Adoptive immunotherapy for cancer - Wiley Online Library

Marco Ruella Michael Kalos

Adoptive immunotherapy for cancer

Authors’ address Marco Ruella1, Michael Kalos1 1 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Summary: Recent clinical success has underscored the potential for immunotherapy based on the adoptive cell transfer (ACT) of engineered T lymphocytes to mediate dramatic, potent, and durable clinical responses. This success has led to the broader evaluation of engineered T-lymphocyte-based adoptive cell therapy to treat a broad range of malignancies. In this review, we summarize concepts, successes, and challenges for the broader development of this promising field, focusing principally on lessons gleaned from immunological principles and clinical thought. We present ACT in the context of integrating T-cell and tumor biology and the broader systemic immune response.

Correspondence to: Michael Kalos Lilly Research Laboratories Eli Lilly and Company 450 East, 29th Street New York, NY 10016, USA Tel.: +1 646-638-5095 e-mail: [email protected] Acknowledgements We are grateful to the multitude of scientists who have laid the foundations for the field, and to the patients who have allowed, through their bravery and generosity, the clinical development of this promising treatment modality. MR is supported by funds from the “Societa Italiana di Ematologia Sperimentale” (SIES), from “Associazione Italiana Pazienti Emopatici” (AIPE) and AFCRI fund number 990-9936-4-9002785043-2433-1410 from the University of Pennsylvania. The authors have read the journal’s policy on disclosure of potential conflict of interest. MR has nothing to declare. MK is named on patents related to CART19 technology with the potential for royalties and milestone payments from the development and commercialization of CART19 technology.

This article is part of a series of reviews covering Adoptive Immunotherapy for Cancer appearing in Volume 257 of Immunological Reviews. Video podcast available Go to www.immunologicalreviews.com to watch an interview with Guest Editor Dr Carl June.

Immunological Reviews 2014 Vol. 257: 14–38 Printed in Singapore. All rights reserved

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews

Keywords: adoptive cell therapy, immunotherapy, chimeric antigen receptor, cancer, immune modulation, tumor microenvironment

Introduction The principal biological role of the immune system is to maintain the integrity of ‘self’. This integrity is maintained principally by eliminating and destroying diseased and infected cells, while ensuring that healthy cells and tissues are not targeted. A vastly complex network of events that bring together both innate and adaptive immune arm through both cell mediated and soluble interactions is responsible for maintaining the integrity of self. The network of cells includes, among others, T and B lymphocytes, natural killer (NK) cells, antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, stromal, endothelial, and epithelial cells. This network of cells is coordinated by a myriad of soluble factors to sense both health and damage and in turn orchestrate the immune system to clear the body of damaged cells and protect the body from infections. Data from patients with inherited immune deficiencies have revealed the critical role for an intact immune system for organismal health, with autoimmune disease and increased risks of developing infections and tumors a consequence of disruptions in immune network functions (1, 2). Broadly speaking, immune cells can be defined as having either effector or regulatory functions in the immune system. The main known effector cells of the immune system are understood to be T and NK lymphocytes, mediating,

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© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

Ruella & Kalos Adoptive immunotherapy for cancer

respectively, adaptive and innate immunity. The variety of cells with immune regulatory activities is considerably broader, and includes cells of lymphoid origin such as CD4+ helper and regulatory cells and B cells, and myeloid origin such monocytes, macrophage, myeloid derived DCs, and myeloid derived suppressor cells. Perhaps not surprisingly given the breadth of the immune system, cells not classically considered to be involved in the immune systems such endothelial, stromal, and epithelial cells have over the past few years been shown to have immune regulatory functions (3–6). The relevance of these cell types in the regulation of a potent immune response is revealed by the fact that tumors often evolve under selective pressure by the immune system to modulate the biology of these cells and blunt anti-tumor immunity (7). Abundant evidence exists to document that the immune system plays an essential role in controlling the growth of tumors. This evidence has been accumulated from the study of animal models with defined immune defects (8), from the increased frequency of cancers in patients with naturally occurring or acquired immune deficiencies (9), and also from the observation that tumors often evolve to become less visible to the immune system. Early efforts to target cancer through manipulating the immune system in fact predated the ‘discovery’ of the immune system, most famously by William Coley who applied heat killed streptococci (‘Coley’s toxin’) at tumor sites to induce inflammation with consequent tumor regressions (10). More recently, the tuberculosis vaccine of Calmette-Guerin (BCG) has been used with success to treat localized bladder neoplasm (11), while adjuvants based on bacterial components that trigger immunity via engagement of Toll-like-receptors (TLRs) on immune cells are essential components of FDAapproved vaccine preparations. Direct evidence of the potency of effector T cells to target and eradicate tumor cells was demonstrated through the clinical application of donor lymphocyte infusion (DLI) to treat leukemia after allogeneic hematopoietic stem cell transplantation (HSCT), with the Graft-versus-leukemia (GVL) effect mediated by alloreactive donor T cells leading to strong anti-leukemic responses in a significant portion of relapsing patients (12, 13). As the field of immunotherapy has unfolded over the past two decades, a number of strategies have been evaluated to target cancer. Such immunotherapeutic strategies have included a plethora of vaccine-based strategies to trigger anti-tumor cellular and humoral immunity, antibody-based strategies to mediate complement and © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

NK-dependent anti-tumor cytolytic activity, block inhibitory receptors, or modulate the tumor microenvironment (TMA) (14–19). Unfortunately, each of these approaches has been compromised by the impact of central and peripheral immune tolerance, which presents a fundamental impediment for the effective targeting of tumor cells by immune effector cells. Specifically, central tolerance, by deleting the T-cell repertoire to self-antigens often overexpressed or aberrantly expressed by tumors, effectively results in the blunting of the potent anti-tumor T cell repertoire to such antigens. On the other hand, peripheral tolerance, commonly exploited by tumors, blunts the ability of existing effector lymphocytes (T cells, NK cells) to target tumor cells by establishing an immune-suppressive state. The relevance of peripheral tolerance in blunting the antitumor immune response has been highlighted over the past few years in clinical trials to evaluate antibodies which block immune-modulating molecules. In these trials, blocking the immune modulatory activity of cytotoxic T-lymphocyte antigen-4 (CTLA4) (CD152) and programmed death-1 (PD1) (CD279) has resulted in potent anti-tumor activity, with document long lasting complete responses in a subset of patients (20–22); these results have led to the first-in-class approval of an anti-CTLA4 antibody (ipilumimab/Yervoy), and the likely approval of PD1-modulating agents in the near future. Overcoming central tolerance poses a unique challenge, since the impact of central tolerance is the lack of a potent anti-tumor T-cell repertoire to self-antigens. One approach to overcome central tolerance is the transfer into patients of potent effector cells, as exemplified by the efficacy of alloreactive T cells in the context of allogeneic cell transplantation. Practical limitations in terms of availability of suitable donors as well as GVH-related toxicity issues have limited the broader use of allogeneic cell transplantation. One alternative to allogeneic cell transplantation is adoptive cell transfer (ACT), which involves the infusion into patients of autologous lymphocytes following ex vivo expansion. Initial efforts to apply ACT in tumor immunotherapy involved transfer of bulk T-cell populations into patients, under the premise that ex vivo manipulation of cells had the potential to lower activation thresholds of potential tumor-reactive cells as well as to expand tumor-specific T cells. The promise of this approach was revealed in clinical trials which showed that this strategy had the potential to rapidly reconstitute host immunity (23–25). Recent advances in the ability to effectively engineer T lymphocytes have provided an

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unprecedented opportunity to be able to expand on the potential of T-cell transfer and have ushered in what promises to be a golden age for T-cell-based immunotherapy based on principles of synthetic biology (26) (Fig. 1). Indeed, clinical results from recent trials employing engineered T cells are demonstrating the dramatic potency of this approach to target cancer (27–31). This review focuses on principles of ACT in the context of recent conceptual and technical advances that are driving the field in this emerging era of synthetic biology. We describe the state of the art for the field with regard to cell types, engineering approaches, and clinical experience. Importantly, we focus on viewing ACT in the context of the systemic immune response, the biological context within which infused T cells are operative. A brief history of ACT The potential to apply ACT as a therapeutic approach was first evaluated in rodent models over 50 years ago (32). The first clinical experience in humans was reported from the Seattle group in 1991 in the context of cytomegalovirus (CMV) prophylaxis during immune reconstitution post transplantation, where CMV-specific T-cell clones were expanded ex vivo and infused into patients at high risk from CMV infection, resulting in the establishment of an anti-CMV protective immune response (33). This seminal report demonstrated that the fundamental requirements for successful ACT, namely ex vivo manipulation, expansion, and reinfusion into patients were technically feasible, and precipitated concerted efforts to apply ACT to target malignancy. Early efforts to target malignancy focused on the use of tumor-infiltrating lymphocytes (TILs) pioneered by the Rosenberg group at NCI (34), the use of bulk populations of expanded lymphocytes (23), as well as the use of tumor antigen-specific clones, isolated following considerable tour-de-force efforts (35, 36). These approaches revealed a number of significant points with regard to the clinical implementation of ACT: (i) T-cell- based immunotherapeutic approaches had the potential to effect potent antitumor activity; (ii) tumor targeting strategies would need to incorporate and address biological considerations for both the T cell (e.g. the need for cosimulation) and the tumor (e.g. immunosuppression); and (iii) robust and practical implementation of ACT to target tumors would require significant improvements in the ability to rapidly generate large and potent populations of tumor-specific lymphocytes.

