruiting
NCT02159716
+
+
+
‘On-target off-organ’ activation may not always be harmful but also sustain the long-term therapeutic effect of CAR T cells. In the case of targeting CD19 in the treatment of leukemia, patients suffer from a lasting depletion of the B cell compartment [38,48] . Those CD19 + healthy B cells restimulate by CAR engagement the anti-CD19 CAR T cells and provide costimulatory ligands to boost the antitumor response. Since the targeted tumor and healthy cells reside in the same compartment repetitive restimulation and migration into the same niche come together to sustain activation and persistence of the anti-CD19 CAR T cells. This particular situation is not the case for the majority of solid tumors. Beside a target dependent cancer cell attack, antigen independent destruction of tumor stroma is required to control solid cancer lesions. The stroma consists of a variety of host cells including connective tissue cells, tumor fibroblasts, vascular endothelial cells and oth-
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ers. Targeting connective tissue cells in solid tumors by targeting fibroblast activation protein in addition to cancer cell targeting showed superior in an experimental model [53] . A second experimental model also points to the relevance of stroma targeting for the treatment of solid cancer lesions. Destruction of large established tumors relies upon targeting of stroma cells by IFN-γ and requires strong CD28 costimulation [54] . CAR T cells thereby act by both target-dependent killing of cancer cells and by indirect mechanisms which require the action of T-cell produced IFN-γ on the tumor stroma. This is in accordance to the observation that IFN-γ is crucial in preventing tumor relapse in a syngeneic model [55] . T-cell trafficking, infiltration & repression
As a fundamental prerequisite for therapeutic efficacy, adoptively transferred T cells need to traffic to the tumor lesion. While trafficking to the sites of disease
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539
Review Abken was shown in patients with leukemia [48,49,56] , only preclinical mouse data are available for T-cell targeting toward solid cancer lesions. Simultaneous bioluminescence imaging of cancer and T cells revealed the accumulation and persistence of systemically applied CAR T cells at the site of transplanted cancer [57] demonstrating that CAR T cells can traffic to solid cancer lesions. Trafficking is the result of a wide range of molecular interactions and depends on a number of soluble and cell bound factors, in particular chemokines. However, extensive amplification of modified T cells ex vivo may alter the panel of expressed chemokine receptors which are required for trafficking. Transgenic co-expression of CXCR2 (CXCL1 receptor) improves trafficking to melanoma [58] and co-expression of CCR2b the migration of CAR T cells to neuroblastoma [59] . On the other hand, the panel of chemokines produced by a variety of solid tumors does not favor T-cell infiltration into the tumor bed. In contrast, specific receptors like the endothelin B receptor prevent T-cell infiltration as shown for ovarian tumors [60] . Blocking those receptors is assumed to improve T-cell infiltration into the tumor lesion. Once cancer cell-specific T cells accumulate in the vicinity, they do not efficiently infiltrate into the tumor lesion. This became obvious when anti-ErbB2 CAR T cells did not control progression of metastases of a breast cancer patient although disseminated cancer cells were efficiently eliminated [61] . One conclusion is that CAR T cells did not sufficiently evade vasculature to penetrate the tumor tissue. This situation is aimed to be improved by targeting VEGF receptor-2, which is overexpressed by tumor-associated endothelial cells; indeed, anti-VEGF-R CAR T cells produced repression of vascularized tumors in an experimental model [62] . Normalization of vasculature by low-dose angiogenesis inhibitors, rather than vascular destruction, also improves the antitumor efficacy [63] and may be more efficacious in the long term. The antitumor activity of CAR T cells correlates, at least to a large extend, with the persistence and sustained activation of modified T cells [37,38,48] . Conditioning chemotherapy prior adoptive cell transfer improves CAR T-cell persistence [49] . Administration of low dose IL-2 helps to improve T-cell persistence as seen in early trials using first-generation CAR T cells [64] . In addition, costimulation improves persistence of modified T cells; in particular, CD28-ζ CAR T cells persisted longer in patients than ζ CAR T cells [56] . T cells with a 4-1BB-ζ CAR persisted more than 9 months after adoptive transfer producing a significant therapeutic effect [48] . CAR T-cell persistence can also be improved by using virus specific T cells which engage viral antigens through
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Immunotherapy (2015) 7(5)