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Accordingly, the principal focus in the field of ACT shifted toward a better understanding of the properties of the effector cells that were important to mediate and drive the observed anti-tumor activity and the development of improved approaches to isolate, manipulate, and expand such potent effector T cells. Immune effector cells in ACT Two effector cell types, T lymphocytes and NK cells have been principally evaluated for ACT. The essential properties of these cells that have supported their evaluation have been the fact that these cells are capable of cytotoxic targeting, and that these cells can be readily isolated and manipulated ex vivo. Table 1 summarizes the advantages and disadvantages for each of the effector cell types that have been evaluated to date in ACT-based approaches against cancer. Classical NK cells (CD3 CD56+) represent about 20– 30% of circulating lymphocytes. As part of the innate immune system, NK cells characterized by rapid and potent cytolytic activation against virus-infected or transformed cells, a potential advantage for ACT. A potential advantage of NK cells is that their principle targets are cells that lack class I, often a phenotype selected for in tumor cells; on the other hand the presence of class I on tumors poses interesting challenges for broad implementation of NK cellbased therapies. In the context of ACT, classical NK cells, both as primary cells and cell lines, have been evaluated in a number of different settings and using a variety of ex vivo expansion protocols. Classical NK cells have been tested in ACT non-modified in the context of haplo-identical HSCT (37), or more recently following genetic modification to express immune-modulatory cytokines (38). Unfortunately, despite numerous attempts, these approaches led to at best modest clinical activity (39, 40). A new and intriguing approach involves gene engineering of NK cells to express chimeric antigen receptors (CARs), and these exciting studies are now starting to be evaluated in early phase clinical trials (41). Smaller lymphocytes subsets that represent intermediate phenotypes between classical NK cells and T cells have also been evaluated in the context of ACT. NK T cells (NKT) (CD3+CD56+), iNKT cells (CD3+CD56+Va24-Ja18), and cytokine-induced killer cells (CIK) (CD3+CD56+) distinguished by Lu and Negrin (42), are each ‘niche’ effector lymphocyte subpopulations with potential advantages, summarized in Table 1, in the context of ACT (43). NKT cells have been shown to modulate immune responses © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


A Anti-VEGF-R Anti-αvβ3 integrin

Treg

NK

T B

B

CART

CART homing and

trafficking C A R T

NKT

D

C

haTCRT

CART

Tumor cell

F Treg

MDSC

Tumor cell Lymphodepletion Chemothererapy Conditioning Radiation

CART

MDSC

H

TIL B

haTCRT

G

Treg

Tumor cell

CART

Treg

B

TAM

Treg

Tumor cell

TIL B

Checkpoint inhibitors Autocrine cytokine augmentation

haTCRT

Anti-FAP E

I

B

Treg depletion

Tumor cell Tumor cell

MDSC

NKT

Anti-NKG2D

MDSC

Molecules : Surface target epitope

Treg

B

MDSC

CART

Effector cells CART

NKT

TAM

: CAR-engineered T cell

NK

Regulatory cells MDSC

: Myeloid-Derived Suppressor Cell

Other cells Tumor cell

: Tumor Cell

: MHC:peptide complex : Endogenously processed epitope

haTCRT

: Affinity-enhanced TCRengineered T cell

: Endothelial cell TAM

: Tumor Associated Macrophage NK

: Natural Killer cell

NKT

: Natural Killer T cell

: CAR : Endogenous TCR complex

: Tumor infiltrating lymphocyte

: Affinity-enhanced TCR complex

: Tumor Associated Fibroblast Treg

: Regulatory T cell B

: B cell

Fig. 1. Adoptive cell transfer (ACT) approaches to effect anti-tumor immunity. (A) Engineering T cells to target tumor endothelium facilitates the entrance of immune cells on tumor site and inhibit angiogenesis. (B) Expression of chemokines (CXCR2, CCR2B) on the surface of T cells favors homing to tumor site. (C) chemotherapy or radiotherapy before ACT enhance T cells cytotoxicity and persistence by both target tumor and tumor associated immunosuppressive cells [myeloid-derived suppressor cells (MDSC), Treg, tumor-associated macrophages (TAM)]. (D) Chimeric antigen receptor engineered T cells (CART) can target TAA and kill tumor cells without the need of HLA presentation. (E) TAA that are processed and presented in HLA on the surface of tumor cells are recognized by tumor-infiltrating lymphocytes (TIL) and haTCRT. (F) Targeting tumor associated FAP+ fibroblasts that may suppress T-cell homing and anti-tumor activity in the tumor environment. (G) Ex vivo depletion of Tregs reduces the number of these inhibitory cells in the infusion product. (H) Checkpoint inhibitors (e.g. anti-CTLA-4 and anti-PD-1) and the increased expression of activating cytokines (e.g. IL-12) enhance T-cell cytotoxic function. (I) Targeting NKG2D on immune cells re-modulates tumor environment, reducing the immunosuppressive milieu. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

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Table 1. General properties of among tumor-infiltrating lymphocytes (TIL), TCR engineered T cells (TCRT), high affinity TCR engineered T cells (haTCRT), and chimeric antigen receptor engineered T cells (CART) Parameter

TIL

TCRT

haTCRT

CART

Tissue of origin Ease of generation Efficiency of generation Ex vivo manipulation time Genetic modification Antigens targeted Antigenic specificity Need for antigen processing and HLA presentation Effector functionality Proven clinical efficacy Potential for Off tissue toxicity Potential for Off target toxicity Feasibility for commercialization

Tumor Low Moderate 17–30 days Not necessary All Undefined Yes Undefined Melanoma Low Low Low

Peripheral blood Moderate* High 8–12 days Required All Defined Yes Low Not clear Low Low Low

Peripheral blood Moderate* High 8–12 days Required All Defined Yes High Solid tumors High High Moderate

Peripheral blood Moderate High 8–12 days Required Surface only Defined No Very high B-cell neoplasms High Low High

*

Once original TCR has been identified.

against cancer and stimulate effector cell functionality (44), and they have additionally been reported to be capable of overcoming peripheral tolerance (45). A potentially important advantage of iNKT cells is the fact that they recognize a unique glycol-molecule (agal-Cer) in the context of the CD1d molecule, which can be used to both specifically expand these cells ex vivo and also to stimulate their expansion in patients (46). Additional subsets of NKT cells have been defined, but to date have not been evaluated in ACT (47). cd T cells (CD3+ TCR a b , TCR c+d+), are a population of T cells very rare in peripheral blood (4–10%) but substantially enriched in areas of mucosal immunity such as gut (48). cd T cells recognize a wide variety of targets including stress-induced antigens in a classical MHC-unrestricted manner and manifest lytic activity and pro-inflammatory cytokine secretion. A major population of cd T cells expresses an invariant Vc9Vd2 T-cell receptor, and has been shown respond to isopentenyl pyrophosphate (IPP), a product of the mevalonate pathway which is dysregulated in tumor cells. These lymphocyte subsets are currently tested for ACT, both unmodified following bulk expansion ex vivo (49) and also following gene engineering using CARs (50). abT cells (CD3+TCR a+b+) are the most abundant T-cell population in the blood, the most extensively studied in general, and the most robustly evaluated for ACT. Both effector (CD8+) and helper (CD4+) T lymphocytes have been evaluated in ACT in every conceivable combination, obtained from the peripheral blood or from tumor sites, either as bulk populations or antigen-specific clones, or following genetic modification to express tumor re-targeting receptors.

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Genetically unmodified T cells One of the first clinical applications of ACT was the infusion of cells termed lymphokine-induced killer cells (LIK), which were generated from patient peripheral blood samples by exposure to high doses of interleukin-2 (IL-2). Although positive clinical responses were reported in these studies, these could not be dissociated from the effects of the co-infusion of high doses of IL-2 (51, 52). Nevertheless, the data generated from LIK-based strategies facilitated the development of next generation approaches, specifically the evaluation of lymphocytes isolated from tumor biopsies (TILs) and bulk T lymphocytes activated ex vivo prior to infusion. The premise behind TIL-based approaches is that T lymphocytes in biopsy specimens are enriched for anti-tumor reactivity but have become functionally anergized, and that ex vivo culture of these cells results in their re-activation. TILbased strategies were developed principally in the context of melanoma, thanks to the pioneering efforts of the NCI group. TILs cultured are selected for anti-tumor activity following ex vivo culture and recognize tumors in an MHCrestricted manner via their TCR. Initial studies showed the feasibility of this approach with expansion of unselected TILs from subcutaneous and lymph node melanomas with subsequent re-infusion together with IL-2; although the response rate for these initial studies was about 30%, TIL persistence was poor (53, 54). To improve long-term persistence of infused cells, follow-up clinical studies evaluated the use of non-myeloablative lymphodepleting chemotherapy (cyclophosphamide and fludarabine) prior to TIL infusion followed by high doses of IL-2, under the premise that competition for niches and homeostatic cytokines were impediments to long-term persistence of infused cells. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


Incorporation of the lymphodepleting regimen improved clinical responses to 50%, with enhanced TIL in vivo expansion and persistence (55, 56). In a recent update, 13% of the patients treated with this regimen experienced complete regressions ongoing beyond 5 years (57). To evaluate the effect of enhanced lymphodepletion, two pilot trials were conducted with total body irradiation (TBI). These treatments resulted in clinical response rate of 49–72% (58). A major impediment with early TIL trials was that the ex vivo generation of TIL cultures was extremely labor intensive, with the generation from each patient of multiple TIL sublines, screening for tumor specific activity against autologous or HLA-matched tumor cell lines, and long expansion cycles. As a result, more than a half of patients were excluded from this treatment. The development of shorter expansion protocols and more streamlined screening approaches has considerably improved the broader implementation of this approach (59). An ongoing phase 2 study is comparing CD8+ enriched short-term cultured TILs plus high dose bolus IL-2 after non-myeloablative chemotherapy against high dose IL-2 alone in metastatic melanoma. The use of TIL therapy in a broader sense is currently limited by a number of factors: (i) tumor specimens are not readily available in many cancers, especially in the metastatic setting where biopsies are not often obtainable; (ii) TILs are reproducibly detectable only in a minority of cancers (mostly melanoma and renal cancer); (iii) expansion protocols remain relatively labor-intensive, expensive, time consuming, and difficult to standardize; (iv) current conditioning regimens are not feasible in all patients; and (v) TIL functionality may revert to the immune-suppressed state when infused TIL re-enter TMA. Parallel effort to TIL-based approaches involved the ex vivo expansion of bulk T lymphocytes from the peripheral blood of cancer patients followed by subsequent re-infusion with the support of IL-2. This approach was aimed to expand a population of activated cells with a lowered triggering threshold and thus with enhanced potential to be triggered by tumor cells, as well as potentially trigger and expand tumor-reactive lymphocytes rendered anergic as a result of peripheral tolerance. Clinical activity was observed in this approach, with rapid functional immune reconstitution observed in patients, and the establishment of optimized timelines for T-cell infusion post lymphodepletion (23). In a follow-up study, transfer of bulk activated T cells was associated to the development of autologous GVHD with rash and colitis in about 25% of treated patients, demonstrating the potency of this approach (24). © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

Genetically retargeted T cells The fundamental premise behind genetic retargeting of T cells is the fact that the endogenous potent tumor-specific T-cell repertoire has been compromised as a consequence of central tolerance. Indeed, comparative analyses have demonstrated that TCRs against tumor have substantially lower antigen affinity (approximately 0.5 logs) compared with TCRs directed against virus-derived antigens (60, 61), providing at least partial explanation for the lack of clinical efficacy of approaches directed to triggering the self-antigen-reactive T-cell repertoire. Two related approaches have been developed to genetically redirect T cells: (i) the transfer of affinity-enhanced tumor-specific TCRs and (ii) the transfer of synthetic CARs. Recent clinical experience has suggested that both of these approaches can mediate potent anti-tumor activity. The ‘canonical’ TCR is composed of a complex of at least six polypeptide chains (a, b, c, d, e, and f) which assemble on the surface of T cells. The a and b chains form the primary binding domain of the TCR, which recognizes intracellularly processed peptides presented on the surface of target cells by proteins of the MHC. Accessory costimulatory and adhesion molecules augment the strength and quality of the TCR-MHC peptide interaction leading to productive T-cell engagement. Initial efforts to evaluate TCR transfer involved transfer of self-antigen specific non-enhanced TCR the genetic transfer of TCR ab chains specific for selfantigens (62, 63). These pioneering efforts demonstrated the proof of principle that TCR-based T-cell engineering was feasible, but also highlighted the need to generate more potent TCR to overcome the consequences of central tolerance on TCR affinity. Approaches to generate more potent TCRs have included the vaccination of human HLA transgenic mice with target tumor antigens to generate high affinity human antigen-specific TCRs, and this approach has been used to isolate high affinity TCRs against MDM-2 (64), p53 (65), CEA (66), gp100 (67), and MAGE-A3 (68). More recently animals transgenic for human HLA and TCR have been used to generate high-affinity human ab TCRs (69). Other approaches to generate high affinity TCRs have involved taking advantage of gender-restricted expression of target antigens (such as prostate, ovary) and the lack of tolerance to these antigens in the opposite gender (70), selection of specific T-cell clones from a polyclonal pool of graft-versus-tumor reactive T cells (71). Early clinical results using affinity enhanced TCRs have highlighted the promise of this approach with dramatic anti-tumor activity observed

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in a number of cases (72–74). However, as discussed further below, the introduction of TCRs with non-physiologically enhanced affinity to self-antigens perhaps predictably has led to increased issues related to off target toxicity in normal tissues (66, 67, 75), an issue of considerable concern that has to be addressed for full implementation of this approach. Beyond TCR affinity enhancement approaches, other efforts to generate higher affinity tumor-specific TCRs have involved efforts to increase the number of TCRs on the surface of T cells (72, 73, 76), increasing introduced TCR expression levels (77–80), and a number of strategies to minimize a and b chains mispairing with endogenous chains (81–89). Although TCR-based ACT approaches hold considerable promise as exemplified by early clinical trials, they do possess certain inherent limitations. More specifically, the HLA-restricted specificity of engineered TCR limits the fraction of potential patients to those expressing the relevant HLA allele recognized by the T cells, and the fact that tumors often downmodulate HLA alleles and/or alter proteasomal processing pathways provides for the real possibility of antigen escape variants and lack of complete responses. CARs are synthetic receptors that combine the extracellular single-chain variable fragment (scFv) of an antibody with a transmembrane (TM) domain and intracellular signaling domains derived from molecules involved in T-cell signaling. Essentially all current CARs utilize the signaling domain from the CD3f chain and additionally can contain signaling domains from molecules involved in T-cell activation or costimulation. Since the scFv domain binds directly to target cell surface epitopes CAR-based strategies bypass the need for MHC-restricted antigen presentation and are thus insensitive to tumor escape mechanism related to HLA downmodulation and altered processing escape-mechanisms (90). CARs bypass most of the T-cell triggering limitations related to epitope density, since the scFv of the monoclonal antibody is characterized by high-affinity for the target and additionally each surface target molecule presents a triggering epitope. CAR-based redirection represents a near universal ‘off-the-shelf method to generate large numbers of antigen-specific helper and cytotoxic T cells. As described in more detail below, a key to this successful implementation of CAR-based approaches is the identification of antigens with surface expression on tumor cells and absent expression on normal healthy tissues. The concept of CARs was introduced by Eshhar and colleagues in 1989 (91). Since then, many groups have evaluated the possibility to redirect T cells using antibody-based

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scFv of an antibody fused to the CD3f or less commonly, Fc receptor c (FcRc) signaling domains. The scFv are usually derived from murine antibodies and thus have the potential to trigger immune rejection responses by the host; current strategies involved the use of humanized or fully human scFv to mitigate this possibility (92). Engineering the optimal signaling domain combination for maximal biological activity has been the focus of extensive preclinical and more recently clinical activity. The most intensively studied signaling domains have been those associated with robust effector or costimulatory activity. Initial (i.e. first generation) CARs contained only the CD3f chain signaling domain. Although such CARs were capable of re-directing T cells to target antigens in vitro demonstrating the proof of concept that T cells could be engineered using CAR-based approaches, clinical trials based on such CARs demonstrated limited efficacy in a variety of cancers, such as neuroblastoma (93), non-Hodgkin’s lymphoma (94), renal cancer (95), and ovarian cancer (96). A recurring theme in these studies was limited in vivo expansion, modest antitumor activity, and lack of in vivo persistence. To overcome the limitations with first generation CARs, second generation CARs were developed and evaluated. Second generation CARs incorporate additional signaling domains from costimulatory and accessory functional T-cell molecules, in attempts to enhance in vivo functionality, potency, expansion capacity, and persistence. The first costimulatory molecule to be included in the CAR construct was CD28, and this led to a dramatic increase of the IL-2 production and killing capacity of the T cells (97). Positioning the CD28 domain 5′ to the CD3f domain was shown to me most effective (97, 98). Moreover, inclusion of the CD28 domain was shown to enhance resistance to suppression by regulatory T cells (99, 100). Many other signaling domains have subsequently been evaluated preclinically, including 4-1BB (CD137) (101, 102), OX-40 (CD134) (103, 104), CD244 (105), CD27 (106), and inducible costimulator (ICOS) (98, 107). In preclinical models, second generation CAR T cells have been shown to mediate enhanced functionalities including expansion and anti-tumor activity (102). CARs containing the 4-1BB domain have been shown to result in improved in vivo persistence, antitumor activity and tumor localization compared with first generation CAR or CAR containing the CD28 domain (100, 101). Inclusion of a CD27 signaling domain to CAR may also enhance in vivo persistence and improve cytotoxic effect against xenogeneic tumor (106), while inclusion of the ICOS signaling domain has been shown to drive human © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