their TCR and thereby obtain necessary costimulatory signals to survive and amplify. By means of this strategy, Epstein-Barr virus (EBV) specific, autologous T cells engineered with a CAR persisted longer in a 6-week period after infusion than T cells from the same patient and engineered with the same CAR [65] . The stage of maturation of adoptively transferred T cells also impacts their persistence in the tumor tissue. Since the frequency of CD4 + CD45RO + CD62L + memory T cells in the transferred T-cell product goes along with improved T-cell persistence [13] forthcoming trials aim at using CD62L enriched T cells for CAR modification. Once migrated into the solid tumor lesion, CAR T cells face a highly suppressive environment which is a strong barrier preventing immune surveillance [66,67] . Various cellular and soluble compartments are involved, including Treg cells, myeloid derived suppressor cells, M2-type macrophages and ligands to repressive receptors on T cells such as PD-L1, CTLA-4, B7-H family members or FasL. In this situation strategies are required to make CAR T cells more resistant to repression. For instance, the anti-tumor activity of CAR T cells in presence of Treg cells is improved in an experimental model by abrogation of IL-2 release through mutation of the CD28 CAR signaling domain [68] . Blocking the PD-1/PD-L1 may furthermore improve the antitumor activity of redirected T cells. Contact to tumor cells with PD-L1 increases PD-1 on CAR T cells [69] ; a blocking anti-PD-1 antibody improves CAR T-cell activity in a preclinical model. The recent approval of the anti-PD-1 antibody pembrolizumab by the Food and Drug Administration will move the field toward testing combination therapy of CAR T cells together with PD-1 blockade. Immune cell suppression is also mediated by cytokines like TGF-β which suppresses T-cell amplification, to a lower degree cytokine release and cytolytic activity, resulting in a reduced antitumor response in the long term. CD28 costimulation provided by a second-generation CAR overcomes TGF-β repression resulting in a more pronounced tumor cell killing than by CD3ζ CAR T cells [70] . However, the best costimulation to tackle the complex network of immune repression still needs to be explored in relevant models and validated in clinical trials. Conclusion & future perspective So far, strategies to overcome the major hurdles for the success in the treatment of solid cancer lesions need to be empirically defined in forthcoming trials with respect to both safety and efficacy. Some modifications of the current concept are outlined below. A major goal of clinical activity in the field needs therefore to be testing CAR T cells against a broad
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Adoptive therapy with CAR redirected T cells: the challenges in targeting solid tumors
range of tumors in order to optimize protocols and treatment schedules, to avoid autoimmunity and to improve persistence and function of anti-tumor T cells in the repressive tumor environment. In this context, it needs to be addressed whether one prototype CAR design can be used to drive optimal clinical responses toward the broad variety of solid tumor entities. Targeting one antigen may select for escape variants of cancer cells which give rise to relapse; simultaneous targeting of two antigens or ideally of a tumor-driver antigen will be required. Combinatorial antigen recognition is thought to improve targeting selectivity and to reduce tumor relapse and autoimmunity. T cells are engineered with two CARs recognizing two different targets on cancer cells, one CAR providing the primary signal, the other costimulation; both signals complement when both targets on cancer cells are simultaneously engaged [71] . Currently, the strategy is merely empiric and a rationale design of the co-operating CARs needs to be explored in more detail. On the other hand, outgrowing cancer cells which lost target expression makes the tumor invisible to CAR T cells. This situation is frequently seen in solid tumors; the recruitment of the innate immune system to attack those cancer cells in an antigen-independent fashion is thought to be a strategy to prevent tumor
Review
relapse after initial tumor reduction. T cells are modified with a CAR alongside with an inducible expression vector for transgenic IL-12 to release high IL-12 levels when the CAR T cells engage their cognate antigen [4,72] . As a consequence deposited IL-12 produces a locally restricted proinflammatory response by innate cells which results in the elimination of those cancer cells which are invisible to CAR T cells [73] . Such TRUCK cells, in other words, CAR T cells engineered with an additional payload, may strengthen and fine-tune the overall immune response against solid tumors; other immune-modulating molecules need to be explored in detail to optimize the efficacy of CAR T cells toward solid tumors. Improving therapeutic efficacy of CAR T cells may require lower pretreatment intensities and lower numbers of T cells to be applied thereby potentially reducing the overall toxicities. T cells with stem celllike properties [74] may be ideal with this respect due to their strong engraftment potential after application. The general assumption is that fewer CAR T cells with a less differentiated phenotype will show similar therapeutic efficacy as high-doses of T cells with a more matured phenotype. Potentially, preconditioning may be reduced when applying those cells compared with the currently used matured central memory T cells.