T cells to a Th17 phenotype (108). Second generation CARs, where costimulatory molecules are included in trans, have also been developed, enabling the ability to more precisely potentially modulate CAR functionality (109). Most current clinical trials evaluate second generation CAR designs (110). Third generation CARs are characterized by the addition of a second costimulatory domain to further improve T-cell expansion, cytotoxicity, and in vivo persistence. Third generation CARs combining CD28 with 4-1BB, and CD28 with OX-40 have been evaluated (111–113). Conceptually, with this approach the threshold of T-cell activation is further reduced in 3rd generation CARs leading to further enhanced potency but also increased potential for low avidity off target activation. In terms of CAR design, both the spacer/hinge and the TM domains have the potential to influence CAR function. In fact the hinge region mediates flexibility and it is important for the positioning of the binding scFv domain (114, 115). A number of studies have shown that the TM domain is critical for effective CAR expression on the surface of the cell, and accordingly many different TM domains have been evaluated including CD28 (116), OX40 (116), CD3f (117), FceRIc (118), CD7 (119), CD4 (102, 103), H2-Kb (120), and CD8 (101, 121). Inclusion of the CD28 TM domain was found to result in a higher expression of CAR in comparison with OX40 and CD3z TM domains (116). Other reports suggested that intracellular rather than TM domains modulate CAR surface expression (122). With regard to the hinge region, IgG1, IgG4, IgD, and CD8 domains have been mostly evaluated (123, 124). An additional important consideration for CAR design is the length of the hinge region, which has been shown to influence the quality of the interaction between the T cell and target, depending on the location of the target epitope on the target antigen (125, 126). Until more specific rules to guide this process are defined, this important issue will likely require empiric testing of multiple CARs against each epitope being targeted. The relative position of the epitope in the target protein also appears to be relevant in terms of CAR engineered T cells (CART) potency. For example, CD22 target epitopes that are proximal to the cell membrane trigger more potent lytic activity compared with distal epitopes (126, 127), while other studies showed longer spacer domains increased the potency of 5T4- and NCAM-specific CAR that recognized membrane proximal epitopes, potentially by providing increased flexibility to the CAR (125). Indeed, the distance between the T cell and the tumor cell is influenced by the combination between the position of the epitope and the length of the © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

CAR molecule that is mainly related to spacer regions, and this is able to influence the entity of the activation and killing activity. CAR-engineered T cells have been generated against many different TAA for both solid and hematologic malignancies: CD19 (128–131), CD20 (94, 132), CD33 (133, 134), B-cell maturation antigen (135), CD22 (127), CD23 (136), CD30 (137, 138), CD38 (139), CD44v6 (140), ROR-1 (141), j-light chain (142), Lewis Y antigen (143, 144), NKp30 (145), TAG-72 (146), CD70 (147), Carboxy-anhydrase-IX (CA-IX) (95, 148), human epidermal growth factor receptor 2 (ErbB-2/Her-2/Neu) (149–153), disialoganglioside 2 (GD2) (154–157), GD3 (158), L1CAM (CD171) (93), VEGF-R2 (159), EGFR (107, 160, 161), MUC-1 (124), MUC-16 (162), prostate-specific membrane antigen (PSMA) (97, 163, 164), prostate stem cell antigen (PSCA) (165, 166), 5T4 oncofetal antigen (h5T4) (167), NCAM (125), mesothelin (111, 168), fibroblast activating protein (FAP), folate receptor-a (96, 169), NKG2D (170–172), IL11 receptor a-chain (173), CEA (115, 174, 175), IL-13Ra2 (117, 176), and erythropoietin-producing hepatocellular carcinoma A2 (EphA2) (177). As described in more detail below, a small number of CARs are currently being tested in early phase clinical trials, with in some cases dramatic and exciting results that highlight the promise of this approach against cancer. A number of groups have initiated efforts to overcome the need for patient-specific products for ACT. Attempts to generate ‘universal donor T cells’ have involved the use of zinc-fingers nucleases to eliminate the expression of the endogenous TCRs (178). Although this area of research is still nascent and many issues will need to be identified and resolved, it has the potential, combined with efforts to reduce the allo-recognition of engineered T cells for example by targeted knockdown of class I and class II loci to bypass the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. A novel approach to simultaneously target multiple targets on tumors is the development of bi-specific CAR. Such moieties, coined as ‘TanCAR’ simultaneously recognize two tumor restricted antigens with synergistic enhancement of effector functionality when both antigens are present on target cells; T cells engineered with TanCAR retained cytolytic ability with loss of one of the target molecules and were superior to single CAR engineered T cells in the control of established experimental tumors in animal models (179). TanCAR or related approaches have the potential to

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counteract the existence of antigen-loss variants that often exist in tumors; however, such approaches may have enhanced potential for off-tissue toxicity, discussed more fully below. An interesting strategy to mitigate off-tissue toxicity is to develop combinatorial CAR constructs that transduce an activating signal only when encountering a specific combination of antigens. Kloss et al. (180) recently described a novel approach by which T cells are co-transduced with a CAR that provides suboptimal activation upon binding of one antigen and a chimeric co-stimulatory receptor (CCR) that recognizes a second antigen. Using the prostate tumor antigens PSMA and PSCA as model antigens, this group demonstrated that combinatorial CAR-engineered T cells eradicated tumor cells that expressed both but not individual antigens (180). A novel strategy to expand the recognition specificity potential of CAR T cells utilizes a biotin-binding immune receptor (BBIR), which consists of an extracellular modified avidin domain linked to an intracellular T-cell signaling domain; target-specific antibodies can then be loaded onto BBIR T cells through streptavidin cross-linking (181). Development of BBIR T cells may allow the sequential or simultaneous targeting of a broad combination of distinct antigens. A recent report has described the development of CARs that target the intracellular antigen WT-1 using a scFv that is able to recognize MHC class I + peptide complexes (182). Although still extremely preliminary, this approach has the potential to overcome the limitation of CAR-based strategies to surface antigens and greatly extend the universe of target antigens available for CAR-based approaches. On the other hand, advantages of CAR-based strategies described above and related to target epitope density and independence from HLA expression and processing defects will likely prove to be impediments for the success of this approach. Beyond the introduction of antigen-re-targeting receptors, gene-engineering efforts have also involved attempts to selectively target T cells to sites of tumor using chemokine and homing receptors such as CXCR2 or CCR2B, which when expressed on engineered T cells have been shown facilitate the homing of T cells to tumor (137, 183–185). Engineering of T cells to express immune modulatory cytokines, such as for example IL-12, has been shown to improve the efficacy of ACT in animal models, at least in part by increasing the expression of Fas within tumor-infiltrating macrophages, DCs, and myeloid-derived suppressor cells (MDSCs) (186). Expression of single chain IL-12 by

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engineered T cells has been shown to enhance the efficacy of low doses of MHC-restricted TILs, at least in part by re-programming tumor associated myeloid cells to crosspresent tumor-associated antigens (187); interestingly, this approach eliminated the need for lymphodepletion before CAR therapy (188). Other approaches that involve efforts to modulation expression of cytokines, such as IL-12 by T cells, are also currently being evaluated (189). Clearly, the ability to at-will engineer T cells to express desired immune-modulating soluble factors and receptors provides an incredibly exciting and diverse menu of opportunities to develop more potent, and disease targeted options for ACT, while also enabling the modulation of SAE associated with ACT-collateral immune activation. T-cell subsets for ACT Over the past few years it has become evident that T-cell function may be influenced by many other parameters beyond antigen specificity. The nature of the T cell that is used for adoptive immunotherapy is highly correlated with clinical results (190). Initially, it was assumed that the ideal T cells for adoptive immunotherapy would be effector T cells (Teff), due to their ability to respond robustly to antigen stimulation as measured by cytotoxicity. However, a number of early clinical studies showed that highly differentiated tumor specific Teff CD8+ T cells engrafted poorly and are less capable of tumor killing compared with heterogeneous population of TILs (191, 192). Retrospective analyses from clinical trials revealed that patients infused with less differentiated cell products had better clinical outcome (193), and follow-up studies in animal models demonstrated that the use of a less differentiated T-cell population for ACT led to better anti-tumor activity, in vivo expansion and persistence (194). These results led to efforts to identify sub-populations of T cells with enhanced in vivo functionality as well as develop in vitro culture approaches that generated less differentiated T-cell products (see below). More recent studies have shown that the frequency of central memory T cells (Tcm) in infusion products correlates with positive clinical response (156). Furthermore, naive T cells (Tn) have shown higher antitumor effect and longer in vivo persistence than Tcm (195) and antigen experienced CD8+ Tcm cells persist longer than effector memory T (Tem) cells (196). More recently a specific T-cell subset, the stem cell memory T cells (Tscm), has been described; Tscm have been described to be capable of self-renewal and to generate potent effectors, this © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


population may be important for adoptive immunotherapy (197). Recent exciting work has also revealed the potential for plasticity in the Tscm repertoire, with data from animal models suggesting that a very small number of the ‘right’ population of engineered T cells may be sufficient to mediate potent anti-tumor activity accompanied by in vivo expansion and differentiation into T-cell subsets (198). Tn, Tcm, and Tscm cells express CD62L and CCR7 and therefore have the potential to home to secondary lymphoid tissues where they can interact with APCs explaining at least in part their better clinical activity. On the other hand, CCR7 cells engineered to express CARs with CD28, OX40, and TCRf costimulatory domains were recently shown to be rescued from apoptosis and result in more efficient antitumor efficacy compared with CCR7+ CAR-engineered T cells (199). A fundamental property of T cells, which mediate potent anti-tumor activity appears to be the ability for in vivo expansion, differentiation, and persistence (28). A number of studies have shown that Teff cells possess a relatively limited proliferative and engraftment potential compared to Tn, Tcm, and Tscm, in part due to limited lifespan associated with shorter telomere length (200–203). Studies using TILs have demonstrated that a short duration in culture, a rapid doubling time and longer T-cell telomere length were correlated with better clinical outcome (53, 57, 204–206). Recent studies indicated that the effector-like CD27 CD8+ cells mediate potent protective immunity and lead to longlived memory cells (207). Finally, recent reports suggest that adoptively transferred Th17 cells demonstrated superior proliferation, persistence, and anti-tumor activity compared with Th1 cells (208, 209). Although data obtained in a number of studies suggest that less differentiated T cells with longer telomeres, such as Tn, Tcm, and Tscm, may be the preferred cells for ACT, there is potential danger of applying reductionist reasoning to an inherently complex biological by selecting a particular T-cell subtype for ACT. There is evidence that CD4+ cells are required for CD8+ memory cell formation that is import for long-term control of the tumor (210, 211). Indeed, the most potent and durable clinical responses to date have been obtained using bulk populations of CD8+ and CD4+ T cells (27–29). Considerable effort has been spent principally by the Baylor group to evaluate the ability to isolate and engineer virus (EBV, CMV)-specific T cells to express CARs, under the premise that these cells are inherently enriched for memory cells and additionally can be maintained in vivo by stimulation © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

through their endogenous TCR (212). Clearly this approach is restricted to patients that have been exposed to the viruses. EBV-specific T cells had been transduced with antiCD30 CARs or anti-GD2 CARs and had been preclinically tested (138, 155). EBV-specific T cells engineered to express a chimeric receptor had also been used to treat nasopharyngeal carcinoma or AML (133, 213). T-cell engineering strategies The development of approaches to at-will engineer T cells prior to ACT has been a fundamental building block for the recent success in the field. Although most preclinical and clinical studies have focused on introducing TCR or CAR re-targeting receptors into T cells (110), more recent studies have begun to evaluate the possibility to introduce T and other immune cell function-modulating molecules in T cells, such as IL-12 (188), IL-15 (214, 215), CCR2 (216), IL-2 (217). Initial successful efforts for engineering T cells utilized pseudotyped c-retroviruses that were capable of infecting primary human T cells and integrating their DNA in the host genome; this approach lead to high efficiency stable transduction of primary T cells that were employed in multiple clinical trials (218, 219). Concerns about retrovirus integration site bias coupled with concerns about long-term retroviral promoter silencing led to the more recent development of lentivirus-based vectors for gene transfer. In addition to the fact that lentiviruses vectors appear to be less susceptible to silencing by host restriction factors and can deliver larger DNA sequences than retroviruses (220), lentivirus vectors have been reported to be able to infect non-cycling cells, particularly when coupled with cytokine ‘pre-activation’ (221, 222). From an oncogenic perspective, significant early and justified concern about the oncogenic potential of retrovirus-based gene therapy as a result of the unfortunate report showing oncogenic transformation of stem cells due to retrovirus integration (223, 224) was mitigated initial in animal models (223, 225) and subsequently in long-term longitudinal retrovirusbased gene-therapy studies now extending beyond 10 years, which confirmed that retrovirus-based gene transfer into human T cells appears to be safe (226, 227). Although lentivirus-based gene transfer has not been implemented as long, the fact that in the natural history of HIV no T-cell oncogenic events have been reported, coupled with the absence of any reports from to-date lentivirus-based clinical trials provide reasonable assurances that this approach will be safe from an oncogenic perspective. Novel integrating

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virus-based delivery systems such as foamy viruses are under development (228) but beyond the scope of this review. Adenovirus-based vectors such as Ad-35 vectors, which result in long-term episomal transgene expression, have also been evaluated in human T cells with high expression efficiencies (229). The relatively high cost for manufacture and release of virus-based vectors has precipitated the development of a variety of non-virus based gene transfer systems. Retrotransposon systems, such as PiggyBac or Sleeping Beauty have shown preclinical promise (230–232). Novel and exciting approaches based on Zinc-finger nuclease, TALEN, and CRISP/Cas9 based-technologies permit to targeted insertion of transgenes or the targeted editing of the T-cell transcriptome and open up the potential to be able to modulate transgene expression and T-cell function at will (229, 231, 233, 234). For some applications, permanent genomic integration of new constructs may not be necessary for therapeutic efficacy or may be useful to mitigate potential toxicity. A novel approach that is currently being investigated to this end is RNA electroporation; this approach allows the rapid transfer of the specific mRNA in T cells allowing a high expression of the specific receptor (122, 235). Since the introduced mRNA is relatively rapidly degraded, this approach results in ‘biodegradable’ engineered T cells. Although RNAengineered T cells have been shown to mediate potent and antigen specific effector functions, multiple infusions are required to compensate for the short in vivo half-life of these cells (236). RNA engineering has been used to deliver transcripts for TCRs, CAR chemokine receptors, or cytokines (215, 237, 238) and is currently being evaluated in clinical trials. The biodegradable nature of this approach provides a considerable safety advantage, in particular when evaluating T cells engineered to express constructs with potential for toxicity. RNA engineering also allows the efficient transfer of multiple transcripts allowing for the ability to engineer T cells to co-express targeting, homing, and immune modulating functionalities. An efficient low cost method for gene transfer to T lymphocytes that includes the use of electroporation and a transposon-based system has recently been described (239). T-cell expansion strategies A fundamental requisite for current ACT strategies is the ability to engineer and expand T cells prior to infusion into patients. As described above, the ‘quality’ of T cells

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employed for ACT appears to be critical for in vivo efficacy. An early approach, based on the pioneering work of Riddell and Greenberg (240), employed antibodies specific to CD3e and cross-linked using allogeneic peripheral blood mononuclear cells, with immortalized B-LCL cells added as feeder cells, and exogenous IL-2. Repetitive stimulation using this approach generated large numbers of antigen-specific T cells that were employed in early trials (192, 241–243), unfortunately without significant success, in retrospect due to the fact that this approach strongly skewed products toward Teff cells (192, 244). A parallel approach, developed by Levine, June and colleagues, employed the use of magnetic beads coated with anti-CD3e and anti-CD28 antibodies to provide costimulation. This approach generates large numbers of T cells with a less differentiated phenotype (245, 246), has been robustly developed (247), and is the approach that has been recently employed to manufacture products with extensive in vivo proliferative potential, potent anti-tumor activity, and long-term functional persistence currently exceeding 3 years in the initial treated patients (27–29, see below). Although early studies suggested that the absolute number of the transferred T cells is almost always correlated with better clinical responses (205), this paradigm is now in question given the dramatic potency achieved with small numbers of cells in a recent trial (27), and the discovery of T cells with stem cell properties (197, 248, 249). Magnetic bead-based approaches have been used for preselection of specific subsets of T cells, such as CD8+ cells for TIL immunotherapy (59), CD4+ T cells for transfer HIV patients (250), CD25+ cells to prevent GVHD after allogeneic HSCT (251), or to select against particular subsets of cells that may be deleterious for in vivo efficacy, such as regulatory T cells (Tregs) (252). Based on preclinical data showing the enhanced persistence and anti-tumor functionality of Tcm cells (196), strategies to isolate CD8+ Tcm cells under GMP conditions have been developed (253). T cells may be also isolated based on for antigen specificity, as discussed above in the context of redirecting virus specific T cells to target tumors (212), and also in the context of utilizing virus-specific T cells to treat viral infections such as CMV (254, 255). The use of artificial antigen-presenting cells (AAPCs), also pioneered by the June group (256), to stimulate T cells under more physiologic conditions provides an interesting alternative approach to expand T cells for ACT. This approach, which has not yet been evaluated clinically, has the potential to provide more physiologic stimulation of © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