Executive summary Concept of CAR-& TRUCK-modified T cells • Chimeric antigen receptor (CAR) T cells show redirected specificity toward predefined targets independently of MHC presentation; TRUCK T cells release a transgenic product in addition upon CAR engagement.
CAR signaling blocks: combined primary & costimulatory signals are superior • The costimulatory domain together with the primary T-cell receptor (TCR)-derived signal orchestrates a distinct pattern of effector functions. • The optimal combination of signals for each T-cell subset is different and needs to be determined in detail.
CAR T-cell production for clinical application • Retro- and lenti-viral vectors are commonly used to modify ex vivo patient’s T cells with CARs. • A fully automated, closed system to amplify T cells under good manufacturing practice conditions is required to establish CAR T-cell therapy for a broad panel of applications.
General problems with CAR T-cell therapy • CARs are immunogenic and provoke a humoral and cellular immune response. • CAR T-cell therapy produces some autoimmunity (on-target off-organ) which is a major challenge when targeting solid cancer. • Cytokine associated toxicity after CAR T-cell activation (cytokine storm) can be severe and life-threatening and may require immune repression and CAR T-cell elimination.
Specific problems in targeting CAR T cells toward solid cancer • A number of early phase trials are initiated to target solid cancer by CAR modified T cells. • Infiltration of solid tumor lesions and persistence of CAR T cells is insufficient and requires additional attention in the near future. • Targeting checkpoint blockade and immune repression may improve efficacy of CAR T-cell therapy in the treatment of solid cancer. • In addition to cancer cells, the tumor stroma needs to be targeted in order to reduce established large tumors.
Future perspective • Strategies to overcome the major hurdles for the success in the treatment of solid cancer lesions need to be empirically defined in forthcoming trials using a more standardized production and clinical trial protocol.
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Review Abken Based on clinical trial data, the current situation is problematic to identify the crucial steps for therapeutic effectiveness since the design of ongoing trials is highly diverse and employs CARs of different modular composition, different T-cell populations and amplification protocols, and applies different preconditioning regimes to the patient. Head-to-head testing in multiple single parameter trials against the same target in the same disease is needed to address the issue [75] . In addition, multicenter trials with a large number of patients are required to address whether CAR T-cell therapy provides benefit with respect to the overall survival and progression-free survival compared with the standard of care. The logistic of such a trial, however, is challenging and requires the optimization of a number of issues, including the production of CAR T cells which is currently performed only in a small number of trial centers and is extremely labor and cost intensive.
Standardization and automation of these processes will help to ensure that many more centers will be able to participate in such evaluation in the near future.
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Financial & competing interests disclosure Work in the author’s laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Deutsche Krebshilfe, Bonn, Else Kröner-Fresenius Stiftung, Bad Homburg v.d.H., Wilhelm Sander-Stiftung, Munich, EU (European Regional Development Fund – Investing in your future), German Federal State North Rhine-Westphalia (NRW) and the Fortune Program of the Medical Faculty of the University of Cologne. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
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