T cells preserving desired differentiation states, and has been adopted by other groups (257–260). Novel strategies involving ex vivo expansion of T cells with cytokines are also currently being investigated. Expansion of CD8+ naive T cells with IL-15 provides the expansion of T cells with the characteristics of Tcm cells, with preserved telomere length (261); compared with T cells expanded with IL-2, the use of IL-15 has been shown to support superior proliferative and anti-tumor activity (262). Expression of IL-15RA on CD8+ T cells has been shown to autonomously enhance the viability and proliferation of primary CD8+ T cells and cytotoxic potential of antigen-specific CD8+ T cells (215). The combined use of IL-15 and IL-7 in the context of anti-CD3/ anti-CD28 bead-based stimulation has been shown to facilitate the ex vivo differentiation and expansion of gene modified CD8+ Tscm cells under GMP compliant conditions (263). IL-21, when used for T cells expansion, leads to the production of minimally differentiated T cells with high proliferative and anti-tumor capacity (264, 265), while reprogramming of CD19-specific T cells with IL-21 signaling has been shown to improve adoptive immunotherapy of B-lineage malignancies (266). IL-4 may also have a role in T-cell expansion, as human T-cells engineered to co-express IL-4 receptor together with a CAR specific for MUC1 exhibited robust capacity to expand in vitro and effect destruction of MUC1-expressing tumors (267). Modulation of the metabolic state of T cells has been shown to influence their phenotype and function; for example, modulation of PI3KAKT-mTOR and Wnt-b-catenin pathways had been shown to control the differentiation status of T cells; co-culture of T cells with mTor inhibitors promotes the formation of memory subsets, while the promotion of the Wnt pathway leads to the formation of Tscm cells with potent anti-tumor activity (268). Modulation of substrate rigidity is another potentially promising approach to control the quality of ex vivo expanded T cells. Early in vitro studies in this area have revealed that modulating substrate rigidity has the potential to impact T-cell activation and proliferation (269). Finally, strategies that involve overexpression of genes involved in survival such as telomerase (270), anti-apoptotic genes (271), as well as the downregulation of pro apoptotic molecules such as Fas (272), or dominant-negative receptors for inhibitory molecules such as dominant negative receptors for TGFb (273) have been evaluated preclinically. Despite the interesting scientific approach of these strategies, concerns regarding safety have been raised and should be excluded before moving to the clinic. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

T-cell ablation strategies A concern with ACT has long been the need to ablate transferred cells, either to counter off target toxicity. To this end, ‘suicide gene strategies’ have been developed. Initial suicide system involved use of the herpes simplex virus (HSV) thymidine kinase (TK), which phosphorylates ganciclovir and acyclovir leading to the generation of toxic triphosphate products and cell death (274). Perhaps predictably, HSV-TK transduction in T cells leads to immunogenic reaction with clearance of TK positive T cells (243, 275). More recently, an elegant system based on the design and use of a caspase-9-driven apoptosis activity was developed; in this approach caspase-9 intracellular signaling domains were fused with a human FK506-binding protein domains; administration of FK506 (tacrolimus) leads to dimerization of caspase-9, activation of apoptotic pathways and potent cell ablation (276, 277). Even more recently an equally elegant system was developed that involved engineering of cells to express a truncated EGFR polypeptide devoid of extracellular ligand-binding and intracellular signaling domains but that retained binding to the anti-EGFR monoclonal antibody Erbitux, enabling both in vitro selection and in vivo ablation of engineered cells (278). Other approaches to specifically eliminate engineered T cells include the co-transduction of CD20 and the use of rituximab to ablate engineered cells by ADC (279). Target antigens for ACT The modern age of cancer immunotherapy arguably began in 1991 with the discovery by Thierry Boon and colleagues of MART-1, the first human cancer antigen (280). Since then, a major area of focus in immunotherapy has been the identification and evaluation, both preclinically and clinically, of candidate target antigens overexpressed or aberrantly expressed by tumors (281). Perhaps predictably, initial efforts in ACT also focused on generating and utilizing T cells directed to such antigens. A plethora of clinical data have now led to the conclusion that targeting self-antigens using ACT likely requires the engineering of enhanced targeting specificity in T cells to overcome the fundamental impact of central tolerance on the endogenous T-cell repertoire (110). Indeed, an unbiased comparison of T cells specific for self-antigens, including cancer testis (CT) antigens, overexpressed by tumors and T cells specific for viral antigens has revealed that the latter have affinities for target antigens approximately one log higher compared with the former (61). As discussed above, engineering efforts have

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focused on affinity enhancement of TCR (282) or the use of high affinity CAR-based strategies. The definition of ideal target antigens is fundamentally different for TCR- and CAR- based strategies. For TCR-based strategies, ideal targets will likely be tumor-specific neoantigens, preferably shared across a significant fraction of tumors to enable systematic tumor targeting, or genderrestricted antigens that can be used to generate high affinity TCR in the non-expressing gender (70). Both of these target classes result in T cells with TCRs that have been shaped by negative selection in the thymus and thus have minimal potential for off target recognition of other tissues. In the case of CARs, because of the potency of this approach, ideal antigens will either have a tumor-specific expression profile, or will be uniquely expressed in non-essential tissues such as prostate, breast, and ovary, which can be removed prior to ACT to enable tumor-specific targeting. Indeed the potency of CAR-based approaches and the need for a relatively high threshold of tumor specificity has been highlighted by reported which have demonstrated significant serious adverse events (SAEs) when targeting antigens with low-level expression in normal tissues such as CAIX and ERBB2 (see below). If necessary, improvements can also be performed on CAR–based designs to enhance affinity and improve potency (141). Taking into account these considerations, precious few antigens have been identified that would qualify as ideal. In terms of neo-antigen targets for TCR, brute force efforts have in fact identified T cells which recognize tumorspecific neo-antigens; however, these neo-antigens appear to in general result from individual tumor-specific genetic events which result in non-self and thus non-tolerized proteins (283–285), with T cells and TCRs which are not amenable to platform development for ACT. In terms of CAR targets, the profound potency of CAR-engineered cells, related at least in part to the fact that each surface molecule for the target protein is a triggering epitope, requires a lower threshold of expression that is considerably lower than that required for therapies; this is best exemplified by the above mentioned SAE with CARs that utilized ScFv derived from clinically approved antibodies. Thus, ideal antigens from a CAR-perspective will have no expression in any non-targeted tissue. Practically speaking, in the emerging field of T lymphocyte engineering to manifest potent and redirected tumor specificity, where the physiologic and evolutionarily selected balance of recognition of self versus non-self is manipulated, the potential for off-target toxicity is a risk which ultimately will need to be addressed using

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comprehensive and innovative preclinical testing followed by evaluation in clinical trials. A conceptually attractive approach involves the development of combinatorial activation strategies, with T-cell activation requiring the simultaneous binding and triggering of multiple target unique CARs (286) and the use of CART ‘cocktails’ that also may help address issues related to the escape of antigen loss variants (287). Animal models, extensively used to evaluate toxicity for more traditional therapeutic agents, are considerably less useful in ACT because of lack of epitope conservation across species and also because they fail to capture the complexity of human T cells interacting with the broader immune system. The clearest demonstration of this is the recent report of SAE using high-affinity TCR redirected T cells against MAGE-3; despite extensive preclinical testing of this affinity enhanced receptor, ACT using cells engineered to express this receptor resulted in two deaths due to cardivascular toxicity as a consequence of off-target recognition of an epitope from titin, a protein expressed in differentiated cardiomyocytes (75, 288). Beyond targeting antigens expressed by tumors, ACT has also been applied to target T cells to molecules expressed by tissues associated with the biology proliferation and survival of tumors. This effort has principally been carried out as part of efforts to target solid tumors, and had brought into juxtaposition the biology of T cells and tumors. Post ACT T cells initially transit through the heart following the natural venous drainage and very quickly are transported to the lungs (289). Localization to lung is a critical initial site for T-cell trafficking due to potential low level expression of target antigens by lung epithelium; indeed, off-target recognition of lung epithelial tissue has been observed in a trial with Her2-specific CART, due to low-level expression of Her2 in lung epithelial cells (153). Rapid liver toxicity has also been recorded in anti-CA IX CART trial due to the expression of this marker in epithelium of the biliary tree (95). Different routes on ACT administration, such as intratumor or intraperitoneum, may reduce the potential toxicity (242, 290), although it apparent that, as expected biologically, T cells subsequently redistribute to secondary organs; indeed, analysis of tissues from post SAE biopsy of the titin-related SAE described above revealed broad lowlevel distribution of infused T cells in the majority of organs evaluated (75). Concern has been raised that T cells may be unable to actively migrate and extravasate into tumor parenchyma, limiting their effectiveness in vivo. Targeting the tumor vasculature using engineered T cells is one possible mechanism to facilitate efflux of T cells into © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


the tumor. Systemic administration of T cells engineered with CAR targeting avb3 integrin, expressed in tumor endothelial cells, led to extensive bleeding in tumor tissues with no evidence of damage to blood vessels in normal tissues; when this approach was coupled with co-delivery of nanoparticles increased deposition of nanoparticles was observed in the tumor (291). Combining ACT with agents that interfere with tumor vasculature such as avastin holds promise in this regard. At the tumor site, T cells face a dynamic and intricate tumor micro-environment (TME) composed of a complex mixture of tumor cells, endothelial cells, stromal cells, fibroblasts, myeloid cells, NKT cells, T cells, and regulatory T cells, and shaped by the selective pressure to support the tumor mass both by providing trophic signals and also by creating a potently anti-inflammatory and immunosuppressive milieu (292–294); these heterogeneous cell types may occupy up to 90% of the tumor mass (295), where they interact via soluble and cell-cell dependent mechanisms with each other, the tumor as well as the effector immune system. Tumor cells can modulate their growth environment through the activity of cytokines and chemokines leading to chemotactic effects on leukocytes, including monocytes and macrophages, suppression of the activity of the immune system, as well as regulation of neovascularization processes (296, 297); accordingly, targeting cells in the microenvironment has the potential to fundamentally disrupt the homeostatic milieu established at tumors thus increasing the effectiveness of cancer-targeted therapies (298). Immune cells, both innate and adaptive, are intimately involved in carcinogenesis by promoting tumor transformation as seen in chronic inflammatory diseases or tumor associated inflammation (299, 300). Tumor cells modify and affect the behavior of tumor infiltrating cells to promote the formation of a pro-neoplastic microenvironment (301). The relevance of the tumor-associated microenvironment for the efficacy of immunotherapeutic approaches has been documented in a number of clinical settings. To enumerate a few examples, the presence of TILs in tumor specimens is correlated with better clinical outcome in cancer patients (302, 303). Patients with cancer have elevated numbers of Tregs in the peripheral blood and within the tumor microenvironment (304), and tumor infiltration by Tregs is associated with poor outcome in many cancer subtypes (305, 306). In colon cancer a T-helper 1 (Th1) immune signature is predictive of better clinical outcome compared with a Th17 signature (305). High frequencies of tumor-associated macrophages (TAMs) are associated with poor prognosis © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

(307, 308). The length of survival among patients with follicular lymphoma correlates with the molecular features of non-malignant immune cells present in the tumor at diagnosis (309). The interplay of engineered T cells with these cell subsets and others is likely to play an important role for the development and establishment of potent and long antitumor immunity, as well as in mediating the immune-based adverse events associated with strong anti-tumor responses observed in recent CAR-based trials (27–29). Immune-suppressive mechanisms at play in the tumor microenvironment involve the suppressive action of regulatory T cells, MDSCs, TAMs and other myeloid-lineage derived suppressive cells, stromal fibroblasts and almost certainly other cell types not yet defined. These cells inhibit Tcell function through the production of immunosuppressive cytokines and other soluble factors, as well as through the expression of surface molecules that bind to inhibitory receptors such as CTLA-4, PD-1, TIM-3, BTLA, and LAG-3, and no doubt others, which are expressed on T cells often post activation and serve to modulate the T-cell response (15, 310). These suppressive cells are conditioned by the tumor microenvironment to become immunosuppressive and are also stimulated to migrate in the tumor site and proliferate (311, 312). MDSCs are directly correlated with chronic inflammation and with the production of IL-1,IL-6, reactive oxygen species (ROS), and nitric oxide (NO), while they also promote hypoxia, lactic acid production, and adenosine accumulation which inhibit APC maturation (313, 314). The presence of TAMs is generally associated with worse prognosis, with type 2 TAMs, which produce immunosuppressive cytokines for Th2 responses, showing poor antigen-presenting capacity and fail to activate T-cellmediated adaptive immunity (315, 316). Recent clinical experience using antibodies that block the function of inhibitory receptors have demonstrated the potency of this mechanism (22, 317, 318); this exciting line of research has led to the approval of the first in class ‘checkpoint inhibitor’ agent, YervoyTM, which blocks the functional activity of CTLA4, and the likely approval in the near future of agents that block the post-activation inhibitory function of PD1 on T cells and PDL1/PDL2 on tumors. An emerging body of clinical evidence is accumulating that correlate the presence of inhibitory receptors such as PD-1 in TILs correlate with distant metastasis and poorer outcome in renal cancer (319) and breast cancers (320). Tregs within the tumor microenvironment have been shown to markedly hinder the anti-tumor efficacy of adoptively transferred tumor-targeted effector T cells (321), and Treg-depleting

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strategies combined with systemic lymphodepletion reduce the number of Tregs in the tumor microenvironment and result in higher anti-tumor activity of T cells (322, 323). Tregs have been shown to also inhibit CART cells in animal models, with prior treatment with cyclophosphamide effectively reversing the suppression (324). Manipulating the microenvironment before T-cell infusion through non-myeloablative chemotherapy and lymphodepletion has been shown to lead to dramatic increases in the persistence of the transferred cells and robust anti-tumor activity (55, 56). The rationale of prior lymphodepletion was based on studies in tumor models in mice that showed improved persistence and anti-tumor activity of transferred T cells following conditioning chemotherapy (325); in these studies, improved engraftment and expansion was correlated with the ablation of resident lymphocytes that presumably competed for cytokines (IL-7 and IL-15) and T-cell growth factors. In particular, IL-15, which can provide a homeostatic growth stimulus for adoptively transferred T cells and can lead to the induction of a memory phenotype with enhanced effector functionality (56, 323, 326), is not normally detectable, but it was detected postlymphodepletion. Lymphodepletion also results in a reduction in the number of regulatory or inhibitory lymphocyte populations present in the tumor (327). Conditioning chemotherapy may also be useful to eliminate or reduce myeloid suppressor cells in the tumor microenvironment, while APCs may be activated by lymphodepletion with the increase of their susceptibility to Toll-like receptor signaling (328). The importance of the lymphodepletion has been confirmed in the clinical setting where most of the patients not receiving lymphodepletion prior to infusion of CART19 did not show any objective response of their malignancy (100, 329). The timing of lymphodepletion and conditioning relative to T-cell infusion and the particular conditioning regimen that are superior is unclear. Early infusion post conditioning seems to be optimal, with infusion on day +2 apparently superior to later time points in term of immune reconstitution after high dose chemotherapy and stem cell infusion (24, 330). A number of regimens have been employed for lymphodepletion, including TBI, fludarabine, and cyclophosphamide, high dose chemotherapy or bendamustine; however, no direct comparison of these protocols is available. Finally, it is possible that the optimal chemotherapeutic regimen will be unique to individual tumor types with disease specific to most effectively target both the microenvironment and the disease. Vasculature is another important mechanistic and structural component of the tumor milieu; accordingly, the targeting of

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tumor angiogenesis has been studied preclinically in the context of ACT, to evaluate the potential for synergistic effects between the two treatment modalities. Adoptive transfer of CAR T lymphocytes against VEGFR-1 delayed tumor growth and formation and inhibited pulmonary metastasis in xenograft models, an effect that was further enhanced by co-transfer of T lymphocytes that expressed IL-15 (331). In a separate study, simultaneous targeting of tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) and the tumor vasculature (VEGFR-2) using ACT revealed synergistic effects in terms of regression of established tumors in mice (332). Tumor stromal fibroblasts (TSFs) are one of the most prominent cell types in the tumor microenvironment of many human cancers such as pancreatic, gastrointestinal, and breast cancers. TSFs appear to play an active role in cancer progression by secreting factors that enhance tumor survival, growth, angiogenesis, and metastasis, in addition to recruiting other tumor-promoting cell types (333, 334). The conversion of TSFs to a tumor-promoting state is characterized by expression of surface proteins such as fibroblast activation protein (FAP), an endopeptidase with expression largely restricted to TSFs. Accordingly, FAP has been evaluated as a target for ACT-based approaches. Unfortunately, as recently reported, although highly reactive anti-FAP CARs had little impact on tumor progression in a variety of syngeneic mouse tumor implantation models, high doses of FAPreactive T cells were observed to induce severe cachexia and dose-limiting bone toxicity (335), underscoring the previously made point about the potency and need to critically evaluate the potential for off-target toxicity of CAR-based approaches. ACT-based strategies that utilize engineered T cells can also be applied to indirectly modulate the host immune system and the tumor microenvironment. For example, T cells engineered to express an NK2GD-targeting CAR were shown to recruit and activate host lymphocytes in a chemokinedependent manner, leading to the development of broader host immune responses (336). Clinical overview of ACT As discussed above, immunotherapy using TILs has a long clinical history, and this approach has provided important mechanistic and clinical insights about the potency of ACT. However, technical issues have prevented the broader implementation of this approach to target cancers. The ability to at-will genetically engineer and ex vivo manipulate and © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


expand potent T cells has brought about what promises to be the beginning of a golden age for ACT, with multiple ongoing clinical efforts, unprecedented positive clinical results, and a real possibility for late stage development and regulatory approval of ACT in the next few years. Here, we present a summary of the most salient clinical results obtained over the past few years that provide insights into the promise and pitfalls this exciting treatment modality. TCR-redirected T cells The first successful clinical experience with TCR redirected T cells was in the setting of melanoma using a low affinity HLA-A2-restricted anti-MART1 (clone DMF4). Two of 17 patients showed an objective response with partial tumor regression with no significant toxicity, with the infused T cells persisting at very low levels for more than 1 year (337). A subsequent trial utilizing T cells that expressed a high affinity anti-MART-1 (clone DMF5) or an anti-gp100 TCR resulted in objective responses in about 25% of the patients. This trial also demonstrated the first example of on target toxicity with 29/36 patients experiencing hearing loss, erythematous skin rash, vitiligo, and/or anterior uveitis due to the destruction of normal tissues that expressed the target antigens (67). A number of clinical trials have been conducted over the past few years to target members of the CT family of antigens. CT antigens are expressed in germ cell tissues but not in adult tissues, excluding the immune privileged testes, and are aberrantly expressed in the cytoplasm of various solid tumor cells, thus representing in principle ideal targets for ACT (338). NY-ESO-1 (New York-esophageal squamous cell carcinoma-1) is one such antigen, expressed in about 80% of synovial carcinoma, 30–40% of breast, urothelial, esophageal, thyroid, prostate, melanoma, hepatic, gastric cancers, and neuroblastomas (34). In a clinical trial at NCI, 4/6 synovial sarcoma patients and 5/11 melanoma patients showed objective clinical response following treatment with affinity-enhanced anti-NY-ESO-1 TCR-redirected T cells; importantly, and perhaps exceptionally - see below, no significant toxicities were recorded (31). A number of additional ACT-based clinical trials to target NY-ESO-1 in multiple solid tumors are currently ongoing. The MAGE (melanoma-associated antigen) group of CT antigens has also been targeted in multiple ACT-based trials. The MAGE family encompasses a large number of sequencerelated members which are expressed at various frequencies in a broad range multiple solid tumor types (339, 340). © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014

Three recent clinical trials targeting members of the MAGE family have served to highlight the previously theoretical safety concerns associated with the potency of TCR-engineered T-cell therapy. In one trial, T cells were engineered to express a TCR generated in HLA-A*0201 transgenic mice (i.e. not sculpted by the human immune system) and that recognized an epitope shared between MAGE-A3, MAGE-A9, and MAGE–A12. Of nine patients treated, five demonstrated objective clinical responses, but three patients demonstrated SAE associated with neural toxicity, including two deaths. Postmortem analysis revealed rare and previously unrecognized expression of MAGE-A12 in brain tissue (341). Two trials which evaluated the use of affinity enhanced HLAA*01-restricted and MAGE-A3-specific TCR to target melanoma and myeloma respectively. The first treated patient in each of these trials experienced SAE associated with cardiotoxicity and each patient died within 7 days of T-cell infusion (75). Extensive and detailed retrospective analysis demonstrated that the affinity enhancement of the TCR resulted in the off target recognition of a related epitope from the protein titin expressed in cardiomyocytes (75). TCRs to target carcino-embryonic antigen (CEA) have been also recently evaluated in ACT. CEA is a glycoprotein involved in cell adhesion and is expressed mainly in gastrointestinal tissue during fetal development. CEA is overexpressed in different cancers, in particular in colorectal cancer. An anti-CEA high affinity TCR was recently employed in a clinical trial to treat metastatic colon cancer. One of three patients treated showed objective response but all patients developed severe on target toxicity with inflammatory colitis. Transient clinical responses as manifested by reductions in circulating levels of CEA were observed in each of these patients (66). Other TCR-based target currently being preclinically evaluated include the human onco-protein MDM-2 (64), the p53 suppressor gene (65, 342), the tyrosinase melanocyte differentiation antigen, the prostate and breast cancer antigen TARP, the telomerase (343) and the Wilms’ tumor 1 antigen (WT-1) (344, 345). A recent clinical trial evaluated the infusion of autologous T cells genetically modified for the expression of a HA1-specific TCR to leukemia patients relapsing after HSCT (346). CAR-redirected T cells The clinical evaluation of CAR-redirected T-cell-based ACT has exploded over the past few years, with more than 25 clinical trials of CART that are actively recruiting, as registered in

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clinicaltrials.gov (as of 15 July 2013). Among these, half are evaluating CART-19 in B neoplasms, three CART against Her2 in GBM and sarcoma, and the remaining different other targets (GD2, j light chain, CD30, EGFR, CD33, CD138, FAP, CEA, mesothelin). Initial clinical studies with 1st generation CART targeting neuroblastoma, lymphoma, renal, and ovarian cancers were disappointing, with limited clinical activity, lack of in vivo expansion and long-term persistence in vivo (93–96). Improvements of CAR engineering led to more positive clinical outcomes and also revealed the potential for SAE. Two studies in particular, targeting carbonic anhydrase -9 (CAIX) in renal carcinoma patients using 2nd generation CARs and ERBB2 in colon cancer using 3rd generation CARs demonstrated considerable on-target off-tissue toxicity, with liver toxicity due to targeting of CA-IX-positive biliary epithelium (148) and death from respiratory distress syndrome due to the activation of T cells presumably in response to low expression of ERBB2 by lung epithelium (153). Other notable studies include the targeting of the glycolipid antigen GD2 on neuroblastoma with Epstein-Barr virus (EBV)specific T cells engineered to express 2nd generation CARs, which resulted in tumor regression or necrosis in 4/8 patients, with one CR and a PR, and persistence of infused cells (155–157, 184). Most of the dramatic recent success of CART-based ACT has been achieved in hematological tumors, in particular targeting the CD19 antigen which is expressed during B-cell development from the early B-cell progenitor (pro-B cell) to the mature B cells and possibly follicular DCs and by almost all B-cell malignancies. The absence of expression of this target in healthy tissues other than B cells has made it an ideal antigen to target for adoptive immunotherapy. Infusion of CD19-specific CART cells targeting CD19 showed to be effective in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and nonHodgkin lymphomas (NHL) (27, 29, 30, 347). The first patients treated with second generation CART19 were at the NCI. These patients received anti-CD19 CARs containing CD28 and CD3f costimulatory domains; this treatment resulted in significant regression of tumor and a partial remission the results of the first eight patients subsequently enrolled in this trial were recently reported, with six of eight patients achieving an objective clinical response, accompanied by transient B-cell ablation (130, 347). A recently published trial (30) from the MSKCC group in ALL applied CART therapy as a bridge toward allogeneic transplant; this study showed activity of infused CART19-28f in

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all five treated patients, including two patients with significant disease burden. The CART treatment was followed by allogeneic cell transplant in 4/5 patients, precluding analysis of long-term efficacy for the CART cells (30). At Baylor College of Medicine, six patients with relapsed or refractory NHL were treated with a combination of first- and secondgeneration CART19 in the absence of lymphodepletion; second generation CARs showed enhanced persistence in this study. Despite transient stabilization of lymphoma in two patients, none showed evidence of sustained tumor regression at the cell doses used (100). Perhaps the most dramatic results to date have been reported from our group at the University of Pennsylvania in the setting of adult CLL (27, 28) and pediatric ALL (29). These studies employ second generation CARs with 4-1BB and CD3f costimulatory domains, and lentivirus-engineered T cells, expanded using anti-CD3 and -CD28 coated beads, and infused 2 days after lymphodepletion. In these studies, CART cells were able to eradicate very large tumor burdens and mediate complete and ongoing complete remissions, with ongoing functional persistence of CART cells now beyond 3 years in 2/3 initial CLL patients and 1/2 ALL patients, as revealed by ongoing B-cell aplasia. SAE of varying severity related to delayed tumor lysis syndrome, cytokine release syndrome, macrophage activation syndrome, and hemophagocytic lymphohistocytosis, all of varying severity are common features for responding patients in these trials. Elevated levels of a number of cytokines and chemokines, including IFNc, IL-6, MIP1a, MIp1b, MCP-1 point to the broad and systemic immune response unleashed by this potent therapy. Tociluzimab, a humanized monoclonal antagonistic antibody against the IL-6 receptor, has been identified and implemented as a key interventive treatment to mitigate the impact of the cytokine cascade (348). Concluding thoughts More than 18 years from the first clinical demonstration of the feasibility of T-cell adoptive transfer (349), the field is poised to capitalize on the multitude of lessons learned as a result of combining the commitment, insights, and innovations of a multitude of scientists combined with the profound bravery and generosity of patients. ACT using engineered T cells is demonstrating dramatic potency in clinical trials, leading to complete and durable responses in patients with late stage and treatment refractory disease, and commercialization of this emerging technology is likely to occur in the next few years (26). © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 257/2014


In this review, we have highlighted and expanded on a number of topics of direct relevance for the application of engineered T cells to ACT, within space and scope constraints. In this regard, a number of topics relevant to the specific implementation of this approach were not fully developed but merit description. Ultimate effective implementation of T-cell-based ACT is likely to require innovation and co-development of at least some of these areas, as summarized here: (i) the issue of targeting aggressive versus indolent disease, exemplified by T-cell inhibitory effects mediated by CLL cells (350), and the effect of indolent diseases on creating an immunosuppressive microenvironment, as illustrated by the differential effect of immune modulating drugs, such as lenalidomide and bortezomib) in the indolent setting; (ii) the issue of targeting solid tumors in general as well as the differential requirements for targeting individual tumor types such as for example pancreatic cancer which has been described as resistant to T-cell infiltration (351). Although the discussion above with regard to tumor microenvironment is related to this topic, tumor type-specific and perhaps even patient-specific issues related to individual tumor biology, architecture, heterogeneity, and metabolism may well impact potency and effectiveness of ACT; accessing the relevance and

resolving such issue is likely to require the application of broad, integrated, and quality supported assay platforms to identify relevant correlations (352). (iii) Tumor burden must be considered. With ACT representing a unique paradigm that involves the in vivo interaction of two viable cell populations, with the potential for T-cell potency, expansion, and persistence paradoxically dependent on higher tumor burdens as reflected by the more profound cytokine elevations observed in the context of higher disease burdens (28–30). (iv) Strategies that combine ACT with the use of agents that impact tumor biology such as demethylating agents (353), tumor signaling, metabolic pathway and cell cycle inhibitors (354–356) will need to be further investigated. The development of such combination strategies, also including checkpoint inhibitor combinations as discussed above, has the potential to unleash the fullbreadth of the immune response against tumors with potentially profound anti-tumor activity. (v) Finally, issues of scale-up, automation, commercialization, and intellectual property (26, 247), addressing at large scale regulatory hurdles unique to cell therapy (357), and identifying and implementing reimbursement models will increasingly need to be addressed as this field further matures and is applied to treat patients at large scale.

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